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Chemical engineering Thesis and Dissertations
2020-03-11
Improving Shelf Life of Injera by Using
Natural Preservatives
Hilemichael, Wendu
http://hdl.handle.net/123456789/10216
Downloaded from DSpace Repository, DSpace Institution's institutional repository
BAHIR DAR UNIVERSITY
BAHIR DAR INSTITUTE OF TECHNOLOGY
SCHOOL OF RESEARCH AND POSTGRADUATE STUDIES
FACULTY OF CHEMICAL AND FOOD ENGINEERING
IMPROVING SHELF LIFE OF INJERA BY USING NATURAL
PRESERVATIVES
By: WENDU HILEMICHAEL
JUNE, 2019
BAHIR DAR, ETHIOPIA
ii
Improving Shelf Life of Injera by Using Natural Preservatives
Wendu Hilemichael
A Thesis
Submitted to School of Research and Post Graduate, Bahir Dar Institute of
Technology, Bahir Dar University in partial fulfillment of the requirement
for the Degree of Master of Science in Chemical Engineering (Process
Engineering Specialization)
June, 2019
Bahir Dar, Ethiopia
iii
DECLARATION
iv
APPROVAL SHEET
v
ACKNOWLEDGEMENTS
First of all, I would like to thank Almighty God who helps me to reach this stage. Next to
this, I would like to express my gratitude to my advisor Dr. Zenamarkos Bantie, for his
invaluable support, encouragement, supervision and useful suggestions throughout my
thesis work and presentation. I also express my special thanks to Mr. Tewodros Andargie
(Ph.D. Candidate) for his help during title selection, idea and material support. Finally, I
express my special thanks to faculty of chemical and food engineering, which facilitates
all the available equipment and the budgets for the purchasing the raw materials and other
expenses.
vi
ABSTRACT
Injera, Ethiopian fermented bread, is made mainly from teff (Eragrostis teff). On average,
two-third of Ethiopian diet consists of injera and it accounts for about two-thirds of the
daily protein intake and has a high nutritional value, as it is rich in calcium, iron and zinc.
Unfortunately, injera has a shelf life of only 3-4 days essentially due to mould (especially
three fungal species Aspergillus niger, Penicillium sp and Rhizopus sp) spoilage.
Nowadays, the use of plant extract as natural preservatives is increasing due to their
antimicrobial activities, beside this they have high acceptability by consumers in regards
to their health effect as well as their flavor and aroma improvement. In this research, the
effect of aqueous and ethanolic extract of garlic, cinnamon, and clove on the shelf life of
injera at different concentrations (2% ,5% and 10%) and different storage temperatures
(30oC and ambient temperature (24±2oC)) were evaluated. The preservatives were added
immediately before baking and their effect on shelf life (microbial quality) and sensory
quality were analyzed. Sensory quality analysis was done using 5-point hedonic scale. For
microbial analysis, total plate count and yeast & mould count were conducted. From the
result obtained, the average sensory quality of the control was 4.4 followed by injera
prepared from teff flour with addition of 2% aqueous extract of garlic and cinnamon with
mean value of 4.2 and 4, respectivelly. Injera prepared from teff flour with addition of
aqueous and ethanolic extract of clove shows the minimum over all acceptability, due to a
bitter flavor of cloves cause by eugenol. Antimicrobial activities of the preservatives
investigated could prolong the shelf life of injera up to 10 days at ambient storage
temperature by using ethanolic extract of clove with 10% addition followed by 10%
addition of aqueous extract of garlic which extend the shelf life of injera up to 8 days. At
30oC the maximum, shelf life of injera observed was 7 days by using ethanolic extract of
clove and aqueous extract of garlic with 10% addition. In general, as the concentration of
spices added increased, the sensory quality decreased whereas the microbial quality
increased and the maximum shelf life of injera were observed at ambient temperature than
30oC.
Key words: Injera, Natural Preservatives, Mould Spoilage, Shelf life, Sensory quality
vii
TABLE OF CONTENTS
DECLARATION ............................................................................................................... iii
APPROVAL SHEET ......................................................................................................... iv
ACKNOWLEDGEMENTS .................................................................................................v
ABSTRACT ....................................................................................................................... vi
TABLE OF CONTENTS .................................................................................................. vii
LIST OF ABBREVIATIONS ..............................................................................................x
LIST OF TABLES ............................................................................................................. xi
LIST OF FIGURES .......................................................................................................... xii
1. INTRODUCTION ...........................................................................................................1
1.1 Background .................................................................................................................1
1.2 Statement of the Problem ............................................................................................3
1.3 Objectives of the Study ...............................................................................................4
1.3.1 General Objective ............................................................................................... 4
1.3.2 Specific Objectives ............................................................................................. 4
1.4 Scope and Limitation of the Study..............................................................................4
1.5 Significance of the Study ............................................................................................4
2. LITERATURE REVIEW ................................................................................................5
2.1 Introduction .................................................................................................................5
2.2 Health Benefits of Teff and Teff-Based Products.......................................................7
2.3 Processing of Injera ....................................................................................................9
2.4 Spoilage and Preservation of Injera ..........................................................................11
2.4.1 Factors that Affect Microbial Growth on Injera .............................................. 11
2.4.2 Methods of Preservation and Extension of Shelf Life of Injera ...................... 16
2.4.3 Anti-Microbial Effect of Some Natural Preservatives ..................................... 17
viii
3. MATERIALS AND METHODS ..................................................................................21
3.1 Raw Materials Used ..................................................................................................21
3.2 Chemicals and Equipment Used ...............................................................................21
3.3 Preparation of Injera and Addition of Preservatives ................................................22
3.4 Preparation of Spice Extract .....................................................................................23
3.5 Sensory Quality Analysis of Injera with Natural Preservatives ...............................23
3.6 Microbiological Analysis of Injera with Natural Preservatives ...............................24
3.7 Experimental Design .................................................................................................25
4. RESULTS AND DISCUSSION ...................................................................................26
4.1 Spice Extract .............................................................................................................26
4.2 Sensory Quality of Injera with Different Natural Preservatives ..............................27
4.2.1 Eye Size and Distribution ................................................................................. 29
4.2.2 Color ................................................................................................................. 30
4.2.3 Flavor ................................................................................................................ 31
4.2.4 Sweetness ......................................................................................................... 32
4.2.5 Bitterness .......................................................................................................... 33
4.2.6 Sourness ............................................................................................................ 34
4.2.7 Overall Acceptability ....................................................................................... 35
4.3 Microbial Quality of Injera with Different Natural Preservatives ............................37
4.3.1 Visual observation of Injera spoilage .............................................................. 37
4.3.2 Microbial Count of Injera with Different Natural Preservatives ..................... 41
4.4 Effect of Temperature and Natural Preservatives on the Shelf Life of Injera ..........44
5. CONCLUSION AND RECOMMENDATION ............................................................46
5.1 Conclusion ................................................................................................................46
5.2 Recommendation ......................................................................................................47
6. REFERENCE ................................................................................................................49
ix
APPENDICES ...................................................................................................................53
Appendix A: Questionnaire Format for Sensory Evaluation .............................................53
Appendix B: ANOVA Table for Sensory Quality .............................................................54
Appendix C: Picture of Sensory Evaluation of Injera by the Panelists .............................58
Appendix D: Storage Condition of Injera .........................................................................59
Appendix E: Visual observation of Injera Spoilage ..........................................................60
Appendix F: Image of Microbial Growth in Petri Dish .....................................................61
x
LIST OF ABBREVIATIONS
ANOVA Analysis of Variance
aw water activity
BAM Bacteriological Analytical Manual
CD Celiac disease
cfu Colony forming units
Eh Oxidation-reduction potential
g Gram
Kg Kilogram
l Liter
ml Milliliter
MAP Modified atmosphere packaging
NA Not Applicable
NC Not Countable
PCA Plate Count Agar
PDA Potato Dextrose Agar
PH Potential of Hydrogen
RH Relative humidity
SPSS Statistical Package for Service Solution
xi
LIST OF TABLES
Table 3.1 Chemicals and equipment used ......................................................................... 21
Table 3.2 Experimental Design ......................................................................................... 25
Table 4.1 Sensory quality of injera prepared from teff flour and preservatives ............... 28
Table 4.2 Visual observation of injera spoilage Stored at Ambient Temperature ........... 39
Table 4.3 Visual observation of injera spoilage Stored at 30oC ....................................... 40
Table 4.4 Total plate count ............................................................................................... 42
Table 4.5 Yeast and Mould Count .................................................................................... 43
xii
LIST OF FIGURES
Figure 2.1 Flow diagram for the preparation of injera ..................................................... 10
Figure 2.2 Image of Raw Garlic ....................................................................................... 18
Figure 2.3 Image of Cinnamon ......................................................................................... 19
Figure 2.4 Image of Clove Buds ....................................................................................... 20
Figure 3.1 Flow diagram for the preparation of injera and addition of natural preservatives
........................................................................................................................................... 22
Figure 4.1 Aqueous and Ethanolic extract of different Spice ........................................... 26
Figure 4.2 Eye size and distribution of injera with spice extract at different concentration
and solvent ........................................................................................................................ 29
Figure 4.3 Color of injera with spice extract at different concentration and solvent ....... 30
Figure 4.4 Flavor of injera with spice extract at different concentration and solvent ...... 31
Figure 4.5 Sweetness of injera with spice extract at different concentration and solvent 32
Figure 4.6 Bitterness of injera with spice extract at different concentration and solvent 33
Figure 4.7 Sourness of injera with spice extract at different concentration and solvent .. 34
Figure 4.8 Over all acceptability of injera with spice extract at different concentration and
solvent ............................................................................................................................... 35
1
1. INTRODUCTION
1.1 Background
Injera is thin, staple Ethiopian fermented traditional bread prepared from grain tef
[Eragrostis tef (Zucc.) Trotter] flour, water and starter (ersho), which is a fluid saved from
previously fermented dough (Ashagrie and Abate, 2012; Bultosa, 2007). The preparation
of teff injera consists of two stages of natural fermentation, which last for about 24 to 72
hours, depending on ambient temperatures. The required ingredients for injera preparation
are the teff flour and water (Ashagrie and Abate, 2012).
The method of baking of injera from its raw materials involves preparing and mixing the
ingredients to make dough, which will ferment and subsequently thinned to a batter. The
batter is then poured onto a hot griddle in a thin layer to cook, and to develop color, flavor
and texture. A higher number of larger eyes and its slightly sour taste due to the acidic (low
pH) nature are the major quality attributes of a good injera (Ashagrie and Abate, 2012;
Mezemir, 2015).
Under the traditional storage condition, injera has a shelf life of only 3-4 days at ambient
temperature essentially due to mould spoilage. Three fungal species (Aspergillus niger,
Penicillium sp and Rhizopus sp) were found to be responsible for injera spoilage.
Penicillium and Rhizopus were more dominant at storage temperature of between 16-200C,
while Aspergillus niger was found to be more dominant at higher temperature of 25-320C
(Ashagrie and Abate, 2012). It is a common practice to discard mouldy injera (Mulaw and
Tesfaye, 2017).
According to Ashagrie and Abate (2012), the shelf life of injera can be extended up to 10-
12 days by using chemical preservatives. Even though, we can extend shelf life of injera
and other food products by using chemical preservatives, the society strongly oppose to
use injera or other food products prepared with chemical preservatives.
2
In addition to customer attitude, certain preservatives, including sulfites and sodium
benzoate, may cause adverse reactions in a small percentage of the population. Sulfites can
cause potentially deadly allergic reactions in sensitive individuals. Sodium benzoate, also
called benzoic acid, can cause asthma, hives and other allergic reactions. It also combined
with ascorbic acid in acidic foods and produce benzene, which may slightly increase the
risk for leukemia and other types of cancer. Nitrates and nitrites, which are often used to
preserve cured meats, may also increase the risk for certain types of cancers (Carocho et
al., 2015).
