Addis Ababa University

School of Graduate Studies

Faculty of Science
Food Science and Nutrition program

IMPROVEMENT OF SHELF LIFE OF INJERA USING CHEMICAL

PRESERVATIVES

BY
ASHAGRIE ZEWDU

A thesis submitted to the School of Graduate studies of Addis Ababa

University in partial fulfillment of the requirement for the Degree of
Master of Science in Food Science and Nutrition.

July, 2009

IMPROVEMENT OF SHELF LIFE OF INJERA USING CHEMICAL

PRESERVATIVES

BY
ASHAGRIE ZEWDU

July, 2009
Addis Ababa

DEDICATION
This work is dedicated to my family who I love most in this world more than anything.
To: My father Zewdu Woldegiorgis (Fazuca)
My mother Yeshareg Bezabh (Mazuca)
My brother Estifanos Zewdu (Adinee)
My sisters Kidest Zewdu (Krro) and Hiwot Zewdu (Dudu)

CONTENTS
DEDICATION............................................................................................................................I
CONTENTS..............................................................................................................................II
LIST OF TABLES....................................................................................................................V
LIST OF FIGURES...............................................................................................................VII
LIST OF ABBREVIATIONS AND ACRONYMS.............................................................VII
ACKNOWLEDGEMENTS.................................................................................................VIII
ABSTRACT..............................................................................................................................X
1. INTRODUCTION..................................................................................................................1
1.1 BACKGROUND.............................................................................................................1
1.2 STATEMENT OF THE PROBLEM ...........................................................................2
1.3 OBJECTIVES OF THE STUDY...................................................................................4
2. LITERATURE REVIEW.....................................................................................................5
2.1 MOULDS.........................................................................................................................5
2.1.1 Food spoilage by moulds ...................................................................................5
2.1.2 Characteristics of moulds...................................................................................6
2.2. FACTORS AFFECTING MICROBIAL GROWTH.................................................8
2.2.1 Intrinsic parameters...........................................................................................8
2.2.2 Extrinsic parameters........................................................................................11
2.2.3 Hurdle technology.............................................................................................13
2.3 METHODS OF PRESERVATION AND EXTENSION OF SHELF LIFE............14
2.3.1 Spoilage and shelf life of food..........................................................................14
2.3.2 Preservation of food..........................................................................................16
2.3.2.1 Major food preservation methods...........................................................16

2.3.2.2 Food preservation with chemicals...........................................................17

2.3.2.2.1 Benzoic acid / Benzoates...............................................................19
2.3.2.2.2 Sorbic acid/ Sorbates....................................................................21
2.3.2.2.3 Propionic acid/ Propionates.........................................................25
2.3.2.2.4 Mode of action weak organic acids.............................................26
2.4 INJERA..........................................................................................................................29
2.4.1 Product description ..............................................................................................29
2.4.2 Processing of injera...............................................................................................31
3 MATERIALS AND METHODS .......................................................................................33
3.1 STUDY SITE.................................................................................................................33
3.2 PREPARATION OF INJERA.....................................................................................33
3.3 MICROBIOLOGICAL ANALYSIS...........................................................................34
3.3.1 Isolation and cultivation of the molds that spoil injera.................................34
3.3.2 Characterization and identification of moulds that spoil injera..................34
3.4 INJERA AGAR MEDIA..............................................................................................35
3.4.1 Preparation of injera sucrose agar media....................................................35
3.4.2 Comparison of ISA with other mycological media.......................................35
3.5 ANALYZING THE EFFECT OF TEMPERATURE .............................................36
3.6 DETERMINATION OF pH AND MOISTURE CONTENT OF INJERA.............36
3.6.1 Sampling method..............................................................................................36
3.6.2 Measurement of pH..........................................................................................36
3.6.3 Moisture content determination......................................................................37
3.7 EVALUATION OF EFFICACY OF CHEMICAL PRESERVATIVES.................37

3.7.1 Addition of chemical preservatives.................................................................37

3.7.2 Comparing the rate of mould invasion...........................................................38
3.7.3 Evaluation of shelf life of injera......................................................................38
3.8 STATISTICAL ANALYSIS.........................................................................................39
4 RESULTS AND DISCUSSION...........................................................................................40
4.1 MOULDS ISOLATED FROM INJERA....................................................................40
4.1.1 Penicillium species............................................................................................41
4.1.2 Aspergillus niger...............................................................................................42
4.1.3 Rhizopus species................................................................................................43
4.2 MEDIA FROM INJERA..............................................................................................45
4.2.1 Growth of moulds on ISA................................................................................45
4.2.2 Effectiveness of ISA......................................................................................... 47
4.3 EFFECT OF TEMPERATURE ON THE GROWTH RATE OF INJERA
MOULDS ..................................................................................................................48
4.4 pH AND MOISTURE CONTENT OF INJERA.......................................................50
4.4.1 pH of injera.......................................................................................................50
4.4.2 Moisture content of injera ...............................................................................52
4.5 EFFICACY OF THE CHEMICAL PRESERVATIVES ON INJERA...................54
4.5.1 Extent of mould invaion on injera...................................................................54
4.5.2 Shelf life of injera with and without preservatives........................................58
5 CONCLUSIONS AND RECOMMENDATIONS..............................................................60
5.1 Conclusions....................................................................................................................60
5.2 Recommendation...........................................................................................................61
REFERENCES....................................................................................................................63
APPENDIX ....................................................... .................................................................72

LIST OF TABLES

Table 1. Major methods of preservation...................................................................................17

Table 2. Summary of some GRAS chemical food preservatives..............................................18
Table 3. Composition of various varieties of injera in terms of 100 g of edible portion..........30
Table 4. Processing of injera from tef ......................................................................................31
Table 5. Rhizopus colony diameter on different media............................................................47
Table 6. Growth rate of injera moulds at different temperature................................................48
Table 7. pH of injera with and without preservatives...............................................................50
Table 8. Moisture content of injera with and without preservatives.........................................52
Table 9. Percentage of the invaded surface as compared to the total surface of injera.............55
Table 10. Shelf life of injera containing preservatives at 20+ 20c............................................59

LIST OF FIGURES
Figure 1. Quality deterioration during storage of foods............................................................15
Figure 2. Structure of benzoic acid and sodium benzoate........................................................20
Figure 3. Struture of most commonly used derivatives of benzoic acid (parabens).................20
Figure 4. Diagrammatic representation of growth of an endospore into vegetable
cells showing stages arrested by minimum inhibitory concentrations of
some food preservatives.............................................................................................23
Figure 5. A schematic representation of the stress response of a yeast cell challenged
with weak organic acids.............................................................................................27
Figure 6. Diffusion of a weak organic acid into a microbial cell, and its dissociation yielding
protons (H+) and potentially toxic anions (A-)..........................................28
Figure 7. Injera in a messhob....................................................................................................32
Figure 8. Injera samples that kept in the laboratory at ambient temperature...........................38
Figure 9. Penicillium isolated from injera................................................................................41
Figure 10. Aspergillus niger isolated from injera...................................................................42
Figure 11. Rhizopus isolated from injera..................................................................................43
Figure 12. Yellowish pigment released by injera moulds after 12 days of storage..................45
Figure 13. Injera sucrose agar medium (ISA)...........................................................................46
Figure 14. Mould invasion of injera with and without preservatives in 12 days......................56

LIST OF ABBREVIATIONS AND ACRONYMS

ADI- Average Daily Intake

aw- Water Activity
CSA- Central Statistical Agency (of Ethiopia)
CYP- Czapeks Solution Agar
Eh or O/R- Oxidation reduction potential
EHNRI- Ethiopian Health and Nutrition Research Institute
FAO-Food and Agriculture Organization
FDA- Food and Drug Administration (of USA)
g- Gram
GRAS- Generally Regarded As Safe
ISA-Injera Sucrose Agar
JECFA- Joint Expert Committee on Food Additives
Kg- Kilogram
l- Litre
MAP- Modified Atmosphere Packaging
MEA- Malt Extract Agar
ml- Millilitre
NA-Nutrient Agar
PDA-Potato Dextrose Agar
ppm- Parts Per Million
SPSS- Statistical Product and Service Solutions
WHO- World Health Organization

ACKNOWLEDGEMENTS

First of all I would like to praise almighty God for always blessing my injera. He always
stands in front of me in all the things I do and he knows what I need.
I would like to express profound gratitude to my advisor, Dr. Dawit Abate, for his valuable
support, encouragement, supervision and useful suggestions throughout this research work.
His moral support and continuous guidance enabled me to complete my work successfully. He
brought out all the best in me and changed me from zero to a hero.
I would also like to thank Dr. Ketema Bacha for his continuous guidance and giving me ideas
that solved some problems I faced while conducting the research. I learn from you respecting
others and devotion.
I would also like to thank Zenebech Aytenew for her help in the laboratory, her help made my
work much easier.
Where would I be without my family? This work is completely dedicated to my family for
their love and support throughout my life. My mother had been the co advisor of this research
since most of the traditional things were made by her at home. My father who had been always
our hero, made the greatest contribution by allowing me to use his car to transport the
messob from home to the research laboratory. My brother, adinee and my sister kiddy had all
been the sponsor of this education and I am very much grateful for all the sacrifice they made
to make my life easier. My other sister, dudu had been the one who shared all my ideas when I
come home from school and her breath taking kiss kept me alive.

My appreciation also goes to the Food science and Nutrition program, especially to the
director of the program Prof. Negussie Reta for checking me and raising vital research
questions that need to addressed.
My special thanks also go to Dr W.R. Abraham, Biotechnology Research Center,
Braunsheweig, Germany, for identifying the moulds we sent to him.
I am grateful to Betty Abebe who did all the secretarial work of this thesis with love.
I would also like to thank Dr. Guiliat Desse and Tilahun Bekele for all the vital comment and
suggestion they gave me to make my paper better.
Finally, I would like to thank all my friends and colleagues who appreciated my work and
contributed a lot in one way or another.

ABSTRACT
Injera is staple Ethiopian fermented bread which can be made from different types of cereals,
particularly from tef (Eragrostis tef). About two-third of Ethiopian diet consists of injera and it
accounts for about two-third of the daily protein intake of Ethiopian population. It has a very
high nutritional value, as it is rich in calcium and iron. Unfortunately, injera has a short shelf
life of 3-4 days essentially due to mould spoilage. The use of weak organic acid as
preservatives is allowed in acidic foods, primarily as mould inhibitor. In this study, the
influence of some commonly used chemical preservatives, benzoic acid, sodium benzoate,
potassium sorbate and calcium propionate was investigated by isolation of moulds before and
during its ambient storage for 12 days. The preservatives were added immediately before
baking at the concentration of 0.1% of benzoic acid/ sodium benzoate, 0.2 % of potassium
sorbate, 0.3 % of calcium propionate and 0. 2 % blend of the four (wt/wt) as recommended by
Food and Drug Administration. Three fungal species: Aspergillus niger, Penicillium sp and
Rhizopus sp found to be responsible in injera spoilage. Penicillium and Rhizopus are more
dominant at the temperature of between 16-200c, while Aspergillus niger is more dominant at
higher temperature of between 25-320c. Injera samples had a pH and moisture content of 3.383.45 and 62-65%, respectively. Antimicrobial activities of the preservatives investigated
prolong the shelf life of injera up to12 days. The effectiveness of preservation was ranked as
sodium benzoate>benzoic acid>potassium sorbate>blend >calcium propionate showing that
benzoates and benzoic acid are the most effective. The incorporation of benzoic acid and its
salt while preparing injera at home or in larger scale processing should be practiced so as to
save a significant amount of injera that is lost due to mould spoilage.

