J.Food Pharm.Sci.

6 (2018) 1 – 6

Available online at www. jfoodpharmsci.com

Review Article

Enzymes Involving in Chocolate Processing

Warsono El Kiyat1, Audrey Monica 1, Noor Qomariyah1, Brian Saputra Manurung2
1
Department of Nutrition and Food Technology, Faculty of Life Sciences, Surya University, Jl. MH Thamrin KM. 2.7,
Tangerang, Banten 15143, Indonesia
2
Department of Biotechnology and Neuroscience, Faculty of Life Sciences, Surya University, Jl. MH Thamrin KM. 2.7,
Tangerang, Banten 15143, Indonesia

ARTICLE INFO ABSTRACT

Indonesia is one of the largest cocoa-producing countries in the world, but the
Received 06/12/2017
productivity of Indonesian cocoa is relatively low compared to other cocoa-
Received in revised form 09/12/2017 producing countries. Many cocoa beans are exported in raw form (raw cocoa
Accepted 11/01/2018 beans) so they do not have high economic value. In addition, cocoa beans also
Available online 24/01/2018Received can not be consumed directly and must be processed first. The processing of
Received in revised form cocoa beans includes curing stage, breaking the fruit, fermentation, soaking
Accepted and washing, drying, sorting, and storage to producing cacao almonds. The
fermenting process of cocoa beans involves the role of the polyphenol oxidase
Available online
to produce flavor precursor compounds. Furthermore, to improve the quality
of the resulting chocolate, protease and carboxypeptidase can be used in the
Email: processing of cacao almonds to enhance flavor precursor compounds forming
[email protected] to produce chocolate with stronger flavor characteristics. Processed
chocolates can be developed into confectionery with a soft or liquid filler by
utilizing invertase to obtain a solid filler during processing that may turn into
liquid during storage.

Keywords : cocoa, chocolate, enzyme, processing.