However, today consumers demand less use of synthetic preservatives, but still they expect
food to be free from microbial growth, toxins and other quality deteriorating factors. Mean-
while product freshness and sensorial qualities must be preserved. The problem for the
food industry is to fulfill the demands of minimum changes in food quality and maximum
security (Nielsen and Rios, 2000).
The growing concern about food safety has recently led to the development of natural
antimicrobials to control food borne pathogens and spoilage bacteria. Spices and their
derivatives such as extract, essential oils, hydrosols and decoctions are one of the most
commonly used natural antimicrobial agents in foods and have been used traditionally for
thousands of years by many cultures for preserving foods and as food additives to enhance
aroma and flavor. Previous Studies confirm that garlic, onion, cinnamon, clove, thyme,
sage, and other spices inhibit the growth of microorganisms in food (Hoque et al., 2008).
Natural plant extracts may provide an alternative to chemical preservatives. Over the years,
much effort has been devoted to the search for new antifungal materials from natural
sources for food preservation (Irkin and Korukluoglu, 2007).
In general, it is realized that, the possibility of using plants as natural preservatives is
achievable and are capable to killing microorganisms that are responsible for deterioration
of food product.
3
1.2 Statement of the Problem
Injera and wot (traditional Ethiopian stew) are two common traditional foods which
consumed by millions of Ethiopians (both in urban and rural areas), in many cases, the
most important food items. The two or three meals of Ethiopians eaten a day are usually
meals of injera and wot (Kebede, 1992).
The most prevalent problems facing to injera consumption is its short shelf life and thereby
becoming unfit for consumption. This problem is due to the growth of moulds and other
microorganisms after baking of injera. In most cases, spoiled injera will be dried and used
as a food which is called firfir. But during drying it loses its freshness as well as it will be
deteriorated by insects, microorganisms and dust. In addition to this, during heating the
toxic substance may be produced.
The other option is using chemical preservatives. Even though chemical preservatives
have been introduced to extend the shelf life of injera up to 10 to 12 days, the chemicals
used for preservation cause health problems and the negative reaction to chemical
preservatives in our society is strongly increasing and refuse foods prepared with chemical
preservatives.
Therefore, actions must be taken to control this problem. One of the best alternatives to
extend the shelf life of injera is by using natural preservatives that have antimicrobial and
antifungal properties. Natural plant extracts as preservatives help to produce a food product
that are safe and preferably free of synthetic additives.
Keeping in view of this fact, this study aims to find out the antimicrobial activity of three
spices including Garlic (Allium sativum), Cinnamon (Cinnamomum zylancium, family
Lauraceae), and clove against pathogenic fungal species were evaluated for their effects on
preserving Ethiopian traditional bread, injera.
4
1.3 Objectives of the Study
1.3.1 General Objective
The general objective of this research is to evaluate the effect of selected natural
preservatives on the shelf life of injera.
1.3.2 Specific Objectives
✓ To investigate the effect of concentration of garlic, Cinnamon, and clove extract
(aqueous and ethanolic extract) on the shelf life of injera.
✓ To evaluate the synergetic effect of temperature and natural preservatives on the shelf
life of injera.
✓ To determine the effect of concentration of garlic, cinnamon, and clove extract
(aqueous and ethanolic) on the sensory quality of injera.
1.4 Scope and Limitation of the Study
The scope of this study is to obtain spice extract from garlic, cinnamon and clove by using
aqueous and ethanolic extraction and to investigate the effect of these spices extract on the
shelf life of injera by storing at different temperature (Ambient temperature and 30oC).
Total plate count and yeast and mould count were used to investigate the microbial analysis
of injera. Moreover, the sensory quality of injera prepared from teff flour by adding
different Spice extract as natural preservatives, were analyzed using five-point hedonic
scale for sensory attributes of eye size and distribution, color, sweetness, sourness,
bitterness, flavor and overall acceptability. Due to the limitation of time only three spices,
two temperature and total plate count and yeast and mould count for microbial analysis
were conducted.
1.5 Significance of the Study
Extending the shelf life of injera by using natural preservatives will significantly affect all
injera makers and consumers of injera. It helps to save energy(fuel) for preparation of
injera by avoiding injera preparation every third days and minimize the work load of
women. In addition, it can also minimize the amount of discarded injera due to spoilage.
Consequently, this study benefits most of the community in general and women, injera
maker and distributer, cafeteria and hotel owner and all injera consumer in particular.
5
2. LITERATURE REVIEW
2.1 Introduction
Majority of the Ethiopian population are dependent on teff (Eragrostis tef (Zucc) trotter)
flour to make injera, a staple food in Ethiopia, although injera could be made from different
cereals (Abraha and Abay, 2017). Even though, the yield potential of the crop is low and
the price of tef is high (Abraha and Abay, 2017), in the northern Ethiopian highlands and
around Addis Ababa, injera is traditionally and preferentially made from the flour of teff ,
but regionally, wheat, barley, sorghum, millet, maize and rice are all incorporated (Cherie
et al., 2018).
About 66% of Ethiopian nutrition covers of injera and it accounts for about two-third of
the daily protein consumption of the Ethiopian population (Mulaw and Tesfaye, 2017).
Injera is also considered as good source of energy, fiber, iron, calcium and vitamins
although the fermentation process during preparation results in significant reduction of
most of the nutrients found in the cereals flour, due to the interactions of nutrients with
antinutritional factors resulting in insoluble complexes with reduced bio-accessibility of
nutrients through binding and entrapment thereby limiting their release from food matrices
(Cherie et al., 2018).
Teff is an indigenous and major food crop in Ethiopia and Eritrea. The whole grain is
ground into flour that can be used as a base ingredient for leavened flatbreads such as
injera, added as a thickening agent to soups and sauces, fermented to make beer and ethnic
beverages, or made into porridge and puddings(do Nascimento et al., 2018). The principal
use of teff grain for human food is the Ethiopian bread injera, a soft porous thin pancake
with a sour taste (Ketema, 1997).
Teff has the largest part of area (23.42%, 2.6 million hectares) under cereal cultivation and
third (after maize and wheat) in terms of grain production (18.57%, 29.9 million quintals)
in Ethiopia (Mulaw and Tesfaye, 2017). Besides, the crop can grow well in moisture stress
and waterlogged conditions; can also grow from sea level up to 2800m, under various
rainfalls, temperature and soil conditions (Ketema, 1997).
6
Interest in teff has increased noticeably due to its very attractive nutritional profile. The
composition of teff shows that it has good mineral content(especially calcium and iron)
and generally higher amount of the essential amino acids (Gebremariam et al., 2014).
Teff has a similar protein content to other more common cereals like wheat. The average
crude protein content of teff is in the range of 8 to 11 percent, similar to other more common
cereals such as wheat. Teff’s fractional protein composition suggests that glutelins (45
percent) and albumins (37 percent) are the major protein storages, while prolamins are a
minor constituent (~ 12 percent) (Adebowale et al., 2011). Teff’s amino acid composition
is well-balanced and contains relatively higher concentrations of lysine, a major limiting
amino acid in cereals, than what is commonly found in other cereals. Similarly, compared
to other cereals, higher contents of isoleucine, leucine, valine, tyrosine, threonine,
methionine, phenylalanine, arginine, alanine, and histidine are found in teff (Baye, 2014).
Teff, perhaps the smallest cereal grain in the world, is composed of complex carbohydrates
(make up 80 percent of the teff grain) with slowly digestible starch, consequently has a low
glycemic index (GI) (a measure of how quickly a food causes our blood sugar levels to
rise). It has a starch content of approximately 73 percent, making teff a starchy cereal
(Baye, 2014). As Bultosa (2007) reported, the amylose content of 13 teff varieties tested
ranged from 20 to 26 percent, comparable to other grains, such as sorghum.
Baye (2014) investigated that teff is a comparatively good source of essential fatty acids
and phytochemicals, such as polyphenols and phytates. The crude fat content of teff is
higher than that of wheat and rice, but lower than maize and sorghum. Teff grains are rich
in unsaturated fatty acids, predominantly oleic acid (32.4 percent) and linoleic acids (23.8
percent). Furthermore, the high dietary fiber along with the relatively good concentration
in phytochemicals makes teff a good contender for a functional food for health promotion
and disease prevention.
There are different varieties of teff that vary in color from light to dark. The three most
common tef colors are white, brown and red tef. Injera from white teff is most preferred
(Cherie et al., 2018).
7
2.2 Health Benefits of Teff and Teff-Based Products
Since food fortification and nutritional supplements can’t be done without side-effects,
adjusting iron intakes with iron-rich foods may be preferred. Teff can be a good alternative
for supplementing of iron to lower iron-deficiency, which is the most widespread
micronutrient deficiency, affecting more than 2 billion people by retarding growth,
impairing mental and psychomotor development, child and maternal morbidity and
mortality, and decreasing immunity and work performance (Baye, 2014).
Alaunyte et al. (2012) showed that by supplementing wheat bread with 30 percent teff
flour, the iron content of the bread is more than doubled. By assuming an average daily
consumption of 200g of teff-enriched bread, it is possible to cover between 42 and 81
percent and 72 and 138 percent of daily intake requirements for iron in women and men,
respectively.
The bioavailability of iron in teff is likely to vary depending on processing. For instance,
during injera fermentation, significant decreases in phytate content results in an ideal
phytate to iron molar ratio. If the bioavailability of iron in teff can be confirmed, teff can
be a very good ingredient for celiac patients not only due to the absence of gluten, but also
for its high iron content (Baye, 2014).
Teff is also a good alternative for population affected by celiac disease (CD) or allergies
find the benefits of a gluten free diet, caused by abnormal Tcell responses to glutens and
gluten-like proteins found in wheat, barley, rye and possibly oats, with a symptom of
diarrhea, abdominal pain, and disturbances in nutrient absorption caused by histological
alterations of the small bowel. (Baye, 2014; Minten et al., 2018).
The only treatment for those with CD available to date is to follow a strict gluten-free diet.
This in practice is difficult, given the abundance of food products containing wheat or other
gluten-containing cereals. Consequently, inadequate intakes of essential nutrients such as
folate and vitamin B12, calcium, iron, and fiber have been observed in those with CD.
Also, a higher percentage of energy intake in such patients was found to be from fat instead
of carbohydrates (Niewinski, 2008),this has a negative impact on their nutritional status.
Therefore, nutrient dense gluten-free alternatives are needed and teff is the best alternative.
8
The other health related benefit of teff is, the high fiber content of the grain which is
particularly important in dealing with diabetes and assisting in blood sugar control.
Several features of teff suggest that its consumption may prevent or control diabetes,
which is increasing alarmingly and has become a major public health problem (Zimmet et
al., 2001). Diets high in whole grains have been associated with a 20 to 30 percent
reduction in the risk of developing type 2 diabetes. Given that, teff is consumed as a whole
grain, similar effects can be expected from the consumption of teff (Baye, 2014).
Related to its small size, the grain cannot be separated into germ, bran and endosperm to
create a variety of other products and this allows teff to yield much higher fiber content
than other grains and makes tef flour high in nutrient value because the bran and germ are
the most nutritious parts. (Piccinin, 2002).
Among macronutrients, the type of carbohydrate and its digestibility play a central role in
glucose levels after eating, and hence on the risk to diabetes. Relative to wheat, teff has a
low glycemic index and thus better suited for diabetic patients. In addition, the relatively
high dietary fiber in teff relative to other common cereals, can decrease fasting blood
glucose levels and, thus, contribute to the prevention and management of diabetes. The
condition of impaired antioxidant status and inflammation have been linked to the
development of insulin resistance and type 2 diabetes. In this regard, the high phytate and
polyphenol content in teff and the associated anti-oxidative property is likely to prevent
and control diabetes (Baye, 2014).