CHAPTER 1
INTRODUCTION
1.1 BACKGROUND
Injera is a thin, fermented Ethiopian traditional bread made from flour, water and starter
(ersho) which is a fluid saved from previously fermented dough. Tef (Eragrostis tef (Zucc)
Trotter) is the most popular grain for making injera; although other grains such as sorghum,
maize, barley, wheat and finger millet are sometimes used. The major quality attribute of a
good injera is its slightly sour flavour (Adamu Zegeye, 1997). Fellow (1997) reported that
normal and typical injera is round, soft, spongy and resilient, about 6 mm thick, 60 cm in
diameter with uniformly spaced honeycomb-like eyes on the top. Injera has a very high
nutritional value, as it is rich in calcium and iron. Wot in the Ethiopian national language
(Amharic) means a stew which is made from plant and animal products is served with injera.
As a result of this, injera is not only a kind of bread it is also an eating utensil (Science of
cooking, n.d). Injera is the undisputed national food of Ethiopians (Blandino et al.,
2003).According to Ball et al., (1996) about two-third of Ethiopian diet consists of injera and
it accounts for about 2/3 of the daily protein intake of Ethiopian population (Arogundade,
2006). Unfortunately, injera storage period does not usually exceed 3 days at ambient
temperature under the traditional storage conditions essentially due to mould spoilage.

Food preservation has been a long lasting desire of human beings (Juneja et al., 2008). It was
the prerequisite to man settling down in one place, instead of moving from place to place in
the never ending hunt for fresh food. The earliest preservation technologies developed were
drying, smoking, chilling and heating. Later on, the art of controlling these technologies was

developed. The use of various compounds such as salt and spices to preserve foods was also
used in ancient times (Zeuthen and Bogh-Soresen, 2003).

Early history is evident of the chemical preservation of food and its various products. The
addition of chemicals to food is not a recent innovation; it is being practiced throughout the
world (Bohra and Pariah, 2006). Food preservatives are added to stop or delay nutritional
losses due to microbiological, enzymatic or chemical changes of foods during its shelf life.
They also prevent consumer hazards due to the presence of microbial toxins or pathogenic
microorganisms and economic losses due to spoilage (Russell and Gould, 2003).

Benzoic acid, sorbic acids, and propionic acid and their respective sodium, potassium and
calcium salts are the most commonly used preservatives in foodstuffs. They are generally used
to inhibit yeast and mold growth, being also effective against a wide range of bacteria. These
compounds are most active in foods of low pH value and essentially ineffective in foods at
neutral pH values (Santini et al., 2009).

1.2 STATEMENT OF THE PROBLEM

Quite a substantial amount of food is lost at household level due to two major reasons. The
first is the direct loss due to microbial spoilage. Though no statistical data is available, it is
assumed that a significant amount of injera is lost due to moulding at household level per
baking cycle. The second type of loss is rather indirect. In many instances, people tend to
consume more than they require because most households cannot afford cooling devices to
extend the keeping quality of the food. Food item, which could last much longer under proper
processing or storage, are consumed, thus lost, in much shorter period of time without the

physiological need to do so. Microbiological studies to improve the keeping quality of

indigenous foods through microbial processing, use of food preservatives or combination of
both would significantly contribute to curb problems of food shortage at house hold level
(Mogessie Ashenafi, 2002; 2006).

Several management tools such as the use of chemical preservatives are available to help
control mold contamination (Higgins and Brinkhaus, 1999). Sorbic acid, propionic acid and
benzoic acid and their potassium, calcium and sodium salts are permitted in breads, cakes,
certain cheese, and other foods, primarily as a mould inhibitor (Jay, 2000). According to
Carson, (2000) it took more than 27 days for bread with chemical preservatives to be 100%
covered by moulds and other microorganism. Mogessie Ashenafi (2006) suggested the
inclusion of sorbates or benzoates in the right proportion after completion of fermentation and
immediately before baking may improve the spoilage of injera due to moulding.

There are no reports on the preservative efficiency of the above chemicals in any of the
traditional Ethiopian fermented foods. This study, therefore, was designed to evaluate the
possibility of using these chemicals at their permissible level to control mould spoilage and
improve shelf life of injera.

1.3 OBJECTIVES OF THE STUDY

1.3.1 General Objective
To improve the shelf life of injera using benzoic acid, sodium benzoate, potassium
sorbate and calcium propionate as preservative.
1.3.2 Specific Objectives
To isolate, cultivate and identify the moulds which are responsible in spoiling injera.
To examine the efficacy of sorbic acid, propionic acid and benzoic acid on the
identified injera moulds.
To evaluate injera based agar media for the cultivation of moulds.

CHAPTER 2
LITERATURE REVIEW
2.1 MOULDS
2.1.1 Food Spoilage by Moulds
Moulds, those dusty little spots found spreading over bread, cheese, books and other things in
the home, cause the loss of millions of dollars to our economy every year and even worse, are
menace to our health (Malloch, 1981). There is an increasing knowledge and understanding of
the role played by moulds in food spoilage. Especially the discovery of mycotoxin production
in foods has highlighted the importance of moulds in food quality. It is, however, only within
the last 510 years that major progresses have been made towards the prevention of spoilage
caused by moulds. This is due to recent international agreements on taxonomy and analytical
methods for food borne moulds, which has led to the discovery, that a specific, very limited
funga (= mycobiota) is responsible for the spoilage of each kind of food. This is called the
associated or critical funga and has been shown to consist of less than ten species (Filtenborg
et al., 1996).
The microbial spoilage of foods may be viewed simply as an attempt by the food biota to carry
out what appears to be their primary role in nature. This should not be taken in the teleological
sense. In spite of their simplicity when compared to higher forms, microorganisms are capable
of carrying out many complex chemical reactions essential to their perpetuation. To do this,
they must obtain nutrients from organic matter, some of which constitutes our food supply
(Jay, 2000).

Moulds are ubiquitous which can be found in a wide variety of environments, such as in
plants, animal products, soil, water and insects. This broad occurrence can be explained by the
fact that moulds can utilize a variety of substrates such as pectines and other carbohydrates,
organic acids, proteins and lipids. Moreover, moulds are relatively tolerant to low pH, low
water activity, low temperature and the presence of preservatives (Huis int Veld, 1996).

In addition to visible spoilage, moulds can also accumulate toxins hazardous to health. It has
now been established that more than 200 different types of moulds do form substances that are
orally toxic to man, when growing in certain foods. Although most research has been carried
out on the metabolites of Aspergilus flavus, it is quite obvious that in addition to the so-called
aflatoxins, many other mycotoxins may be of great significance (Huis int Veld, 1996).
Certain moulds are capable of producing toxic and carcinogenic metabolites. Proliferation of
these organisms on foods must be regarded as a potential health hazard. A further consequence
of such mould growth is economic loss due to poor appearance, off flavours and need for
severe trimming (Skirdal and Eklund, 1993)

2.1.2 Characteristics of Moulds

Molds are filamentous fungi that grow in the form of a tangled mass that spreads rapidly and
may cover several inches of area in 2 to 3 days. The total of the mass or any large portion of it
is referred to as mycelium. Mycelium is composed of branches or filaments referred to as
hyphae (Heritage et al., 1996).

Those of greatest importance in foods multiply by ascospores, zygospores, or conidia. The

ascospores of some genera are notable for their extreme degrees of heat resistance. One group

forms pycnidia or acervuli (small, flask-shaped, fruiting bodies lined with conidiophores).
Arthrospores result from the fragmentation of hyphae in some groups.

There were no radical changes in the systematic of food borne fungi during the 1980s. The
most notable changes involve the discovery of the sexual or perfect states of some well-known
genera and species. In this regard, the ascomycete state is believed by mycologists to be the
more important reproductive state of a fungus, and this state is referred to as the teleomorph.
The species name given to a teleomorph takes precedence over that for the anamorph, the
imperfect or conidial state. Holomorph indicates that both states are known, but the
teleomorph name is used (Jay, 2000).

Some of the genera of moulds that commonly cause spoilage of foods and grains are:
Penicillium, Aspergillus, Eurotium, Fusarium, Endomyces, Rhizopus, Mucor, Monilia
Cladosporium

and

2.2 FACTORS THAT AFFECT MICROBIAL GROWTH ON FOOD

Fungal growth is influenced by a variety of complex interactions between intrinsic and
extrinsic factors (Lopez-Malo et al., 2005).
2.2.1 Intrinsic Parameters
The parameters of plant and animal tissues that are an inherent part of the tissues are referred
to as intrinsic parameters (Jay, 2000, Huis int Veld, 1996). These parameters are Nutrient
content, pH, Moisture content, Oxidation-reduction potential (Eh), Antimicrobial constituents
and Biological structure.

A) Nutrient Content of the Food

Microorganisms can use foods as a source of nutrients and energy as well as to derive the
chemical elements that constituent microbial biomass. The inability of an organism to utilize a
major component of a food material will limit its growth. Thus, the ability to synthesize
starch-degrading enzymes will favour the growth of an organism on cereals and other
farinaceous products. The concentration of key nutrients can, to some extent, determine the
rate of microbial growth (Bohra and Pariah, 2006). Microorganism require a minimum of a
carbon source and energy source, a nitrogen(amino acids and vitamins) source, inorganic
nutrients, water for growth, and these are readily provided by food (Omonigho and
Ikenebomeh, 2000).

B) pH
The acidity or alkalinity of an environment has a profound effect on the activity and stability
of macromolecules such as enzymes, so it is not surprising that the growth and metabolism of
microorganisms are influenced by pH (Bohra and Pariah, 2006). It has been well established
that most microorganisms grow best at pH values around 7.0 (6.6-7.5), whereas few grow
below 4.0. Bacteria tend to be more fastidious in their relationships to pH than molds and
yeasts, with the pathogenic bacteria being the most fastidious (Jay, 2000). According to
(Lucke, 2003) pH is an important factor affecting growth of microorganisms in food because it
affects:
1) Microbial energy metabolism involving the build up of gradients of hydrogen ion
concentration across membrane and
2) Microbial enzyme activity and stability of cellular macromolecules.

C) Moisture Content
One of the oldest methods of preserving foods is drying or desiccation. The preservation of
foods by drying is a direct consequence of removal or binding of moisture, without which
microorganisms do not grow. It is now generally accepted that the water requirements of
microorganisms should be described in terms of the water activity (aw) in the environment
(Jay, 2000). The concept of water activity (aw) introduced by Scott 1957 is the most useful
expression of the water availability for microbial growth and enzyme activity (Abdullah et al.,
2000). This parameter is defined by the ratio of the water vapor pressure of food substrate to
the vapour pressure of pure water at the same temperature aw = p/po, where p is the vapor
pressure of the solution and po is the vapor pressure of the solvent (usually water).

In general, bacteria require higher values of aw for growth than fungi, with gram-negative
bacteria having higher than gram positives. Most spoilage bacteria do not grow below aw =
0.91, whereas spoilage molds can grow as low as 0.80 (Jay, 2000). Salt and sugar have long
been used as effective means of extending shelf life of various products as these solutes bind
water, leaving less water available for the growth of microorganisms. Essentially the water
activity (aw) of the product is reduced, and since most microorganisms require a high water
activity, they are unable to survive (Morris et al., 2004).

D) Oxidation- Reduction Potential (Eh or O/R)

Microorganisms display varying degrees of sensitivity to the oxidation-reduction potential
(O/R, Eh) of their growth medium. The O/R potential of a substrate may be defined generally
as the ease with which the substrate loses or gains electrons. Aerobic microorganisms require
positive Eh values (oxidized) for growth, whereas anaerobes require negative Eh values
(reduced). Among the substances in foods that help to maintain reducing conditions are -SH
groups in meats and ascorbic acid and reducing sugars in fruits and vegetables (Jay, 2000).The
O/R potential of a food is determined by the following:

The characteristic O/R potential of the original food

The poising capacity; that is, the resistance to change in potential of the food
The oxygen tension of the atmosphere about the food
The access that the atmosphere has to the food.

