FoodIntegrity Handbook Compressed
FoodIntegrity Handbook Compressed
FoodIntegrity Handbook Compressed
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Table of contents
Animal Products
Milk and milk products .............................................................................................................................................................................................
33
J. S. Amaral, I. Mafra, A. Pissard, J. A. Fernández Pierna, V. Baeten
Eggs and egg products ......................................................................................................................................................................................... 27
M. Suman, D. Cavanna, M. Zerbini, D. Ricchetti, D. Sanfelici,
E. Cavandoli, L. Mirone
Honey ............................................................................................................................................................................................................................................... 43
K. P. Raezke, E. Jamin, M. Lees
Meat and meat products .................................................................................................................................................................................. 61
E. Valli, M. Petracci, M. Pezzolato, E. Bozzetta
Plant Products
Cereals and cereal-based products – Wheat, Rice ................................................................................................ 101
J.-F. Morin, M. Lees, P. Vermeulen, V. Baeten, E. Maestri, N. Marmiroli
Nuts, nut products and other seeds ............................................................................................................................................ 127
E. Maestri, D. Imperiale, N. Marmiroli
Cocoa, cocoa preparation, chocolate and chocolate-based confectionery ...................
137 137
M. Rektorisova, M. Tomaniova
Plant-derived sugars and sweeteners ....................................................................................................................................... 155
F. Thomas, D. Hammond
- III -
Table of content
Food Additives
Food flavourings ......................................................................................................................................................................................................... 383
E. Jamin, F. Thomas
Determination of species origin of gelatine in foods ........................................................................................ 391
H. H. Grundy
Additional tools for mitigating the risk of food fraud ................................................................................................ 403
J.-F. Morin, M. Lees, P. Rinke, P. Olsen, M. Svorken, S. Elde, P. B. Sørdahl
― ii ―
- IV -
Foreword by Mr. Van Goethem
Director DG Health and Food Safety, European Commission
The past half a century has seen a revolution in the way that
food is produced, processed and marketed. Today, EU citizens
are accustomed to choice, convenience, quality, and
competitive prices when it comes to the food they buy.
The complex nature of our globalized food supply chains and
the economic motivation to provide cheaper food products
have contributed to the growing problem of food fraud, with
recent scandals such as horse meat in beef products drawing
worldwide attention. Fraudsters are becoming increasingly
inventive in the deceptive tactics they are deploying to take advantage of the sophisticated nature
of food supply chain. Because of its complexity and worldwide reach, reining in food fraud requires
a collaborative effort between industry and government agencies. Preventive systems identifying
problems at an early stage, preparedness at all levels and coordination are essential.
In this respect, the European Commission has taken action in creating the EU Food Fraud Network.
EU Member States, supported by a dedicated IT system, can now rapidly exchange information on
potential cross-border fraud. The Knowledge Centre for Food Fraud and Quality created on 13
March 2018 and operated by the Commission's Joint Research Centre complements the EU Food
Fraud Network by providing an interface between science and policy-making.
At industry level, many companies have already implemented ways to counter global fraud
threats, but more needs to be done. Industry alarms should go off whenever a commodity
suddenly floods the market at a too-good-to-be-true price. New analysis tools are appearing to
help alert industry and regulators in real time to potential problems. Some of the responsibility
also falls upon consumers to remain vigilant and speak up when they witness what they believe to
be fraudulent practices. Manufacturers can support this effort by helping consumers identify
issues, giving them the resources to identify fraudulent products so that they know what to look
for to avoid these products. Transparency and data-sharing between national governments,
agencies and industry is key to detect and prevent fraudulent practices. The Food Integrity Project
contributes to this goal by gathering experts from industry, academia, research institutes,
technology providers and a global network of stakeholders. It is an international focal point for
harmonisation and exploitation of research and technology for insuring the integrity of European
food.
Thanks to thorough and detailed guidance, this Food Integrity Handbook will be an important
resource to all of us when looking for information on food authenticity issues and will help to
create a trusted food sector. The authors and all those who contributed to the handbook deserve
special recognition for their work.
-V-
Foreword by Pr. Christopher Elliott OBE
Founder of the Institute for Global Food Safety
Queen's University of Belfast
The first handbook on food authenticity was published 20 years ago and it is
quite remarkable to track the events that have unfolded over this period of
time. Food authenticity or food fraud or as sometimes referred as food
crime has become a widely discussed issue in many parts of the world. The
melamine scandal in China dating back a decade now seems to have been
the trigger to alert many stakeholders in governments, food industries and
more importantly the general public of the impact cheating in the global
food supply system can have.
The new edition of the handbook has sought to address the growing
complexities of food and drink authenticity. It seems the ingenuity of those who set out to cheat
us all knows no bounds. It also seems that virtually everything we eat and drink has some
vulnerability to fraud and that individuals and potentially organised crime gangs will try to exploit
these.
A major element in the fight against fraud is the development, validation and implementation of
novel methodologies that can detect and often quantify the level of cheating that has occurred.
There have been many innovations in analysis over the past two decades and the handbook gives
some excellent examples of these and how they can be applied.
Another interesting and very worthwhile addition to this handbook is the horizon scanning which
the authors have conducted. What will be the major challenges over the next 20 years and how
will analytical science provide some of the solutions.
- VII -
Introduction
The first “handbook” of this type dealing with food authenticity was published in 1998, the result
1
of European collaboration through the EU-funded Concerted Action FAIM which brought together
over fifty scientists from food research institutes, from industry, from regulation authorities and
from private laboratories. Their aim was to review the authenticity issues current at that time and
to investigate the availability of analytical methods to address those concerns. The FAIM handbook
contributors are given a special mention in the Acknowledgements section at the end of this book.
2
Twenty years on, through another, albeit much larger, European funded project, Food Integrity , a
similar group of scientists have collaborated to produce an updated handbook on food
authenticity issues and related analytical methods. A lot has changed in twenty years. The Food
Integrity Handbook is not simply a revised version of the earlier FAIM book but does complement
it in several ways. It has retained a very similar structure, which is repeated throughout the
different chapters. On the other hand, it deals with a wider range of food products; it includes
chapters on eggs and egg products, nuts, nut products and seeds, plant-derived sweeteners,
spices, wines, spirit drinks, tea, flavourings and gelatine, in addition to the main food commodities
- cereals, coffee, dairy products, fish and meat products, fruit juices, honey, oils and fats – dealt
with in the FAIM Handbook. In addition this new Handbook does not have a separate chapter on
the use of Chemometrics in food authenticity studies, which in the FAIM book reviewed some of
the most important and useful concepts in multivariate chemometric/statistical methods. Two
decades ago such concepts were still fairly new in food science whereas today they are widely
used in the analytical field.
The Food Integrity Handbook has been written for food business operators and is primarily aimed
at quality control managers working in food production and to those actors involved in the food
supply chain. It may also be useful to young scientists starting their career in food science and to
students and researchers with little prior knowledge of the area. The first section of this book
provides the definitions of Food Authenticity and the different concerns that constitute Food
3
Fraud, compiled in connexion with the work being undertaken in the Authent-Net project to
establish a European voluntary standard (a CEN Workshop Agreement, or CWA) entitled
"Authenticity in the feed and the food chain - General principles and basic requirements".
1
FAIM : Food Authenticity – Issues and Methodologies. Funded by the European Commission’s Agro-Industrial Research
Programme under project No AIR2-CT94-2452. 1994-1998.
2
Food Integrity - Assuring quality and authenticity in the food chain. Funded by the European Union’s Seventh Framework
Programme for research, technological development and demonstration under grant agreement No 613688. 2014-2018.
3
Authent-Net – Food Authenticity Research Network. Funded under the European Union´s Horizon 2020 research and
innovation programme under grant agreement No 696371.
- IX -
Introduction
Since this Handbook is intended as a simple, searchable guide to food fraud / food authenticity
issues organised by product type, each of the food group chapters follows a similar structure,
making it easier for the reader to find the information they are looking for. The format starts with
a general overview of the product, with a short introduction to the industry sector, its importance
in the global market, and how it may have changed in recent times as a result of consumer
pressure or the implementation of new technology. Food products may be marketed in different
forms, some of which may be linked to a specific manufacturing process or method of agricultural
production. This type of knowledge is important in order to explain certain fraudulent practices,
and essential when interpreting data from analytical tests.
Most food products are defined by a set of specifications, or “standard of identity”, whether in
legislation or in industry sector guidelines. These specifications, which often include specific
compositional characteristics, form the basis for describing a foodstuff and for highlighting any
deviations from this composition that could be due to mislabelling and/or economic adulteration.
Current standards of identity or related legislation, both in the European Union and on the
international level are provided for the different food commodities where available.
The prime focus of this Handbook is of course food authenticity and the analytical solutions
available to address existing concerns. These are described in detail for each food product, starting
with food fraud problems that are currently facing the food industry or have occurred in the past.
Although the main motivation for food fraud is economic, there is increasing concern among both
regulatory authorities and consumers about the potential health risk of a fraudulent practice. For
example a cheap extender might be used that is allergenic, as in the case of nut protein in cumin
spice. Where relevant, examples of the potential threat to human health are provided.
Then follows a review of the analytical methods used to test for authenticity. In this section care
has been taken to highlight those methods that are most commonly used, and in particular, those
that are officially recognised. In recent years, increasingly sophisticated techniques and
instrumentation have been developed to detect adulteration and misrepresentation. These latest
methods are included where they are accessible to routine use. When comparing the original FAIM
Handbook and this present one, two major areas of analytical investigation stand out as different.
One of these is the use of DNA techniques which, for a majority of the food products dealt with in
the FAIM handbook, were only mentioned under the section on “potential methodologies” as
techniques requiring further efforts before being accepted as routine procedures. As can be seen
in this book, DNA techniques are now routinely and officially used for authentication purposes.
One example is the verification of specific Basmati rice varieties from India and Pakistan that,
under EU Regulations, are granted a zero rate of import duty on presentation of an authenticity
certificate based on DNA analysis. The other major difference is the breakthrough in untargeted
methods. These approaches employ various spectroscopic and/or chromatographic techniques,
which can provide an entire analytical profile of a food product which can then be used to judge its
authenticity. The most established in this area is based on NIR (Near Infrared) spectroscopy which
is now widely used in various sectors and is particularly suitable as a rapid method for the at-line
1
and on-line use (see for example the chapter on Cereals). A further example employs H-NMR
(Nuclear Magnetic Resonance) screening which, provided that appropriate statistical models have
been established from authentic material beforehand, can evaluate a large number of analytical
parameters related to quality and authenticity, simultaneously and in a few minutes (see the
example given in the chapter on fruit juices).
One of the purposes of this Handbook is to help small and medium companies with setting up food
fraud mitigation plans and therefore a section on additional tools for mitigating food fraud risk has
been included towards the end of the document. This section provides information on the
― ii ―
-X-
Introduction
different approaches to evaluate food fraud vulnerability, the importance of traceability in the
food supply chain, and a “best practice” example of sector specific food fraud mitigation.
But what will the future look like in the next twenty years? The authors of each of the food
product chapters have been asked to give their expert opinion on potential authenticity issues to
look out for in the future, such as those that may not be economically viable now but may become
so due to changing geopolitical situations, the effects of climate change and so on. They also
provide an insight into where current research on authenticity techniques is heading and which
analytical methods are on the horizon.
Food Fraud has been around a long time but following several highly mediatised incidents such as
the milk and infant formula contaminated with melamine in 2008 and the horsemeat scandal in
2013, all authenticity issues have become big news. Regulators and customers now require food
operators to keep abreast of any potential risks and to regularly assess their raw material and
ingredient supply chains for vulnerability to food fraud. It is hoped that this Food Integrity
Handbook will be a useful companion to help the food industry achieve this aim.
― iii ―
- XI -
Definition of food fraud
and food authenticity
The notion of food fraud and food authenticity received increased focus as a field of investigation
by the research community and the food industry in the late 2000s, following highly mediatised
crises such as the melamine scandal in China in 2007 and Horsegate in 2013, with adverse impact
on vulnerable consumers for the former. These incidents led stakeholders to request a clear
definition of food fraud including the identification of the different types of fraud as a first step
toward combatting these practices.
Some early work was carried out in the United States by the Grocery Manufacturers Association
[1], Michigan State University [2] and the US Pharmacopeial Convention (USP) [3]. After the
horsemeat scandal, a series of high level reports was published by some national health
authorities [4–6]. They all highlighted the importance of the standardisation of the terms related
to food fraud.
In 2012, a ‘Food Fraud Think Tank’ was set up with the support of the Global Food Safety Initiative
(GFSI), an public-private initiative, to explore food fraud issues. It published a document on food
fraud mitigation in which it provided some definitions of the different types of fraud [7]. This
collaborative work, some members of which took part in the FoodIntegrity project over the
following years, is the basis of this chapter. Most of the definitions used in the following pages
refer to it.
During another European research project named Authent-Net [8], a standardisation initiative
within the framework of a "CEN/CENELEC Workshop Agreement" (CWA) has been launched in
order to set up a first consensus-based terminology of authenticity and food fraud [9]. This
working group has received input from scientists, industry organisations and other ongoing
research projects, and in particular from FoodIntegrity. It has made terms and concepts related to
food fraud clearer and more accurate, thus enriching the GFSI definitions. It is expected that this
work will lay down the basis for future internationally standardised definitions.
The Codex Alimentarius Commission has established an electronic working group (eWG), chaired
by the Islamic Republic of Iran and co-chaired by Canada and the European Union, whose mission
has included the clarification of the definitions of food integrity, food authenticity, food fraud and
economically motivated adulteration (EMA) in relation to Codex Committee on Food Import and
Export Inspection and Certification Systems (CCFICS) texts. They have published a position paper
[10] where key elements identified underlying these notions have been identified and definitions
developed. This position paper will be used as a basis for initiating new work in this area, so as to
provide guidance on how to assure the authenticity of food by minimising vulnerability to fraud
and mitigating the consequences of food fraud.
- XIII -
Definition of food fraud and food authenticity
Intentional
Food Fraud adulteration
Unintentional
adulteration
Food Safety
Accidental
Food borne illness
Figure 1: Difference between food fraud, food defense, food safety and food quality. Food fraud.
Adapted from Food Fraud Think Tank [7]
But when the action is intentional, then the behaviour can be considered a crime. When the
motivation of the criminal is to harm people, the type of action falls in the field of "Food defence"
according to the GFSI Food Fraud Think Tank. It can be even qualified as terrorism when the action
aims at gravely disturbing public order. When the intention is only economical, the action can be
considered as food fraud.
The notion of economic gain has been endorsed in the CWA: it is stated that "financial gain is the
most common motivation for food fraud", as well as the intentional factor. The definition of food
fraud that has been agreed on in this document is:
Food fraud: an action "intentionally causing a mismatch between food product claims and actual
food product characteristics, either by deliberately making claims known to be false or by
deliberately omitting to make claims that should have been made."
As most food products are produced and sold according to relevant regulations and requirements,
food fraud also occurs when some aspect of the production violates these requirements or
regulations.
― ii ―
- XIV -
Definition of food fraud and food authenticity
In the same way, the definition of an "Authentic food product" given by the CWA is very close to
that of food fraud:
An authentic food product is "a food product where there is a match between the actual food
product characteristics and the corresponding food product claims; when the food product actually
is what the claim says that it is."
It should be noted that the definition of food fraud developed by Codex Alimentarius position
paper identified as key elements: deliberate intent, deception, financial gain and misre-
presentation. The document provided by the eWG considers food fraud as being intimately linked
to food integrity: food fraud is 'any deliberate action of businesses or individuals to deceive others
in regards to the integrity of food to gain undue advantage'.
The document also distinguishes 'food authenticity' and 'food integrity': both are a status of a food
product, but the former is the state of being 'not altered or modified with respect to expected
characteristics including, safety, quality, and nutrition', while the latter is the state of being
'genuine and undisputed in its nature, origin, identity, and claims, and to meet expected
properties'.
One of the most common frauds is adulteration. According to the CWA, it is:
"A type of food fraud which includes the intentional addition of a foreign or inferior substance or
element; especially to prepare for sale by replacing more valuable with less valuable or inert
ingredients."
This practice is sometimes referred to as Economically Motivated Adulteration (EMA). This term is
defined in the Codex Alimentarius position paper. It is recognised as 'a subset of food fraud'.
Different types of adulteration can occur in food products. Their definition is given below in italic.
Substitution is the "process of replacing a nutrient, an ingredient or part of a food (often one with
high value), with another nutrient, ingredient or part of food (often one with lower value)."
Examples of substitutions are substituting low value fish species for how value fish species when
selling processed products (fillets, fish pies, etc.), substituting milk protein with hydrolysed leather
protein or sunflower oil partially substituted with mineral oil.
Dilution is 'the process of mixing a liquid ingredient (solute) with high value with a liquid of lower
value'. The action addition of water to Not-from-concentrate (NFC) fruit juice or to milk is an
example of this.
Unapproved enhancement is the "process of adding unknown and undeclared compounds to food
products in order to enhance their quality attributes". The melamine in milk falls under this
category, as adulteration with melamine in milk products aimed at enhancing nitrogen content in
― iii ―
- XV -
Definition of food fraud and food authenticity
already diluted milk. Use of unauthorised additives, such as Sudan dyes in spices, is another
example of unauthorised enhancement.
Concealment is the "process of hiding the low quality of food ingredients or products". Injecting
poultry with hormones to conceal disease is an example of this, as well as meat treated with
carbon monoxide.
The other types of food frauds identified by the GFSI Food fraud Think Tank have not been
specifically defined in the CWA. However, they are commonly used in a number of scientific
publications, including this book. A definition of these terms was drafted by the FoodIntegrity
experts when they designed the FoodIntegrity Knowledge Base (see the dedicated chapter of this
book).
Grey market: this term includes production, theft, and diversion involving unauthorised sales
channels for products. An example of this is the sale of excess unreported product when there are
production agreements or quotas for the product and the product in question is deliberately
produced in excess of these. A fish product originating from illegal, unreported, and unregulated
(IUU) fishing is another example. This term also applies when there is a geographical restriction on
the sale and distribution of the product, and the product in question is deliberately sold or
distributed in other areas; this is often referred to as “grey market” sales.
Counterfeit is a case when where Intellectual Property Rights (IPR) infringement is in effect. This
could include any or all aspects of the other product or packaging being fully replicated, for
instance the process of copying the brand name, packaging concept or processing method for
economic gain. Imitation wines and spirits with fake labels of a popular brand is a classical example
(see the chapter on Spirits).
Adulterations
Substitution
Dilution Concealment
FOOD
Unapproved
FRAUD
Mislabelling
enhancement
Grey
Counterfeit
Market
Figure 2: Terminology of food frauds. Adapted from Food Fraud Think Tank [7]
― iv ―
- XVI -
Definition of food fraud and food authenticity
Mislabelling is a special case of food fraud. It concerns the process of putting false claims on
packaging for economic gain. Selling farmed salmon as wild salmon, or conventional fresh produce
as organic are examples of this fraud. Expiry date modifications fall under this category. However,
mislabelling may apply to all forms of food fraud: to be efficient, a fraudulent product must indeed
be "mislabelled" to be purchased by a buyer. But the expression is mainly used to indicate
distortion of the information provided on the label.
The Codex Alimentarius position paper has also identified seven different types of food fraud.
Although their designations are slightly different ('simulation' is used instead of 'concealment', for
instance), they overlap and are consistent with the definition of the GFSI and CEN Workshop
Agreement.
Bibliographic references
1. Grocery Manufacturer Association (GMA) (2010). – Consumer product fraud: deterrence and detection. Available at:
https://www.gmaonline.org/downloads/research-and-reports/consumerproductfraud.pdf.
2. Spink J. & Moyer D.C. (2011). – Defining the Public Health Threat of Food Fraud. J. Food Sci., 76 (9), R157–R163.
doi:10.1111/j.1750-3841.2011.02417.x.
3. Moore J.C., Spink J. & Lipp M. (2012). – Development and Application of a Database of Food Ingredient Fraud and
Economically Motivated Adulteration from 1980 to 2010. J. Food Sci., 77 (4), R118–R126. doi:10.1111/j.1750-
3841.2012.02657.x.
4. European Parliament (2013). – Report on the food crisis, fraud in the food chain and the control thereof.
2013/2091(INI). Available at: http://www.europarl.europa.eu/sides/getDoc.do?pubRef=-//EP//TEXT+REPORT+A7-
2013-0434+0+DOC+XML+V0//EN&language=fr.
5. Elliott C. (2014). – Elliott Review into the Integrity and Assurance of Food Supply Networks – Final Report. A National
Food Crime Prevention Framework. Available at:
https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/350726/elliot-
review-final-report-july2014.pdf.
6. Johnson R. (2014). – Food Fraud and “Economically Motivated Adulteration” of Food and Food Ingredients. Congr.
Res. Serv. CRG, R43358. Available at: https://fas.org/sgp/crs/misc/R43358.pdf.
7. Global Food Safety Initiative (GFSI) (2014). – MyGFSI - Food Fraud Mitigation. Available at:
https://www.mygfsi.com/files/Information_Kit/GFSI_GMaP_FoodFraud.pdf.
8. Authent-Net project. H2020 coordination and support action. Grant agreement n° 696371 Available at:
http://www.authent-net.eu/.
9. CEN WS/86 - Authenticity in the feed and food chain – General principles and basic requirements (To be published).
Available at: https://www.cen.eu/work/areas/food/Pages/WS86.aspx.
10. Codex Alimentarius (2018). – Discussion paper on food integrity and food authenticity - Joint FAO/WHO food
standards programme. Codex committee on food import and export inspection and certification systems. Twenty-
Fourth Session. Brisbane, Australia, 22 - 26October 2018. CX/FICS 18/24/7. Available at: http://www.fao.org/fao-
who-codexalimentarius/sh-
proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FMeetings%252FCX-
733-24%252FWorking%2BDocuments%252Ffc24_07e.pdf.
―v―
- XVII -
Milk and milk products
Joana S. Amaral*
Centro de Investigação de Montanha (CIMO), Instituto Politécnico de Bragança, Portugal
REQUIMTE-LAQV, Faculdade de Farmácia, Universidade do Porto, Portugal
*E-mail corresponding author: [email protected]
Isabel Mafra*
REQUIMTE-LAQV, Faculdade de Farmácia, Universidade do Porto, Portugal
*E-mail corresponding author: [email protected]
https://doi.org/10.32741/fihb.1.milk
-3-
Milk and milk products
1. Product Identity
1.1. Definition of the product and manufacturing process
According to the Codex Alimentarius, milk is the normal mammary secretion of milking animals,
without either addition to it or extraction from it, intended for consumption as liquid milk or for
further processing, while a milk product refers to a product obtained by any processing of milk,
which may contain food additives, and other ingredients functionally necessary for the processing.
Milk and milk products encompass a wide range of products consumed worldwide including liquid
milk, fermented milks and products thereof, cheeses, butter, ghee and dairy fat spreads,
condensed milk, evaporated milk, cream, milk and cream powders, whey products and casein.
Liquid milk, including raw milk and products such as pasteurised, skimmed, ultra-high-temperature
(UHT) and fortified milk, is the most consumed, processed and marketed dairy product [4].
Fermented milk, obtained using suitable microorganisms, is generally used to produce dairy
products such as yoghurt and kefir, among others. Cheese is the ripened or unripened soft, semi-
hard, hard, or extra-hard product, obtained through the coagulation of milk protein by rennet,
other suitable coagulating agents or processing technologies, and in which the whey
protein/casein ratio does not exceed that of milk [5]. During this process, whey is also obtained,
corresponding to the liquid part that remains after the separation of the curd. Whey can be used
for several purposes such as the preparation of whey cheese, whey powder, whey drinks and for
different industrial purposes [4]. Butter, ghee and dairy spreads are fatty milk products in the form
of a water-in-oil emulsion. Cream is the fluid milk product comparatively rich in fat, in the form of
an emulsion of fat-in-skimmed milk, obtained by physical separation from milk [4,6], and can give
raise to a wide range of products such as whipping cream, whipped cream, acidified cream, among
others. Condensed and evaporated milks are both obtained from the partial removal of water
from whole or skimmed milk, with the first being frequently used in the form of sweetened
condensed milk. Milk powders are obtained from the dehydration of milk and include several
products such as whole milk powder, partly skimmed milk powder, skimmed milk powder and
cream powder.
―2―
-4-
Milk and milk products
1.2.2. EU Legislation
General principles and requirements of food law, establishing the European Food Safety Authority
and laying down procedures in matters of food safety have been stated in Regulation (EC) No
178/2002 [12]. The general rules for food business operators on the hygiene of foodstuffs have
been laid down in Regulation (EC) No 852/2004 [13]. Specific hygiene rules for food of animal
origin are covered by Regulation (EC) No 853/2004 where a section is specifically dedicated to raw
milk and dairy products (Annex III, Section IX). Finally, Commission Regulation (EC) No 1664/2006
deals with measures for certain products of animal origin intended for human consumption, in
particular the testing for raw and heat-treated milk [14].
Rules on the common organization of the market in milk and milk products for drinking milk
(Council Regulation (EC) No 1153/2007) are now considered in Regulation (EU) No 1308/2013 of
the European Parliament and of the Council of 17 December 2013 [15].
Milk can easily be contaminated by micro-organisms that are naturally present in the environment
or which originate from diverse human activities. Therefore milk and dairy products have been
extensively covered in EU legislation. The European Parliament and Council have established
specific rules for the organisation of official controls on products of animal origin intended for
human consumption (Regulation (EC) No 854/2004) [16].
Regarding quality evaluation, the Commission has published methods for the analysis and quality
evaluation of milk and milk products eligible for public intervention and aid for private storage
(Commission Implementing Regulation (EU) 2018/150 of 30 January 2018) [17]. More specifically,
the Commission has issued new rules on caseins and caseinates intended for human consumption
with EU Directive 2015/2203 [18].
Requirements on microbiological criteria have been amended by Commission Regulation (EC) No
1441/2007 concerning microbiological criteria for foodstuffs with regards to milk and dairy
products and Commission Regulation (EU) No 365/2010 on microbiological criteria for foodstuffs
as regards Enterobacteriaceae in pasteurized milk and other pasteurized liquid dairy products and
Listeria monocytogenes in food grade salt [19, 20].
The Commission has also ruled on maximum residue levels for several pesticides in or on certain
products (Commission Regulation (EU) 2018/686 and 2018/687 of 4 May 2018) [21,22]. Very
recently, a Commission Implementing Regulation (EU) 2018/555 concerning a coordinated
multiannual control programme of the Union for 2019, 2020 and 2021 has been established to
ensure compliance with maximum residue levels and to assess consumer exposure to pesticide
residues in and on food of plant and animal origin; the date of entry into force is however
unknown at the date of writing this text (pending notification) [23].
―3―
-5-
Milk and milk products
1.2.5. US Regulation
In the United States (US), the Department of Agriculture (USDA) and the Food and Drug
Administration (FDA) regulate milk production and its guidelines are some of the strictest in the
industrialised world. Farmers, processors and government agencies all work together to ensure
the milk is safe and of the highest quality. The US FDA edits Guidance Documents and Regulatory
Information including Coded Memoranda Issued by the Milk Safety Branch, Interstate Milk
Shipments and Dairy HACCP.
The Pasteurized Milk Ordinance (PMO) of the US FDA serves as the basic milk sanitation standard
for National Conference on Interstate Milk Shipments (NCIMS) members (all 50 states and Puerto
Rico). Pasteurized Milk Ordinance, revised in 2015, presents the most current developments in
milk sanitation for ‘Grade A’ milk and milk products [27].
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2. Authenticity issues
2.1. Identification of current authenticity issues
Milk has been considered one of the seven foods most vulnerable to economically motivated
adulteration. Due to the high demand for milk and the value of some dairy products, fraud in the
dairy industry has become a widespread problem and a real concern for many consumers and
authorities since adulteration invariably reduces product quality and may introduce hazards that
can jeopardise health. Over the last decades, there has been an increasing interest in the quality
evaluation and authentication of milk and milk products, in order to ensure consumer protection,
avoid unfair competition among producers and improve general confidence in the sector.
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milk species other than cow, in particular for goat’s milk, due to its superior nutritional features
and other aspects such as its attractive odour and taste, and superior digestibility when compared
to cow’s milk [36]. Additionally, according to some authors, goat’s milk can be a possible
alternative to cow’s milk because it is considered less allergenic [37]. In this case, the undeclared
presence of cow’s milk could be a potential health risk for allergic consumers. Nevertheless, due to
protein similarity, people allergic to cow's milk proteins can be affected by milk from any species,
which demonstrates the importance for correct labelling.
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Milk and milk products
Southern European countries, specific animal or vegetable rennet should be used [41]. In general,
lamb or kid rennet is preferred in the case of some sheep and/or goat PDO cheeses, such as Roncal
cheese in Spain, Pecorino Romano and Fiore Sardo cheeses in Italy and Feta cheese in Greece,
among others. In the case of the Italian PDO cheese Pecorino Romano, the specifications mention
that, besides using exclusively lamb rennet paste, the fourth-stomachs used to produce this rennet
should also come from animals raised in the PDO geographical area [42]. On the other hand, other
PDO cheeses such as Azeitão, Serpa and Évora cheeses in Portugal, prefer the use of specific
vegetable rennet, namely that from Cynara cardunculus. Portuguese sheep milk PDO cheeses,
when compared to other cheeses from the same species, generally present a creamy semi-soft
texture and exquisite flavour, these characteristics being attributed in part to the vegetable
coagulant used, which is very proteolytic [43]. Thus, when specifications of PDO cheeses stipulate
the origin of rennet used for manufacturing, the use of another type of rennet, such as those from
microbial origin, constitutes an adulteration and the characteristics of the final product may even
be different, since the use of a specific rennet is frequently associated to particular characteristics
of the cheese.
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(2003) and Gonçalves et al. (2012) proposed multiplex PCR assays to detect cow, goat, sheep and
buffalo species in dairy products [87,88]. The development of duplex PCR assays enabled the
detection and quantification of cow’s milk in sheep’s [34] and goat’s [35] cheeses.
Real-time PCR has been the technique of choice in many laboratories for species identification and
food authentication, including for dairy products. The combination of high sensitivity, specificity,
reproducibility and quantitative analysis are major advantages of real-time PCR. Additionally, the
amplification of short DNA fragments (100-200 bp) is a major benefit when analysing highly
processed foods [89]. Several authors have proposed real-time PCR assays with TaqMan probes to
detect cow’s milk in dairy products [90-94]. The simultaneous detection of several species in dairy
products has also been succeeded by multiplex real-time PCR assays [95,96].
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Milk and milk products
to variation occurring as a result of natural events (e.g. weather, climate, disease etc.) during
growth or the production of primary foods or to batch-to-batch variations in processed foods or
food ingredients. Interrogation of signals from sufficiently large and characteristic sample
collections by mathematical techniques can detect primary foods which are not what they claim to
be or processed foods that do not comply with a declared specification. Vibrational spectroscopy
methods are suitable for implementation in factories and milk parlours as they allow on-line
control and the screening of a high number of samples by unit of time. Fingerprinting methods are
also of interest to regulatory bodies because they enable rapid preventative action to be taken. It
should be noted that, despite the many studies demonstrating their potential, the application of
fingerprinting methods in routine analysis and food authenticity surveillance still remains limited
[105].
Until now, untargeted analysis has been associated mainly with direct analysis techniques, such as
mass spectrometric-based metabolomics or isotope-assisted methods. Only a few studies have
linked untargeted analysis with vibrational spectroscopic methods [106]. Moore et al. [107]
developed non-targeted screening tools to detect adulteration in skimmed milk powder using NIR
spectroscopy and Xu et al. developed a method for the untargeted detection of protein
adulteration in yogurt by removing unwanted variations in pure yogurt [108]. In all these cases, the
approach involved building statistical models based on the measured fingerprints of a large
representative set of normal and abnormal samples, and then applying these models to unknown
samples in order to characterise them. More recently, Fernández Pierna et al. have developed a
moving window based PCA method using vibrational spectroscopic data. The PCA spectral score
residuals are evaluated and used to define thresholds to be applied to the spectral score residuals
of unknown samples [109]. The method was applied to study milk contaminated with melamine.
Since the discovery of melamine contamination in infant milk formula in China, strict regulations
have been enforced throughout the world and many papers have been published on the use of
such methods as wet chemistry, chromatography, mass spectrometry and vibrational spectroscopy
to detect melamine in both raw and powdered milk. In this study, liquid UHT milk was
contaminated with melamine at various levels ranging from 0.01 % to 1 % (100 – 10 000 ppm) and
measured using Fourier Transform Mid-Infrared (FT-MIR) spectrometry. Samples spiked at levels
higher than 100 ppm were easily detected using this method, which would not have been possible
using classical techniques such as Mahalanobis distance, usually applied as an outlier detection
method.
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Milk and milk products
maize in the animal’s diet and corresponding mislabelling of dairy products declared as being
produced by pastured animals or PDO cheeses for which the diet of the animal has an established
maximum amount of maize in the diet [114]. The analysis of mineral and trace elements coupled
with the development of classification models based on chemometrics have also been applied for
the differentiation of the type of milk production, namely organic versus conventional [115].
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Milk and milk products
5. Conclusion
During the last decade, cultural and social shifts have occurred in developed societies with
consumers becoming increasingly aware about subjects such as biodiversity, climate change and
ecological footprint. Therefore, one can expect a growth in the number of consumers willing to
spend more money on certain food products, such as specialties produced according to traditional
processes and organic foods. In this sense, possible adulterations in the dairy industry that may
occur in a near future include mislabelling of the organic origin of milk and milk products and of
the breed origin of milk used in the production of some PDO products, such as cheeses. Several
PDO milk products, mostly cheeses, besides requiring the use of milk from specific animal species
also specify the animal breed. Among several other examples, Spanish Manchego cheese must be
produced from sheep’s milk of the Manchega breed and the Portuguese Terrincho cheese
produced from sheep’s milk of the Churra da Terra Quente breed. Since some traditional breeds
specified in PDO products are less productive compared to others that are more frequently used, a
possible fraud could imply the use of milk from the same animal species but from a different breed
to that specified for a particular milk product.
Looking into the future, there are several trends in milk and milk products authentication, one of
those being the use of untargeted approaches such as spectroscopic techniques. In recent years,
food safety has become an increased concern for consumers due to several important crises
related directly or indirectly to human health. Most of the studies published have attempted to
develop analytical procedures based on spectroscopic techniques to characterise/authenticate
milk or milk products and at the same time detect the presence of any possible known
contaminant or adulterant before reaching the food chain. Until now, statistical tools have been
used to interpret multivariate data obtained from the spectroscopic analysis of different products
and this has led to the creation of some decision rules. These enable verification of compliance
against specifications in order to decide whether to reject or accept the product. However, the
challenge will be to exploit the huge amount of information contained in the data generated by
such spectroscopic techniques but taking into account the concept of data-driven discovery or
untargeted analysis. New crises of adulteration/contamination with illegal ingredients other than
known ones continue to occur from time to time. By relying solely on targeted analysis methods,
adulteration could get out of control and analysis would become trapped in a cycle of
‘adulteration, targeted analysis, and new adulteration’, and so on. In contrast to targeted analysis,
which uses information from known possible unusual ingredients, an untargeted experiment
registers all information within a certain correlation/similarity, including data from new products.
Untargeted detection methods are therefore required for screening products for a range of known
and unknown adulterants. Untargeted analysis will mean alerts can be given more rapidly and
fraud detected more easily
Vibrational spectroscopic methods are based on measuring the amount of electromagnetic
radiation absorbed by a sample according to the Beer-Lambert law and can be very useful when
authenticity and quality controls need to be established at both the laboratory and the industry
levels, as they can be applied at the point where products are delivered to factories or during the
production process. They are rapid with almost no sample preparation; they do not use chemical
reagents and do not require skilled staff. However, fingerprinting methods are not confirmatory
techniques, and therefore are not used in official control and have no weight in a judicial court.
Nevertheless, such methods could to be interesting for regulatory bodies as they would enable
preventative actions to be taken rapidly. Spectroscopic methods are increasingly presented as new
approaches for at-line, on-line and in-line control of authentication of food products. As
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mentioned, these techniques are already routinely used in the industry to control both raw
materials and finished products for specific production standards as a common authenticity issue
and it is expected that they will be increasingly used in a near future. The main limitation of the
spectroscopic approach is the requirement for large datasets to calibrate any given instrument.
Also, a main challenge facing the spectroscopists is to extract the information in such a way that it
can be used in qualitative and quantitative analysis. NIR spectra can contain thousands of
absorbance values at defined wavelengths (i.e. variables) and the challenge is to characterise the
spectral data set and isolate the variables that can be correlated with the information of interest
(i.e. authenticity issue) [116]. To achieve this goal, a wide range of chemometric tools are at the
disposal of the analyst who has to select the most appropriate according to the specific aims of the
method and the characteristics of the dataset. Among the many methods proposed for the
authentication of food products, spectroscopic methods seem to be the preferred ones to flag
suspicious samples before, during and after the production of a food product. The real future
challenge for spectroscopic techniques will be the demonstration of their daily use in the industry
and the marketplace for food product authentication.
More recently, other novel and advanced techniques, such as real-time PCR coupled with High
Resolution Melting (HRM) analysis, Loop-Mediated Isothermal Amplification (LAMP), next
generation sequencing (NGS) and biosensors have emerged and are being applied to milk
authenticity testing [81]. By relying on isothermal amplification of DNA, LAMP presents several
advantages, namely its simplicity, speed and the fact that is does not require specialised
equipment such as thermocyclers [117]. This, as well as the possibility of being integrated on
microfluidic devices, allows for its portability, giving this technology great potential for use as a
screening tool. NGS technologies have changed the way in which DNA can be analysed by
increasing sequencing throughput by several orders of magnitude. NGS combined with DNA
barcoding has been termed metabarcoding, which relies on the use of universal PCR primers to
amplify, massively, one or more taxonomically informative targets. Recently, Ion Torrent NGS
technology was successfully applied for the identification of species in dairy products by
sequencing targeted mtDNA fragments [118]. Although the cost of NGS platforms is still very high,
this technology presents several advantages regarding species identification for food
authentication, and its use is expected to increase in the near future.
Due to their small size and high integration, biosensors are simple to operate with and generally
capable of fast measurements. Therefore one can also expect their increased used in the dairy
industry with multiple applications [119].
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66. International Organization for Standardization/International Dairy Federation. (2011). - Standard ISO/TS 17193| IDF
208:2011, Milk - Determination of the lactoperoxidase activity - Photometric method (Reference method).
67. Goulden J.D.S. (1964). - The Infra Red Milk Analyser. J. Soc. Dairy Technol., 17 (1), 28-31.
68. Grappin R. & Jenet R. (1976). - Essais de l’appareil Milko-Scan 300 utilisé pour le dosage en série de la matière grasse
et des proteins du lait. Le Lait, 56, 498-520.
69. Ghosh P.K. & Jayas D.S. (2009). - Use of spectroscopic data for automation in food processing industry. Sens.
Instrumen. Food Qual. 3, 3–11.
70. Asselain M., Manifacier D. & Agnet Y. (1996). - Method and Apparatus for the Spectrophotometric Assay of Aqueous
Liquids. Foss Electric A/S, assignee. European Pat. No. 588892.
71. Kaylegian K.E. (2007). - Lipolysis and proteolysis of modified and producer milks used for calibration of mid-infrared
milk analyzers. J Dairy Sci., 90 (2), 602-615.
72. Bonfatti V., Di Martino G. & Carnier P. (2011). - Effectiveness of mid-infrared spectroscopy for the prediction of
detailed protein composition and contents of protein genetic variants of individual milk of Simmental cows. J. Dairy
Sci., 94, 5776–85.
73. Soyeurt H., Bruwier D., Romnee J.M., Gengler N., Bertozzi C., Veselko D. & Dardenne P. (2009). - Potential estimation
of major mineral contents in cow milk using mid-infrared spectrometry. J. Dairy Sci., 92, 2444–2454.
74. Van Knegsel A.T.M., Van der Drift S.G.A., Horneman M., De Roos A.P.W., Kemp B. & Graat E.A.M. (2010). - Short
communication: ketone body concentration in milk determined by Fourier transform infrared spectroscopy: value for
the detection of hyperketonemia in dairy cows. J. Dairy Sci., 93, 3065–3069.
75. Soyeurt H., Colinet F.G., Arnould V.M.R., Dardenne P., Bertozzi C., Renaville R., Portetelle D. & Gengler N. (2007). -
Genetic variability of lactoferrin content estimated by mid-infrared spectrometry in bovine milk. J. Dairy Sci., 90,
4443–4450.
76. Rutten M.J.M., Bovenhuis H., Hettinga K.A., Van Valenberg H.J.F. & Van Arendonk J.A.M. (2009). - Predicting bovine
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77. Soyeurt H., Dehareng F., Gengler N., McParland S., Wall E., Berry D.P., Coffey M. & Dardenne P. (2011). - Mid-infrared
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78. Mohammed R., Khorasani R.G., Goonewardene L.A., Kramer J.K.G. & Kennelly, J.J. (2011). - Persistency of milk trans-
18:1 isomers and rumenic acid in Holstein cows over a full lactation. Can. J. Anim. Sci., 91 (1), 147-167.
79. Friggens N.C., Ridder C. & Lovendahl P. (2007). - On the use of milk composition measures to predict the energy
balance of dairy cows. J. Dairy Sci., 90, 5453-5467.
80. Grelet C., Fernández Pierna J.A., Dardenne P., Baeten V. & Dehareng F. (2015). - Standardization of milk mid-infrared
spectra from a European dairy network. J. Dairy Sci., 98(4), 2150-2160.
81. Kalogianni D. P. (2018). - DNA-based analytical methods for milk authentication. Eur. Food Res. Technol., 244(5), 775–
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82. López-Calleja I., González I., Fajardo V., Rodríguez M.A., Hernández P.E., García T. & Martín R. (2004). - Rapid
Detection of Cows' Milk in Sheeps' and Goats' Milk by a Species-Specific Polymerase Chain Reaction Technique. J.
Dairy Sci., 87, 2839-2845.
83. López-Calleja I., González I., Fajardo V., Martín I., Hernández P.E., García T., & Martín R. (2005a). - Application of
Polymerase Chain Reaction to Detect Adulteration of Sheep's Milk with Goats' Milk. J. Dairy Sci., 88, 3115-3120.
84. Golinelli L.P., Carvalho A.C., Casaes R.S., Lopes C.S.C., Deliza R., Paschoalin V.M.F. & Silva J. T. (2014). - Sensory
analysis and species-specific PCR detect bovine milk adulteration of frescal (fresh) goat cheese. J. Dairy Sci., 97(11),
6693-6699.
85. Di Pinto A., Terio,V., Marchetti P., Bottaro M., Mottola A., Bozzo G., Bonerba E., Ceci E. & Tantillo, G. (2017). - DNA-
based approach for species identification of goat-milk products. Food Chem., 229, 93-97.
86. López-Calleja I., Alonso I. G., Fajardo V., Rodríguez M.A., Hernández P.E., García T. & Martin R. (2005b). - PCR
detection of cows' milk in water buffalo milk and mozzarella cheese. Int. Dairy J., 15, 1122-1129.
87. Bottero M.T., Civera T., Nucera D., Rosati S., Sacchi P. & Turi R. M. (2003). - A multiplex polymerase chain reaction for
the identification of cows', goats' and sheeps' milk in dairy products. Int. Dairy J., 13, 277-282.
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88. Gonçalves J., Pereira F., Amorim A. & van Asch B. (2012). - New Method for the Simultaneous Identification of Cow,
Sheep, Goat, and Water Buffalo in Dairy Products by Analysis of Short Species-Specific Mitochondrial DNA Targets. J.
Agric. Food Chem., 60(42), 10480-10485.
89. Navarro E., Serrano-Heras G., Castaño M.J. & Solera J. (2015). Real-time PCR detection chemistry. Clin. Chim. Acta,
439, 231-250.
90. Lopez-Calleja I., Gonzalez I., Fajardo V., Martin I., Hernandez P.E., Garcia T. & Martin R. (2007). - Real-time TaqMan
PCR for quantitative detection of cows' milk in ewes' milk mixtures. Int. Dairy J., 17(7), 729-736.
91. Lopparelli R.M., Cardazzo B., Balzan S., Giaccone V. & Novelli E. (2007). - Real-Time TaqMan Polymerase Chain
Reaction Detection and Quantification of Cow DNA in Pure Water Buffalo Mozzarella Cheese: Method Validation and
Its Application on Commercial Samples. J. Agric. Food Chem., 55(9), 3429-3434.
92. Zhang C.-L., Fowler M.R., Scott N.W., Lawson G. & Slater A. (2007). - A TaqMan real-time PCR system for the
identification and quantification of bovine DNA in meats, milks and cheeses. Food Control, 18, 1149-1158.
93. Dalmasso A., Civera T., La Neve F. & Bottero M.T. (2011). - Simultaneous detection of cow and buffalo milk in
mozzarella cheese by Real-Time PCR assay. Food Chem., 124(1), 362-366.
94. Di Pinto A., Conversano N.C., Forte V.T., Novello L. & Tantillo G.M. (2004). - Detection of cow milk in buffalo
"mozzarella" by polymerase chain reaction (PCR) assay. J. Food Quality, 27, 428-435.
95. Drummond M.G., Brasil B.S.A.F., Dalsecco L.S., Brasil R.S.A.F., Teixeira L.V. & Oliveira D.A.A. (2013). - A versatile real-
time PCR method to quantify bovine contamination in buffalo products. Food Control, 29(1), 131-137.
96. Rentsch J., Weibel S., Ruf J., Eugster A., Beck K. & Köppel R. (2013). - Interlaboratory validation of two multiplex
quantitative real-time PCR methods to determine species DNA of cow, sheep and goat as a measure of milk
proportions in cheese. Eur. Food Res. Technol., 236(1), 217-227.
97. Cozzolino .R, Passalacquas, Salemi S. & Garozzo D. (2002). - Identification of adulteration in water buffalo mozzarella
and in ewe cheese by using whey proteins as biomarkers and matrix-assisted laser desorption/ionization mass
spectrometry. J. Mass Spectrom., 37, 985–91.
98. Calvano C.D., De Ceglie C., Monopoli A. & Zambonin C.G. (2012). - Detection of sheep and goat milk adulterations by
direct MALDI-TOF MS analysis of milk tryptic digests. J. Mass Spectrom., 47(9),1141–9.
99. Caira S, Pinto G, Nicolai MA, Chianese L & Addeo F. (2016). - Simultaneously tracing the geographical origin and
presence of bovine milk in Italian water buffalo Mozzarellacheese using MALDI-TOF data of casein signature
peptides. Ana.l Bioanal. Chem., 408(20), 5609–5621.
100. Cuollo M, Caira S, Fierro O, Pinto G, Picariello G & Addeo F. (2010). - Toward milk speciation through the monitoring
of casein proteotypic peptides. Rapid Commun. Mass Spectrom., 24(11), 1687–1696.
101. De Noni I. & Resmini P. (2005). - Identification of rennet-whey solids in “traditional butter” by means of HPLC/ESI-MS
of non-glycosylated caseinomacropeptide A. Food Chem., 93(1), 65-72.
102. Abbas O. Dardenne P. & Baeten V. (2012). - Near-Infrared, Mid-Infrared, and Raman Spectroscopy In: Chemical
Analysis of Food: Techniques and Applications, Pico Y. Burlington, Elsevier Science, 59-91.
103. Baeten V., Rogez H., Fernández Pierna J.A., Vermeulen P. &Dardenne P. (2015). - Vibrational Spectroscopy Methods
for the Rapid Control of Agro- Food Products. In Handbook of Food Analysis (3rd Edition). (Eds. Toldra & Nollet).
Volume II, Chapter 32, pp. 591-614.
104. Vermeulen P., Fernández Pierna J.A., Abbas O., Rogez H., Davrieux F. & Baeten V. (2017). - Authentication and
Traceability of Agricultural and Food Products Using Vibrational Spectroscopy In: Food Traceability and Authenticity:
Analytical Techniques, Montet D. and Ray RC. USA, Biology series, CRC press, 450.
105. Riedl J., Esslinger S. and Fauhl-Hassek C. (2015). - Review of validation and reporting of non-targeted fingerprinting
approaches for food authentication, Anal. Chim. Acta, 885, 17-32.
106. Baeten V., Vermeulen P., Fernández Pierna, J.A. and Dardenne, P. (2014). - From targeted to untargeted detection of
contaminants and foreign bodies in food and feed using NIR spectroscopy. New Food, 17(3), 16-23.
107. Moore J.C., Ganguly A., Smeller J.; Botros L., Mossoba M. and Bergana, M.M. (2012). - NIR news, 23, 9-11.
108. Xu L., Yan S., Cai C., Wang Z. and Yu X. (2013). - The Feasibility of Using Near-Infrared Spectroscopy and
Chemometrics for Untargeted Detection of Protein Adulteration in Yogurt: Removing Unwanted Variations in Pure
Yogurt. J. Anal. Methods Chem., 1-9.
109. Fernández Pierna J.A., Vincke D., Baeten V., Grelet C., Dehareng F. and Dardenne P. (2016). - Use of a multivariate
moving window PCA for the untargeted detection of contaminants in agro-food products, as exemplified by the
detection of melamine levels in milk using vibrational spectroscopy. Chemom. Intell. Lab. Syst., 152, 157-162.
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110. Abbas O., Zadravec M., Baeten V., Mikuš T., Lešić T., Vulić A., Prpić J., Jemeršić L., Pleadin J. (2018). - Analytical
methods used for the authentication of food of animal origin. Food Chem., 246, 6–17.
111. Nečemer M., Potocnik D., Ogrinc N. (2016). - Discrimination between Slovenian cow, goat and sheep milk and cheese
according to geographical origin using a combination of elemental content and stable isotope data. J. Food Comp.
Anal., 52, 16–23.
112. Camin, F., Wietzerbin, K., Cortes, A.B., Haberhauer, G., Lees, M., Versini, G., 2004. - Application of multielement
stable isotope ratio analysis to the characterization of French, Italian, and Spanish cheeses. J. Agric. Food Chem. 52,
6592–6601.
113. Commission Implementing Regulation (EU) No 584/2011 of 17 June 2011 approving non-minor amendments to the
specification for a name entered in the register of protected designations of origin and protected geographical
indications (Grana Padano (PDO)). Official Journal of the European Union L160, 65–70. Available from: https://eur-
lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32011R0584&from=EN).
114. Camin F., Perini M., Colombari G., Bontempo L., Versini G. (2008). - Influence of dietary composition on the carbon,
nitrogen, oxygen and hydrogen stable isotope ratios of milk. Rapid Commun. Mass Spectrom., 22, 1690–1696.
115. Rodríguez-Bermúdez R., López-Alonso M., Miranda M., Fouz R., Orjales I., Herrero-Latorre C. (2018). - Chemometric
authentication of the organic status of milk on the basis of trace element content. Food Chem., 240, 686–693.
116. Baeten V. & Aparicio Ruiz, R. (2000). Edible oils and fats authentication by Fourier transform Raman spectrometry.
Biotechnol. Agron. Soc. Environ., 4 (4), 196-203.
117. Deb R., Sengar G.S., Singh U., Kumar S., Alyethodi R.R., Alex R., Raja T. V., Das A. K. & Prakash B. (2016). Application
of a loop-mediated isothermal amplification assay for rapid detection of cow components adulterated in buffalo
milk/meat. Mol. Biotechnol., 58(12), 850–860.
118. Ribani A., Schiavo G., Utzeri V.J., Bertolini F., Geraci C., Bovo S. & Fontanesi L. (2018). - Application of next generation
semiconductor based sequencing for species identification in dairy products. Food Chem., 246, 90–98.
119. Kulkarni A.S., Joshi D.C. & Tagalpallewar G.P. (2014). Biosensors for food and dairy industry. Asian J. Dairy Food Res.,
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Eggs and egg products
Michele Suman*, Daniele Cavanna, Michele Zerbini, Diego Ricchetti,
Damiano Sanfelici, Elisa Cavandoli, Leonardo Mirone
Barilla G.R. F.lli SpA, Parma, Italy
*E-mail corresponding author: [email protected]
The main producing countries are the European Union (EU), China and North-Central America.
Given the perishable nature of both shell eggs and liquid egg products, the flow of trade in these
goods between different countries is limited and most of the production is fully dedicated to
internal markets. This may vary in case of big issues such as the outbreak of Avian Influenza in the
http://doi.org/10.32741/fihb.2.eggs
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US that led the entire country to import liquid products from the EU. Powder products are a
different case, especially albumen which has specific properties for use in bakery products. These
can be commercialised without concern for their expiry dates, but may face some trade
restrictions between countries (i.e. not all European countries can export to the US).
Most suppliers operate a supply chain in which the different steps are vertically integrated: the
feed-mill, the shelling and heat treatment plant and farms (either fully owned or under an
agistment contract). There has been a steady decrease in spot market purchasing of eggs due to
traceability requirements.
The increasing importance of animal welfare has put the entire sector under even more pressure
and it has had to adapt to new regulations especially in the EU (2012) and also to consumer
demand to eradicate the use of cages by suppliers. In response to these issues, the market has
reacted in order to tackle the increasing demand for suitable farms with better animal welfare,
and in addition to ensure a more transparent supply chain and thus prevent any other possible
scandals that have affected the market in the past.
1. Product Identity
1.1. Definition of the product and manufacturing process
The concept of egg products is related to all the forms of presentation of the egg: yolk, albumen or
a mix of both. The term “Egg Products” refers to processed or convenience forms of eggs obtained
by processing shell eggs: egg products include whole eggs, egg whites, and egg yolks in frozen,
pasteurised and refrigerated liquid and dried forms available in a number of different product
formulations. In particular, the food industry is interested in high quality egg products in a
liquefied form, obtained from eggs shelled within 4 days and which have undergone
homogenisation and pasteurisation: their use is mainly related to the preparation of egg pasta and
bakery products [1].
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Eggs and egg products
1.1.1.2. Cage-free
In the EU, this type of egg production includes barns, free-range, organic (in the UK, systems must
be free-range if they are to be labelled as organic) and aviary systems. Non-cage systems may be
single or multi-tier (up to four levels), with or without outdoor access.
In free-range systems, hens are housed to a similar standard as the barn or aviary. In addition, they
have constant daytime access to an outside range with vegetation. Each hen must have at least
2
4 m of space.
The European Union Council Directive 1999/74/EC stipulates that non-cage systems must provide
the following:
2
● A maximum stocking density of 9 hens/m of “usable” space
● If more than one level is used, a height of at least 45 cm must exist between the levels
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Eggs and egg products
2
● One nest for every seven hens (or 1 m of nest space for every 120 hens if group nests are
used)
● Litter (e.g. wood shavings) covering at least one-third of the floor surface, providing at
2
least 250 cm of littered area per hen
● 15 cm of perching space per hen.
In addition to these requirements, free-range systems must also provide the following:
2
● One hectare of outdoor range for every 2,500 hens (equivalent to 4 m per hen; at least
2
2.5 m per hen must be available if rotation of the outdoor range is practiced)
● Continuous access during the day to this open-air range, which must be “mainly covered
with vegetation”
FILTERING
REFRIGERATION
STORAGE OR RAW
LIQUID EGGS
HOMOGENISATION
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Eggs and egg products
Liquid egg products coming from this process are commonly delivered to food companies in
refrigerated tanks and their quality and food safety characteristics (chemical, physical parameters
included in related technical specifications) are carefully controlled by the producers before their
release and by the customers on reception and before use.
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Eggs and egg products
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Eggs and egg products
2. Authenticity issues
2.1. Identification of current authenticity issues
This section concerns pasteurised eggs used in food preparations, starting from liquid eggs already
shelled, provided by suppliers located in the EU with integrated supply chains for farms, feed mills
and transformation plants.
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Eggs and egg products
Artificial colorants
Artificial colorants are allowed but some supply chains claim to be free from artificial colorants.
Eggs intermixing is possible at farm level, feed mill and at the transformation facility.
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Eggs and egg products
The significant microbial growth that occurs in a shelled egg, before the pasteurisation process,
causes the formation of different microbial metabolites and leads to a significant alteration of the
original enzymatic properties.
Egg products that have been restored through thermal pasteurization must respect in particular
−1
these two microbial parameters: total viable mesophilic bacteria, max 5 log CFU g ;
−1
Enterobacteriaceae count, max 2 log CFU g .
Microbiological analysis can be performed following specifications reported in ISO and AOAC
official documents [8–10].
In general, total viable mesophilic bacteria are enumerated using spread plates of plate count agar
incubated at 30 °C for 72 h; Enterobacteriaceae counts are determined by using violet red bile
glucose agar with a double layer, incubated at 37 °C for 24 h.
The egg contains a series of organic acids such as succinic and lactic acids, the presence of which is
directly correlated to microbial quality and which cannot be altered through thermal restoration
actions [11].
3-hydroxybutyric acid is a specific indicator of fertilised, incubated eggs. Succinic acid is used to
evaluate microbial spoilage. Lactic acid is increased in both cases, and can be used to screen egg
products for suitability for human consumption.
The amounts of lactic and succinic acids in high quality liquid fresh egg products are usually not
−1
higher than 200 and 5 mg kg dry egg, respectively [12]. Currently, the legal European Union limit
−1
is: lactic acid ≤ 1000 mg kg dry egg [4,13–15].
The level of 3-hydroxybutyric acid, again following European legislation, must not be higher than
−1
10 mg kg dry egg.
A gas chromatographic approach can be used as analytical method for routine testing of these egg
carboxylic acids to indicate pre-pasteurisation spoilage of egg products: NaOMe is used for
methylation; the carboxylate esters are separated by gas chromatography on a 5% dimethyl
siloxane column under gradient temperature [16].
Also available on the market are several enzymatic tests designed to carry out the quantitative
determination of lactic, succinic and 3-hydroxybutyric acids in egg products. Sample preparation
and analysis can be done using an UV–vis spectrophotometer and following kit instructions /
recommendations and other corresponding studies reported in literature [17].
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Eggs and egg products
presents several rapid non-destructive methods able to assess this parameter: both NIR [18,20,21]
and Vis-IR [22,23] spectroscopies coupled with chemometric data treatment are able to detect
this fraud directly on the shell egg.
At the same time, researchers have attempted to identify volatile components that contribute to
the egg’s unique flavours and aromas, working with different extraction and analytical techniques
(steam-distillation, solvent extraction, purge and trap, etc.): several aldehydes, aromatic
compounds and sulphur compounds have been identified in greatest concentrations [24]. In
particular, methyl-sulphide compounds are strictly related to deterioration and the perception of
unacceptable odours in whole eggs [25]. In most cases these methods are interesting to
demonstrate all the potential compounds that can be emitted from eggs, but often the
corresponding necessary heating procedures may produce an excess of volatiles which is not
representative of the real situation of an egg product which is refrigerated and evaluated by a
sensory panel at room temperature or after a short treatment at 30–40 °C.
An alternative strategy to sensing the global profile of organic volatiles emitted by eggs can
potentially be achieved by using artificial olfactory systems (AOS), also called “electronic noses”.
Currently, the application of AOS has been encouraged thanks to the outstanding developments
which sensor technology and data processing systems have undergone over the last 20 years
[26,27].
Furthermore, in the last years other tests based on hyperspectral imaging are presented as other
non-destructive ways to solve the same problem [28].
Recently, a fast-GC electronic nose was presented as a rapid way to collect the volatile fingerprint
of hen eggs; subsequently, thanks to chemometric data treatment, eggs were clearly separated
according to their storage time, and a prediction of this factor was calculated and validated with a
PLS model [29].
Other interesting approaches for freshness evaluation use the intrinsic fluorescence of thick
albumen and egg yolk [30] or the quantification of S-Ovalbumin [31]; in addition, a rapid
colorimetric test based on the reaction between albumen and 3,3’,5,5’-tetramethylbenzidine is
mentioned in the literature [32].
Different confirmatory techniques are presented, for instance the evaluation of albumen freshness
combining the results obtained with NIR and NMR spectroscopies [33].
Another important issue is the discrimination between organic and conventional eggs and the
common approach reported in literature is the evaluation of the carotenoids fingerprint with HPLC
analysis, usually followed by chemometric elaboration [34].
Stable isotope analysis has been explored in the past and it seems that the isotopic composition of
egg components depends on the diet consumed by the laying hen [35] and on the farming
conditions [36]; on the contrary, ratios are not influenced by the pasteurisation process [37].
However, further studies are required before considering this technique a robust tool for egg
traceability.
Incubated eggs fraudulently added to fresh egg products are usually detected by exploiting
enzymatic assays with 3-hydroxybutyric acid as specific molecular marker [38], as previously
indicated. However, it seems that the combination of this legislated marker with the presence of
uracil (generated as a consequence of high microbial contamination) could provide a more robust
evaluation of the hygienic quality of the products [39].
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Eggs and egg products
The analytical method used up to now for uracil determination in eggs is the one presented by
Morris [40].
Another possible adulteration is the undeclared addition of dyes to eggs or derivatives which can
be detected using liquid chromatography coupled to mass spectrometry [41].
A growing issue of the last years is the introduction of melamine (that results in an apparent
increase of the protein content) in eggs. The literature presents portable instruments that, thanks
to an approach based on surface-enhanced Raman spectroscopy [42], are able to detect this fraud;
however, chromatographic techniques are also widely used [43–45].
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Eggs and egg products
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Eggs and egg products
5. Conclusion
Eggs and egg-derived products are widely used in the food industry. In addition, there is a clear
and growing need for a more transparent supply chain in order to reduce the risks involving
product authenticity and traceability: in fact, especially during the last ten years the sector has
faced many different issues linked to food fraud.
The food industry requires fast and robust methods for the acceptance or rejection of a batch
before its introduction in the production chain but, in parallel, confirmatory methods are required
for quality and authenticity certifications.
Eggs contain a series of metabolites, such as some specific organic acids, whose presence is
directly correlated to the freshness and microbial quality and which cannot be altered through
thermal restoration actions: gas chromatographic and enzymatic methods are available for their
quantitative evaluation.
Future directions will involve finding and validating new analytical solutions to detect non-declared
addition of dyes/additives, to categorise different farming approaches and to discriminate
between organic and conventional eggs (through LC-MS, IRMS, etc.) and enlarging the range of
non-destructive methods (mainly based on optical and spectroscopic measurements).
Alternative emerging strategies deal with HRMS metabolomics and sensing organic volatiles
patterns by using electronic noses/ion mobility or similar instruments, then creating predictive
models able to collect the global fingerprint of the products in relation to potential fraud issues.
6. Bibliographic references
1. Rossi M. (1998). – Proprietà funzionali degli ovoprodotti. Riv. Avicoltura, 67, 28–34.
2. Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the
general principles and requirements of food law, establishing the European Food Safety Authority and laying down
procedures in matters of food safety (10AD). Off. J. Eur. Union, L31, 1–24.
3. Regulation (EC) No 852/2004 of the European Parliament and of the Council of 29 April 2004 on the hygiene of
foodstuffs (2004). Off. J. Eur. Union, L139, 1–54.
4. Regulation (EC) No 853/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific
hygiene rules for food of animal origin (2004). Off. J. Eur. Union, L139, 55–205.
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8. ISO Standard (2013). – Microbiology of the food chain -- Horizontal method for the enumeration of microorganisms -
- Part 1: Colony count at 30 degrees C by the pour plate technique. ISO 4833-1:2013. Available at:
https://www.iso.org/standard/53728.html.
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10. ISO Standard (1993). – Microbiology — General guidance for the enumeration of Enterobacteriaceae without
resuscitation — MPN technique and colony-count technique. ISO 7402:1993. Available at:
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11. Elenbaas, H., Stouten P., Steverink A. & Uijttenboogaart T. (1985). – W. Balters (Ed.), Proceedings of Third European
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spectroscopy and multivariate data analysis. Innov. Food Sci. Emerg. Technol., 12 (2), 182–186.
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synthesis of hyperspectral imaging and multivariate analysis. J. Food Eng., 157, 41–48.
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(10), 3733–3746.
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relative humidity. Anal. Chim. Acta, 582 (1), 83–91.
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an indicator. Poult. Sci., 91 (3), 739–743.
32. Rossi M., Hidalgo A. & Pompei C. (2001). – Reaction between albumen and 3,3’,5,5’-tetramethylbenzidine as a
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Albumen freshness assessment by combining visible near-infrared transmission and low-resolution proton nuclear
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34. Ruth S. van, Alewijn M., Rogers K., Newton-Smith E., Tena N., Bollen M. & Koot A. (2011). – Authentication of organic
and conventional eggs by carotenoid profiling. Food Chem., 126 (3), 1299–1305.
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36. Rogers K. (2009). – Stable Isotopes as a Tool To Differentiate Eggs Laid by Caged, Barn, Free Range, and Organic Hens.
J. Agric. Food Chem., 57 (10), 4236–4242.
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preparation. Food Chem., 136, 1551–1556.
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used as ingredient in fresh egg pasta. Food Chem., 87, 313–319.
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quality in egg products. J. Agric. Food Chem., 56, 1289–1297.
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chromatography. J. Chromatogr., 394, 408–413.
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products by ultra-high performance liquid chromatography tandem mass spectrometry. J. Chromatogr. B, 879 (24),
2416–2422.
42. Cheng Y. & Dong Y. (2011). – Screening melamine contaminant in eggs with portable surface-enhanced Raman
Spectroscopy on gold nanosubstrate. Food Control, 22 (5), 685–689.
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determination of melamine in egg by gas chromatography-mass spectrometry and ultra-performance liquid
chromatography-tandem mass spectrometry. Anal. Chim. Acta, 651 (2), 196–200.
46. Cavanna D., Zanardi S., Dall’Asta C. & Suman M. (2019). – Ion mobility spectrometry coupled to gas chromatography:
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47. Cavanna D., Catellani D., Dall’Asta C. & Suman M. (2018). – Egg product freshness evaluation: a metabolomic
approach. J. Mass Spectrom.
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Honey
Kurt-Peter Raezke
Eurofins Food Integrity Control Service, Ritterhude, Germany
*E-mail corresponding author: [email protected]
https://doi.org/10.32741/fihb.3.honey
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Honey
companies with dumping honey imports from China, including some that were adulterated with
unauthorised residues of antibiotics. This incident was considered to be just “the tip of the
iceberg” in honey fraud [4].
In the same year in Europe, in the aftermath of the horsemeat scandal, the EU included honey in a
top ten list of food products most at risk of food fraud, putting further focus on honey
adulteration. However prior to these events, the honey industry sector had been well aware of the
concerns of economically motivated adulteration of honey, particularly given the ease with which
sugar syrups can be added and premium honey diluted with cheaper types. This has led to
considerable efforts being undertaken by various trade bodies such as Apimondia (the
International Federation of Beekeepers’ Association), the IHC (International Honey Commission)
and the IHEO (International Honey Exporters Organisation) to control the presence of fraudulent
product in the market place.
In addition to the diversity of countries exporting honey, often from remote regions with little or
no transparency of supply, the practice of beekeeping itself is also under threat. During the last
few decades, intensive agriculture and the use of pesticides resulting in a reduction and/or
contamination of available areas for bee foraging and the emergence of new bee diseases have all
led to a decline in traditional beekeeping activities. The availability of cheap, often fraudulent
products in the market resulting in lower prices for domestic honey, has also pushed in the same
direction. The ensuing decline in bees, which pollinate a large portion of global food production,
poses a serious threat to the food chain. In the EU, it is estimated that pollinators, including honey
bees, bumblebees and wild bees, contribute at least EUR 22 billion each year to the European
agriculture industry. They ensure pollination for over 80 % of crops and wild plants in Europe [5].
Alarm bells have sounded particularly in Europe and North and South America. An up-to-date
review of the current situation on the international honey market is given in reference [6].
1. Product Identity
1.1. Definition of the product and manufacturing process
Honey is primarily a concentrated solution of sugars, composed mainly of glucose and fructose,
together with other components such as organic acids, enzymes, vitamins, acetylcholine,
flavonoids, minerals is trace quantities [7]. Honey production itself must be considered at two
levels, taking into account both the collection and processing of plant fluids by the bees [8], and
the extraction and processing of honey by beekeepers and honey packers. The latter includes a
number of processing steps which vary according to the unique characteristics of the honey being
processed. In general the production process follows six main steps: extraction, dehumidification,
liquefaction and blending, heating, pasteurisation, crystallisation, and final packing. Reference [9]
provides a detailed description of each of these steps. The INPhO (Information on Post-harvest
Operations) of the United Nations FAO (Food and Agriculture Organisation) have produced a
Honey Processing toolkit [10] to help in the setting up of a honey infrastructure.
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Honey
According to Decision 2011/163/EU [12] it is mandatory for non-EU countries that want to export
honey to EU Member States to be listed on a third country list in accordance with article 29 of
Council Directive 96/23/EC [13].
The general food-labelling rules laid down in Directive 2000/13/EC [14] also apply to honey but are
subject to certain conditions. In particular the country of origin where the honey has been
harvested should be included on the label. In addition, the labelling of filtered honeys and baker's
honeys is mandatory for every transaction on the bulk market.