In general, the health benefits of teff grain include its ability to help with weight loss,
increase circulation, improve bone health, support proper growth and development, boost
the immune system, aid people with Celiac disease, manage diabetic symptoms, optimize
digestion, and increase heart health. Therefore, injera prepared from teff flour is also
considered as good sources of energy, fiber, iron, calcium and vitamins(Cherie et al.,
2018).
9
2.3 Processing of Injera
Cereal grains had been one of man’s earliest sources of food. One way of processing the
grains into food is through fermentation. The fermentation process of teff injera takes about
three days, depending on ambient temperatures. Temperature in the highlands of Ethiopia
is generally between 17 and 250c. (Gashe, 1985).
As shown in figure 2.1, teff injera is prepared at household by mixing 1 kg of teff flour
with 1.5L of clean water (w/w) and 16% of starter (homemade ersho) by the weight of the
flour and kneaded by hand in a bowl in the traditional way. Then, the resultant dough is
allowed to ferment for 3 days at ambient temperature. After this primary fermentation, the
surface water (an acidic yellowish liquid on the surface of the dough) formed on the top of
the dough is discarded, to minimize the sour test of injera. For every 1kg of original flour,
200ml of the fermented mixture is mixed with 400 ml of water and brought to boil
(traditionally known as ‘absit’ (a dough enhancer) making, which ensures that injera will
have the proper texture and consistency and the dough-rising and gas formation processes
are enhanced so they occur in a short time (Mezemir, 2015)). Then the ‘absit’ is cooled to
about 450C before it has been added into the main part of the dough. The main dough is
then thinned by adding water equal to the original weight of the flour and stirred for 15
min. The batter is left covered for 2h for secondary fermentation. After 2 hours, the absit
is added to the thinned dough and mixed very well (known as batter making). The batter is
left for about 30 min to rise (the second fermentation), before baking commenced. Some
more water is added to thin down and form the right batter consistency. Finally, about half
a liter of batter is poured onto the hot clay griddle in a circular motion from the outside,
working towards the center. After 2 to 3 min of cooking using traditional baking equipment
(metad), the injera is removed and stored in a traditional basket container messob (Mulaw
and Tesfaye, 2017).
10
Mixing
INJERA
Baking
Teff flour
Knead to form
dough Discard the
surface water
Allow to ferment for 3 days at ambient Temperature
(Primary fermentation)
Allow standing for 2h at ambient
temperature
(Secondary fermentation)
Add water
Water (1:1.5 (w/w))
Mixing
16 % (w/w) of
starter
(homemade ersho)
200ml Dough
Kg of flour
400ml Water
Kg of flour
Boiling
Absit
cooling to 45oc
Mixing
Figure 2.1 Flow diagram for the preparation of injera
11
2.4 Spoilage and Preservation of Injera
Injera spoilage can be defined as any sensory change (tactile, visual, olfactory or flavor)
which the consumer considers to be unacceptable (Rawat, 2015). Spoilage, or other
changes that lead to loss of shelf life, may occur at any of the many stages along food
processing, between the acquisition of raw materials and the eventual consumption of a
finished product. 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
(Gould, 1996).
Injera spoils due to the growth of yeast and moulds and other microorganisms after baking
of injera, which gives undesirable flavor and color for injera, and becoming unfit for
consumption. Deterioration of foods is a result of several factors such as insect damage,
physical damage, indigenous enzyme activity in the animal or plant tissue or by microbial
infections (King, 2009; Rawat, 2015).
Microorganisms are thus the most important cause of deterioration of food, especially in
injera is activity and growth of microorganisms. The kinds and numbers of microorganisms
that are present in food depend primarily on the type of food and degree of contamination.
Therefore, preservation of foods is primarily connected with prevention or reducing of
activity of microorganisms (King, 2009).
2.4.1 Factors that Affect Microbial Growth on Injera
The type of bacterial flora developed in each fermented food depends on intrinsic factors
such as water activity, pH, salt concentration, availability of oxygen, composition of the
food matrix, and extrinsic factors such as temperature, relative humidity and other
parameters (Ashenafi, 2006).
2.4.1.1 Intrinsic Parameters
The parameters of plant and animal tissues that are an inherent part of the tissues are
referred to as intrinsic parameters. These parameters are pH, Moisture content, Oxidation-
reduction potential (Eh), Nutrient content, Antimicrobial constituents and Biological
structure (Hamad, 2012; Jay, 2000).
12
A) PH
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 moulds and yeasts, with the pathogenic bacteria being the most
fastidious (Jay, 2000).
B) Moisture Content
Microorganisms need free water to grow in food products. The control of the moisture
content in foods is one of the oldest exploited preservation strategies. The preservation of
foods by drying is a direct consequence of removal or binding of moisture, without which
microorganisms do not grow. Food microbiologists generally describe the water
requirements of microorganisms in terms of the water activity (aw) of the food or
environment. Water activity is defined as the ratio of water vapor pressure of the food
substrate to the vapor pressure of pure water at the same temperature (Moral et al., 2017).
Jay (2000) described that water activity is related to relative humidity (RH) in the following
way: RH = 100 × aw. Pure water has an aw of 1.00, a 22% NaCl solution (w/v) has an aw
of 0.86, and a saturated solution of NaCl has an aw of 0.75.
Most fresh foods, such as fresh meat, vegetables, and fruits, have aw values that are close
to the optimum growth level of most microorganisms (0.97 - 0.99). In general, bacteria
require higher values of aw for growth than fungi, with Gram-negative bacteria having
higher requirements than Gram positives. Most spoilage bacteria do not grow below aw =
0.91, whereas spoilage moulds can grow as low as 0.80. Just as yeasts and moulds grow
over a wider pH range than bacteria, the same is true for aw. The lowest reported value for
foodborne bacteria is 0.75 for halophiles (literally, “salt-loving”), whereas xerophilic
(“dry-loving”) moulds and osmophilic (preferring high osmotic pressures) yeasts have
been reported to grow at aw values of 0.65 and 0.61, respectively The aw can be manipulated
in foods by a number of means, including addition of solutes such as salt or sugar, physical
removal of water through drying or baking, or binding of water to various macromolecular
components in the food (Jay, 2000; Moral et al., 2017).
13
C) Oxidation–Reduction Potential
It has been known for decades that microorganisms display varying degrees of sensitivity
to the oxidation–reduction potential (Eh) of their growth medium. The oxidation–reduction
potential of a substrate may be defined generally as the ease with which the substrate loses
or gains electrons. When an element or compound loses electrons, the substrate is oxidized,
whereas a substrate that gains electrons becomes reduced. Therefore, a substance that
readily gives up electrons is a good reducing agent, and one that readily takes up electrons
is a good oxidizing agent. Aerobic microorganisms require positive Eh values (oxidized)
for growth, whereas anaerobes require negative Eh values (reduced). With respect to Eh
requirements of microorganisms, some bacteria require reduced conditions for growth
initiation (Eh of about −200 mV), whereas others require a positive Eh for growth (Jay,
2000).
D) Nutrient Content
Microorganisms require certain basic nutrients for growth and maintenance of metabolic
functions. The amount and type of nutrients required range widely depending on the
microorganism. These nutrients include water, a source of energy, nitrogen, vitamins, and
minerals (Jay, 2000; Moral et al., 2017).
E) Antimicrobial Constituents
The stability of some foods against attack by microorganisms is due to the presence of
certain naturally occurring substances that possess and express antimicrobial activity.
Some plant 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 (Jay, 2000).
F) Biological Structures
The natural covering of some foods provides excellent protection against the entry and
subsequent damage by spoilage organisms. The biological structure of the testa of seeds,
the outer covering of fruits, the shell of nuts, the hide of animals, and the shells of eggs is
14
sufficient to prevent the entry of all organisms (Jay, 2000).Since injera has no biological
coverage it susceptible for microbial spoilage.
Taken together, these six intrinsic parameters represent nature’s way of preserving food
from microorganisms. By determining the extent to which each exists in a given food, one
can predict the general types of microorganisms that are likely to grow and, consequently,
the overall stability of this particular food (Jay, 2000).
2.4.1.2 Extrinsic Parameters
The extrinsic parameters of foods are not substrate dependent. They are those properties of
the storage environment that affect both the foods and microorganisms in the food. Those
of greatest importance to the welfare of foodborne organisms are temperature of storage,
relative humidity of environment, presence and concentration of gases, and presence and
activities of other microorganisms (Jay, 2000).
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 −34◦C; the
highest reaches 113◦C. It is usual to place microorganisms into three groups based on their
temperature requirements for growth. Microorganisms that grow well at or below 7◦C and
have their optimum between 20◦C and 30◦C are referred to as psychrotrophs. Those that
grow well between 20◦C and 45◦C with optima between 30◦C and 40◦C are referred to as
mesophiles, whereas microorganisms that grow well at and above 45◦C with optima
between 55◦C and 65◦C are referred to as thermophiles (Jay, 2000).
According to Ashagrie and Abate (2012) microorganisms that are responsible for injera
spoilage grow in a temperature range of 25-320C for Aspergillus niger ,and 16-20oC for
Penicillium sp & Rhizopus sp. In general, injera spoiling microorganisms can be
categorized as psychrotrophs and mesophiles.
Just as moulds are able to grow over wider ranges of pH, osmotic pressure, and nutrient
content, they are also able to grow over wide ranges of temperature. Which indicates that
to keep the nutritional value of injera, storage temperature must be seriously taken into
account. Although, it would seem desirable to store all foods at refrigerator temperatures
15
or below, this is not always best for the maintenance of desirable quality in some foods
(Jay, 2000).
B) Relative Humidity of Environment
The RH of the storage environment is important both from the standpoint of aw within
foods and the growth of microorganisms at the surfaces. When the aw of a food is set at
0.60, it is important that this food be stored under conditions of RH that do not allow the
food to pick up moisture from the air and thereby increase its own surface and subsurface
aw to a point where microbial growth can occur. When foods with low aw values are placed
in environments of high RH, the foods pick up moisture until equilibrium is established.
Likewise, foods with a high aw lose moisture when placed in an environment of low RH.
There is a relationship between RH and temperature that should be considered in selecting
proper storage environments for foods i.e. the higher the temperature, the lower the RH,
and vice versa(Jay, 2000). Foods, such as injera that undergo surface spoilage by moulds,
yeasts, and certain bacteria should be stored under conditions of low RH for better shelf
life.
C) Presence and Concentration of Gases in the Environment
Carbon dioxide (CO2) is the single most important atmospheric gas that is used to control
microorganisms in foods. It along with O2 are the two most important gases in modified
atmosphere packaged (MAP) foods (Jay, 2000).
Ozone (O3) is the other atmospheric gas that has antimicrobial properties, and it has been
tried over a number of decades as an agent to extend the shelf life of certain foods. It has
been shown to be effective against a variety of microorganisms, but because it is a strong
oxidizing agent, it should not be used on high-lipid-content foods since it would cause an
increase in rancidity (Jay, 2000).
D) Presence and Activities of Other Microorganisms
Some foodborne organisms produce substances that are either inhibitory or lethal to others;
these include antibiotics, bacteriocins, hydrogen peroxide, and organic acids (Jay, 2000).
16
2.4.2 Methods of Preservation and Extension of Shelf Life of Injera
Injera preservation is an action or a method of maintaining chemical and biological process
of foods at a desired level of properties or nature for their maximum benefits by regulating
the pH value and settling the redox potential of the food. Moreover, it is also used as the
preservatives that hinder the development of the microbial in the food product or to avoid
food spoilage (Rahman, 2007; Sharif et al., 2017).
Each step of handling, processing, storage, and distribution affects the characteristics of
food, which may be desirable or undesirable. The preservation process will restrain the
development of microbes such as bacteria and fungi. In this light, the aims of food
preservations include maintaining sensory quality and nutritional value, to reduce the
wastage of food, to maintain a product’s accessibility for a longer time, and to preserve the
food materials during transportation, and finally, to ease the handling of food materials
(Rahman, 2007; Sharif et al., 2017).
