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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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53

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