―3―
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Honey
2. Moisture content
● in general not more than 20 %
● heather (Calluna) and baker's honey in general not more than 23 %
● baker's honey from heather (Calluna) not more than 25 %
3. Water-insoluble content
● in general not more than 0.1 g/100 g
● pressed honey not more than 0.5 g/100 g
4. Electrical conductivity
● honey not listed below, and blends of these honeys not more than 0.8 mS/cm
● honeydew and chestnut honey and blends of these except with those not less than 0.8 mS/cm
listed below
● Exceptions: strawberry tree (Arbutus unedo), bell heather (Erica), no limit defined
eucalyptus, lime (Tilia spp.), ling heather (Calluna vulgaris), manuka or
jelly bush (leptospermum), tea tree (Melaleuca spp.)
5. Free acid
● in general not more than 50 milli-equivalents
acid per 1000 grams
● baker's honey not more than 80 milli-equivalents
―4―
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Honey
Under its requirements for essential composition and quality factors, the Codex Standard also
stipulates that honey sold as such should not have added to it any food ingredient, including food
additives, and should not have begun to ferment or effervesce. No pollen or constituent particular
to honey may be removed except where this is unavoidable in the removal of foreign inorganic or
organic matter. And chemical or biochemicals treatments to influence honey crystallisation are not
permitted.
The Codex Standard also provides acceptable ranges for moisture, sugars and water insoluble
solids contents. It provides guidelines for sampling and analysis, as well as for labelling, with clear
recommendations for how the honey should be designated.
2. Authenticity issues
2.1. Identification of current authenticity issues
In general honey needs to meet the given definitions and fixed specifications. Questions of
authentication occur on two levels. Firstly, 'pure' honey may have been extended by addition of
sugar, syrup and/or water. Secondly, if the honey has a more detailed description indicating
botanical, geographical or topological origin, the description may be false even though the product
is pure honey. There are other possible incorrect descriptions and information such as health
claims, if it is 'organic', has 'antibacterial activity' and so on which are difficult to evaluate.
―5―
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Honey
composition. However these methods are limited when required to identify sugar addition by
different types of syrups from different botanical sources.
Most bulk sweetening materials are derived from cane sugar, beet sugar or by the hydrolysis of
starch. The starch is often derived from maize but new sources such as rice are now easily
available on the market. Some forms of rice syrup have even been bio-chemically engineered to
make them more difficult to detect.
―6―
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Honey
―7―
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Honey
Bellunesi, della Lunigiana (PDO); from Spain (Mel de Galicia (PGI); Mel Villuercas-Ibores, de
Liébana, de Tenerife, de Granada, de la Alcarria (PDO). These special labels are recognised by the
consumer and command a premium price.
―8―
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Honey
3.1.3.1. Melissopalynology
Melissopalynology, or pollen analysis, is an essential part of honey authenticity testing. Pollens
grains from different types of plants have a distinctive morphology that can be identified by
microscopic examination [7]. The technique, which requires expert judgement, is used to
determine the country of origin by linking pollen type to the characteristic flora of the
geographical source, or to verify authenticity when a particular botanical origin is claimed.
The pollen count can be used to estimate the proportions of nectar present. However the method
does have some limitations, mainly due to the natural variability of amounts of pollen from
botanical sources. For example, in some cases the specific pollen may be 'under-represented' such
as for citrus and lavender, whereas for others, such as forget-me-not, the pollen is 'over-
represented'.
Pollen is also available as a product and the determined adulterator could filter out all the pollen
and add back pollen of choice.
Despite its limitations however, pollen analysis is still a useful method to control country of origin.
A review of harmonised methods of melissopalynology is given in reference [27].
value for honey which on average is around -25.4 ‰. This difference has been used very
successfully to detect adulteration in honey [29,30], and is an official AOAC Method 998-12. In this
method the C/ C ratio, expressed as G C of the whole honey is measured by SIRA (stable
13 12 13
isotope ratio analysis) and compared to the G C value of the protein isolated from the honey. The
13
difference between these values is a measure of the C4 sugar content of the honey, provided that
both honey and protein have been analysed on the same instrument [31,32].
Addition of syrups derived from beet and other plants utilising the Calvin or C 3 metabolic pathway
remains a considerable analytical challenge. Further solutions are described in the following
sections.
―9―
- 51 -
Honey
― 10 ―
- 52 -
Honey
1
TRACE in which the stable isotope ratios of the elements carbon, nitrogen, sulphur and hydrogen
were measured in the protein fraction extracted from honey produced in 20 European areas [46].
The honey protein fraction was specifically chosen since it is part of the preparation in the method
to detect added C4 sugar (as described above) and less easy to manipulate. The study
demonstrated that both hydrogen and carbon isotopes in honey protein are correlated to
precipitation and climate. The sulphur stable isotope composition of the honey protein is clearly
influenced by the geology of the rock underlying the soil in which the flora grew, and from which
the bees foraged nectar and pollen. Despite the natural variability of the product and the similarity
of geological and climatic conditions across the countries investigated, the study concluded that
the four stable isotope ratios considered here, measured on honey protein can be applied to verify
the origin of honey.
1
TRACE Project. Tracing the origin of food. 2005-2009. Funded by the European Commission under the 6th Framework
Programme.
― 11 ―
- 53 -
Honey
NIR spectroscopy is particularly useful as a rapid and non-destructive method for the detection of
honey adulteration. Combined with chemometric data treatment the technique has been used to
discriminate honey adulterated with high fructose corn syrup (HFCS) demonstrating its potential as
a screening method for quality monitoring [52].
― 12 ―
- 54 -
Honey
Analytical
Indicative data, analyte or parameter Authenticity issue or information
technique
― 13 ―
- 55 -
Honey
5. Conclusion
Today honey and bee products are continuously recognised as pure natural products. The
importance of these products is confirmed by the extent of current controls on honey authenticity
at every stage of the global supply chain. The composition of honey, the treatment with veterinary
drugs and contamination by pesticides and other contaminants are closely monitored.
With the daily news reporting repeatedly about food fraud, adulteration of honey and other bee
products will still draw significant attention in the future. It is the responsibility of all stakeholders
of the honey supply chain to optimise existing control mechanisms.
As mentioned earlier, the European Commission started an action plan to tackle food fraud late in
2013. As a follow-up in 2015, the European Commission launched a coordinated control plan on
honey authenticity in which its Joint Research Centre carried out analyses to detect honey
adulteration with exogenous sugars (Commission Recommendation C(2015) 1558 [56]). The aim of
the control plan was to establish the prevalence on the European Union market of: (a) honey
mislabelled with regard to its geographical and/or botanical origin and (b) products declared or
presented as honey although containing exogenous sugars or sugar products. 2 264 honey samples
were collected at all stages of the supply chain; the majority of the samples came from retailers.
More than 10 % of the honey samples checked by EA/LC-IRMS did not conform to published
benchmark purity criteria indicating that foreign sugars may have been added. Around 20 % of
honey either declared as blends of EU honeys, or unblended honeys bearing a geographical
reference related to an EU Member State or a third country were suspected to contain added
sugar.
The published report demonstrates the need for further investigation by all stakeholders of the
honey industry to ensure authentic honey and to justify the trust of the consumer in this natural
product.
An outlook is given in the Meeting Report of the Technical Round Table on Honey Authentication
[57]. The participants agreed on:
● A critical review of the current definition of identity and purity criteria of honey is
necessary.
● Acceptance / rejection criteria for authenticating honey are needed.
● An appropriate analysis of the vulnerability of the honey supply chain should be done and
an improved traceability system implemented.
● Screening methods should be developed to economise testing.
● Analytical methods to detect emerging fraud cases should be developed and already
existing methods should be validated.
● A mechanism for providing quality assurance tools should be established.
● Chemical and biological characteristics of genuine honeys (including blends), bee feeding
products, and products from inappropriate practices should be generated and stored in a
publicly available database.
In addition to those methods that are already regulated, it will be important to regulate methods
that are more suitable to tackle food fraud. One idea will be the evaluation of EA/LC-IRMS as an
official method for honey authenticity controls in future. If this happens authorities should be able
to claim “non-authentic” honey more often which will have a significant impact on the whole
production chain of honey.
― 14 ―
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Honey
With its wide screening potential, the NMR profiling technique is now also widely used for
authenticity checks. More generally, non-targeted methods will be a major add-on to the existing
approaches for anticipating new fraudulent practices in the future.
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41. White J.W. (1966). – Methyl Anthranilate Content of Citrus Honey. J. Food Sci., 31 (1), 102–104. doi:10.1111/j.1365-
2621.1966.tb15421.x.
42. Tette P.A.S., Guidi L.R., Bastos E.M.A.F., Fernandes C. & Gloria M.B.A. (2017). – Synephrine – A potential biomarker
for orange honey authenticity. Food Chem., 229, 527–533. doi:10.1016/j.foodchem.2017.02.108.
43. Elflein L. & Raezke K.P. (2008). – Improved detection of honey adulteration by measuring differences between13C/
12C stable carbon isotope ratios of protein and sugar compounds with a combination of elemental analyzer - isotope
ratio mass spectrometry and liquid chromatography - isotope ratio mass spectrometry (δ13C-EA/LC-IRMS).
Apidologie, 39 (5), 574–587. doi:10.1051/apido:2008042.
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Honey
44. Krummen M., Hilkert A.W., Juchelka D., Duhr A., Schlüter H.J. & Pesch R. (2004). – A new concept for isotope ratio
monitoring liquid chromatography/mass spectrometry: New concept for isotope ratio monitoring LC/MS. Rapid
Commun. Mass Spectrom., 18 (19), 2260–2266. doi:10.1002/rcm.1620.
45. Cabañero A.I., Recio J.L. & Rupérez M. (2006). – Liquid Chromatography Coupled to Isotope Ratio Mass
Spectrometry: A New Perspective on Honey Adulteration Detection. J. Agric. Food Chem., 54 (26), 9719–9727.
doi:10.1021/jf062067x.
46. Schellenberg A., Chmielus S., Schlicht C., Camin F., Perini M., Bontempo L., Heinrich K., Kelly S.D., Rossmann A.,
Thomas F., Jamin E. & Horacek M. (2010). – Multielement stable isotope ratios (H, C, N, S) of honey from different
European regions. Food Chem., 121 (3), 770–777. doi:10.1016/j.foodchem.2009.12.082.
47. Sobrino-Gregorio L., Vilanova S., Prohens J. & Escriche I. (2019). – Detection of honey adulteration by conventional
and real-time PCR. Food Control, 95, 57–62. doi:10.1016/j.foodcont.2018.07.037.
48. Utzeri V.J., Ribani A. & Fontanesi L. (2018). – Authentication of honey based on a DNA method to differentiate Apis
mellifera subspecies: Application to Sicilian honey bee (A. m. siciliana) and Iberian honey bee (A. m. iberiensis)
honeys. Food Control, 91, 294–301. doi:10.1016/j.foodcont.2018.04.010.
49. Soares S., Grazina L., Costa J., Amaral J.S., Oliveira M.B.P.P. & Mafra I. (2018). – Botanical authentication of lavender
(Lavandula spp.) honey by a novel DNA-barcoding approach coupled to high resolution melting analysis. Food
Control, 86, 367–373. doi:10.1016/j.foodcont.2017.11.046.
50. Soares S., Grazina L., Mafra I., Costa J., Pinto M.A., Duc H.P., Oliveira M.B.P.P. & Amaral J.S. (2018). – Novel diagnostic
tools for Asian (Apis cerana) and European (Apis mellifera) honey authentication. Food Res. Int. Ott. Ont, 105, 686–
693. doi:10.1016/j.foodres.2017.11.081.
51. Pita-Calvo C. & Vázquez M. (2017). – Differences between honeydew and blossom honeys: A review. Trends Food Sci.
Technol., 59, 79–87. doi:10.1016/j.tifs.2016.11.015.
52. Ferreiro-González M., Espada-Bellido E., Guillén-Cueto L., Palma M., Barroso C.G. & Barbero G.F. (2018). – Rapid
quantification of honey adulteration by visible-near infrared spectroscopy combined with chemometrics. Talanta,
188, 288–292. doi:10.1016/j.talanta.2018.05.095.
53. Spiteri M., Jamin E., Thomas F., Rebours A., Lees M., Rogers K.M. & Rutledge D.N. (2015). – Fast and global
authenticity screening of honey using 1H-NMR profiling. Food Chem., 189, 60–66.
doi:10.1016/j.foodchem.2014.11.099.
54. Spiteri M., Rogers K.M., Jamin E., Thomas F., Guyader S., Lees M. & Rutledge D.N. (2017). – Combination of 1H NMR
and chemometrics to discriminate manuka honey from other floral honey types from Oceania. Food Chem., 217,
766–772. doi:10.1016/j.foodchem.2016.09.027.
55. Phipps R. (2018). – International Honey Market. Am. Bee J., January, 23.
56. DG SANTE Health and Food Safety (2015). – Commission Recommendation C(2015) 1558 of 12.3.2015 on a
coordinated control plan with a view to establishing the prevalence of fraudulent practices in the marketing of
certain foods. , 1–4.
57. JRC-Geel (2018). – Technical Round Table on Honey Authentication - Meeting report. , 1–19.
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Meat and meat products
Enrico Valli*, Massimiliano Petracci
Department of Agricultural and Food Sciences, Alma Mater Studiorum, Università di Bologna, Italy
*E-mail corresponding author: [email protected]
Figure 1: Evolution of global meat production from 1970 to 2016 (Own design, data source: Faostat)
https://doi.org/10.32741/fihb.4.meat
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Meat and meat products
In the last few years, world bovine meat production has been increasing at a modest pace. The
United States are the major bovine meat producing country in the world, with 11 million tons
(Figure 2). The second producer is Brazil, with 9 million tons, with a herd expansion encouraged by
international trade, despite a reduction in domestic demand. The European Union (EU) is the third
beef producer (almost 8 million tons), followed by China, India and Argentina. In 2016, China
produced about 55 million metric tons of pork which accounted for 47 % of total world production.
The EU is the second world producer with almost 24 million tons followed by Vietnam, Brazil and
the Russian Federation. The biggest poultry meat producers are the United States, with almost 21
million tons a year, followed by China, with 19 million tons, the EU and Brazil with about 14 million
tons (Figure 2).
Global meat production is projected to be 13 % higher in 2026 relative to the base period (2014-
16). This compares with an increase of almost 20 % in the previous decade (Figure 1). Developing
countries are projected to account for the vast majority of the total increase, with a more intensive
use of feed in the production process. Poultry meat is the primary driver of the growth in total
meat production in response to expanding global demand for this more affordable animal protein
compared to red meats. Low production costs and lower product prices have contributed to
making poultry the meat of choice both for producers and consumers in developing countries. In
the bovine meat sector, cow herds are being rebuilt in several major producing regions, but the
decline in cattle slaughter in these regions is projected to be offset by higher carcass weights. Pork
production will also increase after 2017, driven by slow herd expansion in China.
Figure 2: Countries with the greatest share of additional meat production by meat type [1]
In the EU-28, pork is by far the main meat produced, followed by chicken meat and beef (Figure 3).
In the EU, beef is mainly produced from cattle breeds grown specifically for their meat, but it can
also come from dairy cattle. France (19.0 %), Germany (14.7 %) and the United Kingdom (11.7 %)
accounted for almost half (46 %) of the total EU-28 beef production (Figure 4).
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Figure 3: Production share of main meat produced in EU on 2016 (Own design, data source: Eurostat)
As for pork meat production, Germany produced around one quarter (23.9 %) of the EU-28’s pig
meat in 2016, while Spain produced one sixth (17.9 %) of the EU-28 total, equal to 23 million tons.
Finally, Poland, France, the United Kingdom, Spain and Germany each contributed between 10 and
15 % to the EU-28 production of poultry meat in 2016 (Figure 4).
Figure 4: EU Countries with the greatest share of production by meat type on 2016 (Own design, data source: Eurostat)
Global meat apparent consumption per capita is expected to stagnate at 34.6 kg by 2026, an
increase of less than half a kilogram compared to the base period (Figure 5). Beef consumption will
gradually increase over the next ten years. By 2026, and relative to the base period, it is expected
to increase by almost 6 % in developed countries, whereas in developing regions it is expected to
increase by approximately 17 %. In per capita terms, beef consumption in the developing world
remains low relative to developed countries, at about one-third in volume terms. High population
numbers in Asia remain a major driver of growth, combined with the positive perception of
Chinese buyers that bovine and ovine meat are healthier and disease-free; the result is an
expected 44 % increase in beef consumed in Asia over the next decade [1].
Pork consumption on a per capita basis declines marginally over the outlook period with
consumption in most developed countries reaching saturation levels (Figure 5). Among the
developing countries, significant regional differences are evident in per capita pork consumption.
Growth is sustained in Argentina, Brazil, Mexico, and Uruguay, albeit at a generally slower rate
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than the past decade. Pork consumption has grown rapidly over the past few years in Latin
America, fuelled by increased domestic production, improved quality, and favourable relative
prices that have positioned pork as one of the favoured meats, along with poultry. Conversely,
many countries with favourable economic conditions and expanding meat consumption do not
traditionally consume high levels of pork relative to other meats, resulting in stagnant and even
declining consumption on a per capita basis at the regional level. Population expansion still
supports growth in total pork consumption in these regions [1].
Consumption of poultry meat increases regardless of region or income level. Per capita
consumption will grow, even in the developed world, but growth rates will remain slightly higher in
developing regions. Worldwide, poultry grew rapidly and surpassed pork as the preferred animal
protein in 2016. This will remain the case during the outlook period and, of all the additional meat
consumed over the next decade, poultry is expected to account for almost 45 % (Figure 5) [1].
Per capita consumption of meat is expected to slightly increase in the EU overall from 69.1 to 70.7
kilograms by 2026, whereas the individual big five countries (Italy, UK, Spain, France and Germany)
are predicted to experience a decrease in consumption (Figure 5).
1. Product Identity
1.1. Definition of the product and manufacturing process
The first distinction is between fresh and processed meat. Fresh meat is defined as meat having
undergone no treatment other than chilling and freezing, while processed meat is a very broad
category of many different types of products, all defined by having undergone at least one further
processing or preparation step such as, i.e. grinding, adding an ingredient or cooking, which
changes the appearance, texture or taste. The main classes of processed meat are described
below:
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Meat and meat products
● Minced meat: boneless meat reduced in fragments which contains less than 1 % salt;
● Mechanically separated meat: obtained by removing meat from bones using mechanical
devices (high-pressure application machinery) that contribute to the loss or modification
of muscle-fibrous meat texture;
● Desinewed meat: obtained by removing sinews, tendons, cartilages and thicker collagen
using mechanical devices (low-pressure application machinery) without modification of
muscle-fibrous meat texture.
● Meat preparations: fresh meat (including fragments), containing flavourings, additives or
subjected to treatments that do not modify the muscle-fibrous texture;
● Meat products: processed products derived from processed meat or further processing of
other meat products subjected to treatments that modify the muscle-fibrous texture.
There are many meat products that are produced in different countries, but it is possible to
categorise them in six groups, considering the processing technology used:
● Fresh processed meat products: products that are composed of muscle mixed fragments
with different amounts of animal fat. They are salted, and small quantities of non-meat
ingredients are added to improve taste and binding. All ingredients are added fresh and
some of these products are filled in casings. They are cooked or fried immediately prior to
consumption (e.g. hamburgers).
● Formed meat: products which may give the impression that they are made of a whole
piece of meat, but actually consist of different pieces combined together by other
ingredients, including food additives and food enzymes or by other means.
● Cured meat products: products that are submitted to a curing process and treated with
small amounts of nitrite. These products are divided in two groups:
o Cured raw meat: products that undergo a process of curing, fermentation and
ripening in controlled conditions without any heat treatment (e.g. raw cured
beef);
o Cured cooked meat: products that undergo a curing process and then are
submitted to heat treatment (e.g. cooked pork ham).
● Raw-cooked meat products: products composed of muscle meat, fat and non-meat
ingredients which are reduced in fragments, mixed and portioned before being submitted
to heat treatment (e.g. meat loaf);
● Precooked: cooked meat products; products composed of muscle trimmings, fatty tissues,
meat from the head of the animal, animal skin, blood, liver and other edible parts, which
undergo two different heating processes - precooking of raw materials and cooking of the
finished product mix (e.g. corned beef);
● Raw fermented sausages: uncooked meat products obtained by a mixture of lean and
fatty tissues combined with salts, nitrite, sugars, spices and other non-meat ingredients
filled into casings. They are submitted to a fermentation process (drying and ripening) to
obtain the typical flavour and are consumed raw (e.g. salami).
● Dried meat products: lean meat that undergoes a process of drying in natural or artificial
conditions to prolong its shelf-life (e.g. dried meat strips or flat pieces).
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Table 1: Meat definitions according to EU Legislation
EC Basic definition of meat Animal carcass components specifically Basis of meat content declaration
Regulation excluded from the definition
Regulation Meat Genital organs of either female or male Not appropriate
(EC) n. Edible parts of the animals (including blood): animals, except testicles; urinary organs,
853/2004 - Domestic ungulates (domestic bovine including Bubalus and Bison species, porcine, except the kidneys and the bladder; the
ovine and caprine animals, and domestic solipeds); cartilage of the larynx, the trachea and the
- Poultry (farmed birds, including birds that are not considered as domestic, but extra-lobular bronchi; eyes and eyelids; the
which are farmed as domestic animals, with the exception of ratites); external auditory meatus; horn tissue; and in
- Lagomorphs (rabbits, hares and rodents); poultry, the head – except the comb and the
- Wild game (wild ungulates, lagomorphs and birds, as well as other land mammals ears, the wattles and caruncles – the
that are hunted for human consumption); oesophagus, the crop, the intestines and the
- Farmed game: farmed ratites and other farmed land mammals. genital organs.
Meat can be defined "Fresh meat" if it has not undergone any preserving process
other than chilling, freezing or quick-freezing, including meat that is vacuum-
wrapped or wrapped in a controlled atmosphere.
Carcass
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Body of an animal after slaughter and dressing. The definition of ‘carcass’ for bovine,
pigs, sheep, goat and poultry is reported in Regulation (EC) no. 1165/2008.
Offal
Fresh meat other than that of the carcass, including viscera and blood
Viscera
Organs of the thoracic, abdominal and pelvic cavities, as well as the trachea and
oesophagus and, in birds, the crop
Meat preparations The same for meat and minced meat.
Fresh meat, including meat that has been reduced to fragments (minced meat),
which has had other foodstuffs, seasonings or additives added to it or which has
undergone processes insufficient to modify the internal muscle fibre structure of the
meat and thus to eliminate the characteristics of fresh meat.
Meat products The same for meat.
Processed products resulting from the processing of meat or from the further
processing of such processed products, so that the cut surface shows that the
product no longer has the characteristics of fresh meat
Meat and meat products
EC Basic definition of meat Animal carcass components specifically Basis of meat content declaration
Regulation excluded from the definition
Minced meat Raw material used to prepare minced meat
Boned meat that has been minced into fragments and contains less than 1 % salt. must not derive from: scrap cuttings and
Raw material used to prepare minced meat must derive from skeletal muscle, scrap trimmings (other than whole muscle
including adherent fatty tissues. cuttings); mechanically separated meat; meat
containing bone fragments or skin; or meat of
Meat and meat products
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the production (Reg. EC/999/2001 [4]).
Regulation Meat Mechanically separated meat If maximum limits are exceeded, but
(EC) no. For labelling purpose, the term “meat” is referred to: skeletal muscles of mammalian all their criteria for the definition of
1169/2011 and bird species (*) recognised ‘meat’ are satisfied, the ‘… meat’
as fit for human consumption content must be adjusted
with naturally included or downwards accordingly, and the list
adherent tissue, where the of ingredients must mention, in
total fat and connective tissue addition to the term ‘… meat’, the
content does not exceed the presence of fat and/or connective
values indicated below and tissue.
where the meat constitutes an Meat species is required on the
ingredient of another food: label unless indicated by the
product name.
EC Basic definition of meat Animal carcass components specifically Basis of meat content declaration
Regulation excluded from the definition
Minced meat Mechanically separated meat In the case of minced meat and
Specific requirements concerning the designation of minced meat: meat preparations made from pre-
prepared minced meat, except for
sausages and sausage meat, the
label must indicate the appropriate
% of fat and collagen in meat
protein (i.e. “% of fat under.....”, “ %
of collagen in meat under....”)
Meat species is required on the
label unless indicated by the
product name.
Formed meat
Meat products, meat preparations which may give the impression that they are
made of a whole piece of meat, but actually consist of different pieces combined
together by other ingredients, including food additives and food enzymes or by other
means
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Table 2: Rules about the labelling of meat products within European Union
2. Authenticity issues
2.1. Identification of current authenticity issues
In general beef is the main added-value meat product that is the most widely traded, and
therefore where major authenticity problems can occur. This is reflected in the list of authenticity
topics below. However, some authenticity issues do concern other meat types such as poultry or
lamb. These are mentioned where relevant.
Food fraud is a global issue which damages the reputation of companies, disrupts markets and
erodes consumer confidence. Food fraud surfaces more frequently in certain supply chains and
that of meat is always present. The importance of studies covering these topics is mainly related to
economic issues associated with fraud in high-value foods like beef, with cheaper ingredients
added. However other fraudulent practices in the meat industry could occur such as: 1) the origin
of meat and the animal feeding regime (as in the case of certified regional products of poultry and
lamb, for example); 2) substitution of meat ingredients by other animal species, tissues, fat or
proteins; 3) modification of the processing methods of meat products; and 4) addition of non-meat
components such as water or additives.
2.1.1. Substitution
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Meat and meat products
Cheap animal protein might be fraudulently used to substitute more expensive animal protein.
Casein is by far the most commonly used milk protein, sometimes in combination with excessive
amounts of water and polyphosphates. Whey proteins are also used for this purpose.
Vegetable protein such as cheap and readily available soy is probably one of the most commonly
used proteins: in recent years, the addition of soybean protein as a raw material replacing red
meat in burgers for example has increased significantly due to its functional characteristics (which
include increased water and fat binding capacity, emulsification ability), and improved
organoleptic properties, such as appearance, (smooth texture, and cutability), nutritional value, as
well as its low price. For these same reasons the addition of vegetable protein can be carried out
fraudulently, leading to a potential safety concern due to its allergenic properties.
Another special sanitary issue has been the use of gluten which causes intolerance reactions in
some individuals. Microbial proteins have been developed for use in foods but are not widely used
in meat products.
Finally, the addition of melamine and urea to meat products is an unlawful method of increasing
the apparent protein content [6].
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Meat and meat products
many meat-derived products, where it is less easily detected, without declaring it on the label. EU
regulations exclude mechanically recovered meat from the definition of meat and it should be
separately identified in the ingredients list when it is used in meat products.
2.1.2.1. Additives
The purpose of Regulation 1333/2008 [13] and further amendments is to harmonise the use of
specific preservatives in food products and it gives a list of both authorised and prohibited
additives for certain foods, including some traditional meat products. The use of colours (Council
Directive 94/35/EC [13]), antioxidants, preservatives and flavourings is generally not allowed in
fresh unprocessed meat because they mask spoilage. Similarly, many meat products and
preparations have restricted the use of these additives for the same reason.
2.1.2.2. Water
Water is the cheapest extender of meat and meat products and the water-holding capacity of
meat proteins facilitates the binding of water. While the practice of 'enhancing', 'injecting' or
'plumping' has been around since the 1970s, particularly in the chicken industry, it is becoming a
subject of concern in recent years. While many believe injecting meat with salt water helps give
the product some added juiciness, there are some unpleasant truths about this practice. Besides
the increase in product weight, both salt water or contaminated water represent a safety risk, due
in the first instance to an unknown uptake of high quantities of sodium and in the second to the
presence of pathogens in case of polluted water. When the amount of water is greater than 5 % of
the finished product, the EU Regulation requires water to be declared in the ingredient list.
Although the amount of water added to cured meats can be very different, very few countries
have a requirement for a quantitative declaration of added water. However the debate continues
to make consumers aware of the possible fraudulent addition [14].
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Meat and meat products
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Meat and meat products
3.1.1. Substitution
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Meat and meat products
3.1.2.1. Additives
Nitrites and nitrates
The methods consist in colorimetric and spectrophotometric determinations:
● Determination of nitrites content in cured meats by a colorimetric method [33];
● Determination of nitrites content meat and meat products (reference method) [34];
● Determination of nitrates content in meat and meat products by a colorimetric
method [35];
● Determination of nitrates and nitrites content in meat by a spectroscopic method [36];
● Determination of nitrites and nitrate content in meat and meat products by
spectrophotometric determination after enzymatic reduction of nitrate to nitrite [37];
● Determination of nitrites content in meat and meat products, processed meat and poultry
products, canned corned beef, cooked cured chopped meat, cooked cured pork shoulder,
cooked cured ham and luncheon meat by a colorimetric methods [28], different AOAC
and ISO methods.
Ascorbic acid
Determination of total vitamin C in food – semiautomated fluorimetric method [38].
Phosphorus and polyphosphates
Different principles are on the basis of these methods, ranging from spectrophotometry to
gravimetry:
● Determination of total phosphorus content in meat and meat products (reference
method) [39];
● Determination of total phosphorous content in meat and meat products by spectrometric
method [40] (this standard was last reviewed and confirmed in 2001);
● Determination of linear condensed phosphates in meat and meat products by thin layer
chromatographic separation [41];
● Determination of total phosphorus content by gravimetric method [42];
● Determination of phosphorus in meat and meat products by spectroscopic method [43].
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Meat and meat products
Colouring agents
Detection of synthetic, water-soluble colouring agents in meat and meat products by a thin layer
chromatographic method [44].
Sulphur dioxide
Detection of sulphurous acid (free form) in meat by a titrimetric method [45].
Preservatives
Detection of preservatives (sorbates, ascorbates, benzoates, sulphites) in ground meat by a
spectroscopic method [46].
3.1.2.2. Water
The official methods for the determination of water content are basically based on the measure of
the loss in mass obtained for a sample under specific conditions, such as different kind of heat
treatments, divided by the mass of the test portion; moisture content is expressed as a percentage
by mass. NMR analysis can be applied as well:
● Determination of moisture content in meat and meat products (reference method) [47];
● Determination of moisture in meat and meat products by air drying [48];
● Determination of moisture and fat by microwave and NMR analysis [49].
3.2.1. Substitution
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Meat and meat products
measure total nitrogen content (e.g. Kjeldahl and Dumas) are not able to discriminate between
nitrogen atoms derived from proteins or chemical compounds, thus chromatographic techniques
are employed (HPLC or GC usually coupled to mass spectrometry).
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Meat and meat products
3.2.2.1. Additives
Many additives could be fraudulently added to meat. Among these, colouring agents, flavours and
preservatives can be detected using HPLC and GC, while fibrinopeptides A and B from thrombin
addition are identified and quantified by HPLC.
3.2.2.2. Water
Water could be added to meat in order to increase its weight; thus, extraneous water in meat can
be determined by measuring water and protein content, using several methods that are more or
less sophisticated (simple determination in oven, NMR, etc.) and also through the determination
of the water/protein ratio.
3.2.3.2. Fresh versus thawed meat, organic versus conventional meat and
feed intake
Microscopy analysis can be used to differentiate fresh versus thawed meat. The method which is
validated for poultry meat, is based on the principle that thawed meat present microscopic
alteration of muscles fibres which can be related to freezing temperatures.
There are several analytical strategies in the literature showing the possibility to differentiate
between animals bred using organic or conventional farming systems, as well as to determine feed
intake, however these are not routinely used in the industry.
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Meat and meat products
Table 3: Official methods for authenticity testing of meat and meat products
PROFIT (poultry rapid overnight field Antibodies and antigens Species substitution
identification test)
Thin layer chromatographic Linear condensed phosphates Addition of phosphorus and polyphosphates
separation
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Meat and meat products
Table 4: Non-official commonly used methods for authenticity testing of meat and meat products
5. Conclusion
Considering the growing demand for meat, related fraud is expected to represent an ongoing
challenge in future years. The analytical tools to detect meat fraud will need to be improved based
on a number of different strategies. First, those analytical procedures that are not included in
existing standards (determination of additives, use of molecular techniques to determine species
substitutions) need to be standardised and validated. Standardised methods need to be revised,
such as the EU reference method to determine hydroxyproline content in meat. This is a simple
spectrophotometric technique, while other more advanced ones such as LC-MS/MS are available
but not recognised as reference techniques. Multi-screening and untargeted methods further
development to detect simultaneously different and unknown adulterants. And finally, innovative
analytical approaches have to be developed and validated to propose solutions for different old
and emerging issues directly linked to fraud such as:
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Meat and meat products
● Characterizing different animal breeds, using a larger data set to build effective models
(NIR techniques);
● Determining animal feed intake, since the current analysis based on carotenoid content in
fat and blood are influenced by other factors such as breed, gender, lactation and rumen
environment;
● Determining the slaughter age of animals;
● Assessing animal welfare condition related to intensive vs traditional farming practices;
● Distinguishing different meat cuts (a possible solution could be the evaluation of collagen
content that varies among different meat cuts, considering that visual inspection is useful
only to differentiate primary beef cuts);
● Quantifying vegetable fat as adulterant in meat, not only revealing its presence by
phytosterols detection;
● Establishing the geographic origin of meat, since the simple identification of breed may
not be effective since individual breeds can be raised in different countries despite their
origin;
● Detecting animal fat from different undeclared species;
● Developing methods to identify fresh-thawed products that are applicable to ground meat
and temperatures higher than -12°C (the HADH method is not applicable to ground meat
because the grinding process causes similar alterations to those induced by freezing and it
is able to detect frozen-thawed meat only if the freezing temperature has been -12°C or
below).
● Setting up reliable methods to detect mechanically deboned meat (MDM) and to
distinguish among low pressure vs. high pressure MDM in meat products.
6. Bibliographic references
1. OCDE, OECD & FAO (2018). – OECD-FAO Agricultural Outlook (Edition 2018).
doi:https://doi.org/https://doi.org/10.1787/d4bae583-en.
2. Regulation (EC) No 853/2004 of the European Parliament and of the Council of 29 April 2004 laying down specific
hygiene rules for food of animal origin (2004). Off. J. Eur. Union, L139, 55–205.
3. Regulation (EU) No 1169/2011 of the European Parliament and of the Council of 25 October 2011 on the provision of
food information to consumers (2011). Off. J. Eur. Union, L304, 18–63.
4. Regulation (EC) No 999/2001 of the European Parliament and of the Council of 22 May 2001 laying down rules for
the prevention, control and eradication of certain transmissible spongiform encephalopathies (2001). Off. J. Eur.
Union, L147, 1–40.
5. Cavin C., Cottenet G., Blancpain C., Bessaire T., Frank N. & Zbinden P. (2016). – Food Adulteration: From Vulnerability
Assessment to New Analytical Solutions. doi:info:doi/10.2533/chimia.2016.329.
6. Ballin N.Z. (2010). – Authentication of meat and meat products. Meat Sci., 86 (3), 577–587.
doi:10.1016/j.meatsci.2010.06.001.
7. Cengiz E. & Gokoglu N. (2007). – Effects of fat reduction and fat replacer addition on some quality characteristics of
frankfurter-type sausages. Int. J. Food Sci. Technol., 42 (3), 366–372. doi:10.1111/j.1365-2621.2006.01357.x.
8. Rahmati S., Julkapli N.M., Yehye W.A. & Basirun W.J. (2016). – Identification of meat origin in food products–A
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9. EFSA Panel on Biological Hazards (BIOHAZ) (2013). – Scientific Opinion on the public health risks related to
mechanically separated meat (MSM) derived from poultry and swine. EFSA J., 11 (3), 3137.
doi:10.2903/j.efsa.2013.3137.
10. Catillo G., Moioli B., Napolitano F. & Steri R. (2018). – Identification of genomic regions harboring diversity between
Holstein and two local endangered breeds, Modenese and Maremmana. Livest. Sci., 216, 75–83.
doi:10.1016/j.livsci.2018.07.011.
11. Gokulakrishnan P., Kumar R.R., Sharma B.D., Mendiratta S.K., Malav O. & Sharma D. (2015). – Determination of Sex
Origin of Meat and Meat Products on the DNA Basis: A Review. Crit. Rev. Food Sci. Nutr., 55 (10), 1303–1314.
doi:10.1080/10408398.2012.690095.
12. Mir N.A., Rafiq A., Kumar F., Singh V. & Shukla V. (2017). – Determinants of broiler chicken meat quality and factors
affecting them: a review. J. Food Sci. Technol., 54 (10), 2997–3009. doi:10.1007/s13197-017-2789-z.
13. Council Regulation (EC) No 1047/2009 of 19 October 2009 amending Regulation (EC) No 1234/2007 establishing a
common organisation of agricultural markets as regards the marketing standards for poultrymeat (2009). Off. J. Eur.
Union, L290, 1–3.
14. Food Safety Authority of Ireland (2005). – Investigation of the composition and labelling of chicken breast
fillets from the Netherlands imported into Ireland. Available at:
https://www.fsai.ie/uploadedFiles/Monitoring_and_Enforcement/Monitoring/Surveillance/Poultry_Labelling_Repor
t2.pdf.
15. Dave D., Ghaly A.E., Dave D. & Ghaly A.E. (2011). – Meat Spoilage Mechanisms and Preservation Techniques: A
Critical Review. Am. J. Agric. Biol. Sci., 6 (4), 486–510. doi:10.3844/ajabssp.2011.486.510.
16. Leygonie C., Britz T.J. & Hoffman L.C. (2012). – Impact of freezing and thawing on the quality of meat: Review. Meat
Sci., 91 (2), 93–98. doi:10.1016/j.meatsci.2012.01.013.
17. Council Regulation (EC) No 1099/2009 of 24 September 2009 on the protection of animals at the time of killing
(2009). Off. J. Eur. Union, L303, 1–30.
18. D’amico P., Vitelli N., Cenci Goga B., Nucera D., Pedonese F., Guidi A. & Armani A. (2017). – Meat from cattle
slaughtered without stunning sold in the conventional market without appropriate labelling: A case study in Italy.
Meat Sci., 134, 1–6. doi:10.1016/j.meatsci.2017.07.011.
19. Monahan F.J., Schmidt O. & Moloney A.P. (2018). – Meat provenance: Authentication of geographical origin and
dietary background of meat. Meat Sci., 144, 2–14. doi:10.1016/j.meatsci.2018.05.008.
20. Średnicka-Tober D., Barański M., Seal C., Sanderson R., Benbrook C., Steinshamn H., Gromadzka-Ostrowska J.,
Rembiałkowska E., Skwarło-Sońta K., Eyre M., Cozzi G., Larsen M.K., Jordon T., Niggli U., Sakowski T., Calder P.C.,
Burdge G.C., Sotiraki S., Stefanakis A., Yolcu H., Stergiadis S., Chatzidimitriou E., Butler G., Stewart G. & Leifert C.
(2016). – Composition differences between organic and conventional meat: a systematic literature review and meta-
analysis. Br. J. Nutr., 115 (6), 994–1011. doi:10.1017/S0007114515005073.
21. Sapkota A.R., Lefferts L.Y., McKenzie S. & Walker P. (2007). – What do we feed to food-production animals? A review
of animal feed ingredients and their potential impacts on human health. Environ. Health Perspect., 115 (5), 663–670.
doi:10.1289/ehp.9760.
22. AOAC International – Beef and Poultry Adulteration of Meat Products. AOAC 987.06. Available at:
www.eoma.aoac.org/methods/info.asp?ID=16876.
23. ISO Standard (1978). – Meat and meat products — Determination of nitrogen content (Reference method). ISO
937:1978. Available at: https://www.iso.org/standard/5356.html.
24. AOAC International – Nitrogen in meat. AOAC 928.08.
25. AOAC International – Crude Protein in Meat and Meat Products Including Pet Foods. AOAC 992.15. Available at:
http://www.eoma.aoac.org/methods/info.asp?ID=16519%20.
26. AOAC International – Crude Protein in Meat. AOAC 981.10. Available at:
http://www.eoma.aoac.org/methods/info.asp?ID=16570.
27. AOAC International (2011). – Protein in Raw and Processed Meats. Automated Dye-Binding Method. AOAC 2011.04-
2011. Available at:
http://www.aoacofficialmethod.org/index.php?main_page=product_info&cPath=1&products_id=2983.
28. Codex Alimentarius (1999). – Recommended Methods of Analysis and Sampling. PART A – METHODS OF ANALYSIS BY
COMMODITY CATEGORIES AND NAMES, Processed Meat and Poultry Products and Soups and Broths. CODEX STAN
234-1999, p.52-54.
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Meat and meat products
29. AOAC International – Soy Protein in Raw and Heat-Processed Meat Products. AOAC 988.10. Available at:
http://eoma.aoac.org/methods/info.asp?ID=16859.
30. ISO Standard (1973). – Meat and meat products — Determination of total fat content. ISO 1443:1973. Available at:
https://www.iso.org/standard/5356.html.
31. AOAC International – Fat (Crude) or Ether Extract in Meat. AOAC 960.39. Available at:
http://www.eoma.aoac.org/methods/info.asp?ID=16128.
32. AOAC International – Fat (Crude) in Meat and Meat Products. AOAC 991.36. Available at:
http://www.eoma.aoac.org/methods/info.asp?ID=16281.
33. AOAC International – Nitrites in Cured Meat. AOAC 973.31.
34. ISO Standard (1975). – Meat and meat products — Determination of nitrite content (Reference method). ISO
2918:1975. Available at: https://www.iso.org/standard/7961.html.
35. ISO Standard (1975). – Meat and meat products — Determination of nitrate content (Reference method). ISO
3091:1975. Available at: https://www.iso.org/standard/8231.html.
36. AOAC International – Nitrates and Nitrites in Meat. AOAC 935.48.
37. Codex Alimentarius (1998). – Foodstuffs - Determination of nitrate and/or nitrite content - Part 3, Spectrometric
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12014-3:1998-06.
38. AOAC International (1985). – Vitamin C(Total) in Food - Semiautomated Fluorometric Method. AOAC 984.26-1985.
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39. ISO Standard (1974). – Meat and meat products — Determination of total phosphorus content (Reference method).
ISO 2294:1974. Available at: https://www.iso.org/standard/7120.html.
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method. ISO 13730:1996. Available at: https://www.iso.org/standard/22789.html.
41. ISO Standard (1980). – Meat and meat products — Detection of polyphosphates. ISO 5553:1980. Available at:
https://www.iso.org/standard/11620.html.
42. AOAC International – Phosphorus (Total) in Meat. AOAC 969.31. Available at:
http://www.eoma.aoac.org/methods/info.asp?ID=16332.
43. AOAC International – Phosphorus in Meat and Meat Products. AOAC 991.27. Available at:
http://www.eoma.aoac.org/methods/info.asp?ID=16417.
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chromatography. ISO 13496:2000. Available at: https://www.iso.org/standard/21237.html.
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http://www.eoma.aoac.org/methods/info.asp?ID=9328.
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2008.06. Available at: http://www.eoma.aoac.org/methods/info.asp?ID=49193.
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four Italian beef cattle breeds. Meat Sci., 80 (2), 389–395. doi:10.1016/j.meatsci.2008.01.001.
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Development of breed identification markers derived from AFLP in beef cattle. Meat Sci., 67 (2), 275–280.
doi:10.1016/j.meatsci.2003.10.016.
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Fish, seafood and related products
Elena Maestri*, Davide Imperiale, Luigi Parmigiani, Nelson Marmiroli
SITEIA.PARMA, University of Parma, Italy
*E-mail corresponding author: [email protected]
http://doi.org/10.32741/fihb.5.fish
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1. Product Identity
1.1. Definition of the product and manufacturing process
The commercial designation for seafood products is under the heading 03 in the CN code,
Commission Implementing Regulation (EU) 2017/1925 [6].
0302 is for “Fish, fresh or chilled, excluding fish fillets and other fish meat of heading 0304” and
includes all types of fish: Salmonidae, flat fish, tunas, herrings, cod families, tilapias, and also the
offal of fish.
0303 is for “Fish, frozen, excluding fish fillets and other fish meat of heading 0304” including again
the same types of fish.
0304 is for “Fish fillets and other fish meat (whether or not minced), fresh, chilled or frozen”
0305 is for “Fish, dried, salted or in brine; smoked fish, whether or not cooked before or during the
smoking process; flours, meals and pellets of fish, fit for human consumption”
0306 is for “Crustaceans, whether in shell or not, live, fresh, chilled, frozen, dried, salted or in
brine; smoked crustaceans, whether in shell or not, whether or not cooked before or during the
smoking process; crustaceans, in shell, cooked by steaming or by boiling in water, whether or not
chilled, frozen, dried, salted or in brine; flours, meals and pellets of crustaceans, fit for human
consumption” and includes lobsters, crabs, shrimps, crayfish
0307 is for “Molluscs, whether in shell or not, live, fresh, chilled, frozen, dried, salted or in brine;
smoked molluscs, whether in shell or not, whether or not cooked before or during the smoking
process; flours, meals and pellets of molluscs, fit for human consumption” and includes oysters,
scallops, mussels, cuttle fish and squid, octopus, snails, abalone and others.
0308 is for “Aquatic invertebrates other than crustaceans and molluscs, live, fresh, chilled, frozen,
dried, salted or in brine; smoked aquatic invertebrates other than crustaceans and molluscs,
whether or not cooked before or during the smoking process; flours, meals and pellets of aquatic
invertebrates other than crustaceans and molluscs, fit for human consumption” like sea
cucumbers, sea urchins, jellyfish.
The presence on the market of material which is in the shape of fillets or minced flesh, and
material which has been subjected to curing and processing, freezing, smoking, drying, opens
possibilities for fraudulent or accidental substitution and mislabelling.
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Fish, seafood and related products
The European Union has a legislation on seafood labelling, Regulation EU 1379/2013, requiring
indication of commercial designation, scientific name, method of production (caught, farmed),
geographical origin (catch area, body of water, country), fishing-gear category [11]. This is
associated to the traceability requirements of the General Food Law Regulation 178/2002 [12].
Other voluntary information is allowed about dates of catching, environmental or social
information, and nutritional content.
In the USA, the U.S. Food and Drug Administration has produced and maintains a list of Acceptable
Market Names which are allowed for seafood species [13].
2. Authenticity issues
2.1. Identification of current authenticity issues
The main problem for seafood authenticity is mislabelling for the species name, or species
substitution [14]. Indication of the species is an obligation in most labelling requirements.
However, particularly in processed products where visual recognition is not possible, the identity
of the animal can be counterfeited. Usually, there is an economic motivation, substituting
expensive and valued material with other species of lesser value or from illegal fishing. A further
problem is the fact that many seafood species are marketed under a shared name (“umbrella”
term) encompassing different species and/or genera; translation into local languages adds more
problems.
A second important issue concerns geographical origin, connected to the FAO fishing zones. When
this is declared on the label, it might be a fraudulent declaration to cover for IUU fishery or to
mask a species substitution. Similarly, a declaration about the fishing gear may raise the price of
the food product and be a fraud.
Processing or treatment can be falsely declared on the label, as in the case of freeze/thaw process
to sell fresh fish.
Additives can also be fraudulent, as in the case of tuna added with vegetables extracts, salts or
carbon monoxide to change the colour and make it look fresher.
Sustainability is a new issue which generates opportunities for fraud, when declarations about
place and way of fishing are untrue.
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Fish, seafood and related products
False declaration about the cold chain, or the freezing and thawing of products, may be hazardous
due to development of microbes and possible infections.
The recent Minamata Mercury Convention has highlighted the problem of mercury pollution in fish
and seafood. Mercury is transformed into the neurotoxic form methylmercury (MeHg) mainly in
aquatic environments, and from animal to animal it accumulates along the food chain. Humans are
exposed to MeHg through consumption of predator fish like tuna and swordfish, therefore a
correct labelling of the species name is important for an informed choice. The area of origin might
also be important in determining the levels of MeHg, but in this case it is hardly expected that
consumers might recognize the issue when purchasing fish [15,16].
Mislabelling for the geographical origin could become a health threat in case the seafood comes
from polluted areas due to radioactivity, or for the use of veterinary drugs allowed in some
countries and not in others.
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Fish, seafood and related products
ratio mass spectrometry) on the fish oil and choline from the lipid fraction extracted from the fish
1
COFAWS – Confirmation of the Origin of Farmed and Wild Salmon and other fish. Part funded by the European
Commission under the “Fight against Fraud” action and by the UK Food Standards Agency.
―5―
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Fish, seafood and related products
muscle [37]. These parameters successfully separated wild and farmed salmon both from known
origins and unknown market samples. The technique has since been used to check mislabelling in
the UK market. It has since been extended to other fish such as bream, cod, bass.
Other studies have been reported in the literature including a chemometrics approach addressing
the global chemical composition (trace elements, stable isotopes, fatty acids) has been recently
suggested [40,41]. Stable isotope ratios for carbon, nitrogen and oxygen have also been suggested
as a means for discriminating wild from farmed fish, and organic from intensive production, based
on differences in the feed origin [38,42]. A combination of isotope determination and other
profiling methods, e.g. trace elements or fatty acids, could be more effective. Isotopes of
Strontium could be indicative of geographic provenance, since this element is present together
with calcium in bones and calcified materials of seafood [43].
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Fish, seafood and related products
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Fish, seafood and related products
5. Conclusion
FAO [14] has identified the main needs to combat food fraud in the seafood sector: (i) reaching
agreements on names of products and species; (ii) introducing mandatory labelling; (iii) improving
the systems for official control of food; (iv) improving systems for food safety in production; (v)
adding new Codex guidelines.
It is widely recognised [1] that seafood is essential for healthy nutrition, providing nutrients,
micronutrients, vitamins. The steadily increasing consumption shows how public awareness has
grown. For pregnant women and children, particularly in low/middle income countries, seafood
contributes to development of the nervous system and is an accessible source of animal protein.
This can increase the exposure to methylmercury leading to risks for neurotoxicity [15].
Since fish and seafood are highly perishable, the transportation to consumers, in long supply
chains, provides logistic challenges and risks for health. Consumers nowadays require innovative
ways for chilling, preserving, delivering seafood, and in this area authenticity or fraud issues might
arise. Control of the cold chain and traceability with Universal Identifiers will be an area for
development, e.g. by blockchain technology [1].
Pollution will surely become more relevant, particularly considering abandoned, lost, discarded
fishing gear (ALDFG) and microplastics, on which knowledge is still missing. Fishery will also be
impacted by climate change and extreme weather events, requiring adaptation measures.
Aquaculture is included in the strategy for Climate Smart Agriculture, aiming to increase or
maintain production and mitigating impacts. Climate change will affect stocks worldwide, opening
the possibility for fraudulent behaviour in declarations on species or geographic origin.
Sustainability of fishing is also connected to climate change and geographical origin.
Considering the commercialisation of transgenic salmon in Canada, a possible additional
requirement for analytical methods will concern the traceability of transgenic material [56].
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geographic origins of jumbo squid ( Dosidicus gigas ). Rapid Commun. Mass Spectrom., 32 (7), 583–589.
doi:10.1002/rcm.8071.
40. Wang Y.V., Wan A.H.L., Lock E.J., Andersen N., Winter-Schuh C. & Larsen T. (2018). – Know your fish: A novel
compound-specific isotope approach for tracing wild and farmed salmon. Food Chem., 256, 380–389.
doi:10.1016/j.foodchem.2018.02.095.
41. Chaguri M.P., Maulvault A.L., Costa S., Gonçalves A., Nunes M.L., Carvalho M.L., Sant’ana L.S., Bandarra N. &
Marques A. (2017). – Chemometrics tools to distinguish wild and farmed meagre ( Argyrosomus regius ). J. Food
Process. Preserv., 41 (6), e13312. doi:10.1111/jfpp.13312.
42. Camin F., Bontempo L., Perini M. & Piasentier E. (2016). – Stable Isotope Ratio Analysis for Assessing the Authenticity
of Food of Animal Origin. Compr. Rev. Food Sci. Food Saf., 15 (5), 868–877. doi:10.1111/1541-4337.12219.
43. Baffi C. & Trincherini P.R. (2016). – Food traceability using the 87Sr/86Sr isotopic ratio mass spectrometry. Eur. Food
Res. Technol., 242 (9), 1411–1439. doi:10.1007/s00217-016-2712-2.
44. Kim M.R., Kwon K., Jung Y.K. & Kang T.S. (2018). – A rapid real-time PCR method to differentiate between mottled
skate ( Beringraja pulchra ) and other skate and ray species. Food Chem., 255, 112–119.
doi:10.1016/j.foodchem.2018.02.056.
45. Veneza I., Silva R. da, Sampaio I., Schneider H. & Gomes G. (2017). – Molecular protocol for authentication of
snappers (Lutjanidae-Perciformes) based on multiplex PCR. Food Chem., 232, 36–42.
doi:10.1016/j.foodchem.2017.03.007.
46. Marín A., Villegas-Llerena C., Fujimoto T. & Arai K. (2017). – Novel decaplex PCR assay for simultaneous detection of
scallop species with species-specific primers targeting highly variable 5ʹ end of the 16S rRNA gene. Aquac. Res., 48
(3), 920–930. doi:10.1111/are.12935.
47. Fernandes T.J.R., Costa J., Oliveira M.B.P.P. & Mafra I. (2018). – COI barcode-HRM as a novel approach for the
discrimination of hake species. Fish. Res., 197, 50–59. doi:10.1016/j.fishres.2017.09.014.
48. Grassi S., Casiraghi E. & Alamprese C. (2018). – Handheld NIR device: A non-targeted approach to assess authenticity
of fish fillets and patties. Food Chem., 243, 382–388. doi:10.1016/j.foodchem.2017.09.145.
49. Graziano S., Gullì M. & Marmiroli N. (2017). – Development and validation of a SYBR-Green I Real-Time PCR test to
detect bivalves including Mytilus species in foods. Int. J. Food Sci. Technol., 52 (7), 1567–1575.
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50. Bojolly D., Doyen P., Le Fur B., Christaki U., Verrez-Bagnis V. & Grard T. (2017). – Development of a qPCR Method for
the Identification and Quantification of Two Closely Related Tuna Species, Bigeye Tuna ( Thunnus obesus ) and
Yellowfin Tuna ( Thunnus albacares ), in Canned Tuna. J. Agric. Food Chem., 65 (4), 913–920.
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51. Ortea I., O’Connor G. & Maquet A. (2016). – Review on proteomics for food authentication. J. Proteomics, 147, 212–
225. doi:10.1016/j.jprot.2016.06.033.
52. Walker C.C., Lassitter C.L., Lynn S.N., Ford C.B., Rademacher K.R., Ruple A.D. & Bell J.W. (2017). – Rapid Seafood
Species Identification Using Chip-Based Capillary Electrophoresis and Protein Pattern Matching. J. AOAC Int., 100 (5),
1500–1510. doi:10.5740/jaoacint.17-0178.
53. Stahl A. & Schröder U. (2017). – Development of a MALDI–TOF MS-Based Protein Fingerprint Database of Common
Food Fish Allowing Fast and Reliable Identification of Fraud and Substitution. J. Agric. Food Chem., 65 (34), 7519–
7527. doi:10.1021/acs.jafc.7b02826.
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1.
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Cereals and cereal-based products
Jean-François Morin*, Michele Lees
Eurofins Analytics France, Nantes, France
*E-mail corresponding author: [email protected]
https://doi.org/10.32741/fihb.6.cereals
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Figure 1: Phylogenetic relationships of the cereal species and subspecies mentioned in this chapter (nomenclature reported according to http://www.ars-grin.gov)
Wheat and related products
1. Product identity
1.1. Definition of the product and manufacturing process
Wheat is widely grown around the world under diverse climatic conditions and has been the staple
food of the major civilisations in Europe, Asia and North Africa for 8000 years. Of the many species
of wheat that make up the genus Triticum, the most widely grown is common wheat, Triticum
aestivum. The second most cultivated species after common wheat is durum wheat, also known as
pasta wheat (Triticum turgidum subsp. durum).
Within each species there are a number of cultivars or varieties that can be placed into a number
of groups or types; these may be acceptable botanical groups based on grain or plant
characteristics, e.g. red and white grained, hard and soft grain textures, spring and winter types, or
groups based on other attributes such as baking performance or gluten characteristics. The harder
the wheat, the higher the protein content in the flour. Soft, low protein wheats are used for cakes,
pastries, biscuits and oriental noodles, whereas hard, high protein wheats are used in making
bread. Durum wheat is used for pasta and noodles.
Wheat production in 2016 accounted for 672.7 million tonnes worldwide [2]. In 2017, production
by the 28 EU Member States was 152.6 million tonnes, approximately 22 % of the worldwide
production. Of this European production, 93.6 % (142.8 million tonnes) was soft wheat with durum
wheat accounting for the remainder (6.3 %; 9.6 million tonnes) [3]. Production of soft wheat was
concentrated in France (25.3 %), Germany (17.0 %) and the UK (10.3 %). Poland, Romania and
Hungary produced 8.0, 6.9 and 4.4 % respectively. In the case of durum wheat, Italy accounted for
45.4 % of total production; other major producing countries were France (21.7 %), Greece
(13.0 %), and Spain (12.6 %).
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and baby foods for infants and young children lays down requirements for the composition of such
products, including cereal, protein, carbohydrate, mineral and vitamin contents. Further
compositional and labelling rules for processed cereal-based food in the EU Regulation on food for
specific groups [8].
At national level, regulations, directives, recommendations are in application in each European
country. They concern the production and sale of cereals, milling products, bread and pasta. Some
of them, dedicated to authentication, can be found in the regulations section of the FARNHub tool
[9]. For example, in Italian regulations, the presidential decree N° 187, dated 9 February 2001 [10],
stipulates that durum wheat milling products may contain up to three per cent of soft wheat flour.
More general regulations at national level can also be found on the EU-N-Lex website [11].
The upstream cereals sector concerning the seeds is also legislated by regulations defining the
production of new varieties, their registration and varietal purity. Council Directive 66/402/EEC of
14 June 1966 on the marketing of cereal seeds [12] establishes rules, amongst others, on the
production, packaging, sampling, sealing and marking in order to ensure the identity of the
certified seeds. This Directive has been amended several times and in particular by Commission
Directive 2009/74/EC of 26 June 2009 [13] as regards certain Annexes to Directive 66/402/EEC in
the light of developments of scientific and technical knowledge regarding seed purity.
From the point of view of the general public, consumers are showing increasing interest for
different qualities of bread produced from cereals such as spelt (T. spelta), emmer (T. dicoccum),
einkorn (T. monococcum). In order to preserve quality food products coming from particular
geographical areas and to protect consumers against imitations and false information, the
European Commission has defined, via Regulations [14] several quality labels, among which are
the Protected Designation of Origin (PDO) and Protected Geographical Indication (PGI) labels.
Products such as Farro di Monteleone di Spoleto (emmer) produced in Italy-Umbria (PDO), Farro
della Garfagnana (emmer) produced in Italy-Tuscany (PGI) and Petit épeautre de Haute
Provence (einkorn) produced in France (PGI) are protected by these European labels. Other cereal
products such as Epeautre d’Ardennes (spelt) produced in Ardennes in Belgium are protected by
regional labels based on specifications defined by the spelt sector [15].
In addition to the legislation, the cereal sector is managed by standards defining the best practices
in cereal sampling and quality analytical control. These are described in section 3.1.1. below.
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Wheat and related products
2. Authenticity issues
2.1. Identification of current authenticity issues
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Wheat and related products
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Wheat and related products
1
The ICC is an international network of cereal scientists and technologists dedicated to the improvement in safety and
quality of cereal-based foods, one of its missions in the validation and standardization of suitable test methods.
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Wheat and related products
the form of individual ground kernels, flour, farina or semolina, by the separation of gliadin
proteins. The separated protein components are visible from the stained polyacrylamide gels and
compared to a variety catalogue established for major wheat varieties. This method is in common
use in many countries.
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Wheat and related products
method has been favourably compared with the more commonly used ELISA method. A positive
QC-PCR signal and a negative ELISA result indicates a possible gliadin-free wheat starch addition
whereas the opposite situation indicates a possible addition of wheat-free gliadin as a food
additive [36].
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Wheat and related products
level according to the morphological profile, the NIR spectral profile, the protein content and
vitreousness [39].
This NIR technology has also been explored on other species such as barley, maize, and rice to
identify and discriminate varieties [38,39,44].
2
FP6 TRACE Project. Tracing the origin of food. 2005-2009. Funded by the European Commission under the 6th Framework
Programme.
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Wheat and related products
5. Conclusion
Potential authenticity issues in the future are likely to come from new products becoming available
in the market. The “pseudo-cereals” such as quinoa (Amaranthaceae), buckwheat (Lamiaceae) and
chia (Lamiaceae) are becoming increasing popular amongst consumers due to perceived health
benefits [52,53]. As these products command a higher price, there is the possibility that
adulteration or mislabelling will occur [54].
Fraud on wheat seed coating can also be cited as a potential issue. At the current time, no rapid
method exists that is able to assess the coating of cereals seeds. Kernel by kernel analysis by NIR
could be a way to address this potential fraud [55].
As regards the future of analytical methods, improvement is likely to be seen in the progress in
technology and instrumentation. Biomolecular methods remain the most powerful for
differentiating between the different cereals or different varieties of cereals. As the technology
surrounding DNA-based methods progresses, moving toward rapid throughput screening and
efficient instrumentation at an accessible cost, these techniques will be the methods of choice for
unambiguous discrimination. New tools based on proteomics can improve the application of
protein-based identification of species, cultivars or genotypes. Proteomic analysis of glutenins can
be used to detect allelic variants and quality-related issues in durum wheat flours [56].
Over the last few years, a growing number of handheld instruments based on near-infrared
spectroscopy including imaging systems, have appeared on the market. They are particularly
characterised by their compact appearance, ease of use, the ability to be controlled using a
wireless connection via a tablet or a smartphone. It is expected that innovative technology will be
used in order to get integrated NIR systems (spectral information) combined with imaging analysis
techniques (morphological information), sampling systems (representative information), and GPS
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Wheat and related products
devices (geolocated information). Some of them include predictive models for the simultaneous
determination of different quality parameters of the products. Other are connected through the
cloud to a central database and software making remote prediction of these parameters.
Beside the NIR sensors for solid/liquid measurement, new NIR sensors for gas analysis or other
sensors based on alternative spectroscopic techniques (mid-infrared, Raman, terahertz, nuclear
magnetic resonance etc.) are emerging on the market.
These new, smaller and low-cost instruments compared to conventional infrared devices should
answer the forthcoming challenges, in terms of precision agriculture, quality control and fraud
detection to improve authenticity and processing issues on food always more sophisticated [57].
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54. Shotts M.L., Plans Pujolras M., Rossell C. & Rodriguez-Saona L. (2018). – Authentication of indigenous flours (Quinoa,
Amaranth and kañiwa) from the Andean region using a portable ATR-Infrared device in combination with pattern
recognition analysis. J. Cereal Sci., 82, 65–72. doi:10.1016/j.jcs.2018.04.005.
55. Vermeulen P., Flemal P., Pigeon O., Dardenne P., Fernández Pierna J.A. & Baeten V. (2017). – Assessment of pesticide
coating on cereal seeds by near infrared hyperspectral imaging. J Spectr. Imaging, 6 (1a), 1–7.
56. Visioli G., Comastri A., Imperiale D., Paredi G., Faccini A. & Marmiroli N. (2016). – Gel-Based and Gel-Free Analytical
Methods for the Detection of HMW-GS and LMW-GS in Wheat Flour. Food Anal. Methods, 9 (2), 469–476.
doi:10.1007/s12161-015-0218-3.
57. Baeten V., Pierna J.A.F., Lecler B., Abbas O., Vincke D., Minet O., Vermeulen P. & Dardenne P. (2016). – Near Infrared
Spectroscopy for Food and Feed: A Mature Technique. NIR News, 27 (1), 4–6. doi:10.1255/nirn.1573.
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1. Product Identity
Estimated world production of paddy rice in 2016 was 741.0 million tonnes [1]. Of this, Asia
accounted for over 90.1 %, Americas for 4.9 %, Africa for 4.4 % and the 28 EU Member States for
about 0.4 %. The three main producers, China, India and Indonesia, produced more than 60 % of
the world‘s rice. Within the EU, Italy, Spain, Greece, Portugal, France, Bulgaria, Romania and
Hungary are rice producers. Italy accounts for 52.6 % of the total European production in 2017,
followed by Spain with 28.0 %, Greece with 6.5 % and Portugal with 5.6 % [2].
The majority of the world’s paddy rice is consumed in Asia where it is produced. In the
international rice trade, a relatively small number of exporting countries, notably Thailand,
Vietnam and India, interacts with a large number of importing countries in Asia, in Africa and also
in Europe [3].
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milled rice from all countries producing more than 0.1 % of the world’s rice. However, consumer
tastes are changing, and in a highly competitive market with stringent quality requirements and
some varieties prized above others, problems of adulteration can occur.
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1.2.3. EU Regulations
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Commission Regulation (EU) No 1272/2009 [9] lays down common detailed rules for buying-in and
selling of agricultural products under public intervention. However, rice is only accepted into
intervention if it complies with certain eligibility criteria (quality specifications), related to moisture
content, milling yield, defects in the grains, miscellaneous impurities, grains of other rice varieties.
As regards trade with third countries, Commission Regulation (EC) No 1342/2003 [10] lays down
specific rules for the system of import and export licences for cereals and rice. Following
international agreements under WTO or bilateral negotiations, various Tariff Rate Quotas (TRQs)
allow rice imports at low or even zero duty. These are detailed in Commission Regulation (EU) No
1273/2011 [11] which are reopened on 1 January each year and apply specifically to the country of
origin of the imported rice.
In addition, for broken rice used in the production of infant foods, a specific tariff quota for 1000
tonnes at zero duty is available through Commission Regulation (EU) No 480/2012 [12].
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2. Authenticity issues
2.1. Identification of current authenticity issues
Both cultivar and cultivation area are major factors in determining the market price of rice. Hence,
the main authenticity issues are the substitution of one variety or cultivar with another, or the
mislabelling of the geographical origin of the rice.
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The most well-known case of adulteration involved rice-derived products and the addition of
melamine and melamine-related products in rice protein concentrate. This occurred in 2007, when
melamine and cyanuric acid were found in products labelled as rice protein concentrate being
used in the production of pet food. These products had been added to increase the apparent
protein content. The contaminated pet food led to the sickness and death in some cases of pet
dogs and cats in the USA [21].
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Two major targets for geographical authentication are Basmati and Thai rice varieties. For the
former, a combination of 10 rare earth elements ((La, Ce, Pr, Nd, Sm, Eu, Gd, Dy, Er, Yb) and the
87 86
isotope ratio of strontium ( Sr/ Sr) were used as tracers for differentiating Indian Basmati rice
from the other countries of origin[45]. Discrimination of Thai jasmine rice (Khao Dawk Mali 105)
cultivated in five different regions was achieved using 9 elements (As, Mg, Cl, Al, Br, Mn, K, Rb and
13 15 18
Zn) and stable isotopes δ C, δ N, and δ O with 100 % correct classification [46].
5. Conclusion
The main authenticity challenge in the future will be likely to concern the differentiation of rice
varieties. There is currently a huge diversity of rice varieties available, with new varieties being
developed all the time. Some of these varieties command premium prices because of their
particular cooking characteristics or flavour profile. Others fall under negotiated trade tariff
categories granting low or zero duty on imports. GM technology is being investigated for the
biofortification (enhanced folate, zinc and iron content). A further example is Golden Rice [47], a
variety of Oryza sativa that has been genetically engineered to biosynthesis beta-carotene, a
precursor of vitamin A, in the edible parts of rice. This variety has been accepted as safe by a
number of governments around the world but because of the stiff resistance to GM technology,
the product is not yet available.
Today the most complete and comprehensive analytical tool for rice authentication is the
combination of DNA-based methods to confirm variety together with stable isotope ratio analysis
with multi-element analysis to verify provenance. In the future, technological progress particularly
in the instrumentation used will greatly improve the ease-of-use of these techniques. A current
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example is the use of modern sequencing, known as Next-Generation Sequencing (NGS), or high-
throughput sequencing, which enables the analyst to sequence DNA and RNA much more quickly
and cheaply than before.
Today’s focus is shifting more and more towards this type of untargeted approach. Other
1
techniques investigated in this respect include H NMR, which has been studied as a means of
discriminating rice from different regions in China [48], multi-platform MS-based metabolomics
and multivariate analysis for the geographical origin [49] and a LC-MS untargeted approach to
distinguish between organic and conventional rice [50]. These approaches offer the potential of
rapid authentication methods or of a short-cut to identifying suitable markers for use in
authenticity testing.
6. Bibliographic references
1. FAOSTAT Available at: http://fao.org/faostat/en#data.
2. European Commission – Eurostat Database. Available at: http://ec.europa/eurostat/web/agriculture/data/database.
3. Ricepedia – The online authority on rice. Available at: http://ricepedia/org/.
4. Juliano B.O. & Villareal C.P. (1993). – Grain quality evaluation of world rices. IRRI, International Rice Research
Institute, Manila, Philippines.