There are various methods that can be used to preserve food, including conventional
methods and modern preservation technology. Sharif et al. (2017) listed that some of the
preservation methods includes thermal or heat processing, drying, pickling (preservation
in common salts or vinegar), freezing, high-pressure processing, edible coating technique
and so on.
Food preservatives are divided into two classes natural preservatives and artificial or
chemical preservatives (Synthetic preservatives). These preservatives are further classified
as antimicrobial and antioxidant agents (Sharif et al., 2017).
Extending shelf life of injera by using chemical preservatives (Synthetic preservatives),
was studied by Ashagrie and Abate (2012) and good results were observed as extending up
to 10 to 12 days. But, extending shelf life of injera by using natural preservatives was not
previously conducted.
17
2.4.3 Anti-Microbial Effect of Some Natural Preservatives
Some plant, typically spices can be used as a source of natural antimicrobial agents
because, it has lots of important bioactive antimicrobial agent for preservation. In addition
to this, they have high demand by consumers because, they have been exposed and aware
about the effect of using synthetic preservatives. Moreover, compounds that derived from
natural sources contain phenolic, terpenes and alkaloid. These compounds usually present
in certain parts or all parts of the plant (Sharif et al., 2017).
Spices have been defined as plant substances from indigenous or exotic origin, aromatic or
with strong taste, used to enhance the taste of foods. Spices include leaves (bay, mint,
rosemary, coriander, laurel, oregano), flowers (clove), bulbs (garlic, onion), fruits (cumin,
red chilli, black pepper), stems (coriander, cinnamon), rhizomes (ginger) and other plant
parts. Although, spices have been well known for their medicinal, preservative and
antioxidant properties, they have been currently used with primary purpose of enhancing
the flavor of foods rather than extending shelf-life.
The antibacterial activity of spices may differ between strains within the same species.
Moreover, the antimicrobial properties of spices may differ depending on the form of
spices added, such as fresh, dried, or extracted forms and also differ depending on the
harvesting seasons and between geographical sources. However, Hoque et al. (2008)
indicates that the essential oils of spices are more strongly antibacterial than is accounted
for by the additive effect of their major antimicrobial components; minor components to
play a significant role.
2.4.3.1 Anti-Microbial Effect of Garlic (Allium Sativum)
Garlic is one of the most popular of the herbs and spices for many reasons. One is the flavor
it adds to dishes and the other is the health benefits it offers. Allium genus has over 500
members, each differing in maturing, color and taste, but with similar biochemical,
phytochemical and nutraceutical content (Irkin and Korukluoglu, 2007).
Garlic is a large annual plant of the Liliaceae family, which grows in most of Africa and in
Ethiopia. Ethiopian garlic is used in traditional medicine for infectious disease and some
other cases. They are generally used to inhibit yeast and mould growth, being also effective
against a wide range of bacteria (Pundir and Jain, 2010).
18
Figure 2.2 Image of Raw Garlic
There is an extensive literature on the antibacterial effects of fresh garlic juice, aqueous
and alcoholic extracts, lyophilized powders, steam distilled oil and other commercial
preparations of garlic. The antibacterial effects of garlic and other allium vegetables more
recently, were studied by Daka (2011).
Diseases that may be helped or prevented by garlic’s medicinal actions include
Alzheimer’s disease, cancer, cardiovascular disease (including atherosclerosis, strokes,
hypertension, thrombosis and hyperlipidemias) children’s conditions, dermatologic
applications, stress, and infections. Some researches point to possible benefits in diabetes,
drug toxicity, and osteoporosis.
Garlic (Allium sativum) was revered to possess anti-viral, anti-bacterial, anti-fungal
activities and include the powerful antioxidants, sulfur and other numerous phenolic
compounds which arouse significant interests (Irkin and Korukluoglu, 2007).
Abdel-Hafez and El-Said (1997) analyzed the effect of garlic and onion extract on the
mycoflora of pepper, cinnamon and rosemary and reported effectiveness of garlic extract
up to 0.25% (v/v) to inhibit the growth of Aspergillus flavus, A. fumigatus, A. niger, A.
ochraceus, A. terreus, Penicillium chrysogenum, P. puberulum, P. citrinum, P.
corylophilum, Rhizopus stolonifer, Stachybotrys chartarum, Eurotium
chevalieri and Emericella nidulans. Onion extract at all assayed concentrations (0.625,
0.250 and 0.125% v/v) did not present significant inhibitory effect when compared to the
control assay.
19
2.4.3.2 Antimicrobial Activity of Cinnamon (Cinnamomum Zylancium)
Cinnamon is a spice tree containing several bioactive compounds that can be used against
a wide range of microorganisms. Cinnamon bark crude extract has constantly been reported
to have antifungal activity. This activity was attributed mainly to the presence of
cinnamaldehyde and eugenol compounds. Eucalyptus as well contains several chemical
compounds that play several roles in the plant such as defense against insect, vertebrate
herbivores and protection against UV radiation and against cold stress. Both cinnamon
barks and eucalyptus leaves represent important source of compounds like flavonoids,
tannins, glycosides, saponines, alkaloids and essential oils with biological activities such
as bacteriostatic, fungistatic and anti-inflammatory. Terpenoids, which form most of the
essential oil giving eucalyptus foliage its characteristic smell. Cinnamon and Eucalyptus
species possess a strong antimicrobial potential and their volatile oils are used as
antibacterial and antifungal agents in creams, soaps and toothpastes (Irkin and
Korukluoglu, 2007; Mahmoud, 2012).
Figure 2.3 Image of Cinnamon
The essential oil is primarily composed of 65% to 80% cinnamaldehyde and lesser amounts
of other phenols and terpenes, including eugenol, trans-cinnamic acid, hydroxy
cinnamaldehyde, o-methoxy cinnamaldehyde, cinnamyl alcohol and its acetate, limonene,
α-terpineol, tannins, mucilage, oligomeric procyanidins, and trace amounts of coumarin.
Differing material origins and extraction techniques are reported to alter the chemical
composition of the extracts, and hence may impact the intended medicinal (and
experimental) effects (Irkin and Korukluoglu, 2007).
20
Cinnamon extracts have been shown to exert in vitro activity against some common human
pathogens and fungicidal activity against plant pathogens. In vitro inhibition of bacterial
endotoxin has been demonstrated by an unidentified component in cinnamon bark. The
essential oils of cinnamon were shown to halt mycelial growth and aflatoxin synthesis
in Aspergillus parasiticus at a concentration of only 0.1% (Mahmoud, 2012).
2.4.3.3 Antimicrobial Activity of Cloves (Syzygium aromaticum)
Cloves are the aromatic flower buds of a tree in the family Myrtaceae, Syzygium
aromaticum and are commonly used as a spice. This plant represents one of the richest
sources of phenolic compounds such as eugenol, eugenol acetate and gallic acid and
possess great potential for pharmaceutical, cosmetic, food and agricultural applications. Its
essential oil extracts kill many Gram positive and Gram-negative bacteria including some
fungi. The antimicrobial activity of clove is attributable to eugenol, oleic acids and lipids
found in its essential oils.
Clove has been used medicinally in the field of oriental herbal medicine and as a culinary
spice. Plant's flower bud is used for both flavoring and from which the essential oil is
extracted. The characteristics of its essential oil is volatile nature and strong aromatic odors.
Therefore, the essential oil of clove has been widely used in aromatherapy and cosmetics.
It has been also used as a natural anesthetic in dentistry. Direct application of essential oil
of clove was effective against Aspergillus parasitica, Candida albicans, and Cryptococcus
neoformans (Chee and Lee, 2007).
The work by Pandey et al. (2013) shows that methanol extracts of spices (clove, ajwain,
turmeric, black pepper and dalchini) given high antifungal activity against different fungi
(Aspergillus niger and Trichoderma sp).
Figure 2.4 Image of Clove Buds
21
3. MATERIALS AND METHODS
3.1 Raw Materials Used
The main raw materials used in this study, for preparation of injera are teff flour, water
and homemade ersho (used as a source of starter culture to initiate fermentation). Garlic,
clove and cinnamon were used as a natural preservative for extending the shelf life of
injera.
3.2 Chemicals and Equipment Used
In order to accomplish this research, the following main chemicals and instruments were
used as shown in table 3.1.
Table 3.1 Chemicals and equipment used
Name of Chemical or equipment Uses
Dough mixer For mixing of teff flour, water, ersho and other ingredients.
Heating oven For drying of spices.
Heating incubator For the growth of microorganisms
Autoclave For sterilization of laboratory equipment and media
Colony counter For counting of microbial colony
Storage(mesobe) For storing of injera under acceptable condition
Electric stove (mitad): For baking of injera
Potato Dextrose Agar (PDA) For yeast-moulds growth.
Plate Count Agar (PCA) For total aerobic bacteria growth.
Maximum Recovery Diluent For dilution of sample.
22
3.3 Preparation of Injera and Addition of Preservatives
The teff injera samples were prepared at Bahir Dar University in food technology and
process lab following the same way as that of the traditional method explained under
section 2.3. As shown in figure 3.1 below, once the batter was prepared the aqueous and
ethanolic extract of garlic, clove, cinnamon and blend of these three spices, were added at
different concentrations (2%,5%, and 10%) immediately before baking. Finally, injera was
baked for each sample and stored in a traditional basket container called messob for further
analysis.
Figure 3.1 Flow diagram for the preparation of injera and addition of natural preservatives
23
3.4 Preparation of Spice Extract
Fresh garlic, cinnamon, and clove were purchased from a retail food store at Bahir Dar
market. Then, the freshly collected spices were cleaned or thoroughly washed with tap
water followed by sterile distilled water. The spices were dried in an oven at 50°C for 4
hours followed by grinding in to powder. Then the powdered materials were stored in air
tight jars in refrigerator at 4°C. Distilled water and 97% ethanol were used as a solvent for
preparation of spice extract and a total of six extracts (three aqueous and three ethanolic)
were prepared from the three spices.
The extract preparation was done according to Pundir and Jain (2010); for both aqueous
and ethanolic extraction, 100 g of powdered plant materials were dissolved in 300 ml
sterilized distilled water and 300ml of 97% ethanol, respectively . Then the mixture was
allowed to settle at room temperature for 24 hours in a sterile flask covered with aluminum
foil to avoid evaporation and then was filtered through muslin cloth. After filtration, the
extracts were ready to use as preservatives.
3.5 Sensory Quality Analysis of Injera with Natural Preservatives
The prepared injera was stored in mesob (traditional storage facility made of woven grass
straw) by cutting/slicing into quarter of the whole injera with sharp and neat knife to
prevent contamination among different injera samples.
Ghebrehiwot et al. (2016) suggested that the sensory evaluation of injera should be
conducted within two hours after baking, and in this research the sensory acceptability of
injera prepared from teff flour with aqueous and ethanolic extract of garlic, cinnamon,
clove and combination of these three spices, at three different concentration ( 2%, 5% and
10%) including the control were evaluated by using 10 panelists after two hours of baking.
All the panelists are frequent consumers of injera and the age range of the panelists were
20-45 in order to fill the score card properly.
24
At the beginning of sensory evaluation, the panelists were given an instruction and
explanation about the objective of the research and how they could fill the score card based
on their sensory evaluation. Then the panelists were allowed to evaluate the different
sensory attributes of injera which are eye size and distribution, color, flavor, sweetness,
bitterness, sourness and overall acceptability. Once the panelists filled the questioner, their
result were analyzed by using Microsoft excel 2016 and Statistical Package for Service
Solution (SPSS version 20).
Since the panelist were not fully trained, to make the analysis consistent, a simple five-
point hedonic scale (1=dislike extremely, 2=dislike, 3=neither like nor dislike, 4= like, 5=
like extremely) were used for each sensory attribute.