E) Antimicrobial Constituents
The stability of some foods against attack by microorganisms is due to the presence of certain
naturally occurring substances that have been shown to have antimicrobial activity. Some
species are known to contain essential oils that possess antimicrobial activity. Among these
are eugenol in cloves, allicin in garlic, cinnamic aldehyde and eugenol in cinnamon, allyl
isothiocyanate in mustard, eugenol and thymol in sage, and carvacrol (isothymol) and thymol
in oregano. Cow'
s milk contains several antimicrobial substances, including lactoferrin,
conglutinin, and the lactoperoxidase system (Jay, 2000).
F) Biological Structures
The natural covering of some foods provides excellent protection against the entry and
subsequent damage by spoilage organisms. In this category are such structures as the testa of
seeds, the outer covering of fruits, the shell of nuts, the hide of animals, and the shells of eggs.
In the case of nuts such as pecans and walnuts, the shell or covering is sufficient to prevent the
entry of all organisms. Once cracked, nutmeats are subject to spoilage by molds. The outer
shell and membranes of eggs, if intact, prevent the entry of nearly all microorganisms when
stored under the proper conditions of humidity and temperature (Jay, 2000).
2.2.2 Extrinsic Parameters
According to Huis int Veld (1996) and Jay (2000) extrinsic parameters of foods are those
properties of the storage environment that affect both the foods and their microorganisms.
Those of greatest importance to the welfare of food borne organisms are temperature of
storage, relative humidity of environment, presence and concentration of gases and presence
and activities of other microorganisms

A) Temperature of Storage
Microorganisms, individually and as a group, grow over a very wide range of temperatures.
The lowest temperature at which a microorganism has been reported to grow is -34C; the
highest is somewhere in excess of 1000C. It is customary to place microorganisms into three
groups based on their temperature requirements for growth. Those organisms that grow well at
or below 7C and have their optimum between 200C and 300C are referred to as
psychrotrophs. Those that grow well between 200C and 45C with optima between 300C and
400C are referred to as mesophiles, whereas those that grow well at and above 450C with
optima between 55C and 65C are referred to as thermophiles (Jay, 2000).
B) Relative Humidity of Environment
Relative humidity is essentially a measure of the water activity of the gas phase. When food
commodities have a lower water activity are stored in an atmosphere of high relative humidity
water will transfer from the gas phase to the food. It may take a very long time for the bulk of
the commodity to increase in water activity, but condensation may occur on surface giving rise
to localized regions of high water activity. It is in such regions that propagules, which have
remained viable, but unable to grow, may now germinate and grow. Once microorganisms
have started to grow and become physiologically active they usually produce water as an end
product of respiration. Thus they increase the water activity of their own immediate
environment so that microorganisms are able to grow and spoil a food, which was initially
considered to be microbiologically stable (Jay, 2000).

C) Presence and Concentration of Gases

Oxygen is the most important gas in contact with food. Its presence and its influence on redox
potential are important determinants of microbial associations that develop their rate of
growth. The inhibitory effect of carbon dioxide on microbial growth is applied in modified
atmosphere packing (MAP) of food and is an advantageous consequence of its use at elevated
pressures in carbonated mineral waters and soft drinks (Bohra and Pariah, 2006). Since molds
are strictly aerobic, for the attainment of long shelf lives the levels of residual O2 must be kept
below 1% (Guynot et al., 2003; 2004).

D) Presence and Activities of Other Microorganisms

Some food borne organisms produce substances that are either inhibitory or lethal to others;
these include antibiotics, bacteriocins, hydrogen peroxide, and organic acids (Jay, 2000).

2.2.3 Hurdle Technology

Under intrinsic and extrinsic parameters, the effect of single factors on the welfare of
microorganisms is presented. In the hurdle concept, multiple factors or techniques are
employed to affect the control of microorganisms in foods. Barrier technology, combination
preservation, and combined methods are among some of the other descriptions of this concept.
Foods that employ the hurdle concept in their formulation would embody a series of the above
parameters, thus making for a multi targeted approach to preventing germination and growth
of microorganisms. In order to grow, the organism must "hurdle" a series of barriers (Jay,
2000; Lombard et al, 2000; Knochel and Gould, 1995).

2.3 METHODS OF PRESERVATION AND EXTENSION OF SHELF LIFE OF FOOD

2.3.1 Spoilage and Shelf life of food
With few exceptions all foods lose quality and potential shelf life at some rate following
harvest, slaughter or manufacture in a manner that is very dependent on food type,
composition, formulation (for manufactured foods), packaging and storage conditions.
Spoilage, or other changes that lead to loss of shelf life, may occur at any of the many stages
between the acquisition of raw materials and the eventual consumption of a finished product
(Gould, 1996). The shelf-life of a product is defined as the expected time duration that a
product will remain organoleptically acceptable. It is a function of holding temperature and the
number of microorganisms remaining in it after processing (Doughari et al., 2007).

The principal reactions that lead to spoilage and that are consequently also the principal targets
for effective preservation and controls are well known, and relatively few. They include some
that are essentially physical, some that are chemical, some that are enzymic and some that are
microbiological (Huis int Veld, 1996, Morris et al., 2004). The high increase in food
deterioration is due to contamination with micro-organism since the entire environment in
which we live is colonized by micro-organisms (Akpan and Kovo, 2005).

The schematic representation of the complex mechanisms of food is presented in Figure 1.

External conditions
Storage conditions
Humidity Gas atmosphere (O2, N2, CO2)
Packaging (materials)
Light
Productpppp
Product
conditions
Initial quality

Intrinsic factors

Raw materials

Ingredients
Hygienic processing

Rancid flavour
Warmed-over flavour
Change of texture

Discoloration
Production of off-flavours:
Rancid flavour
Warmed over flavour
Putrid flavour
Sour flavour
Loss of Nutrients
Production of toxics
Changes of texture

Figure 1. Quality deterioration during storage of foods

(Adapted from Huis int Veld, 1996)

Putrid flavour
Sour Flavors
Slime
Gas

2.3.2 Preservation of Food

Food preservation is an action or a method of maintaining food at desirable level of properties
or nature for their maximum benefits (Rahman, 2007) .All foods begin to spoil as soon as they
are harvested or slaughtered. Some spoilage is caused by such microorganisms as bacteria and
moulds. Other spoilage resulted from chemical changes within the food itself due to natural
processes such as enzyme action or oxidation (Jay, 2000). Preservation is based firstly on the
delay or prevention of microbial growth. It must therefore operate through those factors that
most effectively influence the growth and survival of microorganisms. Such factors are not
numerous. They include a number of essentially physical factors, some predominantly
chemical ones and some microbial ones which depend on the nature of the microorganisms
that are present. These factors have been categorized in a number of ways but the most widely
quoted categorizations (Huis int Veld, 1996) separate the major factors into intrinsic factors,
and extrinsic factors (section 2.2).

2.3.3 Major Food Preservation Technologies

The major food preservation techniques that are employed are therefore all based on a
relatively limited set of factors, so that their range is necessarily limited also (Gould, 1996;
Morris et al., 2004; Jay, 2000). The major food preservation methods are summarized in Table
1 below.

Cold

Heat

Drying

Fermentation

Physical

Chemical

Freezing

Cooking

Tunnel

Alcoholic

Filtration

Sugar, alt

Chilling

Pasteurization

Solar

Acetic

Separation

spices, acid

Canning

Spray

Lactic

Distillation

preservatives

Vaccum

Irradiation

Freeze Drying

Concentration

e.g. benzoates,
nitrites

Modified/controlle
d atmosphere
packing (MAP)

Additives e.g.
antioxidants

Table 1. Major methods of preservation (Adapted from Huis int Veld, 1996)
2.3.2.2 Food Preservation with Chemicals
According to Jay 2000, the use of chemicals to prevent or delay the spoilage of foods derives
in part from the fact that such compounds are used with great success in the treatment of
diseases of humans, animals, and plants. This is not to imply that any and all chemotherapeutic
compounds can or should be used as food preservatives. Although a large number of
chemicals have been described that show potential as food preservatives, only a relatively
small number are allowed in food products, due in large part to the strict rules of safety
adhered to by the Food and Drug Administration (FDA) and to a lesser extent to the fact that
not all compounds that show antimicrobial activity in vitro do so when added to certain foods.
Below are described those compounds most widely used, their modes of action where known,
and the types of foods in which they are used. Those chemical preservatives Generally
Recognized as Safe (GRAS) are summarized in Table 2.

Table 2.Summary of some GRAS chemical food preservatives (Adapted from Jay, 2000)
Preservatives

Maximum
Tolerance

Organisms Affected

Foods

Propionic acid/
propionates

0.32%

Molds

Bread, cakes, some

cheeses, rope inhibitor in
bread dough

Sorbic
acid/Sorbates

0.2%

Molds

Hard cheeses, figs, syrups,

salad dressings, jellies,
cakes

Benzoic
acid/benzoates

0.1 %

Yeasts and molds

Parabens*

0.1 %

Yeasts and molds

Margarine, pickle relishes,

apple cider, soft drinks,
tomato catsup, salad
dressings
Bakery products, soft drinks,
pickles, salad
dressings

SO2/sulfites

200-300 ppm

Insects,
microorganisms

Ethylene/propylene 700 ppm

oxides

Yeasts, molds, vermin

Molasses, dried fruits, wine

making, lemon juice (not
to be used in meats or
other foods recognized as
sources of thiamine)
Fumigant for spices, nuts

Sodium diacetate

0.32%

Molds

Bread

Nisin

1%

Lactics, clostridia

Certain pasteurized cheese

spreads

Dehydroacetic acid 65 ppm

Insects

Pesticide on strawberries,
squash

Sodium nitrite

120 ppm

Clostridia

Meat-curing preparations

Ethyl formate

15-220 ppm

Yeasts and molds

Dried fruits, nuts

Note: GRAS (generally recognized as safe) per Section 201 (32) (s) of the U.S. Food, Drug,
and Cosmetic Act as amended.
*Methyl-, propyl-, and heptyl-esters of p-hydroxybenzoic acid.
As formic acid

2.3.2.2.1 Benzoic acid / Benzoates

Benzoic acid and its salt have been widely used in the food industry for many years as
important food preservatives in order to inhibit various bacteria, yeasts and fungi growth in
acidic media. They are also used in other products, such as pharmaceuticals and cosmetics. As
one kind of common chemical, 638,000 tons of benzoic acid is approximately produced
globally per year (Qi et al., 2009). According to Jay 2000, sodium benzoate was the first
chemical preservative permitted in foods by the FDA in 1908 as a preservative in certain foods
and it continues in wide use today in a large number of foods.

Benzoic acid is an effective antimicrobial agent for the purpose of preservation. However,
sodium benzoate is more effective and preferred because it is approximately 200 times more
soluble than benzoic acid. The soft drink industry is the largest user of benzoate as a
preservative due to the amount of high fructose corn syrup in many carbonated beverages
(DINOX, 2004).
Benzoic acid has been widely employed as an antimicrobial agent in foods and it occurs
naturally in cranberries, prunes, cinnamon and cloves. It is well suited for acid foods such as
fruit juices, carbonated beverages, pickles and sauerkraut. Benzoic acid has been found to
cause no deleterious effect when used in small amounts. It is, however, readily eliminated
from the body after conjugation with glycine to form hippuric acid (Doughari et al., 2007).

Figure 2. Structure of benzoic acid and sodium benzoate

Benzoic acid (C6H5COOH) and its sodium salt (C7H5NaO2), along with the esters of phydroxybenzoic acid (parabens) are permissible in foods up to 0.1%. The approved derivatives
of benzoic acid have structural formulas as noted below:

Figure 3. Struture of most commonly used derivatives of benzoic acid (parabens)

The parabens appear to be more effective against molds than against yeasts. As in the case of
bacteria, the propyl derivative appears to be the most effective where 100 ppm or less is
capable of inhibiting some yeasts and molds, whereas for heptyl- and methyl parabens, 50-200
and 500-1,000 ppm respectively, are required. Like benzoic acid and its sodium salt, the
methyl- and propyl parabens are permissible in foods up to 0.1%, and heptyl parabens is

permitted in beers to a maximum of 12 ppm and up to 20 ppm in fruit drinks and beverages.
The pK for these compounds is around 8.47, and their antimicrobial activity is not increased to
the same degree as for benzoate with the lowering of pH as noted. They have been reported to
be effective at pH values up to 8.0 (Jay, 2000; Ibekwe et al., 2007).