5. Bael K. (2017). – Rice starch as a unique, natural and invaluable food source. New Food Mag. Available at:
https://www.newfoodmagazine.com/article/33430/beneo-rice-starch/.
6. ISO Standard (2011). – Rice Specification. ISO 7301:2011, 19.
7. Joint FAO/WHO Codex Alimentarius Commission – Codex Standard for rice. Codex STAN 198-1995.
8. Regulation (EU) No 1308/2013 of the European Parliament and of the Council of 17 December 2013 establishing a
common organisation of the markets in agricultural products (2013). Off. J. Eur. Union, L347, 671–854.
9. Regulation (EU) No 1272/2009 of 11 December 2009 laying down common detailed rules for the implementation of
Council Regulation (EC) No 1234/2007 as regards buying-in and selling of agricultural products under public
intervention (2009). Off. J. Eur. Union, L349, 1–68.
10. Regulation (EC) No 1342/2003 of 28 July 2003 laying down special detailed rules for the application of the system of
import and export licences for cereals and rice (2003). Off. J. Eur. Union, L189, 12–29.
11. Commission Implementing Regulation (EU) No 1273/2011 of 7 December 2011 opening and providing for the
administration of certain tariff quotas for imports of rice and broken rice (2011). Off. J. Eur. Union, L325, 6–23.
12. Commission Implementing Regulation (EU) No 480/2012 of 7 June 2012 opening and providing for the management
of a tariff quota for broken rice of CN code 10064000 for production of food preparations of CN code 19011000
(2013). Eur. Comm., L148. Available at: http://data.europa.eu/eli/reg_impl/2012/480/2013-07-01.
13. Regulation (EU) No 1169/2011 of the European parliament and of The Council of 25 October 2011 on the
provision of food information to consumers (2011). Off. J. Eur. Union, L304, 18–63.
14. Commission Implementing Regulation (EU) No 706/2014 of 25 June 2014 amending Regulation (EC) No 972/2006 as
regards the import duty applicable to Basmati rice (2014). Off. J. Eur. Union, L186, 54–55.
15. APEDA Agricultural and Processed Food Products Export Development Authority, India (2017). – Notified varieties of
Basmati rice. Available at: http://www.apeda.gov.in/apedawebsite/SubHead_Products/Basmati_Rice.htm.
16. National Bureau of Agricultural Commodity and Food Standards & Ministry of Agriculture and Cooperatives (2003). –
Thai Agricultural Standard TAS 4000-2003.
17. foodmanufacture.co.uk – Risotto rice is targeted by fraudsters. foodmanufacture.co.uk. Available at:
https://www.foodmanufacture.co.uk/Article/2015/07/02/Food-fraudsters-target-risotto.
18. Feng X., Zhang Q., Cong P. & Zhu Z. (2013). – Preliminary study on classification of rice and detection of paraffin in
the adulterated samples by Raman spectroscopy combined with multivariate analysis. Talanta, 115, 548–555.
doi:10.1016/j.talanta.2013.05.072.
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19. Fake Rice? Chromatography Searches for a Grain of Truth Chromatography Today Available at:
https://www.chromatographytoday.com/news/gc-mdgc-gc-
ms/32/breaking_news/fake_rice_chromatography_searches_for_a_grain_of_truth/35561.
20. SecuringIndustry.com - Philippines deploy scanner to spot “plastic rice” Available at:
https://www.securingindustry.com/food-and-beverage/philippines-deploy-scanner-to-spot-plastic-rice-
/s104/a2505/#.W2FxKLg6_IU.
21. Medicine C. for V. – Recalls & Withdrawals - Melamine Pet Food Recall of 2007. Available at:
https://www.fda.gov/AnimalVeterinary/SafetyHealth/RecallsWithdrawals/ucm129575.htm.
22. AACC International Approved Methods -AACC Method 61-01.01. Amylograph Method for Milled Rice Available at:
http://methods.aaccnet.org/summaries/61-01-01.aspx.
23. AACC International Approved Methods -AACC Method 61-03.01. Amylose Content of Milled Rice Available at:
http://methods.aaccnet.org/summaries/61-03-01.aspx.
24. AACC International Approved Methods - AACC Method 38-52.01. Gluten in Rice Flour and Rice-Based Products by
G12 Sandwich ELISA Assay Available at: http://methods.aaccnet.org/summaries/38-52-01.aspx.
25. Tittlemier (2009). – Background Paper on Methods for the Analysis of Melamine and Related Compounds in Foods
and Animal Feeds.
26. Nutrition C. for F.S. and A. – Laboratory Methods - Laboratory Information Bulletin: LIB 4423 Melamine and Related
Compounds. Available at: https://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm071759.htm.
27. Sood B.C. & Siddiq E.A. (1978). – A Rapid Technique for Scent Determination in Rice. Indian J. Genet. Plant Breed., 38
(2), 268–275.
28. Widjaja R., Craske J.D. & Wootton M. (1996). – Changes in Volatile Components of Paddy, Brown and White Fragrant
Rice During Storage. J. Sci. Food Agric., 71 (2), 218–224. doi:10.1002/(SICI)1097-0010(199606)71:2<218::AID-
JSFA570>3.0.CO;2-5.
29. Saini N., Jain N., Jain S. & Jain R.K. (2004). – Assessment of genetic diversity within and among Basmati and non-
Basmati rice varieties using AFLP, ISSR and SSR markers. Euphytica, 140 (3), 133–146. doi:10.1007/s10681-004-2510-
y.
30. Pal S., Jain S., Saini N. & Aarti J.R.K. (2004). – Identification of microsatellite markers for differentiating some Basmati
and non-Basmati rice varieties. Indian J. Biotechnol., 3, 519–526.
31. Ganopoulos I., Argiriou A. & Tsaftaris A. (2011). – Adulterations in Basmati rice detected quantitatively by combined
use of microsatellite and fragrance typing with High Resolution Melting (HRM) analysis. Food Chem., 129 (2), 652–
659. doi:10.1016/j.foodchem.2011.04.109.
32. Vemireddy L.R., Satyavathi V.V., Siddiq E.A. & Nagaraju J. (2015). – Review of methods for the detection and
quantification of adulteration of rice: Basmati as a case study. J. Food Sci. Technol., 52 (6), 3187–3202.
doi:10.1007/s13197-014-1579-0.
33. 2017 Basmati Code of Practice - Rice Association Available at:
http://www.riceassociation.org.uk/content/1/47/2017-basmati-code-of-practice.html.
34. Locate methods for authenticity testing Available at: http://www.foodauthenticity.uk/methods.
35. Nader W.F., Brendel T. & Schubbert R. (2016). – 2 - Advances in DNA Fingerprinting for Food Authenticity Testing. . In
Advances in Food Authenticity Testing (G. Downey, ed), Woodhead Publishing. pp 7–33doi:10.1016/B978-0-08-
100220-9.00002-3.
36. Wu Y., Zhang Z., Chen Y., Wang B., Yang G. & Yang W. (2009). – Authentication of Thailand jasmine rice using RAPD
and SCAR methods. Eur. Food Res. Technol., 229 (3), 515–521. doi:10.1007/s00217-009-1072-6.
37. Cirillo A., Del Gaudio S., Di Bernardo G., Galderisi U., Cascino A. & Cipollaro M. (2009). – Molecular characterization
of Italian rice cultivars. Eur. Food Res. Technol., 228 (6), 875–881. doi:10.1007/s00217-008-1000-1.
38. Chung I.M., Kim J.K., Prabakaran M., Yang J.H. & Kim S.H. (2015). – Authenticity of Rice (Oryza sativa L.) Geographical
Origin based on Analysis of C, N, O, and S Stable Isotope Ratios: A preliminary case report in Korea, China, and
Philippine. J. Sci. Food Agric., 96. doi:10.1002/jsfa.7363.
39. Chung I.M., Park S.K., Lee K.J., An M.J., Lee J.H., Oh Y.T. & Kim S.H. (2017). – Authenticity testing of environment-
friendly Korean rice (Oryza sativa L.) using carbon and nitrogen stable isotope ratio analysis. Food Chem., 234, 425–
430. doi:10.1016/j.foodchem.2017.05.014.
40. Chen T., Zhao Y., Zhang W., Yang S., Ye Z. & Zhang G. (2016). – Variation of the light stable isotopes in the superior
and inferior grains of rice (Oryza sativa L.) with different geographical origins. Food Chem., 209, 95–98.
doi:10.1016/j.foodchem.2016.04.029.
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41. Gonzálvez A., Armenta S. & Guardia M. de la (2011). – Geographical traceability of “Arròs de Valencia” rice grain
based on mineral element composition. Food Chem., 126 (3), 1254–1260. doi:10.1016/j.foodchem.2010.11.032.
42. Barbosa R.M., Paula E.S. de, Paulelli A.C., Moore A.F., Souza J.M.O., Batista B.L., Campiglia A.D. & Barbosa F. (2016). –
Recognition of organic rice samples based on trace elements and support vector machines. J. Food Compos. Anal.,
45, 95–100. doi:10.1016/j.jfca.2015.09.010.
43. Kelly S., Baxter M., Chapman S., Rhodes C., Dennis J. & Brereton P. (2002). – The application of isotopic and
elemental analysis to determine the geographical origin of premium long grain rice. Eur. Food Res. Technol., 214 (1),
72–78. doi:10.1007/s002170100400.
44. Chung I.M., Kim J.K., Lee K.J., Park S.K., Lee J.H., Son N.Y., Jin Y.I. & Kim S.H. (2018). – Geographic authentication of
Asian rice (Oryza sativa L.) using multi-elemental and stable isotopic data combined with multivariate analysis. Food
Chem., 240, 840–849. doi:10.1016/j.foodchem.2017.08.023.
45. Lagad R.A., Singh S.K. & Rai V.K. (2017). – Rare earth elements and 87Sr/86Sr isotopic characterization of Indian
Basmati rice as potential tool for its geographical authenticity. Food Chem., 217, 254–265.
doi:10.1016/j.foodchem.2016.08.094.
46. Kukusamude C. & Kongsri S. (2018). – Elemental and isotopic profiling of Thai jasmine rice (Khao Dawk Mali 105) for
discrimination of geographical origins in Thung Kula Rong Hai area, Thailand. Food Control, 91, 357–364.
doi:10.1016/j.foodcont.2018.04.018.
47. The Golden Rice Project Available at: http://goldenrice.org/.
48. Huo Y., Kamal G.M., Wang J., Liu H., Zhang G., Hu Z., Anwar F. & Du H. (2017). – 1H NMR-based metabolomics for
discrimination of rice from different geographical origins of China. J. Cereal Sci., 76, 243–252.
doi:10.1016/j.jcs.2017.07.002.
49. Lim D.K., Mo C., Lee J.H., Long N.P., Dong Z., Li J., Lim J. & Kwon S.W. (2018). – The integration of multi-platform MS-
based metabolomics and multivariate analysis for the geographical origin discrimination of Oryza sativa L. J. Food
Drug Anal., 26 (2), 769–777. doi:10.1016/j.jfda.2017.09.004.
50. Xiao R., Ma Y., Zhang D. & Qian L. (2018). – Discrimination of conventional and organic rice using untargeted LC-MS-
based metabolomics. J. Cereal Sci., 82, 73–81. doi:10.1016/j.jcs.2018.05.012.
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Nuts, nut products and other seeds
Elena Maestri*, Davide Imperiale, Nelson Marmiroli
SITEIA.PARMA, University of Parma, Italy
*E-mail corresponding author: [email protected]
https://doi.org/10.32741/fihb.7.nuts
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1. Product Identity
1.1. Definition of the product and manufacturing process
There is no accepted definition of "nuts" in food commodities. The fruits most commonly
considered as nuts include the following (with indication of CN code, cf. Commission Implementing
Regulation (EU) 2017/1925 of 12 October 2017 [3]):
Almond (code 0802 11-12): seed of the species Prunus dulcis, (or Prunus amygdalus) after removal
of the fleshy hull (stone fruit). They can be marketed inshell, without shell as kernels, or as
blanched kernels after removal of the tegument of the kernel (episperm). There are sweet almond
and bitter almonds, containing amygdalin, a glycoside which releases hydrocyanic acid. USA is the
major producer followed by Australia and Spain.
Brazil nut (code 0801 21 00-22 00): nuts of the tree Bertholletia excelsa, either shelled or after
cracking of the shell. The shell is extremely hard and woody, and the kernel is enveloped by a
brown seed coat. Bolivia is the major producer, followed by Peru and Brazil; UK is the major
importer. The yield is highly dependent on environmental conditions.
Cashew nut (code 0801 31 00-32 00): the nut with the hard shell, also called anacardium, is at the
bottom of the fruit (cashew apple) produced by the plant Anacardium occidentale. The main
producer is Western Africa (Cote d'Ivoire), followed by India and Vietnam.
Chestnut (code 0802 41 00-42 00): these are the fruits of trees from the genus Castanea, mainly C.
sativa. The husk is spiky and breaks open spontaneously, revealing 1-3 fruits; the fruit has a kernel,
a thin skin and brown pericarp. The main producer is China, followed by Turkey and Italy.
Hazelnut, or filbert (code 0802 21 00-22 00): nuts of the species Corylus avellana and C. maxima,
free from the husk, sold shelled or after cracking of the shell. Over half of the global production
comes from Turkey, followed by Italy, which is also the main importer.
Macadamia nut (code 0802 61 00-62 00): nuts of the species Macadamia integrifolia, M.
tetraphylla, M. ternifolia growing in hot subtropical climates. After drying and shell cracking, the
kernels can be dry-roasted or oil-roasted. Used in confectionery, baking, ice cream, snacks.
Production is concentrated in Australia, South Africa and Kenya.
Peanut (cod 2008 11): also called groundnuts, they are leguminous fruits of the plant Arachis
hypogaea, growing underground. They have a thin shell, which is in fact the pod, containing
generally two kernels. China is the world major producer, followed by India and Nigeria.
Pecan nut (code 0802 90 10): the seed comes from the tree Carya illinoensis and is encased in a
husk. It can be consumed fresh or used in cooking. The main producers are Mexico and USA.
Pine nut (code 0802 90 50): decorticated kernels of different species of Gymnosperm, Pinus: e.g.
pinea, koraiensis, sibirica, yunnanensis, wallichiana, gerardiana, pumila. The main producers are in
Asia, China, North Korea, Russian Federation. Italy leads the production of Mediterranean pine
nut.
Pistachio nut (code 0802 51 00, 52 00): the kernels are in the single-seeded stone fruit of the tree
Pistacia vera, with a brown seed coat and brilliant green kernel. They are marketed in shell, raw or
salted, sugared, flavoured. The major producer is the USA, followed by Iran and Turkey.
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Walnut (code 0802 31 00-32 00): nuts of the tree Juglans regia, enclosed in a shell made of two
halves, free from the outer green and fleshy husk, sold in the shell or after cracking of the shell.
China and USA are the major producers.
Nuts are generally harvested by shaking trees, in processes which can be mechanised or
performed manually. The fruits are then washed and dried. Some nuts are marketed as such, some
undergo bleaching of the shell to improve the appearance, and others are taken out from the shell
by cracking. Storage varies, with some nuts being more durable than others. New technologies are
actively improving drying and processing, except in cases where natural production requires sun-
drying.
They are commonly sold to the food manufacturing industry, as ingredients, to be processed, and
repackaged. The chocolate industry is the largest user of many edible nuts, also playing on their
health benefits. Breakfast cereals and energy bars involve also edible nuts in their formulations.
The snack industry uses large quantities of edible nuts, particularly peanuts. Other industries
involved concern bakery, ice creams, nut butter, nut milk, and even pet food. The increase in the
use of edible nuts is due to new information about health attributes and claims, the availability of
new types of nuts, and new processing and flavouring possibilities.
Increasingly there are requirements for sustainable products in appealing to consumers,
particularly when edible nuts come from developing countries.
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CODEX has produced a comprehensive document on the Code of hygienic practice for tree nuts,
CAC/RCP 6-1972, on the cultivation, processing, shelling etc. A separate document covers practices
for peanuts, CAC/RCP 22-1979 [12]. A more recent document concerns the Code of practice for the
prevention and reduction of aflatoxin contamination in tree nuts, CAC/RCP 59-2005 [13].
The Transport Information Service [14] provides relevant information and specifications about the
transport of nuts and the possible problems which may arise to damage the products. The website
Standards Map [15] provides a tool for recovering the information on sustainable trade, linking to
the different standards on this subject.
2. Authenticity issues
2.1. Identification of current authenticity issues
The main authenticity issue for tree nuts is probably the country of origin, since this indication is
usually mandatory on the label. Indication of the species is also obligatory and relevant,
particularly for problems linked to allergies: substitution of nuts from different species can
negatively affect allergy patients, if undeclared. The indications on the label can also include the
crop year and the variety. The year of harvest is important because older nuts are more prone to
infestation and rancidity.
A possibility for fraud exists in the declaration for organic products, which should be produced and
processed with specific techniques, including the use of crop rotation, specific crop protection and
fertilisation substances.
Additionally, nuts are used in Jewish cuisine and the Kosher certification is another possible source
for fraudulent declarations.
The increase in demand, combined with problematic harvests, can lead to the shortage of some
edible nuts, and this could open the way to counterfeiting. In the case of Brazil nuts, which are
collected in the Amazonian forest, their availability from year to year is not predictable.
Protected denominations (PGI or PDO) for cultivars of some edible nuts are particularly
appreciated and can be subject to fraud. One such example is the hazelnut "Tonda Gentile delle
Langhe" (Nocciola Piemonte, PGI since 1996) from Italy. Other relevant PDO examples in Europe
concern: chestnuts (Portugal and Italy), almonds (Portugal), walnuts (France, Italy).
Some PDO or PGI productions requiring nuts as ingredients are also subjected to fraud. One
relevant example is Ligurian pesto, a sauce made with basil containing pine nuts as a highly
relevant ingredient. In this case, Pinus pinea is the species of origin, but other pine species could
be used since recognition by visual inspection is impossible. Mislabelling of pine nuts is relevant for
the taste, but also for health effects (see below).
Hazelnut paste is an important ingredient in confectionery, and also the ingredient for appreciated
hazelnut spreads for direct consumption (e.g. Nutella). The percentage of hazelnuts in the paste is
a critical quality issue and dilution or substitution with artificial compounds or with other
ingredients are fraudulent practices.
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The presence of lipids and fatty acids in nuts and nut products can be a problem in some analytical
techniques, but it is also a distinctive feature of nuts. Methods based on analysis of fatty acids or
other metabolites have been developed to check for the authenticity of the species or of the
cultivar, even in highly processed foods [21–23].It is widely considered that these kind of markers
can provide indications about the geographic origin of food products, whereas DNA markers
cannot be used for this purpose [24,25]. They could also provide indications about the year of
harvest, but no method has currently been developed.
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5. Conclusion
The worldwide trend in consumption of nuts, because of their health benefits, supports a
sustainable growth for this market. New literature reporting on the beneficial effects of
unsaturated fatty acids and other components of some nuts are leading to their introduction in
diets for patients or people at risk of cardiovascular diseases. Milk substitutes from nuts, and nuts
substituting meat, will be appealing to vegans. Savoury snacks are already widely used, but they
are constantly increasing because of health effects, new processing ideas, and availability of
several nut types. In view of this, the increased consumption could lead to fraud opportunities.
Additionally, since the chemical composition of nuts depends on species and geographic origin,
fraudulent labelling for provenance could be envisaged. Current methods can effectively recognise
species and cultivars, but recognition of provenance is more difficult.
Consumer interest in sustainability of provenance and fair trade are particularly increasing in the
EU market, and this also applies to fruit and nuts. Certification of these schemes will probably
become more common, creating potential opportunities for frauds. However, methods to
establish the correctness of such claims are not yet available.
In the case of nuts, climate change is expected to have highly negative and unpredictable effects.
The areas for production, often in tropical countries, will be heavily affected by changes in growing
seasons, temperature and rainfall. Additionally, new pathogens and infectious diseases are
expected to appear. Currently, pine nuts are defined as the “caviar of plants” because they are
becoming increasingly rare, mainly due to an increase in pathogens worldwide, and to
deforestation and changes in climate conditions. The yield and safety of the products will
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therefore be affected in a negative way, and this will probably require additional methods for
traceability and inspection. In Ligurian pesto, for instance, the pine nuts required by the original
recipe are sometimes substituted with cashews. Fraud control will therefore require methods for
species recognition, but also methods for provenance recognition, which are not completely
developed.
The certification of organic cultivation will also become more important. This market is growing
continuously, and the production cannot keep up with the pace. This will also be an opportunity
for fraud. Unfortunately analytical controls on organic cultivation are not well developed at the
moment.
6. Bibliographic references
1. United States Department of Agriculture - Forest Service – Nuts. Available at:
https://www.fs.fed.us/wildflowers/ethnobotany/food/nuts.shtml.
2. Weinberger T. & Sicherer S. (2018). – Current perspectives on tree nut allergy: a review. J. Asthma Allergy, 11, 41–51.
doi:10.2147/JAA.S141636.
3. Commission Implementing Regulation (EU) 2017/1925 of 12 October 2017 amending Annex I to Council Regulation
(EEC) No 2658/87 on the tariff and statistical nomenclature and on the Common Customs Tariff (2017). Off. J. Eur.
Union, L282, 1–958.
4. UNECE – Dry and Dried Produce - Standards - Trade. Available at:
http://www.unece.org/trade/agr/standard/dry/ddp-standards.html.
5. OECD – Brochures - OECD Fruit and Vegetables Scheme. Available at: http://www.oecd.org/agriculture/fruit-
vegetables/publications/brochures/.
6. Regulation (EU) No 1169/2011 of the European parliament and of The Council of 25 October 2011 on the
provision of food information to consumers (2011). Off. J. Eur. Union, L304, 18–63.
7. Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in
foodstuffs (2006). Off. J. Eur. Union, L364, 5–24.
8. Commission Regulation (EU) No 165/2010 of 26 February 2010 amending Regulation (EC) No 1881/2006 setting
maximum levels for certain contaminants in foodstuffs as regards aflatoxins (2010). Off. J. Eur. Union, L50, 8–12.
9. European Commission – RASFF - Food and Feed Safety Alerts - Food Safety. Food Saf. Available at:
https://webgate.ec.europa.eu/rasff-window/portal/.
10. ISO Standard (1982). – Fruits — Nomenclature — First list. ISO 1990-1:1982. Available at:
https://www.iso.org/standard/6726.html.
11. ISO Standard (1991). – Dry fruits and dried fruits — Definitions and nomenclature. ISO 4125:1991. Available at:
https://www.iso.org/standard/9877.html.
12. Codex Alimentarius (1979). – Code of hygienic practice for groundnuts (peanuts). CAC/RCP 22-1979. Available at:
http://www.fao.org/fao-who-codexalimentarius/sh-
proxy/en/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCAC
%2BRCP%2B22-1979%252FCXP_022e.pdf.
13. Codex Alimentarius (2005). – Code of practice for the prevention and reduction of aflatoxin contamination in tree
nuts. CAC/RCP 59-2005. Available at: http://www.fao.org/fao-who-codexalimentarius/sh-
proxy/fr/?lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FStandards%252FCAC
%2BRCP%2B59-2005%252FCXP_059e.pdf.
14. Cargo site map Available at: http://www.tis-gdv.de/tis_e/ware/inhaltx.htm#6.
15. Identify Voluntary Sustainability Standards to start your sustainable trade journey! Available at:
http://standardsmap.org/identify2.aspx.
16. Safenut Project - Home Available at: http://www.stdf-safenutproject.com/.
17. Awan H., Pettenella D., Awan H.U.M. & Pettenella D. (2017). – Pine Nuts: A Review of Recent Sanitary Conditions and
Market Development. Forests, 8 (10), 367. doi:10.3390/f8100367.
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18. Center for Food Safety and Applied Nutrition – Laboratory Methods - MPM: V-10. Nuts and Nut Products Methods.
Available at: https://www.fda.gov/food/foodscienceresearch/laboratorymethods/ucm084406.htm.
19. Rabadán A., Pardo J.E., Gómez R., Alvarruiz A. & Álvarez-Ortí M. (2017). – Usefulness of physical parameters for
pistachio cultivar differentiation. Sci. Hortic., 222, 7–11. doi:10.1016/j.scienta.2017.04.034.
20. Akkak A., Boccacci P. & Botta R. (2008). – An Efficient DNA-Extraction Protocol for Nut Seeds. J. Food Qual., 31 (4),
549–557. doi:10.1111/j.1745-4557.2008.00219.x.
21. Čížková H., Rajchl A., Šnebergrová J. & Voldřich M. (2013). – Filbertone as a marker for the assessment of hazelnut
spread quality. Czech J. Food Sci., 31, 81–87. doi:10.17221/493/2011-CJFS.
22. Barreira J.C.M., Alves R.C., Casal S., Ferreira I.C.F.R., Oliveira M.B.P.P. & Pereira J.A. (2009). – Vitamin E Profile as a
Reliable Authenticity Discrimination Factor between Chestnut (Castanea sativa Mill.) Cultivars. J. Agric. Food Chem.,
57 (12), 5524–5528. doi:10.1021/jf900435y.
23. Ruiz del Castillo M.L., Gómez Caballero E., Blanch G.P. & Herraiz M. (2002). – Enantiomeric composition of filbertone
in hazelnuts and hazelnut oils from different geographical origins. J. Am. Oil Chem. Soc., 79 (6), 589–592.
doi:10.1007/s11746-002-0527-1.
24. Manfredi M., Robotti E., Quasso F., Mazzucco E., Calabrese G. & Marengo E. (2018). – Fast classification of hazelnut
cultivars through portable infrared spectroscopy and chemometrics. Spectrochim. Acta. A. Mol. Biomol. Spectrosc.,
189, 427–435. doi:10.1016/j.saa.2017.08.050.
25. Lerma-García M.J., Cortés V., Talens P. & Barat J.M. (2018). – Chapter Six - Variety Discrimination of Fruits, Edible
Plants, and Other Foodstuffs and Beverages by Infrared Spectroscopy. . In Comprehensive Analytical Chemistry (J.
Lopes & C. Sousa, eds), Elsevier. pp 127–163doi:10.1016/bs.coac.2018.03.004.
26. Brüning P., Haase I., Matissek R. & Fischer M. (2011). – Marzipan: Polymerase Chain Reaction-Driven Methods for
Authenticity Control. J. Agric. Food Chem., 59 (22), 11910–11917. doi:10.1021/jf202484a.
27. Ballin N.Z. & Mikkelsen K. (2016). – Polymerase chain reaction and chemometrics detected several Pinus species
including Pinus armandii involved in pine nut syndrome. Food Control, 64, 234–239.
doi:10.1016/j.foodcont.2015.12.036.
28. Nader W., Brendel T. & Schubbert R. (2013). – DNA-analysis: Enhancing the control of food authenticity through
emerging technologies. Agro Food Ind. Hi Tech, 24 (1), 42–46.
29. López-Calleja I.M., Cruz S. de la, Pegels N., González I., García T. & Martín R. (2013). – High resolution TaqMan real-
time PCR approach to detect hazelnut DNA encoding for ITS rDNA in foods. Food Chem., 141 (3), 1872–1880.
doi:10.1016/j.foodchem.2013.05.076.
30. Pafundo S., Gullì M. & Marmiroli N. (2010). – Multiplex real-time PCR using SYBR® GreenERTM for the detection of
DNA allergens in food. Anal. Bioanal. Chem., 396 (5), 1831–1839. doi:10.1007/s00216-009-3419-z.
31. Christopoulou S., Karaiskou S. & Kalogianni D.P. (2017). – Microbead-based simultaneous fluorometric detection of
three nut allergens. Microchim. Acta, 185 (1), 13. doi:10.1007/s00604-017-2559-7.
32. Sheu S.C., Tsou P.C., Lien Y.Y. & Lee M.S. (2018). – Development of loop-mediated isothermal amplification (LAMP)
assays for the rapid detection of allergic peanut in processed food. Food Chem., 257, 67–74.
doi:10.1016/j.foodchem.2018.02.124.
33. Esteki M., Farajmand B., Amanifar S., Barkhordari R., Ahadiyan Z., Dashtaki E., Mohammadlou M. & Vander Heyden
Y. (2017). – Classification and authentication of Iranian walnuts according to their geographical origin based on gas
chromatographic fatty acid fingerprint analysis using pattern recognition methods. Chemom. Intell. Lab. Syst., 171,
251–258. doi:10.1016/j.chemolab.2017.10.014.
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Cocoa, cocoa preparation, chocolate and
chocolate-based confectionery
Michaela Rektorisova, Monika Tomaniova*
University of Chemistry and Technology, Prague, Czech Republic
*E-mail corresponding author: [email protected]
https://doi.org/10.32741/fihb.8.cocoa
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Africa and Asia, a typical smallholder cocoa farm covers only 2 to 5 hectares of land. In a
meaningful concept of sustainability, consumption is of equal importance to production. A
sustainable world cocoa economy implies an integrated value chain in which all stakeholders
develop and promote appropriate policies to achieve levels of production, processing and
consumption that are economically viable, environmentally sound and socially responsible for the
benefit of present and future generations, with the aim of improving productivity and profitability
in the cocoa value chain for all stakeholders concerned, in particular for the smallholder producers.
Basic principles are given in the Cocoa Agreement by the International Cocoa Organisation
describing arrangements between producing and consuming countries to safeguard markets and
raise average prices to stabilise trade, supplies and prices of cocoa [11]. CAOBISCO (Association of
Chocolate, Biscuit and Confectionery Industries of Europe), the European Cocoa Association (ECA)
and the Federation of Cocoa Commerce (FCC) are committed to working towards more sustainable
cocoa which complies with such requirements for benefit of the consumer, the manufacturer and
the farmer [10].
To support the sustainability of cocoa production, independent certification schemes have been
established to provide increased transparency and responsibility in cocoa supply chains, providing
farmers with the resources they need and helping them to manage their farms professionally, and
in turn be rewarded for sustainable production and for providing consumers with products they
can enjoy and trust. Examples of these certifications are Fairtrade [12], UTZ Certified [13] and
Rainforest Alliance Certified [14].
1. Product Identity
1.1. Definition of the product and manufacturing process
A wide range of cocoa products originates from the seeds of the cocoa tree, Theobroma cacao L.
Cocoa trees are grown in a narrow band around the equator (approximately 20° north and 20°
south), which goes through four continents: Africa, Asia, Australia and Oceania, and South
America. Africa produces 73 % of the world production, followed by America with less than 17 %,
and Asia and Oceania at about 10 % [15]. For a long time, most cocoa production has been
concentrated in 7 countries: Ivory Coast, Ghana, Indonesia, Cameroon, Nigeria, Brazil and Ecuador.
The cocoa tree has four main varieties (some of which are bound to a particular geographical
region) and several hybrids, each of which possesses a unique potential for flavour development.
In terms of world trade, the quality of cocoa beans is divided into two categories (i) ‘fine’ or
‘flavour’ and (ii) ‘bulk’ or ‘ordinary’. The difference between ‘fine’ or ‘flavour’ cocoa and ‘bulk’
cocoa is in the flavour rather than in other quality factors [16]. Forastero accounts for the most of
‘bulk’ cocoa production and is referred to as a basic variety. Criollo, Trinitario and a rare variety
Nacional (last producing well-known Arriba beans) are considered ‘fine or flavour’ cocoas and are
used for gourmet chocolates [1,5,17]. However, it is not only variety that influences flavour
development, which is also affected by other factors such as a growing locality and conditions
during growth and harvesting. Moreover, the final flavour and taste of cocoa products are highly
dependent on individual processing stages and conditions. The processing is thus very important
for final product quality, though it may not be necessarily related to its authenticity. While the
term quality has different associations, authenticity is always strictly related to true product
identity. Especially in the case of chocolate, consumers may have different preferences and
expectations, often in relation to their geographical regions. When assessing chocolate
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authenticity, tracing the initial ingredients may be much harder due to these differences in
manufacturing processes and their complexity [5].
To understand the identity of individual cocoa products, it is important to firstly explain the cocoa
manufacturing process. This can be divided into two stages, firstly cocoa processing, and, secondly,
chocolate manufacturing. However, both of them are often directly connected since most cocoa is
used to make chocolate. After harvest, cocoa beans are released from cocoa pods (fruit) and then
cleaned of any extraneous matter by blowers and sieves. The fresh cocoa beans are then left to
ferment under the action of naturally present yeasts. During this process, which is especially
important for the development of main flavour precursors, the beans change colour from purple
to brown. To prevent the growth of moulds and reduce microbial contamination, the fermented
beans are dried and then roasted. During roasting, the beans also gain additional flavour.
Loosened hard shells are then removed from the beans (winnowing) to reveal cocoa nibs (or,
alternatively, deshelling may be performed prior to the roasting). The nibs are ground to a
homogenous paste called cocoa liquor (or by a number of other terms, such as cocoa mass, cocoa
paste, chocolate paste). This paste can be used directly in products such as chocolate or pressed to
separate cocoa butter (fat) from cocoa solids (cocoa cake). Crushing the cocoa cake will result in
natural cocoa powder. An optional process, typical for Dutch cocoa powder, contains an alkalising
step using potassium or sodium carbonate, which leads to lower acidity, a darker colour, more
intense flavour, milder taste and better dispersibility in water.
Cocoa is traded at different stages of this process and intermediates/products may differ
significantly in composition; thus, the process of authentication is a complex procedure involving
various steps for different products.
In chocolate manufacturing, the first step is to mix all its ingredients together while applying
moderate heat to melt the cocoa butter. Additional steps, such as refining and conching, are
carried out to achieve a smooth texture and intense flavour. Finally, a tempering step occurs which
is important to obtain good surface gloss, a snap and a stable structure resistant to fat bloom. All
manufacturing processes have a strong influence on the final product quality and, due to their
complexity, can make authentication very difficult [5,17,18]. Regarding the ingredients used within
the manufacturing process, there are three basic types of chocolate: dark, milk, and white. Dark
chocolate is a complex food product in which sugar crystals and non-fat cocoa particles are
surrounded by a continuous phase of crystalline and liquid cocoa butter. Milk chocolate is a
complex rheological system having solid particles (non-fat cocoa, milk and sugar particles)
dispersed in cocoa butter, which represents the fat phase [19]. White chocolate has a similar
composition to that of milk chocolate, but the cocoa is represented exclusively by cocoa butter.
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difference, though, is that it has authorised the use of other vegetable fats in chocolate, up to a
level of 5 %, which previously had only been acceptable in seven Member states, such as the
United Kingdom or Austria. However, only six specified fats, the so-called cocoa butter equivalents
(CBEs), without any enzymatic modifications, can be used (illipe, palm-oil, sal, shea, kokum gurgi
and mango kernel), together with the mention “contains vegetable fats in addition to cocoa
butter” on the product label. The list of specified products has also been reduced together with
their detailed descriptions, such as “cocoa beans”, “cocoa nibs”, “cocoa mass” or “cocoa press
cake”. Products specified in the new Directive are “cocoa butter”, “cocoa powder”, “drinking
chocolate”, “milk chocolate”, “family milk chocolate”, “white chocolate” among others. According
to the definitions, for instance, cocoa butter is described as the fat obtained from cocoa beans or
parts of cocoa beans with the specified content of free fatty acids and unsaponifiable matter; and
chocolate is defined simply as the product obtained from cocoa products and sugars which
contains not less than 35 % total dry cocoa solids, including not less than 18 % cocoa butter and
not less than 14 % dry non-fat cocoa solids. There are slight differences when the name is
supplemented by any of the specified words (such as vermicelli, flakes, couverture, gianduja nut).
For most cocoa and chocolate products, their labelling must indicate their total dry cocoa solids
content. Moreover, the Directive authorises the addition of other edible substances (with the
exception of flour, starch or animal fat other than milk fat) up to 40 % of the total weight of
finished chocolate products, while the content of cocoa butter and cocoa solids still has to be
calculated after deducting these substances. Various flavourings, if they do not imitate the taste of
chocolate or milk, may also be added to several cocoa/chocolate products. The use and amount of
sugar in chocolate products are no longer restricted; any sugars intended for human consumption
can be used. Additives that are applicable for cocoa and chocolate products are specified in a
separate, general document on food additives, Regulation (EC) No. 1333/2008 [22] as amended.
This document lists various additives authorised for certain foods and specifies their maximum
levels in the products (or their use according to the principle of quantum satis (q.s.), meaning the
minimum level for achieving the desired effect). For cocoa and chocolate products, common
additives are emulsifiers and acidity regulators. Of emulsifiers, lecithins or mono- and di-glycerides
of fatty acids are applicable at q.s. levels, while polyglycerol polyricinoleate has a maximum level
of 5 000 mg/kg and ammonium phosphatides of 10 000 mg/kg. Acidity regulators include
carbonates, hydroxides, magnesium oxide and citric acid. More attractive surface gloss can be
achieved by the use of glazing agents, such as gum arabic, carnauba wax, shellac or pectins.
Products with reduced energy or no added sugar can contain various polyols (e.g. sorbitol,
mannitol, maltitol) or sweeteners (e.g. aspartame, acesulfame K, saccharin, sucralose and steviol
glycosides). No food colour is permitted in cocoa and chocolate products. The EU Directive does
not recommend any methods of analysis.
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additives listed in the Standard. Following this general description, Codex specifies chocolate types
and their composition (chocolate or, alternatively, bitter sweet chocolate, semi-sweet chocolate,
dark chocolate, chocolate fondant; sweet chocolate; couverture chocolate; milk chocolate; etc.).
The addition of other edible foodstuffs is limited to 40 % and other vegetable fats to 5 %, as in the
EU Directive, but the nature of these fats is not further specified. Furthermore, Codex
recommends some internationally recognised analytical methods (for example, for the
determination of fat content, cocoa shell, free fatty acids or moisture) published by the
Association of Official Analytical Chemists (AOAC), the International Union of Pure and Applied
Chemistry (IUPAC) or the International Confectionery Association (ICA, formerly International
Office of Cocoa, Chocolate and Sugar Confectionery, IOCCC).
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high quality cocoa and chocolate products, and includes also the sampling, assessment of physical
quality and flavour of cocoa beans, manufacturing procedures, or storage conditions [25].
For the use by Customs authorities, statistical agencies and other regulatory bodies, all
commodities are classified and coded by the Harmonised Commodity Description and Coding
System (“Harmonised System”) developed by the World Customs Organization [26]. The
Harmonised System is applied worldwide to facilitate the international trade and monitor and
control the import and export as well as for the purposes of customs tariffs and taxes. The
Combined Nomenclature was established by Council Regulation (EEC) No 2658/87 on tariffs and
statistical nomenclature and is updated every year; the latest version is now available as EU
Regulation No 2017/1925 [27]. In the Combined Nomenclature, the codes of the Harmonised
System are used. There are different categories for cocoa and cocoa preparations, beginning with
the number 18 (e.g. 18 06 10 for cocoa powder, containing added sugar or other sweetening
matter), while white chocolate is classified as sugar confectionery and coded as 17 04 90 30.
At the end of this chapter, it is worth mentioning the famous Swiss and Belgian chocolate. Both
these terms are related to the product manufacturing country. The term ‘Belgian chocolate’ was
introduced in 2008 in the ‘Belgian Chocolate Code’ of The Royal Belgian Association of the Biscuit,
Chocolate, Pralines and confectionary (Choprabisco) [28] which is an agreement between Belgium
manufacturers and has no legal weight. The only criterion for chocolate to be called Belgian is that
the complete process of mixing, refining and conching is carried out in Belgium. ‘Switzerland’,
‘Swiss’ or ‘Suisse’ chocolate are the trademarks registered by the Association of Swiss Chocolate
Manufacturers (Chocosuisse) [29] which are used for products manufactured in the Switzerland
under specific technical guidelines.
2. Authenticity issues
2.1. Identification of current authenticity issues
In ISO standard 2451:2017 and FCC Quality Rules[23] related to cocoa beans, the term adulteration
is defined as the “alteration of the composition of a lot of cocoa by any means whatsoever”; a lot
is defined as “quantity of cocoa beans in bags or in bulk established at any point in the cocoa
supply chain and from which primary samples and/or incremental samples are to be drawn for
quality analysis purposes”. For whole beans, the possibilities of adulteration are rather limited,
involving the presence or addition of foreign matter or of cocoa beans of poor quality (insufficient
deshelling, defective beans). These issues can be quite easily recognized by simple visual
inspection or other simple tests. On the other hand, the authentication of different complex
products can become a highly demanding task. The most straightforward examples of product
adulteration are inappropriate labelling, substitution of valued materials by cheaper ones, and the
addition of undeclared or unauthorised materials/substances.
Among various cocoa materials, cocoa butter is considered the most important by-product of
cocoa beans due to its unique physical and chemical characteristics and to its specific functional
properties compared to other fats, such as brittleness at room temperature, fast melting at body
temperature [30]. Since fat, especially its amount, is very important for the sensory properties of a
product, cocoa butter may be “diluted” by other fats rather than used in lower amounts than
declared. In addition to the economic reasons, such dilution may be motivated by certain
technological advantages, such as increased stability [31]. Among various fats, cocoa butter
equivalents (CBEs) are most suitable for mixing with cocoa butter in unlimited quantities due to
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their similar physical and chemical properties [30]. As mentioned above, in the EU only six CBEs
can be added in a limited amount and declared on the product label. Such addition has to be
declared by an informative statement on the labelling, in addition to the listing among the
ingredients. For other products, any presence of undeclared fat is considered adulteration. This is
not limited to vegetable fats, since animal fats, lard or tallow, are used in the adulteration of cocoa
butter, mostly in developing countries. This type of adulteration is also of religious concern [32].
Moreover, the quality of cocoa butter intended for the human consumption is strictly defined in
various standards; any use of cocoa butter or cocoa fat of poor quality would be categorised as
adulteration. For instance, a higher level of free fatty acids in cocoa butter indicates that cocoa
beans or cocoa butter have not been handled properly (cocoa beans are diseased or damaged,
stored or transported in poor conditions). Such cocoa butter can negatively affect a flavour as well
as crystallisation properties (snap, melting properties). In both the EC Directive 2000/36 [21] and
the Codex Alimentarius, a level of free fatty acids of 1.75 % is specified as the maximum amount.
Unsaponifiable matter is another parameter that is often used to assess fat quality and purity. This
includes all compounds that, after saponification, are insoluble in water but soluble in fat. These
are those compounds frequently found dissolved in fats and oils that cannot be saponified by the
usual caustic treatment but are soluble in ordinary fat and oil solvents. They are mainly various
natural components of fat (e.g. sterols, pigments, terpenic alcohols, higher aliphatic alcohols,
hydrocarbons), the amount of which is characteristic for particular fat; however, unsaponifiables
also include contaminants, such as mineral oil hydrocarbons, which come from transport
materials, lubricants, fuels, exhaust fumes or debris from tyres [10,33].
Various cocoa and chocolate products may be adulterated by the use of improperly processed
cocoa beans, which contain higher amounts of cocoa shell or germ, or other plant materials than
those that occur naturally, or have higher moisture content. Interestingly, not all standards or
legislation include the limitation of cocoa shell content; nevertheless, Codex limits the shell
content in cocoa mass and cocoa cake to a maximum of 5 % by weigh calculated on the fat-free dry
matter. Additional dilution of cocoa and cocoa products may be achieved by the intentional
admixture of starch, flour, dextrins or various powdered materials, such as peanut shells, chestnut
shells, soybean meal, sesame meal, carob and non-fermented cocoa beans [34].
Since chocolate is a more complex product, possibilities of its adulteration are increasing. Although
the presence of some undeclared components (milk, peanuts, and nuts) may be unintentional,
other ones may be added to reduce production costs or to increase the palatability of the product.
The latter, for instance, may be achieved by the addition of milk. Milk fat not only influences the
sensory properties (taste, softer texture) of products, its addition significantly improves resistance
to bloom [35]. Improved stability, thus appearance and attractiveness, can be also achieved by the
admixture of some foreign vegetable fats into cocoa butter [30,36]. In addition to the alteration of
fat content or composition, the content of non-fat cocoa solids may decrease, resulting in a value
which is non-compliant either with the legislative limit or, when summed together with cocoa
butter, with total dry cocoa solids on product label. The lower cocoa content can then be easily
adjusted by increasing the major and cheapest chocolate ingredient, sugar or any of the materials
mentioned above.
In the EU [21], the addition of up to 40 % of other edible substances to chocolate brings the
possibility of decreasing the mass of cocoa needed for the production of chocolate without
affecting the cocoa percentage on the labelling (this value, and also the minimum requirement for
cocoa butter and dry non-fat cocoa solids, are calculated after deducting the weight of such
substances). If these substances are not clearly visible and do form a homogenous matter with the
chocolate, consumers may not be aware of the difference between such a product and a common
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chocolate with an identical cocoa percentage on the label. Examples of products which benefit
from the rule of calculating cocoa solids for the label are those with the addition of dietary fibre
(e.g. inulin, used also as an alternative to sucrose as a sweetener to develop sugar reduced
chocolate products) [37].
Particularly vulnerable to adulteration are the various specialty (or premium) cocoa and chocolate
products, which are characterised by a higher market price. Recently, the rapid increase in
consumer demand for such products has opened a new, very lucrative area for fraudulent
practices. Consumers have become especially interested in premium chocolates with a variety of
exotic ingredients, chocolates made from single-origin cocoa beans, such as those from Ghana,
Ecuador or Venezuela, products that have declaration of ‘fine and flavour’ cocoa content, or
products with a certain type of certification (e.g. Fairtrade, UTZ, organic), or production (e.g. raw,
with reduced sugar, glycaemic index or cariogenicity) [38]. Geographical Indications (GIs), first
introduced in the EU, are also increasingly used as a marketing tool to differentiate agri-food
products, including cocoa beans, in the globalised marketplace. While the majority of origin-
specific products are produced in EU countries, some of the developing countries where the cocoa
beans are produced have also successfully implemented GIs and origin-based and quality
differentiation strategies. Examples from the international cocoa markets for ‘fine and flavour’
cocoa, vulnerable to fraudulent practices, include Arriba from Ecuador, and Chuao from Venezuela
[39,40].
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Other attributes considered for the authenticity testing of cocoa and chocolate products are
related to their essential composition and quality factors [41]. For most cocoa and chocolate
products, analytical methods are then related to the two main components of cocoa beans: cocoa
butter, and non-fat cocoa solids.
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For chocolate, another basic parameter is total dry cocoa solids content, which is then calculated
as a sum of cocoa butter content and non-fat cocoa solids content.
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TAG determination by comparing with GC-FID. Other techniques that have been reported as
appropriate for this task are MALDI-TOF-MS (Matrix Assisted Laser Desorption/Ionisation coupled
with mass spectrometry) [51,52], silver ion LC-MS [53] and Fourier transform infrared
spectroscopy (FTIR); the latter for the quantification of lard content [32] or other vegetable fats in
cocoa butter [54]. The potential of different analytical approaches for the reliable quantification of
foreign fats in real samples in food control laboratories is still being investigated. Indeed, such
quantification is complicated due to a high variability in cocoa butter composition (caused for
example by differences in geographical origin and processing) as well as by the variability in
admixed fats.
To determine cocoa shell content, the standard photometric method has been criticised for its low
sensitivity and selectivity. Alternatively, an HPLC-FLD method for the determination of fatty acid
tryptamides (behenic acid tryptamide, lignoceric acid tryptamide) as indicators for cocoa shell was
first described by Münch and Schieberle in 1999 [55] and in 2000 published as optimised for
routine analyses. Using a high number of various cocoa products (cocoa nibs, cocoa shells, cocoa
liquors, cocoa powders, cocoa butters, cocoa pods), the use of this method was further evaluated
by Janßen and Matissek [56]. Another detection technique, GC-FID, has been shown to be suitable
for these indicators [57], thereby making this approach applicable in a wide range of laboratories.
Colorimetry and photoacoustic spectroscopy have been reported as a suitable tool for the
determination of non-fat cocoa solids in dark chocolates [58], thermogravimetry for the
characterization of milk and dark chocolates [19,59].
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Cocoa, cocoa preparation, chocolate and chocolate-based confectionery
Physical testing Physical properties of cocoa Substitution of high quality raw material, cocoa beans
beans
Sensory analysis Off-flavour, aroma profile Substitution of high quality raw material, cocoa beans
Gravimetry (acid hydrolysis Total fat in all cocoa products Decreased fat content: substitution of cocoa butter
followed by Soxhlet extraction) with non-fat components
GC-FID Triacylglycerols Cocoa butter equivalents in cocoa butter or chocolate
Titrimetry (alkalimetry) Total free fatty acids Substitution of cocoa butter
GC-FID Fatty acids Substitution of cocoa butter
GC-FID, MALDI-TOF-MS, NARP- Fat composition Substitution of cocoa butter
LC-MS, silver ion LC-MS, FTIR
LC-UV, LC-MS Non-fat cocoa solids in cocoa Substitution (lower content of cocoa solids)
products based on theobromine
and caffeine content
Gravimetry (saponification Unsaponifiable matter Substitution
followed by extraction)
Enzymatic (diastase) Starch Substitution
LC-FLD, GC-FID Behenic acid tryptamide, Substitution (cocoa shell / shell processing
lignoceric acid tryptamide contamination in cocoa products)
GC-MS, HS-PTR-MS Volatile organic compounds Geographical or botanical origin
Substitution (mono-variety/region products)
IRMS, 1H-NMR Stable isotope composition Geographical or botanical origin
Substitution (mono-variety/region products)
ICP-MS Trace elements Geographical or botanical origin
Substitution (mono-variety/region products)
LC-ESI-Q-TOF, GCxGC-FID, LC- Proteins and oligopeptides Geographical or botanical origin
ESI-MS, MALDI-TOF-MS profiles Substitution (mono-variety/region products)
NIR Spectral information Geographical or botanical origin
Substitution (mono-variety/region products)
LC-MS Phenolic compounds Geographical or botanical origin
Substitution (mono-variety/region products)
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5. Conclusion
Food fraud in the cocoa beans processing sector is influenced by the increasing price of cocoa as a
consequence of the production size, the influence of weather conditions, pests or diseases and a
higher demand for cocoa. In addition to social and ethical aspects for less developed countries
where cocoa beans are primarily produced, government support for the production and
certification of premium products (e.g. geographical indication) must be sustained for this sector
to continue developing.
The main challenge to the authentication of cocoa beans and cocoa-based products is the inherent
compositional variability due to differences in variety, geographical origin, and also processing
techniques.
Nowadays, non-targeted analysis (fingerprinting) to assess the authenticity of a suspicious sample
by comparing it to an authentic one is a novel approach that is particularly useful in differentiating
geographical origin, genotype and production technology by using fingerprints of cocoa and cocoa-
based products obtained by various analytical approaches, such as LC-MS, GC-MS, proteomic,
peptidomic, elemental, and combined with appropriate chemometric tools. However, the
availability of well-designed, specific and extensive compositional databases, reference materials
and reliable, validated analytical protocols are needed.
Due to the large number of different fats, especially artificially prepared mixtures, which can be
used as alternatives to cocoa butter, it is difficult to assess fat composition based on only a few
physical or chemical parameters. Moreover, the use of several analytical procedures to assess the
quality of cocoa butter is time consuming. Thus, lipidomic fingerprinting appears to be an
interesting approach for the future.
Analytical methods enabling the determination of various parameters according to the legislation
within a single analytical run are desirable, with the aim of increasing sample throughput in control
labs. Supercritical fluid chromatography (SFC), coupled with UV detection, refractometric
detection (RID), evaporative light scattering detection (ELSD), FID or mass spectrometric (MS)
detection for fat composition, saccharides profile and purine alkaloids content might be a solution.
Instead of conventional analytical methods, the application of ‘omic’ technologies also represents
new trends for the future. Plants produce a considerable amount of chemically diverse
metabolites. Differences observed in the composition of metabolites of a particular species or
cultivar of the plant are determined by various genetic and environmental factors. They cause
small variations, such as within one fruit tree, between fruit from the marginal and inner parts of
the crown, as well as medium or larger differences (due to different soil types and other climatic
conditions given by the region). Geographic origin has a significant influence on metabolite
composition and is an important attribute for determining the quality and price of many foods
including cocoa-based products. The natural variability of metabolites thus provides reliable
information on the origin and authenticity of food. Another important factor for the quality of food
of plant origin are the conditions of harvesting and storage of crops. Even after the harvest,
intense metabolic processes are underway, and plant materials can be degraded by a misuse.
Other changes can occur during cocoa beans processing. Metabolomics might be a useful tool for
finding the conditions that will be optimal for maintaining quality.
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Plant-derived sugars and sweeteners
Freddy Thomas*, David Hammond
Eurofins Analytics France, Nantes, France
*E-mail corresponding author: [email protected]
https://doi.org/10.32741/fihb.9.sugar
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also the addition of more “taps” to the recent plantings, which has become possible as these new
trees mature and this allows more taps to be added to each tree.
12 000
10 000
8 000
6 000
4 000
2 000
Figure 1: Maple syrup production data for Quebec province in 1 000’s of US gallons from 1997 to 2014
1. Product Identity
1.1. Definition of the product and manufacturing process
The products covered in this chapter are all sugar containing extracts produced from plants. For
the purpose of this chapter they will be divided into a number of classes.
“Tree” waters: The products discussed here are produced from the sap collected from a tree in the
early spring. The liquid is subjected to little additional treatment; apart from processes to limit
microbiological changes e.g. ultrafiltration and/or pasteurisation. Two products will be considered
here which are extracted from maple or birch trees. There are other “tree” waters like bamboo,
but this will not be covered here due to a lack of background information on the product.
However, the authenticity issues identified for maple and birch waters may also be applicable to
this material.
“Tree” syrups: These products are also produced from tree saps but are concentrated by simple
heat or using a pre-concentration step {such as ultrafiltration or freeze concentration} followed by
heating to remove most of the water. The most common of these tree syrups is derived from
maple sap and the syrup typically has a Brix (sugar) content around 66 %. The former is often used
as a topping for pancakes and waffles for instance, whereas birch syrup is often used more in
savoury dishes.
Another type of “tree” syrup is extracted from coconut flower spikes. Unlike the true tree syrups,
such as maple, this material is actually extracted from the flower spike from which the coconuts
themselves will develop later. Once the sap is collected the water is again removed from the sap
by heating to give either a syrup (ca 65 %) or coconut sugar if the Brix is increased to a higher level
that promotes crystallisation of the sugars on cooling.
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Plant-derived sugars and sweeteners
Agave syrups: These products are syrups derived from a range of succulents of the “agave” family
(e.g. americana, tequilana and salmiana). These syrups are produced by the extraction of the
storage carbohydrate (inulin) from the agave pinas and its subsequent hydrolysis to liberate a
fructose rich syrup which is then concentrated to ca 70 Brix.
Plant derived sugars: Bulk sucrose can be prepared from two sources either cane or beet. The
former is produced in hot climates like Brazil and the Caribbean whereas beet sucrose is produced
in more temperate climates such as Northern Europe, USA, Canada, Russia and Turkey.
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Plant-derived sugars and sweeteners
By convention, US and Canadian Standards, the minimum Brix that is acceptable for this product is
66 %. Unlike Birch syrup the majority of the Brix in maple syrup is sucrose (98 to 99 %) with only
very low levels (< 1 %) of glucose and fructose being detected.
Figure 2: Palm flower spike being cut prior to collection of sap for coconut sugar production
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Plant-derived sugars and sweeteners
sugar is an additional product from this plant as most of these palms are grown for date
production. The nipa palm (Nypa fruticans) is also commonly used for syrup production on the
coastlines and tropical regions of the Indian and Pacific Oceans. It is the only palm that will tolerate
high water levels, as seen in mangrove swamps, where often only its leaves and flowers are seen
above the water level. Finally, the sugar palm (Arenga pinnata) can be used in the coastal and
tropical regions of Asia, around China and Indonesia for syrup/sugar production. The sugars are all
derived from a tap on the flower spikes of these plants and are analogous to coconut blossom
syrup/sugar. There is no standard for these products.
Figure 3: Agave “pinas” after leaf stripping showing relative size (ca 40 to 60 kg)
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Plant-derived sugars and sweeteners
The finalised agave syrup will be a clear pale yellow/yellow product with a Brix of ca 70 %. The
majority of the soluble solids of these products are the simple sugars, fructose and glucose, which
are the major components of inulin. Typically, the fructose to glucose ratio is around 10.0 in these
materials. Sucrose and other disaccharides are also present in the syrup, but at low
concentrations, with variable levels of the polyols mannitol and inositol are also seen.
Although there is no International standard for agave syrup, Mexico published a non-binding
standard (NMX) for this material in 2008 [7] as a preliminary step towards the introduction of a
binding/mandatory norm (NOM), which was adopted in 2016 [8]. Since then, any agave syrup
exported from Mexico has to comply with this standard.
2. Authenticity issues
2.1. Identification of current authenticity issues
These products, along with most fruit juices and honey, are sold on the basis of their sugar content
(Brix). This presents an opportunity for an unscrupulous supplier to adulterate their products as
there are always other cheaper sugar sources than can be used as a substitute for the sugar
materials extracted from the plant (tree, palm or succulent). This substitution has been found to
occur from time to time and some examples of these will be detailed here. Presented in Figure 4
are typical prices for the adulterants that have been used to extend these types of products
together with the costs for the authentic materials (where prices for bulk purchases are available).
8 000
7 000
6 000
5 000
4 000
3 000
2 000 Price ($/T) {100%}
1 000
0
Figure 4: Nominal prices for “real” products and potential adulterants in 2016
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Plant-derived sugars and sweeteners
Due to the high difference in price between the potential adulterant and the authentic vegetable
sugars (a ratio of 1 to 10), the relative additional profit that an unscrupulous supplier can make by
extending the authentic material by 20 % with an appropriate type of sugar is clear so there is a
significant driving force if someone wants to cheat.
The typical materials that have been used to extend these products are cane and/or beet sucrose,
high fructose syrups from starch (corn and/or rice), invert syrups from cane or beet and unrefined
sugar.
The material selected to carry out the adulteration has depended on:
a) availability of another cheaper sugar source,
b) the knowledge and sophistication of the adulterator,
c) surveillance operations that have taken place on this product.
The substitution of part or all of maple syrup with cheaper materials is an issue that occurs from
time to time. Recently the maple syrup producers of nine US producer states have petitioned the
FDA to look at a number of products that appear to them as misrepresenting their contents [7,8].
Under US law only products containing a minimum of 10 % maple syrup can use the term “Maple”
in their name. The FDA subsequently published a “note to consumers” to advise them to study the
label of products carefully so they were not misled [11].
There have also been other cases in the US where products containing very little maple syrup
present themselves in the manner in which they are marketed as if they contain a higher level of
maple syrup [12]. In a study published in 2010 the authors [13] looked at reported cases of
adulterations of food from 1980 to 2010 and found that there were 16 cases of the adulteration of
maple syrup over that period, which represented ca 2 % of all of the examples they had examined.
Around a similar time, there was a small-scale study carried out in the UK on palm sugar. This
identified that there were samples of this sugar on sale in the UK which were misrepresented as
13
they were blends of palm sugar and cane/corn sugars. Here δ C values were shown to be in the
region of -13 to -15 ‰ [14], which clearly indicated that the majority of the sugar was actually
derived from cane/corn rather than being isolated from the palm.
One reason for the publication of the Mexican standard for agave syrup was the relatively high
number of cases of syrup detected in both Europe and the US that clearly showed the presence of
sugars derived from other sources (cane and corn) rather than being fully derived from agave [15].
The authors are unaware of any published examples of the sale of adulterated maple or birch
waters but there is certainly a risk that these products could be extended by unscrupulous
suppliers.
―7―
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Plant-derived sugars and sweeteners
―8―
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Plant-derived sugars and sweeteners
3) CAM (Crassulacean Acid Metabolism) pathway: The final route is used by a range of
specialist plants that have to limit water loss during the day. This means that they shut
their stomata, holes in the underside of their leaves, through which they absorb carbon
13
dioxide during the day and so it is fixed at night. In these plants the isotope ratio (δ C) is
typically between -12 and -15 ‰. Plants in this category are succulents mainly, e.g.
pineapples and agave.
2 1 18 16
Plants also accumulate different levels of hydrogen ( H/ H) and oxygen isotopes ( O/ O)
2 1
dependant on climatic and geographic conditions. The use of ( H/ H) sometimes allows the
differentiation of natural sugars from exogenous sugars, derived from a C3 source (beet/rice), that
have been added to extend a product. The hydrogen isotopic pair together with oxygen isotopes
has also been used to help differentiate between not from concentrate (NFC) and from
concentrate fruit juices (FC).
13
All tree and palm saps are extracted from plants that use the C 3 pathway and so show δ C ca -25
‰. Therefore the use of carbon isotopic analysis allows the addition of cane sucrose, cane invert
and corn-based syrups (glucose and HFCS) to be detected when it is added to maple
syrup/sap/sugar and coconut flower sugar/syrup at a level of ca 10 % and higher. This sort of
detection level is lower than would be possible when using just conventional sugar analysis which
has to allow for the natural variation in the glucose and fructose levels seen in these products.
However, as agave is a succulent it uses the CAM pathway to fix carbon dioxide and means that
standard carbon isotopic analysis is not sensitive enough to differentiate between 100 % agave
syrups and materials adulterated with corn syrups.
There are two methods that can be applied here that have been validated on maple syrup. The
first of these, AOAC 984.23 [19] {“whole sample method”}. This uses simple combustion of the
sample to carbon dioxide which is then analysed by isotope ratio mass spectrometry. Although the
AOAC method uses an off-line combustion of the sample and then introduction of the carbon
dioxide produced into the mass spectrometer, a continuous flow system is now normally used
where each sample is combusted in turn and the liberated carbon dioxide is directly fed into an
isotope ratio mass spectrometer (IRMS) by a stream of carrier gas (N 2) (EA-IRMS).
The second method {“ethanol method”}, AOAC 2004.01, uses a similar detection method but
involves the initial fermentation of the sugars into ethanol (EtOH), which is then recovered by
13
careful distillation using a spinning band column. The ethanol is then subjected to C isotopic
analysis using an IRMS machine [20]. The two methods have similar sensitivities, with a detection
13
limit in the region of a 10 % addition of a C 4-derived sugar but give rise to very different δ C
values. This difference arises from the loss of some carbon atoms, as carbon dioxide, during the
fermentation step, which means that it is critical that the correct “judgement criteria” are used to
assess a sample e.g. the judgement criteria must be ones developed using the same analysis
procedure e.g. “whole sample” or “ethanol” procedure.
13
The sensitivity of the C IRMS method for the detection of added C 4 sugar to maple syrup/sugar
13
can be improved by using malic acid as an internal standard [21]. Here the δ C values detected for
the sugars and the malic acid isolated from the sample are compared. A similar improvement in
sensitivity of the detection of added C4 sugars to palm sugar has been published by Kelly [22]. This
uses a similar approach to that given in the “internal standard” method published by AOAC
(998.12) [23]. Here the protein contained in the sample is precipitated and washed to remove any
13
bound sugars. After drying this material is then subjected to combustion and the δ C ratio is
13
determined using IRMS. This value is then compared with the δ C value obtained on the “sugar”
portion or on the whole sample, with no pre-treatment. These two values should be close to each.
―9―
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Plant-derived sugars and sweeteners
If the percentage of C4 sugars calculated using equation (1), as defined in the AOAC method, is
larger than 7 % then the sample can be considered as adulterated with a C 4 sugar source.
ఋ భయ ିఋ భయ ೞ
ܥସ ݏݎܽ݃ݑݏΨ ൌ ൈ ͳͲͲ (1)
ఋ భయ ିሺିଽǤሻ
13 13 13 13
δ CP = C ratio seen in “protein” fraction, δ CS = C ratio seen in the sugar
13
The addition of cane sugar or HFCS to agave syrup (CAM) cannot easily be detected by C-IRMS as
13
the three plants share similar global δ C values around -11 to -12 ‰. This means that other routes
must be applied to detect this type of adulteration, which will be discussed in Section 3.2.
If a C4 sugar (sucrose or cane invert), is added to a tree sap derived material, the C4 derived sugars
are concentrated in one of the components (either sucrose or glucose and fructose respectively)
and this causes a disturbance in the carbon isotope ratios and means that the detection level is
roughly halved over the global EA-IRMS approach. In work carried out using liquid chromatography
linked with elemental analysis and isotope ratio mass spectrometry LC-EA-IRMS on honey [24] and
agave syrups it has been reported that the carbon isotope ratios for glucose and fructose were
much closer, within 0.5 ‰ for agave syrups, than seen in fruit juices, suggesting that this route
offers a method to detect the adulteration of agave syrups with C4 derived sugars. However due to
the complexity and the low implementation of this hyphenated technique in food control
laboratories, even if the intra-lab uncertainty of this technique is similar to the current EA-IRMS
technique, the inter-lab uncertainty is still higher at the date of this publication.
The methods discussed above only address the addition of C 4 sugars to products derived from tree
saps (maple, birch and palm products {syrup/sugar/sap}). However, if the unscrupulous supplier
13
uses beet sucrose, which is derived from a C 3 plant, there will be no derivation in the δ C isotope
ratio as seen with cane sucrose. Here detecting this type of adulteration requires a different
method.
The level of deuterium at the methyl site (D/H) 1 of the EtOH, produced during the fermentation of
the sugars in the maple syrup, has been found to be very useful. The analysis is carried out using
2
Deuterium-Nuclear Magnetic Resonance ( H-NMR) spectroscopy. The EtOH is again recovered
from the fermentation broth by careful distillation using a spinning band column as mentioned
13
above for C measurements. In fact, if when employing this SNIF-NMR® method the EtOH-IRMS
method is also used, it is possible to detect added exogenous sugars derived from both C3 and C4
sources. This is described for maple syrup in the AOAC Official method 2000.19 [25]. By combining
2
H-SNIF-NMR® and IRMS on the ethanol probe, it is therefore possible to detect addition of both
beet and cane sugars/syrup in C3 syrups such as maple as illustrated in Figure 5 below.
13
Although not normally used in this sense, the C-IRMS and SNIF-NMR® methods can be used to
determine whether sucrose is correctly described as “cane” or “beet”. In the former case for pure
13
cane sucrose the δ C value would be ca -11 ‰ and the (D/H)1 value would be around 110 ppm,
13
whereas if sucrose derived from beet was added much lower δ C and (D/H)1 values would be
detected. Conversely with beet sucrose, if cane sucrose had been added to the product it would
13
show a less negative δ C value and the (D/H)1 value would be higher. These two isotopic methods
are the only two procedures that can be used to differentiate these two products. Similarly with
13
starch derived syrups for which the corn or rice source can be differentiated from their δ C values.
― 10 ―
- 164 -
Plant-derived sugars and sweeteners
― 11 ―
- 165 -
Plant-derived sugars and sweeteners
Here the sugar/syrup/sap is freeze-dried to remove the water. The sugars are then derivatised
using pyridine/trimethylsilylimidazole mixture (4:1) with heating. The samples are then injected on
to a non-polar Capillary-GC column (DB 5) and the peaks are detected using a flame ionisation
detector (FID).
Although high fructose syrups from starch (e.g. corn and rice) do not make good adulterants for
maple or coconut flower syrups/sugar — as their addition will reduce the sucrose level while also
increasing the levels of glucose and fructose — they have been used to extend these types of
products because of their low price and large availability. If this type of material is added to maple
syrup it is detected by the presence of maltose and isomaltose, as shown in Figure 6.
Maltose
Isomaltose
Here an addition of HFCS at a level of around 2 % is often detectable, which is much better than
13
can be achieved using HPLC, to look at the simple sugar levels, or by C-IRMS. The Cap-GC
procedure can also detect the presence of invert syrup when added to maple syrup/sugar and
coconut flower syrup/sugar. Again there are two peaks that show the presence of the exogenous
sugars in the product.
The Cap-GC method can also be used to analyse agave syrups for the addition of HFCS. Unlike the
case of maple syrup, HFCS makes a “better” extender for agave syrup as they:
a) Are rich in fructose, similar to agave syrups,
13
b) Share a similar global δ C value to that of agave as they are produced from a CAM plant.
Both features make them hard to detect by other analytical procedures.
When adulterated with HFCS, two signals corresponding to isomaltose appear in a flat zone, which
are not present in typical Cap-GC profile for agave syrup (see Figure 7).
HPAEC-PAD can also be used to look for the presence of exogenous gluco-oligosaccharides that
maybe present if a starch derived syrup is added to these syrups [26]. This uses a different column,
with a lower retentivity (Dionex PA-100), to that used for the mannitol/inositol quantification
discussed above. This method can be complementary to the Cap-GC method discussed above but
looks at a different set of compounds.
― 12 ―
- 166 -
Plant-derived sugars and sweeteners
Isomaltose
Figure 7: Portion of a Cap-GC profile for agave syrup adulterated with HFCS
Although the Cap-GC method offers one of the fastest and most sensitive routes to detect sugar
syrup addition to these types of products, it will not detect the addition of either cane or beet
13
sucrose to maple or coconut flower syrup. The former maybe detectable using C-IRMS, if the
addition level is high enough, whereas beet sucrose addition can similarly be detected using SNIF-
NMR®. This reinforces the need to use more than one method in screening samples to ensure any
adulterations are detected.