3.6 Microbiological Analysis of Injera with Natural Preservatives
The microbial analysis, i.e. total plate count and yeast-mould count were conducted
according to Bacteriological Analytical Manual (BAM). Total aerobic plates count was
determined by pour plate technique and yeasts-moulds count was determined by spreading
technique after suspending 10g injera samples in 90mL of maximum recovery. Serial
dilutions of the suspensions up to 10-5 dilution were prepared by using maximum recovery
and from 10-4 and 10-5 dilution, 1mL and 0.1 mL samples were transferred into a separate
duplicate sterile petri plate for total plate count (pouring technique) and yeast-mould count
(spreading technique), respectively.
For yeasts-moulds counts, Potato Dextrose Agar (PDA) was aseptically poured into the
plates which contains 0.1 mL samples and after incubation aerobically at 25°C for 3 to 5
days yeast-moulds were enumerated on plates bearing 30 to 300 colonies using colony
counter as colony forming units/g of injera (cfu/g). For total aerobic plate count the Plate
Count Agar (PCA) was used and incubated aerobically at 30°C for 72 h and the total
aerobic bacteria were enumerated on plates bearing 20 to 200 colonies using colony counter
as cfu/g (Girma et al., 2013).
25
3.7 Experimental Design
Completely randomized design (CRD) were used for the experimental design for the
sensory and microbial analysis. The effect of the preservatives namely garlic, cinnamon,
clove and their blend at concentrations of 2%,5%, and 10% and extraction methods i.e.
aqueous and ethanolic extraction on the shelf life and sensory quality of injera were
designed as shown in table 3.1 below. As stated under section 3.2, once the spice extracts
were prepared by using aqueous and ethanolic extraction method, to see the combination
effect of selected spices, equal amount by volume of garlic, cinnamon and clove of each
extraction methods were blended separately, and at the end blend of aqueous and blend of
ethanolic extract were obtained. Effect of temperature (Ambient temperature and 30oC) on
the shelf life of injera were also included in the experimental design.
Table 3.2 Experimental Design
Temperature
Ambient Temperature 30oC
Concentration (%) Garlic Cinnamon Clove Blend Garlic Cinnamon Clove Blend
2 1R 2R 3R 4R 1X 2X 3X 4X
5 1S 2S 3S 4S 1Y 2Y 3Y 4Y
10 1T 2T 3T 4T 1Z 2Z 3Z 4Z
0 control control
Therefore, the total number of runs=4(3 Spices+1 blend) *2(Temp)*3(Conc)*2(solvent)
+ 1(control)*2(Temp)
=50 runs
26
4. RESULTS AND DISCUSSION
4.1 Spice Extract
After 24 hours of soaking at ambient temperature and removal of the precipitate by filtering
in muslin cloth, the extracts were obtained with different color and flavor, and the color is
shown in figure 4.1 below. The different extraction method i.e. aqueous and ethanolic
extraction methods had different effects on different spices. From aqueous and ethanolic
extraction methods, ethanol extraction gives clear extract with better settlement of the mud
while, the aqueous extraction produces extract with unsettled dispersion.
Figure 4.1 Aqueous and Ethanolic extract of different Spice
Ethanolic Extract
of Clove Ethanolic Extract
of Garlic Ethanolic Extract
of Cinnamon
Aqueous Extract
of Cinnamon
Aqueous Extract
of Clove Aqueous Extract
of Garlic
27
4.2 Sensory Quality of Injera with Different Natural Preservatives
Sensory evaluation is defined as the examination of a product (e.g., foods and beverages)
through the evaluation of the attributes traceable by one or more of the five human senses
i.e. taste, smell, touch, sight, and hearing. It is used in food science to objectively analyze
food quality. In many cases, it is an indispensable tool because it allows for the objective
determination of whether or not consumers will accept a novel food product (Ghebrehiwot
et al., 2016).
The average sensory quality of injera prepared from teff flour and different preservatives
at different concentration were recorded in table 4.1 below.
In addition to the mean value, as the Analysis of Variance (ANOVA) table in the Appendix
B shows that, there is a significant difference of concentration of spice extract, type of
spice, and type of solvent, on the sensory attributes of injera which are eye size and
distribution, color, flavor, sweetness ,bitterness, sourness, and overall acceptability of
injera. Exceptionally, spice extract has no significant effect on sweetness and sourness,
that of type of spice extract has no significant effect on sourness. As indicated in the
ANOVA table, the interaction effect of concentration and type of spice, concentration and
type of solvent (except for flavor), type of spice and type of solvent (except for eye size
and distribution, color and sweetness), as well as the overall interaction i.e. concentration,
type of spice and type of solvent (except for eye size and distribution) don’t show a
significant difference on the sensory attributes of injera.
28
Table 4.1 Sensory quality of Injera prepared from teff flour and preservatives
Average Sensory Result
Sensory
Parameters
Concentration Control Garlic
Water
Garlic
Ethanol
Cinnamon
Water
Cinnamon
Ethanol
Clove
Water
Clove
Ethanol
Mixed
Water
Mixed
Ethanol
Eye size
Distribution
2%
4.9
4.2 4.4 4.2 3.8 3.6 4.5 3.2 2.1
5% 4.8 3.9 3.8 3.3 4.5 3.8 4.1 4.3
10% 4.4 3 3.6 2.9 3.6 3.9 4 3.6
Color 2%
4.7
4.7 4.7 4.2 3.9 2.8 4.2 3.8 4
5% 4.7 4.4 3.9 3.6 2.2 3.4 2.9 3.8
10% 4.7 4.1 3.1 3 1.4 3.2 2.5 3.5
Flavor 2%
4.4
4.3 3.5 3.9 3.7 3.1 2.6 3.9 3.2
5% 3.1 3.1 3.1 3.3 3.1 2.7 3.3 3.1
10% 3.3 2.6 3.2 2.6 3.1 2 3 2.4
Sweetness 2%
4.0
3.8 3.6 3.5 3 3.4 2.3 3.6 3.5
5% 3.4 3.4 3 2.8 3.6 2.1 3 3.2
10% 3.7 3 3.6 2.8 3.4 1.7 2.9 2.4
Bitterness 2%
4.3
4.1 3.7 3.9 3.7 3.6 2.9 3.9 3.7
5% 3.7 3.8 3.6 3.5 3.2 2.5 3.3 3.3
10% 3.9 3.3 3.6 3.1 2.8 1.3 3.4 2.3
Sourness 2%
4.2
3.5 3.5 3.7 3.2 3.7 2.8 3.4 3.1
5% 3.2 3.1 2.6 2.4 2.9 2.3 2.8 2.7
10% 3.3 3.1 3.3 3.2 3.2 2.1 3.3 2.4
Overall
Acceptability
2%
4.4
4.2 3.7 4 3.6 3.1 2.7 3.4 3.3
5% 3.7 3.7 3.3 3.2 3.3 2.4 3.2 3.3
10% 3.9 3.1 3.4 2.9 2.8 1.8 3.1 2.5
29
4.2.1 Eye Size and Distribution
Eye size and distribution (appearance of injera) is one of the most important quality
parameters, which refers to the quality of the eyes (cells) of the honeycomb-like structure
of the top surface of injera formed during cooking due to escaping CO2 bubble
(Ghebrehiwot et al., 2016). A higher number of larger eyes is a very desirable attribute of
an attractive injera. As shown in table 4.1 and figure 4.2 most of the spice extract added
in to injera as preservatives show good eye quality with the highest eye quality in control
followed by aqueous extract of garlic with 5% addition and the minimum eye quality is
recorded for combination of ethanolic extract of the spices with 2% addition. As shown in
the figure 4.2 below, injera prepared from addition of aqueous extract of garlic, clove and
mixture of spices as well as ethanolic extract of mixture of spice showed maximum eye
size and distribution at 5% concentration and all the others spice added injera showed
maximum eye size and distribution at 2% concentration. This implies that good eye size
and distribution can be observed at low concentrations. There is also a significant
difference between the type of solvent on the eye quality of injera and an aqueous extract
of spice gets more acceptance by the panelists than ethanolic extract.
In addition to this, as the ANOVA table in the Appendix B shows, concentration, type of
spice, type of solvent, as well as interaction of concentration and type of spice, type of
spice and type of solvent, and overall interaction of concentration, type of spice, and type
of solvent have shown a significant effect on eye size and distribution of injera at
confidence interval of 95%.
0
1
2
3
4
5
Control Garlic
Water
Garlic
Ethanol
Cinnamon
Water
Cinnamon
Ethanol
Clove
Water
Clove
Ethanol
Mixed
Water
Mixed
Ethanol
Mea
n v
alue
of
Eye
Siz
e an
d
Dis
trib
uti
on
Type of Preservatives
Eye size and Distribution 2%
5%
10%
Figure 4.2 Eye size and distribution of injera with spice extract at different
concentration and solvent
30
4.2.2 Color
The color of injera also affects the appearance of the injera in relation to its aesthetic
appeal. In areas where injera is consumed as a staple food especially in Eritrea and
Ethiopia, people prefer their injera be white in color (Ghebrehiwot et al., 2016). Most of
the spice extract added in to injera as preservatives have changed the color of injera,
specially the aqueous extract of clove has changed the color of injera in to black color, due
to reddish-black color of clove, which were not acceptable by most panelists, because it
was found to be in contradiction to the common acceptable white color of teff injera. As
shown in table 4.1 and figure 4.3, except for aqueous extract of garlic, all the other spice
added injera, the quality of color decrease significantly as concentration increase from 2%
to 10%. The most preferred color was observed on the injera prepared by adding aqueous
extract of garlic and least preferred color was observed in aqueous extract of clove. In
addition to this, as the ANOVA table in the Appendix B show, concentration, type of spice,
type of solvent, as well as interaction of type of spice and type of solvent, have shown a
significant effect on color of injera at confidence interval of 95%.
0
1
2
3
4
5
Control Garlic
Water
Garlic
Ethanol
Cinnamon
Water
Cinnamon
Ethanol
Clove
Water
Clove
Ethanol
Mixed
Water
Mixed
Ethanol
Mea
n v
alue
of
colo
ur
Type of Preservatives
Colour2%
5%
10%
Figure 4.3 Color of injera with spice extract at different concentration and solvent
31
4.2.3 Flavor
Fermentation process have been renowned by the ability to improve the organoleptic
properties by making different flavors in different foods. As shown in table 4.1 and figure
4.4, the control one scores the highest acceptance for flavor and the minimum score were
observed in injera prepared by adding 10% concentration of ethanolic extract of clove, due
to the presence of eugenol in clove, which is responsible for pungent, bitter, astringent
flavor. As the concentration of spice is increased from 2% to 10% the flavor of spices added
injera gets less acceptance by the panelists. In addition to this, as shown in figure 4.4 there
is a significant difference between aqueous extract and ethanolic extract of the spice and
the ethanolic extract of spice gets less acceptance for flavor by the panelists. Moreover, as
the ANOVA table in Appendix B show, concentration, type of spice, type of solvent, as
well as interaction of concentration and type of solvent have shown a significant effect on
flavor of injera at confidence interval of 95%. In general, most of the natural preservatives
added to injera in this study were found to be acceptable for flavor by panelists, except for
ethanolic extract of clove and ethanolic extract of combination of spices.
0
1
2
3
4
5
Control Garlic
Water
Garlic
Ethanol
Cinnamon
Water
Cinnamon
Ethanol
Clove
Water
Clove
Ethanol
Mixed
Water
Mixed
Ethanol
Mea
n V
alu
e o
f F
lavour
Type of Preservatives
Flavour2%
5%
10%
Figure 4.4 Flavor of injera with spice extract at different concentration and solvent
32
4.2.4 Sweetness
As shown in table 4.1 and figure 4.5, the control one scores the maximum acceptance for
sweetness of injera by the panelists and injera prepared by adding ethanolic extract of
clove as a preservative score the minimum acceptance for sweetness, due to the bitter taste
of eugenol found in clove. In addition to this, there is a significant difference between
aqueous extract and ethanolic extract of the spice added in to injera and the ethanolic
extract of spice scores less acceptance for sweetness by the panelists. Moreover, as the
ANOVA table in Appendix B show, type of spice, type of solvent, as well as interaction of
type of spice and type of solvent have shown a significant effect on sweetness of injera at
confidence interval of 95%.