The antimicrobial activity of benzoate is related to pH, the greatest activity being at low pH
values. The antimicrobial activity resides in the undissociated molecule. These compounds are
most active at the lowest pH values of foods and essentially ineffective at neutral values. The
pK of benzoate is 4.20 and at a pH of 4.00, 60% of the compound is undissociated, whereas at
a pH of 6.0, only 1.5% is undissociated. This results in the restriction of benzoic acid and its
sodium salts to high-acid products such as apple cider, soft drinks, tomato catsup, and salad
dressings. High acidity alone is generally sufficient to prevent growth of bacteria in these
foods but not that of certain molds and yeasts. As used in acidic foods, benzoate acts
essentially as a mold and yeast inhibitor, although it is effective against some bacteria in the
50- to 500- ppm range. Against yeasts and molds at around pH 5.0-6.0, from 100 to 500 ppm
are effective in inhibiting the former, whereas for the latter, from 30 to 300 ppm are inhibitory
(Jay, 2000; Akpan and Kovo, 2005)
2.3.2.2.2 Sorbic acid/ Sorbates
Sorbic acid (CH-CH=CH-CH=CH-COOH) is employed as a food preservative, usually as the
calcium, sodium, or potassium salt (Jay, 2000; Venturini et al., 2002 and Santini et al., 2009).
Sorbic acid and its potassium salt are the most widely used forms of the compounds and are
collectively known as sorbates. Sorbate was first patented by Gooding 1945 as an antifungal
agent, and has been used to a growing extent to protect a variety of foods against spoilage by

microorganisms (Razavi-Rohani and Griffiths, 1999). Sorbic acid was approved by FDA for
use as a food preservative in 1955 (Jay, 2000). Potassium salt is commonly used because it is
more stable. Furthermore, its greater solubility extends the use of sorbate to solutions
appropriate for dipping and spraying (Gonzalez-Fandos et al., 2005). These compounds are
permissible in foods at levels not to exceed 0.2 %. The widest use of sorbates is as fungistats
in products such as cheeses, bakery products, fruit juices, beverages, salad dressings, and the
like ( Jay 2000). The acceptable daily intake (ADI) values, determined by the Joint
FAO/WHO Expert Committee on Food Additives (JECFA), is 25 mg/kg of body mass for
sorbic acid and sorbates salts(Santini et al., 2009).

The sorbates are primarily effective against molds and yeasts, but research has shown them to
be effective against a wide range of bacteria (Jay, 2000, Venturini et al., 2002 and Santini et
al., 2009). In general, the catalase-positive cocci are more sensitive than the catalase
negatives, and aerobes are more sensitive than anaerobes. The resistance of the lactic acid
bacteria to sorbate, especially at pH 4.5 or above, permits its use as a fungistatic in products
that undergo lactic fermentations.

In the case of molds, inhibition may be due to inhibition of the dehydrogenase enzyme system.
Against germinating endospores, sorbate prevents the outgrowth of vegetative cells (Figure 5).

Figure 4. Diagrammatic representation of growth of an endospore into vegetable cells

showing stages arrested by minimum inhibitory concentrations of some food
preservatives (Source: From Jay 2000)

Inhibition of microorganisms by sorbates is dependent on microbial types, species, and strains

and on environmental factors. There are reports that lower pH and higher NaCl concentrations
increase the inhibitory effect of sorbates against fungi (Razavi-Rohani and Griffiths, 1999).
Some moulds are sensitive to the action of sorbic acid, while others can grow in the presence
of large amounts, metabolize or degrade it. For example some penicillia degraded sorbic acid
and produced 1,3-pentadiene, a volatile compound with an extremely strong kerosene-like
odour (Skirdal and Eklund, 1993).

As with other food preservatives, the use of sorbate has advantages as well as limitations, but
when used with proper planning and under the correct conditions, the advantages outweigh the
disadvantages (Razavi-Rohani and Griffiths, 1999). Sorbic acid and its salts have several
advantages as food preservatives. Initially thought to have only antimycotic activity, they are
now known to also inhibit a wide range of bacteria, particularly aerobic catalase-positive
organisms. Effective concentrations do not normally alter product taste or odor. These

preservatives are also considered harmless (Gonzalez-Fandos et al., 2005). Sorbic acid and its
derivatives are commonly used to inhibit mold growth and extend the shelf life of several
foods because of physiological harmlessness and organoleptic neutrality (Venturini et al.,
2002). Other studies showed that sorbic acid has low toxicity, explained by the fact that it is
rapidly metabolised by pathways similar to those of other fatty acids. In humans a few cases of
idiosyncratic intolerance to sorbic acid have been reported (non-immunological contact
urticaria and pseudo-allergy). For the above mentioned reasons, sorbic acid and sorbates salts
(especially potassium sorbate) have become the leading preservatives for a wide variety of
food products (Santini et al., 2009).

Like sodium benzoate, they are more effective in acid foods than in neutral foods and tend to
be on par with the benzoates as fungal inhibitors. Sorbic acid works best below a pH of 6.0
and is generally ineffective above pH 6.5. These compounds are more effective than sodium
benzoate between pH 4.0 and 6.0. At pH values of 3.0 and below, the sorbates are slightly
more effective than the propionates but about the same as sodium benzoate. The pK of sorbate
is 4.80, and at a pH of 4.0, 86% of the compound is undissociated, whereas at a pH of 6.0,
only 6% is undissociated (Jay, 2000; Skirdal and Eklund, 1993). Therefore, the antimicrobial
activity of sorbates is known to be pH dependent and its antimicrobial effectiveness increases
as the pH value approaches its dissociation constant (Razavi-Rohani and Griffiths, 1999).
Sorbic acid can be employed in cakes at higher levels than propionates without imparting
flavour to the product (Jay, 2000).

2.3.2.2.3 Propionic acid/Propionates

Propionic acid is a three-carbon organic acid with the structure CH3CH2COOH. This acid and
its calcium and sodium salts are permitted in breads, cakes, certain cheese, and other foods
(Jay 2000). The antimicrobial effect of propionic acid has been known since 1913. Propionic
acids and their salts are primarily inhibitory to molds; however, some species of molds are
resistant and can grow in media containing substantial amounts of propionic acid and its salts
(Razavi-Rohani and Griffiths, 1999). They tend to be highly specific against moulds, with the
inhibitory action being primarily fungistatic rather than fungicidal. Propionic acid is employed
also as a "rope" inhibitor in bread dough (Jay, 2000). Propionic acid has previously been
shown to inhibit moulds and Bacillus spores, but not yeasts to a large extent, and has therefore
been the traditional chemical of choice for bread preservation (Ryan et al., 2005).
Similar to other lipophilic acid preservatives, the optimum pH level for the inhibitory effect of
propionic acid is around 5.0, with a maximum effective pH of 6.0. Propionic acid was most
effective at lower pHs, but propionic acid was never as effective as sorbic acid at any of the
pHs tested. The antifungal activity of sodium and calcium propionate did not approach
propionic acid in effectiveness (Razavi-Rohani and Griffiths, 1999). The tendency toward
dissociation is low with this compound and its salts, and they are consequently active in lowacid foods. With respect to the antimicrobial mode of action of propionates, they act in a
manner similar to that of benzoate and sorbate. The pK of propionate is 4.87 and at a pH of
4.00, 88% of the compound is undissociated, whereas at a pH of 6.0, only 6.7% remains
undissociated. The undissociated molecule of this lipophilic acid is necessary for its
antimicrobial activity (Jay, 2000). The mode of action of propionic acid is similar as noted
below with benzoic acid and sorbic acid on section 2.3.2.2.4.

Concentrations of propionic acid and propionates ranging from 8 to 12% were effective in
controlling mold growth on the surface of cheese and butter (Razavi-Rohani and Griffiths,
1999). Legislation implemented under the European Parliament and Council Directive No.
95/2/EC requires that Propionic acid may only be added to bread in a concentration not
exceeding 3000 ppm. However, recent studies have shown that under these conditions
Propionic acid is not effective against common bread spoilage organisms. Additionally, a
reduction of preservatives to sub-inhibitory levels might stimulate the growth of spoilage fungi
and/or mycotoxin production. Recent trends in the bakery industry have included the desire for
high-quality foods, which are minimally processed and do not contain chemical preservatives,
thus increasing the interest toward natural preservation systems (Ryan et al., 2008).

2.3.2.2.4 Mode of Action of Weak Organic Acids

As lipophilic acids sorbate, benzoate, and propionate appear to inhibit microbial cells by the
same general mechanism. The mechanism involves the proton motive force (PMF). Briefly,
hydrogen ions (protons) and hydroxyl ions are separated by the cytoplasmic membrane, with
the former, outside the cell, giving rise to acidic pH and the latter, inside the cell, giving rise to
pH near neutrality. The membrane gradient thus created represents electrochemical potential
that the cell employs in the active transport of some compounds such as amino acids. Weak
lipophilic acids act as protonophores. After diffusing across the membrane, the undissociated
molecule ionizes inside the cell and lowers intracellular pH. This result in a weakening of the
transmembrane gradient such that amino acid transport is affected adversely (Figure 6).
Although alteration of the PMF by lipophilic acids has wide support, other factors may be
involved in their mode of action (Jay, 2000; Gould, 1996; Brul and Coote, 1999).

Figure 5. A schematic representation of the stress response of a yeast cell challenged with
weak organic acids (Adapted from Brul and Coote, 1999)

According to Gould (1996) the element important in their modes of action is the dissociation
constant of the acid, because it is the undissociated form that is most lipophilic and therefore
most readily permeates the membrane, and it is the pH value and the dissociation constant
together that determine the proportion of the acid that is in this form. The pK values of the
common weak organic acid preservatives range from 4.2 (benzoic) to 4.87 (propionic), so that
at pH values much above these, activity is greatly reduced.

The inhibition by organic acids has been attributed to the protonated form of these acids,
which are uncharged and may therefore cross biological membranes (Figure 7). The resulting
inhibition of growth may be due to acidification of the cytoplasm and/or accumulation of
anions inside the cell (Sahlin, 1999).

Figure 6. Diffusion of a weak organic acid into a microbial cell and its dissociation
yielding protons (H+) and potentially toxic anions (A-)
(Adapted from Sahlin, 1999)
The ability of an acid to inhibit bacteria depends principally on the pKa of the acid: the higher
the pKa of the acid, the greater the proportion of undissociated acid, and the more inhibitory
the acid is likely to be. The degree of dissociation of weak acids in acidic environment can be
calculated using the Hendersen-Hasselbalch equation:

pH= pKa +log [Ac-]

[HAc]

2.4 INJERA
2.4.1 Product Description
Injera is Ethiopian fermented bread made from different types of cereals. It is large, flat, round
and uniformly spaced honeycomb-like eyes, each measuring about 60 cm in diameter and the
base has a smooth surface. Injera looks whitish cream, reddish brown or brown depending on
the type of cereal flour used (Fellow, 1997). Injera can be made from tef (Eragrostis tef),
wheat, barley, sorghum, or maize or a combination of some of these cereals. Whenever the
soil type and rainfall patterns are suitable for the cultivation of tef, injera from tef is more
favoured than from the other cereals (Mogessie Ashenafi, 2006). According to EHNRI (1997)
the various types of injera produced from the different varieties of cereals do not have
significant variation in their calorie, moisture, protein, carbohydrate, or phosphorus nutrients
(Table 3). Significant variations are, however, observed in the other nutrients.
According to CSA (2008) report, tef [Eragrostis tef (Zucc) Trotter] has the largest share of
area (23.42 %, 2.6 million hectares) under cereal cultivation and third (i.e. after maize and
wheat) in terms of grain production (18.57 %, 29.9 million quintals) in Ethiopia. Tef grain
commands premium price among other cereals cultivated in Ethiopia. There is a growing
interest on tef grain utilizations because of nutritional merits (whole grain); the protein is
essentially free of gluten the type found in wheat and it accounts (Arogundade, 2006) for
about 2/3 of the daily protein intake in the diet of Ethiopian population. The grain proteins are
also presumed easily digestible because prolamins are very small. Tef grain micronutrient is
also apparently high, particularly in iron, a result of agronomic practices used in Ethiopia and
fermentation on injera making. Because of this, the prevalence of iron deficient anaemia
among tef injera consumers in Ethiopia is low (Geremew Bultosa, 2007).