1. Introduction cocoa beans raw materials cannot be consumed directly

Indonesia is one of the largest cocoa producing as raw materials, because of its composition and taste
countries in the world, but the productivity is still lower must be addressed. An important phase of cocoa
compared to other cocoa producing countries. processing is fermentation that reduces bitterness and
Compared with the processed chocolate products, the fermentation. In addition, fermentation can also improve
original exports of cocoa is more, so the economic value the mouthfeel and cocoa, with a flat chocolate aroma,
is small. In addition to cocoa beans, other types of and lower levels of impurities and acidity (Kim et al.,
exports include cocoa shells, cocoa paste, cocoa butter, 2010). The fermentation process involves altering the
cocoa powder, the rest are chunky chocolate products enzymatic action of cocoa's chemical compounds that
and other processed products. According to Central initially provide undesirable properties associated with
Bureau Statistics of Indonesia issued by Director General the desired characteristics of compounds such as typical
of Plantations, in 2015 Indonesia's cocoa exports chocolate flavor compounds.
amounted to 350,730 tonnes valued at US$ In addition to the processing of cocoa beans, the
1,316,867,000. The number increased by 333,679 tons enzymes used in the chocolate processing can also be
over the previous year. cocoa processed in advanced processes, and the
Cocoa has astringent bitterness by the content of chocolate processed into candies. Each stage of
tannin and polyphenols (Paembong, 2012). Chocolate, processing involves different enzymes and compounds.
Therefore, it is necessary to do research on the role of
 Warsono El Kiyat, et al., / J.Food Pharm. Sci. 6 (2018), 1 – 6 2
enzymes that play a role in the mechanism of chocolate Cocoa plants include three varieties, namely Criolo,
as well as the functioning of these enzymes. Forastero and Trinitario (hybrids). Criolo is also known as
fine cocoa or noble cocoa. At present, the use of Criolo
2. Chocolate processed is few, but chocolate varieties dominate the
Chocolate is one of the most widely known foods, market until the mid-eighteenth Century, also known as
both as confectionery and as a flavor of other foods. In the form of cocoa, cacao ordinary or bulk cocoa is the
general, chocolate has a sweet taste due to the addition largest breed, the two types processing and planting of
of sugar, but naturally, chocolate has a bitter taste. small chocolate handles. Criolo and Forastero hybrids
Commercially, chocolate can be in the form of a solid, produce Trinitario. Repairs in cocoa plantations are a
liquid, pasta or powder. The widespread popularity of must-do and are trimming weak, weeding tree branches
chocolate has made chocolate a part of many traditions to protect, fertilize, water and pest insecticide-
and cultures, especially in Europe, for example on Easter eradicating pesticides (Secretary General of Department
and Valentine's Day. of Industry, 2007).
According to its history, chocolate was originally
introduced into the modern civilization from the Mayan Table 1. Classification of cacao plants
in Mexican by Spanish colonists and became popular Classification Scientific name
among the aristocrats of the 17th century. In 1828, Division Spermatophyta
Conrad Marriott London, a Dutch chemist, developed Subdivision Angiospermae
the Dutch method of separating fat from the cocoa Class Dicotyledonae
beans process, which was subsequently called cocoa Subclass Dialypetalae
butter. Subsequently, in 1847, the British company Order Malvales
Fletcher and Son (now part of Cadbury and Billion Family Sterculiaceae
International) will produce the first chocolate bar in full
Genus Theobroma
by mixing cocoa butter with cacao and sugar. Thirty
Species Theobroma cacao L.
years later, the collaboration between Swiss innovator
Source: Jurniati(2013)
Nestle and Swiss chocolate Daniel Pete produced the
first milk chocolate mixed with powdered milk. Rudolf
The young cocoa greens whitey, when they mature
Swiss lotus, a Swiss inventor, further develops chocolate
yellowish and young cacao fruit is red, while fully mature
refining techniques that can enhance the flavor and
is orange (Wahyudi, 2008). The cocoa fruit consists of
texture of chocolate in 1879. Although it may seem
four parts, namely skin, placenta, pulp, and seeds. A
simpler, chocolate requires considerably more complex
mature cocoa can have 30-40 seeds covered with white
processing involving a variety of complex chemical
paste or mucus (Jurniati, 2013). According to NSAI
reactions (Tannenbaum, 2004).
(2008), cocoa beans are divided into five groups based
on the weight of the seed, and the water content of AA,
3. Cacao
A, B, C and S in cocoa beans is an important property of
According to its etymology, the name cocoa or
storage in affecting its durability. If the moisture
cocoa is derived from the term "Kakava" in the Mixe-
content, they are highly susceptible to fungal or insect
Zoquean language group and is highly regarded as
damage. However, if the water content is too low, cocoa
"God's food." At the same time, the Aztec first
beans can easily become brittle or broken.
discovered chocolate drink called “cacahuatl”, meaning
Pests, which usually attacking cocoa beans is C.
cocoa water. Thus, in 1735, Linneas gave the scientific
cramerella, cause wrinkling, small, dark, and hard-to-
name Theobroma cacao. (Tannenbaum, 2004). The name
separate from the flesh of fruit (Azim et al., 2016). In
chocolate itself is considered to be chocolate by the