Figure 4.5 Sweetness of injera with spice extract at different concentration and solvent
0
1
2
3
4
5
Control GarlicWater
GarlicEthanol
CinnamonWater
CinnamonEthanol
CloveWater
CloveEthanol
MixedWater
MixedEthanol
Mea
n v
alue
of
Sw
eetn
ess
Type of Preservatives
Sweetness
2%
5%
10%
33
4.2.5 Bitterness
Addition of spice extract has significant effect on the bitterness of injera and as shown in
table 4.1 and figure 4.6, the control one scores the maximum acceptance by the panelists
and injera prepared by addition of clove as a preservative score the minimum acceptance
for bitterness. The ethanolic extract of clove with 10% addition develops a bitter taste due
to the bitter taste of eugenol found in clove and most panelist dislikes the bitterness of
injera prepared by addition of ethanolic extract of clove. As the concentration of spice
added were increased from 2% to 10% the bitterness of injera were also increased and the
acceptance were decreased. In addition to this as shown in figure 4.6 there is a significant
difference between aqueous extract and ethanolic extract of the spice added in to injera
and the ethanolic extract of spice scores less acceptance for bitterness by the panelists.
Moreover, as the ANOVA table in Appendix B show, concentration, type of spice, and
type of solvent have shown a significant effect on the bitterness of injera at confidence
interval of 95%.
Figure 4.6 Bitterness of injera with spice extract at different concentration and solvent
0
1
2
3
4
5
Control GarlicWater
GarlicEthanol
CinnamonWater
CinnamonEthanol
CloveWater
CloveEthanol
MixedWater
MixedEthanol
Mea
n v
alue
of
Bit
tern
ess
Type of Preservatives
Bitterness
2%
5%
10%
34
4.2.6 Sourness
The major quality attribute of a good injera is its slightly sour taste, which is due to the
acidic (low pH) nature of injera (Ashagrie and Abate, 2012). As shown in table 4.1 and
figure 4.7, the control one showed the maximum acceptance for sourness and the minimum
score were observed in ethanolic extract of clove with 10%, and 5% addition followed by
5% ethanolic extract of cinnamon and 10% addition of a combination of ethanolic extract
of spices. In addition to this as shown in figure 4.7 there is a significant difference between
aqueous extract and ethanolic extract of the spice and the ethanolic extract of spice scores
less acceptance for sourness by the panelists. Moreover, as the ANOVA table in Appendix
B show, type of solvent has shown a significant effect on sourness of injera at confidence
interval of 95%.
Figure 4.7 Sourness of injera with spice extract at different concentration and solvent
0
1
2
3
4
5
Control GarlicWater
GarlicEthanol
CinnamonWater
CinnamonEthanol
CloveWater
CloveEthanol
MixedWater
MixedEthanol
Mea
n v
alu
e o
f S
ou
rnes
s
Type of Preservatives
Sourness2%
5%
10%
35
4.2.7 Overall Acceptability
Overall acceptability refers to the combinations of evaluations by consumers or panelists
of a product. In this study, results showed that there was a statistically significant difference
(P < 0.05) in the overall acceptability of the injera samples prepared by addition of different
spices extract. As shown in table 4.1, the control (without addition of any preservatives)
was the most acceptable with mean value of 4.4, which is consistent with the control value
of Ghebrehiwot et al. (2016) results with mean value of 4.2. Next to control, injera
prepared from teff flour with addition of 2% aqueous extract of garlic and cinnamon scores
the maximum value with mean score of 4.2 and 4, respectivelly. Injera prepared from teff
flour with addition of aqueous and ethanolic extract of clove showed the minimum over all
acceptability. As shown in the figure 4.8, as the concentration of spices increased from 2%
to 10% the overall acceptapility had decreased. There is also a significant difference
between the type of solvent used for preparation of spice on the overall acceptability of
injera and an aqueous extract of spice scores more acceptance by the panelists than the
ethanolic extract of spice. In addition to this, as the ANOVA table in Appendix B show,
concentration, type of spice, and type of solvent have shown a significant effect on over
acceptability of injera at confidence interval of 95%.
Figure 4.8 Over all acceptability of injera with spice extract at different concentration and solvent
0
1
2
3
4
5
Control GarlicWater
GarlicEthanol
CinnamonWater
CinnamonEthanol
CloveWater
CloveEthanol
MixedWater
MixedEthanol
Mea
n V
alu
e o
f o
ver
all
Acc
epta
bil
ity
Type of Preservatives
Overall Acceptability 2%
5%
10%
36
In general, as the sensory quality analysis shows, the selected natural preservatives namely
garlic, cinnamon and clove, have a significant effect on the sensory quality of injera.
Garlic has a pungent smell and its own characteristic flavor, which has a strong, spicy
flavor that mellows and sweetens considerably with cooking. In this study, both aqueous
and ethanolic extract of garlic added in to injera as natural preservatives, gets good
acceptance by the panelists, especially aqueous extract of garlic scores the highest sensory
acceptance than other natural preservatives added in to injera. When a whole garlic bulb is
used, it has very little aroma and when a piece is cut or bruised the allinase is released
giving it the pungent, spicy and mellow smell.
Cinnamaldehyde and eugenol, which are found in cinnamon and clove, respectively had
also showed a significant effect on the sensory quality of injera. Injera prepared by adding
cinnamon as natural preservatives, showed a moderate acceptance especial ethanolic
extract of cinnamon scored a relatively minimum acceptance by the panelists than aqueous
extract of cinnamon, and this may be due the higher solubility of cinnamaldehyde in alcohol
(ethanol) than water, which causes a warm, sweet, spicy odor and pungent taste.
Injera prepared by adding clove as natural preservatives, especially ethanolic extract of
clove scored a minimum acceptance by the panelists, and this may be due to the bitterness,
and astringency flavor of eugenol found in clove. Since eugenol is more soluble in alcohol
(ethanol) than water, the ethanolic extract of clove showed more effect on the sensory quality
of injera than aqueous extract of clove.
From the overall sensory acceptability, even though most panelists preferred injera prepared
from without addition of any natural preservatives (control), an aqueous extract of garlic,
aqueous extract of cinnamon and ethanolic extract of garlic showed a good acceptance by
the panelists at 2% concentration addition with mean value of 4.2,4, and 3.7, respectively.
Moreover, concentration of spice extract, type of spice, and type of solvent had shown a
significant effect (at P ≤ 0.05) on the sensory attributes of injera which are eye size and
distribution, color, flavor, sweetness, bitterness, sourness, and overall acceptability of
injera.
37
4.3 Microbial Quality of Injera with Different Natural Preservatives
4.3.1 Visual observation of Injera spoilage
One of the method in which the shelf life of injera can be determined is by using visual
observation i.e. the day in which mould was visible to the naked eye (Ashagrie and Abate,
2012). The effect of selected natural preservatives with aqueous and ethanolic extraction
with different concentration on the shelf life of injera, were determined by visual
observation and shown in table 4.2 and 4.3, for storage condition of ambient temperature
and 30oc, respectively.
Investigation of the antimicrobial activity of the preservatives tested in this study revealed
that most natural preservatives were effective against injera spoiling microorganisms. This
was shown by the reduction in percentage of mould invasion of the injera samples
containing preservatives as compared to the control.
The shelf life of injera without preservatives (control) was 3 days at ambient temperature
(average temperature during conducting the experiment was 24±2oC), which is consistent
with Ashagrie and Abate (2012) results which was 3-4 days at a temperature of 20±2oC.
For 30oC storage temperature the shelf life of injera without preservatives is only 2 days,
and this is due to the effect of temperature on the growth of microorganisms.
Injera stored at ambient temperature, as shown in table 4.2, shows a longer storage time
(up to 10 days) by using ethanolic extract of clove with 10% addition followed by 10%
addition of aqueous extract of garlic which extended the shelf life of injera up to 8 days.
Ethanolic extract of cinnamon and garlic at 10% concentration, ethanolic extract of clove
at 5% concentration, and a mixture of ethanolic extract of spices with 10% addition also
showed a significant effect on the shelf life of injera by extending 6 up to 7 days.
Furthermore, aqueous extract of garlic at 5% concentration, a mixture of aqueous extract
of spices at 10% concentration and a mixture of ethanol extract of spices with 5% addition
showed some effect on the shelf life of injera by extending up to 5 days.
38
Samples without preservatives (control) and all aqueous and ethanolic extraction of tested
natural preservatives at 2% concentration, however, exhibited the highest mould growth
from day 1-10 of preservation and consequently, had the poorest preserving potential as
compared to other preservatives. This is due to the effect of amount of preservatives added
on the shelf life of injera. An aqueous extract of cinnamon and clove also doesn’t show
any preservatives effect on the shelf life of injera and this may be due to the less solubility
of essential oils (cinnamaldehyde from cinnamon, and eugenol from clove) in water than
alcohol (Rahman, 2007).
At 30oC storage temperature, as shown in table 4.3, the effect of selected natural
preservatives on the shelf life of injera showed relatively shorter storage time compared to
ambient temperature. This is due to, the effect of temperature on the growth of
microorganisms that are responsible to spoilage of injera and these microorganisms grow
more preferably at higher temperature (30oC) than lower temperature(24±2oC). And the
maximum, shelf life of injera observed was 7 days by using ethanolic extract of clove and
aqueous extract of garlic with 10% addition followed by ethanolic extract of garlic,
cinnamon and a mixture of ethanolic extract of spices at 10% concentration, which extend
the shelf life of injera up to 6 days. Ethanolic extract of clove and a mixture of spices as
well as aqueous extract of garlic with 5% addition had also showed some effect on the shelf
life of injera by extending up to 4-5 days.
In addition to the type of solvent used to prepare the spice extract and storage temperature,
the concentration added had also a significant effect on the shelf life of injera and a longer
shelf life were observed at higher concentration i.e. 10% addition and there was no
significant effect on the shelf life of injera with 2% addition.
In general, from visual observation in which the mould was visible, the maximum effect
of spice extract on the shelf life injera were observed in ethanolic extraction (due to the
higher solubility of essential oils on alcohol than water) with 10% addition and low storage
temperature.