Table 3. Composition of various varieties of injera in terms of 100 grams of edible portion (EHNRI 1997)
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2.4.2 Processing of Injera

Cereal grains had been one of mans earliest sources of food. One way of processing the
grains into food is through fermentation (Taiwo, 2009 and Blandino et al., 2003). The
preparation of tef injera consists of two stages of natural fermentation, which last for about 24
to 72 hours, depending on ambient temperatures. Temperature in the highlands of Ethiopia is
generally between 17 and 250c. The only required ingredients are the tef flour and water
(Berhanu Abegaz Gashe, 1985). The method of processing of injera from its raw materials to
the final product is summarized by Fellow, (1997) as described below on Table 4. It involves
preparing and mixing the ingredients to dough, which is fermented and subsequently thinned
to a batter. The batter is then poured onto a hot griddle in a thin layer to cook, developing its
colour, flavour and texture.
Table 4. Processing of injera from tef (Source: Fellow, 1997)
Process

Notes

Raw materials

Tef (Eragrostis tef) is an indigenous cereal for making injera. Other

cereals which may be used are sorghum, millet, barely, wheat or a
combination of cereals.

Clean

All impurities are removed by hand and winnowed in the case of

sorghum, millet, barely, and wheat. Tef is simply winnowed and sifted
through a fine sieve.

Hull

Sorghum, barley and wheat are usually dampened and pounded

traditionally in a wooden mortar and pestle to remove the bran.
Mechanical hullers are also available.

Grind

The sifted tef is ground through a stone mill.

Mixing and first

Mix one part of flour, two parts of water and about 16 percent ersho (a

fermentation

starter saved from previously fermented dough) by weight of the flour.

Mix very well and leave it to ferment for three days.

Thin and heat

Discard the surface water formed on the top of the dough. For every 1kg
of original flour, take about 200ml of the fermented mixture and add
twice as much water, mix and bring to a boil (traditionally known as
absit making). It should be cooled to about 460c/1150F before it is
mixed into the main part of the dough. Thin the main dough by adding
water equal to the original weight of the flour.

Batter making and

Add the absit to the thinned dough and mix very well (known as batter

second fermentation

making). Leave the batter for about 30 minute to rise (the second
fermentation), before baking commences. A small portion or the batter is
saved to serve as a starter (ersho) for the next batch.

Griddle

Injera is griddled by pouring about two-thirds of a litre of the batter onto

the hot greased electrical metad (injera griddle made of clay) using
circular motion from the outside towards the center. It is cooked in about
2-3 minutes at a temperature of the metad reaching 90-950c.Rapeseed oil
is used to grease the metad between each one.

Store

Several layers of injera can be stored in a messhob (traditional straw

basket) with a tight cover for three days in a cool, dry, ventilated place.

Figure 7. Injera in a Messhob

CHAPTER 3
MATERIALS AND METHODS
3.1 STUDY SITE
Experiments were carried out from October 2008-April 2009 in the Mycological Research
Laboratory of the Biology Department and the Food Microbiology Laboratory of Food
Science and Nutrition Program, at the Science Faculty, Addis Ababa University. Addis Ababa
is the capital city of Ethiopia with the altitude of 2444 meter above sea level with a mean
annual rainfall of 1196mm and located 9001 N and 038045 E. The minimum and maximum
temperatures were 9.9 0c and 24.60c, respectively (Fissha Itana & Olsson, 2004).

3.2 PREPARATION OF INJERA

The tef injera samples were prepared at home in almost the same way as reported by Fellow
(1997), except that the art of my mother was also included in this traditional method.
Accordingly, the tef flour mixed 1:2 (w/w) with clean water and 16 % of ersho by the weight
of the flour was kneaded by hand in a bowl in the traditional way. The resultant dough was
allowed to ferment for 3 days at ambient temperature. After this primary fermentation, the
dough was mixed 1:3 (v/v) with boiling water, and heated for 15min with continuous stirring.
The hot cooked dough (absit) was then mixed back into the fermenting dough, and sufficient
clean water was added to make a batter. The batter was left covered for 2 h of secondary
fermentation. Some more clean water was added to thin down and form the right consistency
batter. Finally, about half a litre of batter was poured onto the hot clay griddle in a circular
motion from the outside, working towards the centre. After 2-3 min of cooking using electric

injera baking equipment (metad), it was removed and stored in a basket called messhob.
The injera was then transported from home to Addis Ababa University, Faculty of Science:
Food Microbiological laboratory of Food science and Nutrition program for further study.

3.3 MICROBIOLOGICAL ANALYSIS

3.3.1 Isolation and Cultivation of Moulds that Spoil Injera
The injera samples were kept in the laboratory at ambient temperature for at least 4-5 days
until moulds starts to appear visually on it. After 4 or 5 days of storage at ambient
temperature, depending upon their difference in color and other morphology, the spoiling
moulds were directly transferred into a growth media of potato dextrose agar ( PDA) and
Injera sucrose agar (ISA)[as described in section 3.5] amended with 60mg/l Chloramphenicol,
in order to suppress the growth of bacteria. The cultures were incubated at room temperature
to induce the growth of mycelium and fruiting structures (spores) for five to seven days. To
get a pure culture, each of the emerging mycelium or spores was transferred to fresh growth
media. The pure colony from the isolation were incubated again at ambient temperature and
kept in slant at refrigeration at 40c for further identification.
3.3.2 Characterization and Identification of Moulds that Spoil Injera
The identification of the fungal isolates was based on morphological characterization that
emphasizes on colony characteristics, conidial features and vegetative compatibility of isolates
within genus level. For this purpose slide cultures were prepared for each of the isolates.
Identification to the genus level was made according to Barnett & Hunter, (1972). In addition

to these, the fungal isolates were sent to Biotechnology Researches Institute, Braunshweig,
Germany, for confirmation of their identity.
3.4 INJERA AGAR MEDIA
3.4.1 Preparation of Injera Sucrose Agar (ISA) Media
Freshly baked injera was sundried and changed to derkosh (dried injera). The derkosh was
powdered using mortar and pestle, 100g of the powder was mixed with 500ml of water and
kept in a shaker for 15 minutes. The injera broth was filtered using cheese cotton cloth,
proportional to the volume of the broth 2 % sucrose and 2 % agar was then added. It was
mixed and heated until boiling. After sterilization (15 minutes at 1210c), the media was poured
on Petri dishes. The ISA (Injera sucrose agar) media was kept in a refrigerator for future
practical studies on fungi and for cultivation of the injera moulds.
3.4.2 Comparison of ISA with other Mycological Media
For evaluating the effectiveness of injera sucrose agar media as compared to other
mycological media, Aspergillus niger, Aspergillus flavus, Rhizopus and Penicillium were sub
cultured on PDA at ambient temperature for 3-4 days. A sterile cork borer (6-mm diameter)
was used to bore and transfer each young culture to the newly prepared media (Gupta et al.,
2000) of potato dextrose agar, malt extract agar, nutrient agar, czapeks solution agar and
injera sucrose agar media. The plates were then incubated at ambient temperature. The
evaluations were carried out by means of daily measurement of the colony diameter, starting
at 24 h after the experiment began and finishing when the entire plate surface of the control
treatment was covered by the fungus.

3.5 ANALYZING THE EFFECT OF TEMPERATURE

Temperature optima for the common injera moulds were investigated. Growth and sporulation
of Aspergillus niger, Aspergillus flavus, penicillium and Rhizopus were assessed by
inoculating them on injera sucrose agar media. The cultures were incubated at temperatures of
40c (refrigeration temp), 160c (room temperature during the experiment days), 200c, 250c and
320c. The growth (colony size) of each of the moulds was determined as no growth, + slight
mould growth, ++ moderate mould growth and +++ high mould growth after 5 days of
incubation. A duplicate of each Petri dishes were used in each temperature treatments.
3.6 DETERMINATION OF pH AND MOISTURE CONTENT OF INJERA
3.6.1 Sampling Method
From each of the baking cycles, samples were taken to determine pH and moisture content of
injera. The sampling was done by taking pieces of injera from every quarter of the injera roll
and blending all together.

3.6.2 Measurement of pH
An electronic pH meter (JENWAY MODEL 370 pH/mV METER, England) was used to
measure p. After calibration using standard solutions at pH 4 and 7, each injera suspension (10
g of ground injera was added to 100 g distilled water and the dispersion was homogenised
using a shaker) was measured.

3.6.3 Moisture Content Determination

The moisture content was determined by placing a weighed quantity (5g) of part of the injera
samples first at 650c. Further drying was done at temperature of 1000C in an air oven until
constant weight was recorded (AOAC, 2000). Moisture content was expressed as:
%Moisture content

M initial M final X 100%

M initial

3.7 EVALUATION OF EFFICACY OF CHEMICAL PRESERVATIVES

3.7.1 Addition of Chemical Preservatives
Injera was prepared in the same way as described in section 3.2. Each of the chemical
preservatives were added on the injera batters at the concentration of 0.3 % calcium
propionate, 0.2 % potassium sorbate, 0.1 % benzoic acid and sodium benzoate and 0.2 %
blend of the four just immediately before baking. The concentration of preservatives in breads
is 0.3% of final weight for calcium propionate, 0.1% for sodium benzoate and benzoic acid
and 0.2% for potassium sorbate. These values correspond to 1.5 grams of propionate per
pound of flour, 0.5 grams of benzoate and 1grams of sorbate per pound of flour (Jay, 2000).
After the baking process, the experimental and control samples were stored in the laboratory at
ambient temperature. (The chemicals were added immediately before baking to prevent the
chemicals from retarding the 20 fermentation of the injera preparation process).

Figure 8. Injera samples that kept in the laboratory at ambient temperature

3.7.2 Comparing the Rate of Mould Invasion
Mould outgrowth on each of the injera treatments was monitored daily over a period of 12
days (injera rolls were left at room temperature) and photos of each was taken. Injera spoilage
was evaluated based on the percentage of the total surface area of each roll where fungal
outgrowth occurred. The measurement of the mould outgrowth from the photos was done
using the Photoshop elements of Adobe Photoshop Version 10.0. An injera rolls was deemed
positive if more than 1% of the total surface area was covered with fungi (Ryan et al., 2008).
3.7.3 Evaluation of Shelf Life of Injera
The injera samples were examined for visible signs of moulds growth on the crust every day.
The microbial shelf life is defined as the period in days in which the spoilage caused by
microorganisms was first observed. The shelf life was expressed in relation to the
corresponding control (Katsinis et al., 2008).

3.8 STATISTICAL ANALYSIS

The statistical analysis of the colony diameter of moulds at different media by compare means
one way ANOVA (P<0.05) and the line graph relating mould invasion (%) over time were
conducted using SPSS version 15.0. Differences between media were determined by using
LSD.