addition, Moniliophtora perniciosa can also lead to the
name of Aztec tribal civilization found by Spanish
broom disease, which reduces the quality of cacao beans
explorers.
and causes huge losses in the cocoa industry in Brazil.
The main component of chocolate processing is
Pest attacks on cocoa plants may affect the quality and
cocoa beans. The main habitat for cocoa plants is in the
quantity of cocoa beans and add additional production
tropical forest. Cocoa consists of five parts, namely
costs to separate good cocoa and bad cocoa beans. The
leaves, stems, lateral roots, roots and cocoa fruit
damage can also be caused by larvae.
(Prihastanti et al., 2015). Cocoa is a group of caulifloris, a
plant that grows flowers from a stem or branch. Cocoa
4. Cacao Processing
plant classification is shown in Table 1.
One of the most common cocoa processed
Cocoa was first found in Central and South America
products is chocolate. Cocoa processing will determine
and then spread to all areas along the equator 10-20
the final quality of the product, both of the form of taste
degrees. Cocoa plants have large stems and branches,
and aroma, as well as the reduction of bitterness and
and small flowers produce large pieces of fruit that can
taste. Processed cocoa is a seven-phase chocolate
reach 1 kilogram in size. Each cocoa fruit has 30 to 40
product that cures, crushed fruit, fermented, soaked,
seeds surrounded by the flesh of the fruit. It is believed
washed, dried, sorted and stored (Secretary General of
that humans are first attracted to cocoa due to the flesh
Department of Industry, 2007).
of the fruit is sweet (Tannenbaum, 2004).
Curation of cocoa fruit is designed to make the
maturity of fruit is more uniform and promote seed
 Warsono El Kiyat, et al., / J.Food Pharm. Sci. 6 (2018), 1 – 6 3
release from the fruit. Curing continues for 5-7 days and reduces the formation of fermentation compounds and
is done in the shade. During the curing process, cocoa flavoring compounds. Cocoa almonds, the initial
beans produce a unique brown flavor on the final treatment for fermentation and drying, are usually
product by known important germination processes carried out directly on the plantation to prevent spoilage
(Jurniati, 2013). Breaking the fruit is to remove the cocoa of cocoa beans for distribution to the industry. However,
beans from the fruit. After removal, cocoa beans are this treatment was done in direct plantations not under
fermented for several days. Fermentation is performed controlled conditions, resulting in many changes in
to obtain seed color characteristics, increase flavor and cocoa almonds that were sufficient to allow enzymatic
taste, and improve seed pieces (Sari, 2016). However, reactions. In the absence of enzymatic reactions, the
the chemical processes that occur during cocoa cocoa almonds obtained with low quality. Enzymatic
fermentation remain unclear and remain a research reactions play important roles in protein hydrolysis in
topic. cocoa almonds to produce flavoring precursor
There are two ways of fermentation, fermentation compounds are not formed during the fermentation
and bamboo basket fermentation. Cocoa beans are process. Therefore, in the production of a standardized
placed into a bamboo box or basket and covered with chocolate mass, the addition enzyme can be used in the
banana leaves (Secretary General of Department of treatment of cocoa (Gray, 2011).
Industry, 2007). Incomplete or too short fermentations The development of food processing technology
result in grainy and gray grain texture, while too long will has made the development of chocolate products an
result in moldy and fragile seeds, reducing the quality of important part of the candy industry. Also sold
the beans. During fermentation, proteins and peptides separately, chocolate is also commonly marketed with
react with polyphenols and produce brown color other products such as crackers, or with stuffing.
(Diansari et al., 2016). Variation adding in chocolate processing products can
The next step is to soak and wash the seeds to stop increase the economic value of chocolate. This goal can
the fermentation process. The cocoa beans are washed be achieved through the use of enzymes produced in
to remove residual fermentation mucus that inhibits the chocolate products that have special characteristics that
drying process. Seed drying can be accomplished by increase consumer buying interest (Chandrasekaran et
drying or heating until the water content reaches 7-8%. al., 2016).
During the drying process, water, volatile compounds,
and low-boiling acidic compounds drastically decreased, 5.1. Polyphenol oxidase
producing cacao beans with poor bitter and astringent Polyphenol oxidase (1,2-benzenediol:
taste (Secretary General of Department of Industry, oxidoreductase oxygen; EC 1.10.3.1), or often referred as
2007). After that, cocoa beans are sorted according to phenolase, catechol oxidase, monophenol oxidase,
their quality and stored. cresolase, or catecholase, was first discovered in 1856 by
There are cocoa beans which are processed without Schoenbein on fungi as aerobic oxidation reaction
fermentation. Cocoa beans can only be washed and catalyst of certain compounds in plants (Putra et al.,
dried using sunlight. However, the resulting cocoa beans 2010; Singh et al., 2015). Polyphenol oxidase ( PPO) can
have one drawback, such as the unique brown aroma is be found in many plant tissues, as well as some fungi
not strong, the seeds feel bitter (Schwan and Wheals, especially those producing brown silk), and in some