Figure 8: Plot of 13C site-specific values of ethanol detected using 13C-SNIF-NMR for agave and adulterants [30]
― 13 ―
- 167 -
Plant-derived sugars and sweeteners
The detection limit for this technique to detect the presence of cane/corn sugars in agave syrup is
around 15 %. This is much higher than is possible, in some cases, using Cap-GC for HFCS, however,
as mentioned above cane invert is only readily detectable using this approach. Cane sucrose
addition to agave could be detected by elevated levels of sucrose, which are relatively low in agave
syrups. However, some agave syrups naturally contain low, but significant levels of, inulobiose (1-
O-β-D-fructofuranosyl-D-fructose) which may be seen to elute very close to sucrose on many
chromatographic systems and caution needs to be taken in interpretation these results.
― 14 ―
- 168 -
Plant-derived sugars and sweeteners
Isotope ratio mass spectrometry (EA- Sugar and malic acid δ13C values Addition of C4 derived sugars to products
IRMS, LC-IRMS) (refined procedure)$
Isotope ratio mass spectrometry (EA- Sugar and protein δ13C values Addition of C4 derived sugars to products
IRMS, LC-IRMS) (refined procedure)#
Quantitative deuterium nuclear Deuterium level at CH3 site of EtOH Detection of the addition of exogenous C3
magnetic spectroscopy (liberated by fermentation of sugars) derived sugars
(2H-SNIF-NMR)
Quantitative carbon 13-nuclear Relative levels of 13C isotope at CH3 Detection of the addition of added
magnetic spectroscopy and CH2 positions of EtOH (liberated cane/corn sugars to agave syrup£
(13C-SNIF-NMR)® by fermentation of sugars)
High pH anion exchange Presence of gluco-oligosaccharides Addition of starch derived syrups
chromatography linked with
electrochemical detection (HPAEC-
PAD)
High pH anion exchange Levels of mannitol and inositol Detection of dilution of agave syrup
chromatography linked with
electrochemical detection (HPAEC-
PAD)
Capillary Gas-Chromatography Presence of marker disaccharides for Addition of exogenous sugar syrups (from
(Cap-GC) sugar syrups starch (glucose and HFS) & invert syrup)
* Levels are defined in Mexican Standard for agave syrups. $ excludes agave syrup. £ not applicable to maple, birch and
coconut flower syrups/sugars/saps. # applicable to coconut flower sugar
― 15 ―
- 169 -
Plant-derived sugars and sweeteners
5. Conclusion
The main component of all the products covered in this chapter is sugar. Unfortunately as there
are always cheaper sources of sugar that can be used by unscrupulous producers to extend these
materials there is a sizeable driving force to “cheat” and make an elicit profit. The route chosen by
an unscrupulous producer will depend on the product in question, availability of adulterant and
their skill/experience. Most of these products are mainly sucrose and so the use of cane or beet
sucrose could be expected.
However, this is not always the case and HFCS is a material which is often chosen instead. This
addition will be detectable in maple and coconut flower syrups by distortion of the sugar profile,
presence of unusual oligosaccharides by Cap-GC and HPAEC-PAD and dilution of other components
13
plus a shift in the δ C value.
Birch sap/syrup on the other hand shows low levels of sucrose and roughly equal levels of glucose
and fructose, so addition of HFCS to this type of material will show little changes in the sugar
levels. However, the addition of this type of material will still be detectable by IRMS & Cap-GC
analysis.
2
Detection of beet and cane sucrose addition to maple sugar/syrup/sap should be detectable by H-
SNIF-NMR®. However, due to the much wider distribution pattern for the production of birch
sap/syrup/sugar this may make the use of SNIF-NMR less sensitive for this product.
13
As agave is a CAM plant its δ C value seen in the sugars of the product are very different from the
other products but are similar to sugars derived from cane/corn using the global method. The
13
presence of HFCS in agave is detectable by Cap-GC or by using isotopic methods C-SNIF-NMR.
13
The origin of sucrose (cane/beet) and glucose syrups (corn/rice) can be achieved using C-IRMS
and/or SNIF-NMR®.
There are many methods that can be applied to these products, but isotopic methods generally
provide one of the best opportunities to detect extension of these products with cheaper sugar
sources.
About 5 or more years ago a new screening method was introduced for the analysis of fruit juices
1
using H-NMR [40]. This method will be discussed in more detail in the chapter on fruit juices. It
has also been successfully applied to the analysis of honey to detect sugar additions and it is likely
that the same procedure could be applicable to the analysis of agave, maple, birch and coconut
flower sugar but this is a “work in progress” for these products.
As unscrupulous suppliers are always looking for new avenues by which they can to extend their
products without being detected this will always present a challenge to the analyst. It may be that
they will detect new ways to prepare syrups so that they will not carry the markers in use today to
detect their addition to these sugar-based products.
As there is significant ecological pressure on suppliers to reduce the new planting of palm trees in
Asia which are used for palm oil production, it is highly likely that this may also extend to palm
flower sugar/syrup in the future if the popularity of this product grows. In Mexico there is a
growing demand for agave pinas for syrup production. However, the supply of pinas is limited at
present and there is pressure on prices and availability of the raw material for their production.
The availability of these materials must be shared between both syrup and spirit producers
(Tequila and Mezcal) and it is likely that raw material prices will remain high while syrup demand
remains high/increases.
― 16 ―
- 170 -
Plant-derived sugars and sweeteners
6. Bibliographic references
1. Agriculture and Agri-Food Canada (2013). – Horticulture Sector Reports - Production figures for maple syrup from
Canada. Available at: http://www.agr.gc.ca/eng/industry-markets-and-trade/canadian-agri-food-sector-
intelligence/horticulture/horticulture-sector-reports/?id=1368482338314.
2. Government of Canada C.F.I.A. (2014). – Labelling Requirements for Maple Products. Available at:
http://www.inspection.gc.ca/food/labelling/food-labelling-for-industry/maple-
products/eng/1392414400422/1392414462687?chap=0.
3. NAPSI certified Maple water NAPSI Certif. Maple Water. Available at: http://www.napsi.ca?lang=en.
4. US standard for maple syrup CFR - Code Fed. Regul. Title 21. Available at:
https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=168.140.
5. Vermont mapel product regulations (2013).
6. Coconut sap sugar - Grading and classification (2010). Available at: http://coconutbenefits.com/wp-
content/uploads/PNS-BAFPS-76-2010-Coconut-Sap-Sugar.pdf.
7. Mexican standard for agave syrup specifications; Norma Mexicana NMX-FF-110-SCFI-2008 (2008).
8. Mexican Standard for agave syrup relative to the characteristics of health, agri-food quality, authenticity labelling
and evaluation of the conformity of agave syrup (NOM-003-SAGARPA-2016) (2016). Available at:
http://dof.gob.mx/nota_detalle.php?codigo=5461591&fecha=18/11/2016.
9. Codex Alimentarius (1999). – Standards for sugars - Codex Stan 212. Available at:
http://www.fao.org/input/download/standards/338/CXS_212e_u.pdf.
10. Summaries of EU legislation on sugars (2018). Available at: https://eur-lex.europa.eu/legal-
content/EN/TXT/?uri=LEGISSUM%3Al21130.
11. Commissioner O. of the (2016). – What’s in a Name? What Every Consumer Should Know About Foods and Flavors.
Available at: https://www.fda.gov/ForConsumers/ConsumerUpdates/ucm521518.htm.
12. Maple syrup producers: Fake flavors nothing like the real thing - Chicago Tribune (2016). Available at:
https://web.archive.org/web/20160324205329/http://www.chicagotribune.com/business/ct-maple-syrup-
producers-fda-20160216-story.html.
13. Vermont: Log Cabin All Natural Syrup not the real deal (2010). Mercury News. Available at:
https://www.mercurynews.com/2010/09/09/vermont-log-cabin-all-natural-syrup-not-the-real-deal/.
14. Moore J.C., Spink J. & Lipp M. (2012). – Development and Application of a Database of Food Ingredient Fraud and
Economically Motivated Adulteration from 1980 to 2010. J. Food Sci., 77 (4), R118–R126. doi:10.1111/j.1750-
3841.2012.02657.x.
15. Jahromi R., Reimann L., Thomas F., Jamin E. & Hammond D.A. (2014). – Critical Assessment of Methodologies used
for the Characterization of Agave Syrups - A Eurofins white paper. Available at:
https://cdnmedia.eurofins.com/european-west/media/92008/doc_aau11u_agave_white_paper_eurofins.pdf.
16. Icumsa methods for sugar analysis Available at: http://www.icumsa.org/index.php?id=174.
17. AOAC methods for sugar analysis. Available at: http://www.aoac.org.
18. Site-specific deuterium/hydrogen (D/H) ratios in vanillin. Site-specific natural isotope fractionation-nuclear magnetic
resonance (SNIF-NMR) spectromety (2006). Available at:
http://www.aoacofficialmethod.org/index.php?main_page=product_info&cPath=1&products_id=1711.
19. AOAC 984.23-1988, Corn syrup and cane sugar in maple syrup. Carbon ratio mass spectrometric method (1988).
Available at: http://www.aoacofficialmethod.org/index.php?main_page=product_info&cPath=1&products_id=2454.
20. AOAC 2004.01-2004, Carbon stable isotope ratio of ethanol derived from fruit juices and maple syrups. Isotope ratio
mass spectrometry (IRMS) (2004). Available at:
http://www.aoacofficialmethod.org/index.php?main_page=product_info&cPath=1&products_id=81.
21. Tremblay P. & Paquin R. (2007). – Improved detection of sugar addition to maple syrup using malic acid as internal
standard and in 13C isotope ratio mass spectrometry (IRMS). J. Agric. Food Chem., 55 (2), 197–203.
doi:10.1021/jf062413a.
22. Kelly S. (2009). – Identification of an internal isotopic reference compound in palm sugar to improve the detection of
cane sugar addition - Final report. UK Food Standards Agency. Available at:
http://randd.defra.gov.uk/Document.aspx?Document=Q01127IFRxPalmSugarIIRfinalreport.pdf.
― 17 ―
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Plant-derived sugars and sweeteners
23. AOAC 998.12-1998, C-4 plant sugars in honey. Internal standard stable carbon isotope ratio method (1998). Available
at: http://www.aoacofficialmethod.org/index.php?main_page=product_info&cPath=1&products_id=49.
24. Elflein L. & Raezke K.P. (2008). – Improved detection of honey adulteration by measuring differences between
13C/12C stable carbon isotope ratios of protein and sugar compounds with a combination of elemental analyzer —
isotope ratio mass spectrometry and liquid chromatography — isotope ratio mass spectrometry (δ13C-EA/LC-IRMS).
Apidologie, 39 (5), 574–587. doi:10.1051/apido:2008042.
25. AOAC 2000.19-2000, Beet or cane sugar in maple syrup. Site-specific natural isotope fractionation-nuclear magnetic
resonance (SNIF-NMR) method (2000). Available at:
http://www.aoacofficialmethod.org/index.php?main_page=product_info&cPath=1&products_id=2651.
26. Willems J.L. & Low N.H. (2012). – Major carbohydrate, polyol, and oligosaccharide profiles of agave syrup.
Application of this data to authenticity analysis. J. Agric. Food Chem., 60 (35), 8745–8754. doi:10.1021/jf3027342.
27. Low N.H. (1995). – Apple and orange juice authenticity analysis by capillary gas chromatography with flame
ionization detection. Fuit Process., 11, 362–367.
28. Low N.H. (1996). – Detection of high fructose syrup from inulin in apple juice by capillary gas chromatography. Fuit
Process., 4, 135–139.
29. Detection of Syrup Addition to Juices by Capillary Gas Chromatography - International Fruit Juice Union
Recommendation #4 (2005).
30. Thomas F., Randet C., Gilbert A., Silvestre V., Jamin E., Akoka S., Remaud G., Segebarth N. & Guillou C. (2010). –
Improved characterization of the botanical origin of sugar by carbon-13 SNIF-NMR applied to ethanol. J. Agric. Food
Chem., 58 (22), 11580–11585. doi:10.1021/jf102983v.
31. AOAC 986.13-1989(1996), Quinic,Malic,and Citric acids in cranberry juice cocktail and apple juice. Liquid
chromatographic method (1996). Available at:
http://www.aoacofficialmethod.org/index.php?main_page=product_info&cPath=1&products_id=980.
32. DIN (1994). – Fruit and vegetable juices - Enzymatic determination of citric acid (citrate) content - NADH
spectrometric method. DIN EN 1137:1994.
33. DIN (1994). – Fruit and vegetable juices - Enzymatic determination of D-glucose and D-fructose content - NADPH
spectrometric method. DIN EN 1140:1994.
34. DIN (1994). – Fruit and vegetable juices - Enzymatic determination of D-isocitric acid content - NADPH spectrometric
method. DIN EN 1138:1994.
35. DIN (1994). – Fruit and vegetable juices - Enzymatic determination of L-malic acid (L-malate) content - NADH
spectrometric method. DIN EN 1139:1994.
36. DIN (1996). – Fruit and vegetable juices - Enzymatic determination of sucrose content - NADP spectrometric method.
DIN EN 12146:1997.
37. DIN (1999). – Fruit and vegetable juices - Enzymatic determination of D- and L-lactic acid (lactate) content - NAD
spectrometric method. DIN EN 12631:1999.
38. DIN (1999). – This European Standard specifies an enzymatic method for the determination of the total content of
acetic acid or acetate salts in fruit and vegetable juices and related products. DIN EN 12632:1999.
39. DIN (1997). – Fruit and vegetable juices - Enzymatic determination of D-malic acid content - NAD spectrometric
method. DIN EN 12138:1997.
40. Rinke P., Moitrier S., Humpfer E., Keller S., Moertter M., Godejohann M., Hoffmann G., Schaefer H. & Spraul M.
(2007). – An 1H-NMR technique for high throughput screening in quality and authenticity control of fruit juice and
fruit juice raw materials - SGF-profiling. Fruit Process., 1, 10–18.
― 18 ―
- 172 -
Spices
Pamela Galvin-King, Simon A. Haughey*, Christopher T. Elliott
Institute for Global Food Security, Queen's University Belfast, United Kingdom
*E-mail corresponding author: [email protected]
1. Product Identity
1.1. Definition of the product and manufacturing process
1.1.1. FAO
According to the FAO, spices can be defined as “vegetable products used for flavouring, seasoning
and imparting aroma in foods”. Herbs, considered a subset of spices, are leafy spices, and some,
like dill and coriander, can provide both spice seeds and leafy herbs [1].
https://doi.org/10.32741/fihb.10.spices
- 173 -
Spices
―2―
- 174 -
Spices
2. Authenticity issues
2.1. Identification of current authenticity issues
―3―
- 175 -
Spices
meets objectives, a test method in an accredited laboratory, and supply chain verification
measures which may include pre-delivery of samples prior to purchase for approval, or evidence of
authenticity from an accredited laboratory [10]. The prevention of fraud is not in detecting each
individual fraud and controlling one type, but reducing the vulnerabilities, as the fraudsters are
always evolving and looking for their next crime [14]. The herb and spice industry has been a
victim of EMA on numerous occasions. Table 1 focuses on examples where substitution
adulteration occurred with various herbs and spices.
―4―
- 176 -
Spices
The addition of colour to spices to improve their value is a common occurrence. Colour can
influence the perception of food and stimulate appetite, therefore, increase the value of a product
[38]. The addition of colourants to foodstuffs dates back to at least 1500 BCE, and up until the
middle of the 19th century, ingredients such as the spice saffron was added for a decorative effect
in certain foodstuffs [38]. Natural dyes were commonly used in food around this time, however, as
the 1900s began, the use of synthetic dyes became the colouring of choice with ease of
production, less expense and superior colouring ability [38].
As with other types of food adulteration, there is a likelihood that certain synthetic dyes may be a
threat to public health, and historical records show that injuries and even death occurred following
ingestion of toxic colourants [38]. Allergic and asthmatic reactions as well as DNA damage have
also been reported [39]. Therefore, the use of most synthetic dyes is forbidden in Europe. The two
main types of dyes that may be illegally added to food include azo dyes and triphenylmethanes
[40]. Examples of these illegal azo dyes include Sudan I, II, III, IV, para red, orange II, methyl yellow
and rhodamine B. Malachite green and its metabolite leucomalachite green are examples of
triphenylmethane dyes considered genotoxic and/or carcinogenic.
In May 2003, Sudan 1 was found to be illegally present in chilli powder and foods containing chilli
powder in the EU [40]. Following this event, in 2005 and 2006, numerous tests were carried out for
the presence of illegal dyes by the UK Food Standards Agency (FSA) [41]. Regulatory legislation was
put in place following the scandal, and member states were required to monitor high risk products
and provide analytical reports for the presence or absence of Sudan dyes as an emergency
measure in the European Commission Decision 2005/402/EC [42]. This legislation was later
repealed in the European Commission Regulation (EC) No. 669/2009 [43] to a less intensive testing
regime due to a reduction in the presence of Sudan dyes.
Legislation varies in different countries, which can cause problems for importers and exporters
[41]. In the EU, Regulation (EC) No. 1333/2008 [44] on food additives was developed “…with a view
to… ensuring a high level of protection of human health and a high level of consumer
protection….” With regard to food colours, there are currently 25 natural, and 15 synthetic dyes on
Annex II of this regulation that can be allowed in food [41]. The US FDA regulates food additives in
the US. To indicate the variation between countries, three synthetic dyes approved in the US are
not approved in the EU, and nine synthetic food colours in the EU are not approved in the US [41].
There is still a continued risk of adulteration with dyes in spices.
Spice Adulteration
Red Pepper Chili Sudan 1, Sudan 4, Metanil Yellow, Sudan 3, Oil Orange SS, Rhodamine B, Auramine
powder O, Orange II, Dimethyl Yellow, Fast Garnet GBC, Malachite Green, Allura Red
Paprika powder Sudan 1, Sudan 4, Acid Black 1, Orange II, Annatto
Turmeric powder Sudan 1, Mentanil Yellow, Orange II, Lead Chromate
Sumac Amaranth Red, Basic Red 46
Curry powder Auramine O, Chrysoidin (Basic Orange II)
Saffron flower Acid Orange II, Mentanil Yellow, Sudan I, Ponceau 4R, Ponceau 6R
Cayenne pepper Crystal Violet
Five spice powder Auramine O
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The results in Table 2 summarises reported cases of adulteration of spices with dyes from 2013 to
2017 in the US. In this work the most common dyes reported were Sudan 1 and Sudan 4. These
results indicate that adulteration with dyes is ongoing. Continued surveillance of spices to detect
and prevent adulteration with dyes is vital to the herb and spice industry as well as the safety of
consumers. Health risks can occur alongside both substitution and addition adulteration. They can
cause more than an economic threat to the consumer.
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Chinese star anise (Illicium verum) is infused in teas to relieve the symptoms of colic in children.
The adulteration of Chinese star anise with Japanese star anise (Illicium anisatum) has in previous
years resulted in the intoxication of children. Japanese star anise looks similar to Chinese star
anise, and they are often even more difficult to distinguish as they can be sold in broken or ground
form. Therefore, chemical analysis is required to distinguish them. Japanese star anise contains
neurotoxins and can result in a child having neurological and gastrointestinal problems [28].
Papaya seeds have been used to adulterate and bulk black pepper. However, these papaya seeds
can cause liver and stomach problems, and therefore pose a health risk to the unsuspecting
consumer [27].
Turmeric can contain various adulterants that threaten public health. Yellow chalk powder has
been used to add bulk to turmeric as it is a cheap material [37,50]. This adulterated product
however can cause swelling of the face, loss of appetite, nausea and vomiting. Curcuma zedoaria
can be used to adulterate turmeric [36], and was found to have toxic effects in rats and chickens
by Latif et al. if not processed properly [51]. Lead chromate added to turmeric was used as a dye as
well as a bulking powder. Over exposure to lead can cause delayed mental and physical
development [52].
In a case reported in the Times of India [23], poor grade fennel seeds were coated with waste
marble dust and dye, and mixed in with the cumin product. In this case, it was the treatment of the
fraudulent product that caused the public health risk rather, than the fennel seeds themselves.
The use of other plant cuttings such as olive leaves in the adulteration of oregano [19] can also
pose a health risk to the consumer. As these leaves are not produced for consumption, it is
unknown how these cuttings may be treated. In the case of olive leaves in particular, evidence of
pesticides can be found (Elliott, C- personal communication). Pesticide residues pose a health risk,
and hazards such as toxicity, carcinogenicity and mutagenicity are associated with them [53].
There are many possible risks with food adulteration. Therefore, it is vital that there is adequate
policing of the supply chains and the food industry to deter and try to prevent any fraud before it
is too late. Illegal dyes are a constant threat to the international food industry and are found
intermittently, as indicated by the alerts in Rapid Alert System for Food and Feed (RASFF) [54].
Examples from RASFF and the possible health impacts can be seen in Table 4.
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It is vital that authentication testing is carried out to detect cases of economic fraud and to verify
that preventative measures are effectively in place [10]. This prevention not only maintains quality
and consumer trust, but also helps to prevent the possibility of public health risk [55].
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of oregano with Cistus incanus L., Rubus caesius L., and Rhus coriaria L. [21], 1 % for the detection
of olive leaves in oregano [61] and a limit of detection (LOD) of 10 g/kg for the presence of
Curcuma zeodoaria / Curcuma malabarica in turmeric [36] indicate this. However, a limitation of
SCAR-PCR is the need for sequence data for the PCR primers design [61].
DNA barcoding is a relatively new method that was first developed in 2003 and is based on the
variability within a standard region of the genome, the ‘DNA barcode’ [62]. It has become
increasingly used since its development, and there is successful evidence of this method in the
detection of adulterants in herbs and spices. This method has been used for the detection of
adulterants in saffron [63], and chilli adulteration in black pepper [25]. DNA barcoding is a fast,
reliable sensitive method for a wide range of food commodities, and even strongly processed
foods and there is also the possibility of building reference databases to improve the chances of it
becoming a routine test for food quality, and traceability [64].
DNA purity and integrity are concerning with regard to DNA barcodes, which, can be a limitation of
the test. Poor quality DNA may reduce amplification success of DNA barcodes [65]. DNA barcoding
also relies on the availability of sequence libraries to reference against [66].
Whole genome sequencing is becoming a possibility and it has potential for the detection of food
adulteration with Next Generation Sequencing (NGS). However, so far, little work in this area has
been carried out with the complex work flow and high costs associated with this method [67].
The methods for the detection of adulteration in herbs and spices using DNA analysis described
are qualitative. Quantitative methods often result in high measurement uncertainty, although
advancements in PCR technologies are improving in this way [67]. Overall, the limitations with
DNA analysis may include poor integrity and purity of the DNA, poor efficiency of the extraction,
and the risk of contamination is a concern with these methods. Also, low level accidental
contamination can be misinterpreted as intentional substitution.
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the detection of a characteristic marker of olive leaves, the phenolic compound oleuropein, in
both oregano and sage with the use of Liquid Chromatography-Electrospray Ionization Mass
Spectrometry (LC-ESI-MS/MS). This compound oleuropein was later found to be also present in
myrtle leaves by Wielogorska et al. [69]. Similarly, the use of untargeted Ultra High Performance
Liquid Chromatography coupled to High Resolution Mass Spectrometry (UHPLC-HRMS) merged
with chemometrics, OPLS-DA proved to be a successful powerful tool in determining products
from the PDO of saffron [33]. Falsely declared saffron from a PDO can be used in substitution of
the authentic product.
GC-MS is another method that has been used to detect possible adulterants such as with the study
carried out in 2015 investigating detection methods for known fruit adulterants in fennel seed
[71]. Essential oils of fennel seed and two adulterants were profiled, and distinct differences
between fennel seed and two of its adulterants were observed. Bononi, Fiordalise and Tateo were
able to use GC-MS to detect olive leaves in oregano and sage by using GC-MS with a detection limit
of 1 % [72]. The benefits of this method included the ease of use and reproducibility of the results.
However, with regard to the detection of adulteration in herbs and spices, an issue that may occur
with the use of GC-MS is that, only the volatile oils are investigated. Therefore, the addition of
volatile oils to a product may cheat the GC-MS adulteration detection method.
ICP-MS along with PCA and Canonical Discriminant Analysis (CDA) was a method developed to
detect falsely declared Szegdi paprika (PDO) [32]. The Sr isotopic composition and the multi-
elemental analysis are indicative of paprika from the region.
Upgrades in mass spectrometry involve the use of real time analysis of samples by directly
introducing the samples to the mass spectrometer. Ambient mass spectrometry is a relatively new
analytical technique that gives comparable results to conventional techniques without complex
sample preparation [73]. Examples of its use include the detection of the adulterant Japanese star
anise in Chinese star anise using Direct Analysis Real Time-High Resolution Mass Spectrometry
(DART-HRMS) by detecting the presence of anisatin [74]. Advances on this method involves the
use of direct plant spray combined with orbitrap-HRMS [75]. This method can detect between the
neurotoxic Japanese star anise and the Chinese star anise in seconds, and without sample pre-
treatment. DART ionisation has slightly higher selectivity, no solvents added and the absence of
high voltages when compared to direct plant spray. The benefits of direct plant spray over DART
ionisation include the low cost, lower standard deviations and simplicity. Direct plant spray and
DART ionisation techniques are more successful qualitative methods than quantitative methods.
Currently the disadvantages of mass spectrometry in comparison to spectroscopy are the cost and
the requirement of a laboratory setting and highly trained analysts. However, advances to
overcome this are ongoing with aims to miniaturize the instrumentation, and for the data to be
presented so that it is easily interpreted. However, these developments require further
optimization and are not readily available [68]. Similarly to spectroscopy, the validation procedure
for non-targeted methods in mass spectrometry has not been standardised. This can reduce
consistency between laboratories.
3.3. Spectroscopy
Vibrational spectroscopies, along with chemometrics, have become well known as rapid, non-
destructive, fingerprinting techniques and are valuable screening tools in the detection of
adulteration / authentication in the food industry. A range of spectroscopic analytical techniques
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used in the food industry include FTIR, Fourier Transform Near infrared (FT-NIR), Raman,
Hyperspectral Imaging (HSI) [76] and Nuclear Magnetic Resonance (NMR) [77].
In the detection of adulteration of herbs and spices for economic gain, a number of spectroscopic
methods continue to be developed. Work has been carried out to develop competent models to
detect cornstarch in garlic powder by FTIR [76] and onion powder by FTIR and NIR [78]. Raman has
also been used to detect cornstarch in onion powder and garlic or ginger powder [79,80]. Starch
may be added to white powders such as garlic and onion powder to add bulk to the product. In
these studies, a quantitative model was built using the algorithm Partial Least Squares Regression
(PLSR) in chemometrics. The Raman, FTIR and NIR spectral data based models described here are
capable of detecting adulteration in onion powder, garlic and ginger with starch up to 35 %.
In a study by Black et al. on the detection of adulteration in oregano, FTIR was used alongside the
confirmatory technique LC-HRMS [19]. Following the identification of biomarkers for both oregano
and its adulterants, and the development of spectroscopic classification models using the
unsupervised PCA and supervised OPLS-DA chemometric algorithms, a rapid screening method and
confirmatory method was developed. The benefit of this method was that a number of different
adulterants could be added to the database that was used to build the model. The developed
screening technique therefore was robust and could identify numerous adulterants at each
screening in the survey that was subsequently carried out. The results of the survey indicated that
adulteration was ongoing, but also, it displayed the use of a rapid screening technique to help the
fight against food fraud. Further development on these analytical techniques was carried out with
the development of targeted quantitative methods using FTIR with PLSR and LC-MS/MS for the
detection of adulteration in oregano [69].
Raman and FTIR methods analyse the sample in the mid infrared region of the electromagnetic
spectrum. The spectral data consist of sharp bands representing inelastic scattering, or
information on the fundamental vibrations of the sample respectively. This is in comparison to the
vibrational overtones and combination peaks of the NIR, which does not provide as much
information [68]. However, in the detection of starch in onion powder, NIR with PLSR chemometric
algorithm was determined the most suitable method [78]. NIR has the ability to penetrate deeper
into the sample and therefore is more suitable for bulk samples that have little or no sample
preparation. Raman has advantages over NIR and FTIR as it is not affected by water, and inorganic
materials can be analysed more easily. Analysis through packaging or glass is also a possibility [79].
Recent improvements to Raman also include the use of Surface Enhanced Raman Scattering (SERS)
and Spatially Offset Raman Spectroscopy (SORS) which has shown its ability to detect counterfeit
products through packaging [68].
1
The use of Proton Nuclear Magnetic Resonance ( H-NMR) combined with chemometrics (PCA,
OPLS-DA, O2PLS-DA) was investigated and was proven successful at determining the quality and
authenticity of saffron [81], allowing the detection of common adulterants such as Sudan dyes
[82], other dyers mixed with stabilizing agents or bio-adulterants such as gardenia, safflower or
curcuma [77] whose specific markers have been identified. Additionally to these targeted studies
(use of markers), some untargeted approaches coupled to chemometrics were also developed to
assess saffron authenticity and detect the presence of unexpected adulterants [83]. That sort of
approach can be transferrable to other spices, given the availability of consequent authentic
database. Quantitative metabolomics analysis were also performed to distinguish cinnamon
varieties and showed encouraging results [84]. Others spices such as safflower [85] were also
1
studied by NMR. H-NMR was shown to give reproducible results rapidly, however, this technique
requires solvent extraction and is then limited to extracted metabolites. Additionally only organic
compounds are visible with this technique. Further work carried out using DRIFTS on FTIR
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minimized the process of sample preparation and proved to be successful along with PLS-DA
classification and quantitative PLSR models at detecting six known saffron adulterants [86].
Although these spectroscopy methods are often successful on their own, further developments
are being made to improve the methods by:
1) Combining data: Wang et al. [87] carried out a study that improved FTIR and NIR results for the
detection of the adulterant Iuicium lanceolatum A.C. Smith (ILACS) in Chinese star anise. This
method involved combining the NIR and FTIR spectral data and the use of PCA and Linear
Discriminant Analysis (LDA) chemometric techniques. Although the FTIR performed better than
NIR in this study when analysed separately, the classification results from the combined approach
proved to be even more successful.
2) Increasing sensitivity: Vermaak et al. [88] used hyperspectral imaging with PCA and PLS-DA to
distinguish between the neurotoxic Japanese star anise and Chinese star anise. This emerging
method incorporates spectroscopy and imaging to produce both spatial and spectral data from a
sample [89] (Gowen, O'Donnell, Cullen, Downey and Frias, 2007). This method is also non-
destructive and rapid with the added advantage that with the acquisition of several predictions on
the sample, the statistics are better [88]. The quantification of adulterants, buckwheat or millet, in
ground black pepper was carried out using FTIR and NIR with hyperspectral imaging with PLSR
chemometrics. NIR with hyperspectral imaging was seen to produce the best calibrations which, in
this case was largely to do with the larger sample area used with NIR, and the spatial information
from the imaging system used with it [90]. Galaxy Scientific’s Classical Least Squares (CLS)-based
Advanced-ID algorithm has been developed to detect screening samples to a level as low as 0.01 %
[91]. When it was used to detect paprika adulterants, it detected Sudan 1 dye at 0.1 %, tomato
skin at 0.5 % and brick dust at 5 %.
3) Analysis through packaging: Terahertz spectroscopy was used to overcome the barrier of
common packaging materials such as plastics and papers [37]. This method is a promising non-
intrusive technique that was used for the detection of yellow chalk powder in turmeric.
It is apparent that further improvements and developments are ongoing with the use of
spectroscopy. Developments seen in benchtop spectroscopic instruments are also being
transferred to handheld devices. An added benefit as discussed by Ellis et al. [68] would be to use
the advantages of the NIR and FTIR combined, and developed into a handheld device. Overall, the
ability to transfer this technology to portable and handheld devices allows the user to determine
authenticity in the field, and can focus on vulnerable points of the supply chain. This not only
allows improvements in traceability and detection of fraud, but at a basic level, it can also act as a
deterrent. If food fraud criminals are aware of this possibility, they may be less likely to take the
risks of committing a crime in the first place.
Limitations of spectroscopy must not be overlooked. Spectroscopy is used as a rapid screening
technique and therefore, further investigations may need to be carried out by confirmatory
techniques that require more expertise, time and cost more, such as mass spectrometry. This is
also true when building models using chemometrics, the purity of samples needs to be assured in
order to build accurate models. Another limitation of spectroscopy, as a non-targeted method, is
the lack of a standardised validation procedure for all laboratories.
Following a review of more than sixty scientific publications, it was found that spectroscopic
techniques are the major analytical techniques used to determine adulteration of herbs and spices
in high concentrations [92]. Overall, these techniques provide a good first point of control in the
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fight against food fraud. Although the use of other confirmatory techniques such as mass
spectrometry may be required in some circumstances, the bulk of screening herbs and spices for
EMA are possible with spectroscopy.
Although not a spectroscopic technique, an analytical screening technique called the ‘electronic
nose’, capable of detecting aroma fingerprints, was used alongside PCA and Artificial Neural
Networks (ANN) to detect adulteration in saffron. This technique was found to be promising, as
detection was possible at higher than 10 % adulteration, enough to detect EMA [34].
3.5. Chemometrics
Chemometrics is used to improve the chemical data obtained from analytical instruments and to
correlate the properties of samples with the use of mathematics and statistical methods [76].
Chemometrics has been used in the calibration analysis of spectroscopic and spectrometric data. It
has been used with both targeted and untargeted methods to detect the presence of fraud in food
or to determine authenticity [92]. The use of pre-processing is carried out in chemometrics to
amplify desirable information from raw data and reduce the effects of undesirable information in
the spectra. There are three key stages in the use of chemometrics, data pre-processing,
development of a robust model, and the validation of a model and the analysis of results. Two
commonly used pre-processing techniques include scatter correction methods, and spectral
derivatives. Scatter corrective techniques can include Multiplicative Scatter Correction (MSC),
Standard Normal Variate (SNV) and, normalisation to reduce the effects of physical variability
caused by scattering [93]. The two commonly used spectral derivatives are Norris-Williams (N-W)
and Savitzky-Golay (S-G). The spectral derivatives aim to smooth the spectra without reducing the
signal to noise ratio in the spectra too much.
The analysis of adulteration using spectroscopy and in some cases mass spectrometry requires
further investigation with chemometrics. The most common algorithms used for the
determination of authenticity or the detection of fraud are the classification/discrimination
algorithms such as the unsupervised PCA, and the supervised LDA, PLS-DA or OPLS-DA. For the
quantification of adulterant in a sample, PLSR analysis is used frequently.
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Chemo-
Ingredient Adulterant Reference Detection Methods
metrics
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Chemo-
Ingredient Adulterant Reference Detection Methods
metrics
Oregano Olive leaves, myrtle leaves, hazelnut leaves, [69] LC-MS/MS, FTIR PLSR
sumac
Oregano Olive leaves [70] LC-ESI-MS/MS
Sage Olive leaves [70] LC-ESI-MS/MS
Oregano Olive leaves [72] GC/MS
Paprika Falsely declared Szegedi paprika substituted [32] ICP-MS PCA, CDA
for Szegedi Füszerpaprika PDO
Oregano Olive leaves, myrtle leaves, cistus, hazelnut [19] FTIR , LC-HRMS PCA, OPLS-
leaves, sumac DA
Garlic Cornstarch [76,78] Raman, FTIR PLSR
Ginger Cornstarch [78] Raman PLSR
Onion Powder Cornstarch [78,79] Raman, FT-NIR, FTIR PLSR
1
Saffron Crocus sativus stamens, turmeric, safflower, [77] H-NMR PCA, OPLS-
gardenia DA, O2PLS-
DA
Saffron Crocus sativus stamens, calendula, [86] DRIFTS-FTIR PLS-DA,
safflower, turmeric, buddleja, and gardenia PLSR
Chinese star ILACS [87] NIR/MIR LDA, PCA
anise
Chinese star Japanese star anise [88] SWIR-HIS PCA, PLS-
anise DA
Black pepper Buckwheat or millet [90] NIR hyperspectral imaging, PLSR
FTIR
Paprika Tomato skins, brick dust [91] FT-NIR & Advanced-ID
algorithm
Turmeric Yellow chalk powder [37] Terahertz spectroscopy
Saffron Safflower dyed corn stigma [34] Electronic Nose PCA, ANN
5. Conclusion
It is evident that EMA is a constant threat in the growing herb and spice industry. Cases of fraud
have an economic impact on the industry as well as reducing consumer confidence. Potential
public health risks following adulteration, such as the case of nut protein in cumin and paprika, are
a major concern in the industry. Advances in DNA analysis include the use of SCAR-PCR and DNA
barcoding provide faster and cheaper methods of analysis. Further advancement may include the
use of NGS as it moves into the area of food fraud. Mass spectrometry, commonly used for the
detection of food fraud is also improving by becoming faster and cheaper with the introduction of
ambient techniques. Spectroscopic methods along with chemometric techniques are increasingly
being used in the fight against food fraud and offer a rapid, robust screening technique that is cost
effective and requires little expertise. There is an increasing need for screening techniques that
can detect EMA over a range of products in the growing herb and spice industry.
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Acknowledgements
The authors would like to thank Food Control for the permission to use the original review article
as the basis for this chapter [95]. The original article was published in full as follows:
Pamela Galvin-King, Simon A. Haughey, Christopher T. Elliott (2018), Herb and spice fraud; the
drivers, challenges and detection, Food Control, 88, 85-97.
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Saffron
Natalia Moratalla-López, Amaya Zalacain, Maria José Bagur,
Maria Rosario Salinas, Gonzalo L. Alonso*
Universidad de Castilla-La Mancha, Albacete, Spain
*E-mail corresponding author: [email protected]
https://doi.org/10.32741/fihb.11.saffron
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1. Product Identity
1.1. Definition of the product and manufacturing process
The flower of Crocus sativus grows from a corm in late October and early November in the
northern hemisphere, and in late April and early May in the southern hemisphere. It is picked
manually in the field, and depending on the tradition of the production area, either at dawn with
the flower closed, or at noon when the flower is open. The stigma and part of the style are
separated from the rest of the flower, to a greater or lesser extent according to tradition.
Subsequently, the stigmas are dried either using a direct flameless heat source or by leaving them
for several days in the sun or in the shade, depending on each producing area. In some places
there is a flower market, where the farmer who collects the flowers is not the one who produces
the spice. All these factors mean that there is great diversity among the products obtained in
different areas and, therefore, different quality products [3].
In La Mancha saffron, the three filaments are joined together with a small part of the yellowish-
white style that contributes nothing. Italian saffron is very similar and although in Greece stigmas
are also held together, Greek saffron is usually accompanied by flower pollen which contributes
other flavour and aroma characteristics. In Iran, traditionally, the stigma is accompanied by a long
part of the style and is dried in the sun or in the shade, as in Morocco. In India, the stigmas, after
being separated, are rubbed together to obtain a homogenous darker colour and therefore
quality.
Saffron is marketed for its colour, flavour and aroma with major metabolites that determine the
quality of saffron. These are currently controlled by ISO 3632 [5] and used in all commercial
transactions with saffron. The substances responsible for the colouring properties of saffron are
the glycosidic esters of the carotenoid dicarboxylic crocetin (2E, 4E, 6E, 8E, 10E, 12E, 14E)-
2,6,11,15-tetramethylhexadeca-2,4,6,8,10,12,14-heptaenedioic acid, C20H24O4). The glycosides
bound to crocetin are gentiobiose, glucose, neopolitanose and triglycose [6–9], which in saffron
are found in their trans (majority) and cis (minority) forms. All these compounds are referred to in
the literature as crocins, although in fact crocin is only trans-crocetin di (β-D-gentiobiosyl) ester.
Figure 1 describes the names that have been accepted in recent years by the scientific community.
The colouring strength of the spice depends on the concentration of these compounds, which
ranges between 16-28 % in the dried stigma of Crocus sativus L., reaching concentrations up to 30
% in some years.
The substance responsible for the characteristic bitter taste of saffron is picrocrocin (4- (β-D-
glucopyranosyloxy)-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde, C16H26O7). Up to now, this
compound has not been detected in any other raw material, whether from plant or animal origin,
and it is therefore considered to be a molecular marker of true saffron [11]. Its concentration is
usually between 7-16 % [12], although in some samples it can reach 20 %.
With respect to saffron aroma, safranal (2,6,6-trimethyl-1,3-cyclohexadiene-1-carboxaldehyde,
C10H14O) is the main compound [13] and the aglycone of picrocrocin. It has been detected in very
few plant products and can be also generated when certain carotenoids undergo a thermal
process. Safranal concentration is much lower than crocins and picrocrocin, usually between 0.1-
0.6 % [14].
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Saffron
Figure 1: Simplified names of the glycosidic esters, crocins, of the carotenoid crocetin introduced by Carmona et al. [3].
Meaning of the abbreviations: t is Triglucose; G is Gentiobiose; n is Neapolitanose; g is Glucose [10]
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Saffron
2. Authenticity issues
The main problem for the saffron consumer is the lack of knowledge about the shape of the
product, which is the reason why some plant products such as Carthamus tinctorius are on offer on
the market that are not true saffron. Also by not knowing the product in certain regions, it is easy
to confuse consumers with fibres coloured with artificial dyes.
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Saffron
would be more expensive than saffron itself. In addition, given that the picrocrocin content has to
be higher than 7 % to be considered saffron, and that this compound is a molecular marker since it
is only found in saffron, the fraud would be even more expensive. In other words, simply by
changing the method of determining the quality of the spice, all types of fraud could be avoided.
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Saffron
Figure 2: Saffron fingerprint obtained by HPLC, chromatograms at 440, 330 and 250 nm, and their UV-vis spectrum. The
peaks corresponding to the major metabolites (crocins, safranal and picrocrocin) are indicated
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Saffron
5. Conclusion
To control the quality of saffron and avoid adulteration, it is necessary to introduce analytical
methodologies in the compulsory standards, as well as in ISO 3632, which determine in detail the
metabolites: crocins, picrocrocin and safranal, responsible for colour, taste and aroma,
respectively. At this time and in the near future the methodology based on HPLC-DAD is the most
appropriate, fastest and cheapest.
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Saffron
6. Bibliographic references
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Publishing Limited, Philadelphia, PA, USA. pp 469–498doi:10.1533/9780857095671.469.
3. Carmona M., Zalacain A. & Alonso G.L. (2006). – The Chemical Composition of Saffron: Color, Taste and Aroma.
Bomarzo SL, Albacete, Spain.
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metabolomics of some Iranian saffron (Crocus sativus L.) accessions. Ind. Crops Prod., 118, 26–29.
doi:10.1016/j.indcrop.2018.03.024.
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3632-1:2011. Available at: https://www.iso.org/fr/standard/44523.html.
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Related Compounds Present in Crocus sativus Stigmas and Gardenia jasminoides Fruits. Tentative Identification of
Seven New Compounds by LC-ESI-MS. J. Agric. Food Chem., 54 (3), 973–979. doi:10.1021/jf052297w.
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Zusammensetzung im Safran. Helv. Chim. Acta, 58 (6), 1608–1620. doi:10.1002/hlca.19750580615.
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plant extract using high-performance liquid chromatography-UV-visible photodiode-array detection-mass
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– Saffron: An Old Medicinal Plant and a Potential Novel Functional Food. Molecules, 23 (1), 30.
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(2010). – Picrocrocin Content and Quality Categories in Different (345) Worldwide Samples of Saffron (Crocus sativus
L.). J. Agric. Food Chem., 58 (2), 1305–1312. doi:10.1021/jf903336t.
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saffron. Food Chem., 221, 838–843. doi:10.1016/j.foodchem.2016.11.089.
15. Carmona M., Sánchez A.M., Ferreres F., Zalacain A., Tomás-Barberán F. & Alonso G.L. (2007). – Identification of the
flavonoid fraction in saffron spice by LC/DAD/MS/MS: Comparative study of samples from different geographical
origins. Food Chem., 100 (2), 445–450. doi:10.1016/j.foodchem.2005.09.065.
16. Maggi L., Carmona M., Campo C.P. del, Kanakis C.D., Anastasaki E., Tarantilis P.A., Polissiou M.G. & Alonso G.L.
(2009). – Worldwide market screening of saffron volatile composition. J. Sci. Food Agric., 89 (11), 1950–1954.
doi:10.1002/jsfa.3679.
17. García-Rodríguez V.M., Serrano-Díaz J., Tarantilis P.A., López-Córcoles H., Carmona M. & Alonso G.L. (2014). –
Determination of Saffron Quality by High-Performance Liquid Chromatography. J. Agric. Food Chem., 62 (32), 8068–
8074. doi:10.1021/jf5019356.
18. Publication of an application for registration pursuant to Article 6(2) of Council Regulation (EEC) No 2081/92 on the
protection of geographical indications and designations of origin - Krokos Kozanis (1998). Off. J. Eur. Communities, C
207, 2–5.
19. Publication of an application for registration pursuant to Article 6(2) of Regulation (EEC) No 2081/92 on the
protection of geographical indications and designations of origin - Azafrán de la Mancha (2000). Off. J. Eur.
Communities, C 173, 4–8.
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20. Publication of an application for registration pursuant to Article 6(2) of Regulation (EEC) No 2081/92 on the
protection of geographical indications and designations of origin - PDO Zafferano dell’Aquila. (2004). Off. J. Eur.
Union, C 93, 23–26.
21. Commission Regulation (EC) No 205/2005 of 4 February 2005 supplementing the Annex to Regulation (EC) No
2400/96 on the entry of certain names in the Register of protected designations of origin and protected geographical
indications (Valdemone — [PDO], Queso Ibores — [PDO], Pera de Jumilla — [PDO], Aceite de Terra Alta or Oli de
Terra Alta — [PDO], Sierra de Cádiz — [PDO], Requeijão Serra da Estrela — [PDO], Zafferano dell’Aquila — [PDO],
Zafferano di San Gimignano — [PDO], Mantecadas de Astorga — [PGI] and Pan de Cea — [PGI]) (2005). Off. J. Eur.
Union, L33, 6–7.
22. Commission Regulation (EC) No 98/2009 of 2 February 2009 entering certain names in the register of protected
designations of origin and protected geographical indications (Aceite de La Alcarria (PDO), Radicchio di Verona (PGI),
Zafferano di Sardegna (PDO), Huîtres Marennes Oléron (PGI)) (2009). Off. J. Eur. Union, L33, 8–9.
23. Alonso G.L., Varón R., Navarro F. & Gómez R. (1988). – Algunos detalles históricos sobre el azafrán. Ens. Rev. Fac.
Educ. Albacete, 2, 223–230.
24. Ordoudi S.A., Cagliani L.R., Melidou D., Tsimidou M.Z. & Consonni R. (2017). – Uncovering a challenging case of
adulterated commercial saffron. Food Control, 81, 147–155. doi:10.1016/j.foodcont.2017.05.046.
25. Sánchez A.M., Maggi L., Carmona M. & Alonso G.L. (2011). – Authentication of Saffron Spice ( Crocus sativus L.). . In
Progress in Authentication of Food and Wine (S.E. Ebeler, G.R. Takeoka & P. Winterhalter, eds), American Chemical
Society, Washington, DC. pp 309–331doi:10.1021/bk-2011-1081.ch022.
26. Aramburu A.Z. (2018). – Detection of the most sophisticated saffron fraud with the latest technologies: current
fraudulent practices using Gardenia jasminoides extracts. Acta Hortic., (1200), 205–212.
doi:10.17660/ActaHortic.2018.1200.34.
27. Zalacain A., Ordoudi S.A., Blázquez I., Díaz-Plaza E.M., Carmona M., Tsimidou M.Z. & Alonso G.L. (2005). – Screening
method for the detection of artificial colours in saffron using derivative UV-Vis spectrometry after precipitation of
crocetin. Food Addit. Contam., 22 (7), 607–615. doi:10.1080/02652030500150051.
28. Zalacain A., Ordoudi S.A., Díaz-Plaza E.M., Carmona M., Blázquez I., Tsimidou M.Z. & Alonso G.L. (2005). – Near-
Infrared Spectroscopy in Saffron Quality Control: Determination of Chemical Composition and Geographical Origin. J.
Agric. Food Chem., 53 (24), 9337–9341. doi:10.1021/jf050846s.
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Federica Camin*, Luana Bontempo
Unità Tracciabilità -Dipartimento Qualità Alimentare e Nutrizione
Fondazione Edmund Mach, Trento, Italy
*E-mail corresponding author: [email protected]
Roberto Larcher*
Experimental and Technological Services Department, Technology Transfer Centre
Fondazione Edmund Mach, Trento, Italy
*E-mail corresponding author: [email protected]
Carsten Fauhl-Hassek*
Bundesinstitut für Risikobewertung, Berlin, Germany
*E-mail corresponding author: [email protected]
Freddy Thomas*
Eurofins Analytics France, Nantes, France
*E-mail corresponding author: [email protected]
https://doi.org/10.32741/fihb.12.wine
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For its intoxicating and exciting properties, rapidly wine became far more than an ordinary
beverage, and was often used as a ritual libation for priests and royalty in religious ceremonies, or
as a votive offering to gods. Later, the expansion of the Greek civilization, and also that of the
Roman Empire, led to the diffusion of the cult of Dionysus (or Bacchus for the Romans), the god of
wine, and the vine growing culture, in all the coastal regions around the Mediterranean Sea. Under
Celtic and Roman influence, viticulture was then introduced to the continental European
temperate regions, notably to France and Germany. After the fall of the Roman civilization, when
Europe was afflicted by mass migration and invasions, inside scattered monasteries was seeded
and nursed the first embryo of modern winemaking knowledge.
Nowadays, the European Union is the world's largest wine producer and consumer, with roughly
70 % of global production and 60 % of global consumption. All 27 EU member states produce wine
to some extent, and each has its own language, traditions and wine classifications. World wine
production was around 246.7 mhl in 2017 (OIV report), with Italy, France and Spain as the leading
world producers.
According to the European Commission’s Directorate General for Agriculture and Rural
Development (DG AGRI), European wine production can vary a lot from year to year (with yields
ranging from +20 % to -20 %), highly influenced by weather conditions and/or the sanitary
conditions of the vines. This has an important impact on price levels and hence on the number and
types of adulteration. The price of wine also depends on its production area and label.
Wine exports are increasing year by year and accounted in 2017 for over 25 % of the volumes
produced, whereas imports remain constant. Five main destinations (USA, Switzerland, Japan,
Canada, China-Hong Kong) account for up 70 % in value of all wines exported outside the EU.
Outside Europe, the main wine producer is the USA, followed by Australia and China. Wine
production in China is increasing year by year, from being absent in 2005 and taking its place as
the world’s 6th largest wine producer in 2016.
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1. Product Identity
1.1. Definition of the product and manufacturing process
The most relevant constituents of must and wine are water, carbohydrates, acids, alcohols,
phenolics, nitrogenous compounds (proteins, amino acids and ammonium salts), inorganic
substances (metals and anions) and flavours. The chemical composition of grapes is affected by
many factors, particularly grape variety or cultivar, environmental factors such as climate and soil
(the concept of ‘terroir’), viticultural management and seasonal variations (the concept of
‘vintage’), and also on the variability of winemaking practices.
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wine is periodically transferred into new containers, such as stainless-steel tanks or oak barrels.
Wine can be also clarified using fining agents and filtration equipment.
Aging and Bottling
The ageing of wine, using variable periods of maturation in oak barrels or of aging in glass bottles,
represents a crucial winemaking step, potentially able to improve the fineness of wine, making its
aroma and taste more complex and pleasing to consumers. A shorter aging in steel tanks before
bottling is instead commonly used for fresh white wines.
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2. Authenticity issues
2.1. Identification of current authenticity issues
Food and beverage authenticity issues fall into one of the following categories:
i. Non-compliance with the established legislative standards,
ii. Adulteration of high value products, through substitution by cheaper but similar
ingredients or extension adulterant
iii. Misdescription and/or mislabelling of geographical, botanical or species origin.
In the case of wine/must, category (i) corresponds to the non-compliance with the legislative
reference standards and limits of European regulations and OIV, Codex and specification rules of
each PDO or IGP in terms of the chemical-physical composition of the product. Some examples are
given in Table 1. The authenticity of the samples is determined by using quantitative analyses
which quantify the amount of the compounds present: if the actual values are outside the limits
quoted in the table, the samples are non-authentic.
The category (ii) relates to the unpermitted addition of exogenous sugars and water in order to
increase the alcoholic degree and the yield of the product, and the unpermitted addition of
exogenous compounds, such as flavours, glycerol, dyes, tartaric acid and CO 2 in order to improve
the poor quality of the product.
In these cases, the authenticity of the product is evaluated using analytical approaches able to
trace the source of the compound (from grape, from exogenous products or synthetic). Maximum
acceptable limits do not exist, but a reference database on the basis of the analysis of authentic
samples has to be built.
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Table 1: Maximum acceptable limits of various substances contained in wine (mainly from Compendium of International
Methods of Analysis-OIV, 2015/1 Issue)
Misdescription and mislabelling (iii) concern false declaration of origin and grape variety, harvest
year and wine category. The aim of this adulteration is to give premium price and value to
products with low quality.
In addition, for these types of adulteration, reference databases have to be built on the basis of
the analysis of authentic samples in order to define the ranges of values that are characteristic of a
particular production area, vintage or variety.
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3.1.1. Sugars
The determination of fermenting sugars in must and wine represents a fundamental issue for
oenology. Different approaches are provided: the most practical for use in the winery, but not very
accurate, is the determination of reducing sugars as an estimation of fermentable ones. It is
indeed of the lowest category. The determination of glucose and fructose by an enzymatic
method, and the determination of sugars, including glycerol and sucrose, by HPLC, are both
regarded as being of superior accuracy and selectivity, and are considered as belonging to category
II. Of a lower classification are the two approaches that use differential pH sensors for the joint
determination of glucose and fructose or, separately, of glucose, fructose and sucrose.
Polyols derived from sugars and residual sugars in dry wines (fructose, glucose, mannitol, sorbitol,
dulcitol, and mesoinositol) are determined using gas chromatography after formation of their
trimethylsilylated derivatives.
The source of sugar (whether from grape or from cane or beet) is determined using Site Specific
Nuclear Isotope Fractionation Nuclear Magnetic Resonance (SNIF-NMR) which determines the
deuterium distribution and the D/H ratios in the methylic and methylenic sites of ethanol derived
from the fermentation of grape musts, concentrated grape musts, grape sugar (rectified
13 12
concentrated grape musts) and wines. The C/ C isotope ratios of glucose, fructose, glycerol,
ethanol in products of vitivinicultural origin (dry wine, sweet wine, grape juice, and rectified
concentrated must) are determined by HPLC/IRMS. This method belongs to category II for glucose,
fructose and glycerol, and III for ethanol.
3.1.2. Alcohols
Accurate measurement of alcoholic strength (by volume) was, for a long time, both a technical
challenge and a practical need for establishing the commercial value of wine. Two methods
(categories I and IV) are available. The first measures the alcoholic strength of wine determining
the density of its distillate using, alternatively, a pycnometer, an electronic densimeter, or a
hydrostatic balance. The second method, definitely less accurate, uses a hydrometer or
refractometer to determine the alcoholic strength of the wine distillate.
Two possible methods for methanol quantitation are also considered. The first determines
methanol in the wine distillate using GC/FID, while the second measures it on the base of the
violet colour intensity at 575 nm after its oxidation to formaldehyde by potassium permanganate
and reaction with chromotropic acid in a sulphuric medium.
13 12
In this section are also reported 2 isotopic methods. The first determines the C/ C isotope ratio
of wine ethanol or that obtained through the fermentation of musts, concentrated musts or grape
sugar by IRMS, enabling the detection and quantification of sugars of C4 origin (sugar cane or corn
isoglucose) which are added to products derived from grapes. The second method is for the
13 12
determination of the C/ C isotope ratio of glycerol in wines by GC/C or HPLC coupled to IRMS,
and which is used to detect the addition of glycerol from maize (C 4 plant) or from synthesis (fossil
sources) to wines or to spirit drinks.
Moreover, the absolute content of glycerol in wine can be investigated using two different
approaches: one method based on the colorimetric measure at 480 nm of the reaction products of
formaldehyde, obtained by the oxidation of glycerol, with phloroglucinol; or using an enzymatic
approach.
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3.1.3. Acids
Total and volatile acidity (and their difference, fixed acidity) methods both belong to category I,
and are based on titrimetric measurements, directly or after distillation of the wine.
For the determination of the single organic acids, several chromatographic approaches are
proposed: by thin-layer chromatography (sorbic acid); by HPLC (tartaric, malic, shikimic, lactic,
acetic, citric, succinic and fumaric acids; sorbic, benzoic and salicylic acids; shikimic acid; L-ascorbic
acid and D-iso-ascorbic acid); by GC (sorbic acid); by Capillary Electrophoresis (sorbic acid; tartaric,
malic and lactic acids) and by ionic chromatography (malic, citric and tartaric acids).
Enzymatic methods are also provided for the selective measuring of enantiomeric forms (D-lactic
and L-lactic acids, D-malic and L-malic acids, L-ascorbic acid) and citric acid.
14
A method for the identification of L- tartaric acid origin (plant or fossil) using C activity is also
proposed.
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proteins from fining agent can be detected in wine applying the direct and indirect ELISA methods.
Immunological methods of immunoprinting are also available for testing the presence of unstable
proteins in white wines.
These methods allow in the majority of cases to detect adulteration linked to the (i) category: non-
compliance with the established legislative standards, based on the comparison of data with
reference limits (cf. Table 1). Some of these methods are also used commonly to verify other types
of adulteration belonging to the other 2 categories (cf. section 3.2.1).
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sugar and water addition as well as mislabelling, on the basis of a comparison of data with an
official reference databank, set up according to the current European Regulation 555/2008.
According to this, every year a number of samples that are representative of the wine production
of each Member State are officially collected by the relevant national competent authority. The
sampling design has to take into account both the geographical distribution and the harvest period
due to the geographical and climatic variability of the isotopic values. For each sample, about 10 kg
of fresh grapes are harvested, vinified under controlled conditions and the resulting reference
wines analysed in accredited laboratories. The data plus a number of metadata related both to the
harvest and the vinification are registered in one official databank that is managed by the
European Directorate General, Joint Research Centre (DG JRC). The isotope databank comprises
reference data for each year. This allows definition of limits for authentic wines and musts in terms
of isotopic data, for each country, each sub-area (e.g. region) and each protected designation of
origin (PDO-IGP) as well as general limits [3].
Recently the effect of some oenological practices, such as dealcoholisation, grape withering and
the stopping of fermentation on these isotopic ratios has been investigated. The variations in wine
water G O and G C ethanol encountered have to be considered when interpreting the isotopic
18 13
v/v dealcoholisation. The drop in G O water is mainly caused by isotopic diffusion, which involves
18
H2 O migration from the wine to the extracting solution. The increase of G C is due to the fact
18 13
13 12
that C has vapour pressure lower than the C, and this causes a prevalent transfer of ethanol
12
with C.
Withering involves postharvest drying of grapes and can be performed in a dedicated ventilated or
unventilated fruit drying room, (called a “fruttaio”) during autumn-winter, or withering grapes on
the plant (‘plein-air’). In both cases, during this period the grapes lose water, and this causes a
variation of wine water G O [6]. In ‘fruttaio’, G O decreases significantly from fresh to dry grapes,
18 18
temperature and is due to a chemical exchange between grape water and atmospheric water
vapour according to equilibrium isotope fractionation. For Passito produced ‘en plein air’, G O
18
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increased with withering in southern Italy, since, due to the relatively higher temperature in these
areas, kinetic evapo-transpiration takes place.
Stopping of alcoholic fermentation up to 4.5–10 % of ethanol, is used for the production of some
traditional Italian sweet wines (such as Moscato d’Asti) in order to leave a pleasant amount of
residual sugar in the wine. It was found [7] that the G C and, in particular, the (D/H)II values of
13
ethanol of wine were positively related to the stage of fermentation, while (D/H) I and G O of
18
ethanol were not. The partially fermented musts were characterized by lower isotopic values,
which, in the case of (D/H) II, are outside the range of variability of natural wines.
Moreover, more innovative isotopic methods, based on the analysis of the stable isotope ratio of
other elements or of other components have been developed.
The G O of wine ethanol was measured directly on dry-ethanol using TC/EA-IRMS in pyrolysis
18
conditions, after having trapped residual water using a molecular sieve [8]. It was found to be
significantly correlated with the G O of wine water and can therefore be considered as an internal
18
reference to improve the detection of wine watering, as is the case for fruit juice [9].As the
addition of water to wine changes only the G O of water and not that of ethanol, the watering of
18
wine changes this relationship, which can then fall outside the threshold value, even if the water
G O is not outside the limit defined by the wine databank. Thus, measuring the G O of ethanol
18 18
addition to wine [10]. The compound specific analysis of the main higher alcohols in wine showed
indeed a strong relationship between their G C and that of ethanol, that might help to identify
13
exogenous ethanol sources. However, additional experiments verifying the possible refinement
were not performed.
Recently also a method to measure G N in must, wine and in the extracted proline was developed
15
[11]. For proline, the most abundant amino acid in grape and wine and not used by yeast as
nitrogen source, G N was measured after N-acetylisopropyl derivatisation using gas
15
15
chromatography − combustion − isotope ratio mass spectrometry (GC-C-IRMS). δ N values of
leaves, grapes, wine and particularly must and wine proline were found to be related to those of
15
δ N in the growing soil. The addition of inorganic or organic adjuvants was able to influence the
15 15
δ N of bulk wine, but not the δ N of wine proline, which is therefore the best marker for tracing
the geographical origin of wine.
A GC-c-IRMS method for analysing vanillin in distillates after dichloromethane extraction was
developed [12]. Storage in oak barrels release different degradation products such as vanillin,
which plays an important role in the flavour and aroma of the distillates. The addition of vanillin, as
well as other aroma compounds, of different origins is prohibited by European law. G C values are
13
able to distinguish natural vanillin extracts (-21.0 ‰ to -19.3 ‰), vanillin from lignin and also from
tannin (-29.5 ‰ to -26.7 ‰) and synthetic vanillin (-32.6 ‰ to -29.3 ‰).
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In 1994, Latorre et al. [15] differentiated the PDO Rias Baixas Spanish wine from Galicia from its
imitations. Pattern recognition analysis, performed on ICP-MS data, revealed that Li and Rb were
the most discriminating variables. Similar studies were carried out by Baxter et al. [16] on wines
from different regions of Spain and England. Taylor et al. [17] studied soils and wines from the
Canada’s two major wine-producing regions. They found that, among trace elements, strontium
was able to differentiate both soils and wines from the two regions. The fingerprint of REE was
kept unaltered in the passage soil–grapes–must, while fractionation occurred in wine [18] after the
clarification with bentonites. In addition, analysis of Moscato musts from 102 samples showed that
is possible to classify their geographic origin, building a basis for identification of possible addition
of foreign musts.
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coumaroylated anthocyanins (Rac/cou) and the sum of acylated anthocyanins (Sac) in the
assessment of the variety ([18]. For example, it has been noted that Pinot Noir grapes contain no
acylated anthocyanins and this feature of burgundy wines is successfully applied for their variety
control. For example, the German speciality “Weißherbst” which is a rosé wine produced from 100
% Pinot Noir grapes, should show no significant proportion of acetylated anthocyanins. It should
be noted, however, that measurement uncertainty of the wine in question, the typical authorized
blending (e.g. 15 % in the EU) and, in the case of sweetened wines, the addition of further
products (such as must), should all be considered appropriately before drawing conclusions on the
variety in question.In addition, the ageing of wine gives rise to the degradation and
polymerisation of the anthocyanins which leads to the absence of the analytes.
Brunello di Montalcino, one of the flagship products of Italian oenology, must be produced from
Sangiovese grapes grown in Montalcino, a specific area in Tuscany. Sangiovese grapes are poor in
acetylated anthocyanins, one property that in principle promotes the makes it possible to
authenticate these premium wines by analysis of the anthocyanins, but as these wines typically
aged up to 10 years, sophisticated mass spectrometric approaches give more reliable results as
shown by Arapitsas et al. 2012 [24].
One example related to grape variety fraud -the so-called “Pinotgate” incident- was uncovered
2010 in California where Pinot Noir wine imported from France sold in the United States was
identified to contain large amounts of Merlot and Syrah [25]. According to information available,
the wine was first suspected because of its sensory properties.
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Valley, Loire Valley ; Italy : Piemonte, Toscana, Sicilia, Puglia ; Spain : La Rioja, Ribera des Duero)
and also to control the major varieties (Red : Pinot Noir, Tempranillo,Garnacha Tinta, Syrah,
Merlot Noir, Cabernet Sauvignon, Sangiovese, Nebbiolo, Montepulciano, Primitivo, Dornfelder,
Portugieser Blau, Zweigeltrebe Blau ; White : Chardonnay Blanc, Sauvignon Blanc, Riesling, Pinot
Blanc/Gris, Silvaner, Verdejo, Mueller Thurgau, Veltliner, Moscatel, Welschriesling. This analytical
technique gives information on unforeseen deviations and is a multivariate untargeted analysis
useful for a screening control of the market. Interlaboratory comparison is monitored with a
dedicated Proficiency Testing Scheme, Pro-PTS, organised by Eurofins Analytics France which
controls not only the quantitative parameters but also the interpretation of the sample.
The Wine screener provides additional answers in the control of the authenticity of wines, and in
combination with other methods, such as stable isotope analysis as described above, it can offer a
performant solution using data fusion. The benefit of fusing NMR data with alternative techniques
has been provided by [27]. The authors evaluated the combination of discrete isotopic data with
the untargeted NMR spectrum to have better control of wines. Both techniques are known to
1
provide useful information to the characterization of wine: H NMR spectroscopy can be used to
build robust classification models for grape variety, year of vintage and geographical origin, while
stable isotope ratio analysis is a good source of chemical information for the authenticity
assessment of food products. By combining these two methodologies, an improvement of
classification rates of wine was achieved: 100 % for the determination of geographical origin (60–
1
70 % correct prediction was obtained with stable isotope data alone and 82–89 % with H NMR
1
spectroscopy) and 99 % for the vintage of wine (from 88 to 97 % with H NMR).
3.3.2. MS metabolomics
Metabolomics represents one of the most recent analytical approaches used in wine
authentication. Since wine is a very complex matrix and all its metabolites are physically and
chemically diverse, it is not possible to identify all of them in a single platform measurement.
Therefore, it is necessary to use different, complementary analytical techniques. Besides NMR
mentioned above, mass spectrometry (MS) is frequently used, either ambient or coupled to
separation techniques such as gas chromatography (GC) and liquid chromatography (LC) [28–30].
Among mass spectrometry techniques used in wine metabolomics, LC-MS is the most common. It
is suitable for determination of non-volatile, thermolabile compounds (e.g. phenolic compounds).
One of its main advantages is, that before analysis of the wine, no pre-treatment or extraction of
the sample is necessary. However sometimes, simple steps like filtration, dilution or pre-
concentration of the sample might be desirable [28].
LC provides metabolite separation based on the different distribution between the mobile (liquid)
and stationary phases. For this purpose, the LC system can be equipped with different types of
columns, although usually reverse phase columns are used. The ionisation sources frequently used
in conjunction with LC-MS are electrospray ionisation (ESI), atmospheric pressure chemical
ionisation (APCI), or atmospheric pressure photoionisation (APPI), however, ESI is the most
common. Considering that most metabolites ionise in one ionisation mode (positive or negative),
not in both, it is necessary to analyse the samples in both of them, in order to cover a wider
metabolome. After ionisation, ions pass through the mass analyser. Since the metabolomics
approach is focused on characterisation of the entire composition of small metabolites
(metabolome), mass analysers capable of whole metabolome analysis within single analytical run
(with good dynamic range, fast scan speed, sensitivity and high mass resolution and mass
accuracy) such as Time of flight (TOF) and orbital ion traps are usually used. The resolution
achieved is closely associated with the ability of the instrument to measure the accurate mass,
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which is crucial for the identification of unknown compounds. The mass error is usually below 5
ppm in the case of TOFs and below 2 ppm in the case of orbital ion traps. Extensive technological
improvements have been achieved with the new generation of hybrid instruments, Q-TOFs and Q-
Orbitraps, allowing performance of the specific ion fragmentation, bringing an additional
dimension by enabling the identification of unknown compounds (MS/MS spectra, i.e. spectra of
fragment ions by HRMS) [31]. It should be mentioned that by metabolomics analysis large amount
of data is obtained. In order to extract valuable information from the data, pre-treatment steps
(data mining, retention time and m/z alignment etc.) and also effective statistical software tools
are required for effective data handling (not only for LC-MS, but also for GC-MS and ambient MS)
[28,30,32,33] .
LC-MS in combination with metabolomics could be used for different wine authentication
purposes, such as classification/discrimination of wine samples according to their variety [34–36],
origin/producer [34,37], vintage [34], and quality [34].
Since in most of the cases, this goal is achieved using statistical evaluation of data, marker
identification does not represent a necessary step in non-targeted metabolomics studies aimed at
sample classification/discrimination [36]. However sometimes, it might be useful to know the
identity of a compound related to the sample differentiation. In the following paragraph, examples
of several markers are listed.