CHAPTER 4
RESULTS AND DISCUSSION
4.1 MOULDS ISOLATED FROM INJERA
Under favourable conditions moulds can grow on a large variety of substrates. In bakery
processing, the most common type of microbial spoilage is mould growth and in many cases it
is the major factor governing shelf-life (Marin, 2005). The most wide spread and probably
most important moulds, in terms of biodeterioration of bakery products are species of
Aspergillus, Penicillium, Eurotium and Fusarium (Guynot et al., 2005; Gerez et al., 2009).
Other species, such as those of Cladosporium, Mucor and Rhizopus, have been found less
frequently (Guynot et al., 2003). According to Filtenborg et al., (1996) each individual food
type is normally infected by a limited number of fungi.
In this study, small white fungal colonies (visible to the naked eye) appeared on injera after
staying 4-5 days of storage in a messhob. Accordingly, the small colonies gradually grew and
started to show different kind of colony color on sporulation. The injera moulds were isolated
at different times of storage at ambient temperature and detected by microscopic evaluation,
after observing their morphological characteristics of the colony. It was then found that three
types of fungal species that belonged to the genera of Penicillium, Aspergillus (Aspergillus
niger) and Rhizopus species were responsible in spoiling injera, although their extent of
growth varied depending up on the ambient temperature of the day (The effect of temperature
on the growth rate of the injera moulds was discussed on section 4.3). The three fungi isolated
from injera were discussed below:

4.1.1 Penicillium species

Penicillium is a deuteromycetes which is greenish or blue green colonies; conidia in long
chains on repeatedly branched conidiophores resembling a brush like head (Fig.9d) (penicllus)
as described by Aneja, (2003). Penicillium species greenish colonies were seen on injera
(Fig.9a), on PDA (Fig.9b) and also on sterile injera plated on a Petri dish (Fig.9c). According
to Gock et al., (2003) penicillium species are xerophilic fungi that frequently bring about
spoilage of intermediate and low water activity baked goods.

a) Penicillium growing on injera

c) Penicillium grown on sterile injera plate

b) Penicillium growing on PDA

d) Microscopic brush like structure of

Penicillium

Figure 9. Penicillium isolated from injera

4.1.2 Aspergillus niger

Aspergillus niger is very common imperfect fungi with typically black colony.
Microscopically (Fig.10d) conidiophores arising from a foot-cell, catenate (basipetal) conidia
on phialides (1or 2 series) on vesicle as described by Aneja (2003). In this study also
Aspergillus niger black colonies were seen on injera (Fig.10a), on PDA (Fig.10b) and on a
sterile injera plated on a Petri dish (Fig.10c). Further Aspergillus niger black colonies became
more dominant and visible to the naked eye when the ambient temperature got warmer.
Similarly Shur and Nielsen, (2004) also reported that Aspergillus niger is more favoured in
warmer climates.

a) Aspergillus niger grown on injera

c) Aspergillus niger grown on sterile injera plate

b) Aspergillus niger grown on PDA

d) Microscopic structure of A. niger

Figure10. Aspergillus niger isolated from injera

4.1.3 Rhizopus species

Rhizopus is member of the common bread moulds. Its colony on injera (Fig.11a), potato
dextrose agar (Fig.11b) and on sterile injera plated on a Petri dish (Fig.11c) was white to dark
grey. As described by Aneja (2003) rhizopus has a nonseptate mycelium with root like
rhizoids; black columellate, sporangiophores, in clusters.

a) Rhizopus grown on injera

c) Rhizopus grown on sterile injera plate

b) Rhizopus grown on PDA

d) Microscopic structure of Rhizopus

Figure11. Rhizopus isolated from injera

The overall analysis of the identification and isolation process implied various causes for the
growth of the moulds on injera. As clearly described by Marin et al., (2002) and Gock et al.,
(2003) the baking temperature is generally sufficient to destroy these organisms,
contamination might arise from mould spores derived from the atmosphere or from surfaces
during the cooling, finishing and wrapping procedures Therefore, good hygienic practices
should be carried out with respect to food handlers, raw materials, equipment and premises. In
relation to this the fermenting vat should be well cleaned after each batch of injera making.
Potable water should be used. The straw basket (messhob) used to store injera should have
tight covers, and be kept in a ventilated, cool, dry places, raised of the floor. Packing also
protects to some extent against insects, animals and soils. When empty, the baskets should
regularly be cleaned and stored, raised off the floor in a dry place (Fellow, 1997).
Apart from the repelling sight of visible growth, fungi are also responsible for off-flavour
formation and the production of mycotoxins and allergenic compounds (Gutierrez et al.,
2009). In this study a yellowish pigment on the surface of a spoiled injera had been observed
when injera was kept for 12 days (Figure 12). Since both Penicillium and Aspergillus sp. are
toxin producing moulds, a spoiled injera could also be a source of toxic compounds that might
be menace to human health, although further investigation is needed to know for sure whether
this yellowish pigment released by this moulds was actually a toxin or not.

Figure12. Yellowish pigment released by injera moulds after 12 days of storage

4.2 MEDIA FROM INJERA

4.2.1 Growth of Moulds on Injera Sucrose Agar (ISA) Medium
The medium prepared from injera with sugar supplement, Injera sucrose agar (ISA) was found
to be effective in supporting the growth of moulds such as Aspergillus niger, Penicillium,
Aspergillus flavus and Rhizopus. In this experiment injera sucrose agar media had been used
as any other kind mycological media for the cultivation of injera moulds due to its
effectiveness and cheaper price. The injera sucrose agar media were also used to evaluate the
effect of temperature on the growth rate of injera moulds which was discussed in section 4.3.

Figure13. Injera sucrose agar media (ISA)

Whenever a particular mould is needed, it must be supplied with some form of organic carbon
for energy, a source of nitrogen for protein and vitamin synthesis, and several minerals
(Malloch, 1981) and these could be easily provided by injera. EHNRI, 1997 reported that
injera in general can be considered as a good source of energy, fibre, iron and vitamins (Table
1). This might be also one of the reason that injera was spoiled easily after 3 days of storage at
ambient temperature.
ISA medium was used in this study for getting a pure culture of each of the moulds for
identification. Since, many moulds have quite specific nutrient requirements and are
specialized to use materials that other fungi use with difficulty or not at all. We can take
advantage of this for the isolation of fungi by presenting a particular substance to the
environment for colonization and then later recovering it for isolation of the fungi that
occupied it (Malloch, 1981). This way of isolating fungi is usually called baits (baiting). ISA
had used as a bait in this study to favoured only moulds that could utilize injera as their source
of energy.
The contribution of injera sucrose agar media in this study had been also for founding out the
growth temperature range of injera moulds so as to use the appropriate incubation temperature

while cultivating them. Accordingly using the result that was found in section 4.3, Penicillium
and Rhizopus species were incubated at ambient temperature while Aspergillus niger was
incubated at 250c and more.
4.2.2 Effectiveness of Injera Sucrose Agar (ISA) Medium
The media prepared from injera, injera sucrose agar media was found to be as effective as
other mycological media. Table 5 shows the colony diameter of Rhizopus on media of potato
dextrose agar (PDA), Czapeks solution agar (CZP), Nutrient agar (NA), Malt extract agar
(MEA) and Injera sucrose agar (ISA) on day 1-5. Although the colony diameter of Aspergillus
niger, Aspergillus flavus and Penicillium were difficult to measure due to their non uniform
outgrowth on all the media.
Table 5. Rhizopus colony diameter on different media
Colony diameter of in mm on day 1-5
Media
Type

PDA

46

66

78

100

MEA

36

52

70

100

CYP

42

70

80

100

NA

32

50

72

100

ISA

16

38

62

100

The ANOVA (Appendix 1) showed that the colony diameter of rhizopus grown on injera
sucrose was not significantly (p<0.05) different from the colony diameter of the other
mycological media evaluated in this study.

Injera sucrose agar media had been the cheapest and conducive way in cultivating those
moulds isolated from a spoiled injera. However, the effectiveness of injera sucrose agar media
to support the growth of other types of moulds should be evaluated further in order to use it as
one kind of mycological media.
4.3 EFFECT OF TEMPERATURE ON THE GROWTH RATE OF INJERA MOULDS
Temperature is generally considered to be one of the most important environmental factors
that seriously affect not only the growth and germination of spoilage moulds in vitro, but also
in vivo (Tian and Bertolini, 1995). This study revealed that Penicillium and Rhizopus were
more dominant in spoiling injera at lower temperature, while Aspergillus niger grew much
faster as the temperature gets higher (25-320c). None of the moulds grew when the
temperature was kept at 40c (Table 6). The reduced activity of a fungus at lower temperature
may have corresponded with reduction of nutrient utilization, so that the fungus used the
colonized substrate more slowly (Ibid, 1995).
TABLE 6. Growth rates of injera moulds on ISA medium at different temperature
Growth of moulds under different temperature ( 0 c)
0

Moulds

4c

16 c

20 c

25 c

32 c

A.niger

++

+++

Penicillium spp

+++

++

Rhizopus spp

+++

++

No growth at all
++ Moderate mould growth

+ Slight mould growth

+++ High mould growth

Aspergillus niger grew in a very fast and furious way as the storage temprature rised from 250c
to 320c. Tyson & Fullerton (2004) mentioned that Aspergillus niger require a higher
temperature with an optimum temperature range of 28-34C for its growth. Similarly Shur and
Nielsen (2004) also confirmed that Aspergillus niger is favoured more in warmer climates.

From literature most fungi are mesophilic and are inhibited or killed at unusually high or low
temperatures. There are some, however; that actually require unusual temperatures:
thermophiles (>400c) and psychrophilic (150c or less) (Malloch, 1981). Obviously from the
analysis of the study the moulds spoiling injera were mesophilies since their growth
temperature range was between 16 and 320c. One of the possible explanations for the isolated
moulds to be only mesophilies could be related to climatic condition of the study site, where a
moderate climatic condition of maximum temperature of 24.6 0c was recorded. According to
fellow, (1997) contamination of moulds is less important in cool highland regions than warm
lowland regions. With a significant altitude difference in Ethiopia, obviously the dominant
mould spoiling injera might vary from the colder to the hotter parts of the country.
In the final analysis temperature could be one of the hurdles that could be applied to prolong
the shelf life of injera in addition to the use of the preservatives.

4.4 pH AND MOISTURE CONTENT OF INJERA

4.4.1 pH of Injera
Results of this investigation showed that injera was generally acidic irrespective of the type of
preservatives added (Table 7). The pH value of the samples stored at ambient temperature
ranges from 3.45-3.38. The pHs were 3.38 for the sample with benzoic acid, 3.39 for the
sample with sodium benzoate, 3.45 for the sample with potassium sorbate and 3.44 for the
sample with calcium propionate. The pH of the control sample without preservatives and
blend of all the four preservatives sample were 3.40 and 3.38 respectively. The results
indicated no variation in temperature (24.70c) for all the samples. The acidity decreased
slightly for the samples containing benzoic acid, sodium benzoate and blend of the four, while
it slightly increased for the samples containing potassium sorbate and calcium propionate as
compared to the control.
TABLE 7. pH of injera with and without preservatives
Injera samples

pH

Control

3.40

0.1 % benzoic acid

3.38

0.1 % sodium benzoate

3.39

0.2 % Potassium sorbate

3.45

0.3 % calcium propionate

3.44

0.2 % Blend of the four

3.38

The pH values obtained, when compared with the pH of bread which is mostly between 4.7
and 7.4 (FDA, 2007) was quite low. The high acidity of injera could account for the low
numbers and few types of organisms isolated, although the isolates have been found to be
associated with food spoilage as described by Ryan, et al., (2008). This might implied that the
moulds that spoiled injera were only those which could overcome such acidic environment
like Penicillium and Aspergillus species.
Gould (1996) reported that it is the pH value of the food and dissociation constant of the acid
that determine the proportion of the acid that is in the undissociated form. The antimicrobial
effect of the undissociated acid is much stronger than the dissociated acid because the
undissociated form is lipophilic and therefore most readily permeates the microbial membrane.
In the present study therefore, one of the reasons for the better effectiveness of benzoic acid
(discussed in section 4.5) in preserving injera than any of the preservatives could be due to the
lower pH of injera in addition to its dissociation constant (pKa). According to Shur and
Nielsen (2004) the pKa value of propionic acid, sorbic acid and benzoic acid are 4.88, 4.76
and 4.2, respectively which demonstrate the more acidic nature of benzoic acid than sorbic and
propionic acid.

4.4.2 Moisture Content of Injera

The moisture content of fresh injera samples (as soon as it was baked) were ranging from 6265% (Table 8). There was no significant difference in the moisture content of injera with and
without preservatives. However, there was a gradual decrease in the moisture content as the
number of days of storage gone from the 1st day to the 5th day at ambient temperature.
TABLE 8. Moisture Content of the Injera with and without preservatives
Injera samples

Moisture content
1st day

5th day

Control

64.2 %

63.6 %

0.1 % benzoic acid

63.7 %

62.3 %

0.1 % sodium benzoate

63.4 %

62.5 %

0.2 % Potassium sorbate

64.3 %

61.8 %

0.3 % calcium propionate

64.4 %

63. 1 %

0.2 % Blend of the four

63.5 %

61.2 %

According to Guynot et al., (2003) even if bakery products are important staple foods in most
countries and cultures, mold growth and drying are two problems that limit the shelf life of
both high and intermediate- moisture bakery products. In this study also the moisture content
of injera had decreased significantly when kept for 5 days.
The moisture content of the control injera obtained in this study was comparable with the
value reported in the Ethiopian Food composition table (Table 3) of Ethiopian Health and
Research Institute of Ethiopia (EHNRI, 1997) although this study concentrated on injera
baked at home. From the composition table it can be seen that injera made from any of the
cereals has higher moisture content than any of the breads.