39
Table 4.2 Visual observation of Injera spoilage Stored at Ambient Temperature
Where - - - - - No growth at all - -+++ High mould growth
- - - - + Slight mould growth -++++ Very high mould growth
- - - ++ Moderate mould growth +++++ Extremely high mould growth
The visual observation is indicated in Appendix E
Sample Concn Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10
Control 2% - - - - - - - - - - - - - ++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
5% - - - - - - - - - - - - - ++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
10% - - - - - - - - - - - - - ++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
Garlic
Water 2% - - - - - - - - - - - - - ++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
5% - - - - - - - - - - - - - - - - - - - + - - - ++ - - +++ +++++ +++++ +++++ +++++
10% - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + - - - ++
Garlic
Ethanol 2% - - - - - - - - - - - - - ++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
5% - - - - - - - - - - - - - - - - - - - + - - +++ +++++ +++++ +++++ +++++ +++++
10% - - - - - - - - - - - - - - - - - - - + - - - - + - - - ++ - - + + + +++++ +++++ +++++
Cinnamon
Water 2% - - - - - - - - - - - - - ++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
5% - - - - - - - - - - - - - ++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
10% - - - - - - - - - - - - - ++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
Cinnamon
Ethanol 2% - - - - - - - - - - - - - ++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
5% - - - - - - - - - - - - - - - - - - ++ - - +++ +++++ +++++ +++++ +++++ +++++
10% - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + - - - + + - - +++ +++++ +++++
Clove
Water 2% - - - - - - - - - - - - - ++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
5% - - - - - - - - - - - - - ++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
10% - - - - - - - - - - - - - ++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
Clove
Ethanol 2% - - - - - - - - - - - - - ++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
5% - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + - - - ++ - - +++ +++++ +++++
10% - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - +
Mixed
Water 2% - - - - - - - - - - - - - ++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
5% - - - - - - - - - - - - - ++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
10% - - - - - - - - - - - - - - - - - - - + - - - ++ - ++++ +++++ +++++ +++++ +++++
Mixed
Ethanol 2% - - - - - - - - - - - - - ++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
5% - - - - - - - - - - - - - - + - - - - + - - - ++ - - +++ +++++ +++++ +++++ +++++
10% - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + - -+++ +++++ +++++ +++++
40
Table 4.3 Visual observation of Injera spoilage Stored at 30oC
Where - - - - - No growth at all - -+++ High mould growth
- - - - + Slight mould growth -++++ Very high mould growth
- - - ++ Moderate mould growth +++++ Extremely high mould growth
The visual observation is indicated in Appendix E
Sample Concn Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10
Control 2% - - - - - - - - - + +++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
5% - - - - - - - - - + +++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
10% - - - - - - - - - + +++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
Garlic
Water 2% - - - - - - - - - + +++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
5% - - - - - - - - - - - - - - - - - - ++ - ++++ +++++ +++++ +++++ +++++ +++++
10% - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ++++ +++++ +++++
Garlic
Ethanol 2% - - - - - - - - - + +++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
5% - - - - - - - - - - - - - - + - - +++ -++++ +++++ +++++ +++++ +++++ +++++
10% - - - - - - - - - - - - - - - - - - - + - - - - + - - - ++ +++++ +++++ +++++ +++++
Cinnamon
Water 2% - - - - - - - - - + +++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
5% - - - - - - - - - + +++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
10% - - - - - - - - - + +++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
Cinnamon
Ethanol 2% - - - - - - - - - + +++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
5% - - - - - - - - - - - - - ++ - - +++ +++++ +++++ +++++ +++++ +++++ +++++
10% - - - - - - - - - - - - - - - - - - - - - - - - + - - - ++ - ++++ +++++ +++++ +++++
Clove
Water 2% - - - - - - - - - + +++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
5% - - - - - - - - - + +++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
10% - - - - - - - - - + +++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
Clove
Ethanol 2% - - - - - - - - - + +++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
5% - - - - - - - - - - - - - - - - - - - + - - - ++ - - +++ +++++ +++++ +++++ +++++
10% - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -+ - - +++ +++++ +++++
Mixed
Water 2% - - - - - - - - - + +++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
5% - - - - - - - - - + +++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
10% - - - - - - - - - + +++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
Mixed
Ethanol 2% - - - - - - - - - + +++++ +++++ +++++ +++++ +++++ +++++ +++++ +++++
5% - - - - - - - - - - - - - - + - - - ++ - - +++ +++++ +++++ +++++ +++++ +++++
10% - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + +++++ +++++ +++++ +++++
41
4.3.2 Microbial Count of Injera with Different Natural Preservatives
The other method in which the shelf life of injera was determined is by conducting the
microbial count such as total plate count and yeast and mould count (Girma et al., 2013).
The effect of selected natural preservatives with aqueous and ethanolic extract on total
plate count and yeast and mould count of injera were shown in table 4.4 and 4.5,
respectively.
As the microbial result shows, except for aqueous extract of garlic and ethanolic extract
of cinnamon and clove at 10% concentration, all the other natural preservative added in to
injera doesn’t show any preservatives effect after seven-day ambient temperature storage.
This may be due to, the less solubility of essential oils ( cinnamaldehyde from cinnamon,
and eugenol from clove) in water than alcohol (Rahman, 2007). In addition to this, the
amount of preservatives added have also significant role on the shelf life of injera i.e. 10%
addition of preservatives shows longer shelf life than 5% and 2%.
Since they were completely spoiled and were not suitable for human consumption, the
microbial analysis of all-natural preservatives added at 2% and 5% concentration were not
conducted after seven-day at ambient temperature storage and recorded as NA (not
applicable) for microbial analysis or it is useless to conduct microbial analysis.
As shown in the table 4.4, at ambient storage temperature, the minimum total plate count
on tenth day were observed in aqueous extract of garlic and ethanolic extract of clove at
10% concentration addition with total plate count of 6.60*106 cfu/g and 8.86*106 cfu/g,
respectively. The total plate count of all other natural preservative added in to injera were
not countable on the tenth day storage time and recorded as NC (Not Countable). Similarly,
at 30oC storage temperature the minimum total plate count on tenth day was observed only
in ethanolic extract of clove with 10% concentration addition with total plate count of
12.98*106 cfu/g and all the other natural preservatives added injera were not countable.
As shown in the table 4.5 at ambient storage temperature the minimum yeast and mould
count on tenth day was observed in aqueous extract of garlic at 10% concentration addition
with yeast and mould count of 6.17*106 cfu/g and all other natural preservatives added
injera were not countable on the tenth day storage time and recorded as NC.
42
At 30oC storage temperature the yeast and mould count of all-natural preservatives added
injera were not countable on tenth day storage time. On the seventh day storage time the
minimum yeast and mould count was observed in ethanolic extract of clove at 10%
concentration addition with yeast and mould count of 1.98*106 cfu/g.
Table 4.4 Total plate count
Where NA Not Applicable NC Not countable
Average Total plate count for Injera Stored at Ambient Temperature (106 cfu/g)
Day
Concn
Control
Garlic
Water
Garlic
Ethanol
Cinnamon
Water
Cinnamon
Ethanol
Clove
Water
Clove
Ethanol
Mixed
Water
Mixed
Ethanol
Day 3 2% 2.56 1.99 4.56 4.06 3.69 5.71 2.42 3.01 4.53
5% 3.78 1.74 4.42 4.96 3.62 5.1 2.14 1.85 2.17
10% 2.31 0.69 1.56 2.67 1.83 1.89 0.84 3.43 3.86
Day 5 2% 4.60 4.53 10.95 NC NC 6.76 4.12 4.06 5.80
5% 14.30 2.49 NC NC 3.635 9.4 2.65 5.75 2.81
10% 8.60 1.00 6.84 9.35 2.74 3.06 1.34 7.15 7.47
Day 7 2% NA NA NA NA NA NA NA NA NA
5% NA NA NA NA NA NA NA NA NA
10% NC 3.74 NC NC 7.09 NC 2.43 NC NC
Day 10 2% NA NA NA NA NA NA NA NA NA
5% NA NA NA NA NA NA NA NA NA
10% NC 6.60 NC NC NC NC 8.86 NC NC
Average Total plate count for Injera Stored at 30oC (106 cfu/g)
Day 3 2% 3.28 1.59 1.71 6.01 1.38 5.30 2.72 8.02 3.80
5% 3.06 1.37 2.14 5.66 1.29 5.02 3.76 2.56 10.25
10% 2.82 1.19 0.47 NC 0.725 NC 2.15 2.69 2.63
Day 5 2% 9.95 8.53 9.20 6.84 NC NC 5.05 9.31 NC
5% 7.16 6.40 4.43 5.82 8.75 9.97 NC 7.30 13.48
10% 4.26 1.87 0.79 NC 10.05 NC 2.89 NC 4.42
Day 7 2% NA NA NA NA NA NA NA NA NA
5% NA NA NA NA NA NA NA NA NA
10% NC NC 4.27 NC NC NC 3.93 NC 6.25
Day 10 2% NA NA NA NA NA NA NA NA NA
5% NA NA NA NA NA NA NA NA NA
10% NC NC NC NC NC NC 12.98 NC NC
43
Table 4.5 Yeast and Mould Count
Where NA Not Applicable NC Not countable
Average Yeast and Mould Count for Injera with preservatives Stored at Ambient Temperature (106
cfu/g)
Day
Concn
Control
Garlic
Water
Garlic
Ethanol
Cinnamon
Water
Cinnamon
Ethanol
Clove
Water
Clove
Ethanol
Mixed
Water
Mixed
Ethanol
Day 3 2% 3.61 0.89 1.39 2.24 3.47 1.68 1.11 2.36 2.89
5% 4.8 0.37 0.25 1.14 3.21 1.21 0.23 1.16 0.52
10% 9.55 0.23 0.62 4.5 0.17 1.04 0.12 0.99 0.23
Day 5 2% NC NC NC NC NC NC NC NC NC
5% NC 1.49 NC NC 3.64 9.40 2.65 11.5 2.81
10% NC 3.11 5.1 10.6 7.2 NC 0.51 12.4 1.82
Day 7 2% NA NA NA NA NA NA NA NA NA
5% NA NA NA NA NA NA NA NA NA
10% NC 4.02 NC NC NC NC 5.48 NC NC
Day 10 2% NA NA NA NA NA NA NA NA NA
5% NA NA NA NA NA NA NA NA NA
10% NC 6.17 NC NC NC NC NC NC NC
Average Yeast and Mould Count for Injera with preservatives Stored at 30oC (106 cfu/g)
Day 3 2% 4.29 5.60 3.04 6.20 5.80 3.80 2.01 NC 1.17
5% NC 1.79 1.18 5.80 1.43 2.62 1.95 2.56 0.91
10% NC 1.56 0.27 NC 0.55 NC 0.43 2.5 0.31
Day 5 2% NC NC NC NC NC NC NC NC NC
5% NC 4.50 6.50 NC 2.87 NC 1.53 NC NC
10% NC 1.59 1.33 NC 6.3 NC 1.61 NC 2.59
Day 7 2% NA NA NA NA NA NA NA NA NA
5% NA NA NA NA NA NA NA NA NA
10% NC NC NC NC NC NC 1.98 NC NC
Day 10 2% NA NA NA NA NA NA NA NA NA
5% NA NA NA NA NA NA NA NA NA
10% NC NC NC NC NC NC NC NC NC
44
In general, as the microbial quality analysis shows, the selected natural preservatives
namely garlic, cinnamon and clove, have a significant effect on the shelf life of injera,
especially ethanolic extract showed a better preservatives effect than aqueous extract of
spice.
The potent antimicrobial activity of clove and cinnamon is due to the presence of eugenol
and cinnamaldehyde, respectively. These are the phenolic components of clove and
cinnamon, which render them effective against different micro‐organisms. Eugenol and
cinnamaldehyde, a major constituent of clove and cinnamon were found to limit the growth
of yeast and mold in injera, especially the ethanolic extract of clove and cinnamon showed
a good preservatives effect, due to the high solubility of eugenol and cinnamaldehyde in
alcohol than water.
Similarly, garlic was also effective against the growth of injera spoiling micro‐organisms.
This could be contributed to allicin, one of the active principal components of freshly
crushed garlic homogenates.
4.4 Effect of Temperature and Natural Preservatives on the Shelf
Life of Injera
Among all factors that affected microbial growth, temperature is one of the most important
factors directly affecting the growth of microorganisms in foods. Evaluating the effect of
temperature on microbial growth is of paramount important in predicting microbiology and
shelf life of a product (Huang et al., 2011). According to Ashagrie and Abate (2012) studies
Penicillium and Rhizopus were more dominant in spoiling injera at lower temperature(16-
200c), while Aspergillus niger grow much faster as the temperature gets higher (25-320c).
None of the moulds grew when the temperature was kept at 4oC.
Injera containing different natural preservatives show different microbial effect depending
upon the storage temperature. Both the total plate count and yeast and mould count shows
significant difference on selected temperature, i.e. ambient temperature (average
temperature during conducting the experiment was 24±2oC) and 30oC. As the temperature
increased from ambient temperature to 30oC, the minimum total plate count observed in
aqueous extract of garlic increased from 6.60*106 cfu/g to 12.98*106 cfu/g and ethanolic
45
extract of clove increased from 8.86*106 cfu/g to uncountable state. Similarly, the
minimum yeast and mould count observed in aqueous extract of garlic with 10%
concentration addition increased from 6.17*106 cfu/g to uncountable state when the
temperature was increased from ambient temperature to 30oC.