In the study of Rubert et al. [35], astilbin was identified as a marker of Pinot Noir and different
flavonol glucosides as markers of Merlot and Tempranillo (among varieties Pinot Noir, Tempranillo,
Merlot, Shiraz, Riesling, Sauvignon Blanc, Silvaner and Chardonnay Blanc). In the study carried out
by Roullier-Gall et al. [37], polyphenols, fatty acids, carbohydrates, and amino acids were identified
as markers of different wine samples according to the geographical origin/producer (among
Chablis, Meursault 1, Meursault 2, and Corton Charlemagne wines).
Another technique frequently used in metabolomics is GC-MS. Unlike LC-MS, this technique is
limited to detection of thermostable, sufficiently volatile compounds. Therefore, the main
drawback of GC-MS-based metabolomics is the need for sample handling prior to the analysis. The
most important of these are procedures enhancing the volatility and the thermal stability of the
metabolites (e.g. derivatisation) and procedures (extraction processes) isolating metabolites and
enhancing their concentration (e.g. liquid-liquid extraction - LLE, solid-phase extraction - SPE, solid-
phase microextraction - SPME) [28,38].
As with LC-MS, a chromatographic separation based on different distribution between two phases
is used. However this time, the mobile phase is gaseous. The ionisation source dominantly used in
conjunction with GC-MS is electron ionisation (EI) with an ionisation energy of 70 eV. In
combination with standardised protocols of data acquisition, the use of EI results in reproducible,
rich fragmentation mass spectra. These can then be recorded in large user libraries (e.g. NIST 14
Mass Spectral Library) or compared (matched) with mass spectra (and other additional
information) already present in the libraries, in order to confirm the compound identification. This
is undoubtedly one of the main advantages of the GC-EI-MS-based techniques [30,38].
Since the requirements for the mass analysers capabilities in GC-MS are similar to LC-MS, mass
analysers such as TOF or hybrid Q-TOF are suitable for wine metabolomics. However, the most
frequently used mass analyser (due to its relatively low price, high sensitivity and good dynamic
range) in GC-MS-based metabolomics is the quadrupole [30,38].
GC-MS in combination with metabolomics is often used to authenticate wine variety [39,40],
producer[39] and vintage [39]. In the following paragraph, examples of several markers are listed.
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Wine and must
In the study of Kruzlicova et al. [39], various terpenes and alcohols (e.g. α-terpineol, linalool, 1-
hexanol and (E)-3-hexen-1-ol) were identified to be the most important markers for wine
classification according to the wine variety (among varieties Welsch Riesling, Gruener Veltliner and
Mueller Thurgau) Most important markers for wine classification according to the producer/origin
(producers located in West and South West Slovakia) were esters, alcohols and carboxylic acids
(e.g. diethyl succinate, 2-ethylphenylacetate, (E)-3-hexen-1-ol, 1-hexanol and hexanoic acid) and
according to the vintage (years 1996, 1997 and 1998) were alcohols, esters and carboxylic acids
(e.g. (E)-3-hexen-1-ol, hexanoic acid, 1-hexanol and ethyl-3-hydroxy butanoate).
Ambient mass spectrometry represents a group of MS-based techniques, which are not coupled
with chromatographic separation. In other words, analytes are directly injected / transferred into
the mass spectrometer, without prior separation. In comparison with the GC- and LC-MS based
approaches, ambient MS is far less informative. It is not capable of isomer separation,
quantification of individual metabolites is less accurate and it does not provide additional data
such as retention time/factor/index etc. Also, the absence of chromatographic separation prior to
MS analyses may increase matrix effects and cause ionisation suppression. However, ambient MS
is much faster than GC- and LC-MS-based approaches and for large sample sets analysis it is often
the only possible/reasonable option [30].
The ionisation sources frequently used in ambient MS are desorption ESI (DESI), extractive ESI
(EESI), direct analysis in real time (DART) or matrix-assisted laser desorption / ionization (MALDI).
For the purposes of ambient MS analysis, advanced high-resolution tandem MS instruments such
as Fourier-transform ion cyclotron resonance MS (FT-ICR-MS), TOF-MS or Orbitrap MS are usually
used [30,41].
Ambient MS was used for example for the authentication of wine variety or to detect adulteration
of wine by illegal wine mixing or by colouring [42]. In the following paragraph, examples of several
markers are listed.
In the study of Hartmanova et al. [42], different anthocyanins (e.g. malvidin-3,5-diglucoside,
malvidin-3-acetylglucoside and peonidin-3-acetylglucoside) were used for authentication of wine
according to the variety.
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Wine and must
concentrated musts that may have been used. The potential of a genetic traceability approach in a
such heterogeneous matrix, indeed, is almost unlimited, since the molecular analysis would enable
the identification, not only of the grape varieties from which it has been produced, but also the
yeasts and/or bacteria strains used for fermentation and to establish if genetically modified
organisms (GMO) have been used [43–45]. Thus, the development of genetic analysis would make
possible the traceability of a wine at all levels and in all stages of the winemaking process.
However, winemaking implies several processing steps which limit the quantity and quality of DNA
available in wine. On one hand the DNase from the microorganisms degrade DNA during
fermentation generating denatured and fragmented residues. On the other hand, decantation,
clarification, filtration and other fining treatments may contribute to the decrease of the final DNA
concentration available. In addition, the co-existence of polysaccharides and proteins interfere
with DNA isolation, and other substances — such as polyphenols — act as inhibitors of the
polymerase chain reaction (PCR) methodology used for genetic fingerprinting analysis.
Genotyping for grapevine varietal identification can be roughly described in four main steps: DNA
isolation from plant material, DNA markers amplification by PCR, analysis of the PCR products by
capillary gel electrophoresis and results interpretation [46]. This methodology was first applied for
grape juice varietal identification by [47], and then for varietal wine authentication by Siret el al.
[48,49], who analysed experimental wines from the start to the end of fermentation. These
authors performed successful varietal identification by SSR genotyping in musts, but reported
difficulties for the authentication of the cultivars in finished wines due to the scarce DNA isolated.
Successive studies have been performed in order to improve DNA isolation from wine, but in all
cases, although varietal identification of musts was possible, reproducibility problems for the
systematic authentication of finished experimental and/or commercial wines were always
reported again due to the extraction of low DNA quantity and quality from a wine matrix [50–60].
Analysis of other marker types, such as chloroplast SSR markers or Single Nucleotide
Polymorphism (SNP) markers, has been proposed [51,59]. Multivarietal must mixtures and
blended experimental wines have been analysed as well to detect the discriminatory power of the
DNA marker technology for determining the varieties used in the mixtures [49–51,55,61]. Although
it could be determined if more than one variety was used, the identification of unknown additional
cultivars used became impossible, especially when the blends consisted of more than two
varieties. Preliminary results obtained using a TaqMan SNP genotyping approach highlighted the
potential of Real Time PCR for wine varietal authentication and quantification [59]. The TaqMan
assay is much more sensitive than SSR genotyping, not only because it requires a smaller amount
of DNA for the analysis, but also because it is based on the analysis of cultivar-specific SNPs.
Moreover, this method is more sensitive and precise for relative quantification of each variety in a
mixture because is based on specific allele probes.
Despite all the studies performed up to date, the main limiting factor for the development of a
standard method for wine varietal authentication remains the quality of grape nucleic acids
extracted from wine. The PCR amplification of shorter fragments of DNA that allows access to
minute traces of nucleic material seems more promising at least for authentication purposes.
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Wine and must
Aphrometer CO2
HPLC Biogenic amines
Colorimetry, titrimetry, Anions
gravimetry
AAS, FP, GFAA, ICP-MS Cations
Direct & indirect ELISA Proteins Residues of allergenic proteins from fining agent
TLC Saccharine, dulcin, cyclamate Use of artificial sweeteners
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Wine and must
5. Conclusion
The authenticity of oenological products appears to be well guaranteed by a complex and robust
analytical control system. However, the high value of these commodities generates continuous
attack to their genuineness. Botanical and geographical, as well as varietal origins, probably
represent the main issues for the sector. Further innovative methods using isotope, mineral and
metabolomic profiles integrated with DNA molecular analysis can represent the future of this
challenge. Availability of specific and extensive compositional databases and of validated and
recognised analytical protocols are required. However only a higher awareness of these new
approaches from the competent governmental control bodies and courts will make it possible to
reach a superior control of frauds and mislabelling.
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Spirit drinks
Ian Goodall*, Shona Harrison, Rebecca Eccles, Peter Cockburn
The Scotch Whisky Research Institute, Edinburgh, United Kingdom
*E-mail corresponding author: [email protected]
Monika Tomaniova
Department of Food Analysis and Nutrition
University of Chemistry and Technology, Prague, Czech Republic
E-mail corresponding author: [email protected]
https://doi.org/10.32741/fihb.13.spirits
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counterfeiter. In the UK, it has been estimated that the Treasury loses GBP 1.3 billion annually
through alcohol fraud [9].
An effective legal framework for tackling the production and marketing of fraudulent spirit drinks
requires two elements. The first element is a clear and enforceable set of definitions of the spirit
drinks categories, including their production processes and specific analytical and organoleptic
characters. The second element is a range of appropriate analytical methods that will help confirm
that a suspect spirit drink product meets its labelling claims, according to the legally established
definitions. These two elements, as well as an overview to the common spirit drink frauds such a
system is designed to tackle, are explored in the rest of the chapter.
1. Product Identity
1.1. Definition of the product and manufacturing process
Spirit drinks can be simply defined as alcohol beverages created from the distillation of fermented
agricultural raw materials. Exact terminology and definitions will vary on jurisdiction, but these key
elements, along with the requirement that the distillate be intended for human consumption
(potable), will be common to most markets.
1
Regulation 110/2008 is, at the time of writing, currently undergoing revision. However, it is assumed that most, if not all
of the points noted in this chapter will be retained in any forthcoming legislation.
2
With the exception of egg liqueur or advocaat or avocat or advokat where the minimum strength is 14 %.
3
The EU regulation does not specifically state that EAAO has to be a distillate of agricultural ethanol, but (given the
minimum strength) this is effectively the case.
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Spirit drinks
designed to be low in compounds other than ethanol and water, and consequently neutral in its
flavour profile. The last category of distillate is a “distillate of agricultural origin”. This covers any
agricultural distillate that does not meet the criteria for a spirit drink or ethyl alcohol of agricultural
origin. Spirit drinks can be created directly from a distillation of naturally fermented products, or
can be produced from appropriate treatments of EAAO, distillates of agricultural origin or other
spirit drinks [4, Article 2].
Within the Spirit Drinks Regulation [4, Annex II] there are 46 defined categories of spirit drinks. The
first 14 of these have certain restrictions placed on their production [4, Article 5]. These include
the sole use of the raw material contained within the category definition for the production of
alcohol, a prohibition on the use of EAAO and flavourings, and restrictions in relation to colouring
and sweetening. Examples of such spirits include rum, whisky and brandy. Unless otherwise stated
in their category definitions, the remaining 32 categories may use any agricultural raw material as
the origin for the alcohol, EAAO, as well as any permitted flavourings, colourings and sweeteners.
All alcoholic beverages which meet the definition of one of the 46 spirit drink categories must
“bear in their description, presentation and labelling the sales denomination assigned therein”; for
those spirit drinks that do not fall into one of the 46 categories, they “shall bear in their
description, presentation and labelling the sales denomination ‘spirit drink’.” [4, Article 9(1-2)].
It is notable that the European legislation for the definition of spirit categories are typically process
definitions. Whilst all spirit drinks categories specify minimum alcohol strength, few additional
analytical parameters are set in the legislation against which compliance can be judged - examples
include:
● limits for anethole concentration in pastis, pastis de Marseille, sambuca and Mistrà;
● limits for sugars in various liqueurs and the spirit drink Berenburg/Beerenburg; and
● a minimum egg yolk content in Egg liqueur/advocaat/avocat/advokat or liqueur with egg.
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available on the Her Majesty’s Revenue and Customs website [15], as well as a register of all
brands produced at verified sites. Such a scheme offers both protection against fraud, and a
market opportunity for guaranteeing authenticity, and has been looked at by other EU spirit
producers, e.g. Swedish vodka and Dutch gin [16].
1.2.3.1. Canada
The Canadian Food and Drug Regulations [18] define 8 different spirit categories (whisky, rum, gin,
brandy, liqueurs and spirituous cordials, vodka, tequila and mezcal), although provision is made for
the protection of a number of geographical indications, such as Scotch Whisky, Bourbon Whiskey,
Cognac, Armagnac and Grappa. The Canadian definition of whisk(e)y is an example of a
specification that approximates closely to the European Union definition.
4
This is ethyl alcohol of agricultural origin (EAAO), a highly rectified distillate meeting specific technical definitions and
requirements (Regulation 110/2008, Annex I(1)), including possessing a minimum alcoholic strength of 96.0% v/v.
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1.2.3.3. Australia
Other jurisdictions have much looser definitions than those seen above. An example is Australia,
where there are certain provisions under the Australia New Zealand Food Standards Code which
govern spirits where these are manufactured or imported into Australia. Principally, Standard 2.7.5
[20] defines brandy, liqueurs and spirits in general. All definitions are light on details of production,
in particular the definition for spirits (2.7.5-2), given that only 2 categories are specifically referred
to in the same legislation. This states that a spirit means an alcoholic beverage consisting of:
(a) a potable alcoholic distillate, including whisky, brandy, rum, gin, vodka and tequila,
produced by distillation of fermented liquor derived from food sources, so as to have the
taste, aroma and other characteristics generally attributable to that particular spirit; or
(b) such a distillate with any of the following added during production:
(i) water;
(ii) sugars;
(iii) honey;
(iv) spices.
In addition, all spirits have a minimum alcohol strength of 37 % alcohol by volume (2.7.5-3). As can
be seen, such a definition, for whisk(e)y say, provides little in the way of specifics about the
production methods of the spirit, so long as a subjective organoleptic assessment indicates the
standard of identity has been met, and there is a lower minimum strength than in Europe. In
addition, a number of additives such as sugar, honey and spices are permitted under the
definition, contrary to European legislation for whisk(e)y.
Some additional process information can be found in the Excise Act 1901 [21, Section 77FI] and the
Customs Act 1901 [22, Section 105A]. However, these are similarly light on detail compared to
their European definitions. Both pieces of legislation provide minimum maturation requirements
for brandy, rum and whisky. However, these are limited to the requirements that these spirit types
are stored for a minimum of 2 years in wood. They also define the materials for the production of
brandy (grape wine), rum (a fermented liquor derived from the products of sugar cane) and whisky
(a fermented liquor of a mash of cereal grain).
However, the Australia New Zealand Food Standards Code does contain explicit protection for
spirit drink geographical indications, including a specific requirement that products produced in
accordance with a geographical indication, but shipped and bottled elsewhere, must meet the
minimum alcohol strengths of the laws relevant to the geographical indication.
1.2.3.4. India
India has (based on the regulations to be enforced from April 2019 onwards) a number of spirit
category definitions [23], including brandy, gin, rum, vodka, liqueurs/cordials/aperitifs and
whisk(e)y. Like other jurisdictions, a number of culturally significant definitions are included:
country liquors, fenny and pot distilled spirits. A key example of the conflict between different
cultural perceptions of a spirit category can be seen in the whisky definition [23, Section 2.8]).
Whilst placing an emphasis on cereal being the raw material for whisky production, it is clear that
whisky can also be made from neutral spirit, which can be made from fruits, vegetables, molasses
or any other source of carbohydrates of agricultural origin, as well as grains and has a minimum
alcohol strength of 96 % alcohol by volume [23, Section 1.2.9]. This is a clear contradiction to most
other definitions of whisk(e)y, which require a cereal substrate, a maximum distillation strength
(to retain an appropriate level of organoleptic character from the raw material) or both.
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India is also a country that has imposed a number of analytical limits on the spirit beverages it
defines [23 Table 1]. These are individually tailored to each category of spirit. Some of these limits
are obviously intended to act as a general restriction on compounds of public health concern, such
as the levels for heavy metals, although it is unclear why spirits produced according to good
distilling practice should ever be at risk of exceeding such limits, and thus why such category-based
limits are required.
The inclusion of other varying limits based on spirit category (such as total esters and higher
alcohols) are more typical of quality-based specifications, but unlike the European regulations that
relate limits to some characteristic of a particular product category (e.g. the high sugar content of
liqueurs or the specific flavouring requirements of aniseed spirits such as Pastis) these are applied
to each category in turn. Thus, the limits in effect represent an attempt to provide an analytical
definition to a product category. Such limits, whilst seemingly providing some guidance as to
appropriate analytical range for authentic products, should be treated with caution. Whilst usually
covering a large proportion of a category, they do not always include all the various styles and
variations contained within a spirit drink category definition. They can therefore act as misleading
guides to authentic database ranges and may also restrict trade in genuine products.
2. Authenticity issues
2.1. Identification of current authenticity issues
5
Counterfeit alcohol is just one type of illegal alcohol. For information on the correct terminology to use when discussing
legal and illegal alcohol refer to the resources produced by The International Alliance for Responsible Drinking [24], in
particular the section on taxonomy of the alcohol market [25].
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The two cans shown in Figure 1 claimed to be Scotch Whisky and are examples of generic
counterfeits, trading on the goodwill associated with that geographical indication. These products
were canned in Austria and sold in the Middle East. They were manufactured from industrial
alcohol and flavouring. In total, it has been estimated that 15 million of these cans were sold over
a period of a few years, which provides an indication of the scale of some of these spirit drink
counterfeiting operations.
For both generic and brand counterfeiting, the liquid inside the bottle is often the extension
(dilution) or replacement of the authentic product with: (i) water; (ii) cheaper locally produced
spirit, (iii) neutral alcohol (a highly rectified spirit lacking in flavour, used as a base to produce
many genuine spirits) or (iv) an alternative alcohol. These products may also contain added
sweetening or flavourings to mask the inferior flavour of the counterfeit spirit or to mimic aromas
of the genuine spirit.
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neutral spirit or ethyl alcohol of agricultural origin; synthetic alcohol; some alternative alcohol such
as methanol; or industrial alcohol.
The alcohol used for extension or substitution may be from non-permitted agricultural substrates,
i.e. the botanical origin of the alcohol is incorrect. For example, rum can only be created from
sugar cane by-products or sugarcane juice, according to most spirit drinks legislation. The
identification of alcohol from different agricultural origins will signify a fraudulent product [33,
pages 18-19]. Whilst constituents of the distillates of the incorrect agricultural origin may make
their presence detectable in a fraudulent product [34] much work has been undertaken on the use
of the stable isotopic ratios of ethanol and water to detect this fraud [35-38]. Isotope ratios are of
particular importance when the product is fraudulently substituted or diluted with highly rectified
neutral spirit and for reasons of natural variability the levels of components in the authentic
product are insufficient to detect this practice [39]).
The use of alternative alcohols added to potable ethanol from agricultural substrates is particularly
attractive to spirit drink counterfeiters, since there is no excise duty to be paid. Synthetic alcohol
has been used to produce counterfeit spirits. For example, tequila made from synthetic alcohol
(probably derived from petroleum) has been identified [33, page 20], as has the falsification of
vodka using synthetic ethanol [40]. Denatured alcohol is used in a number of industrial
applications. This product, exempt from excise duty after the addition of specific chemicals
(denaturants) designed to render the alcohol non-potable, has also been used as the base alcohol
for counterfeit products [33, page 10]). Whilst the denaturants are often added to specifically
mark a product as denatured alcohol, counterfeiters will often attempt to remove these
compounds, thus recovering the alcohol in an unmarked form and making its presence in
counterfeit spirits hard to identify [41].
2.1.4. Additives
Counterfeit products may also contain added sweetening or flavourings to mask the inferior
flavour of the counterfeit spirit or to mimic aromas of the genuine spirit. Depending on the
legislation relevant to the spirit category, these additives may not be permitted in the genuine
products. An example of this is where sugars are illegally added to whisky [31].
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spirits. To contain the problem the Czech authorities temporarily banned the consumption of
spirits above 20 % alcohol by volume. At least 36 deaths were related to this incident [29].
Another health risk that can arise is from metals used in illicit stills and other production materials
that are unfit to come into contact with food products. Genuine producers will take steps to
prevent any unwanted contamination from metals and plastics or other food contact materials
that could leach into the final products. Counterfeiters are either unaware of these risks or are not
concerned enough for the health of their customers. Where industrial alcohol has been
substituted into the food chain similar concerns occur, with the added health impact of the
chemicals used to denature the alcohol. Elevated levels of metals in illicit alcohol include metals
such as lead, arsenic and mercury. These have been linked to makeshift illicit distilling apparatus
using a variety of reused metal components that may leach harmful toxins into the distillate,
including the aforementioned metals [43,46-48].
Chloroform has been detected at high levels in illegally produced alcoholic products [49], creating
an increased risk to the public. This could be due to a process used by some counterfeiters to
remove the common denaturant denatonium benzoate, which has a unpalatable bitter taste, from
denatured alcohol, via the addition of hypochlorite [50,51]; chloroform is known to be a product of
hypochlorite and ethanol [52]. Other denaturants may also have health impacts, to a greater or
lesser degree. The impact of methanol has already been noted; other compounds, whilst not
exhibiting acute toxicity would still be regarded as unwanted contaminants of toxicological
significance [43].
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Category authentication, brand authentication and screening technologies all have their specific
uses and applications. The aim of this section is to assist in spirit drink authentication by providing
supplemental information, references and guidance for analytical methods commonly employed.
The officially recognised methods referred to herein relate to three main sources of reference
methods: Commission Regulation (EC) No 2870/2000 (Union reference methods of analysis) [13],
the OIV Compendium of International Methods of Spirit Beverages of Viticultural Origin [53] and
the AOAC International Official Methods of Analysis [54], which are typically the methods of
analysis referred to by the U.S. Alcohol and Tobacco Tax and Trade Bureau (TTB). The methods in
the OIV are often aligned with those in the Union reference methods. Some national markets will
have their own official methods, which will often be variations of the techniques referred to in the
above standards. Where significantly differently methods are employed, it would be advisable to
demonstrate equivalency to the common methods referred to here.
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Spectroscopic methods involving near-infrared (NIR) are commonly used within the industry to
provide alcohol strengths, since real strengths can be obtained for many distilled spirit matrices
without the need for a distillation step. However, only the OIV currently has an official method
detailing the application of NIR spectrometers (OIV-MA-BS-08). This method of determining the
real alcoholic strength is based on the physical principle of the spectral analysis of materials with
absorption bands in the near infrared range. The key point about the use of such apparatus is that,
as noted in the method, the NIR equipment needs to be appropriately calibrated and verified
against an appropriate reference set of samples, measured using an approved reference method
for real strength as referred to above.
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3.1.4. Sugars
Sugars may be found in a variety of different spirit types. The individual composition and levels
observed will be related to the spirit category and how that spirit is produced. In the EU
(Regulation No 110/2008), some spirit categories such as liqueurs require the addition of a
minimum concentration of sugars for sweetening; others allow the addition of sweetening
sufficient to round off the final taste of the product. Still other categories (e.g. whisky) prevent any
sweetening by the addition of permitted carbohydrate sources. Trace levels of certain sugars can
be naturally present in some spirits as a result of the post-distillation manufacturing procedures of
maturation and addition of caramel colouring.
It is common for counterfeiters to add sugars to poorly produced, fraudulent products to try and
improve the taste or mimic the natural sweetness of a genuine product. To confirm if sugars are
present naturally as opposed to adulteration, the sugar profile of the suspect product should be
compared with the known ranges and ratios encountered within the spirit category or brand. For
example, analysis of genuine Scotch Whisky products has shown that, where sucrose is present,
the level is considerably less than the concentration of glucose and fructose [31].
Liquid chromatography (LC) with Refractive Index (RI) detection is a common technique for sugars
analysis. This technique is principally used for quality control of distilled spirits containing high
(g/L) levels of sugar content such as liqueurs and pastis. The Union reference method (Annex VIII)
and the OIV method (OIV-MA-BS-11) for measurement of total sugars (glucose, fructose, sucrose,
maltose and lactose) are aligned. LC-RI is not suitable for identifying sugars adulteration in spirit
categories that contain low levels of sugars such as vodka, gin and whisky. A much more effective
method is Ion Chromatography (IC), typically used in conjunction with a pulsed electrochemical
detector (PAD) [31]. This technique can allow trace levels of individual sugars present naturally in
certain spirits to be distinguished from higher levels that can only be achieved by adulteration.
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3.1.6. Metals
There are several recognised methods for the analysis of metals in distilled spirits. This reflects
both quality control measures and potential concern from external regulators about the levels of
such metals in the food chain. (Based on the low risk presented by the sector’s products, however,
the EU assigns no analytical limits to distilled spirits.) The OIV has four recognised methods for
metals analysis by atomic absorption spectroscopy (AAS): calcium, copper, iron and lead (OIV-MA-
BS-29 to OIV-MA-BS-32). The AOAC also has methods for distilled spirits using similar methods
such as atomic absorption techniques for copper (967.08) and iron (970.12).
Modern laboratories however can employ a variety of techniques to measure metal ions. Typically,
they will employ methods that allows the detection of a number of metals within the same
analysis, for example inductively coupled plasma optical emission spectrometry (ICP-OES) [64],
inductively coupled plasma mass spectrometry (ICP-MS) and IC. Brand owners often have to
measure the concentrations of a number of metals to complete certificates of analysis for markets
outside the EU. Hence, a database of genuine products can be built up by brand owners, where
expected ranges for a number of metals can be set and compared against unknown or suspect
samples. This technique is naturally more challenging for generic authentication, particularly
where the product is not limited to being bottled in a particular location.
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been added, knowledge of the individual spirit category and its production practices is required.
Knowledge of common flavourings, flavouring carriers and additives used in the food and drinks
industry will also assist in the detection of counterfeit products.
If a suspected flavouring additive is detected, it is often beneficial to identify if the chemical
compound is synthetic (man-made). Synthetic compounds are not found in nature hence they will
not be naturally occurring as part of standard production practises [68]. Flavouring additives often
need only to be present at trace levels to be able to influence the aroma and flavour of a product,
therefore sensitive techniques are required. GC-MS and LC-MS are commonly used for the
detection of volatile and non-volatile flavouring additive compounds. The analysis of anethole has
already been noted as a necessary flavouring constituent of aniseed flavoured products; however
its presence in other spirits, such as whisky, would indicate a non-genuine product. Other
examples of added flavouring seen in counterfeit products include the synthetic flavouring ethyl
vanillin [69] and the flavouring carrier propane-1,2-diol [40].
For certain spirit categories and brands, it will be necessary to extend the range of compound
information over and above that provided by the standard major volatile congeners and
maturation related congeners methods that are often employed for spirit drink authentication.
Such extension will be category specific; for example, the characterisation of gin brands, which
uses EAAO (neutral spirit) as its base, is typically free of most major volatile congeners listed, bar
methanol. Different entities, such as a range of terpenic compounds, will be more suitable for
brand authentication [70]. GC-MS and LC-MS will often be used to increase both the range and
sensitivity of compound information obtained from the volatile and non-volatile fractions of a
spirit, thus improving differentiation, but also increasing complexity of analysis. MS based
techniques (GC, LC or direct injection) can also be used for fingerprinting/non-targeted analysis
[71,72]. This will require the creation of large databases gathered from genuine products as well as
the use of multivariate statistical analysis. Such techniques have the advantage of identifying when
a profile deviates from the expected and may identify the contaminants, or lack of expected
congeners, resulting from adulteration/counterfeiting.
3.2.3. Denaturants
Ethanol is produced on a large scale for a variety of industrial uses. To aid in the differentiation of
potable alcohol from industrial alcohol and its products, ethanol is “denatured” to make the liquid
non-potable and excise duty exempt. The denaturants can act as useful markers for identifying
instances where industrial alcohol may have been used in the production of illicit spirits. The
chemicals used, and the proportions of denaturants, have traditionally varied by country. In 2008,
the EU Commission started a review that has led to a reduction and harmonisation of denaturants
in use within Europe. A new “Euro” denaturant formulation is now established, designed to help
prevent fraud. This consists of isopropanol, methyl ethyl ketone and denatonium benzoate [73].
Denaturants vary in the ease with which they can be differentiated from constituents of spirit
drinks; they will also vary considerably depending on location. Outside the EU, different
formulations will be used. In the US, these can be found in Title 27 of the Code of Federal
Regulations Part 21 [19]. Methanol has been a commonly used denaturant, which can be
potentially fatal contaminant in a fraudulent spirit. In addition to the denaturants themselves,
secondary markers resulting from attempts made by counterfeiters to remove denaturants from
industrial alcohol may indicate denatured alcohol in a fraudulent spirit [51]. Methods of analysis
used to detect denaturants will be targeted to the specific alcohol denaturants. The OIV already
have a method for isopropanol; the Customs Laboratory European Network is also due to
implement methods to measure the three constituents of the “Euro” denaturant.
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5. Conclusion
There are considerable financial incentives to create fraudulent spirit drink products. The prices
commanded by premium spirit drinks and excise duty combine to offer a lucrative opportunity,
especially where excise exempt alcohol can be used in its creation. Excessive taxation is often
quoted as being a key contributor to the production and consumption of illicit alcohol. For
example, in Indonesia many local people cannot afford to purchase genuine spirit drinks as they
are heavily taxed, leaving them to risk drinking unregulated products. In 2018 more than 100
people in Indonesia were killed by one poisoning outbreak [74]. Another potential issue is the ease
with which denatured alcohol can enter the potable supply chain. Efforts are being made to
address this, such as the changes in European legislation designed to reduce the wide range of
denaturants in use, and to focus on formulations which prove hard to remove.
The detection of counterfeit spirit drinks can be challenging. Spirit drinks are characterized by two
major constituents: ethanol and water. The other compounds present, which provide
differentiation in terms of flavour and identity, are generally present at low levels. Many
compounds, such as proteins and DNA that are associated with the raw materials (cereals, grapes
etc.), are removed during the distillation step. As a result, techniques used to identify counterfeit
spirit drinks are typically based on profiles of flavour and other constituents present at trace levels
(ppm to ppb), such as the measurement of major volatile or maturation related congeners. Certain
properties of the whole spirit, such as pH, UV spectrum and alcohol strength can however prove
useful in identifying frauds.
Looking to the future, there are several trends apparent in spirit drink authentication. The first is
the drive for portability in analytical measurements, allowing rapid evaluations to take place at key
points in the supply chain, for example at point of sale. Portable pH and conductivity meters can
already be employed [75]. The use of portable UV-Vis for brand authentication is common, but it
can also be used for detection of specific compounds such as sugars [76]. Raman and NIR
spectroscopy are also being explored for their potential [77-79]; the opportunity of analysis
through spirit drinks bottles using such techniques is an attractive option for fraud detection.
Another trend is the increasing availability of more conventional laboratory techniques in
machines with a smaller footprint. These offer the potential for the both the quantitative profiling
of key marker compounds (of either genuine or counterfeit products) where chromatography is
involved [80] or a rapid assessment of authenticity based on a chemometric model of a particular
brand or category [72]. Finally, advances in laboratory authentication of spirit drinks will most
likely result in more detailed analysis (increased number of compounds and/or increased
sensitivity) becoming more routine and more rapid. The application of NMR as a routine technique
for both targeted and untargeted analysis of spirit drinks is one possibility [81].
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46 Gerhardt R.E., Crecelius E.A. & Hudson J.B. (1980). – Trace element content of moonshine. Archives of Environmental
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47 Holstege C.P., Ferguson J.D., Wolf C.E., Baer A.B. & Poklis A. (2004). – Analysis of moonshine for contaminants.
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48 Tobiassen R.M. (2014). – The “fake alcohol” situation in the United States: the impact of culture, market economics,
and the current regulatory systems. Available at: http://www.centerforalcoholpolicy.org/wp-
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50 Jackson D.S., Crockett D.F. & Wolnik K.A. (2006). – The indirect detection of bleach (sodium hypochlorite) in
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51 Hosnedl T., Ondrousek S. & Mazac J. (2007). – The determination of degradation products of denatonium benzoate
(Bitrex) in alcoholic beverages. In 3rd International Workshop on Alcoholic Beverages Authentication, Stresa (Italy),
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52 Ledgard J. (2014). – Kings Chemistry Guide™. Third edn., Uvkchem, United States. 165-169.
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54 AOAC (2016). – Official methods of analysis of AOAC International. 20th edn., AOAC International, USA.
55 European Parliament and Council (2011). – Regulation (EU) No 1169/2011 of the European Parliament and of the
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56 OIML (1973). – International alcoholometric tables, Bureau International de Metrologie Legale, France.
57 Lehtonen M. (1983). – High performance liquid chromatographic determination of nonvolatile phenolic compounds
in matured distilled alcoholic beverages. Journal of AOAC International, 66 (1), 71-78.
58 Puech J.-L. (1988). – Phenolic compounds in oak wood extracts used in the ageing of brandies. Journal of Scientific
Food Agriculture, 42, 165-172. doi: 10.1002/jsfa.2740420209.
59 Conner J.M., Reid K.J.G. & Jack F. (2003). – Chapter 7: Maturation and blending. In Whisky technology production and
marketing (I. Russell, ed), Academic Press, London: UK. 211-242 doi: 10.1016/B978-012669202-0.50024-5.
60 Reid K.J.G., Owen C.D., Conner J.M. & Goodall I.C. (2004). – Chapter 6: New methods for detecting counterfeit or
adulterated whiskies. In Distilled Spirits: Tradition and Innovation - Proceedings of the Worldwide Distilled Spirits
Conference, Eds: J.H. Bryce & G.G. Stewart, Nottingham University Press, Nottingham: UK. pp 33-40.
61 Bauer-Christoph C., Wachter H., Christoph N., Roßmann A. & Adam L. (1997). – Assignment of raw material and
authentication of spirits by gas chromatography, hydrogen- and carbon-isotope ratio measurements. I. Analytical
methods and results of a study of commercial products. Zeitschrift fur Lebensmittel-Untersuchung und -Forschung,
204, 445-452. doi: 10.1007/s002170050111
62 OIV (2018). – Compendium of international methods of wine and must analysis vol. 1. International Organisation of
Vine and Wine, Paris. Available at: http://www.oiv.int/public/medias/5772/compendium-2018-en-vol1.pdf.
63 Aguilar-Cisneros B.O., Lopez M.G., Richling E., Heckel F. & Schreier P. (2002). – Tequila authenticity assessment by
headspace SPME-HRGC-IRMS analysis of 13C/12C and 18O/16O ratios of ethanol. Journal of Agricultural and Food
Chemistry, 50, 7520-7523. doi: 10.1021/jf0207777.
64 Rettie A.B. & Hayward E.M. (2016). – Determination of metals by inductively coupled plasma - optical emission
spectroscopy (ICP-OES). In Distilled Spirits: Future Challenges, New Solutions - Proceedings of the 5th Worldwide
Distilled Spirits Conference, Eds: I. Goodall, R. Fotheringham, D. Murray, R.A. Speers & G.M. Walker, Context,
Packington: UK. pp 423-426.
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65 Dunbar E., Cook G.T., Murdoch I., Xu S. & Fabel D. (2018). – Chapter 29: Identification of fraudulent-age whiskies
using accelerator mass spectrometry (AMS) radiocarbon (14C) analyses. In Distilled Spirits: Local Roots, Global Reach:
Delivering Distilling Expertise to the World - Proceedings of the 6th Worldwide Distilled Spirits Conference, Eds: F.
Jack, D. Dabrowska, S. Davies, M. Garden, D. Maskell & D. Murray, Context, Packington: UK. pp 147-151.
66 Contreras U., Barbosa-García O., Pichardo-Molina J.L., Ramos-Ortíz G., Maldonado J.L., Meneses-Nava M.A., Ornelas-
Soto N.E. & López-de-Alba P.L. (2010). – Screening method for identification of adulterate and fake tequilas by using
UV–VIS spectroscopy and chemometrics. Food Research International, 43 (10), 2356-2362. doi:
10.1016/j.foodres.2010.09.001.
67 Bauer-Christoph C., Dreßler S., Sahiri T. & Sahiri M. (2018). – Authentication of spirits. Deutsche Lebensmittel-
Rundschau, 114 (2), 46-50.
68 Eccles R. (2018). – Chapter 67: The development of methods to detect the addition of flavourings in counterfeit
whisky. In Distilled Spirits: Local Roots, Global Reach: Delivering Distilling Expertise to the World - Proceedings of the
6th Worldwide Distilled Spirits Conference, Eds: F. Jack, D. Dabrowska, S. Davies, M. Garden, D. Maskell & D. Murray,
Context, Packington: UK. pp 333-334.
69 Martin G.G., Symonds P., Lees M. & Martin M.L. (1995). – Chapter 15: Authenticity of fermented beverages. In
Fermented Beverage Production (A.G.H. Lea & J.R. Piggott, eds), Blackie Academic & Professional, Glasgow: UK doi:
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70 Aylott R.I. (1995). – Analytical strategies to confirm gin authenticity. Journal of the Association of Public Analysts, 31,
179-192.
71 Collins T.S., Zweigenbaum J. & Ebeler S.E. (2014). – Profiling of nonvolatiles in whiskeys using ultra high pressure
liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS). Food Chemistry, 163, 186-
196. doi: 10.1016/j.foodchem.2014.04.095
72 Teodoro J.A.R., Pereira H.V., Sena M.M., Piccin E., Zacca J.J. & Augusti R. (2017). – Paper spray mass spectrometry
and chemometric tools for a fast and reliable identification of counterfeit blended Scottish whiskies. Food Chemistry,
237, 1058-1064. doi: 10.1016/j.foodchem.2017.06.062.
73 European Commission (2017). – Commission Implementing Regulation (EU) 2017/1112 of 22 June 2017 amending
Regulation (EC) No 3199/93 on the mutual recognition of procedures for the complete denaturing of alcohol for the
purposes of exemption from excise duty. Journal, L162 (23.06.2017), 22-26. Available at: https://eur-
lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32017R1112&from=EN.
74 Minelle B. (2018). – Bootleg liquor kills more than 100 in Indonesia. Sky News. Available at:
https://news.sky.com/story/bootleg-liquor-kills-more-than-100-in-indonesia-11326068.
75 Lachenmeier D.W., Schmidt B. & Bretschneider T. (2007). – Rapid and mobile brand authentication of vodka using
conductivity measurement. Microchimica Acta, 160 (1), 283-289. doi: 10.1007/s00604-007-0825-9.
76 Glancy S., Cockburn P., Eccles R. & Goodall I. (2018). – Chapter 65: Developing rapid analysis methods to identify
counterfeit spirits. In Distilled Spirits: Local Roots, Global Reach: Delivering Distilling Expertise to the World -
Proceedings of the 6th Worldwide Distilled Spirits Conference, Eds: F. Jack, D. Dabrowska, S. Davies, M. Garden, D.
Maskell & D. Murray, Context, Packington: UK. pp 325-327.
77 Ellis D.I., Eccles R., Xu Y., Griffen J., Muhamadali H., Matousek P., Goodall I. & Goodacre R. (2017). – Through-
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handheld SORS device. Scientific Reports, 7 (1), 12082. doi: 10.1038/s41598-017-12263-0.
78 Kiefer J. & Cromwell A.L. (2017). – Analysis of single malt Scotch Whisky using Raman spectroscopy. Analytical
Methods, 9 (3), 511-518. doi: 10.1039/C6AY02907H.
79 Nordon A., Mills A., Burn R.T., Cusick F.M. & Littlejohn D. (2005). – Comparison of non-invasive NIR and Raman
spectrometries for determination of alcohol content of spirits. Analytica Chimica Acta, 548 (1-2), 148-158. doi:
10.1016/j.aca.2005.05.067.
80 Cockburn P. (2016). – Method development for identification of adulterated spirits using field portable GC/MS.
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using-Field-Portable-GC-MS_012711_01.pdf.
81 Kuballa T., Hausler T., Okaru A.O., Neufeld M., Abuga K.O., Kibwage I.O., Rehm J., Luy B., Walch S.G. & Lachenmeier
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Fruit juices
Peter Rinke*
SGF International e.V., Nieder-Olm, Germany
*E-mail corresponding author: [email protected]
Eric Jamin*
Eurofins Analytics France, Nantes, France
*E-mail corresponding author: [email protected]
https://doi.org/10.32741/fihb.14.juices
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1. Product Identity
1.1. Definition of the product and manufacturing process
The type of fruit juice found on the market is generally conditioned by the processing method used
to get the product from its growing region to the supermarket shelf and by the specific regulations
in force in the country where it is sold. Fruit juice is traded either as natural strength juice or purée
or as concentrated fruit juice or purée from which the water has been extracted. The latter greatly
reduces storage space requirements and cuts transport costs considerably. The concentrate is
stored at very low temperatures or in aseptic drums and transported in bulk from the production
area to the main markets where it is reconstituted to single strength juice by adding water. The
juice is then pasteurised, bottled and sold as “100 % fruit juice made from concentrate” or “100 %
fruit juice made with concentrated fruit juice”.
Natural strength fruit juice is obtained directly from the fruit, pasteurised and bottled ready to be
sold or kept in sterile tanks at low temperature for packing at a later date. It is sold as “100 % fruit
juice” and sometimes as “direct juice”.
Chilled (refrigerated) short shelf life juices
● Chilled freshly squeezed juice: single strength juice not made from juice concentrate with
a shelf life of between 3 days and 3 weeks depending on fruit type, storage temperature
0-5 °C
● Pasteurised 100 % (direct) juice: single strength juice not made from concentrate with
shelf life of about 24 days
● Pasteurised juice made with concentrated fruit juice: reconstituted from juice concentrate
(generally frozen), shelf life of about 24 days.
Pasteurised, ambient juices
● Pasteurised direct juice/freshly squeezed: long shelf life (6 to 12 months depending on
fruit type)
● Pasteurised juice made with concentrated fruit juice: reconstituted from frozen
concentrate with a long shelf life (6 to 12 months depending on fruit type).
Fruit nectars are also popular in Europe. These are blends of fruit juices (between 25 – 90 % juice
content depending on the fruit type), water and sugar.
Fruit purées and pulps are ideal raw materials for soft fruits such as strawberry and raspberry that
are prone to physical damage during transport. Such products are used in fruit juice blends, drinks
and nectars as well as in jam and marmalade manufacture.
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Fruit juices
including post-harvest surface treatments applied in accordance with the applicable provisions of
the Codex Alimentarius Commission.
[….]
The juice is prepared by suitable processes, which maintain the essential physical, chemical,
organoleptical and nutritional characteristics of the juices of the fruit from which it comes…”
Definitions given by the Codex Alimentarius can be taken as a general basis for export purposes.
However these guidelines may differ from those of specific countries.
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Fruit juices
2. Authenticity issues
2.1. Identification of current authenticity issues
There are various potential frauds possible as regards fruit juices. The most important authenticity
issues are listed below:
● Water addition: there is a natural variation in fruit juices for the ratio between soluble dry
matter and water. However it is not allowed to add water, even if the fixed minimum
density or Brix are lower than any naturally obtained product not from concentrate. Also,
for reconstituted juice from concentrate, legislation stipulates that the quantity of water
added to reconstitute the juice must be the same as that removed during concentration.
As mentioned earlier, the AIJN, has laid down guidelines for minimum density and its
corresponding Brix value, a measure of the soluble solids content. One of the simplest
forms of adulteration is dilution of a concentrate to below the permitted minimum Brix.
● Sugar addition: As a commodity, sugar is much cheaper than fruit juice, and therefore its
addition to the latter to increase Brix values can be an economic advantage. Sugar can be
added as beet, cane or corn sugars, or as modified sugar syrups such HFCS (high fructose
corn syrup) or BMIS (beet medium invert syrup).
● Complete or partial replacement of juice by juices made from concentrate: in some
countries consumer preference has shifted in recent years to freshly squeezed or not
from concentrate (NFC) juices, conferring a higher premium on these products. It is
therefore not permitted to pass reconstituted juices off as NFC or to add a proportion of
water or reconstituted juice to the direct product.
● Added products from undeclared cheaper fruits: the prices of certain fruit types can
fluctuate widely from one season to another, affected by poor harvests, gluts, and trade
regulations. The addition of a cheaper fruit alternative to stretch one in short supply
and/or high demand is another fairly common form of adulteration. Examples include the
addition of orange to passion fruit, apple to red fruit, grape to pomegranate
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Fruit juices
● Addition of undeclared ascorbic acid/vitamin C: some fruit types are naturally high in
vitamin C and use this as a selling point.
● Addition of undeclared organic acids (e.g. citric acid, malic acid): in some cases the
acidity of a juice can be corrected by the addition of organic acids within the limits
tolerated by the legislation, and with suitable mention on the label. Specific practices such
as addition of malic acid to apple juice is not permitted.
● Addition of flavour compounds (natural or synthetic): authenticity issues arise when the
product claiming to be "natural" contains flavours not from the named fruit or that have
been chemically synthesised. Food fraud through addition of unauthorised flavour
compounds is covered in the “flavourings” chapter.
● Colourings (e.g. anthocyanin extracts, cochenille red, beetroot): the colour of fruit juices
is an important part of the product's appeal. Colourings may be added to meet colour
intensity specifications, to restore natural colour lost during processing conditions or
storage, or to darken colour when the authentic product has been extended with a less
pigmented fruit type.
● Addition or over-proportional use of fruit extracts which were produced by non-
authorised technology (water extractable solids): water extractable solids is the material
obtained from washing the remaining pulp and cell membrane material after extraction of
orange juice. Its overuse in juice is not permitted under the legal definition of the EU for
citrus fruits.
● Texture influencing agents (e.g. pectins): texturisers can enhance body and mouthfeel in
juices that are less than 100 % fruit. This is authorised for specific fruit types only and
provided that the compounds are mentioned on the product label.
● Declaration of wrong origin: the country of origin of a fruit product can become an
authenticity issue if it is falsely declared on the juice label or in the product's trade
specifications. This could be the concern of customs and excise authorities, if the fruit
origin in question is subject to preferential import duties. Certain geographical origins
may also carry a premium on the market
● Declaration of wrong fruit variety: this could be an issue where a specific variety is prized
for its flavour or processing qualities
In particular if the fruit content in the falsified product is lower than in an authentic one the fraud
could be covered up by adding other ingredients to adjust the analytical profile to the expected
picture of an authentic product. Therefore minerals, organic acids, amino acids or a combination of
different materials from other fruits can be added as part of the fraudulent practice.
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Fruit juices
An orange juice concentrate is diluted with deionised well water and after respective flavour
addition commercialised as juice not from concentrate. In this case an analysis of the isotope ratio
18 16
O/ O of the water in the juice can detect the fraud. To avoid being discovered, the water used
for dilution could be replaced by the dishonest juice producer by water obtained from the
18 16
concentration process of grape juice. The O/ O isotope ratio – which is in many companies the
routinely applied control parameter - would then be insufficient to detect the fraud. The risk
increases that the fraud remains undiscovered. However ²²as most grape juices used for the
production of concentrate are stabilised by sulfiting, the presence of sulphur dioxide (SO2) in the
final product is possible. SO2 is listed as an allergen and represents a health risk for sensitive
consumers.
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Fruit juices
Table 1: Available IFU methods and recommendations for fruit juice authenticity testing
IFU Method
IFU 01 Determination of Relative Density (Pycnometer Method)
IFU 01A Relative Density (Method using Density Meter)
IFU 02 Determination of Ethanol by Gas Chromatography
IFU 03 Determination of Titratable Acidity
IFU 05 Determination of Volatile Acids
IFU 07A Determination of Total Sulphur Dioxide (SO2)
IFU 08 Determination of Soluble Solids (Indirect Method by Refractometry)
IFU 09 Determination of Ash
IFU 10 Determination of Ash Alkalinity
IFU 11 Determination of pH Value
IFU 12 Determination of Hydroxymethylfurfural (HMF)
IFU 12A Microbiogical detection of taint alicyclobacillus
IFU 17A Determination of ascorbic acid by HPLC
IFU 18 Fermentation Test (Screening Test for the Presence of Preservatives)
IFU 21 Determination of L-Malic Acid, Enzymatic
IFU 22 Determination of Citric Acid, (enzymatic)
IFU 24 Detection of Artificial Water-Soluble Artificial Colorants
IFU 25 Organoleptic Examination
IFU 26 Determination of Pectin
IFU 28 Determination of Total Nitrogen
IFU 30 Determination of Formol Number
IFU 33 Determination of Sodium, Potassium, Calcium and Magnesium
IFU 36 Determination of Sulphate
IFU 37 Determination of Chloride
IFU 45 Determination of Essential Oils (Bromate Method)
IFU 46 Determination of Pectin Esterase (PE) Activity in Citrus Juices and their Concentrates
IFU 49 Determination of Proline
IFU 50 Determination of Phosphate
IFU 52 Determination of Alcohol, Enzymatic
IFU 53 Determination of Lactic Acid, Enzymatic
IFU 54 Determination of D-Isocitric Acid, Enzymatic
IFU 55 Determination of Glucose and Fructose, Enzymatic
IFU 56 Determination of Sucrose, Enzymatic
IFU 57 Determination of Free Amino Acids
IFU 58 Determination of Hesperidin and Naringin, HPLC
IFU 59 Determination of Total Carotenoids and Individual Carotenoid Groups
IFU 60 Determination of Centrifugable Pulp
IFU 61 Determination of Total Dry Matter
IFU 62 D-Sorbitol (Enzymatic)
IFU 63 Preservatives (HPLC)
IFU 64 D-malic Acid (Enzymatic)
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Fruit juices
IFU Method
IFU 65 Tartaric Acid in Grape Juice (HPLC)
IFU 66 Acetic Acid (Enzymatic Method)
IFU 67 Determination of Sugars and Sorbitol (HPLC)
IFU 68 Test for Pectin (Turbidimetric)
IFU 69 Determination of Hydroxymethylfurfural (HPLC)
IFU 70 Cell Content of Pulps and Juices
IFU 71 Anthocyanins by HPLC
IFU 72 Fumaric Acid (HPLC)
IFU 73 Detection of Starch in Fruit Juices
IFU 74 Determination of Nitrate by Ion Chromatography
IFU 76 Determination of D-Gluconic Acid in Grape Juice (Enzymatic)
IFU 77 Determination of Glycerol in Grape Juice (Enzymatic)
IFU 78 Determination of Galacturonic Acid using High Performance Anion Exchange Chromatography
IFU 79 Measurement of Polyols in Fruit and Vegetable Juices using Electrochemical detection
IFU 80 Measurement of the Colour of Clear and Hazy Juices (Spectrophotometric Method)
IFU 81 Determination of Ergosterol by HPLC (Provisional)
IFU 82 Determination of Nitrate (Provisional)
IFU 83 Colour measurement of blood orange juices
IFU 84 Stability test for clarified juices
IFU Recommendations
IFU R01 Detection of Invert Syrup Addition by Oligosaccharide Analysis
IFU R02 Recommendation for the Determination of Patulin
IFU R03 The Use of Isotopic Procedures in the Analysis of Fruit Juices
IFU R04 Detection of Syrup Addition to Juices by Capillary Gas Chromatography
IFU R05 Recommendation for Vitamin C Analysis
IFU R06 Determination of Heavy Metals in Fruit Juices
IFU R07 Recommendations for Turbidity Measurements
IFU R08 Recommendations for Analysis of High Intensity Sweeteners
IFU R09 Recommendation for Colour Measurements in Cloudy Juices
IFU R10 Recommendations for Analysis of Ochratoxin in Fruit Juices
IFU R12 Methods for the Conformation of Country of Origin
IFU R13 The use of DNA Methods in the analysis of fruit juices, purees and concentrates
IFU R14 Recommendation. Methods to assess the organic or bio nature of juices
IFU R15 Recommendations, Basic quality systems for juice laboratories
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Fruit juices
Reference Application
EN 1131:1994 Determination of relative density
EN 1132:1994 Determination of the pH-value
EN 1133:1994 Determination of the formol number
EN 1134:1994 Determination of sodium, potassium, calcium and magnesium content by atomic absorption
spectrometry (AAS)
EN 1135:1994 Determination of ash
EN 1136:1994 Determination of phosphorus content - Spectrometric method
EN 1137:1994 Enzymatic determination of citric acid (citrate) content - NADH spectrometric method
EN 1138:1994 Enzymatic determination of L-malic acid (L-malate) content - NADH spectrometric method
EN 1139:1994 Enzymatic determination of D-isocitric acid content - NADPH spectrometric method
EN 1140:1994 Enzymatic determination of D-glucose and D-fructose content - NADPH spectrometric method
EN 1141:1994 Spectrometric determination of proline content
EN 1142:1994 Determination of the sulphate content
EN 12133:1997 Determination of chloride content - Potentiometric titration method
EN 12134:1997 Determination of centrifugable pulp content
EN 12136:1997 Determination of total carotenoid content and individual carotenoid fractions
EN 12137:1997 Determination of tartaric acid in grape juices - Method by high performance liquid
chromatography
EN 12138:1997 Enzymatic determination of D-malic acid content - NAD spectrometric method
EN 12143:1996 Estimation of soluble solids content - Refractometric method
EN 12144:1996 Determination of total alkalinity of ash - Titrimetric method
EN 12145:1996 Determination of total dry matter - Gravimetric method with loss of mass on drying
EN 12146:1996 Enzymatic determination of sucrose content - NADP spectrometric method
EN 12147:1996 Determination of titratable acidity
EN 12148:1996 Determination of hesperidin and naringin in citrus juices - Method using high performance liquid
chromatography
EN 12630:1999 Determination of glucose, fructose, sorbitol and sucrose contents - Method using high
performance liquid chromatography
EN 12631:1999 Enzymatic determination of D- and L-lactic acid (lactate) content - NAD spectrometric method
EN 12632:1999 Enzymatic determination of acetic acid (acetate) content - NAD Spectrometric method
EN 12742:1999 Determination of the free amino acids content - Liquid chromatographic method
ENV 12140:1999 Determination of the stable carbon isotope ratio (13C/12C) of sugars from fruits juices - Method
using isotope ratio mass spectrometry
ENV 12141:1996 Determination of the stable oxygen isotope (18O/16O) of water from fruit juices - Method using
isotope ratio mass spectrometry
ENV 12142:1996 Determination of the stable hydrogen isotope ratio (2H/1H) of water from fruit juices - Method
using isotope ratio mass spectrometry
ENV 13070:1998 Determination of the stable carbon isotope ratio (13C/12C) in the pulp of fruit juices - Method
using isotope ratio mass spectrometry
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Table 3: Available AOAC methods and recommendations for fruit juice authenticity testing
Reference Application
2004.01-2004 Carbon stable isotope ratio of ethanol derived from Fruit Juices and Maple Syrups
2005.02-2005 Total Monometric Anthocyanin Pigment Content
969.30-1980(1998) Organic acids (foreign) in fruit juices.
971.18-1980 Carbohydrates in fruit juices. Gas chromatography
981.09-1983(1997) Carbon stable isotope ratio of apple juice
982.21-1983(1997) Carbon stable isotope ratio of orange juice
986.13-1989(1996) Quinic, Malic and Citric acids in cranberry juice cocktail and apple juice. Liquid chromatography
986.14-2008 Orange Pulpwash and/or Added H2O in Processed Florida Orange Juice Spectral Characterization
991.30-1994 Polydimethylsiloxane in pineapple juice. Atomic Absorption
991.46-2008 Glycerol in wine and grape juice. Liquid chromatography
992.09-1997 Sugar-Beet-Derived Syrups Frozen Concentrated Orange Juice δ18O Measurements in Water
Stable Isotope Ratio Mass Spectrometric Method
993.05-1997 L-malic/total malic acid ratio in apple juice.
995.06-1998 D-malic acid in apple juice. Liquid chromatography
995.17-1998 Beet Sugar In Fruit Juices Site Specific Natural Isotope Fractionation– Nuclear Magnetic
Resonance Method
999.05-1999(2002) Naringin and neohesperidin in orange juice. Liquid chromatography
― 10 ―
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Fruit juices
For all analyses the quality and specificity of reference databases is important. A critical
consideration is recommended in this regard. In many cases region-specific reference data help to
better recognise food fraud or to avoid false positive interpretation. Regional differences are
mainly due to weather conditions, cultivated varieties and process techniques. If one of these
parameters changes, then an incorrect analytical evaluation could result. However experience has
shown that such changes are relatively seldom, and when they do, the reference databases must
be adjusted by addition of appropriate authentic reference samples when they do occur.
To obtain an overall profile of the fruit juice, the best choice today would be, where available, an
untargeted proton NMR juice screening combined with selected conventional parameters. If the
NMR screening is not possible, a larger set of conventional analyses is needed to cover a maximum
of fraud possibilities. The comparison in table 4 below shows how conventional parameters can be
replaced by the NMR screening for a minimum scope to check the authenticity for apple and
orange juice. Some conventional parameters are not necessary because the NMR screening gives
the same information as these parameters.
Wherever possible, positive fraud detection should be confirmed by at least a second analytical
approach.
Table 4: Extract from the SGF-Conformity matrix for apple and orange juice/-concentrate. The columns indicate the
analytical order with or without SGF-ProfilingTM
Apple Orange
Analysis Without SGF With SGF Without SGF With SGF
TM TM TM TM
Profiling Profiling Profiling Profiling
TM
SGF-Profiling x x
Relative density 20/20 x x x x
Brix (table) x x x x
Soluble solids x x
Glucose x x
Fructose x x
Sucrose x x
Titrat. acidity expr. as tart. acid pH 7.0 x x x x
Titrat. acidity expr. as citric acid pH 8.1 x x x x
L-malic acid x x
Citric acid x x x
Isocitric acid x x
L-ascorbic acid x x
Sodium x x x x
Potassium x x
Calcium x x x x
Magnesium x x
Nitrate x x x x
Phosphate x x
Sorbitol x x
Formol number x x
Proline x
Water-soluble pectins x
Lactic acid x x
HMF (5-hydroxmethylfurfural) x x
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― 12 ―
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Table 6: Parameters which can be automatically quantified from proton-NMR spectrum of SGF ProfilingTM
Calculated values
glucose/fructose % sucrose total sugar malic acid/quinic acid
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Fruit juices
Every laboratory and its analysts have to develop a way of judging the whole analytical pattern as
such and not only value by value. Therefore no standardised procedure exists, however hereafter
follows an attempt by the chapter’s authors try to give a rough description of one way of
proceeding.
1. Check the Brix value or density and Brix/acidity ratio.
- High values of Brix or density in single strength products stand for high sugar content
and high degree of ripeness. Ripe products would probably tend to have a low acid
content and high Brix/acidity ratio. Particularities of certain varieties should be taken
into account.
- Single strength products have natural variations, official reference values are
generally set at the lower limit. If products from one source are regularly close to this
minimum level without variation, a systematic standardization through water
addition is to be considered.
2. Check the sugar profile.
- The glucose/fructose ratio is specific for different fruit types.
- The glucose/fructose ratio generally decreases with microbiological spoilage. Check
consistency with other metabolites indicative of spoilage (e.g. lactic acid, ethanol,
volatiles acids)
- Sucrose content is typical for different fruit types, some fruits do not have any. The
step of invertase deactivation in the process flow plays an important role.
- A product which has undergone heat stress, a long storage time or inappropriate
transport conditions can show sucrose inversion to glucose and sucrose in equal
quantities. Inversion is also favoured by high acid content.
3. Check the sugar free extract and its relation to the total amount of sugar.
- The sugar free extract contains all soluble compounds which are not the main sugar
compounds glucose, fructose and sucrose. This would decrease if external sugar is
added.
- The relation between total sugar and the sugar free extract would shift towards the
sugar content for products which are supposed to derive from very ripe fruits.
4. Try to explain the sugar free extract with available data.
- An analytical profile will never cover the total soluble extract. Depending on the type
of product and the chosen analytical parameters the gap between measurable
compounds of the sugar free extract and the total sugar free extract is more or less
great. However, the sum of measured concentrations for organic acids, minerals,
sorbitol (if present) could show experienced analysts whether the usually expected
gap is in the natural range. If the gap is too small, an adjustment of the analytical
profile to mask a low fruit content would be expected.
- If the gap is too big unexpected compounds must be present (e.g. sorbitol, solubles
from mash extraction, starch or other polysaccharides)
5. Check relation of compounds inside the sugar free extract.
- Group of organic acids: the ratio of citric acid and D-isocitric acid is typical for every
fruit type. High values indicate the addition of citric acid.
- Group of minerals: higher sodium and nitrate values can indicate the presence of
minerals from process water or process agents. Regional exceptions are possible.
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Fruit juices
- In particular, for products not from concentrate high values of sodium or nitrate –
even below the AIJN-Code of Practice maximum guide values – could hint at added
water or the use of reconstituted concentrates.
- Group of flavonoids, anthocyanins: different fruit types have different patterns.
6. Check individual marker substances. Examples:
- Some substances are untypical for certain fruit types, e.g. sorbitol, sucrose,
- Some substances can indicate the presence of another fruit type, e.g tartaric acid
indicates grape, phloridzin indicates apple, arbutin indicates pear, naringin indicates
grapefruit.
- Lactic acid and ethanol indicate fermentation.
- 5-hydroxy-methylfurfural (HMF) is a typical Maillard product and indicates heat stress
and/or long-term storage in inappropriate conditions.
There is no obligation to follow this list in this order or limited to the afore-mentioned examples.
However it represents a useful approach to interpretation for the less experienced analyst, while
building up his/her own referential of importance for any parameter. Table 7 provides an idea of
which authenticity aspect a parameter could contribute to.
An authenticity check based on the overall analytical profile is particularly efficient if the analyst
has a good idea about the product: its ripeness, applied process, microbiological status, etc. Meta
data information with influence to the analytical profile can be counter checked.
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Fruit juices
Table 7: Choice of conventional parameters and their use for interpretation (frequent analytical targets are indicated;
fruit and product type specific deviations are possible)
Organic
Fruit Sugar Foreign Water Technology
Parameter acid
content addition fruit addition (citrus juice)
addition
Brix / Density X
Total titratable acidity X
Potassium X
Formol number X
L-Malic acid X X
D-Malic acid X
Magnesium X
Calcium X X
Phosphorus X
D-isocitric acid X X
Proline X X
Citric acid X X
Ratio citric / isocitric acid X
pH X
Maltose/Isomaltose X
Chloride X
Sulphate X
Glucose X
Fructose X
Ratio Glucose/Fructose X X
Sucrose X X
Total sugar (glucose, fructose,
X
sucrose)
Sucrose versus total sugar X
Sodium X
Nitrate X
Carotenoid profile (diff. fractions) X
Total Carotenoids, E-Carotene X
Sorbitol X X X
Water soluble pectins X
Centrifugeable pulp X
Phlorin X
Ascorbic acid X
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Fruit juices
The example of sorbitol as a marker substance for the presence of undeclared fruit types is
discussed here. It can be used to detect apple, pear, aronia and certain other fruits which might be
added to blackcurrant juice. Apples, pears and aronia naturally contain sorbitol, whereas
blackcurrant does not. Therefore positive detection of sorbitol in blackcurrant juice would clearly
show that the juice is not authentic, but it would not differentiate which type of fruit is the
adulterant. A more fruit specific indicator for added apple juice would be the phenolic compound
phloridzin, which is a typical marker for apple and not present in pears or aronia.
If the marker substances are present in low concentrations, the possibility of unintentional product
cross-contamination must be considered and manufacturing practices should be investigated.
Furthermore, the natural occurrence of traces of sorbitol through naturally present micro flora
should be looked at. A reference value to which a measured concentration remains acceptable is
complex and needs more investigation. Official guidelines such as the AIJN Code of Practice have
to apply relative high uncertainty margins in the benefit of the doubt, whereas individual company
policies can be different.
Marker substances are also used in chromatographic fingerprint methods such as anthocyanin or
flavonoid profiles. The occurrence of fruit specific substances allows the differentiation and
identification of undeclared fruit types present in a sample. Important methods are shown in
Table 9.
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Fruit juices
Furthermore there are also methods which use typical by-products from the production of sugar
syrups as marker substance for added sugar. Such marker substances could be oligosaccharides.
Maltose, maltotriose and higher starch degradation products can be detected with ion HPLC.
Typical by-products of inversion or degradation of polysaccharides give characteristic peak
patterns in GC chromatograms obtained after silylation [18].
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Table 10: Available stable isotope analyses for the authentication of fruit juices
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Fruit juices
5. Conclusion
Due to the complexity of authenticity control in fruit juices an analytical authenticity check is
always a more or less tailor-made combination of different methods. A first establishment of an
overview profile can be followed by analyses with a more precise focus and a lower limit of
detection.
The high number of individual analyses to establish a meaningful overall profile is an economic and
TM
time-consuming handicap. If available, the SGF NMR-Profiling is part of a better alternative. For
the future it can be expected that this technique using modified sample preparation (e.g.
fractionation, concentration) or other techniques with large data treatment like LC/HRMS [31] will
successively enhance fruit juice integrity analyses.
However there will remain the necessity to confirm analytical observation by targeted methods.
Isotopic techniques are likely to fulfil a major part of this need. Internal referencing methods will
play an important role there.
Due to the growing number of production regions for semi-finished goods, agricultural
development and the changing climate the interpretation of analytical results will become more
difficult and specific reference data bases will be increasingly required in the future.
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6. Bibliographic references
1. AIJN (2018). – AIJF European Fruit juice Association - Annual report 2018. Available at:
http://viewer.zmags.com/publication/bc62cfea#/bc62cfea/1.
2. Priyadarshini A. & Priyadarshini A. (2018). – Chapter 2 - Market Dimensions of the Fruit Juice Industry. . In Fruit Juices
(G. Rajauria & B.K. Tiwari, eds), Academic Press, San Diego. pp 15–32doi:10.1016/B978-0-12-802230-6.00002-3.
3. Codex Alimentarius (2005). – General Standard for Fruit Juices and Nectars. CODEX STAN 247-2005, 1–19.
4. Directive 2012/12/EU of the European Parliament and of the Council of 19 April 2012 amending Council Directive
2001/112/EC relating to fruit juices and certain similar products intended for human consumption (2012). Off. J. Eur.
Union, L115, 1–11.
5. AIJN - European Fruit Juice Association Available at: http://www.aijn.org/.
6. Website of SGF International e.V. Available at: https://www.sgf.org/index.php?id=29.
7. U.S. Food and Drug Administration (1983). – Title 21 - Food and Drugs, Chapter I - Food and Drug Administration,
Department of Health and Human Services, Subchapter B - Food For Human Consumption, Part 156 Vegetable Juices.
Available at: https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart=156.
8. U.S. Food and Drug Administration (2011). – 21 CFR 101.30 - Percentage juice declaration for foods purporting to be
beverages that contain fruit or vegetable juice. Available at: https://www.gpo.gov/fdsys/granule/CFR-2011-title21-
vol2/CFR-2011-title21-vol2-sec101-30.
9. U.S. Food and Drug Administration (2001). – 21 CFR 120 - Hazard analysis and critical control point (HACCP) systems.
Available at: https://www.gpo.gov/fdsys/granule/CFR-2011-title21-vol2/CFR-2011-title21-vol2-sec101-30.
10. FDA Food Safety Modernization Act (FSMA) (2011). , Page 124 STAT. 388.
11. Focused Mitigation Strategies to Protect Food Against Intentional Adulteration (2013). Available at:
https://www.regulations.gov/docket?D=FDA-2013-N-1425.
12. U.S. Food and Drug Administration (2017). – Juice HACCP and the FDA Food Safety Modernization Act: Guidance for
Industry.
13. Website of International Fruit and Vegetable Juice Association Available at: https://www.ifu-fruitjuice.com/.
14. Rinke P., Moitrier S., Humpfer E., Keller S., Moertter M., Godejohann M., Hoffmann G., Schaefer H. & Spraul M.
(2007). – An 1H-NMR technique for high throughput screening in quality and authenticity control of fruit juice and
fruit juice raw materials - SGF-profiling. Fruit Process., 1, 10–18.
15. Spraul M., Schütz B., Rinke P., Koswig S., Humpfer E., Schäfer H., Mörtter M., Fang F., Marx U.C. & Minoja A. (2009). –
NMR-Based Multi Parametric Quality Control of Fruit Juices: SGF Profiling. Nutrients, 1 (2), 148–155.
doi:10.3390/nu1020148.
16. Spraul M., Schütz B., Humpfer E., Mörtter M., Schäfer H., Koswig S. & Rinke P. (2009). – Mixture analysis by NMR as
applied to fruit juice quality control. Magn. Reson. Chem., 47 (S1), S130–S137. doi:10.1002/mrc.2528.
17. Ludwig C. & Viant M.R. (2010). – Two-dimensional J-resolved NMR spectroscopy: review of a key methodology in the
metabolomics toolbox. Phytochem. Anal., 21 (1), 22–32. doi:10.1002/pca.1186.
18. Low N.H. (1996). – Determination of fruit juice authenticity by capillary gas chromatography with flame ionization
detection. J. AOAC Int., 79 (3), 724–737.
19. IFU (2017). – Recommendation 3 - The Use of Isotopic Procedures in the Analysis of Fruit Juices (revised version of
2017). Available at: https://www.ifu-fruitjuice.com/list-of-methods.