According to Ayub et al., (2003), the overall mean moisture content of different kinds of bread
made from wheat range from 37-47 % during storage. In the contrary, injera had a higher
moisture content of 62-65% that made injera more perishable than most bread. The other
reason injera was more perishable than bread could be due the larger the surface area of injera
which made it more accessible by fungal spores to land and grew. According to Fellow
(1997), to prevent mould growth the product should be properly cooled and dried before
packing. Hence, any surface moisture will encourage contamination and the injera must not be
handled with wet hands as surface moisture will facilitate mould growth.

4.5 EFFICACY OF THE CHEMICAL PRESERVATIVES ON INJERA

4.5.1 Extent of Mould Invasion on Injera
Investigation of the antimicrobial activity of the preservatives tested in this study revealed that
the chemical preservatives were effective against injera moulds. This was shown by the
reduction in percentage of mould invasion of the samples containing preservatives as
compared to the sample without preservative (Table 9 and Figure 14). Antifungal activity of
preservatives investigated in this study also showed that injera samples containing benzoic
acid and its sodium salt were the most effective of all the preservatives tested. Even after 12
days of preservation, samples containing benzoic acid and its sodium salt had more keeping
quality than samples with other preservatives. Samples without preservatives (control),
however, exhibited the highest mould invasion from day 1-12 of preservation and
consequently, had the poorest keeping quality as compared to those with preservatives.
Chemical preservatives has become an increasingly important practice in modern food
technology with the increase in production of processed and convenience foods. These
preservatives are deliberately added to stop or delay nutritional losses due to microbiological,
enzymatic or chemical changes and thus increasing its shelf-life. Benzoic acid, sorbic acid and
propionic acid are generally effective to control mould and inhibit yeast growth, and against a
wide range of bacterial attack (Tfouni and Toledo, 2002).

TABLE 9. Percentage of the invaded surface as compared to the total surface of injera
Treatments
0
Control

Invasion of injera(%) over a number of days

4
6
8
10

12

12

40

63

92

10

18

0.1 % sodium
benzoate

0.2 % potassium
sorbate

17

41

0.3 % calcium
propionate

57

75

86

0.2 % blend of the

four

10

29

66

0.1 % benzoic acid

Depending up on the type of mould that dominated the injera samples at the given condition
there might be a variation in the degree of invasion that was reported on table 8. If the injera
was first dominantly invaded by for Rhizopus it might invade the whole surface of injera in a
very short period of time due to many zygomycetes are fast growing and coarse which can
take over the culture they are growing in a few days (Malloch, 1981). As clearly depicted in
the graph (Figure14) the sample containing 0.3 % calcium propionate (purple line) had shown
a drastic change than the control (blue line) in its degree of mould invasion between the 6 and
8 day of storage.

key

100

control
benzoic acid (0.1%)
sodium benoate
(0.1%)
Ca-propionate(0.3%)
K-sorbate (0.2%)
blend (0.2%)
control
benzoic acid (0.1%)
sodium benoate
(0.1%)
Ca-propionate(0.3%)
K-sorbate (0.2%)
blend (0.2%)

Mould invasion (%)

80

60

40

20

0
0

10

12

Days of storage

Figure 14. Mould invasion of injera with and without preservatives in 12 days
The effectiveness of the organic acids used in this study generally increase in effectiveness in
the order of propionic acid < sorbic acid < benzoic acid which is in agreement with the results
of Gould, (1996) which reported the order reflects their increasing lipophilicity. The sample
containing benzoic acid and its salt (sodium benzoate) best preserved injera of all the
preservatives tested could be due to the antimicrobial activity of benzoic acid is principally in
the undissociated form and benzoic acid is relatively acidic (pKa =4.2) which it inhibits the
growth of spoilage moulds. It has the ability of the dissociated molecule to interface with
membrane energetic (Akpan & Kovo, 2005). Further it was speculated that effectiveness of
sodium benzoate than benzoic acid could be due to the fact that the salt is 200 times more
soluble than the acid (DIONEX, 2004).

Similarly, the smaller effect of propionic acid in contrast to benzoic acid and sorbic acid
confirms earlier results of Razavi-Rohani and Griffiths, (1999 who reported that even if
propionic acid and its salts are primarily are inhibitory to moulds; some species of moulds are
resistant and grow in media containing substantial amounts of propionic acid and its salts.
Moreover when compare with propionic acid and sodium propionate, calcium propionate (also
used in this study) is least effective on moulds.
Ryan et al., (2008) stated that the optimum pH level for the inhibitory effect of propionic acid
is 5.0 with a maximum effect of pH of 6.0. In contrary to this, the pH of injera containing
calcium propionate was highly acidic (around 3.44) and made it very far from the optimum pH
for it to be effective. In addition to this, the least effectiveness of calcium propionate on injera
could be due to the concentration used. Propionic acid should only be added in bread in a
concentration not exceeding 3000ppm (also the concentration used in this study); recent
studies have shown that under these conditions propionic acid is not effective against common
bread spoilage organisms.
Finally, the combined (blended) effect of the four preservatives used for this study had been
also evaluated. According to Martinez-Flores et al., (2004) the combined action of two or
more preservatives is more effective in inhibiting the growth of microorganism, which could
be due to a synergistic effect among the preservatives added to the product. However in this
study there seem to be no synergistic effect since the blended sample was only more effective
than calcium propionate.

4.5.2 Shelf Life of Injera with and without Preservatives

The effect of chemical preservatives on the shelf life of injera was shown in Table 10 and it
was determined as the day in which mould was visible to the naked eye. The shelf life of
injera without preservatives (control) was 3-4 days while injera containing 0.3 % calcium
propionate was 4-5 days depending upon the storage temperature. Shelf life of injera
containing 0.1 % benzoic acid and sodium benzoate were the better preservatives of all tested
which prolonged the shelf life of injera up to 10 and 12 days respectively. 0.2 % of potassium
sorbate and 0.2 % of blend of the four preservatives had similarly prolonged the shelf life of
injera 6-8 days at 20+20c.
Table 10. Shelf life of injera containing preservatives at 20+ 20c
Injera Treatments

Shelf life ( days)

Control

3-4 days

0.1 % benzoic acid

8-10 days

0.1 % sodium benzoate

10-12 days

0.2 % potassium sorbate

6-8days

0.3 % calcium propionate

4-5 days

0.2 % blend of the four

6-8 days

Fungi are the most common spoilers in bakery products. Commonly a shelf life of 3-4 days
may be expected when they are unpreserved (Ryan et al., 2008). Preservatives may be
microbicidal and kill the target organisms or they may be microbiostatic in which case they
simply prevent them from growing, thus prolonging the shelf life of the product (Jay, 2000).
The preservatives used in this study had prolonged the shelf life of injera from just 3-4 days to

up to 10 days. In these 10 days injera could be consumed before it loses its acceptability, since
the shelf life of a product is the time a product will remain organoleptically acceptable
(Doughari, 2007).

CHAPTER 5
CONCLUSIONS AND RECOMMENDATIONS
5.1 Conclusions
Even though injera is one of the most popular Ethiopian indigenous fermented food, it did not
received the scientific attention it deserves for many years as far as preservation concerned. Its
storage period does not usually exceed 3 days at room temperature. To the best of our
knowledge, there are no previous reports on the use of chemical preservatives in any of the
Ethiopian traditional foods. This study not only identified the moulds that were responsible in
spoiling injera but also evaluated the common chemical preservatives used in the food
industry for the control of injera moulds.
In the present study three fungal species from Penicillium, Rhizopus and Aspergillus
(Aspergillus niger) species were found spoiling injera while keeping it at ambient temperature.
The first two species were more dominant in spoiling injera as the storage temperature lowers
between 16-200c, while Aspergillus niger is more dominant in spoiling injera stored at higher
temperatures.
The study also showed that the shelf life of injera can be extended by the use of preservatives
and storage conditions that will not favour mould outgrowth. Injera sample without
preservatives exhibited the highest percentage of moulds from day 1 to 12 days of preservation
and consequently had the poorest keeping quality as compared to those with preservatives.
Investigation of the antimicrobial activity of the preservatives also revealed that sodium
benzoate was the most effective of all the preservatives tested in inhibiting mould growth
while calcium propionate was least effective of all the preservatives tested.

5.2 Recommendations
In developing country like Ethiopia which is still under a struggle to be food secured,
losing a substantial amount food due to spoilage is a disaster. Applying the result of
this study can contribute a lot in saving a significant amount of injera that is lost in
every household per every baking cycle. Moreover, prolonging the shelf life of injera
can curb the problem faced to export injera to other parts of the world and to the many
Diasporas that miss their home country as well as injera.
As the study was conducted in Addis Ababa with specific climatic condition, further
investigation on the type and kind of moulds that spoil injera on the warmer parts of
Ethiopia should be evaluated.
Further studies on the sensory attributes of injera with preservatives must be
conducted to see if any of the additives has effect on health and nutritional status.
Even if, the use of preservatives is an attractive means to diminish spoilage and insure
food safety, consumers today are not in favour of additives as preservatives and an
urge to reduce the quantities used exists within the bakery industry. Reduction of
preservatives to sub-inhibitory levels has nevertheless been shown to stimulate growth
of spoilage fungi in some cases. As a result of this, the application of the hurdle
technology (combined technology) should be evaluated to minimize the amount of the
chemical preservatives used in injera. So the application of other alternative
preservation methods such as:
Modified atmosphere packaging (MAP),
Irradiation,

Pasteurization of the packaged injera

Natural plant extracts ( also called green chemicals) and
Bio-preservation [the use of microorganisms and/or their metabolites] to
prevent spoilage and extend the shelf life of injera (and other Ethiopian
traditional foods) should also be evaluated.

REFERENCES
Abdullah, N., Nawawi, A., & Othman, I. (2000). Fungal spoilage of starch-based foods in
relation to its water activity(aw). Journal of Stored Products Research , 36:47-54.
Adamu Zegeye. (1997). Acceptability of injera with stewed chicken. Food Quality and
Preference , 8 (4), 293-295.
Akpan, U. G., & Kovo, A. S. (2005). Preservation of passion fruit juice. Leonardo Journal of
Sciences (7), 17-22.
Anjea, K. R. (2003). Experiments in Microbiology, Plant Pathology and Biotechnology (4th
ed.). New Delhi: New Age International.
AOAC.(2000).Official Methods of Analysis (17th ed., Vol. 2). Washington D.C: Association of
Official Analytical chemistry.
Arogundade, L. A. (2006). Functional characterization of tef (Eragostics tef) protein
concentrate: Influence of altered chemical environment on its gelation, foaming, and
water hydration properties. Food Hydrocolloids , 20:831-838.
Ayub, M., Wahab, S., & Durrani, Y. (2003). Effect of water activity (aw), moisture content
and total microbial count on the overall quality of bread. Intrenational Journal of
Agriculture and Biology , 274-278.
Ball, L. J., Sands, D. C., & Newman, R. D. (1996). A biotechnological approach to world
hunger:the use of lysine excreting microbial mutants as inoculum in Ethiopian
bread. Journal of the American Dietetic Association , 96:9(1) A55.