In general, from the selected storage temperature, injera with natural preservatives stored
at lower temperature shows a longer shelf life than higher temperature. This is due to the
reason that microorganisms responsible for injera spoilage are more dominant at a
temperature range of 16-200c for Penicillium and Rhizopus, while Aspergillus niger grow
much faster at a temperature range of 25-320c. Since the ambient temperature during
conducting the experiment was 24±2oC, it doesn’t create favorable condition for injera
spoiling microorganisms.
46
5. CONCLUSION AND RECOMMENDATION
5.1 Conclusion
The sensory quality of injera prepared by adding different natural preservatives shows
that almost all spices have their own flavor, aroma and color. Except for aqueous and
ethanolic extract of clove, the others brought a good flavor and aroma which is acceptable
by majority of the panelists.
Aqueous extract of spices scores a better sensory attribute than ethanolic extract, which
indicates that the type of extraction method plays significant role in the sensory quality of
injera.
The effect of selected spice (garlic, clove, and cinnamon) with aqueous and ethanolic
extraction added in to injera immediately before baking shows a positive effect on the
sensory quality of injera and some spices help to improve the existing quality of injera
like appearance, color, flavor and aroma. On the other hand, ethanolic extract of clove
develop unpleasant sensory attributes which is not accepted by the panelist. Even though
most panelist prefer the control one, aqueous extract of garlic specially at lower
concentration also shows a comparable sensory result with the control, but most panelist
doesn’t prefer the addition of ethanolic extract of clove specially at higher concentration
and this is due to bitter taste arises from high solubility of eugenol in ethanol.
Most of the selected natural preservatives especially aqueous extract of garlic, and
ethanolic extract of cinnamon and clove show good preservatives effect at 10%
concentration and an aqueous extract of cinnamon and clove doesn’t show any
preservatives effect on the shelf life of injera. Increasing the concentration of natural
preservatives added to injera, reduces the microbial count which indicates the
effectiveness of antimicrobial activity of natural preservatives at higher concentration.
Injera made with 10% aqueous extract of garlic and ethanolic extract of clove results an
improved microbial quality which shows a preservative effect up to 10 days.
47
In addition to the type of extraction method and concentration of spice extract, storage
temperature also plays a significant role on the shelf life of injera and based on this study,
injera treated with different natural preservatives shows a good microbial quality at lower
temperature (ambient temperature) than 30oC.
In general, the concentration of spices, type of spice, type of solvent used and storage
temperature have a significant effect on the shelf and the sensory quality of injera. As
concentration of spice added increased, the sensory quality decreased.
The maximum, shelf life of injera were observed at higher concentration and lower
storage temperature i.e. at ambient temperature (24±2oC) than 30oC.
Finally, extending shelf life of injera by using natural preservatives is possible and based
on this study injera prepared by adding aqueous extract of garlic shows better sensory
quality as well as good preservatives effect.
5.2 Recommendation
In developing country like Ethiopia, which is still under a struggle to be food secured,
losing a substantial amount injera due to spoilage is a disaster. Moreover, the popularity
of teff injera is increasing around the world due its gluten free protein, because of this
applying the result of this research can contribute in saving a significant amount of injera
that might lost in every household per every baking cycle.
As the study was conducted at ambient temperature of Bahir Dar (average temperature
during conducting the experiment was 24±2oC) and 30oC further investigation on effect
of natural preservatives on the shelf life of injera at lower storage temperature can be a
good research topic, which can provide essential data for design of cold storage for injera.
In this research the health effect and nutritional states of injera is not investigated.
Therefore, further studies of the health effect and nutritional states of injera prepared by
adding natural preservatives is recommended.
48
The microorganisms that are counted as total plate count and yeast and mould count are
not isolated and further study on isolation of microorganisms is recommended to check
whether the microorganisms are really spoilage or not.
Sometimes some peoples may be allergic to the natural preservatives used in this
research. As a result of this, the application of hurdle technology (combined technology)
should be evaluated to minimize the amount of preservatives added in to injera. So, the
application of other alternative preservation methods such as: design and construction of
low temperature storage for injera (Mesob), Modified atmosphere packaging (MAP)
should also be evaluated.
Some researchers suggest that essential oil is more effective than extracts. Therefore, the
comparative study of essential oil and spice extract of natural preservatives on the shelf
life of injera can also be a good research topic.
Finally, while conducting this research the following research topics are identified i.e.
design and construction of cold storage (Mesob) for injera, improvement of shelf life of
injera using essential oil of spices and preservation of Ethiopian injera using hurdle
technology.
49
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APPENDICES
Appendix A: Questionnaire Format for Sensory Evaluation
Sensory Analysis Please select one of the following options & put the number only at appropriate place on
the table for each sensory parameter.
5. Like very much
4. Like
3. Neither like nor dislike
2. Dislike
1. Dislike very much
Product One
Code of sample Sensory parameter
Eye size and
Distribution
color Flavor sweetness Bitterness Sourness Overall
S0W
S1W
S1E
S2W
S2E
S3W
S3E
S4W
S4E
Comment (if any) ...……...…………………………………………………………………
………………………………………………………………………………………………
………………………………………………………………………………………………
Thank You for coming!!!
54
Appendix B: ANOVA Table for Sensory Quality
ANOVA Table for Sensory Analysis of Injera with Different Natural Preservatives
Source Dependent Variable Type III Sum
of Squares
df Mean
Square
F Sig.
Corrected Model
Eye Size and
Distribution 115.363a 26 4.437 6.898 .000
Color 200.000b 26 7.692 14.291 .000
Flavor 99.000c 26 3.808 5.939 .000
Sweetness 90.785d 26 3.492 3.571 .000
Bitterness 115.467e 26 4.441 3.490 .000
Sourness 77.867f 26 2.995 2.351 .000
Overall 102.652g 26 3.948 7.525 .000
Intercept
Eye Size and
Distribution 3909.171 1 3909.171 6077.597 .000
Color 3546.486 1 3546.486 6588.655 .000
Flavor 2761.310 1 2761.310 4306.793 .000
Sweetness 2603.161 1 2603.161 2662.324 .000
Bitterness 3010.919 1 3010.919 2366.279 .000
Sourness 2566.034 1 2566.034 2014.038 .000
Overall 2900.833 1 2900.833 5528.647 .000
Concentration
Eye Size and
Distribution 4.850 2 2.425 3.770 .024
Color 14.917 2 7.459 13.857 .000
Flavor 13.101 2 6.551 10.217 .000
Sweetness 1.029 2 .514 .526 .592
Bitterness 8.853 2 4.426 3.479 .032
Sourness 7.382 2 3.691 2.897 .057
Overall 4.738 2 2.369 4.515 .012
55
Type_of_Spice
Eye Size and
Distribution 14.146 3 4.715 7.331 .000
Color 88.413 3 29.471 54.751 .000
Flavor 11.767 3 3.922 6.117 .001
Sweetness 16.146 3 5.382 5.504 .001
Bitterness 36.513 3 12.171 9.565 .000
Sourness 6.633 3 2.211 1.735 .160
Overall 34.433 3 11.478 21.875 .000
Type_of_Solvent
Eye Size and
Distribution 8.438 1 8.438 13.118 .000
Color 10.004 1 10.004 18.586 .000
Flavor 13.067 1 13.067 20.380 .000
Sweetness 21.004 1 21.004 21.482 .000
Bitterness 14.504 1 14.504 11.399 .001
Sourness 10.417 1 10.417 8.176 .005
Overall 11.267 1 11.267 21.473 .000
Concentration *
Type_of_Spice
Eye Size and
Distribution 30.442 6 5.074 7.888 .000
Color 5.300 6 .883 1.641 .136
Flavor 4.933 6 .822 1.282 .266
Sweetness 5.467 6 .911 .932 .473
Bitterness 5.800 6 .967 .760 .602
Sourness 4.242 6 .707 .555 .766
Overall 2.042 6 .340 .649 .691
Concentration *
Type_of_Solvent
Eye Size and
Distribution 2.325 2 1.163 1.807 .166
Color .433 2 .217 .403 .669
Flavor 4.433 2 2.217 3.457 .033
56
Sweetness 3.433 2 1.717 1.756 .175
Bitterness 6.033 2 3.017 2.371 .096
Sourness 1.058 2 .529 .415 .661
Overall 2.708 2 1.354 2.581 .078
Type_of_Spice *
Type_of_Solvent
Eye Size and
Distribution 6.413 3 2.138 3.323 .020
Color 31.779 3 10.593 19.680 .000
Flavor 1.700 3 .567 .884 .450
Sweetness 15.179 3 5.060 5.175 .002
Bitterness 4.746 3 1.582 1.243 .295
Sourness 4.883 3 1.628 1.278 .283
Overall 2.633 3 .878 1.673 .173
Concentration *
Type_of_Spice *
Type_of_Solvent
Eye Size and
Distribution 15.375 6 2.563 3.984 .001
Color 3.433 6 .572 1.063 .385
Flavor 1.200 6 .200 .312 .930
Sweetness .933 6 .156 .159 .987
Bitterness 1.267 6 .211 .166 .986
Sourness 1.842 6 .307 .241 .963
Overall 1.692 6 .282 .537 .780
Error
Eye Size and
Distribution 156.300 243 .643
Color 130.800 243 .538
Flavor 155.800 243 .641
Sweetness 237.600 243 .978
Bitterness 309.200 243 1.272
Sourness 309.600 243 1.274
57
Overall 127.500 243 .525
Total
Eye Size and
Distribution 4441.000 270
Color 4094.000 270
Flavor 3136.000 270
Sweetness 3106.000 270
Bitterness 3628.000 270
Sourness 3076.000 270
Overall 3277.000 270
Corrected Total
Eye Size and
Distribution 271.663 269
Color 330.800 269
Flavor 254.800 269
Sweetness 328.385 269
Bitterness 424.667 269
Sourness 387.467 269
Overall 230.152 269
a. R Squared = .425 (Adjusted R Squared = .363)
b. R Squared = .605 (Adjusted R Squared = .562)
c. R Squared = .389 (Adjusted R Squared = .323)
d. R Squared = .276 (Adjusted R Squared = .199)
e. R Squared = .272 (Adjusted R Squared = .194)
f. R Squared = .201 (Adjusted R Squared = .115)
g. R Squared = .446 (Adjusted R Squared = .387)
58
Appendix C: Picture of Sensory Evaluation of Injera by the Panelists
59
Appendix D: Storage Condition of Injera
Injera Storage at Ambient Temperature
Injera Storage at Ambient Temperature
Injera Storage at Ambient Temperature
Injera Storage at Ambient Temperature
Injera Storage at Ambient Temperature
Injera Storage at Ambient Temperature
Injera Storage at Ambient Temperature
Injera Storage at Ambient Temperature
Injera Storage at 30oC
Injera Storage at 30oC
Injera Storage at 30oC
Injera Storage at 30oC
Injera Storage at 30oC
Injera Storage at 30oC
Injera Storage at 30oC
Injera Storage at 30oC
60
Appendix E: Visual observation of Injera Spoilage
Where A = Injera prepared by adding aqueous extract of Garlic
B = Injera prepared by adding ethanolic extract of Garlic
C = Injera prepared without any preservative (Control)
Day 4 (10% concentration)
Day 10 (10% concentration)
Day 6 (10% concentration)
Day 8 (10% concentration)
+++++
+++++
+++++ +++++
+++++
+++++
A
C
B
C
A B
B
A
C
B
C
A
+++++ +++++
+++++
+++++
A
A
B
B
C
C
----- ----+ ----- ---++
----- +++++
---++
+++++
61
Appendix F: Image of Microbial Growth in Petri Dish