20. Jamin E., Gonzalez J., Remaud G., Naulet N., Martin G.G., Weber D., Rossmann A. & Schmidt H.L. (1997). – Improved
detection of sugar addition to apple juices and concentrates using internal standard 13C IRMS. Anal. Chim. Acta, 347
(3), 359–368. doi:10.1016/S0003-2670(97)00189-X.
21. Jamin E., Gonzalez J., Remaud G., Naulet N. & Martin G.G. (1997). – Detection of Exogenous Sugars or Organic Acids
Addition in Pineapple Juices and Concentrates by 13C IRMS Analysis. J. Agric. Food Chem., 45 (10), 3961–3967.
doi:10.1021/jf9701087.
22. Jamin E. (Eurofins L., Gonzalez J., Bengoechea I., Kerneur G., Remaud G. & Naulet N. (1998). – Measurement of
13C/12C ratios of sugars, malic acid, and citric acid as authenticity probes of citrus juices and concentrates. J. AOAC
Int. USA. Available at: http://agris.fao.org/agris-search/search.do?recordID=US1997091119.
23. Day M.P., Correia P. & Hammond D.A. (1998). – 13C-IRIS - A refined method to detect the addition of cane/corn
derived sugars to fruit juices and purees. Fuit Process., (8), 86–90.
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24. González J., Remaud G., Jamin E., Naulet N. & Martin G.G. (1999). – Specific Natural Isotope Profile Studied by
Isotope Ratio Mass Spectrometry (SNIP−IRMS): 13C/12C Ratios of Fructose, Glucose, and Sucrose for Improved
Detection of Sugar Addition to Pineapple Juices and Concentrates. J. Agric. Food Chem., 47 (6), 2316–2321.
doi:10.1021/jf981093v.
25. Jamin E., González J., Bengoechea I., Kerneur G., Remaud G., Iriondo C. & Martin G.G. (1998). – Proteins As
Intermolecular Isotope Reference for Detection of Adulteration of Fruit Juices. J. Agric. Food Chem., 46 (12), 5118–
5123. doi:10.1021/jf980664g.
26. Thomas F., Jamin E. & Hammond D. (2013). – 18O Internal Referencing Method to Detect Water Addition in Wines
and Fruit Juices: Interlaboratory Study. J. AOAC Int., 96 (3), 615–624. doi:10.5740/jaoacint.12-339.
27. Jamin E., Lees M., Fuchs G. & Martin G.G. (2000). – Detection of added L- and D, L- malic acids in apple and cherry
juices – site specific 13-IRMS method. Fuit Process., (11), 434–436.
28. Jamin E. (2009). – Superfruits are they authentic? Fuit Process., (11), 170–175.
29. Thomas F., Randet C., Gilbert A., Silvestre V., Jamin E., Akoka S., Remaud G., Segebarth N. & Guillou C. (2010). –
Improved characterization of the botanical origin of sugar by carbon-13 SNIF-NMR applied to ethanol. J. Agric. Food
Chem., 58 (22), 11580–11585. doi:10.1021/jf102983v.
30. IFU (2018). – Recommendation 13 - The use of DNA Methods in the analysis of fruit juices, purees adnd
concentrates. Available at: https://www.ifu-fruitjuice.com/list-of-methods.
31. Dubin E., Dumas A.S., Rebours A., Jamin E., Ginet J., Lees M. & Rutledge D.N. (2017). – Detection of Blackcurrant
Adulteration by Aronia Berry Using High Resolution Mass Spectrometry, Variable Selection and Combined PLS
Regression Models. Food Anal. Methods, 10 (3), 683–693. doi:10.1007/s12161-016-0638-8.
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Vinegar
Raquel M. Callejón*, Rocío Ríos-Reina, M. Lourdes Morales, Ana M. Troncoso
Departmento de Nutrición y Bromatología, Toxicología y Medicina Legal
Universidad de Sevilla, Spain.
*E-mail corresponding author: [email protected]
Freddy Thomas*
Eurofins Analytics France, Nantes, France
*E-mail corresponding author: [email protected]
Federica Camin*
Unità Tracciabilità -Dipartimento Qualità Alimentare e Nutrizione
Fondazione Edmund Mach, Italy
*E-mail corresponding author: [email protected]
https://doi.org/10.32741/fihb.15.vinegar
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Vinegar
industry are the main driving factors of the vinegar market. It is expected that the global vinegar
market will witness growth both in terms of revenue and volume during the following years.
Growth will come from changing consumer lifestyles and preferences. The interest in cooking
gourmet and ethnic foods have increased among many consumers, thus prompting the sales of
various dressings, most of which use vinegar as one of the key ingredients.
Some premium vinegars are being commercialised worldwide. A typical example of this trend is
the increased consumption and trade of Balsamic Vinegar of Modena (Aceto Balsamico di
Modena). In fact, Italy is the country that exports the most vinegar, providing twice the quantities
of the other main exporters, Germany, Spain and France. Moreover, in terms of revenues, Italian
vinegars are exported at much higher values than Spanish or German vinegars. The situation in
Germany is different, considering that most German vinegar is sold for the pickling or sauce
industry, whereas Spanish exports include also some premium vinegars such as Sherry vinegars
(Vinagre de Jerez).
Sherry vinegars that are included in the European Union’s Protected Designation of Origin (PDO)
framework derive from Sherry wines and are necessarily aged in wood barrels for at least six
months. This aging can be performed by a dynamic system (i.e., passage through different barrels
containing vinegar from different vintages or different ages) or a static system. A more complex
example is Aceto Balsamico, which is either Aceto Balsamico Tradizionale (ABT), regulated by two
different PDO labels (ABT di Modena or ABT di Reggio Emilia), and Aceto Balsamico di Modena,
which has a Protected Geographical Indication (PGI) status. The production of ABT is a long process
that starts with the cooking of the grape must, which increases the sugar concentration to 400-500
g/L. Next, a partial alcoholic fermentation, which is initiated by osmophilic yeasts, produces a
“sweet wine” of approximately 7 % (v/v) ethanol concentration and over 200 g/L of residual
sugars. Then, some mother of vinegar is added to this “sweet wine,” and it is left to be acetified.
Once is acetified, the vinegar is placed in a “bateria” formed by five to seven barrels of different
woods (oak, mulberry, chestnut, cherry, juniper, ash and acacia) and decreasing volumes (from 60
to 15 L), which are filled up to 2/3 of their total volume. This “bateria” is kept for at least 12 years
with a yearly refilling from the previous barrel in a dynamic aging process. During this aging
process, two phenomena occur: the transfer of components from the wood to the ABT and, more
importantly, the concentration of the product and the integration of its components. The final
product can have up to 500 g of sugar per kg of product, 7 % acetic acid (v/v) and 20 g of gluconic
acid per kg. The oxidation of glucose by acetic acid bacteria yields gluconic acid. The result is a
dark, concentrated and thick product sold in 100 mL bottles and with a market value that can
easily reach 100 euros [3,4]. In contrast, Aceto Balsamico di Modena (ABM) is a PGI (Protected
Geographical Indication) salad dressing ingredient now renowned throughout the world, obtained
from cooked and/or concentrated grape must (at least 20 % of the volume), with the addition of at
least 10 % of wine vinegar and a maximum 2 % of caramel for colour stability that is aged at least
two months, not necessarily in barrels [5]. The geographical origin of ABM ingredients is not
specified. However, some of these ABM can be aged for more than three years and are labelled
“Invecchiato” (Aged). Overall, ABM is a cheaper version of ABT that has been popularized all over
the world.
Some Asian vinegars, such as black vinegars from China or “kurosu” from Japan, are produced
from rice and other cereals (including sorghum, wheat, and others) with a very important aging
process in which concentration and thickening occur in a similar manner to ABT.
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1. Product Identity
1.1. Definition of the product and manufacturing process
In general, food regulations consider vinegar the result of a double fermentation (alcoholic and
acetous or acetification) of any sugar substrate, usually agricultural raw materials of plant origin
with the exception of those produced from whey or honey.
In the European Union, the established limits for acidity and residual ethanol content are strictly
set. Thus, the acidity of wine vinegar (acetification obtained exclusively from wine) must be at
least 6 % (w/v), and the maximum residual ethanol allowed is 1.5 % (v/v) [4]. However, the variety
of raw materials used in the production of vinegar is important, ranging from by-products and
agricultural surpluses to high-quality substrates for the most unique and prized vinegars, such as
Sherry vinegar (Spain) and Aceto Balsamico Tradizionale (Italy). There are up to ten types of
vinegars, which include wine, fruit, cider, alcoholic, cereal, malt, malt distillate, honey and whey
vinegars. Undoubtedly, wine vinegar is the most common type in Mediterranean countries,
although the latest gastronomic trends have led to a considerable expansion of the varieties
available in recent years. However, worldwide most of the vinegar produced is “white” vinegar,
that is, vinegar produced directly from diluted alcohol [3]. In Asia, rice vinegar is the most common
type, although other types are also found, many of them following very traditional systems of
production.
Table 1: Different vinegars of the world are classified according to substrate, name and region/country of production and
distribution [6]
Region/Country
Substrate (Raw material) Name
(Production & distribution)
Grape Wine vinegar Global
Balsamic vinegar Global
Red vinegar Global
White vinegar Global
Distilled white vinegar Global
Sherry vinegar Global
Traditional Balsamic vinegar Global
Apple Cider vinegar US, Canada
Different fruits Fruit vinegar East and Southeast Asia
(mango, kaki, berries)
Date Date vinegar Middle East
Coconut Coconut vinegar Tropical Africa
Rice Rice vinegar China, Japan, Korea
Kurosu China, Japan, Korea
Malt Malt vinegar USA, Northern Europe
Distilled malt vinegar USA, Northern Europe
Whey (dairy by-products) Whey vinegar Europe
Honey Honey vinegar Global
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Vinegar
Therefore, vinegars can be classified by their substrates of origin or by their systems of production.
It is necessary to differentiate between those products derived from the double fermentation of a
single fruit (most often grapes or apples) and those that are “flavored” vinegars, that is, vinegars of
various origin with added concentrates of different fruits, flowers, or spices. Although these
“flavoured” vinegars are not considered vinegars in some countries, lax regulations in other
countries allow these products or condiments to be sold as “vinegars”.
The first fermentation is an alcoholic fermentation and transforms sugars or processed starches
into ethanol. This process is performed by yeast, mostly from the species Saccharomyces
cerevisiae, although some other species can also perform the alcoholic fermentation, partially or
totally. The final result is considered the substrate of the second transformation, to convert
ethanol to acetic acid. Although this second process is often referred as “acetic” or “oxidative”
fermentation, it is not biochemically a fermentation but an oxidation. The proper term is thus
“acetification” and involves a two-step oxidation, from ethanol into acetaldehyde and further from
acetaldehyde into acetic acid, whereby the end of this process requires an electron acceptor, with
molecular oxygen being the most common [2]. The microorganisms responsible for this process
are acetic acid bacteria. These bacteria are found on substrates containing sugars and/or alcohol,
such as fruit juice, wine, cider and beer. On these substrates, the sugars and alcohols are
incompletely oxidized, leading to the accumulation of organic acids, such as the production of
acetic acid from ethanol. Although more than 60 species have been described as acetic acid
bacteria, only a limited number of them are involved in the production of vinegar. The species
most commonly found in the production of vinegar are Acetobacter pasteurianus, Komagataei
bactereuropaeus and Komagataei bacterxylinus. Several attempts have been done to have single,
well-defined species of acetic acid bacteria for the production of vinegar, although it has been
concluded that a mixture of at least two species (one of them as “starter” and the other as
“finisher”, with different acetic acid sensitivities) is the most appropriate to be used as inoculum
for the production of vinegar, especially those above 5 % (w/v) acetic acid [7,8].
Vinegars can also be differentiated by their production systems. In traditional vinegars, the
transformation of ethanol into acetic acid is performed by a static culture of acetic acid bacteria at
the interface between the liquid and air. Barrels are filled to 2/3 of their capacity, as to leave an air
chamber, which is kept in contact with the outside air using one opening or various types of
openings. This production system, which is considered to be the traditional method, is called
“surface culture”. A more standardized version of this method, the “Orleans method,” includes
side holes for air circulation and adds a funnel with an extension to the base of the barrel to allow
wine to be added at the bottom of the barrel, preventing the alteration of the "mother of vinegar".
This mother of vinegar is the biofilm formed by the transforming microorganisms, i.e. the acetic
acid bacteria, which develops on the surface due to the need for oxygen. The vinegars produced by
this traditional system are generally considered of high quality because of their organoleptic
complexity, which is mainly due to the metabolism of the acetic acid bacteria and the overlapping
vinegar production with aging. However, this process is very slow, and the production of vinegar
can take from months to years.
To reduce the acetification time, other methods, such as the Schutzenbach systems with
submerged cultures, have been developed. Bacteria are immobilized on wood chips or charcoal,
forming a solid bed on which the vinegar spreads. After passing through the bed of chips, the
vinegar is collected in a container at the bottom and pumped back to the same fixed bed. The
acidity successively increases, and it is possible to obtain vinegar of reasonable quality within a
week.
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Submerged culture systems provide a much faster alternative. These systems rely on suitable
turbines to generate a flow of air bubbles into the wine or alcoholic solution. The oxidative process
occurs in the air-liquid interfaces of the air bubbles. Improvements to this process generally
involve engineering (improving the maintenance and persistence of the bubbles in the liquid, the
uniformity of the bubble size, the recovery of lost aromas, etc.). Vinegar is then produced at a
significantly lower cost, the bacteria act as bioreactors for the transformation of alcohol into acetic
acid, the airflow contributes to a considerable loss of the volatile compounds present in the
original alcoholic solution, resulting in a less complex product from a sensory point of view.
Although early containers for submerged culture processing were made of wood, the usual
containers are stainless steel, which is more hygienic and resistant to acidic conditions. The
limitations can be compensated by subsequent aging in barrels or by submerging wood fragments
or wood chips, which may contribute to the recovery of some of the missing organoleptic
character. Despite the loss in product quality, this methodology has two important advantages:
speed (the vinegar can be produced in cycles of 24 hours, or even shorter) and acidity (the product
can reach concentrations of acetic acid of up to 23-25 %, compared to the 6-13 % achieved with
other systems). Higher acidity helps to reduce transportation costs by reducing water transport.
An important aspect that contributes to the organoleptic quality of vinegars is aging, which
enables the integration of the different compounds in vinegars. The increase in organoleptic
quality during aging is remarkable; in addition to interactions with the wood, a series of chemical
reactions, evaporation, the production of esters, reactions between acids and residual alcohols,
and other processes result in better integration of aromas and metabolites and a reduction in the
pungency of acetic acid.
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Council as regards the title of the food category 12.3 Vinegars. The new title of the food category
12.3 is now: Vinegars and diluted acetic acid (diluted with water to 4-30 % by volume). This
category was renamed because in some Member States only vinegars obtained from the
fermentation of agricultural products are allowed to be named ‘vinegars’. In other Member States,
however, both products obtained from the dilution with water of acetic acid and vinegars obtained
from the fermentation of agricultural products are marketed under the name ‘vinegar’.
Three EU schemes of geographical indications and traditional specialties, known as Protected
Designation of Origin (PDO), Protected Geographical Indication (PGI), and Traditional Specialities
Guaranteed (TSG), promote and protect names of quality agricultural products and foodstuffs.
Products registered under one of the three schemes may be marked with the logo for that scheme
to help identify those products. The schemes are based on the legal framework provided by EU
Regulation No 1151/2012 [13] of the European Parliament and of the Council of 21 November
2012 on quality schemes for agricultural products and foodstuffs. This regulation (enforced within
the EU and being gradually expanded internationally via bilateral agreements between the EU and
non-EU countries) ensures that only products that originate from that particular region are
allowed to be marketed as such. Regarding vinegars, there are currently five PDO registered
categories and one PGI. Among PDOs: three from Spain (Vinagre de Jerez, Vinagre de Montilla-
Moriles, Vinagre de El Condado de Huelva) and two from Italy (Aceto Balsamico Tradizionale di
Modena, Aceto Balsamico Tradizionale di Reggio Emilia). Lastly Aceto Balsamico di Modena is
registered as a PGI.
Currently there is no European trade association of vinegar producers. The Vinegar Institute is the
international trade association representing the vast majority of vinegar manufacturers and
bottlers, mainly those with activities in the USA.
2. Authenticity issues
2.1. Identification of current authenticity issues
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balsamic vinegar as Aceto Balsamico di Modena IGP, there could also be the unfair practice of
adding exogenous sugars to cooked and/or concentrated grape must.
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of decreasing volume [19]. The organoleptic vinegar properties developed during ageing make the
finished product very appealing. Nevertheless, the production time and costs are too excessive to
permit a lucrative trade. Hence, an objective of the vinegar industry is to produce these aged
vinegars with the same organic characteristics related to aging, but making it in the most economic
and rapid way. For these reasons, the vinegar industry has a very real interest in speeding up
ageing if this can be done in a way which does not produce an inferior product or result in the
consumer being misled. In this context, the use of wood chips is being investigated. Moreover,
there is an increasing necessity to develop simple methods able to detect specific metabolites in
vinegars as possible indicators for the ageing process and traditional procedures, in order to
protect the consumers and avoid unfair competitions.
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Within the different types of sensory analysis, the most used are the descriptive test, that is useful
for determining the sensory profile of the samples, and the discriminatory test, which include a
wide range of tests such as triangular test (ISO, 2004, standard 4120) [32] and Paired Comparison
tests (ISO, 1983b, standard 5495) [33], preference test, etc. These methods require a well-trained
testing panel, and concrete and adequate attributes.
3.2.1.2. Viscosity
Viscosity is another important parameter in the sensorial quality of some vinegars such as in the
case of the Traditional Balsamic Vinegar of Modena. Nevertheless, no procedure has yet been
established to determine this objectively, as it is assessed in an empirical manner and wrongly
expressed as physical density.
3.2.1.3. Colour
Colour is one of the most important parameters used by consumers to assess the quality of a food
product. Some studies have described a relationship between some compounds and a darker
colour such as melanoidin, and products from the degradation of sugars and Maillard reactions [3].
A darker colour is also related to a longer aging period in wine vinegars and Traditional Balsamic
vinegar of Modena. Some techniques such as UV-Visible spectrophotometry or excitation-emission
fluorescence or transmission colorimetric techniques are being used with promising results for this
issue [34-36]. However, the colour could be easily modified with the use of grape must caramel or
other additives and no methods haves been officially established to assess and control this
parameter.
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ATR has been applied to investigate its potential as a tool for characterising different categories of
high-quality vinegars by a studying the differences in the spectra. FT-IR spectra have also been
used to predict the sensory score of traditional balsamic vinegar of Modena by the performance of
different partial least squares (PLS) regression models [52] as well obtaining a full calibration
model for organic acids in vinegars [53]. Finally, the technique can also be used to control certain
steps and factors of the production processes in industry, making it possible to carry out necessary
corrective actions without delay [54].
Fluorescence spectroscopy
Fluorescence spectroscopy is also being investigated as an alternative quality control tool for
vinegars, with the same attributes as those mentioned above. Different methods of analysis are
possible, the conventional one being the measurement of the excitation or emission spectra at a
single emission or excitation wavelength, respectively. However, instead of measuring a single
emission spectrum at a selected excitation wavelength, the emission spectra at different excitation
wavelengths can be recorded, in a technique known as excitation-emission fluorescence. The latter
results in a bi-dimensional Excitation- Emission Matrix (EEM), which contains unique information
of each measured sample, having the advantage of containing more information about the
fluorescent species than the conventional excitation and emission spectra separately. Moreover,
the potential of the EEM technique can be improved by applying multivariate methods in the
analysis of the fluorescence results such as Parallel Factor Analysis (PARAFAC) and its combination
with PLS discriminant analysis. PARAFAC is used to decompose fluorescence EEMs into different
independent groups of fluorescence components (fluorophores), as well as their relative
concentration (scores) in each sample. This method extracts the most relevant information from
the data in order to build further robust calibration and/or classification models. For this reason,
this technique has been more widely applied in the study of wine vinegars than the simple
excitation or emission analysis. Thus, Callejón et al. [48] and Ríos-Reina et al. [16] studied
fluorescence excitation–emission spectroscopy combined with suitable multivariate methods. In
these studies, the fluorescence Excitation-Emission Matrices (EEMs) were obtained by varying the
excitation wavelength ranging between 250 and 700 nm (every 5 nm), and recording the emission
spectra from 300 to 800 (every 2 nm). For these measurements, excitation and emission slits were
both set at 5 nm, and the scan rate was fixed to 1200 nm min-1. These studies demonstrated this
method’s ability to characterise and classify three Spanish PDO wine vinegars according to their
protected designation of origin, as well as their categories (aged and sweet) [24; 55]. However,
despite the promising results obtained, is not yet widely in use in this field.
Nuclear Magnetic Resonance (NMR) Spectroscopy
NMR spectroscopy, which has the advantage of being a rapid and non-selective analysis without
any manipulation or derivatisation, has recently achieved general acceptance as a powerful tool
for vinegar quality and authenticity determination. NMR can provide information on chemical
composition, concentration of soluble metabolites and their structure in the samples such as
sugars, acids and flavonoids, with the advantage of providing the best combination of fast data
acquisition and predictive capability. However, the large amount of data needs to be treated by
multivariate methods such as principal component and discriminant analysis with the final
objective of making models able to discriminate authentic and non-authentic vinegars, origins, or
vinegar types.
Different nuclei to which the spectrometer is tuned have been investigated for vinegar
authentication. The most commonly applied NMR technique for origin authentication, and
recently recognised as an official method, is deuterium SNIF-NMR (Site-specific Natural Isotopic
Fractionation studied by nuclear magnetic resonance spectrometry). However, another very used
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1
method with promising results is proton nuclear magnetic resonance ( H-NMR) spectroscopy,
which, combined with multivariate statistical data analysis, has demonstrated its usefulness in the
characterisation of the ageing process and the discrimination of different vinegar types [19,56].
13 1 13
The application of C NMR, two-dimensional H− C heteronuclear multiple-bond correlation
1 13
(HMBC), and H− C heteronuclear single quantum coherence (HSQC) spectra for the
characterisation and discrimination of Balsamic vinegars of Modena in order to obtain an indirect
indicator of authenticity and a quality control tool have also been studied, although to a lesser
extent [57]. It should be also considered that as vinegar samples contain a high amount of water,
optimising water suppression methods is required, since it is one of the elements that most
impacts the overall quality of the spectrum [58]. Moreover, as NMR generates a complex spectrum
containing information on all proton/carbon bearing compounds, multivariate data analysis such
as principal component analysis or discriminant analysis is employed to develop
classification/authentication models.
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5. Conclusion
The issues mentioned in the sections above are those that have already been identified and
remain the most economically viable forms of adulteration at the present time. However, in the
future, there could be more problems that should be kept in mind. These problems will most likely
concern the growing range of new vinegar types, less common nowadays in the market or the
emergence of other food ingredients that can create new, potential areas of deception when used
improperly.
The diversity of vinegars in the market and the increase in demand makes it necessary to
characterise them to establish quality control parameters. The characterisation of the vinegar
covers different objectives including the authentication and classification of the product based on
quality criteria. Consequently, there is an increasing need for investigating reliable analytical
methods able to detect the possible adulterations and frauds as well as to assess the authenticity
of the vinegar.
In recent years, there has been a growing need to develop fast, cheap, robust and effective
analytical methods that do not require much sample manipulation such as sensors and
spectroscopic techniques (e.g. MIR, NIR, Fluorescence, NMR and UV) coupled to chemometric
tools. These techniques take into account both the individual contribution and the interactions of
the different components presented in the vinegar, generating a global fingerprint of a food
product. However, one of the main disadvantages is their ability to recognise just a limited number
of molecules.
Finally, given the complexity of vinegars, and the fact that they are perceived by the consumer in a
global way, they must be evaluated from a multivariate point of view. For this reason, a new trend
in food authentication based on a combination of more than one of the aforementioned
techniques has appeared. This promising methodology known as “data fusion” should be further
studied for vinegar authentication.
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wine vinegars with protected designation of origin by ICP-OES and chemometric approach. Food Control, 75, 203–
210. doi:http://dx.doi.org/10.1016/j.foodcont.2016.12.006.
60 Guerrero, M. I., Herce-Pagliai, C., Cameán, A. M., Troncoso, A. M. & González, A. G. (1997). – Multivariate
characterization of wine vinegars from the south of Spain according to their metallic content. Talanta, 45, 379–386.
doi:10.1016/S0039-9140(97)00139-2.
61 Perini, M. et al. (2014). – Stable Isotope Ratio Analysis for Verifying the Authenticity of Balsamic and Wine Vinegar.
Journal of Agricultural and Food Chemistry, 62, 8197–8203. doi:10.1021/jf5013538.
62 Commission Regulation (EC) (2008). – Commission Regulation (EC) No 555/2008 of 27 June 2008 laying down
detailed rules for implementing Council Regulation (EC) No 479/2008 on the common organisation of the
market in wine as regards support programmes, trade with third countries, production potential and on controls in
the wine sector. Official Journal of the European Union. L 170/1-80.
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Jean-François Morin*, Eric Jamin, Sophie Guyader, Freddy Thomas
Eurofins Analytics France, Nantes, France
*E-mail corresponding author: [email protected]
https://doi.org/10.32741/fihb.16.coffee
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A great variety of coffee products can now be purchased. International coffee trade is conducted
almost exclusively in green coffee. However, consumers are nowadays offered roasted coffee
beans, roasted and ground coffee, as well as liquid and dried coffee extracts (soluble coffee).
Furthermore, coffee can be mixed with coffee substitutes, and also sold as roasted and ground
blends or as dried extracts. Whole-bean roasted coffee may also be soaked with liquid flavouring
agents to produce flavoured coffees. Finally, dried coffee extracts already containing milk solids
(café au lait, cappuccino) exist on the market. Decaffeinated forms of each of these coffee
products are also available.
The different varieties of coffee bean and the region where the coffee is grown may give rise to
products of different qualities that are more or less popular with the consumer. This in turns leads
to price differences on the market and the potential for adulteration or misrepresentation by a
dishonest trader. A popular component of the Western diet, coffee is also an important
commodity in international trade upon which the economies of a number of countries are
particularly dependent. In 2010 the International Coffee Organization (ICO) estimated total coffee
sector employment at about 26 million persons in 52 producing countries[4]. Thus, the coffee
industry itself has devoted considerable time and effort to ensuring both the quality and
authenticity of its product, and to developing suitable analytical techniques for this purpose.
In the last 30 years, the coffee market has seen the emergence of an increasing number of
initiatives related to fair-trade and sustainability. Often marked with a label on the coffee
packaging, these labels certify the sustainability of coffee production and the respect of
smallholder producers by improving their conditions of trade (e.g. more equitable and more stable
prices). In the coffee market, most extended programmes are UTZ Certified and the Rainforest
Alliance, which merged early 2018, and the Max Haavelar Foundation. According to Fairtrade
International, fair-trade coffee farmers produced an estimated 560 900 tonnes of coffee in 2015
(approximately 6 % of the worldwide production).
1. Product Identity
1.1. Definition of the product and manufacturing process
Green coffee may be produced by either a wet or dry process. The wet process involves washing
the coffee cherries and transferring them to depulping machines which remove the outer skin and
most of the pulp. This process leaves some of the pulp mucilage on the parchment shells which
encase the coffee bean and this remaining mucilage is fermented and washed away with clean
water. The beans are then dried and the inner husk known as 'parchment' is broken by rollers and
removed. Further rubbing removes the film or 'silverskin' which closely adheres to the coffee bean.
The dry process involves drying the fresh ripe cherries in the sun for up to three weeks. The dried
coffee cherries are dehulled mechanically to remove the outer skin, pulp, 'parchment' and the
'silverskin' to leave the clean, naked, green coffee beans.
Coffee is usually traded as green coffee beans, a state in which they can be kept without loss of
quality or taste. It is roasted and further processed in the purchasing country. Roasting brings out
the aroma and flavour that is locked inside the green coffee beans. The roasting process involves
the heating of the green beans at about 200 °C, followed by fast cooling to stop the process. Once
roasted, coffee should be used as quickly as possible before the fresh roast flavour begins to
diminish.
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Instant coffee (soluble coffee) is also produced in the coffee growing countries and may be traded
packed ready for retail sale or in bulk for re-packaging in the country of receipt for national
consumption or for further export. Instant coffee is the dried water-extract of roasted, ground
coffee. Roasted, ground coffee is placed into columns known as percolators through which hot
water is fed in a counter-current process. The extract is further concentrated and may be traded in
bulk as such or dried to produce soluble coffee solids. Instant coffee is sold in three forms, which
relate to the drying process of the soluble coffee extract. Instant coffee powder is formed by spray
drying the extract; coffee granules are formed by agglomerating this powder with steam; and
freeze-dried coffee is formed by removing moisture from the extract under vacuum (sublimation)
at much lower temperatures than spray drying. Freeze-drying is more energy expensive but is
gentler on the product as less heat is applied to evaporate the water content. Consequently,
freeze-drying is used for the finer and more expensive blends of instant coffee.
Decaffeinated coffee is produced from green beans. Three different extraction processes slightly
differing from each other are in use in the industry. Basically a solvent is circulated around the
water soaked beans and this causes the caffeine to be released. The most widely used and less
costly is extraction with an organic solvent such as methylene chloride (also known as
dichloromethane) or ethyl acetate, an ester that is found naturally in fruits and vegetables. The
second method is water processing: water is used as a solvent to extract the caffeine. In the third
approach, carbon dioxide in supercritical state under a pressure of 250 to 300 bar circulates
through a bed of green beans. At the end of the process, caffeine content is usually reduced from
1–2 g% to 0.02–0.3 g% [5].
The ICO was formed in 1962 under the auspices of the United Nations. It is a inter-government
body comprising 51 coffee importing and exporting countries which aims through international co-
operation on trade in coffee to achieve economic diversification and development of coffee-
producing countries, increased coffee consumption, price stabilisation and improved economic
relations between coffee exporting and importing countries. The ICO is well regarded for its
statistical services and its role as the international forum for discussing all issues affecting the
world coffee market. It also co-ordinates a number of projects (most of which deal with marketing,
pest/disease/quality problems or sustainability) and holds seminars on issues such as the
environmental aspects of coffee production and the use of the futures market.
The International Coffee Agreement 2007 is the legal agreement which sets out how these
objectives will be met [6]. In this document, the different coffee products are defined for
harmonising data collection, statistics and trade among producing and importing countries. On the
other side, the International Standard Organisation has issued a standard “Coffee and coffee
products – Vocabulary” (ISO 3509:2005) [7] also for setting the definitions of coffee products. The
same terms, such as “Roasted coffee” or “Decaffeinated coffee” can be found in both documents,
but definitions are largely consistent. Roughly ICO definitions are more statistically oriented
whereas ISO focuses more on quality and process.
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Table 1: Comparison of the definition of coffee and coffee products between ICO and ISO
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Among these standards, two of them have a special application to instant coffee authenticity. The
standard “Instant coffee - Criteria for authenticity” (ISO 24114:2011) [8] specifies criteria for
authenticity of soluble (instant) coffee. Its purpose is to identify adulterated soluble coffee,
defined as a “product prepared by the co-extraction or the separate extraction of roasted coffee
beans and of raw or roasted materials other than coffee beans, where the product is sold as pure
soluble coffee and the addition of the non-coffee bean material is not declared on the label”. The
aim is to avoid incorrect declarations that adulterated products with cheaper coffee substitutes
are 100 % pure soluble coffee. The standard focuses on two different parameters: total glucose
and total xylose, the values of which must not exceed certain limits (respectively 2.46 % and
0.45 %) for the instant coffee sample to be declared authentic.
The standard is based on a standardised method looking at the carbohydrate content of the
instant coffee, under the reference “Instant coffee - Determination of free and total carbohydrate
contents - Method using high-performance anion-exchange chromatography” (ISO 11292:1995)
[9]. The free and total carbohydrate profiles in soluble coffee are determined by anion exchange
chromatography with pulsed amperometric detection (AE-PAD).
For roasted coffee, the German standard method “Analysis of coffee and coffee products -
Determination of 16-O-methyl cafestol content of roasted coffee - HPLC-method” (DIN
10779:2011) [10] can also be used for authentication purposes. It is used to quantify the amount
of 16-O-methylcafestol (16-OMC) in roasted beans originally, even if applications to green coffee
beans and coffee brews have also been described in the literature [11]. It is based on the
observation that 16-OMC is present exclusively in Robusta.
1.2.2. EU legislation
Beyond general regulations on food products, such as the General Food Law (Regulation EC
178/2002), the European Union has set up several regulations dealing with coffee products.
The general EU Regulation 1169/2011 [12] on the provision of food information to consumers,
combines two Directives into one legislation: 2000/13/EC - Labelling, presentation and advertising
of foodstuffs, and 90/496/EEC - Nutrition labelling for foodstuffs. Among other themes, it deals
with the labelling of origin. No specific rules have been set up for coffee, the general principle that
“information shall not be misleading” applies. Voluntary provenance labels (i.e. indication where
the green coffee was grown) can be made in relation to product claims such as ‘100 % Brazilian
coffee’.
This regulation also stipulates a list of foods, including the following coffee products, which are
exempted from the requirement of the mandatory nutrition declaration:
● Products covered by Directive 1999/4/EC of the European Parliament and of the Council
of 22 February 1999 relating to coffee extracts and chicory extracts,
● Whole or milled coffee beans and whole or milled decaffeinated coffee beans.
Directive 1999/4/EC [13] relating to coffee extracts and chicory extracts determines which
substances may be added during manufacturing of these products, lays down common rules
concerning the packaging and labelling of such extracts and specifies the conditions under which
particular designations may be used for some of these products. It simplifies the legislation
previously regulated by Directive 77/436/EEC.
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It defines coffee extracts as “the concentrated products obtained by extraction from roasted
coffee beans using only water as the medium of extraction and excluding any process of hydrolysis
involving the addition of an acid or a base”.
In particular it stipulates that “coffee extract must contain only the soluble and aromatic
constituents of coffee”, apart from those insoluble substances which it is technically impossible to
remove, and insoluble oils derived from coffee.
It controls the composition of three types of coffee extracts which differ in terms of their coffee-
based dry matter content:
● Dried coffee extract: not less than 95 % by weight,
● Coffee extract paste: from 70 % to 85 % by weight,
● Liquid coffee extract: from 15 % to 55 % by weight.
Liquid coffee extract is specifically allowed to contain edible sugar provided the sugar content in
the final product does not exceed 12 % by weight. The Directive does not permit coffee extract in
solid or paste to contain any substance other than those derived from its extraction.
This Directive also states that the term 'decaffeinated' can only be applied to coffee extracts which
have an anhydrous caffeine content of not more than 0.3 % by weight of its coffee-based dry
matter content.
The Directive does not cover roast and ground coffee.
According to Directive 2009/32/EC [14], solvents can be used for decaffeination of coffee in the
European Union. There are maximum residue limits restrictions for the extraction solvents such as
methyl acetate (20 mg/kg in the coffee), dichloromethane (2 mg/kg in the roasted coffee) and
ethylmethylketone (20 mg/kg in the coffee). In the United States, according to the FDA, methylene
chloride may be present in coffee as a residue from its use as a solvent at a level not to exceed 10
parts per million in decaffeinated roasted coffee and in decaffeinated soluble coffee extract
(instant coffee) [15].
Directive 2002/67/EC [16] on the labelling of foodstuffs containing quinine,and caffeine sets up
specific rules for protecting consumers and providing them with clear information on the presence
of these compounds.
Where a beverage which is intended for consumption without modification, or after reconstitution
of the concentrated or dried product, contains caffeine, from whatever source, in a proportion in
excess of 150 mg/l, the following message must appear on the label in the same field of vision as
the name under which the product is sold: "High caffeine content". This message shall be followed
by the caffeine content expressed in mg/100 ml.
However, this obligation does not apply to beverages based on coffee, tea or coffee or tea extract
where the name under which the product is sold includes the term "coffee" or "tea".
One Protected designation of origin (PDO) and one protected geographical indication (PGI) have
been granted by the European Union:
● Café de Colombia (PGI) in Regulation (EC) 1050/2007 of 12 September 2007 [17];
● Café de Valdesia (PDO) in Regulation (EU) 2016/1043 of 15 June 2016 [18].
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2. Authenticity issues
2.1. Identification of current authenticity issues
As mentioned previously the two coffee species of commercial importance are Arabica and
Robusta. Producer countries and coffee traders are mainly interested in being able to recognise
the country of origin of coffees, whereas food processors and regulatory authorities are interested
in checking on compliance of the declared composition in commercial blends and in the detection
of adulteration by addition of substitutes or other ingredients
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Coffee buyers or roasters are paying more and more attention to the cultivar of the products they
buy. If some introgressed cultivars are preferred because of specific properties, coffee buyers want
to check if purchased coffee batches actually originate from the expected species. Secondly it has
been shown that introgression can have a negative impact on the cup quality of cultivars derived
from the Timor Hybrid. Consequently, coffee buyers or roasters may wish to assess whether the
coffee they are purchasing comes from introgressed varieties [25]. Finally it has been
demonstrated that the variety characteristics are not stable from one harvest to the next making it
necessary to use at least two harvest dates for each variety [26]. Therefore there might be a
concern about procurement quality and stability in time.
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coffee. It is based on the observation that 16-OMC is present exclusively in Robusta, whereas
other, more abundant diterpenes, such as cafestol and kahweol, cannot be used for this
discrimination.
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models correctly identified all authentic Robusta green coffee beans from Cameroon and Vietnam
and 94 % of those from Indonesia. Moreover, PLS-DA afforded independent models for Robusta
samples from these three countries with sensitivities and specificities of classifications close to
100 % and for Arabica samples from America and Africa with sensitivities of 86 and 70 % and
specificities to the other class of 90 and 97 %, respectively [38].
1 13
Using both H-NMR and C-NMR spectroscopy, it has been shown that metabolite levels in coffee
were significantly different between the Arabica and Robusta species, and secondarily influenced
1
by geographical origins [39]. OPLS-DA models performed on H-NMR data led to a clear separation
of samples according to their origin: fatty acids, chlorogenic acids and lactate and finally acetate
and trigonelline were shown to be the main compounds characterising the American, African and
Asian samples respectively. The analytical approach presented here confirmed the potential of
joint NMR analysis and statistical treatment in coffee authentication [40]. Classification models
were built on aqueous NMR profiles allowing the distinction of 192 coffees on countries or
continents of origin [41]. More precisely, 50 samples of Colombian have been differentiated from
22 Asian, 12 African and 108 other American origins. Although the discrimination was based on the
global fingerprint, fatty acids, acetate and caffeine were identified to having a particular part in the
differentiation. However, some impacts of roasting processes were observed on spectral profile as
well as the post-harvest processes, the ripening periods and the year of harvest.
NIR spectroscopy has also demonstrated its potential in geographical origin authentication. Fourier
transform infrared spectroscopy (FTIR) following solvent extraction permits examination of
molecular variation to distinguish degrees of roast and country of origin, as between Columbia,
Costa Rica, Ethiopia and Kenya [42]. Near-infrared spectroscopy (NIR) has been used to distinguish
geographic origin and genotype of samples grown in Brazil [43].
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differentiation of Arabica and Robusta coffees. Indeed, the reference method DIN 10779:2011 [10]
has proven the existing correlation between the 16-OMC concentration and the Robusta rate in
Arabica roasted coffee. In parallel, kahweol was shown to be a key compound in species
differentiation and was considered as a marker of Arabica species, although this compound is
structurally very close to cafestol, compound present in both species.
1
Blend compositions were determined by H-NMR spectral fingerprints with a high accuracy for 56
mixtures in aqueous solution using Orthogonal - Partial Least Square (OPLS) regression models
[53]. This NMR method was proven to be a substitute for the official method because it requires
only limited preparation, thus avoiding the loss of analytes. It was also shown that this technique
could reach low limits of detection and quantification (5 and 20 mg/kg, respectively). This
performance is adequate to detect the presence of Robusta at percentages lower than 0.9 % and
down to 0.2 %, thus lower than the official method by HPLC (about 2 %) [11]. Furthermore, a
recent paper has proven the presence of 16-OMC, a marker of Robusta, in ground roasted Arabica
coffee in the order of 1-2 % [54]. Consequently the limit of quantification for Robusta content
must be defined at 5 % and 10 % respectively in roasted and green Arabica coffee, in order to
avoid false negative results. Moreover, this recent paper detected 2 doubtful market samples of
Arabica coffee with adulterations at levels up to 30 % (w/w) in a panel of 60 retail purchased
coffees using a limit of detection at 1 % and of quantification at 4 % [54].
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aroma compounds which are formed during roasting. Arabicas contain (due to their high sucrose
content) considerably higher amounts of steam-volatile furans, hydroxymethylfurfural and some
aliphatic sugar degradation products than Robustas [58]. In a recent paper, a comparison between
1
GC-C-IRMS, GC-MS, and H-NMR was carried out to discriminate coffees from Colombia versus
nearby countries (Brazil and Peru). According to the authors, results show that the quality of the
classifiers depends mainly on the number of variables included in the analysis, which does not
favour GC approaches [59].
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racemosa) and one Racemosa (C. racemosa) accession. Six leaf rust resistant Arabica were also
included. Authors concluded that that it is possible to use these SSRs for coffee variety
identification. Single Nucleotide Polymorphism (SNP) has also been studied for identification of
coffee germplasm with good results. A panel of 180 SNPs has been validated on 25 C. arabica and
C. canephora accessions from Puerto Rico [65]. All the Robusta accessions were differentiated, as
well as 10 out the 12 Arabica accessions (the 2 remaining ones were considered as synonymous).
All these tools are available for coffee players to assist in coffee germplasm management, quality
control of planting material propagation, coffee cultivar authentication and protection of varietal
rights in the international coffee community.
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5. Conclusion
Coffee authentication is a major concern for the coffee sector. The product itself, once roasted,
ground or processed as instant coffee, can be easily adulterated. Furthermore coffee is one of the
most appreciated and valued food commodities. Extensive research has been carried out on coffee
authentication over the last few decades with the results that robust authentications methods are
now available for the industry throughout the supply chain in order to ensure that genuine
products are delivered to consumers.
The problem of determining the proportion in blends or the adulteration of Arabica with Robusta
has been addressed and there are techniques that provide a good estimation of mixtures.
However, under pressure of changing climate conditions, new varieties are being created by
breeding Arabica and Robusta cultivars, for instance. Current differentiation between these two
species is becoming more and more complex. New knowledge is needed in the future to ensure
accurate results and to avoid false positives.
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38. Alonso-Salces R.M., Serra F., Reniero F. & HÉberger Ká. (2009). – Botanical and Geographical Characterization of
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48. Oliveira M., Ramos S., Delerue-Matos C. & Morais S. (2015). – Espresso beverages of pure origin coffee: Mineral
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Tea and flavoured tea
Stephanie Heaney*, Tassos Koidis
Institute for Global Food Security, Queen's University Belfast, United Kingdom
*E-mail corresponding author: [email protected]
Jean-François Morin
Eurofins Analytics France, Nantes, France
https://doi.org/10.32741/fihb.17.tea
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In Europe, tea is mainly sold by supermarkets and convenience stores which may sell their own-
label tea as well as the major brands from tea companies. Some retailers specialise in upper-end
tea blends and also pack their own products.
1. Product Identity
1.1. Definition of the product and manufacturing process
Tea is derived solely from the leaves of the plant species Camellia sinensis. There are many stages
in tea processing which transforms the fresh shoots and leaves of the tea plant into dried leaves
for brewing an infusion. Immediately after harvesting the leaves are brought to the factory for
processing. It is the processing procedure that determines the type of tea produced. The main
types of tea produced are; white, black, oolong and green. The manufacturing process for these
types mainly differ in the degree of enzymatic oxidation or interchangeably known as the
‘fermentation’ process. In the processing of green and black teas, the fresh leaves are left to
wither until the moisture content is reduced to a degree which depends of the type of variety of
the tea [8]. The loss of water results in the concentration of polyphenols compounds and a
deterioration of the leaf structural integrity. Withering is important for aroma development [9]
and to prepare the leaf for rolling and/or maceration. If the leaf is still turgid when it is rolled or
macerated this prevents efficient mixing of the cellular components important for initiation of
oxidation or generation of aroma. The withered leaves are rolled and crushed to initiate the
oxidation of tea polyphenols. Green tea leaves are dried after rolling to prevent further chemical
changes. Tea leaves which have been macerated are known as ‘dhool.’ The preparation of the
brew is simple, and involves adding hot water over the processed, dry tea leaves.
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Oolong tea: traditionally from China's Fujian province and Taiwan, oolong tea is produced by
withering, then partial aeration or tumbling [11]. During the tumbling process, the edges of leaf
become bruised and partially oxidised. The drying process is called roasting and sometimes the tea
is heated and dried repeatedly until the process is completed. The degree of fermentation, which
varies according to the chosen oxidation duration, can range from 8–85 %, depending on the
variety and production style. The name oolong tea came into the English language from the
Chinese name which means black dragon tea or dark green teas.
Green tea: for its manufacture, the withered leaf is steamed (Japanese style) or pan fired (Chinese
style) and rolled before drying. This is done to prevent the veins in the leaf breaking and thus
preventing oxidation of the leaf [11]. In green tea processing, once the leaves have been dried,
they can also undergo orthodox rolling or CTC. A brewed green tea is typically green, yellow or
light brown in colour. Most green tea is quite light in colour and only mildly astringent.
White tea: this tea was originally established in Fujian Province, China. Authentic white tea is
produced on a very limited scale, picked for only a few weeks each year in Fujian. It is made from
the unopened buds of Camellia sinensis, which contain fine white filaments on the surface. The
name is associated with these silvery white hairs on the unopened buds. For optimum quality of
white tea, it is essential that shoots and leaves are gently plucked to minimise damage. The buds
plucked are usually shielded from sunlight during growth which results in a reduction of
chlorophyll from sunlight. The brew of white tea is usually very pale in colour. This type of tea is
not as popular as black or green tea. White tea processing involves rapid drying of the freshly
harvested leaves to inactivate the enzymatic reactions [12].
Pu-erh (or Pu'er) tea: it is a variety of aged dark tea which is produced in the Yunnan province of
China. The tea leaves undergo microbial fermentation and oxidation after they are dried and
rolled. The quality of this tea improves with maturation and time [12].
Tea products can be segmented into three different groups according to the quality of the tea.
They are described in more detail in Figure 2.
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2. Authenticity issues
2.1. Identification of current authenticity issues
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concentration decreased [45]. Another study found that in elevated CO2 conditions there was a
decrease in caffeine, free amino acids and an increase in the concentration of the polyphenol
compounds in the tea plant [46]. The gradual change in the ratio of free amino acids to
polyphenols in shoots will ultimately cause deterioration of black tea quality [47]. Research has
demonstrated that higher levels of amino acids can contribute to higher quality green tea and
higher quantities of polyphenol compounds are positively associated with black tea quality [46].
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compounds including: tea catechins, gallic acid, purine alkaloids, theanine in tea because of its high
efficiency and high resolution [51]. The quantification of catechins using HPLC has been used to
detect geographical origin of tea [52–54].
ISO 14502-2 [35] specifies a HPLC method for the determination of the total catechin content of
tea from the summation of 9 individual catechins. It is applicable to both leaf and instant green tea
and has precision limitations to black tea. Gallic acid, theogallin and caffeine can also be
determined by this method. ISO 19563 [36] specifies a HPLC method for the determination of total
theanine in tea. There is currently no standard method for the quantification of theaflavins
however this is under development by ISO.
3.2.2.1. IRMS
Mass spectrometry is one of the most sensitive techniques that can be used for identification of
compounds. IRMS is a rapid, reproducible technique. Stable isotope signatures of both tea leaves
and tea infusions have been investigated to identify the geographical origin in several studies.
Results have demonstrated the potential for IRMS to determine geographical origin in tea samples
[68,69].
3.2.2.2. NMR
NMR has been widely used for metabolic profiling in medicinal plants. It provides a very fast and
detailed analysis of the biomolecular composition of crude extracts. NMR spectrum is a physical
characteristic of a compound and thus highly reproducible. NMR has demonstrated the ability to
identify authenticity issues associated with tea including quality and geographical origin [67].
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3.2.2.3. NIR
NIR spectroscopy is a fast, accurate and non-destructive analytical tool. Many studies have
demonstrated the ability for NIR spectroscopy and multivariate calibration to quantify the
chemical composition of teas and classify tea products into different categories [55,56], varieties
[57–63], age [64] and geographical origin [39,57,65,66]. The NIR applications in tea studies show
great potential for the instrument to be applied in the industry to detect authenticity parameters.
Handheld portable NIR spectrometers could also be implemented online during tea production or
used at tea auctions to verify authentication.
3.3.1.1. E-nose
The E-nose is designed to mimic the mammalian sense of smell by producing a composite response
unique to each odorant. As an important quality factor of tea, aroma depends upon the amount of
volatile organic compounds and their ratios. Compared with the conventional methods, it is an
increasingly reliable, fast, and robust technology. Over the last decade, numerous applications of
E-nose in tea quality detection have been reported and many studies have been dedicated to
improve the capability. Preliminary results from studies have demonstrated the ability of the E-
nose to be a valuable method for targeting potential and future tea authenticity issues including;
tea grades [77,78], types [79], varieties [80,81], categories [82], geographical origin [80,83] and
storage times [84].
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3.3.1.2. E-tongue
The E-tongue is an analytical instrument that artificially reproduces the taste sensation [85]. The
taste of tea infusions is the influential attribute of sensory information. It is an important factor in
both assessing tea quality and classifying tea grades. E-tongue instruments have shown good
precision, accuracy and reliability, but they are time-consuming, destructive and unsuitable for
online monitoring. Thus, this technique opens up new avenues in taste sensing and could be
successfully implemented in the near future for tea analysis. Studies have demonstrated the ability
of the E-tongue to discriminate tea varieties [86,87], geographical origin [88], grades [88–94]and
fermentation degree [95]. The benefits are still unclear over traditional tea tasters as it is time
consuming, not practical and does not give the same resolution as a human taster.
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Sensory analysis Visual, olfactory and taste scores Tea grades, adulteration
Macroscopic observation Visual assessment Adulteration
Sieving Foreign bodies Adulteration
HPLC Catechins and theaflavins Geographical origin
HPTLC Catechin compounds Geographical origin
CE Catechins, caffeine, theanine Geographical origin
and other amino acids
IRMS Catechin and theaflavins Geographical origin
NIR Catechin compounds Tea categories, geographical origin, varieties, age
NMR Amino acids, organic acids, Geographical origin and quality
caffeine and catechins
SSR fingerprinting Genome Varietal identification
SNP fingerprinting Genome EST transcriptome Varietal identification
E-Nose Volatile organic compounds Tea grades, types, varieties, categories, geographical
origin, storage times
E-Tongue Catechins, amino acids and Tea varieties, geographical origin, grades,
caffeine fermentation degree
Multi-spectral imaging Image analysis Tea categories, grades, brands
Hyperspectral Imaging Image analysis Tea grades, quality
Normal Camera Imaging Image analysis Tea grades, varieties, colour
5. Conclusion
Tea is recognised as one of the world’s most popular beverages and the authenticity of tea relies
on many factors linked to the chemical composition of the final product. Global and EU legislation
and standards have been developed to ensure quality and safety of tea products. The most
problematic authenticity issues discovered in the industry to-date include mislabelling of tea
grades and geographical origin.
Wet chemical analysis is currently used for the determination of the main quality parameters of
tea, however, it would be more efficient to use alternative methods which are rapid, non-
destructive and not labour intense. Looking into the future, spectroscopic, electronic or computer
vision sensors would be ideal for detecting authenticity in tea products. Portable sensors could be
utilised in tea factories or by purchasers to determine the chemical composition of the product.
They would be beneficial at auctions to verify the tea leaf grading process and the quality of the
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product. The studies which investigate the use of these rapid techniques have demonstrated early
evidence that these recently developed technologies have been equally reliable as
chromatographic methods which are standard methods used to test for authenticity by regulatory
bodies.
It is significant that enhanced detection methods are developed and utilised in industry to ensure
tea authentication. Food NIR or NMR fingerprinting approaches are expected to become very
effective methods in authentication verification aiming at products with complex compositions
such as teas. These approaches aim to capture as many compounds or features as possible to gain
a comprehensive insight into the composition of the sample. A comparison of authentic samples
may allow revealing mislabelled or adulterated products.
Global climate change will have a great impact on the growth of tea, its end quality and finally on
tea prices. The increase in temperature and extreme weather events pose a major threat to the
resilience of tea production systems. Significant change in climate may result in the major tea
producing countries becoming less suitable for tea cultivation in the future. Therefore there is an
increased risk of adulteration of high value origin teas being blended with cheaper teas.
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Olive oil
Diego Luis García González*, Ramón Aparicio, Ramón Aparicio-Ruiz
Instituto de la Grasa, Consejo Superior de Investigaciones Científicas (CSIC), Sevilla, Spain
*E-mail corresponding author: [email protected]
https://doi.org/10.32741/fihb.18.oliveoil
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oil in the market anymore but a formulation of different cheaper edible oils that can avoid
detection when using trade standards. This procedure is harmful for emerging virgin olive oil
markets whose local consumers buy olive oil for its potential health benefits and they would be
concerned if they receive an adulterated oil instead that does not have these benefits.
Hence, effective control of olive oil adulteration requires tighter controls by exporting countries,
clear definitions for olive oil products and uniform labelling regulations. As regards Analytical
Chemistry, the best solution probably lies in multi-disciplinary studies involving instrumental
methods of chemical and sensory analysis, and mathematical procedures.
Table 1: World production and consumption of olive oil (2015/16) of olive oil by country
Source: www.internationaloliveoil.org.
1. Product Identity
1.1. Definition of the product and manufacturing process
Olive oil is the oil obtained from the fruit of the olive tree (Olea europaea sativa L.) to the
exclusion of oils obtained by solvents or re-esterification procedures and of any mixture with oils
of other kinds [4]. Olive oil is defined in three categories: virgin olive oil, refined olive oil and olive
oil.
Virgin olive oil is the oil obtained from the fruit of the olive tree solely by mechanical or other
physical means under conditions that do not lead to alteration of the oil. The result of the process
is an oil that is chiefly a mixture of glycerides, which are esters of glycerol with fatty acids. In
addition, olive oil contains small quantities of many chemical compounds (Table 2) that are
commonly used in its characterisation and authenticity [6,7]. The generic concept of virgin olive oil
contains four different types: extra-virgin olive oil, virgin olive oil, ordinary virgin olive oil and
lampante virgin olive oil although the category ordinary virgin olive oil is not accepted by all the
regulatory bodies (as for example in the EU).
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Olive oil
Table 2: Ranges of the chemical components of virgin olive oil [8]. Information from fatty acid, triacylglycerides and
squalene are given in percentages while the rest is given in mg/kg. Note: tr, traces; nd, not detected; a, values exclusively
circumscribed to some major Spanish and Italian cultivars
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The free acidity, expressed as oleic acid, and the organoleptic characteristics have been the
parameters used to define these categories according to the trade standard of the International
Olive Council [4]. Extra-virgin olive oil, a gourmet oil highly prized for its delicious flavour, tops all
olive oil categories in terms of the strictest quality parameters.
Refined olive oil is obtained by refining virgin olive oil under conditions which do not lead to
alteration of the initial glyceridic structure. Olive oil is the oil consisting of a blend of virgin and
refined olive oil fit for consumption. Olive pomace oil is obtained by solvent extraction of the olive
residue that remains after mechanical extraction of the virgin olive oil, made edible by refining
methods. There are three different olive-pomace oils: olive-pomace oil, crude olive-pomace oil and
refined olive-pomace oil. The first one is the oil comprising a blend of refined olive-pomace oil and
virgin olive oil. The second is olive-pomace oil intended for refining while the last is the oil
obtained from crude olive-pomace oil by a refining process which does not lead to alterations in
the initial glyceridic structure [4].
Table 3 shows the limits of the parameters for olive oil designations according to the European
Union. This information describes the characteristics of each designation that are not fully
accepted by all the institutions involved in the olive oil business; in fact, there are notable
disagreements [9]. Thus, Australian and South African standards propose values for palmitic, oleic
and linolenic which are different from those of the IOC and the EU whereas the difference with
Codex Alimentarius is in linoleic and gadoleic acids. The limit values for some 4-desmethylsterols
(i.e. campesterol and stigmasterol) differ between IOC trade standards and standards from other
institutions because the concentrations of those compounds are influenced by the latitude and
altitude of olive tree orchards [10]. These scientific explanations have increased the debate about
how olive oils from new producing regions (mostly in the Southern Hemisphere) can be classified
as genuine without compromising the control of adulteration that a change in limits for these
sterols would mean for the rest of world production. Thus, IOC has included decision trees for olive
oils with percentages of campesterol between 4.0 and 4.5. The objective is to classify those oils as
genuine oils, because they are, but without, however, comprising the fight against olive oil fraud;
although no certainty value is associated to the decision tree yet. In addition, some regulations
such as Australian and South African standards have even established a limit higher than 4.5 while
they do not include any limit for total sterols (Australia and South Africa) and erythrodiol plus
uvaol. With the aim of having a single regulation, a harmonisation program between the IOC, the
EU and Codex Alimentarius is under progress.
The content of waxes is another source of disagreement between IOC/EU and the other
institutions. IOC and EU assign different contents of waxes according to the olive oil designation
(extra, virgin, ordinary, lampante, etc.) while the remaining institutions – Codex, USA, California,
Australia and South Africa – give a value (≤ 250 mg/kg) whichever the designation. The maximum
content of stigmastadienes, which can be used to determine the presence of any refined edible oil
in virgin olive oil, is another source of disagreement among IOC/EU and the other institutions.
Thus, the IOC and the EU recently lowered the limit from 0.10 to 0.05 due to modern analytical
instruments have higher sensitivity with excellent values of precision while Codex and USA
standards have values of 0.15 mg/kg.
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Olive oil
Table 3: Limits of the chemical compounds used as parameters for protecting virgin olive oil designations against
potential adulterations with edible oils [11]
Designations (1) (2) (3) (4) (5) (6c) (7) (8) (9d)
Extra-virgin olive oil ≤0.05 ≤0.05 ≥1000 d4.5 d150 d0.05 d│0.2│ B d2.50
Virgin olive oil ≤0.05 ≤0.05 ≥1000 d4.5 d150 d0.05 d│0.2│ B d2.60
Lampante virgin olive oil ≤0.10 ≤0.10 ≥1000 d4.5a d300a d0.50 d│0.3│ C -
Refined olive oil ≤0.20 ≤0.30 ≥1000 d4.5 d350 - d│0.3│ C -
Olive oil ≤0.20 ≤0.30 ≥1000 d4.5 d350 - d│0.3│ B -
Crude olive pomace oil ≤0.20 ≤0.10 ≥2500 >4.5b >350b - d│0.6│ ≤1.4 % -
Refined olive pomace oil ≤0.40 ≤0.35 ≥1800 >4.5 >350 - d│0.5│ d1.4 % -
Olive pomace oil ≤0.40 ≤0.35 ≥1600 >4.5 >350 - d│0.5│ d1.2 % -
Designations (10d) (11d) (12) (13) (14) (15) (16) (17) (18e) (19)
Extra-virgin olive oil d0.22 d0.01 d0.8 d20 d0.5 d0.5 d0.1 d4.0 <Camp t93.0
Virgin olive oil d0.25 d0.01 d2.0 d20 d0.5 d0.5 d0.1 d4.0 <Camp t93.0
Lampante virgin olive oil - - >2.0 >20 d0.5 d0.5 d0.1 d4.0 - t93.0
Refined olive oil d1.25 d0.16 d0.3 d5 d0.5 d0.5 d0.1 d4.0 <Camp t93.0
Olive oil d1.15 d0.15 d1.0 d15 d0.5 d0.5 d0.1 d4.0 <Camp t93.0
Crude olive pomace oil - - no limit no limit d0.5 d0.5 d0.2 d4.0 - t93.0
Refined olive pomace oil d2.00 d0.20 d0.3 d5 d0.5 d0.5 d0.2 d4.0 <Camp t93.0
Olive pomace oil d1.70 d0.18 d1.0 d15 d0.5 d0.5 d0.2 d4.0 <Camp t93.0
Designations (20d) (21d) (22d) (23) (24) (25) (26) (27) (28) (29)
Extra-virgin olive oil Mf>0 Md=0 ≤35 ≤0.03 ≤1.0 ≤0.6 ≤0.5 ≤0.2 ≤0.2 (2)
Virgin olive oil Mf>0 0<Md≤3.5 - ≤0.03 ≤1.0 ≤0.6 ≤0.5 ≤0.2 ≤0.2 (2)
(f)
Lampante virgin olive - Md>3.5 - ≤0.03 ≤1.0 ≤0.6 ≤0.5 ≤0.2 ≤0.2 (2)
Refined olive oil - - - ≤0.03 ≤1.0 ≤0.6 ≤0.5 ≤0.2 ≤0.2 (2)
Olive oil - - - ≤0.03 ≤1.0 ≤0.6 ≤0.5 ≤0.2 ≤0.2 (2)
Crude olive pomace oil - - - ≤0.03 ≤1.0 ≤0.6 ≤0.5 ≤0.3 ≤0.2 (2)
Refined olive pomace - - - ≤0.03 ≤1.0 ≤0.6 ≤0.5 ≤0.3 ≤0.2 (2)
Olive pomace oil - - - ≤0.03 ≤1.0 ≤0.6 ≤0.5 ≤0.3 ≤0.2 (2)
Note: (1): trans-oleic fatty acid (%); (2): Sum of trans-linoleic & linolenic fatty acids (%); (3): Total sterol content (mg/kg);
(4): Erythrodiol and uvaol content (% total sterols); (5): Wax content: C42+C44+C46 for extra virgin and virgin designations
and C40+C42+C44+C46 for the rest of designations (mg/kg); (6): Stigmastadiene content (mg/kg); (7): Difference between
the actual and theoretical ECN42 triacylglycerol content; (8): Content of 2-glyceryl monopalmitate (2P); B, 2P≤0.9 if total
C16:0d14.0 % or 2P≤1.0 if C16:0>14.0 %; C, 2P≤0.9 if C16:0d14.0 % or 2P≤1.1 if C16:0>14.0 %; (9): Absorbency in ultra-
violet at K232; (10): Absorbency in ultra-violet at K270, if cyclohexane is used, and K268 if iso-octane is used; (11): Absorbency
in ultra-violet (ΔK); (12): Free acidity (%m/m expressed in oleic acid); (13): Peroxide value (in milleq. peroxide oxygen per
kg/oil); (14): Δ7-Stigmastenol (%); (15): Cholesterol (%); (16): Brassicasterol (%); (17): Campesterol (%); (18): Stigmasterol
(%); (19): The value of E-Sitosterol is calculated as : '5,23-Stigmastadienol + Clerosterol + E-Sitosterol + Sitostanol + '5-
Avenasterol + '5,24-Stigmastadienol; (20) Organoleptic assessment: median of fruity attribute (Mf); (21) Organoleptic
assessment: median of defect (Md); (22) Fatty acid ethylesters (FAEEs); (23) Myristic acid (% m/m methylesters); (24)
Linolenic acid (% m/m methylesters); (25) Arachidic acid (% m/m methylesters); (26) Eicosenoic acid (% m/m methylesters);
(27) Behenic acid (% m/m methylesters); (28) Lignoceric acid (% m/m methylesters); (29) Other fatty acids (% m/m
methylesters). a, when the oil has a wax content of between 300 mg/kg and 350 mg/kg it is considered a lampante olive oil
if the total aliphatic alcohol content is ≤ 350 mg/kg or if the erythrodiol + uvaol content is ≤ 3.5 %; b, when the oil has a wax
content of between 300 mg/kg and 350 mg/kg it is considered a crude olive pomace oil if the total aliphatic alcohol content
is > 350 mg/kg and the erythrodiol + uvaol content is >3.5 %; c, Total isomers which could (or could not) be separated by
capillary column; d, quality characteristics; e, Camp, campesterol (%); f, or where the median defect is less than or equal to
3,5 and the fruity median is equal to 0; (2), Palmitic: 7.5-20.0; Palmitoleic: 0.3-3.5; Heptadecanoic: ≤ 0.4; Heptadecenoic: ≤
0.6; Stearic: 0.5-5.0; Oleic: 55.0-83.0; Linoleic: 2.5-21.0.
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The presence of re-esterified oils in olive oils is detected by the quantification of 2-glyceryl
monopalmitate, for which maximum admitted percentages depend on the designation. Values
proposed by IOC/EU are lower than those described in the standards supported by Codex
Alimentarius, California, Australia and South Africa. The free acidity and the peroxide value
associated with olive oil designations are stricter in the standards of California than in the rest of
institutions. Finally, virgin olive oil is widely regulated by IOC as regards sensory assessment by a
complete set of documents, which have been copied by all the other institutions. Differences once
again concern the limits for the medians of defects and fruity attribute associated to extra-virgin
and virgin olive oil designations. Thus, the median of defects for VOO has been raised to 3.5 – to
take into account the uncertainty in the classification of the boundaries of virgin and
ordinary/lampante – in the IOC/EU trade standard/regulation whereas this value has not been
changed in the other standards. Another source of disagreement is the fact that the Californian,
Australian and South African standards also consider these values of medians of defects and fruity
attribute for olive pomace and refined oils.
International regulatory bodies have designed their standards with the information supplied by
their delegates though a high percentage of the parameters qualifying the olive oil designations
and the limits for determining their genuineness were initially proposed by the IOC. The limits for
some parameters are, as already described, at the core of the disagreements among international
regulatory bodies because climate conditions affect the chemical and biochemical pathways that
are responsible for quantitative changes in olive oil chemical composition, and today there is an
increasing number of orchards that are not located at the Mediterranean basin as was the case in
the past.
Harmonisation among international institutions is being developed and this activity has been
identified as a priority objective for the present [9]. The harmonisation should come from the
collaboration among regulatory bodies in order to achieve an agreement for some specific
parameters that are currently the subject of debate. Other actions, such as reducing the number of
standard parameters and methodologies for example, would be beneficial for facilitating
international trade as well. Most of the methods were proposed by the IOC specifically for olive
oils although there are alternatives proposed by other institutions (i.e. AOCS, ISO, IUPAC, FOSFA).
2. Authenticity issues
2.1. Identification of current authenticity issues
It has been proved that fraud has been part of commercial transactions, in one manner or another,
since they were practised in the remote past, and today olive oil is still considered a vulnerable
product in terms of authenticity [6,9]. Fraud can mean ruin for many actors in the olive oil market
like farmers and sellers although the consumer is the ultimate one affected by this dishonest
attitude. Mass media, in fact, do not usually distinguish among food actors that intentionally carry
out this illegal and dishonest activity and those that are simply affected by a one-off unintentional
fail in quality control. Thus, the product’s authenticity of the entire food market is called into
question when the mass media publicise news on fraud, with the real risk that consumers might
decide not to consume olive oil any more even though the potential fraud does not pose a threat
to public health. Consumer perception of the product may be affected negatively despite the strict
controls that are imposed on this product today.
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Authenticity has many aspects, from adulteration and mislabelling to characterisation of protected
designations of origin (PDOs). With so many potential issues to be studied, the great number of
olive oil designations, and the large variety that can be used in adulteration, a questionnaire was
prepared in order to obtain a broad opinion of producers, wholesalers, retailers, researchers,
analysts etc. A first survey was collected in 1996, inside FAIM - a European funded project -, and a
second survey was launched in 2016 inside FoodIntegrity –another European funded project- with
updated questions about olive oil authenticity. Table 4 shows how the priorities of olive oil actors
have evolved in the last twenty years. The importance of protecting virgin olive oil designations
has not decreased and there is a great interest in determining the presence of soft-deodorized
virgin olive oil in extra-virgin ones, and in knowing the traceability of the extra-virgin olive oils. The
authenticity of extra-virgin olive oils is still linked to the classification by means of the sensory
assessment (“Panel Test”) [7], the results of which are questioned by some olive oil actors up to
the point that objective methods based on the quantification of volatiles responsible for sensory
descriptors are being studied as a potential alternative or a complementary action to sensory
assessment. Consumer interest in a reliable geographical declaration of extra virgin olive oil
(EVOO) has increased over the last years but not in the expected percentage. The importance of
‘Typicality’ (distinctive production) is revealed in the surveys that were carried out with
information from consumers. However, a PDO may show or not clear differences in characteristics
compared with other PDOs or non-PDOs.
Table 4: Percentages of the importance of authenticity issues according to answers of olive oil actors to surveys launched
in 1996 – European funded project FAIM – and 2016 – European funded project FoodIntegrity
Legend: VOO, Virgin Olive Oil; EVOO, Extra Virgin Olive Oil; ROO, Refined Olive Oil; 1, this market is banned inside producer
countries but it was an increasing market in some non-producer countries, e.g. Holland, Germany; Source: FAIM,
FoodIntegrity project.