Barnett, H. L., & Hunter, B. B. (1972). Illustrated Genera of Imperfect Fungi (3rd ed.).
Minneapolis: Burgess publishing company.
Berhanu Abegaze Gashe. (1985). Involvement of lactic acid bacteria in the fermentation of tef
(Eragrostis tef), an Ethiopian fermented food. Journal of Food Science , 50:800801.
Blandino, A., Al-Aseeri, M. E., Cantero, D., & Webb, C. (2003). Cereal based fermented
foods and beverages. Food Research International , 36:527-543.
Bohra, & Parihar. (2006). Food Microbiology. Jodhpur: AGROBIOS(INDIA).
Brul, S., & Coote, P. (1999). Preservative agents in foods: mode of action and microbial
resistance mechanisms. International Journal of Food Microbiology , 50:1-17.
Carson, N. S. (2006, April 2). How long does it take bread with preservatives to grow mold
than it takes bread without preservatives. Retrieved January 18, 2009, from
http://www.usc.edu/CSSF/history/2006/projects
CSA. (2008). Agricultural Sample Survey: Report on Area and Production of Crops. Addis
Ababa: Central Statstical Agency of Ethiopia.
DIONEX. (2004). Determination of benzoate in liquid food products by reagent free ion
chromatography. Retrieved December 28, 2008, from www.dionex.com/enus/webdocs/7751_AN165_V23.pdf
Doughari, J. H., Alabi, G., & Elmahmood, A. M. (2007). Effect of some chemical
preservatives on the shelf life of sobo drink. African Journal of Microbiology
Research , 2:37-41.

EHNRI. (1997). Food compostion table for use in Ethiopia. Addis Ababa: Ethiopian Health
and Nutrition Research Institute.
FDA. (2007, April). Acidified and Low-Acid Canned Foods. Retrieved May 14, 2009, from
U.S Food and Drug Adminstration: http://www.cfsan.fda.gov/~comm/lacf-phs.html
Fellow, P. (Ed.). (1997). Traditional Foods:Processing for profit. London: Intermediate
Technology Publications.
Filtenborg, O., Frisvad, J. C., & Thrane, U. (1996). Moulds in food spoilage. International
Journal of Food Microbiology , 33(1):85-102.
Fissha Itana, & Olsson, M. (2004). Land degradation in Addis Ababa due to industrial and
urban development. Ethiopian Journal of Development Research , 26:77-100.
Geremew Boultosa. (2007). Physicochemical characterstics of grain and flour in 13 tef
[Eragrostis tef (zucc.) Totter] grain varities. Journal of Applied Science Research ,
3(12):2042-2051.
Gerez, C. L., Torino, M. I., & Valdez, G. F. (2009). Preservation of bread mould spoilage
using lactic acid bacteria with antifungal properties. Food Control , 20:144-148.
Gock, M. A., Hocking, A. D., Jonh, I., Pitt, J. I., & Poulos, P. G. (2003). Influence of
temperature, water activity and pH on growth of some xerophilic fungi.
International Journal of Food Microbiology , 2481:11-19.

Gonzalez-Fandos, E., & Dominguez, J. L. (2007). Effect of potassium sorbate washing on the
growth of Listeria monocytogenes on fresh poultry. Food Control , 18:842-846.
Gould, G. W. (1996). Methods of preservation and extension of shelf life . International
Journal of Food Microbiology , 33:51-64.
Gupta, C., Garg, P. A., Uniyal, R. C., & Kumari, A. (2008). Comparative analysis of the
antimicrobial activity of cinnamon oil and cinnamon extract on some food-borne
microbes. African Journal of Microbiology Research , 2(9):247-251.
Gutierrez, L., Sanchez, C., Batlle, R., & Nerin, C. (2009). New antimicriobial active package
for bakery products. Trends in Food Science and Technology , 20:92-99.
Guynot, M. E., Marin, S., Sanchis, V., & Ramos, J. (2003). Modified atmoshere pacaking for
prevention of mold spoilage of bakery products with differnt pH and water activity.
Journal of Food Protection , 66(10):1864-1872.
Guynot, M. E., Marin, S., Sanchis, V., & Ramos, J. (2004). An attempt to minimize potassium
sorbate concentration in sponge cakes by modified atmosphere packaging
combination to prevent fungal spoilage. Food Microbiology , 21: 449457
Heritage, J., Evans, E. V., & Killington, R. A. (1996). Introductory Microbiology. Cambridge:
Press Syndicate of university of Cambridge.
Higgins, C., & Blunkhaus, F. (1999). Efficacy of several organic acids against moulds.
Journal of Applied Poultry Research , 8:480-487.

Huis in'
t Veld, J. (1996). Microbial and biological spoilage of foods: an overview.
International Journal of Food Microbiology , 33:1-18.
Ibekwe, Sixtus, E., Uwakwe, Augustine, A., Monanu, & Michael, O. (2007). In vivo effects of
sodium benzoate on plasma aspartate amino transferase and alkaline phosphatase of
wistaralbino rats. Scientific Research and Essay , 2(1):10-12.
Ikrin, R., & Koruklouogl, M. (2007). Control of Aspergillus with garlic, onion and leek
extracts. African Journal of Biotechnology , 6(4):384-387.
Jay, J. M. (2000). Modern Food Microbiology (6th ed.). Maryland: Aspen Publishers.
Juneja,V. K., Sheen, S., & Tewari, G. (2008). Food Preservation. In C. L. Wilson, Microbial
food contamination (pp. 347-393). New york: CRC press.
Katsinis, G., Rigas, F., & Doulia, D. (2008). Synergistic effect of chemical preservatives with
ethanol on the micriobial shelf life of bread by factorial design. International
Journal of Food Science and Technology , 43:208-215.
Knochel, S., & Gould, G. (1995). Preservation microbiology and safety. Trends in Food
Science and Technology , 6:134-146.
Lombard, G. E., Weinert, I. G., & Taylor, J. N. (2000). Preservation of South African steamed
bread using hurdle technology. Lebenum -Wiss.U-Technol , 33:138-143.
Lopez-Malo, A., Alzamora, S. M., & Palou, E. (2005). Aspergillus flavus growth in the
presence of chemical preservatives and naturally occuring antimicrobial
compounds. International Journal of Food Microbiology , 99:119-128.

Lucke, F. K. (2003). The control of pH. In P. Zeuthen, & L. Bogh-Soresen, Food preservation
techniques (pp. 107-108). Cambridge: WoodHead Publishing Limited .
Malloch, D. (1981). Moulds:Isolation, Cultivation and Identification. Toronto: University of
Toronto.
Marin, S., Guynot, M. E., Sanchis, V., & Ramos, A. J. (2002). Risk assessment of the use of
sub-optimal levels of weak-acid preservatives in the control of mould growth on
bakery products. International Journal of Food Micriobiology , 79:203-211.
Marin, S., Guynot, M. E., Sanchis, V., & Ramos, A. J. (2005). An attempt to optimize
potassuim sorbate use to preserve low pH (4.5-5.5) intermediate moisture content
products by modelling Eurotium spp. And

penicillium corylophilum growth.

International Journal of Food Microbiology , 101:169-177.

Martrtinez-Flores, H. E., Gaytan-Martinez, M., Figueroa-Cardenas, J. D., Martinez-Bustos, F.,
Reyes-Vega, M. L., & Rodriguez-Vidal, A. (2004). Effect of some preservatives on
the shelf life of corn tortillas obtained from extruded masa. Agrociencia , 38(3):285292.
Mogessie Ashenafi. (2002). The microbiology of Ethiopian foods and beverages:a review.
SINET: Ethiopian Journal of Science , 25(1):97-140.
Mogessie Ashenafi. (2006). A review on the microbiology of indigenous fermented foods and
beverages of Ethiopia. Ethiopian Journal of Biology , 5(2):189-245.

Morris, A., Barnett, A., & Burrows, O. J. (2006, 9 21). Food Preservation. Retrieved Feburary
25, 2009, from www.paho.org/English/CFNI/cfni-caj37No304-art-1.pdf
Omonigho, S. E., & Ikenebomeh, M. J. (2000). Effects of different preservative treatments on
the chemical changes of pounded yam (Dioscorea rotundata) in storage at 28+20c.
Food Chemisty , 68:201-209.
Qi, P., Hong, H., Liang, X., & Liu, D. (2009). Assessment of benzoic acid levels in milk in
China. Food Control , 20:414-418.
Rahman, M. S. (2007). Food preservation:overview. In M. S. Rahman, Hand book of food
preservation (pp.3-17). New York: CRC press.
Razavi-Rohani, S. M., & Griffiths, M. W. (1999). Antifungal effects of sorbic acid and
propionic acid at differnt pH and NaCl conditions. Journal of Food Safety , 19:109120.
Russell, N. J., & Gould, G. W. (2003). Food Preservatives (2nd ed.). Chicago: Springer.
Ryan, L. M., Dal Bello, F., & Arendt, E. K. (2008). The use of sourdough fermented by
antifungal LAB to reduce the amount of calcium propionate in bread. International
Journal of Food Microbiology , 125:274-278.
Sahlin, P. (1999). Fermentation as a method of food processing: production of organic acids,
pH-development and microbial growth in fermenting cereals (Licentiate thesis, May
1999) Lund Institute of Technology, Lund University.

Santini, A. O., Pezza, H. R., Filho, O. C., Sequinel, R., & Pezza, L. (2009). Potentiomertic
sensor for sorbic acid determination in food products. Food Chemisrty , doi:
10.1016/j.foodchem.2009.02.012.
Science of cooking. (n.d). Ethiopian injera. Retrieved November 20, 2008, from
Exploratorium: http://www.exploratorium.edu/cooking/bread/recipe-injera
Shur, K. I., & Nielsen, P. V. (2004). Effect of weak acid preservatives on growth of bakery
product spoilage fungi at different water activities and pH values. International
Journal of Food Microbiology , 95:67-78.
Skirdal, I. M., & Eklund, T. (1993). Microculture model studies on the effect of sorbic acid on
penicillium chryogenum,Cladosporium cladosporiodies and Ulocladium atrum at
different pH levels. Journal of Appiled Bacteriology , 74:191-195.
Taiwo, O. (2009). Physical and nutritive properties of fermented cereal foods. African Journal
of Food Science , 3(2):023-027.
Tfouni, S. V., & Toledo, M. F. (2002). Determination of benzoic and sorbic acids in Brazillian
food. Food Control , 13:117-123.
Tian, S. P., & Bertolini, P. (1995). Effects of low temprature on mycelial growth and spore
germination of Botrytis alli in culture and pathogenicity to stored garlic bulbs. Plant
Pathology , 44:1008-1015.
Tyson, J. L., & Fullerton, R. A. (2004). Effect of soil-borne inoculum on incidence of onion
black mould (Aspergillus niger). New Zealand Plant Protection , 57:138-141.

Venturini, M. M., Blanco, D., & Oria, R. (2002). In vitro antifungal activity of several
antimicriobial compounds against penicillium expansum. Journal of Food
protection , 65(5):834-839.
Zeuthen, P., & Bogh-Soresen, L. (2003). Introduction. In P. Zeuthen, & L. Bogh-Soresen,
Food Preservation Techniques (pp. 1-2). Cambridge: Wood Head publishing
Limited.

APPENDIX 1. ANOVA of Rhizopus colony diameter on different kind of media

DECLARATION

I, the undersigned, declare that this thesis is my original work and that all sources of materials
used for the thesis have been correctly acknowledged.

Name: Ashagrie Zewdu

Signature: _______________
Date: ________________

The thesis has been submitted with my approval as a supervisor.

Name: Dawit Abate (PhD) Signature: ________________
Date: _________________

RESEARCHABLE TITLES
While conducting this research I came across many researchable areas that need to be
addressed in the future. For anyone who is interested I suggested the following titles as
research topics:
1. Improvement of shelf life of injera using parabens (Esters of p-hydroxy benzoic
acid)
2. The use of antifungal LAB to reduce the amount of calcium propionate, benzoic acid
or sorbic acid added on injera
3. An attempt to minimize chemical preservatives added in injera by modified
atmosphere packing combination to prevent fungal spoilage
4. Influence of temperature , water activity and pH on the growth of injera moulds
5. Effect of some naturally occurring antimicrobial compounds on the shelf life of injera
6. Preservation of Ethiopian injera using hurdle technology
7. In vitro study of the efficacy of several organic acids against molds isolated from injera
8. Effectiveness of sorbic acid in Ethiopian dairy products