2004). Once dry, cocoa beans are sometimes also higher animals, including insects and humans. In those
roasted to produce products with better flavor higher plants, this enzyme is used to protect plants from
characteristics. The roasting process led mainly to insects and microorganisms, and when they are injured,
Maillard's reaction to produce color and flavor allowing plants to form melanin-scar tissue that protects
compounds from drought and other attacks. Meanwhile, in insects,
PPO participates in the sclerotization in the exoskeleton
5. Enzyme Utilization on Chocolate Processing (chitin hardening) and melanin formation. In humans,
The main ingredient in making chocolate is cacao PPOs are involved in the pigmentation of the skin, hair,
almonds or dried fermented cacao beans. Although and eyes (Whitaker, 1995).
chocolate has a distinctive flavor, cacao beans have a PPO is stable at a temperature of 25-65°C and has
chocolate-like flavor that is separated from the freshly- an optimum activity at a pH in the range of 5.0-7.0 at a
ripe pods and has a very bitter and astringent taste. In temperature of 25-35°C and a pH of neutral (in the range
order to obtain the desired flavor, cocoa beans must of 6.0-7.0) and can be extracted from various materials,
undergo a curing process that is divided into some of them are plums (Ioniţă et al., 2017), eggplant
fermentation and drying processes. This process leads to (Harish et al., 2017), avocado (George and
the formation of flavor precursors. At the same time, the Christophersen, 2016) and apple (Liu et al., 2015). The
commercial value of cocoa is dependent on the boiling point of the PPO phenolic compound, 181.7°C,
characteristics of cocoa butter and chocolate, making renders the compound resistant to high temperatures,
the correct handling of cocoa beans important for but 300°C (Wiranata et al., 2016) will be damaged if the
determining the quality and economic value of the temperature is too high. PPO activity can be inhibited by
chocolate product (Oliveira et al., 2011). using various compounds including tropolone (Valero et
The main problem encountered in the chocolate al., 1991), dithiothreitol, sodium metabisulfite (Wuyts et
industry is the low quality of cocoa almonds which al., 2006), and potassium pyrosulfite (Signore et al.,
 Warsono El Kiyat, et al., / J.Food Pharm. Sci. 6 (2018), 1 – 6 4
1997). However, the maximal inhibitory activity is shown make chocolates with filling that dominate the market. A
by ascorbic acid, L-cysteine, quercetin (Ioniţă et al., 2017; variety of padding variations and their favor and quality
Bravo and Osorio, 2016). In food, many of the roles of allow chocolate with padding to become an expensive
PPO in enzymatic browning reactions are neither product. The chocolate stuffing can be milk, fruit, beans,
desirable because of the desire in apples and potatoes, etc. But the most popular stuffing is the filling cream
nor desired as in black tea and chocolate (Quesnel and with a unique texture and taste. In order to produce
Jugmohunsingh, 1970). Browning occurs by the PPO such confectionery products, the chocolate filling should
oxidation reaction occurring through the amino acid be solid enough for handling and processing, but
tyrosine to form an o-quinone monophenolic compound softened and creamy when consumed.
ring followed by the polymerization of the pigment o- The technology currently used to achieve this goal
quinone compound that produces the polyphenolic in the confectionery industry is the use of invertase.
compound of black, brown or red (Mayer, 2006). Invertase can convert the sucrose content of the filling
In chocolate production, PPO is used to form the into glucose and fructose. This change allows the filling
flavor compound precursor from the oxidation stage of form to be changed from solid to liquid during storage
the fermentation process and continues to the dry during processing. The very high fructose solubility
stage. Some flavor changes occur when bitterness and properties can help prevent the occurrence of sugar
sourness are reduced due to the interaction between crystals in the fillings so the fillings will continue to have
polymerized polyphenols and proteins. During the drying a soft and creamy texture. Using invertase can also
process, the infiltration of oxygen into the cocoa bean extend the shelf life by maintaining the required product
maximizes the oxidation of the compound epicatechin consistency over time. The conversion of sucrose to
and the pro-cyanidin, producing melanin and glucose and fructose also results in a decrease in Aw
melanoprotein confer typical of brown of chocolate value and minimizes the possibility of microbial
(Hammer, 2013). The study by Lima et al. (2001) and de contamination and growth which can lead to product
Brito et al. (2002) also pointed out that the action of damage (Chandrasekaran et al., 2016).
polyphenol oxidase reduces the polyphenols, tannins
and the bitter and astringent features that affect cocoa 5.3. Protease and carboxypeptidase
beans. The study by Oliveira et al. (2011) showed the better
In general, PPO activity can be carried out at a quality of the chocolate produced by protease and
moisture content of 40 to 10% at 40-60°C (Lopez, 1986). carboxypeptidase (flavor protease) used in the
Optimizing PPO activity in cocoa beans will require processing of cocoa almonds. This enzyme utilization is
rehydration (water back-up), as well as pH, temperature accomplished as a means of overcoming the low-quality
conditions, and time is optimal. Therefore, PPO from problems of cocoa almonds processed early in