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oils that after being mixed cannot be detected with regular methods. However, to commit this
fraud, the fraudster needs to have an advanced knowledge of olive oil chemistry [12]. On the other
hand, these new adulteration issues are described in terms of feasibility from a chemical/analytical
point of view. In other words, in many cases reliable information is not available about the actual
incidence of these frauds and their importance in the market and they are considered, among
other reasons, because they are included in those malpractices that from a theoretical point of
view would pose difficulties in their detection. In order to identify potential issues in olive oil
authentication, those adulteration cases that are described to prove the potential of a new
method/technology but which do not exist in the real world because as they are not economically
viable should be omitted to avoid confusion. That would be the case of mixtures with more
expensive oils or with oils that are easily detected with existing methods (e.g. mixture of virgin
with seed oils).
A new possible adulteration is the addition of soft-deodorised virgin olive oil to extra virgin olive
oils, that are more difficult to be detected and new strategies are needed [13]. Thus, when soft-
deodorisation at low temperatures (<100 °C) is carried out in a virgin olive oil to remove slight
sensory defects, the resulting soft-deodorised oil is the so-called ‘‘deodorato” or ‘‘deodorato soft”.
After undergoing this thermal process, it can no longer be considered ‘virgin’ according to the legal
definition for ‘‘virgin olive oil” [4]. For that reason, any mixture of a VOO with a ‘‘deodorato” is
considered to be a fraud. The proposed chemical parameters for their detection (pyropheophytins,
alkyl esters) has demonstrated not to be infallible so far, so new analytical strategies are needed.
Another relevant authenticity issue that is gaining importance is the authentication of geographical
provenance. Since production today is moving beyond the Mediterranean countries (USA,
Australia, Argentina, Chile, South Africa) etc., and consumers are aware about commercial
transactions between countries, they demand more information about geographical origin. The
fact that provenance is sometimes presented as an additional value to the product (regardless of
actual quality) within a marketing strategy has resulted in an authenticity issue related with
mislabelling. Thus, today, if the declared origin on the label does not match with the new origin,
then it is considered that the oil clearly fails in its integrity. No standard methods exist in this
regard. However, building a large database with major and minor compounds and the
implementation of an expert system have been suggested for geographical characterization. That
was the case of the SEXIA project [10,14]. Today, new alternatives based on non-targeted
techniques are being developed [15].
Despite the strict regulations in force, advanced knowledge of the chemical composition of olive
oil and other edible oils has brought to the table the possibility of building tailored oils designed to
pass all the controls. This possibility has led researchers to consider other authentication strategies
other than those based on existing methods. Since some compounds have been studied on olive
oils and are not included in the standards, they are being tested for potential authentication
purposes.
Other complex authenticity issues are related to the current use of olive oil as an ingredient to be
incorporated in more complex food formulations. Thus, once the olive oil is mixed with other
ingredients (e.g. canned foods in olive oil), the current methods are difficult to apply since the
mixing changes the natural composition of the lipid fraction. Since the addition of virgin olive oil is
claimed on the label as an additional value in the food formulation to attract consumers, the
authentication of the olive oil content is perceived today as an emerging authenticity issue.
Sometimes, even the highest quality designation of virgin olive oil (“extra virgin”) is mentioned on
the label. In this case, evaluating the quality of virgin olive oils in mixtures with other ingredients is
also difficult considering the migration of compounds between ingredients.
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Table 5: Summary of the relevant methods proposed in the international regulations supported by the International
Olive Council (IOC), Codex Alimentarius, EU, USDA, California State (USA), Australia, and South Africa (Source: [9])
Determination Method
Fatty acid composition (EU) 1833/2015 Annex IV; COI/T20/Doc No 33; AOCS Ce 1f-96
Methyl ester preparation: ISO 5509:2000; AOCS Cc 2-66; COI/T20/Doc No 24
Gas Chromatography: ISO 5508:1990; AOCS Ch 2-91
Trans fatty acid content (EU) 1833/2015 Annex IV; COI/T20/Doc No 33; COI/ T20/Doc No 17 Rev 1;
ISO 15304:2002; AOCS Ce 1f-96; AOCS Ch 2a-94 (Rev 2002)
Sterol and triterpene dialcohols composition (EU) 1348/2013 Annex IV; COI/T20/Doc No 30; COI//T20/ Doc No 10 Rev 1;
ISO 12228:1999; AOCS Ch 6-91 -Erythrodiol + uvaol: COI/T20/ Doc No 30 ;
IUPAC 2431
Wax content COI/T20/Doc No 18 ; AOCS Ch 8-02; (EC) 702/2007 Annex IV
Aliphatic and triterpenic alcohol content COI/T20/Doc No 26 Rev1; (EU) 2015/1833 Annex VI
Difference between the actual and COI/T20/Doc No 20 rev 3; COI/T20/ Doc No 23; AOCS Ch 5b-89; (CE) 2472/97
theoretical ECN 42 triacylglycerol content Annex XVIII
Stigmastadiene content COI/T20/Doc No 11/Rev2; COI/T20/Doc No 16/Rev1; ISO 15788-1:1999;
AOCS Cd 26-96; ISO 15788-2:2003(EC) 656/95 Annex XVII
Content of 2-glyceryl monopalmitate COI/T20/Doc No 23; (EC) 702/2007 Annex VII
Unsaponifiable matter ISO 3596:2000; ISO 18069:2000; AOCS Ca 6b-53
Organoleptic characteristics (EU) 1348/2013 Annex V; Amended by (EU) 2016/1227; COI/T20/Doc No 15
α-tocopherol ISO 9936
Waxes and alkyl esters COI/T20/Doc No 28; COI/T.20/Doc. No 33; (EU) No 61/2011 Annex II
Biophenols COI/T20/Doc No 29
Free acidity COI/T20/Doc No 34; (EU) 2016/1227 Annex I; ISO 660(03); AOCS Cd 3d-63;
AOCS Ca 5140
Peroxide value COI/T20/Doc No 35; ISO 3960; (EU) 2016/1784 Annex III; AOCS Cd 8b-90
Absorbency in ultra-violet COI/T20/Doc No 19 Rev 3/Rev 2; ISO 3656; AOCS Ch 5-91; (EU) 2015/1833
Annex III
Moisture and volatile matter ISO 662; AOCS Ca 2c-25
Pyropheophytins ISO 29841:2009
Insoluble impurities in light petroleum ISO 663; AOCS Ca 3a-46
Flash point FOSFA Int. method; ISO 15267:1998
Trace metals copper, iron and nickel ISO 8294
Traces of heavy metals Lead ISO 12193; AOCS Ca 18c-91; AOAC 994.02
Arsenic AOAC 952.13; AOAC 942.17; AOAC 985.16
Traces of halogenated solvents COI/T20/Doc No 8; (EEC) 2568/91 Annex XI
Waxes fatty acid methyl esters and fatty acid COI/T20/Doc No 31 provisional
ethyl esters by GC using 3g of silica gel
Composition of triaclyglycerols and COI/T20/Doc No 32 provisional; ISO 29822
diacylglycerols by GC in vegetable oils
Refractive Index ISO6320:2000; AOCS Cc 7-25
Iodine value (EEC) 2568/91 Annex XVI
Saponifiable value ISO 3657:2002; AOCS Cd 3-25
Fatty acid in the 2-position of triglycerides ISO 6800:1997; AOCS Ch 3-91
Relative density IUPAC 21011
Oxidative stability index AOCS Cd 12b-92
Note: 1, with the appropriate conversion factor; EU, European Union; EC, European Commission; AOCS, American Oil
Chemists Society; ISO, International Organization for Standardization; COI, International Olive Council; FOSFA, Federation of
Oils, Seeds and Fats Associations Ltd; IUPAC, International of Union of Pure and Applied Chemistry.
Sources: IOC: www.internationaloliveoil.org, Codex: www.fao.org/fao-who-codexalimentarius/codex-home/es/, EU:
ec.europa/agriculture/olive-oil_en, USDA: www.ams.usda.gov/grades-standards/olive-oil-andolive-pomace-oil-grades-and-
standards, California State (USA):www.cdfa.ca.gov, Australia: www.aph.gov.au, and South Africa (SANS) www.sabs.co.za/.
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The determination of pyropheophytins (PPP) is another major point of discrepancy between the
IOC and the associations of new olive oil producing countries (Australia, California, South Africa,
New Zealand). The increment in PPP is associated with the presence of energy in terms of light
and/or temperature during the extra virgin olive oil (EVOO) shelf-life, which provides information
on EVOO freshness, a concept that is not accepted for producer countries structured around the
IOC. The analytical method based on the reverse-phase solid-phase extraction (RP-SPE) has a
critical point in the collection of the analytes in 0.2-0.3 mL of acetone because of its high volatility
which suggests that the injection in the HPLC instrument should be as rapid as possible. The
method allows two kinds of elution, with petroleum ether (40-60 °C) or with petroleum ether (40-
60 °C): ethyl ether (9:1) for removing the lipids.
The concentration of ethyl esters of fatty acids (FAEEs) is among the parameters that have been
recently approved by IOC/EU for determining EVOO quality though there is no causal relationship
between the concentration of these compounds and the sensory assessment, which is the official
method for determining whether a virgin olive oil is or is not extra-virgin. The role of FAEEs is not
accepted by international associations other than the IOC.
The development of standard methods is normally a consequence of industrial or commercial
needs and they are established as standards after their validations by collaborative studies. The
lobbying that groups of chemists carry out in the implementation of methods is becoming
increasingly irrational, so the usefulness of some new methods gets more and more preposterous.
However, the requirement of validation as a prerequisite may prevent standard methods providing
unsatisfactory results being put forward.
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Table 6: Main characteristics of alternative techniques proposed for the authentication of olive oils
Characteristics Techniques
Structural & Pattern Recognition NMR, MS, NIR, FTIR, FT-Raman, DSC, TG, SF.
Note: Nuclear magnetic resonance (NMR); near infrared spectroscopy (NIR), Fourier transform infrared spectroscopy (FTIR)
and Fourier transform Raman spectroscopy (FT-Raman); isotope ratio mass spectrometry (IRMS); inductive coupled plasma-
atomic emission spectroscopy (ICP-AES); atomic absorption spectroscopy (AAS); flame absorption spectroscopy (FAAS);
electrothermal atomization-AAS; mass spectrometry (MS), GC-MS, LC-MS and ICP-MS; elemental analyser-pyrolysis-isotope
ratio mass spectrometry (δ2-H-EA-Py-IRMS) and δ2-H-GC-Py-IRMS; bidimensional chromatography (GC×GC, LC×LC);
supercritical fluid chromatography (SFC); synchronous fluorescence (SF); differential scanning calorimetry (DSC) and
simultaneous thermogravimetry (TG).
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Fatty acids are the most abundant biomolecules in olive oil, and they do not allow vibrational
spectroscopy to get information from minor compounds due to the barrier effect that is exerted
by saponifiable matter in the spectra acquisition [30]. Thus, one of the problems in vibrational
spectroscopy is caused by the interferences of the saponifiable matter (fatty acids and TAGs) when
determining unsaponifiable compounds (i.e. sterols) [9]. One solution is to perform a previous
transesterification of the oil [30]. Unfortunately, those minor compounds are the most informative
compounds for detecting habitual and sophisticated adulterations of oils nowadays.
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Table 7: Basic characteristics of non-standard (in-house) methods proposed for some analytical challenges of the olive oil
authenticity issues
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Table 7 (follow-up)
Note: a, checked by the authors at their labs and in the course of collaborative analyses of European funded projects. b, the
best figure reached in the course of collaborative analyses with blind samples. c, the measurement is carried with the entire
oil but if the measurement is of the unsaponifiable matter, 60 min has to be added to the total analytical procedure. d,
foliar fertilizers can contain K, Fe, Mg, Mn, P and Zn in different proportions, together with other elements (i.e. B, Ca),
which can be presented complexed with amino acids such in the cases of Ca, Fe, Mg, Mn and Zn. Fungicides can contain Cu
among other elements. e, other authors have proposed the study of particular geographical production zones by diverse
series of compounds, and data are analysed by an umpteen different number of statistical procedures, either unsupervised
(e.g. PCA, MDS) or supervised (e.g. LDA, PLS).
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(PGIs) safeguarded from fraudulent labelling? Is the authentication of the geographical origin of
olive oil the great forthcoming challenge? How are the proposed non-targeted techniques
managed in a legal framework (in court proceedings)? How can olive oil authenticity benefit from
the new approaches on big data and data management? Should the olive oil market move toward
a common commercialization as daily oil instead of delicatessen marketing?
These are some questions that highlight the current problems in the field of olive oil research and
compel food scientists to bring continue their efforts to solve them and to find new methods. The
solutions to the current problems of olive oil may come from a high level of chemical
characterization. International institutions, led by the IOC, are tackling the influence of climate and
geographical provenance in the chemical composition of some genuine olive oils by means of a
mathematical algorithm so-called Decision Trees. Although the already accepted Decision Trees [4]
do not have mathematical support to their conclusions yet - it is a matter of time that they have a
statistical probability associated to their conclusions -, the results seem to be acceptable.
9
Minimum detectable adulteration %
8
7
6
5
4
3
2
1
0
Figure 1: Minimum detectable percentage of some edible oils from different vegetable origins when they are mixed with
virgin olive oil by applying the methods described in Table 8
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Table 8: Methods and their analytical parameters to be quantified with the objective of detecting the presence of
extraneous edible oils in olive oils.
Authenticity information
Parameter Compounds
(presence of…)
Note: The minimum detectable percentage of adulteration with some of these oils are shown in Figure 1.
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Table 9: The standard methods for quantifying acyl lipids and fatty acids
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Total Fatty acids:
Methylation with cold methanolic solution of KOH or
GC-FID
double methylation in a methanolic medium with alkaline
Column: Capillary (25-100m×0.2-0.8mm×0.1-0.2μm)
and acid catalysis.
Stationary phase: polyglycol, polyester or cyanopropylsilicone
Fatty acids Trans fatty acids: Carrier gas: Hydrogen
GC-FID
Methylation with cold methanolic solution of KOH. Operation conditions: Temperature gradient
Injection mode: split
Fatty acid in the 2-position:
GC-FID Hydrolysis with pancreatic lipase previously and methylation
in a methanolic medium with alkaline and acid catalysis.
Note: GC, Gas Chromatography; FID, Flame Ionisation Detector; HPLC, High Performance Liquid Chromatography; RI, Refractive Index detector; SPE, Solid Phase Extraction.
Table 10: The standard methods for determining minor compounds
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Injection mode: split
Note: GC, Gas Chromatography; FID, Flame Ionisation Detector; HPLC, High Performance Liquid Chromatography; TLC, Thin Layer chromatography.
Olive Oil
5. Conclusion
The criteria defining the authenticity, purity or genuineness of a food product are numerous and vary from one
foodstuff to another although many generic definitions have been proposed. Concerning virgin olive oil, authenticity
issues may be associated with adulteration with other edible oils but also with designation of origin, olive varieties and
with oils that do not meet the requirements of integrity and good practices in labelling. Trade standards, either at
national or international level, define the chemical characteristics of a genuine oil with much more detail compared to
other vegetable oils. However, considering that consumers expect olive oil to be a foodstuff endowed with reputed
sensory and healthy properties, today the authenticity issues of extra virgin olive oil also reach the sensory properties
of the oil, which would be inside the declared designation.
The great interest of researchers in virgin olive oil authentication shown in the last few years, mostly analysing the
chemical/sensory results by mathematical procedures, has led to an improvement in the control of virgin olive oil
adulteration although the new adulterations - e.g. oils with similar chemical composition (hazelnut oil) - represent a
new challenge for researchers. Regardless the endless discussion on olive oil authenticity over the decades, the
continuous achievement of solutions from analytical chemistry has posed serious problems to the fraudster to commit
adulteration and it can be concluded that only the most sophisticated authenticity issues are challenges for the future
(olive oils spiked with soft-deodorised oils or tailored oils).
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33. Z Zou M.Q., Zhang X.F., Xiao-Hua Q.I., Han-Lu M., Dong Y., Chun-Wei L.I.U., Guo X.U.N. & Wang H. (2009). - Rapid authentication of olive oil
adulteration by Raman spectrometry. J. Agric. Food Chem., 57 (14), 6001-6006. doi: 10.1021/jf900217s.
34. El-Abassy R.M., Donfack P. & Materny A. (2009). - Visible Raman spectroscopy for the discrimination of olive oils from different vegetable oils
and the detection of adulteration. J. Raman Spectrosc., 40 (9), 1284-1289. doi: 10.1002/jrs.2279.
35. Dais P. (2013). – Nuclear magnetic resonance: Methodologies and applications. In: Handbook of Olive Oil. Analysis and Properties, second ed.
(R. Aparicio & J. Harwood eds), Springer, New York. pp 395-430 doi: 10.1007/978-1-4614-7777-8_11.
36. Dais P. & Hatzakis E. (2013). - Quality assessment and authentication of virgin olive oil by NMR spectroscopy: A critical review. Anal. Chim.
Acta, 765, 1-27. doi: 10.1016/j.aca.2012.12.003.
37. Mannina L., D’Imperio M., Capitani D., Rezzi S., Guillou C., Mavromoustakos T., Vilchez M.D.M., Fernández A.H., Thomas F. & Aparicio R.
(2009). - 1H NMR-based protocol for the detection of adulterations of refined olive oil with refined hazelnut oil. J. Agric. Food Chem., 57,
11550-11556. doi: 10.1021/jf902426b.
38. García-González D.L., Mannina L., D’Imperio M., Segre A.L. & Aparicio R. (2004). - Using 1H and13C NMR techniques and artificial neural
networks to detect the adulteration of olive oil with hazelnut oil. Eur. Food Res. Technol., 219, 545-548. doi: 10.1007/s00217-004-0996-0.
39. López-Díez E.C., Bianchi G. & Goodacre R. (2003). - Rapid quantitative assessment of the adulteration of virgin olive oils with hazelnut oils
using Raman spectroscopy and chemometrics. J. Agric. Food Chem., 51, 6145-6150. doi: 10.1021/jf034493d.
40. Tay A., Singh R.K., Krishnan S.S. & Gore J.P. (2002). - Authentication of olive oil adulterated with vegetable oils using Fourier transform
infrared spectroscopy. LWT - Food Sci. Technol., 35, 99-103. doi: 10.1006/fstl.2001.0864.
41. Aparicio R. & García-González D.L. (2013). - Olive oil characterization and traceability. In: Handbook of Olive Oil. Analysis and Properties,
second ed. (R. Aparicio & J. Harwood eds), Springer, New York. pp 431-478. doi: 10.1007/978-1-4614-7777-8_12.
42. Aparicio R. & Luna G. (2002). - Characterisation of monovarietal virgin olive oils. Eur. J. Lipid Sci. Technol., 104, 614-627. doi: 10.1002/1438-
9312(200210)104:9/10<614::AID-EJLT614>3.0.CO;2-L.
43. Camin F., Larcher R., Perini M., Bontempo L., Bertoldi D., Gagliano G., Nicolini G. & Versini G. (2010). - Characterisation of authentic Italian
extra-virgin olive oils by stable isotope ratios of C, O and H and mineral composition. Food Chem., 118, 901-909. doi: 10.1016/j.foodchem.
2008.04.059.
44. Chiavaro E., Cerretani L., Matteo A. di, Barnaba C., Bendini A. & Iacumin P. (2011). - Application of a multidisciplinary approach for the
evaluation of traceability of extra virgin olive oil. Eur. J. Lipid Sci. Technol., 113, 1509–1519. doi: 10.1002/ejlt.201100174.
45. Alonso-Salces R.M., Moreno-Rojas J.M., Holland M.V., Reniero F., Guillou C. & Héberger K. (2010). - Virgin olive oil authentication by
ŵƵůƚŝǀĂƌŝĂƚĞĂŶĂůLJƐĞƐŽĨϭ,EDZĨŝŶŐĞƌƉƌŝŶƚƐĂŶĚɶϭϯĐĂŶĚɶϮŚĚĂƚĂ͘ J. Agric. Food Chem., 58, 5586-5596. doi: 10.1021/jf903989b.
46. Llorent-Martínez E.J., Ortega-Barrales P., Fernández-De Córdova M.L., Domínguez-Vidal A. & Ruiz-Medina A. (2011). - Investigation by ICP-MS
of trace element levels in vegetable edible oils produced in Spain. Food Chem., 127, 1257-1262. doi: 10.1016/j.foodchem.2011.01.064.
47. Benincasa C., Lewis J., Perri E., Sindona G. & Tagarelli A. (2007). - Determination of trace element in Italian virgin olive oils and their
characterization according to geographical origin by statistical analysis. Anal. Chim. Acta, 585, 366-370. doi: 10.1016/j.aca.2006.12.040.
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48. Zeiner M., Steffan I. & Cindric I.J. (2005). - Determination of trace elements in olive oil by ICP-AES and ETA-AAS: A pilot study on the
geographical characterization. Microchem. J., 81, 171–176. doi: 10.1016/j.microc.2004.12.002.
49. Beltrán M., Sánchez-Astudillo M., Aparicio R. & García-González D.L. (2015). – Geographical traceability of virgin olive oils from south-western
Spain by their multi-elemental composition. Food Chem., 169, 350-357. doi: 10.1016/j.foodchem.2014.07.104.
50. Bøwadt S. & Aparicio R. (2003). - The detection of the adulteration of olive oil with hazelnut oil: A challenge for the chemist. INFORM -
International News on Fats, Oils and Related Materials, 14 (6), 342-344.
51. Angerosa F., Camera L., Cumitini S., Gleixner G. & Reniero F. (1997). - Carbon Stable Isotopes and Olive Oil Adulteration with Pomace Oil. J.
Agric. Food Chem., 45 (8), 3044–3048. doi: 10.1021/jf960993d.
52. Spangenberg J.E., Macko S.A. & Hunziker J. (1998). - Characterization of Olive Oil by Carbon Isotope Analysis of Individual Fatty Acids:
Implications for Authentication. J. Agric. Food Chem., 46 (10), 4179-4184. doi: 10.1021/jf980183x.
53. Huang J., Norgbey E., Nkrumah P.N., Opoku P.A. & Apreku T.O. (2017). - Detection of corn oil in adulterated olive and soybean oil by carbon
stable isotope analysis. J. Verbrauch. Lebensm., 12 (3), 201-208. doi: 10.1007/s00003-017-1097-x.
54. Royer A., Gerard C., Naulet N., Lees M. & Martin G.J. (1999). - Stable isotope characterization of olive oils. I - Compositional and carbon-13
profiles of fatty acids. J. Am. Oil Chem. Soc., 76 (3), 357-363. doi: 10.1007/s11746-999-0243-8.
55. Camin F., Larcher R., Perini M., Bontempo L., Bertoldi D., Gagliano G., Nicolini G. & Versini G. (2010). - Characterisation of authentic Italian
extra-virgin olive oils by stable isotope ratios of C, O and H and mineral composition. Food Chem., 118 (4), 901-909. doi:
10.1016/j.foodchem.2008.04.059.
56. Camin F., Larcher R., Nicolini G., Bontempo L., Bertoldi D., Perini M., Schlicht C., Schellenberg A., Thomas F., Heinrich K., Voerkelius S.,
Horacek M., Ueckermann H., Froeschl H., Wimmer B., Heiss G., Baxter M., Rossmann A. & Hoogewerff J. (2010). - Isotopic and elemental data
for tracing the origin of European olive oils. J. Agric. Food Chem., 58 (1), 570-577. doi: 10.1021/jf902814s.
57. Faberi A., Marianella R.M., Fuselli F., La Mantia A., Ciardiello F., Montesano C., Mascini M., Sergi M. & Compagnone D. (2014). - Fatty acid
composiƚŝŽŶ ĂŶĚ ɷϭϯ ŽĨ ďƵůŬ ĂŶĚ ŝŶĚŝǀŝĚƵĂů ĨĂƚƚLJ ĂĐŝĚƐ ĂƐ ŵĂƌŬĞƌ ĨŽƌ ĂƵƚŚĞŶƚŝĐĂƚŝŶŐ /ƚĂůŝĂŶ WKͬW'/ ĞdžƚƌĂ ǀŝƌŐŝŶ ŽůŝǀĞ ŽŝůƐ ďLJ ŵĞĂŶs of
isotopic ratio mass spectrometry. J. Mass Spectrom., 49 (9), 840-849. doi: 10.1002/jms.3399.
58. García-González D.L. & Aparicio R. (2010). - Research in olive oil: Challenges for the near future. J. Agric. Food Chem., 58, 12569-12577. doi:
10.1021/jf102735n.
23
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Vegetable oils
Ramón Aparicio, Diego Luis García González, Ramón Aparicio-Ruiz*
Instituto de la Grasa, Consejo Superior de Investigaciones Científicas (CSIC), Sevilla, Spain
*E-mail corresponding author: [email protected]
https://doi.org/10.32741/fihb.19.vegetableoil
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Vegetable oils
sunflower and the Ukrainian steppes [2]. Geographical provenance and the characterization of oils
produced in particular geographical areas are not aspects of minor importance as this information
can help in health warnings (e.g. Ukrainian sunflower oil contamination).
This chapter does not deal with all the vegetable oils but a set of them: arachis, cottonseed,
evening primrose, maize, palm, palm kernel, rapeseed, safflower, soya, sunflower and argan; olive
oil is studied in an independent chapter.
Note: 1 figures from USA Oilseeds: World Markets and Trade, April 2018 (www.fas.usda.gov). In the case of evening
primrose, China accounts for approximately 90 % of world production.
Sources: USDA Foreign Vegetable Oils (www.fas.usda.gov), indexmundi (www.indexmundi.com) and world atlas
(www.worldatlas.com).
1. Product Identity
1.1. Definition of the product and manufacturing process
Codex Alimentarius (henceforth, Codex) defines edible vegetable oils as foodstuffs which are
composed primarily of glycerides of fatty acids being obtained only from vegetable sources. They
may contain small amounts of other lipids, such as phosphatides, of unsaponifiable constituents
and of free fatty acids naturally present in the fat or oil. These kinds of oils are labelled as virgin
oils, cold pressed oils or refined oils according to their manufacturing process. Codex also defines
these designations. Thus, virgin oils are obtained, without altering the nature of the oil, by
mechanical procedures (e.g. expelling or pressing) and the application of heat only; they may have
been purified by washing with water, settling, filtering and centrifuging only. Cold pressed oils,
according to Codex, are obtained, without altering the oil, by mechanical procedures only (e.g.
expelling or pressing) without the application of heat; they may have been purified by washing
with water, settling, filtering and centrifuging only. Refined edible vegetable oils result from
oilseeds or solvent-extracted oils which have undergone a comprehensive processing to be
deacified in one of the following ways: a) with alkali; b) by physical refining or both; c) by miscella
refining using a permitted food grade solvent, followed by bleaching with absorbent earth and/or
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Vegetable oils
activated carbon or both of them and deodorised with steam without using any other chemical
agent; and d) also including the process of degumming using phosphoric/citric acid. Applying
appropriate quality management systems, the refining process produces oils with consistent
quality.
Oilseeds for producing edible vegetable oils selected for this chapter can have different synonyms
for the same name or various species can be associated to the same name. Following definitions of
oilseeds help to oil identity (Codex):
Arachis oil (synonyms: peanut oil; groundnut oil) is derived from groundnuts (seeds of Arachis
hypogaea L.). More than 70 % of this oil is produced in China (50.4 %) and India (20.3 %).
Cottonseed oil is derived from the seeds of various cultivated species of Gossypium spp. China
(26.8 %) and India (25.2 %) produce more than 50 % of this oil.
Evening primrose oil is derived from the seeds of the evening primrose (Oenothera biennis) plant.
Maize oil (synonym: corn oil) is derived from maize germ (the embryos of Zea mays L.). It is
produced all over the world but USA (55.2 %), China (8.2 %) and Turkey (5.9 %) are major producer
countries.
Palm oil is derived from the fleshy mesocarp of the fruit of the oil palm (Elaeis guineensis). Note:
Palm olein is the liquid fraction derived from the fractionation of palm oil while palm stearin is the
high-melting fraction derived from the fractionation of palm oil. Two countries supply more than
80 % of palm oil: Indonesia (55.2 %) and Malaysia (29.4 %).
Palm kernel oil is derived from the kernel of the fruit of the oil palm (Elaeis guineensis). The
production of this oil in Indonesia (54.2 %) and Malaysia (28.3 %) accounts for more than 80 % of
world production.
Rapeseed oil - low erucic acid (synonyms: low erucic acid turnip rape oil; low erucic acid colza oil;
canola oil) is produced from low erucic acid oil-bearing seeds of varieties derived from the Brassica
napus L., Brassica campestris L. and Brassica juncea L. species. The European Union is the major
producer of this oil (40.4 %) followed by China (26.4 %) and Canada (15.5 %).
Safflower seed oil (synonyms: safflower oil; carthamus oil; kurdee oil) is derived from safflower
seeds (seeds of Carthamus tinctorius L.). Safflower seed oil - high oleic acid (synonyms: high oleic
acid safflower oil; high oleic acid carthamus oil; high oleic acid kurdee oil) is produced from high
oleic acid oil-bearing seeds of varieties derived from Carthamus tinctorius. The main producer of
these oils is the USA followed by India and Mexico.
Soya bean oil (synonym: soybean oil) is derived from soya beans (seeds of Glycine max (L.) Merr.).
USA has a dominant position in soybean (39 %) followed by Brazil (23.8 %) and Argentina (17.9 %).
Sunflower seed oil (synonym: sunflower oil) is derived from sunflower seeds (seeds of Helianthus
annuus L.). Sunflower seed oil - high oleic acid (synonym: high oleic acid sunflower oil) is produced
from high oleic acid oil-bearing seeds of varieties derived from sunflower seeds (seeds
of Helianthus annuus L.). Russia is the largest producer of sunflower oil (17.8 %) followed by
Ukraine (16.7 %) and Argentina (14.8 %).
Argan oil is derived from the kernel of the fruit of the spiny argan tree (Argania spinosa). Morocco
is the exclusive producer country of argan oil.
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Vegetable oils
Edible vegetable oils are mainly consumed after being submitted to a refining process although the
market for crude oils - either virgin or cold pressed oils - has recently increased. Refining is a
homogenous well-established process that has peculiarities for some of the selected oils. Thus,
arachis oil cannot be winterised because of its high melting point – it solidifies at 3°C. Besides,
peanut oil may contain aflatoxin B1 that can be removed in the refining process whether the
standard alkali refining and the washing of the oil are used – detoxifying can then lower the
aflatoxin content to 10-14 ppb – so that a subsequent bleaching operation is essential to reduce it
to less than 1 ppb. The refining of safflower oil has the peculiarity of increasing the content of free
sterols – due to the acid-catalysed hydrolysis of steryl esters in the degumming and bleaching
processes – and a significant reduction of the content of total sterols during the bleaching process
because of reduction of esterified sterol fraction. Crude maize oil has a high content of
phosphorous and a wet degumming process at low temperature is recommended; degumming at
10-30 °C results in the removal of more phosphorous than at 70 °C.
The identity of edible vegetable oils usually requires its characterization with information of the
most defining/characteristic physical-chemical parameters. As the refining process may modify the
original chemical composition (e.g. tocopherols, sterols), the changes of which depend on the
refining process used, the following tables show the values related to crude vegetable oils.
The distribution of fatty acid methyl esters (Table 2), which account for more than 95 % of edible
oil chemical composition, is used most frequently to characterize oils and confirm their
authenticity. The second source of information is the composition of sterols, which is the major
series of the unsaponifiable matter of vegetable oils and as important as fatty acids in food
authentication. In fact, some sterols may be unique to an oil (i.e. brassicasterol). Sterol
composition as shown in Table 3 covers 4-desmethylsterols or also so-called phytosterols or simply
sterols. Table 3 also displays the composition of methyl tocols (tocopherols and tocotrienols) not
only because they are powerful lipid-soluble antioxidants and a major dietary component but also
because their profiles can be used to distinguish vegetable oils; e.g., sunflower seed oil is a good
source of α-tocopherol and palm oil of the tocotrienols. In addition to the detailed chemical
composition specification, essential physical-chemical characteristics of selected crude vegetable
oils (relative density, refractive index, saponification value, iodine value, unsaponifiable matter
content) are given in Table 4, which may be also used as identity criteria. Finally, the stable carbon
isotope ratio is also included but only for maize oil as it is believed that this measurement provides
a better means of detecting foreign oils in this type of oil than other more traditional techniques.
1.2.1. EU Legislation
Unlike olive oil, which is extensively covered in EU regulations, there is no specific EU legislation
for other edible vegetable oils. The only vertical legislation relevant in this area, is Directive
76/621/EEC which limits the level of erucic acid permitted in oils and fats intended for human
consumption, and Directive 80/891/EEC that describes the method for the analysis of erucic acid.
National legislation, in particular in the producing countries, defines permitted processing
conditions such as neutralization, bleaching, hydrogenation, deodorization, and so on. In fact, EU
laws do not provide for a “generally acknowledged definition of food fraud” but there is an
extensive EU legislative framework focused on food safety. Only a general guideline is found in EU
regulations requiring that food labelling, advertising, presentation and packaging “shall not
mislead consumers” [3].
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Vegetable oils
2. Authenticity issues
2.1. Identification of current authenticity issues
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Vegetable oils
although the number of documented incidents may be a small fraction of the actual number since
they usually do not result in a food safety risk and consumers often do not notice them.
Groundnut and sunflower seed oils.
The contamination or adulteration of groundnut and sunflower seed oils with cheaper soya bean
oil has been identified for several years in traded oils containing low concentrations of linolenic
acid whereas according to chemical information pure sunflower seed and groundnut oils should be
free of this acid (≤0.3), unlike the high content of this fatty acid in soya bean oils (4.5-10 %). More
recently (November 2015) the adulteration of groundnut oil was reported in India, the second
major producer of this oil, because the demand for this oil had increased leading to an abrupt
spike in its average price. The Consumer Association of India found that a majority of a large set of
groundnut oil samples was adulterated with palmolein and cottonseed oils among other cheaper
oils. Thus, 7 % of the samples contained less than 10 % of groundnut oil and 43 % contained less
than 20 %. Furthermore, the Food and Drug Administration (FDA) of India reported that traders
blended sunflower seed, groundnut and soybean oil with cheaper cottonseed oil. As regards
groundnut, this is one of the major edible oils in China, besides soybean oil and rapeseed oil, but it
is more expensive than the other two making it prone to adulteration. In January 2012 it was
reported that unscrupulous dealers had mixed cottonseed oil and flavour-enhanced soybean oil
and marked it as peanut oil. In addition, oils such as soybean oil, sunflower oil, canola oil, and palm
oil are also blended into peanut oil in several proportions.
Safflower oil
Safflower oil is a high-priced oil, favoured as a result of its high content of linoleic (C18:2) acid and
almost zero content of the easily oxidised linolenic (C18:3) acid. Sunflower seed oil is closely
related to it, with a high content of linoleic acid but a very low content of linolenic acid. The
similarity of the fatty acid composition and other properties of the two oils has meant that it is
difficult to detect the adulteration of safflower seed oil with sunflower seed oil. The main
producers of sunflower seed oil are adjacent to the main producer of safflower oils, which might
not help end this fraudulent practice.
Other adulterations have been described using safflower oils as an adulterant of virgin olive oil in
scientific papers. However, it is unlikely since safflower oil should be refined first, and then the
determination of stigmastadienes would make that adulteration unprofitable.
Palm oil
A major problem in the early 1980s was the so-called Singapore Cocktail. This was a reconstituted
palm oil made by blending unrelated palm stearin and olein fractions. The blend was much more
severely oxidised than unprocessed whole palm oil. Unfortunately, as both the fractions had
originated from Malaysian palm oil and were usually blended back in something approaching the
right ratio, it was almost impossible to prove by conventional analytical methods that the
manipulation had taken place. Another adulteration with a toxic effect is the addition of the
artificial dye Sudan IV to some palm oil brands from Ghana; Sudan IV is known to cause cancer.
The addition of artificial colouring to palm oil is so widely used that it can be rare to buy any palm
oil that has not artificially coloured. In fact, Solvent red 24, which is used in colouring plastics, or
Anatol dye are added to palm oil by the nefarious traders to improve its redness.
The Roundtable on Sustainable Palm Oil has reported that palm oil made with sustainable and
ethical sourcing claims is at high risk of food fraud; it has been claimed that some manufactures of
palm oil use child labour in harvesting or refining processes.
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Vegetable oils
Recently, oleic-enhanced palm oil interesterified with high oleic acid components, followed by
fractionation using a patented MPOB (Malaysian Palm Oil Board) process, has been product.
However name of “high oleic acid palm oil” is still under consideration for Codex (Codex
Alimentarius, REP17/FO-Rev, 3 March 2017) as are value ranges for its chemical compounds and
physical-chemical parameters.
Palm kernel oil
Another concern is the co-mingling of lauric oils, usually palm kernel and coconut oils. These two
oils have closely related chemical compositions (both contain about 47 % lauric acid) and low
levels of unsaturation. Coconut oil usually trades at a higher price, however, so there is a
temptation to adulterate it with small amounts of palm kernel oil. An equally worrying, if less
prevalent, problem is associated with an oil called babassu. This oil is produced mainly in Brazil and
has no international market. However, its chemical and physical properties are similar to those of
palm kernel oil. There has therefore been a temptation to bring babassu oil into the edible oil
trade by blending it with palm kernel oil.
Palm kernel oil can be fractionated into hard and soft fractions in much the same way as palm oil.
In this case, however, it is the hard fraction that is valuable, as a confectionery butter or cocoa
butter substitute. The by-product is palm kernel olein, which, as in the case of palm stearin, has
only a limited number of outlets and it therefore trades at a lower price than whole unfractionated
palm kernel oil. There is a temptation to dispose of the palm kernel olein by blending it into palm
kernel oil. Levels of about 10 % are difficult to detect. If the palm kernel oil is then used for the
production of hydrogenated palm kernel oil, another useful confectionery fat, the addition of up to
40 % prior to hydrogenation is difficult to detect.
Cottonseed oil
A related case of adulteration occurred in 1983, when cottonseed oil was diluted with palm olein
[8]. The incident was widely publicised by the Malaysian Palm Oil Processing Industry at the time
[8,9] since, although the Malaysians had supplied the palm olein, they had done so as part of an
honest transaction and had not been part of the deception. Cotton is one of the top four GMO
crops produced in the world (83 %) - along with soybean (89 %), canola (75 %), and corn (61 %) -
and approx. 90 % of all US cotton is genetically engineered. GM products are not labelled in some
countries since manufacturers are not required by National Safety Authorities to list the existence
nor the quantity of GM food in a producer’s products (i.e. cottonseed oil) on their labels. Thus, it is
a potential, if not actual, authenticity problem for consumers concerned about consuming GM
foods.
―7―
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Table 2: Composition of fatty acids of the selected crude edible vegetable oils from authentic samples. Sources: [4-6]
FAME 1 2 3 4 5 6 7a 7b 8 9a 9b 10 11
C6:0 nd nd nd nd чϬ͘ϴ nd nd nd nd nd nd nd nd
Vegetable oils
C8:0 nd nd nd nd 2.4-6.2 nd nd nd nd nd nd nd nd
C10:0 nd nd nd nd 2.6-5.0 nd nd nd nd nd nd nd nd
C14:0 чϬ͘ϭ 0.6-1.0 чϬ͘ϯ 0.5-2.0 14.0-18.0 чϬ͘Ϯ чϬ͘Ϯ чϬ͘Ϯ чϬ͘Ϯ чϬ͘Ϯ чϬ͘ϭ чϬ͘Ϯ чϬ͘ϭ
C16:0 8.0-14.0 21.4-26.4 8.6-16.5 39.3-47.5 6.5-10.0 2.5-7.0 5.3-8.0 3.6-6.0 8.0-13.5 5.0-7.6 2.6-5.0 11.5-15.0 6.0-10.2
C16:1 чϬ͘Ϯ чϭ͘Ϯ чϬ͘ϱ чϬ͘ϲ чϬ͘Ϯ чϬ͘ϲ чϬ͘Ϯ чϬ͘Ϯ чϬ͘Ϯ чϬ͘ϯ чϬ͘ϭ чϬ͘Ϯ nd
C17:0 чϬ͘ϭ чϬ͘ϭ чϬ͘ϭ чϬ͘Ϯ nd чϬ͘ϯ чϬ͘ϭ чϬ͘ϭ чϬ͘ϭ чϬ͘Ϯ чϬ͘ϭ чϬ͘ϭ nr
C17:1 чϬ͘ϭ чϬ͘ϭ чϬ͘ϭ nd nd чϬ͘ϯ чϬ͘ϭ чϬ͘ϭ чϬ͘ϭ чϬ͘ϭ чϬ͘ϭ nd nr
C18:0 1.0-4.5 2.1-3.3 чϯ͘ϯ 3.5-6.0 1.0-3.0 0.8-3.0 1.9-2.9 1.5-2.4 2.0-5.4 2.7-6.5 2.9-6.2 4.3-7.2 1.5-3.5
C18:1 35.0-69 14.7-21.7 20.0-42.2 36.0-44.0 12.0-19.0 51.0-70.0 8.4-21.3 70.0-83.7 17-30 14.0-39.4 75-90.7 43.0-50.1 5.0-12.1
C18:2 12.0-43.0 46.7-58.2 34.0-65.6 9.0-12.0 1.0-3.5 15.0-30.0 67.8-83.2 9.0-19.9 48.0-59.0 48.3-74.0 2.1-17 23.3-36.0 65.0-85.4
- 366 -
C18:3 чϬ͘ϯ чϬ͘ϰ чϮ͘Ϭ чϬ͘ϱ чϬ͘Ϯ 5.0-14.0 чϬ͘ϭ чϭ͘Ϯ 4.5-11.0 чϬ͘ϯ чϬ͘ϯ чϬ͘ϯ 8.0-14.1
C20:0 1.0-2.0 0.2-0.5 0.3-1.0 чϭ͘Ϭ чϬ͘Ϯ 0.2-1.2 0.2-0.4 0.3-0.6 0.1-0.6 0.1-0.5 0.2-0.5 чϬ͘ϱ чϬ͘ϯ
C20:1 0.7-1.7 чϬ͘ϭ 0.2-0.6 чϬ͘ϰ чϬ͘Ϯ 0.1-4.3 0.1-0.3 0.1-0.5 чϬ͘ϱ чϬ͘ϯ 0.1-0.5 чϬ͘ϲ nr
C22:0 1.5-4.5 чϬ͘ϲ чϬ͘ϱ чϬ͘Ϯ чϬ͘Ϯ чϬ͘ϲ чϭ͘Ϭ чϬ͘ϰ чϬ͘ϳ 0.3-1.5 0.5-1.6 чϬ͘Ϯ nd
C22:1 чϬ͘ϯ чϬ͘ϯ чϬ͘ϯ nd nd чϮ͘Ϭ чϭ͘ϴ чϬ͘ϯ чϬ͘ϯ чϬ͘ϯ чϬ͘ϯ nd nd
C24:0 0.5-2.5 чϬ͘ϭ чϬ͘ϱ nd nd чϬ͘ϯ чϬ͘Ϯ чϬ͘ϯ чϬ͘ϱ чϬ͘ϱ чϬ͘ϱ nd nd
Note: FAME, fatty acid methyl esters. 1, Arachis oil; 2, Cottonseed oil, 3, Maize oil; 4, Palm oil; 5, Palm kernel oil; 6, Rapeseed (Canola) oil; 7a, Safflower oil; 7b, Safflower high oleic oil; 8, Soya oil; 9a, Sunflower oil; 9b,
Sunflower high oleic oil; 10, argan oil; 11, evening primrose oil; nd, non-detectable, defined as <0.05 %; nr, not reported.
Table 3: Levels of 4-desmethylsterols as a percentage of total sterols (in mg/kg), tocopherols and tocotrienols in mg/kg in the selected crude edible vegetable oils from authentic samples. Sources: [4-6]
1 2 3 4 5 6 7a 7b 8 9a 9b 10 11
Cholesterol чϯ͘ϴ 0.7-2.3 0.2-0.6 2.6-6.7 0.6-3.7 чϭ͘ϯ чϬ͘ϳ чϬ͘ϱ 0.2-1.4 чϬ͘ϳ чϬ͘ϱ nd nd
Brassicasterol чϬ͘Ϯ 0.1-0.3 чϬ͘Ϯ nd чϬ͘ϴ 5.0-13.0 чϬ͘ϰ чϮ͘Ϯ чϬ͘ϯ чϬ͘Ϯ чϬ͘ϯ nd nd
Campesterol 12.0-19.8 6.4-14.5 16.0-24.1 18.7-27.5 8.4-12.7 24.7-38.6 9.2-13.3 8.9-19.9 15.8-24.2 6.5-13.0 5.0-13.0 чϬ͘ϰ 8-9
Stigmasterol 5.4-13.2 2.1-6.8 4.3-8.0 8.5-13.9 12.0-16.6 0.2-1.0 4.5-9.6 2.9-8.9 14.9-19.1 6.0-13.0 4.5-13.0 nd nd
ɴ-sitosterol 47.4-69.0 76.0-87.1 54.8-66.6 50.2-62.1 62.6-73.1 45.1-57.9 40.2-50.6 40.1-66.9 47.0-60 50-70 42.0-70 nd 87-90
5
ѐ -avenasterol 5.0-18.8 1.8-7.3 1.5-8.2 чϮ͘ϴ 1.4-9.0 2.5-6.6 0.8-4.8 0.2-8.9 1.5-3.7 чϲ͘ϵ 1.5-6.9 nd чϰ
7
ѐ -stigmastenol чϱ͘ϭ чϭ͘ϰ 0.2-4.2 0.2-2.4 чϮ͘ϭ чϭ͘ϯ 13.7-24.6 3.4-16.4 1.4-5.2 6.5-24.0 6.5-24.0 20.5-35.9 чϮ
7
ѐ -avenasterol чϱ͘ϱ 0.8-3.3 0.3-2.7 чϱ͘ϭ чϭ͘ϰ чϬ͘ϴ 2.2-6.3 чϴ͘ϯ 1.0-4.6 3.0-7.5 чϵ͘Ϭ 1.2-4.4 nd
a
Others чϭ͘ϰ чϭ͘ϱ чϮ͘ϰ nd чϮ͘ϳ чϰ͘Ϯ 0.5-6.4 4.4-11.9 чϭ͘ϴ чϱ͘ϯ 3.5-9.5 59.0-78.0 nr
Total sterols 900-2900 2700-6400 7000-22100 300-700 700-1400 4500-11300 2100-4600 2000-4100 1800-4500 2400-5000 1700-5200 1300-3190 nr
ɲ-tocopherol 49-373 136-674 23-573 4-193 чϰϰ 100-386 234-660 234-660 9-352 403-935 400-1090 14-78 76-356
ɴ-tocopherol чϰϭ чϮϵ чϯϱϲ чϮϯϰ чϮϰϴ чϭϰϬ чϭϳ чϭϯ чϯϲ чϰϱ 10-35 чϱ͘Ϯ nd
- 367 -
ɶ-tocopherol 88-389 138-746 268-2468 чϱϮϲ чϮϱϳ 189-753 чϭϮ чϰϰ 89-2307 чϯϰ 3-30 322-810 187-358
ȴ-tocopherol чϮϮ 0-21 23-75 чϭϮϯ nd чϮϮ nd чϲ 154-932 чϳ͘Ϭ чϭϳ 28-113 чϭϵ
Total (mg/kg) 170-1300 380-1200 330-3720 150-1500 чϮϲϬ 430-2680 240-670 250-700 600-3370 440-1520 450-1120 597-880 263-661
Note: 1, Arachis oil; 2, Cottonseed oil, 3, Maize oil; 4, Palm oil; 5, Palm kernel oil; 6, Rapeseed (Canola) oil; 7a, Safflower oil; 7b, Safflower high oleic oil; 8, Soya oil; 9a, Sunflower oil; 9b, Sunflower high oleic oil; 10, argan
oil; 11, evening primrose oil; nd, non-detectable, defined as <0.05 й͖Ŷƌ͕ŶŽƚƌĞƉŽƌƚĞĚ͘DĂŝnjĞŽŝůĂůƐŽĐŽŶƚĂŝŶƐчϱϮŵŐͬŬŐɴ-tocotrienol; a, stigmasta-8,22-diene-3-ol (3-6 %), spinasterol (34-44 %), schottenol (44-49 %) and
sigmasta-7,24-diene-3-ol (4-7 %).
Vegetable oils
Table 4: Physical-chemical characteristics of the selected crude edible vegetable oils from selected samples. Sources: [4-6]
1 2 3 4 5 6 7a 7b 8 9a 9b 10 11
0.912-0.920 0.918-0.926 0.917-0.925 0.891-0.899 0.899-0.914 0.914-0.920 0.922-0.927 0.913-0.919 0.919-0.925 0.918-0.923 0.909-0.915 0.906-0.919
RD nr
x=20 °C x=20 °C x=20 °C x=50 °C x=40 °C x=20 °C x=20 °C x=20oCa x=20 °C x=20 °C x=25oC x=20 °C
Vegetable oils
SV 187-196 189-198 187-195 190-209 230-254 182-193 186-198 186-194 189-195 188-194 182-194 189.1-199.1 192-198
IV 86-107 100-123 103-135 50.0-55.0 14.1-21.0 105-126 136-148 80-100 124-139 118-141 78-90 91-110 147-155
UM чϭϬ чϭϱ чϮϴ чϭϮ чϭϬ чϮϬ чϭϱ чϭϬ чϭϱ чϭϱ чϭϱ чϭϭ ч2
Note: 1, Arachis oil; 2, Cottonseed oil, 3, Maize oil; 4, Palm oil; 5, Palm kernel oil; 6, Rapeseed (Canola) oil; 7a, Safflower oil; 7b, Safflower high oleic oil; 8, Soya oil; 9a, Sunflower oil; 9b, Sunflower high oleic oil; 10, argan
oil; 11, evening primrose oil; nd, non-detectable, defined as <0.05 %; nr, not reported. RD, relative density (x °C/water at 20 °C); RI, refractive index (ND 40 °C); SV, saponification value (mg KOH/g oil); IV, iodine value; UM,
unsaponifiable matter (g/kg). a, 0.910-0.916 x=25oC; b, 1.460-1.464 at 40oC. Stable carbon isotope ratio for maize oil should oscillate between -13.71 and -16.26.
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Vegetable oils
Maize oil
Maize oil is a premium oil which may also be adulterated but, because its fatty acid composition
overlaps that of other vegetable oils, blending is difficult to detect. Similarly, although maize oil
has a much higher level of sterols than other oils, the ratios of the concentrations of the individual
sterols (useful in resolving purity issues with most other oils) hardly change at all in blends of
maize oil with minor amounts of other vegetable oils. For these reasons, it is difficult to detect
adulteration of maize oil by conventional analytical techniques. This is the case for the reported
addition of cheaper rapeseed oil to maize oil at high percentages which is an economically
motivated adulteration. There are, however, many scientific papers that describe the addition of
maize oil to virgin olive oil which would, in fact, be easily detected by only tasting the hypothetical
mix if crude maize oil is added, or by the quantification of stigmastadienes if refined maize oil is
added to virgin olive oil. The fraud of adding refined maize oil to refined olive oil has been
detected at very low percentages using stable carbon isotope ratio analysis since the beginning of
1990’s. Although any kind of adulteration is always possible if there is a profit for fraudsters, some
types of adulteration put forward by some scientists may not be supported by facts and may be far
from the actual oil market.
Rapeseed and soya-bean oil
Rapeseed and soya-bean oils have similar fatty acid compositions. Although they are among the
cheaper vegetable oils, they are traded at slightly different prices, soya-bean oil usually being the
more expensive. Blends of the two may alleviate a temporary shortage of one or other of the oils
or provide a small commercial advantage but such blends have been difficult to identify when the
levels of addition are below 20 %. Conversely, soya bean oil can be adulterated with rapeseed oil.
The type of adulteration depends on the market demands for one or the other edible oil, and the
tariffs to be paid for the declared edible oil in the destination market. Whenever large tariffs are
payable in the destination country, there is a high risk of cross-border smuggling operations,
including various proportions of the adulterated mixture.
Evening primrose oil (EPO)
EPO is produced, at a high cost, because of its moderate content of gamma-linolenic acid (GLA, i.e.
all cis-6,9,12 octadecatrienoic acid), which is distinguished from normal or alpha-linolenic acid (all
cis-9,12,15 octadecatrienoic acid). GLA is normally formed in the human body by the metabolism
of linoleic acid (all cis-9,12 octadecadienoic acid). However, some people have a deficiency in their
delta-6 desaturase enzyme system leading to a deficiency of GLA and subsequent metabolic
products. It is claimed that this results in a number of human aliments that can be alleviated by
consuming preformed GLA. Thus, evening primrose is produced commercially as a source of GLA
[10]. Borage and blackcurrant seed oils are also considered as valid sources of GLA [11]. As an
unofficial standard requires that EPO should contain at least 10 % GLA, these other oils have been
considered as possible adulterants of evening primrose in order to reach the required
concentration of GLA. Although consumers thus receive the required amount of GLA, it is
fraudulent to describe the oil as pure EPO.
Sunflower seed oil
Although sunflower seed oils are produced at a low price, they have not been free from
adulteration with other food products. In 2017, for example, the Security Service of Ukraine
carried out inspections of sunflower seed oil market operators because it had been alleged that
producers adulterated this oil with chicken fat. In the past, the enzymatic interesterification of lard
and high-oleic sunflower oil was used for the legal development of new products [12], which
― 11 ―
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Vegetable oils
increased the stability of the vegetable oil and its bland flavour in fried foods [13]. During
winter/spring 2007/2008, in Ukraine, nearly 100 000 t of sunflower oil were contaminated with
mineral oil at concentrations often above 1 000 mg/kg. Fortunately, the European Food Safety
Authority (EFSA) concluded that “exposure to such oil, although undesirable, would not be a public
health concern” since no additives for lubricating oils or pesticides were detected – the risk
assessment was exclusively based on the hydrocarbons - but the complete absence of n-alkanes
suggested that the contaminant consisted of a base oil for the manufacture of lubricants or
hydraulic oils [14].
Argan oil
Argan oil is expensive, its current price in Europe is above 100 euros per litre. Thus, such a price is
likely to incite unscrupulous behaviour and illegal practices are not uncommon (i.e. dilution with
olive oil coloured with paprika or other substances). In addition, the detection of its adulteration is
sometimes a complex problem [15]. Historically, detecting such fraud has been difficult because of
the small databases establishing appropriate purity criteria. Today, argan oil is widely sold in
Western-Europe, North-America and Japan, and the set of potential adulterants include now soya
bean and sunflower seed oils among others.
Coconut oil
The current edible oil market shows a fast-rising demand for coconut oil in developed countries
(e.g., USA), whereas at the same time coconut production is falling in producer countries, mostly
the Caribbean and Central America, because of a lethal yellowing disease which is threatening
coconut crops [16]. An immediate consequence has been widespread adulteration and
counterfeiting of retail coconut oil brands, alleged to be occurring in large amounts (50 %) in India
[17]. The expected fraudulent activity could not be circumscribed to the dilution of coconut oils
with cheaper edible oils but also includes aspects such as the misrepresentation of the organic
coconut oil status and country of origin (geographical provenance) or the addition of undeclared
ingredients for flavouring or colouring possibly resulting in an allergen risk.
Oil processing
Oils prepared by mechanical means alone, without the application of extra heat and in the absence
of further processing, are described as cold-pressed. These normally have a fine flavour, depending
on the quality of the seeds used as raw material. They are produced in relatively low yields and are
therefore more expensive. Oils obtained by hot-pressing and/or solvent extraction are obtained in
higher yields and are therefore cheaper. Furthermore, oils can be solvent-extracted from inferior
seeds and then refined and deodorised to give bland, manufactured oils which are even cheaper
than those produced, for example, by hot-pressing from high grade seeds.
Processing of poor-quality oilseeds or poor storage conditions may lead to an unwelcome increase
of free fatty acids (FFA) in the oil. Crude oils are often purchased on contracts that specify a
maximum FFA content. If, as a result of some mishap, an oil has an FFA above the contractual
maximum, there is a temptation to refine part of the oil to remove the FFA and then to blend back
unprocessed oil to give a supposed crude oil that is now within the contractual limit.
As a further consideration, partially hydrogenated oils (PHOs) are going to be banned in several
countries in 2018 (e.g. Final Determination Regarding Partially Hydrogenated Oils, FDA- 80 FR
34650). The demand for PHOs will fall and the price of replacement oils expected to rise, with a
medium risk that unscrupulous suppliers may use PHOs in edible oils and other food materials that
are claimed to be free of such compounds.
― 12 ―
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Vegetable oils
― 13 ―
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Vegetable oils
The combined action of databases with information on genuine crude vegetable oils with
analytical methodologies that are able to detect additions of cheaper edible oils to expensive ones
at low concentrations is making classical adulteration less profitable, at least in developed
countries (e.g., EU, USA, Canada). However, it does not guarantee that the adulteration of seed
oils does not exist if international and national control organisms lift legal barriers.
There are, however, relatively new adulterations like the addition of “gutter oils” to edible oils
[18]. Gutter oils are used edible oils or waste cooking oils, which are collected from restaurant
fryers, grease traps, slaughterhouse waste etc. and re-labelled as normal edible oils.
Unfortunately, the very diverse sources (e.g., processes, kind of oils, mixtures) of the gutter oils
mean that the identification of a good marker for their detection in adulterated edible oils is an
analytical challenge. In addition, the detection of the toxic substances might not be reliable if one
carefully analyses the variability of the origin of the oil and also the fact that these oils are treated
with chemicals prior to being sold back to restaurants in Asia. In September 2014, a scandal was
reported involving 240 tons of gutter oil in Taiwan, some of which may have been exported
overseas. Although, this kind of adulteration is currently limited to Asia, finding it in developed
countries in the next years cannot be ruled out.
Another adulteration that is expected to continue is the illegal colouring of palm oils in developing
nations which remains a challenge for the enforcement authorities. The Migration of populations
often leads to an increase in the imports of food products that were common in the home
countries of the migrants, and these are then bought in markets used by foreign traders. There
have been recent reports (4 March 2018) of palm oil that is been adulterated with artificial
colouring [19] in such markets.
Other consumer concerns that are likely to increase include the demand for sustainably-grown
palm oil, with the high risk that this sustainability status is fraudulently misrepresented.
Consumers in some countries are also worried about consuming edible oils extracted from GM
oilseeds because of possible mislabelling not only of their containers but also as they are
ingredients in a number of food products. The high complexity of national and international
regulations dealing with genetically modified organisms adds furthers difficulties to effective
authentication in this regard.
An attempt at identifying potential issues in authentication should also focus on the abrupt
changes in price and a break in the value balance between edible oils. A common rule is that larger
harvests of an oilseed often lead to lower prices, which usually means a decreased risk of
fraudulent activity for this particular oil; whereas the availability of cheap and abundant amounts
of the oil make it attractive as an adulterant, a diluent or filler. For example, a large harvest of
hazelnuts makes refined hazelnut oil a potential adulterant of olive oil but, on the contrary, a large
harvest of olives may make roasted hazelnut oil the object of adulteration with virgin olive oil.
Food fraud can include economic adulteration, economically motivated adulteration, intentional
adulteration, or food counterfeiting, according to the United States Pharmacopeial Convention
(USP). Given the diversity of possible types of fraud, it is necessary to register real cases of
adulteration to understand the actual incidence of a fraud type and go beyond hypothetical cases
described in analytical studies. Today, the coordination between institutions for registering these
cases is assumed as a critical tool for detecting new fraud types.
― 14 ―
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Vegetable oils
― 15 ―
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Vegetable oils
granulomas in several tissues. Currently modifications in the production process are under
discussion to avoid the sources of this contamination. Similar problems can be identified with
other migrating contaminants, such as phthalates [23].
― 16 ―
- 374 -
Vegetable oils
Determination Method
Fatty acid composition IUPAC 2.301, 2.302 and 2.304 or ISO 5508: 1990 and 5509: 2000 or AOCS Ce
2-66, Ce 1e-91 or Ce 1f-96
Sterol content ISO 12228:1999, or IUPAC 2.403
Tocopherol content IUPAC 2.432 or ISO 9936: 1997 or AOCS Ce 8-89
Total carotenoids BS 684 Section 2.20
Acidity IUPAC 2.201 or ISO 660: 1996 or AOCS Cd 3d-63
Unsaponifiable matter IUPAC 2.401 (part 1-5) or ISO 3596: 2000 or ISO 18609: 2000
Peroxide value IUPAC 2.501 (as amended), AOCS Cd 8b - 90 (97) or ISO 3961: 1998
Matter Volatile at 105°C IUPAC 2.601 or ISO 662: 1998
Arsenic content AOAC 952.13, IUPAC 3.136, AOAC 942.17, or AOAC 985.16
Insoluble impurity IUPAC 2.604 or ISO 663: 2000
Trace metals of copper and iron ISO 8294: 1994, IUPAC 2.631 or AOAC 990.05 or AOCS Ca 18b-91
Determination of traces of heavy metals Lead: IUPAC 2.632, AOAC 994.02 or ISO 12193: 1994 or AOCS Ca 18c-91
Arsenic: AOAC 952.13; AOAC 942.17; AOAC 985.16
Slip point ISO 6321: 1991 and Amendment 1: 1998 for all the edible oils, or AOCS Cc
3b-92 or Ce 3-25 (97) for palm oils only
Crismer value AOCS Cb 4-35 (97) and AOCS Ca 5a-40 (97)
1
Badoiun test AOCS Cb 2-40 (97)
Halphen test AOCS Cb 1-25 (97)
Reichert and Polenske values AOCS Cd 5-40 (97)
Refractive Index IUPAC 2.102 or ISO 6320: 2000 or AOCS Ce 7-25
Iodine value Wijs - according to IUPAC 2.205/1, ISO 3961: 1996, AOAC 993.20, or AOCS
Cd 1d-92 (97), or by calculation - AOCS Cd 1b-87 (97)
Saponifiable value IUPAC 2.202 or ISO 3657: 1988
Soap content BS 684 Section 2.5
Relative density IUPAC 2.101a
Apparent density ISO 6883: 2000a or AOCS Cc 10c-95
Note: a, with the appropriate conversion factor; 1, modified Villavecchia test or sesame seed oil test; AOCS, American Oil
Chemists Society; ISO, International Organization for Standardization; FOSFA, Federation of Oils, Seeds and Fats
Associations Ltd; IUPAC, International of Union of Pure and Applied Chemistry.
The majority of the unsaturated fatty acids that make up edible oils are found in the cis form.
When oils are hardened by hydrogenation (to formulate margarine), or partially hydrogenated to
stabilise against oxidation, there is a conversion of some cis to trans double bonds. FTIR can be
used to determine the trans isomer content of oils and fats with a good agreement with GC results
[24]. Actually, FTIR has demonstrated good performance in this determination and a IUPAC
method was developed with this objective [30]. Raman spectroscopy has also been used to
determine the cis/trans isomer content of edible vegetable oils, as well as to determine the total
unsaturation of oils and margarines. Furthermore, Fourier transform mid infrared (FT-MIR) has
― 17 ―
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Vegetable oils
been used in the detection of adulteration of virgin coconut oil [31], the mixtures of sesame and
corn oils [32], and the presence of lard in some vegetable oils [33] also analysed by differential
scanning calorimetry [34]; a large set of applications is described by [7].
FTIR analysis also provides a rapid means of evaluating the oxidative state of an oil or of
monitoring changes in oils undergoing thermal stress [35]. Rapid methods based on FTIR have also
been developed for the quantitative determinations of the iodine value and saponification
number, free fatty acids, peroxide value and solid fat index.
In terms of oil authenticity, FTIR, NIR and Raman spectroscopy coupled with multivariate analysis
techniques have been used to characterise edible oils, according to their degree of unsaturation
and other characteristics [25,27]. The basis for the discrimination between fats is often the
concentration of unsaturated fatty acids, and different concentrations of linoleic acid in the case of
oils (sunflower, olive and peanut oils) [25,36].
1
H-NMR has also been applied to the study of the triacylglycerol structure of palm oils, seed oils,
some hydrogenated fats and vegetable margarines as, for instance, the adulteration of peanut
oil [37].
13
Quantitative C-NMR data of the acyl profile have been reported to be in good agreement with GC
13 1
for other edible vegetable oils, fats and lipids. Thus, a profiling strategy with C-NMR, H-NMR and
the analysis of results by chemometrics [38,39] has shown satisfactory results in detecting the
presence of different vegetable oils, although always with the involvement of a database that can
affect these results.
Isotopic analysis has already been carried out with respect to the isotope ratio of individual fatty
acids in an oil and it has been shown that there are slight differences [40]. Any contamination of an
oil will upset these slight differences, the nature of the distortion from the established pattern
revealing the cause of the impurity. Thus, for example, carbon isotope ratio was used, in
combination with GC-FID, to detect the presence of corn oil in sesame oil [41].
The technique of site-specific isotope fractionation studied by NMR (SNIF-NMR) has been applied
to alcoholic beverages and fruit juices. There is every possibility that it may also show advantages
in the evaluation of edible seed oils.
The main applications of spectroscopic techniques in authenticating edible oils has been focused
on detecting their presence in olive oil categories as described in the chapter on olive oil.
Sometimes, however, authors imagine a virtual world in which any kind of adulteration can be
possible, some ones even being unprofitable for fraudsters, and they study cases that do not exist
in the real world even being published in reputed scientific journals.
― 18 ―
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Vegetable oils
Fatty acid profile by GC Linolenic acid (C18:3) Contamination of groundnut and sunflower seed
Fatty acid concentration at oils with soybean and rapeseed oils
the triglyceride-2 position
Sterol profile by GC Brassicasterol Detection of rapeseed oil in sunflower seed or
groundnut oils
Tocopherol content by HPLC Gamma-tocopherol Detection of soybean oil in sunflower seed oil
Carbon number triglyceride C60/C58 ratio Detection of sunflower seed oil in safflower seed
composition by HPLC oil
Slip melting point/Iodine values Detection of stearins or oleins in palm oil
Carbon number triglyceride C48 concentration * palmitic Detection of stearins or oleins in palm oil
composition by HPLC acid enrichment factor
ICP-OES Elemental content Detection of olive oil and soybean oil in argan oil
Carbon number triglyceride C50 and C54 Detection of palm olein oil in cottonseed oil
composition by HPLC
Carbon number triglyceride Various purity criteria Mixtures of palm kernel and coconut oils
composition by HPLC
Fatty acid profile by GC and Iodine Oleic acid (C18 :1) Detection of palm kernel olein in palm kernel oil
values
Differential Scanning Calorimetry Thermogram profiles Detection of animal fat in sunflower seed oil
13
Stable isotope analysis C/12C ratios Detection of commercial oils in maize oils and vice
versa
13
Stable isotope analysis C/12C ratios Detection of maize oil in olive, sesame and
soybean oils
Fatty acid profile by GC Oleic and linoleic acids Detection of rapeseed and soybean blends
Fatty acid profile by GC Linoleic and erucic acids Detection of borage oil in evening primrose oil
Fatty acid profile by GC Linoleic and stearidonic acids Detection of blackcurrant seed oil in evening
primrose oil
― 19 ―
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Vegetable oils
5. Conclusion
A large number of the problems in identifying more than 10 % adulteration or contamination of
bulk edible oils have been clarified. However, changes in commercial trade patterns and in
consumer eating habits, together with increasing application of genetic engineering to improve oil
crops mean that tomorrow’s problems may not be the same as those encountered today. Thus,
using genetic modification it is possible to obtain oils from different botanical sources with
chemical characteristics that are similar to those oils that are targets for adulteration. In
consequence, authentication strategies should consider this fact to be efficient in detecting frauds.
As regards analytical developments, the difficulty is that some of the methods, such as sterol
analysis, are long, tedious and therefore expensive. Others, such as some spectroscopic analysis
(e.g. NMR, isotopic analysis), require sophisticated equipment which are only available in a limited
number of laboratories.
The challenge for the future is still the identification and detection of adulteration, but at lower
levels of impurity, and by simpler routine methods. In global terms, the analysts predict that the
greatest increase in vegetable oil consumption will take place in South East Asia and South
America. North America, Australia and Japan will experience a moderate increase in the demand
for vegetable oils mostly as a result of health concerns. Thus, abrupt changes in demand may also
bring some new chances for adulteration that will need to be controlled with coordinated and
efficient tools that combine analytical enhancement and data management. Since vegetable oils
are essential in the human diet and they form part of many food formulations, they cannot be
omitted or substituted by other ingredients. Thus, any problem of fraud is magnified and can have
a significant impact on health and consumer concern. Therefore, an oriented strategy on vegetable
oils authentication is always necessary and it should be on the table of food safety authorities,
without forgetting the news on food frauds reported on media (https://ec.europa.eu/jrc/en/food-
fraud-and-quality/monthly-summary-articles).
6. Bibliographic references
1. Lyubun Y.V., Kosterin P.V., Zakharova E.A., Shcherbakov A.A. & Fedorov E.E. (2002). – Arsenic-contaminated soils.
Phytotoxicity studies with sunflower and sorghum. J. Soils Sediment., 2 (3), 143–147. doi:10.1007/BF02988466.
2. Plourde J.D., Pijanowski B.C. & Pekin B.K. (2013). – Evidence for increased monoculture cropping in the Central
United States. Agric, Ecosyst. Environ., 165, 50–59. doi:10.1016/j.agee.2012.11.011.
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Jee, ed), CRC Press Blackwell Publishing, Boca Raton, FL, pp 95-114.
12. Rodríguez A., Castro E., Salinas M.C., López R. & Miranda M. (2001). – Interesterification of tallow and sunflower oil.
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13. Seriburi V. & Akoh C.C. (1998). – Enzymatic interesterification of lard and high-oleic sunflower oil with candida
antarctica lipase to produce plastic fats. J. Am. Oil Chem. Soc., 75 (10), 1339–1345. doi:10.1007/s11746-998-0181-x.
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15. Ourrach I., Rada M., Pérez-Camino M.C., Benaissa M. & Guinda Á. (2012). – Detection of argan oil adulterated with
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16. Gurr G.M., Johnson A.C., Ash G.J., Wilson B.A.L., Ero M.M., Pilotti C.A., Dewhurst C.F. & You M.S. (2016). – Coconut
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18. Wee H.M., Budiman S.D., Su L.C., Chang M. & Chen R. (2016). – Responsible supply chain management – an analysis
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19. Food Standards Agency. (2018). Surya Foods recalls Mother Africa Palm Oil because it contains illegal dye Sudan IV.
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25. Baeten V. & Aparicio R. (2000). – Edible Oils and Fats Authentication by Fourier Transform Raman Spectrometry.
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functional food oils. Appl. Spectrosc. Rev., 47 (1), 1–13. doi:10.1080/05704928.2011.619020.
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near-infrared spectroscopy. Appl. Spectrosc., 54 (8), 1168-1174. doi: 10.1366/0003702001950733.
28. Javidnia K., Parish M., Karimi S. & Hemmateenejad B. (2013). – Discrimination of edible oils and fats by combination
of multivariate pattern recognition and FT-IR spectroscopy: A comparative study between different modelling
methods. Spectrochim. Acta A, 104, 175–181. doi:10.1016/j.saa.2012.11.067.
29. Voort F.R. van de, Ghetler A., García-González D.L. & Li Y.D. (2008). – Perspectives on quantitative Mid-FTIR
spectroscopy in relation to edible oil and lubricant analysis: Evolution and integration of analytical methodologies.
Food Anal. Methods, 1 (3), 153–163. doi:10.1007/s12161-008-9031-6.
30. IUPAC (1987) Method 2.207 in Standard Methods for the Analysis of Oils and Fats (7th Ed.), Pergamon Press.
31. Rohman A. & Che Man Y.B. (2011). – The use of Fourier transform mid infrared (FT-MIR) spectroscopy for detection
and quantification of adulteration in virgin coconut oil. Food Chem., 129 (2), 583–588.
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32. Yang R., Dong G., Sun X., Yang Y., Liu H., Du Y., Jin H. & Zhang W. (2017). – Discrimination of sesame oil adulterated
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33. Rohman A., Che Man Y.B., Hashim P. & Ismail A. (2011). – FTIR spectroscopy combined with chemometrics for
analysis of lard adulteration in some vegetable oils. CYTA - Journal of Food, 9 (2), 96–101.
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34. Marikkar J.M.N., Dzulkifly M.H., Nadiha M.Z.N. & Man Y.B.C. (2012). – Detection of animal fat contaminations in
sunflower oil by differential scanning calorimetry. Int. J. Food Prop., 15 (3), 683–690.
doi:10.1080/10942912.2010.498544.
35. Tena N., Aparicio R. & García-González D.L. (2018). – PhotooxidationEffect in Liquid Lipid Matrices: Answers from an
Innovative FTIR Spectroscopy Strategy with “mesh Cell” Incubation. J. Agric. Food Chem., 66 (13), 3541–3549.
doi:10.1021/acs.jafc.7b05981.
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37. Zhu W., Wang X. & Chen L. (2017). – Rapid detection of peanut oil adulteration using low-field nuclear magnetic
resonance and chemometrics. Food Chem., 216, 268–274. doi:10.1016/j.foodchem.2016.08.051.
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margarines. Ital. J. Food Sci., 7, 27-36.
39. Guyader S., Thomas F., Portaluri V., Jamin E., Akoka S., Silvestre V., Remaud G. (2018). - Authentication of edible fats
13
and oils by non-targeted C INEPT NMR spectroscopy. Food Control, 91, 216-224.
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40. Woodbury S.E., Evershed R.P., Barry Rossell J., Griffith R.E. & Famell P. (1995). – Detection of Vegetable Oil
Adulteration Using Gas Chromatography Combustion/Isotope Ratio Mass Spectrometry. Anal. Chem., 67 (15), 2685–
2690. doi:10.1021/ac00111a029.
41. Seo H.Y., Ha J., Shin D.B., Shim S.L., No K.M., Kim K.S., Lee K.B. & Han S.B. (2010). – Detection of corn oil in
adulterated sesame oil by chromatography and carbon isotope analysis. J. Am. Oil Chem. Soc., 87 (6), 621–626.
doi:10.1007/s11746-010-1545-6.
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Food flavourings
Eric Jamin*, Freddy Thomas
Eurofins Analytics France, Nantes, France
*E-mail corresponding author: [email protected]
1. Product Identity
1.1. Definition of the product and manufacturing process
The three main categories of flavourings are:
● Essential oils and natural extracts are obtained from natural sources such as flowers,
fruits, etc. The processes used included solvent extraction, steam distillation, etc.
https://doi.org/10.32741/fihb.20.flavourings
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Food flavourings
The above mentioned ‘appropriate physical process’ “shall mean a physical process which does not
intentionally modify the chemical nature of the components of the flavouring, without prejudice to
the listing of traditional food preparation processes in Annex II, and does not involve, inter alia, the
use of singlet oxygen, ozone, inorganic catalysts, metal catalysts, organometallic reagents and/or
UV radiation.”
Besides Europe, national regulations exist in other parts of the world, but they will not be covered
here. A complete review can be found in [1].
2. Authenticity issues
2.1. Identification of current authenticity issues
Thanks to Mother Nature’s gifts on one hand, and human creativity on the other hand, a wide
range of natural sources and processes can be used to produce food flavourings, which are usually
complex mixtures of chemical compounds. Many organic compounds have a flavouring impact,
which is not correlated to their concentration. What makes it even more subtle is that the final
sensory impact depends on all flavour compounds present, their proportions, and even the effect
of the food matrix itself on the perception. Synthetic mixtures may smell as beautiful and natural
as extracts, and conversely some natural sources may produce poor quality aromas. Therefore
using one’s nose is not sufficient for judging the authenticity of an aroma.
Natural flavourings are among high risk ingredients regarding economic food fraud, because of
their high price and the availability of cheaper substitutes. The most commonly encountered fraud
is the addition of synthetic compounds which are chemically identical to the main component(s) of
a given natural flavouring. Typical examples are the addition of synthetic vanillin or para-
hydroxybenzaldehyde to vanilla extracts / aromas, or the addition of synthetic benzaldehyde to
bitter almond oil.
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Food flavourings
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Food flavourings
vanilla beans origin. Not only can this method be applied to flavouring ingredients such as beans,
extracts, and aromas, but also to finished products such as vanilla-flavoured ice-cream, cakes, etc.
After a solvent extraction, applied to the vanilla-flavoured food or ingredient, the volatile
components are separated by GC and the peak of vanillin is selectively submitted to combustion
13 12 13
and C/ C isotopic ratio measurement. Since the C deviation of agricultural vanillin is less
negative than most of its artificial counterparts, it is possible to detect blending or substitution.
However this approach is not sufficient for precisely identifying the artificial sources.
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Food flavourings
5. Conclusion
The world of food flavourings is extremely rich in terms of sources, compounds and sensorial
impacts. Their high commercial value and scarce sources make them prone to economic
adulteration risks. Climatic and political incidents might be aggravating factors to the fraud risk.
The price increase of vanilla beans following the “Enawo” hurricane in Madagascar in 2017 can be
taken as an example: the price, that had already increased over the last decades then suddenly
doubled from USD 200 per kg to USD 425 per kg, causing a lot of trouble in the market. Similarly,
unstable political situations can influence the risk level of supplies for many aroma sources.
When performing authenticity controls, the first question to ask is the precise definition of the
flavouring being used, which leads to some expectations based on legal definitions. Then suitable
analytical method(s) performed by laboratories having access to appropriate reference knowledge
bases should be selected to check whether the composition of the aroma matches with these
expectations.
In many cases also the choice of the appropriate method is governed by technical feasibility, and
R&D work is still on-going to cover unsolved issues. The identification of precursors has made
considerable progress thanks to the use of isotopic methods. One of the most difficult challenges
remains the characterisation of processes used for manufacturing these high value ingredients, as
a given precursor may be transformed into the final flavouring substance through different ways.
The large amount of information generated by the above-mentioned methods can be exploited in
an optimal way using multivariate statistics. And finally, instead of considering only known signals,
one can imagine to use the aroma screening as a non-targeted screening, which could enhance the
possibility to detect unexpected manipulations of flavours.
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Food flavourings
6. Bibliographic references
1. Ziegler H., ed. (2007). – Flavourings: production, composition, applications, regulations. 2., compl. rev. ed, Wiley-VCH,
Weinheim.
2. Regulation (EC) No 1334/2008 of the European Parliament and of the Council of 16 December 2008 on flavourings
and certain food ingredients with flavouring properties for use in and on foods and amending Council Regulation
(EEC) No 1601/91, Regulations (EC) No 2232/96 and (EC) No 110/2008 and Directive 2000/13/EC (2008). Off. J. Eur.
Union, L354, 34–50.
3. IOFU (2012). – International Organization of the Flavor Industry Code of Practice - Effective Date February 29, 2012 -
Update version 1.3. IOFU. Available at: http://www.iofi.org.
4. Directive 2012/12/EU of the European Parliament and of the Council of 19 April 2012 amending Council Directive
2001/112/EC relating to fruit juices and certain similar products intended for human consumption (2012). Off. J. Eur.
Union, L115, 1–11.
5. Wolter C., Gessler A. & Winterhalter P. (2008). – Aspects when Evaluating Apple-Juice Aroma. Fuit Process., (March-
April 2008), 64.
6. Heil M. & Ara V. (2008). – Aroma of Fruit Juices II: Composition and Valuation of Apple Juice Aroma. Fuit Process.,
(May-June 2008), 126.
7. Leeuwen K.A. van, Prenzler P.D., Ryan D. & Camin F. (2014). – Gas Chromatography-Combustion-Isotope Ratio Mass
Spectrometry for Traceability and Authenticity in Foods and Beverages. Compr. Rev. Food Sci. Food Saf., 13 (5), 814–
837. doi:10.1111/1541-4337.12096.
8. Jamin E. & Thomas F. (2017). – SNIF-NMR Applications in an Economic Context: Fraud Detection in Food Products. .
In Modern Magnetic Resonance, Springer, Cham. pp 1–12doi:10.1007/978-3-319-28275-6_103-1.
9. Remaud G.S., Martin Y.L., Martin G.G. & Martin G.J. (1997). – Detection of Sophisticated Adulterations of Natural
Vanilla Flavors and Extracts: Application of the SNIF-NMR Method to Vanillin and p-Hydroxybenzaldehyde. J. Agric.
Food Chem., 45 (3), 859–866. doi:10.1021/jf960518f.
10. Jamin E., Martin F. & Martin G.G. (2007). – Determination of site-specific (deuterium/hydrogen) ratios in vanillin by
2H-nuclear magnetic resonance spectrometry: collaborative study. J. AOAC Int., 90 (1), 187–195.
11. AOAC 2006.05-2006 - Site-specific deuterium/hydrogen (D/H) ratios in vanillin. Site-specific natural isotope
fractionation-nuclear magnetic resonance (SNIF-NMR) spectromety (2006). Available at:
http://www.aoacofficialmethod.org/index.php?main_page=product_info&cPath=1&products_id=2362.
12. Remaud G., Debon A.A., Martin Y. loïc, Martin G.G. & Martin G.J. (1997). – Authentication of Bitter Almond Oil and
Cinnamon Oil: Application of the SNIF-NMR Method to Benzaldehyde. J. Agric. Food Chem., 45 (10), 4042–4048.
doi:10.1021/jf970143d.
13. Martin G.J., Martin M.L., Mabon F. & Bricout J. (1982). – A new method for the identification of the origin of natural
products. Quantitative deuterium NMR at the natural abundance level applied to the characterization of anetholes. J.
Am. Chem. Soc., 104 (9), 2658–2659. doi:10.1021/ja00373a064.
14. Fronza G., Fuganti C., Guillou C., Reniero F. & Joulain D. (1998). – Natural Abundance 2H Nuclear Magnetic
Resonance Study of the Origin of Raspberry Ketone. J. Agric. Food Chem., 46 (1), 248–254. doi:10.1021/jf970530n.
15. Tenailleau E.J., Lancelin P., Robins R.J. & Akoka S. (2004). – Authentication of the Origin of Vanillin Using Quantitative
Natural Abundance 13C NMR. J. Agric. Food Chem., 52 (26), 7782–7787. doi:10.1021/jf048847s.
16. Caytan E., Botosoa E.P., Silvestre V., Robins R.J., Akoka S. & Remaud G.S. (2007). – Accurate Quantitative 13C NMR
Spectroscopy: Repeatability over Time of Site-Specific 13C Isotope Ratio Determination. Anal. Chem., 79 (21), 8266–
8269. doi:10.1021/ac070826k.
17. Caytan E., Remaud G.S., Tenailleau E. & Akoka S. (2007). – Precise and accurate quantitative 13C NMR with reduced
experimental time. Talanta, 71 (3), 1016–1021. doi:10.1016/j.talanta.2006.05.075.
18. Chaintreau A., Fieber W., Sommer H., Gilbert A., Yamada K., Yoshida N., Pagelot A., Moskau D., Moreno A.,
Schleucher J., Reniero F., Holland M., Guillou C., Silvestre V., Akoka S. & Remaud G.S. (2013). – Site-specific 13C
content by quantitative isotopic 13C Nuclear Magnetic Resonance spectrometry: A pilot inter-laboratory study. Anal.
Chim. Acta, 788, 108–113. doi:10.1016/j.aca.2013.06.004.
19. Guyader S., Thomas F., Jamin E., Grand M., Akoka S., Silvestre V. & Remaud G. – Use of 13C and 2H SNIF-NMR®
isotopic fingerprint of vanillin to control precursors and processes. Flavor Fragance J., In press.
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Determination of
species origin of gelatine in foods
Helen H. Grundy*
Fera Science Limited, York, United Kingdom
*E-mail corresponding author: [email protected]
1
https://www.gelatine.org/gelatine/comparison-hydrocolloids.html
2
https://www.gelatine.org/ and https://www.gelatine.org/gme/sustainability.html
https://doi.org/10.32741/fihb.21.gelatine
- 391 -
Species origin of gelatine
1. Product Identity
1.1. Definition of the product and manufacturing process
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Species origin of gelatine
gelatine, although some plants handle more than one species. Since gelatine is prepared from
bone or hide material, there is opportunity for unscrupulous manufacturers to prepare a batch of
gelatine from any animal species if the raw materials were available and label it with an alternative
species origin. This batch could then enter the food chain.
Approximately 80 % of the gelatine prepared in Europe is derived from porcine skins and 15 %
3
from cattle split. The remaining 5 % is prepared from porcine and bovine bones and fish . Globally,
46 % is prepared from porcine hide, 29.4 % from bovine split, 23.1 % from bones and 1.5 % from
other sources including chicken [4]. There is also interest in increasing the amount of fish gelatine
which is manufactured, due partly to its abundance and biodegradability [1,5].
The processes used to prepare gelatine in industry differ depending on the starting material and
examples of the acid, alkali and enzymatic processes used to prepare gelatines are discussed
below. The gelatine yield from the raw starting materials tends to be approximately 10 %.
5%
Porcine skin
15%
Cattle hide
3
https://www.gelatine.org/gelatine/manufacturing.html
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Species origin of gelatine
4
https://www.gelatine.org/gme/mission-and-objectives.html
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Species origin of gelatine
There is no legislation requiring that the animal origin of gelatines is included on food labels, but
many suppliers choose to include this information to better-inform consumers. A paper trail is the
method used to determine the origin of batches of gelatine. Supplier premises may be inspected
by accreditation and certification bodies, including Halal food certification bodies.
2. Authenticity issues
2.1. Identification of current authenticity issues
Regarding authenticity issues, as previously mentioned, due to the high levels of processing
involved in the preparation of gelatines, resulting in gelatine granules or powders with little or no
apparent indicators as to origin, there is potential for deliberate adulteration or accidental
contamination to occur using raw material from animals other than those on the product label.
Since many consumers choose to abstain from certain species due to religious rules or ethical
preferences it is important that methods are available to ensure correct labelling.
Since the physical appearance of gelatines provides little or no significant indicator of animal
origin, it is perceivable that batches of gelatine can be mislabelled or mixed, either fraudulently or
accidentally. Also, certain gelatine manufacturers use the same factory establishments to process
gelatine of different origins and therefore mixing of species can occur at the point of manufacture.
The widespread adulteration of processed beef products with horse in 2013 highlighted that
species-related fraud is present in the food chain. The subsequent Elliott Review (2014) into the
integrity and assurance of food supply networks investigated, amongst other aspects, the ‘causes
of the systemic failure that enabled the horsemeat fraud’. Further highlighting the issue in terms
of gelatine adulteration, highly processed gelatine (hydrolysed collagen) of bovine origin has been
found by the UK Food Standards Agency as a plumping agent in chicken breasts labelled as
containing chicken only (UK Food Standards Agency, 2009). Therefore, methods to determine
species origin of processed products such as gelatine would support the food chain and consumers
by aiding policing against known potential threats. Further, given the religious and ethical
sensitivities regarding the species origin of gelatine, it is important that analytical methods are
available to authenticate the animal origin of gelatines in foods and capsules. While gelatine
manufacturers are audited to support the species authenticity of gelatine, there is still opportunity
for the accidental and deliberate mislabelling, particularly since porcine gelatine is cheaper in
terms of cost than bovine gelatine [2]. Analytical methods which can determine the presence of an
adulterating gelatine present at low levels when mixed with an alternative gelatine are required
with a high level of sensitivity to support food integrity.
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Species origin of gelatine
Due to concerns linked to BSE in 1997, the TSE Advisory Body, in collaboration with the US Food
and Drug Administration, began monitoring the potential risk of transmitting BSE. The disease was
mainly associated with consumption of tissue of the nervous system including skull, brain and
vertebrae. It was recognized that the heat, alkali and filtration treatments used during gelatine
manufacture could be effective in reducing the level of contaminating transmissible spongiform
encephalopathies. In 2002, the Scientific Steering Committee of the European Union (SSC) stated
that the risk associated with bovine gelatine is very low or zero and, in 2004, a team determined
that the acid and alkali processes of gelatine manufacture from bovine bone reduce infectivity to
undetectable levels [8].
Regarding alternative threats to health, from a nutritional point of view, although gelatine is
composed of around 98 % protein (dry weight), it does not contain all essential amino acids and
therefore must be consumed only as part of a balanced diet.
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Species origin of gelatine
community, alternative methodology capable of accurate, specific and precise determination with
high sensitivity across a wide range of food types, which also addresses false positive and false
negative issues in gelatine species determination, is required.
There are emerging technologies which focus on the screening of collagen peptides present in a
food sample. Peptides are often more robust to degradation compared to DNA and whole proteins
which become fragmented and denatured during manufacture. The peptide complement tends to
be comparatively intact for the food material. Peptide mass spectrometry can be used to
determine species-specific peptides. Matrix Assisted Laser Desorption Ionisation Time-of-flight
Mass spectrometry (MALDI-ToF MS) has been used to compare and contrast the collagen peptide
fingerprint of species. Indeed, this technology has been used in research to determine species
markers in ancient archaeological collagen samples, so robust is the collagen protein [17].
In terms of using MALDI-ToF technology to support food integrity, issues regarding sensitivity have
been reported. Pork gelatine could only be determined in bovine gelatine when present at 20 %
(w/w) [18] whereas significantly greater sensitivity is required in the food chain with the UK Food
Standards Agency requiring sensitivity at the 1 % (w/w) level. The early stages of work have also
been carried out using low resolution liquid chromatography mass spectrometry (LC-MS)
instruments using multivariate analysis to compare the mass spectral data for bovine, porcine and
fish gelatine with success on a limited number of samples and sample types tested to date [2].
However, development of methods with integrated confirmatory techniques, such as tandem
mass spectrometry (MS/MS) would prove valuable as a single test to determine origin.
One successful emerging technology, in the form of high-resolution accurate mass liquid
chromatography mass spectrometry (HR LC-MS/MS), can be applied to compare and contrast
samples of high sequence homology to determine species differences. The peptides in a food
sample are separated by nano-flow liquid chromatography. Each peptide is fragmented and then
further fragmented in the mass spectrometer and the accurate mass of each fragment ion is
measured. Algorithms are used to determine the amino acid composition of each peptide and to
ultimately determine species origin, screening for marker peptide.
A full scan (untargeted) high resolution, high accuracy mass spectrometry (HR LC-MS/MS) method
is available for the qualitative determination of the animal origin of gelatine extracted from foods
[19]. Despite the high levels of collagen amino acid sequence homology between bovine and
porcine gelatine, the method can differentiate a wide range of species using a suite of peptides in
a proprietary database which contains species-marker peptides to differentiate not only bovine
and porcine, but also species including equine, ovine, piscine and poultry gelatines amongst others
[20]. In this work, a library of collagen sequences was prepared using molecular mining of
Expressed Sequence Tags (EST) and other databases, coupled with de novo sequencing from thirty-
two different mammalian species, identified peptides which can be used as species markers in
gelatine. Species identity of these peptides was verified by mapping the phylogeny of the peptides
[20]. The quality and verification of the database is critical: unlike for other proteins, so aggressive
is the gelatine manufacturing process, the modifications caused to the collagen protein during
processing cannot necessarily be correctly predicted by conventional proteomics software and
database packages in order to build a database. It is critical that the species specificity of marker
peptides is independently verified by testing a wide range of same-species collagens so that
potentially incorrect sequences are not attributed to species specificity [20]. This HR LC-MS/MS
method has been evaluated on a range of foods. Based on the threshold applied by the UK Food
Standards Agency to the adulteration of processed meats during the horse meat issues of 2013
which required detection of adulterant at 1 % (w/w), this method was evaluated on a range of
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Species origin of gelatine
gelatine-rich foods containing an adulterating gelatine at levels of 0.5 % (w/w). Both the accuracy
and the precision of the method were 100 % and the maximum specificity was also demonstrated
([19], unpublished data). The tissue origin (bone or skin) of the gelatine can also be determined by
this method. Another benefit of untargeted methods is that all the peptide data from a sample can
be archived and interrogated at a later date should the need arise in the future to investigate the
presence of a new species of interest once the peptide sequence data for that species is available.
HR LC-MS/MS methods benefit from confirmatory techniques also, by analysing the fragmentation
patterns of the peptides to verify correct marker peptide, and thus species, assignment. This way,
matrix interferences can be ruled out meaning the false positive rate of the method is not an issue
which is another benefit over techniques such as ELISA or PCR.
Some progress towards developing a quantitative HR LC-MS/MS method has been made by
developing internal standards by oxygen-18 labelling of gelatine marker peptides. The
incorporation of stable isotopes into peptides results in a fixed mass shift with no effect on the
chemical properties of the peptides. Therefore, the relative abundances of the labelled peptides
from different samples can be accurately quantified using HR LC-MS/MS [9]. The method was
developed on pure gelatines and its future application in the food industry relies on developing an
extraction method and accurate measuring method prior to the analysis by HR LC-MS/MS.
Although showing excellent capability to determine the species origin of gelatines, high resolution
accurate mass spectrometry methods require very high initial investment, highly trained personnel
and elevated instrument upkeep costs. These instruments tend to be used more for research
discovery purposes than for the routine analyses which tend to be required to support the food
chain in terms of screening for adulteration. In the future, it is likely that more and more targeted
methods will be developed from the data generated by these HR LC-MS/MS research instruments
to screen for a pre-selected target list of species-specific marker peptides in gelatine food extracts.
Such targeted methods, by Selected Reaction Monitoring mass spectrometry (SRM) are relatively
low cost and are already used routinely to screen for other contaminants in the food chain
including veterinary drugs, pesticide residues, mycotoxins, natural toxins, processing contaminants
including acrylamide and materials which migrate into food from containers and packaging
materials.
A recent evaluation of a targeted SRM mass spectrometry method tested forty-eight food samples
simulating commercial food products and food supplement capsules containing bovine and
porcine gelatine mixtures, alongside relevant positive and negative quality control samples. The
foods were analysed in two ways: to determine the origin of the adulterating gelatine (a) when
present at 1 % of the total mass of the food matrix and (b) when present at 1 % of the total mass
of gelatine within the food matrix. The adulterating gelatine was present at as little as 0.07 % of
the total food sample mass, depending on the food matrix type. The method showed 100 %
accuracy and precision across all samples and the specificity of the method was also of the highest
level, screening for fourteen bovine- and eight porcine-specific markers (Project FA0165,
5
Department for Environment, Food and Rural Affairs, 2018 ). Given that the EU Commissioning
Body advised that the threshold for ‘deliberate adulteration’ of an undeclared meat product is 1 %
w/w, this method is well within this tolerance. A further benefit of this form of technology is that
there is also evidence that targeted mass spectrometry methods such as this one could offer a
greater dynamic range than HR LC-MS/MS methods for quantification of peptides [21]. This is an
aspect worthy of future investigation in relation to gelatine peptides in order to inform as to
whether deliberate adulteration or accidental low concentration contamination may have taken
5
Pending publication, https://www.gov.uk/government/organisations/department-for-environment-food-rural-affairs
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Species origin of gelatine
place. SRM methods offer the benefits of screening for a wide range of known species peptide
markers, screening for the precursor ion of each peptide and, critically, for each of its four
confirmatory product ions, which must all be identified in a product to provide consumers and
producers alike the confidence that the results are correct and not due for example to matrix
interferences.
Polymerase Chain Reaction e.g. Mitochondrial cytochrome b High levels of DNA degradation contribute to the lack
(PCR) gene target of methods to determine the species origin of
gelatine in foods.
High Resolution Accurate Mass Determination of suite of marker Relevant to single species or mixtures of gelatines
Spectrometry peptides for a wide range of extracted from foods, sensitive to 0.5 % or lower, can
species by untargeted analysis. screen for a wide range of species in a single analysis.
Potential to develop quantitative analysis of food
extracts. High financial investment required.
Selected Reaction Monitoring Targeted analysis for a suite of Determination of species from a target list of marker
(SRM) Mass Spectrometry species marker peptides. peptides. Relevant to single species or mixtures of
gelatine extracted from foods, sensitive to 0.07 %
depending on matrix. Potential to develop
quantitative methods. Suitable for routine, high
throughput screening of foods.
5. Conclusion
As markets fluctuate regarding livestock in the food chain, the possibility exists for the carcasses of
any animal species, for example horse, to be used to produce gelatine for financial gain.
There is evidence that adulteration of gelatine is occurring in the food chain and indeed the UK
Food Standards Agency discovered fraudulent use of hydrolysed collagen in plumping agents
added to chicken fillets in 2009. Research work has also discovered mislabelling of gelatines in
food products [2]. Methods are required to distinguish gelatines from different species to support
authenticity and integrity in the food chain and also to inform consumer choice to support ethical
and religious preferences. Gelatine is a highly processed product, manufactured under conditions
of high temperature and long-term exposure to acid or alkali. These conditions cause denaturation
of DNA and protein structure and therefore conventional animal origin determination methods,
such as PCR techniques and ELISA, cannot be applied.
Mass spectrometry methods are emerging for the determination of species origin of gelatine. Full
scan (untargeted) technologies, coupled to strictly curated and independently verified databases,
offer the capability to screen for a range of species in a single qualitative analysis and therefore the
opportunity to uncover unexpected issues in the food chain during routine analysis. The potential
now also exists to develop these methods to allow quantitation. The importance of such food
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6. Bibliographic references
1. Eryılmaz H.S., Işık B.Ş., Demircan E., Memeli Z., Çapanoğlu E. & Erdil D.N. (2017). – Origin Determination and
Differentiation of Gelatin Species of Bovine, Porcine, and Piscine through Analytical Methods. Turk. J. Agric. - Food
Sci. Technol., 5 (5), 507–517. doi:10.24925/turjaf.v5i5.507-517.1077.
2. Jannat B., Ghorbani K., Shafieyan H., Kouchaki S., Behfar A., Sadeghi N., Beyramysoltan S., Rabbani F., Dashtifard S. &
Sadeghi M. (2018). – Gelatin speciation using real-time PCR and analysis of mass spectrometry-based proteomics
datasets. Food Control, 87, 79–87. doi:10.1016/j.foodcont.2017.12.006.
3. Bloom O.T. (1925). – Machine for testing jelly strength of glues, gelatins, and the like. Available at:
https://patents.google.com/patent/US1540979A/en.
4. Karim A.A. & Bhat R. (2009). – Fish gelatin: properties, challenges, and prospects as an alternative to mammalian
gelatins. Food Hydrocoll., 23 (3), 563–576. doi:10.1016/j.foodhyd.2008.07.002.
5. Benbettaïeb N., Karbowiak T., Brachais C.H. & Debeaufort F. (2016). – Impact of electron beam irradiation on fish
gelatin film properties. Food Chem., 195, 11–18. doi:10.1016/j.foodchem.2015.03.034.
6. An R., Jia Y., Wan B., Zhang Y., Dong P., Li J. & Liang X. (2014). – Non-Enzymatic Depurination of Nucleic Acids: Factors
and Mechanisms. PLoS ONE, 9 (12), e115950. doi:10.1371/journal.pone.0115950.
7. Elliott C. (2014). – Elliott review into the integrity and assurance of food supply networks: final report - A national
food crime prevention framework. Available at: https://www.gov.uk/government/publications/elliott-review-into-
the-integrity-and-assurance-of-food-supply-networks-final-report.
8. Grobben A.H., Steele P.J., Somerville R.A. & Taylor D.M. – Inactivation of the bovine-spongiform-encephalopathy
(BSE) agent by the acid and alkaline processes used in the manufacture of bone gelatine. Biotechnol. Appl. Biochem.,
39 (3), 329–338. doi:10.1042/BA20030149.
9. Sha X.M., Tu Z.C., Wang H., Huang T., Duan D.L., He N., Li D.J. & Xiao H. (2014). – Gelatin Quantification by Oxygen-18
Labeling and Liquid Chromatography–High-Resolution Mass Spectrometry. J. Agric. Food Chem., 62 (49), 11840–
11853. doi:10.1021/jf503876a.
10. Hidaka S. & Liu S.Y. (2003). – Effects of gelatins on calcium phosphate precipitation: a possible application for
distinguishing bovine bone gelatin from porcine skin gelatin. J. Food Compos. Anal., 16 (4), 477–483.
doi:10.1016/S0889-1575(02)00174-6.
11. Nemati M., Oveisi M.R., Abdollahi H. & Sabzevari O. (2004). – Differentiation of bovine and porcine gelatins using
principal component analysis. J. Pharm. Biomed. Anal., 34 (3), 485–492. doi:10.1016/S0731-7085(03)00574-0.
12. Shabani H., Mehdizadeh M., Mousavi S.M., Dezfouli E.A., Solgi T., Khodaverdi M., Rabiei M., Rastegar H. & Alebouyeh
M. (2015). – Halal authenticity of gelatin using species-specific PCR. Food Chem., 184, 203–206.
doi:10.1016/j.foodchem.2015.02.140.
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13. Hashim D.M., Man Y.B.C., Norakasha R., Shuhaimi M., Salmah Y. & Syahariza Z.A. (2010). – Potential use of Fourier
transform infrared spectroscopy for differentiation of bovine and porcine gelatins. Food Chem., 118 (3), 856–860.
doi:10.1016/j.foodchem.2009.05.049.
14. Fajardo V., González I., Rojas M., García T. & Martín R. (2010). – A review of current PCR-based methodologies for
the authentication of meats from game animal species. Trends Food Sci. Technol., 21 (8), 408–421.
doi:10.1016/j.tifs.2010.06.002.
15. Abdullah Amqizal H.I., Al-Kahtani H.A., Ismail E.A., Hayat K. & Jaswir I. (2017). – Identification and verification of
porcine DNA in commercial gelatin and gelatin containing processed foods. Food Control, 78, 297–303.
doi:10.1016/j.foodcont.2017.02.024.
16. Demirhan Y., Ulca P. & Senyuva H.Z. (2012). – Detection of porcine DNA in gelatine and gelatine-containing
processed food products—Halal/Kosher authentication. Meat Sci., 90 (3), 686–689.
doi:10.1016/j.meatsci.2011.10.014.
17. Collins M., Buckley M., Grundy H.H., Thomas-Oates J., Wilson J. & Doorn N. van (2010). – ZooMS, the collagen
barcode and fingerprints. Spectrosc. Eur., 22 (2), 6–10.
18. Flaudrops C., Armstrong N., Raoult D. & Chabrière E. (2015). – Determination of the animal origin of meat and gelatin
by MALDI-TOF-MS. J. Food Compos. Anal., 41, 104–112. doi:10.1016/j.jfca.2015.02.009.
19. Grundy H.H., Reece P., Buckley M., Solazzo C.M., Dowle A.A., Ashford D., Charlton A.J., Wadsley M.K. & Collins M.J.
(2016). – A mass spectrometry method for the determination of the species of origin of gelatine in foods and
pharmaceutical products. Food Chem., 190, 276–284. doi:10.1016/j.foodchem.2015.05.054.
20. Buckley M., Collins M., Thomas-Oates J. & Wilson J.C. (2009). – Species identification by analysis of bone collagen
using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom.
RCM, 23 (23), 3843–3854. doi:10.1002/rcm.4316.
21. Kim J.S., Fillmore T.L., Liu T., Robinson E., Hossain M., Champion B.L., Moore R.J., Camp D.G., Smith R.D. & Qian W.J.
(2011). – 18O-labeled proteome reference as global internal standards for targeted quantification by selected
reaction monitoring-mass spectrometry. Mol. Cell. Proteomics MCP, 10 (12), M110.007302.
doi:10.1074/mcp.M110.007302.
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the risk of food fraud
Section 1
Jean-François Morin*, Michèle Lees
Eurofins Analytics France, Nantes, France
*E-mail corresponding author: [email protected]
Section 2
Peter Rinke*
SGF International e.V., Nieder-Olm, Germany
*E-mail corresponding author: [email protected]
Section 3
Petter Olsen, Marianne Svorken*, Silje Elde, Patrick Berg Sørdahl
Nofima, Tromsø, Norway
*E-mail corresponding author: [email protected]
General overview
The primary incentive for carrying out food adulteration and other fraudulent practices is
economic and a desire by the dishonest producer or distributor to make money by passing off
inferior product as one of a higher value. Unlike food defence where tampering of food is carried
out with the aim of harming a company, its employees and even the consumer, the intention of
the food fraudster is not directly to cause a public health threat, although in some cases this may
be an indirect consequence.
There are a large number of potential types of fraud as described in the introduction to this book.
However, they have one aspect in common: their unpredictable nature. This differentiates food
fraud from food safety concerns, where contamination is often unintentional and can be linked to
a specific source (microbiological contamination in food, excessive use of pesticide residues,
mycotoxin production during storage, and so on). Food safety has been the main focus of the food
industry over several decades leading to the globally used HACCP (Hazard Analysis and Critical
Control Points) approach, a documented food safety system to identify and control biological,
physical and chemical hazards in food production. Food fraud on the other hand can occur outside
the company’s processing and distribution system, and therefore outside the scope of the its food
safety management plan.
https://doi.org/10.32741/fihb.22.riskmitigation
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There is growing awareness in the food sector for the need for a preventive approach to mitigate
the risk of food fraud. Whilst analytical methods such as those described in this handbook play an
important role in detecting adulteration, they are not the only solution to preventing food fraud
and sometimes provide no solution at all. A more efficient approach is to look at the entire value
chain and identify not risks but vulnerabilities in the supply chain and of the product itself. This
means taking into account various aspects of the whole chain:
As shown in the above figure, a comprehensive strategy of food fraud mitigation requires placing
the food product or ingredient in an all-round context which includes taking into account previous
food fraud occurrences, where the product has been sourced from, the complexity of the supply
chain involved and the adequacy of traceability within the chain.
Various approaches dealing with all or part of such a strategy have been documented and are
available either as guidelines or as specific vulnerability tools. Details of these are given in Section
2 below.
The area of fruit juice fraud has been addressed over the last 30 years and a sophisticated and
global approach to controlling this sector has been set in place by the industry itself. Section 3 of
this chapter describes the SGF Product Control system, an excellent example of incorporating
supply chain monitoring and appropriated analytical testing on an international scale.
Supply chain traceability is an essential part of the overall strategy to mitigate the risk of food
fraud. Considerable technological progress has been made in this area. A description of the
concept of traceability and the latest tools is given Section 4 of this chapter.
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With these requirements now in place, food companies have been seeking help with implementing
the Vulnerability Assessment required by the CPOs. There are now a number of tools available to
help companies with this that have been developed either independently from or specifically in
reply to the new GFSI requirements for food fraud mitigation. The two main tools that are freely
available to food operators are the US Pharmacopeia (USP) Food Fraud Mitigation Guidance
Document and SSAFE/PwC Vulnerability Assessment tool. These are described below.
It is worth noting that in all cases the tools that been developed are described as “living” or
“dynamic” tools. Food fraud and associated vulnerabilities do not remain static but evolve over
time, often influenced by changing environmental conditions, the opening up of new markets,
fluctuating economic conditions, the appearance of new adulterants, and so on. It is therefore
important that the vulnerability assessment process is carried out on a regular basis.
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Step 2 then identifies the impact that the food fraud event might have both on the food company
and on its wider environment; the premise being that while all foods and food ingredients are
possible targets of fraud, not all will impact either public health, consumer confidence or the
company’s economic situation.
Figure 2: Four steps of the USP Food Fraud Mitigation Guidance Document.
©2015 U.S. Pharmacopeial Convention (USP)
The results of steps 1 and 2 are then brought together in a “Vulnerability Characterization Matrix”
(see Figure 3) to assess overall vulnerabilities and provide an indication of where further fraud
mitigation measures are required (Step 4).
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with Wageningen UR and VU University Amsterdam they developed a science-based tool to assess
a company’s food fraud vulnerabilities. This is available as a free tool, to be used by food operators
across the food supply chain, irrespective of size, geographical location or type of food business. It
can be downloaded as an Excel file from www.ssafe-food.org or completed online by visiting
www.pwc.com/foodfraud.
The SSAFE/PwC Tool has several components starting with a general information sheet in which
the user can enter details of the company and the person or team responsible for filling in the
questionnaire. It also provides a decision tree that can be used as a pre-filter to help prioritize
where the tool should be applied. Its main part is a set of fifty assessment questions structured in
two dimensions.
The first dimension explores those elements linked to potential criminal behaviour:
● Opportunities: these include the potential for fraud such as the type of product or
process and previous fraud history, and the nature of the supply chain.
● Motivations: these relate to organizational aspects such as the business culture of the
company, its economic situation and that of its customers and suppliers, and any
evidence of previous offenses.
● Control measures: these include mitigation and contingency control measures, with
questions on whether internal or external controls are in place, and whether these are
hard or soft controls.
The user provides answers to the different questions by assessing their associated risk levels (low,
moderate, and high).
The second dimension brings into play the company and its external environment, such as its
suppliers, customers, and supply chain. How these two dimensions the key elements link together
is shown in Figure 4.
Figure 4: The SSAFE VA tool showing the environment of the company and the three elements of food fraud
Sourced from: Introduction to SSAFE Food Fraud Vulnerability Assessment Tool, December 2015
Once the questionnaire is complete, the tool provides a set of spider webs giving both an overview
and a detailed assessment of the findings. Although it does not provide specific recommendations
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for mitigation techniques, an overall final report does identify certain areas of vulnerability and
this can point the company in the right direction to address the potential risks.
Documentation accompanying the tool vulnerability assessment tool also has a full list for further
reading, providing references to other tools and to external sources where more information can
be found.
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Food fraud is a recognized safety risk for consumers and effective strategies to mitigate this risk
are required, including a vulnerability assessment of purchased ingredients and suitable analytical
checks. However, food fraud detection calls on particular competencies and means which are not
always available at the different links in the supply chain. Criminal energy is often spent on
reducing the detectability of fraud and special intelligence is necessary to stay ahead in a constant
race between the fraudster and control techniques. Thus there are good arguments to centralise
the necessary competencies in a pre-competitive approach to assist raw material purchasing
companies as much as possible in this task. Although processing companies are not completely
dispensed from carrying out any fraud control, sector specific monitoring systems can reduce
significantly the risk of purchasing falsified products and assure fair competition. As a best practice
example the control system which is operated by SGF International e.V. (SGF), formerly
“Schutzgemeinschaft der Fruchtsaftindustrie e.V.” [11] is discussed in this paper.
The Voluntary Control System (VCS) of SGF was established as a company certification system. It
started much earlier than other international food certification systems such as the GFSI certified
standards or ISO 22000 [12,13] which have gained importance since the scandals such as dioxin
and BSE in the nineties. The non-profit organisation SGF [14] was founded in 1974 in Germany by
the fruit juice industry. The initial motivation of fruit juice companies to set up the VCS was the
wish to combat unfair competition in the marketplace and avoid negative headlines when food
fraud incidents came to light. Therefore, control structures were established which have focussed
on authenticity and legal compliance right from the very beginning.
It soon became obvious that major food fraud risks were linked to processed semi-finished goods
purchased from third parties. For this reason the VCS extended controls along the whole value
chain from the first fruit processing step to the distribution to consumers. Farming activities have
less potential for food fraud and were not included. Checks of traceability and plant specific
technology were intensified successively as support for the interpretation of analytical results. A
worldwide unique combination of product and system control thus developed. This includes co-
operating independent control systems for consumer goods in a number of European countries.
This paper will focus on food fraud control and not discuss the positive effect of the VCS on other
quality aspects, food safety and hygiene.
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Companies agree to both announced and unannounced audits during normal working days. They
also allow SGF auditors to check any production or traceability record.
SGF is in charge of scheduling the control plan and orders audits. Every supplier is audited at least
once a year. If considered necessary, for example, if any doubt about the conformity of products
from any producer exists or if post controls for already solved issues should be carried out, SGF can
increase the frequency of audits or inspections for one specific supplier.
Auditors are trained by SGF and follow an integrity programme.
Every participating company keeps a retained sample from every production unit, every reception
of semi-finished goods and every delivery to customers, from which the auditor selects samples for
analytical controls. Advice on what to sample is provided by SGF headquarters in function of the
specific situation of a company. Specially targeted sampling is carried out when an investigation is
underway. An average of about 10 samples per audit are sealed by the auditor and send to SGF
headquarters. Analyses are carried out in different independent qualified laboratories to stay
flexible in the choice of methods and to benefit from the judgement of independent experts. A
legal evaluation is requested from laboratories for analysed samples. If there are reasons to doubt
the authenticity of any product, a previously defined procedure for further analytical confirmation
is applied.
By covering all links in the supply chain, the identity of retained samples along the whole value
chain can be counter checked by comparison with samples taken at both supplier and customer
from the same batch. The interpretation of analytical data can be fine-tuned if it appears that the
processing conditions have influenced the analytical profile and can be taken into account.
Furthermore, auditors are instructed to take authentic reference material from the running
production and to document their history. These samples are used to maintain a worldwide
unique analytical reference data base for fruit and vegetable juices. Such samples can also be
provided to laboratories to help them develop and test new analytical approaches. The support of
analytical development is part of SGF’s tasks.
Both analytical results and traceability documentation are evaluated by specialists at SGF’s
headquarters.
If controls are considered as satisfactory or if required corrective actions have been carried out,
the producer is listed as an approved supplier on the SGF-internet member portal which is updated
daily.
VCS rules for participating companies also include the purchase of semi-finished goods from SGF
approved suppliers with priority or alternatively to apply an extended analytical scope to assure
conformity. Such analyses create significant costs and are an additional motivation for suppliers to
join the system and to benefit from a list of additional services which are not discussed here.
Products from companies which are not actively participating in the certification scheme are
controlled too. Sampling of semi-finished goods from non-participants of the VCS can be carried
out during audits at participants who purchase from these sources or who have received
commercial samples. Finished products are taken from retail outlets.
In other certification systems food fraud is seen as one safety and quality risk to be controlled by a
single company. Thus, only products from one company and their direct suppliers are submitted to
controls.
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The major advantage of centralised and independent controls combined with corrective actions is
the assurance of fair competition and a clean market. When a purchasing company detects food
fraud, the subsequent consequences generally only have an effect on a single supplier-customer-
relation. Unscrupulous suppliers remain in the market with adulterated products and harm fair
competition and food safety. Therefore general market controls are part of the mandate that SGF
had received from VCS participants. The matrix in Table 1 summarises the economic impact of
possible frauds.
The VCS is recognized by the industry as control body because the system acts independently. The
management and administration of the system must be structured accordingly. Other functions of
an industry association such as lobby work in legislation processes and standard setting cannot be
carried out by the control system if it is to maintain its neutrality and trust within the industry. The
size and economic status of any company should not make any difference when food fraud is
detected. For SGF the structure as shown in Figure 6 guarantees this requirement.
Table 1: Impact of food fraud to individual companies and the whole industry
Liability
Food safety risk
Company related Less competitive
Official reprimand No negative impact
risks purchasing conditions
Recalls
Damaged brand image
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As an overriding principle, all company related control results and corrective actions remain
confidential between the company concerned and SGF operational headquarters and any
problems are discussed solely between these two parties. No names or details are reported or
transmitted to third parties. Thus, no direct relationship with the company’s customers or with the
authorities are affected. This allows constructive solving of any problem to assure that fraud
practice is stopped. Furthermore, a tight and targeted follow up through SGF post controls ensures
the effectiveness of corrective actions.
Only in a very few cases, for example if in a court case or if official notification for a detected
health risk is required, does it become necessary for the operational headquarters to break
confidentiality. These exceptional decisions are the responsibility of the board of directors who
would be informed about the identity of parties involved.
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The non-respect of VCS system rules by participants leads also to corrective actions.
Figure 8 shows the different corrective actions. Depending on the seriousness of the case, first one
of the internal measures is applied. For food fraud, this is generally the request to sign a negative
covenant independently if the company is an SGF member or not. With such a covenant the
company confirms that it will refrain from the detected fraud practices and agrees to a dissuasive
penalty fee for each case of repetition. If a chosen measure is not successful the next stronger step
is initiated. The system is also applied for other quality problems than food fraud, e.g. production
errors or increased food spoilage.
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However, the setting-up of a control system would require a certain starting investment and,
above all, its acceptance by the industry branch. Only if a major share of market players is in
support of its implementation and accept to abide by the rules of conduct, can such a system be
rolled out successfully. To limit costs, benefitting from experience of existing control
infrastructures is recommended. Limiting it to a defined region for finished goods and/or a
reduced product scope could facilitate the start of a new system.
At the end of the day, companies will minimise their own costs incurred in carrying out
vulnerability assessments and product control thanks to the advantage of centralisation.
Additional market controls would lead to fair competition and fewer risk of scandals. System rules
and control mandate must be defined exactly and agreed by all participants. Important points are
listed in Table 2 and Table 3.
Rules are defined and agreed by active Rules must be accepted by a major share of the market. Industry reality must
participants be taken into account.
Stimulation to trade and purchase Participants must have an interest to purchase semi-finished goods from
products from participants of the system system approved suppliers preferably.
Enhanced controls when purchasing from No system can be hermetically closed. Therefore the system must include
outside of the system goods from non-controlled suppliers to ensure sufficient protection against
food fraud.
Whole chain approach Authenticity control is more efficient with cross checks along the whole supply
chain.
Assure pre-competitiveness Antitrust rules are prerequisite.
Mandate Comment
Analyses (product checks) Analyses are necessary to check products and confirm frauds.
Audits (system checks) Traceability data and knowledge about applied technology and specific
circumstances allow refined evaluation of analytical results.
Whole market control Controls must cover the whole market to assure fair competition.
Positive communication Blacklisting harms the willingness of defrauding companies to carry out corrective
actions. Only publication of achieved certification or approval of companies is
recommended.
Maintain confidentiality Constructive work on corrective actions is possible only if the companies concerned
are sure about their anonymity with respect to customers and competitors.
Corrective actions The system must tend to stop the danger of repetition for any detected source of
food fraud.
Development of analytical methods The system must support the best use and development of applicable analytical
science. Access to efficient methods and updated information for market players is
important. (e.g. publication of reference databases).
Development of control intelligence Horizon scanning of fraud possibilities is required for efficient control work. Product
specific experience to investigate and to detect fraud must be built up.
Combination with other services Synergies with other branch specific services can be useful. Therefore, pre-
(facultative) competitive character must be maintained.
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the conversion factors and other possible sources of discrepancies, an interview was conducted
with a company that produces and sells various types of fish products. Among other factors, the
interview revealed that the discrepancy in product properties is largely dependent on the product
in question. The discrepancy relating to weight, condition and conservation is much higher in the
production of highly processed products such as saltfish and clipfish (cod that has been both salted
and dried) with a long storage time, than in the production of fresh fish. In general, the more
complex the production, the higher the discrepancy. It is also in the production phase of the
product that discrepancies are most likely to occur, not in the export and sale of products.
Input Output
600 000
500 000
400 000
Tonnes
300 000
200 000
100 000
-
2010 2011 2012 2013 2014 2015 2016 2017
Year
Figure 10: Discrepancy in tonnes between input and output when mass balancing cod in Norway
The conversion factors used to convert product weight into living weight stands out as a significant
source of error, but there are also several other factors that can explain why this discrepancy
seems to appear annually. Table 4 shows the identified main sources of discrepancies, the
different reasons why they occur and the associated responsibility.
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Additional tools for mitigating the risk of food fraud
The findings from this case study shows that while public record requirements in the Norwegian
fishing industry covers a wide range of topics, only a few can be used to trace a product or to
identify a discrepancy. The case study shows that tracing claims like origin, time/date and
ownership through the production is possible provided there are good systems for recording these
properties. Properties like weight, conservation and product condition are more difficult to trace
as they may change during the production. As weight often is related to catch volume and illegal,
unregulated and unreported (IUU) fishing, this claim is of special interest.
If there are recordings of both input and output in a production, a MFA is of high relevance.
However, the case study shows that the reliability is highly dependent on industry structure, the
complexity in production, data availability, and data quality. Further, whilst the analysis shows that
there is a gap between input and output, it does not identify whether this gap is due to
unintentional actions (e.g. production errors, manual error, etc.) or if it is due to criminal activity.
As the quantitative approach described above does not identify the source of the discrepancy, it
must be supplemented by a qualitative approach, either in-depth interviews with industry actors
or more cost-effective methods such as questionnaires or phone interviews, the former used in
[27]. These methods can be used to identify weak points in the supply chain, such as those
described above, relating to production complexity, conversion factors between product types,
etc.
With the MFA-approach being highly dependent on data availability and data quality, it is useful
within industries with many control points, but less so in cases where product registrations are
few. As the case study shows, the discrepancy can be comparatively higher for highly processed
products than products that undergo a much simpler production. For products that undergo a
relatively substantial transformation during production, control points throughout the production
process itself would be necessary to better account for discrepancies due to inherent product
characteristics.
4. Bibliographic references
1. MyGFSI - What is GFSI Available at: www.mygfsi.com/about-us/about-gfsi/what-is-gfsi.html.
2. Food Fraud Think Tank (2012).
3. MyGFSI - GFSI Releases New Edition of Benchmarking Requirements Available at:
www.mygfsi.com/news-resources/news/press-releases/654-gfsi-releases-new-edition-of-
benchmarking-requirements.html.
4. The United States Pharmacopeial Convention – Food Fraud Mitigation Guidance - Appendix XVII General
Tests and Assays.
5. EMAlert Available at: https://emalert.org/.
6. European Commission – RASFF - Food and Feed Safety Alerts - Food Safety. Food Saf. Available at:
https://webgate.ec.europa.eu/rasff-window/portal/.
7. Food Fraud Database Available at: http://www.foodfraud.org/.
8. FAIR: Food Adulteration Incidents Registry
9. FIDES (Focused Integration of Data for Early Signals) Available at:
https://foodprotection.umn.edu/innovations/food-systems.fides.
10. FoodIntegrity project (2018). – FoodIntegrity Knowledge Base. Available at:
https://secure.fera.defra.gov.uk/foodintegrity/wp2.
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Additional tools for mitigating the risk of food fraud
11. Guidance on Food Fraud Mitigation - Version 1 (2018). FSCC 22000. Available at:
http://www.fssc22000.com/documents/graphics/version-4-1-downloads/fssc-22000-guidance-on-food-
fraud-final-100418.pdf.
12. MyGFSI - Global Food Safety Initiative MyGFSI. Available at: http://www.mygfsi.com/.
13. ISO 22000 Food safety management Available at: https://www.iso.org/iso-22000-food-safety-
management.html.
14. Website of SGF International e.V. Available at: https://www.sgf.org/index.php?id=29.
15. Rinke P., Moitrier S., Humpfer E., Keller S., Moertter M., Godejohann M., Hoffmann G., Schaefer H. &
Spraul M. (2007). – An 1H-NMR technique for high throughput screening in quality and authenticity
control of fruit juice and fruit juice raw materials - SGF-profiling. Fruit Process., 1, 10–18.
16. Spraul M., Schütz B., Rinke P., Koswig S., Humpfer E., Schäfer H., Mörtter M., Fang F., Marx U.C. &
Minoja A. (2009). – NMR-Based Multi Parametric Quality Control of Fruit Juices: SGF Profiling. Nutrients,
1 (2), 148–155. doi:10.3390/nu1020148.
17. Website of International Fruit and Vegetable Juice Association Available at: https://www.ifu-
fruitjuice.com/.
18. Herbst S. (2017). – The SGF International e.V. as a cornerstone of a risk based quality management
system for companies of the worldwide fruit juice industry.
19. Olsen P. & Borit M. (2013). – How to define traceability. Trends Food Sci. Technol., 29 (2), 142–150.
doi:10.1016/j.tifs.2012.10.003.
20. Swan M. (2015). – Blockchain: blueprint for a new economy. First edition, O’Reilly, Beijing Cambridge
Farnham Köln Sebastopol Tokyo.
21. Tian F. (2016). – An agri-food supply chain traceability system for China based on RFID & blockchain
technology. 2016 13th Int. Conf. Serv. Syst. Serv. Manag. ICSSSM, , 1–6.
22. Brunner P.H. & Rechberger H. (2016). – Handbook of Material Flow Analysis: For Environmental,
Resource, and Waste Engineers. 2nd ed., CRC Press.
23. Dewulf J. & Langenhove H.V., eds. (2007). – Renewables-based technology: sustainability assessment.
Reprinted, Wiley, Chichester.
24. Bringezu S. & Moriguchi Y. – Material flow analysis. . In A handbook of industrial ecology, Ayres, R. &
Ayres, L. pp 79–90
25. Gould O. & Colwill J. (2015). – A framework for material flow assessment in manufacturing systems. J.
Ind. Prod. Eng., 32 (1), 55–66. doi:10.1080/21681015.2014.1000403.
26. Christophersen J.G. (2011). – Organisert fiskerikriminalitet I et nordatlantisk perspektiv. Rapport til
Fiskeri- og kystdepartemente.
27. Svorken M. & Hermansen Ø. (2014). – Urapportert fiske I torskefiskeriene. Resultater fra
spørreundersøkelse om juk. NOFIMA.
28. Svorken M., Sørdahl P.B. & Elde S. (2018). – Identifying discrepancies in the wild caught fisheries
through a mixed method approach – is it possible? Forthcoming.
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The FoodIntegrity Knowledge Base
Jean François Morin*
Eurofins Analytics France, Nantes, France
*E-mail corresponding author: [email protected]
A whole Work Package of the FoodIntegrity project has been dedicated to the design and creation
of a comprehensive Knowledge Base linking each food product, its potential integrity issues and
appropriate solutions for detection.
For use by industry and regulatory authorities, this tool will make it possible to identify, easily and
rapidly, potential food fraud threats to a given food product or ingredient and the existing
solutions. The Knowledge Base contains a wealth of information including the type of the
fraudulent practice, the analytical methods available, their use and performance criteria, and the
availability of reference data with links to literature or standards and open-access databases.
Currently hosted under the umbrella of the FoodIntegrity website [1], a transfer to a European
organisation is ongoing in order to ensure its sustainable use in the future.
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FoodIntegrity Knowledge Base
standardisation organisations such as the International Organisation of Vine and Wine (OIV) in the
wine industry or professional associations such as the International Federation of Fruit Juice
Producers (IFU) for juices. Indications if the method has been transferred to several laboratories
and is available in routine analysis have been added: this allows users, especially from industry, to
assess how easily they will be able to access the method. The Knowledge Base contains a field
where the code of the Combined Nomenclature, the systematic list of commodities in use in the
EU for classifying goods [3], is stored. This field allows better categorisation for statistics and,
acting as a primary key, make future interaction possible with other resources or tools. For easier
comparison between methods, information is recorded in a standardised way. When information
cannot be stored in the Knowledge Base, links to other sources are provided.
Datasets containing for instance raw data from analytical devices are also attached to some of the
entries. Nowadays different analytical approaches are used for food authenticity testing. The
majority require a comparison to reliable authentic data to judge the compliance of a food sample.
It is of enormous benefit to any organisation carrying out food fraud testing, or embarking on the
development of new analytical methodology, to get access to this kind of information. The use of
reference data is even more crucial where fingerprinting or profiling approaches are used.
Thanks to public funding from the European Commission and to the work of the FoodIntegrity
partners, access to this resource will be free of charge for all users. Access to knowledge will be
possible for any stakeholder, whether a food manufacturing SME, a researcher in the field of
authenticity or a civil servant working in a food safety organisation.
Finally the Knowledge Base will act as a European focal point on analytical methods in the field of
food fraud standing above the vast array of information that exists in a number of private and
public analytical data bases. It will be open to users from any country. The fact that such data sets
exist at a single point of reference will benefit most organisations.
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FoodIntegrity Knowledge Base
For standardisation organisations, the Knowledge Base will help to determine gaps in the
coverage of food fraud detection by standards. Furthermore future candidates for standardisation
or methods which need extended validation will also be identified.
The Knowledge Base will also be useful for food testing laboratories, whether academic or private.
Thanks to the description and to the links to scientific papers containing the full description of the
protocol, analytical methods can be easily implemented in routine practice. The service portfolio
of these companies will expand, allowing a better monitoring of fraud risk in industry. Furthermore
it will enable proficiency tests to be organised among more and more laboratories, facilitating
dissemination and recognition of these methods throughout the European Union. For research
laboratories, access to authentic and standardised datasets of analytical methods which can be
reused will promote the development of new and improved analytical methods.
Ultimately the Knowledge Base will be used by all stakeholders as a knowledge reference in the
field of food fraud. It will help to disseminate the idea that food fraud is not inevitable and that
tools exist to tackle it.
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FoodIntegrity Knowledge Base
Acknowledgments
The FoodIntegrity knowledge Base is the collaborative result of the work of 19 European
organisations from the FoodIntegrity project:
FERA, EUROFINS, JRC IRMM, BFR, SITEIA.UNIPR , CRA-W, FiBL, , UCPH, DLO, VSCHT Praha, FEM,
UCLM, BARILLA, TEAGASC, Isolab GmbH, CSIC, FAO, SOLTUB, SWRI.
Bibliographic references
1. FoodIntegrity project (2018). – FoodIntegrity Knowledge Base. Available at:
https://secure.fera.defra.gov.uk/foodintegrity/wp2.
2. Global Food Safety Initiative (GFSI) (2014). – MyGFSI - Food Fraud Mitigation. Available at:
https://www.mygfsi.com/files/Information_Kit/GFSI_GMaP_FoodFraud.pdf.
3. Commission Implementing Regulation (EU) 2017/1925 of 12 October 2017 amending Annex I to Council
Regulation (EEC) No 2658/87 on the tariff and statistical nomenclature and on the Common Customs
Tariff (2017). Off. J. Eur. Union, L282, 1–958.
4. MyGFSI - GFSI Releases New Edition of Benchmarking Requirements Available at:
www.mygfsi.com/news-resources/news/press-releases/654-gfsi-releases-new-edition-of-
benchmarking-requirements.html.
5. European Commission – RASFF - Food and Feed Safety Alerts - Food Safety. Food Saf. Available at:
https://webgate.ec.europa.eu/rasff-window/portal/.
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Further reading
James Donarski*
Fera Science Limited, York, UK
*E-mail corresponding author: [email protected]
The FoodIntegrity Network, a group that had members representing the key stakeholders
impacted by food fraud (regulators, industry, academics, research providers and customers) was
used to identify key areas where scientific guidance was required. The output from this was the
production of several opinion papers. The aim of the scientific opinions produced as part of the
FoodIntegrity project was to provide independent scientific advice and communicate to all
interested parties on topical issues concerning food integrity. The scientific opinions provide
objective, science-based advice, and clear and coherent communication, grounded in the most up-
to-date scientific knowledge and data. These opinions are recommended as further reading. The
titles, an abridged abstract and the corresponding author are contained below. At the time of
writing, several of these papers are under peer review and it is expected that these will be
available in open access journals at the time of reading.
Stable isotope techniques for verifying the declared geographical origin of food in legal cases
Corresponding Author: Federica Camin (Department of Food Quality and Nutrition, Research and
Innovation Centre, Fondazione Edmund Mach (FEM))
Consumers are increasingly interested in the provenance of the foods and European laws require
protection against the mislabelling of premium foods. Methods for testing authenticity require
robust analytical techniques that can be utilised by the various regulatory authorities. Of the many
techniques, the most widely-used method is stable isotope ratio analysis. Scope and approach:
Focus is on the use of stable isotope ratios of H, C, N, O, S and Sr for verifying the geographical
origin of food, cross-referencing it with examples of legal cases. State of the art including rules for
building an authentic sample reference database (commonly called databank) and for interpreting
the results obtained in actual cases is described. The overall objective is to provide stakeholders
and competent authorities dealing with fraud, with a best-practice guide for its use. Key findings
and conclusions: Stable isotope ratios can differentiate foods on the basis of their geographical
origin and, especially for light elements, can be measured reliably in routine work in different
matrices and compared successfully between different laboratories. Examples of legal applications
are grape products, orange juices, olive oil, cheese, butter, caviar. Sometimes, the cases are not
brought directly to the court, but before further verifications (e.g. paper traceability, forensic
accounting) are conducted. The system can satisfy the court when a robust databank of authentic
samples exists, the methods used are officially recognized, validated and accredited, and the
expert demonstrates that the conclusions are sufficiently robust and reliable to stand up to the
required level of proof.
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Further reading
Role of analytical testing for food fraud risk mitigation – principles of cost-benefit determination
for analytical fraud testing
Corresponding Author: Francis Butler (University College Dublin)
Food fraud is of high concern to the food industry. A multitude of analytical technologies exist to
detect fraud. However, this testing is often expensive. Available databases detailing fraud
occurrences were systematically examined to determine how frequently analytical testing
triggered fraud detection. A framework was developed for deciding when to implement analytical
testing programmes for fraud and a framework to consider the economic costs of fraud and the
benefits of its early detection. Current regulatory issues relating to food fraud detection are
explored as well as some of the main factors associated with statistical sampling for fraud
detection.
What are the scientific challenges in moving from targeted to non-targeted methods for food
fraud testing and how can they be addressed? – Spectroscopy case study
Corresponding Author: Terry F. McGrath (Institute for Global Food Security, ASSET Technology
Centre, School of Biological Sciences, Queen's University Belfast, Northern Ireland, United Kingdom)
Background: The authenticity of foodstuffs and associated fraud has become an important area. It
is estimated that global food fraud costs approximately $US49b annually. In relation to testing for
this malpractice, analytical technologies exist to detect fraud but are usually expensive and lab
based. However, recently there has been a move towards non-targeted methods as means for
detecting food fraud but the question arises if these techniques will ever be accepted as routine.
Scope and approach: In this opinion paper, many aspects relating to the role of non-targeted
spectroscopy based methods for food fraud detection are considered: (i) a review of the current
non-targeted spectroscopic methods to include the general differences with targeted techniques;
(ii) overview of in-house validation procedures including samples, data processing and
chemometric techniques with a view to recommending a harmonized procedure; (iii) quality
assessments including QC samples, ring trials and reference materials; (iv) use of “big data”
including recording, validation, sharing and joint usage of databases.
Key findings and conclusions: In order to keep pace with those who perpetrate food fraud there is
clearly a need for robust and reliable non-targeted methods that are available to many
stakeholders. Key challenges faced by the research and routine testing communities include: a lack
of guidelines and legislation governing both the development and validation of non-targeted
methodologies, no common definition of terms, difficulty in obtaining authentic samples with full
traceability for model building; the lack of a single chemometric modelling software that offers all
the algorithms required by developers.
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Further reading
The scientific challenges in moving from targeted to non-targeted mass spectrometric methods
for food fraud analysis: A proposed validation workflow to bring about a harmonized approach
Corresponding Author: Michele Suman (Barilla Advanced Laboratory ResearchParma, Italy)
Background: Detecting and measuring food fraud is a challenging analytical task since a very wide
range of food ingredients and types may be adulterated by numerous potential adulterants, many
of which are yet unknown. To date most of the methods applied for the control of food fraud are
targeted methods, which are focused on the detection of one or a few classes of known
compounds.
Scope and approach: There is an increasing availability of solutions and applications based on high
resolution mass spectrometry (HRMS), allowing parallel non-targeted approaches, novel
compound identification and retrospective data analysis. For these types of methods sample-
handling must be minimal to allow the inclusion of as many as possible chemical categories.
However data-handling of such methods is much more demanding, together with the potential
requirement to integrate multiplatform data as well as conducting data fusion. To allow the
processing of massive amounts of information based on the separation techniques and mass
spectrometry approaches employed, effective software tools capable of rapid data mining
procedures must be employed and metabolomics based approaches does appear to be the correct
way forward. To verify the relevance of modelling results, appropriate model validation is essential
for non-targeted approaches, confirming the significance of the chemical markers identified.
Key findings and conclusions: The present paper is devoted to review and assess the current state
of the art with regards non-targeted mass spectrometry in food fraud detection within many food
matrices and to propose a harmonized workflow for all such applications.
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Further reading
The future of NGS (next generation sequencing) analysis in testing food authenticity
Corresponding Author: Edward Haynes (Fera Science Ltd, York, UK)
Food authenticity is a big concern for consumers, food authorities and food producers and
processors, since incorrect food labelling and other types of fraudulent practices have been
demonstrated to negatively affect the confidence and even the safety of the final consumer.
European Union regulation (EU) No. 1169/2011 requires that consumers should be appropriately
informed regarding the food they consume. This is vital in order to achieve a high level of health
protection and to guarantee their right to information, as well as to protect the businesses of
scrupulous producers from unfair competition. Consumers’ choices can be influenced by health,
economic, environmental, social and ethical considerations. In fact, the general dictionary
definition of “authenticity” is “the quality of being authentic, trustworthy, or genuine”, and the
relevant dictionary definitions of “authentic” include “not false or copied; genuine; real” and
“having an origin supported by unquestionable evidence; authenticated; verified”. More
specifically regarding food authenticity, a recently produced CEN standard defines authenticity in a
food and feed context as the match between the food product characteristics and the
corresponding food product claims (CEN WS86). These labelling requirements, which are legally
specified and differ depending on the product, may include the scientific name or breed, and
production method (e.g. organic, free-range, wild-caught etc.). However, other features of the
product can also be included by producers to inform the consumer, including (i) ethical issues
(halal, vegetarian, etc.), (ii) nutritional composition (vitamins, omega 3, etc.), (iii) the area where
the product was caught or farmed (for sustainability reasons, or with particular regard to EU
legislation regarding protected designation of origin (PDO), protected geographical indication
(PGI), traditional specialities guaranteed (TSG) etc.), (iv) status of the product (such as whether the
product has been previously frozen and defrosted) and (v) the presence of undeclared ingredients
that can also represent a health risk for the consumer (allergens such as gluten, nuts, etc.).
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Acknowledgements
The editors would like to thank warmly the FoodIntegrity project management team, especially
Claire Sykes, Paul Brereton and Monika Tomaniova for their help in making this book a reality.
They would also like to thank Edgar Morales Ortega for his help in compiling the references of
numerous chapters and Stéphanie Guillet for having shared her knowledge in genotyping and
molecular biology.
The authors of the Food Integrity Handbook gratefully acknowledge the previous work compiled in
the book “FAIM Food Authenticity – Issues and Methodologies” (EU-funded Concerted Action
AIR3-CT94-2452, 1994 – 1997). Published in 1998, this early work inspired both the format and
content of this new Handbook on Food Authenticity. The contributors to the FAIM document are
listed below together with their organisations at the time the book was published.
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Acknowledgements
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