previous cocoa beans should be isolated and plantations. The results show that application of
characterized by its optimal conditions (Kim et al., 2010). Flavourzyme for the treatment of cocoa albumen
Measurement of PPO activity can be accomplished by produces a flavorful precursor compound with a better
reducing the concentration of phenol or catechol with flavor due to a sufficient amount of flavoring resulting
quinone formation by increasing reaction time (Murniati from the hydrolysis reaction by Flavourzyme. This
et al., 2014). research has also shown that the potential of almonds
processed for cocoa is considered to be of low quality,
5.2. Invertase to produce a quality of 50% better with sensory quality
Invertase (β-fructofuranosidase, EC 3.2.1.26) or also chocolate.
commonly known as saccharase, glucosidase and Carboxypeptidase (EC 3.4.16-3.4.18) is responsible
invertase are enzymes that catalyze the hydrolysis of for breaking the peptide bond of the carboxyl terminus
sucrose. Thus, the result of the hydrolysis reaction of of a compound of a protein or peptide.
sucrose in the form of a mixture of fructose and glucose Karboksipeptidase, also known as beta-glucanase, works
is referred to as inverted syrup. Invertase breaks down at optimum pH in the range of 5.5 and 45-50°C of
sucrose by cleaving O-C bonds in the fructose portion temperature. For commercial purposes,
(Schiweck et al., 2012). carboxypeptidases are commonly obtained from
Invertase works optimally at 60°C and pH 4.5. For Aspergillus oryzae cultures. In addition to
industrial purposes, invertase is usually extracted from microorganisms, carboxypeptidases are also naturally
yeast, but it is also naturally produced by honeybees that found in various plant seeds and play a role in the seed
produce nectar from honey. In the human body, germination. At the same time, it assists in digestion,
invertase acts as an immune enhancer, as an antioxidant, protein modification (eg, in the synthesis of insulin), and
as an antiseptic, and as a treatment for some cases of regulate biological processes including production of
patients with bone and stomach cancer. In the growth hormone, clotting, wound healing, reproduction,
meantime, in plants, sucrase plays a role in regulating and various other treatments in humans (Whitehurst's
osmosis, metabolism, supporting growth and fighting and van Oort's 2009). Carboxypeptidase is also useful in
viral attacks. Invertase activity can be inhibited by the medical field for the treatment of apoptosis and
fructose in the form of Hg2+, Ag+, Ca2+, Cu2+ ions and adipogenesis.
furanose (Kulshrestha et al., 2013). Flavourzyme itself is a combination of several
In the confectionery industry, invertase is used to enzymes produced by Novozymes, an international
 Warsono El Kiyat, et al., / J.Food Pharm. Sci. 6 (2018), 1 – 6 5
biotechnology company headquartered in Denmark. Harish, B.B.N., Wilfred, A. and Venkatesh, Y.P. 2017. Emerging
Commercially, the Flavourzyme produced by Aspergillus food allergens: Identification of polyphenol oxidase as an
oryzae results in the extraction of peptides and the important allergen in eggplant (Solanum melongena L.).”
Immunobiol. 222 (2): 155-163.
hydrolysis of proteins is marketed, both in the industry
Ioniţă, E., Gurgu, L. Aprodu, I., Stănciuc, N., Dalmadi, I., Bahrim,
and in research. Several enzymes constitute the
G. and Râpeanu G. 2017. Characterization, purification,
estimated flavor protease component, but studies by and temperature/pressure stability of polyphenol oxidase
Merz et al. (2015) showed that eight major components extracted from plums (Prunus domestica). Process
of Flavourzyme are two aminopeptidases, two types of Biochem. 56: 177-185.
dipeptidyl peptidases, tripeptidases, The alpha-amylase Jurniati, J., 2013. Pola sebaran karakteristik fisik biji kakao
ATCC 42149 / RIB 40 of the Aspergillus strains is a mold of (Theobroma cacao L.) berdasarkan posisi buah pada
koji. The optimal conditions for each enzyme are pohon, Thesis, Universitas Hasanuddin, Indonesia.
different so that the processing conditions and the best Kulshrestha, S., Tyagi, P., Sindhi, V. and Yadavilli, K.S. 2013.
Invertase and its applications - A brief review. J. Pharm.
quality of chocolate-flavored cocoa almonds have yet to
Res. 7 (9): 792–797.
be further studied to produce the product.
Lima, E.D.P.A., Pastore, G.M., Barbery, S.D.F., Garcia N.H.P., de
Brito, E.S., Lima, C.A.A. 2001. Obtaining and use of
6. Conclusion polyphenol oxidase enzyme extracted from ripe custard
There are many enzymes used in chocolate apple (Annona squamosa l.) pulp on the cocoa
processing, PPO, invertase, and a combination of (Theobroma cocoa l.) nibs in taste improvement. Rev.
protease and carboxypeptidase. During cocoa bean Bras. Frut. 23: 709–13.
fermentation, PPO functions during the formation of Liu, F., Yao, P., Guo, X., Liu Z., Guo, X. and Xu, B. 2015.
flavor-precursor compounds, producing cocoa almonds. Characterization of Prophenoloxidase in Resisting
Adverse Stresses in Apis cerana cerana. Env. Anal. Chem.
The fermented and dried cocoa beans (cocoa almonds)
2 (3):1-6.
can then be treated by adding Flavourzyme and
Lopez, A.S. 1986. Chemical change occurring during the
carboxypeptidase to improve the quality of the processing of cacao. Proceeding of The Cacao
chocolate produced by disrupting the peptides in cocoa Biotechnology Symposium. Department of Food
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the chocolate fillings in the form of liquid cream can be Pennsylvania, USA.
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