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ADDIS ABABA UNIVERSITY

SCHOOL OF GRADUATE STUDIES

TECHNOLOGY INSTITUTE

DEPARTMENT OF CHEMICAL ENGINEERING EVALUATION OF ETHIOPIAN THYME (THYMUS SCHIMPERI R.)

ANTIOXIDANT ACTIVITY AND ITS PRESERVATIVE EFFECT ON SOME FOOD PRODUCTS

A Thesis submitted to the school of Graduate Studies of Addis Ababa University in partial fulfillment of the requirements for the Degree of Master of Science in

Chemical Engineering (Food Engineering) By:

Gebrehana Ashine

Advisor:

Dr. Eng. Shimelis Admasu (Associate Prof.)

June, 2012

Addis Ababa, Ethiopia

i

ADDIS ABABA UNIVERSITY

SCHOOL OF GRADUATE STUDIES

TECHNOLOGY INSTITUTE

DEPARTMENT OF CHEMICAL ENGINEERING EVALUATION OF ETHIOPIAN THYME (THYMUS SCHIMPERI R.)

ANTIOXIDANT ACTIVITY AND ITS PRESERVATIVE EFFECT ON SOME FOOD PRODUCTS

A Thesis submitted to the school of Graduate Studies of Addis Ababa University in partial fulfillment of the requirements for the Degree of Master of Science in Chemical Engineering (Food Engineering)

By

Gebrehana Ashine

Approved by the Examining Board

_________________________________ _____________ (Chairman, Department’s Graduate Committee) Dr.Eng. Shimelis Admassu (Associate Prof.) _________________________________ _____________ (Advisor)

_________________________________ _____________

(Internal Examiner)

_________________________________

(External Examiner)

_____________

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Acknowledgments

My first and foremost gratitude goes to my advisor, Dr. Eng. Shimelis Admasu, for his

unreserved advice and continuous encouragement, guidance and valuable suggestions in every

step of the thesis work. Without his kindness and understanding, this piece of work would not

have come into completion.

Further thanks are extended to the staff of chemical and food engineering School, Technology

Institute of Bahir Dar University, notably Ato Biresaw Demelash, Ato Tadele Andarige, and Ato

Mengistu Gizaw for their cooperation during laboratory work.

My sincere gratitude is due to Ato Mulugeta Belayihun, Ato Derese Mekonnen, Habtamu

Asimare, Tuamelisan Shumye and other friends for their encouragement and friendly support

during the course of the study. I am very grateful for my family members, Ato Alayu

Hailemariam and Ato Eshetu Adelahu for their encouragement and loving support. I highly

admire my fiancée Dr. Sosina Tarekegn and thank her for her encouragement throughout the

period of my study.

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Table of Contents

Chapter Title Pages

Title page i Acknowledgement ii Table of contents iii List of tables vi List of figures vii List of abbreviations

Abstract viii

ix 1 Introduction 1

1.1. Background 1

1.2. Statement of the problem 4

1.3. Objectives 6

1.4. Scope of the study 6

2 Literature Review 7

2.1. Distribution of thyme in Ethiopia 7

2.2. Chemical composition and use of thyme 7

2.3. The development of oxidative rancidity in foods 9

2.4. Antioxidants and their effects in food preservation 12

2.5. Antioxidants and health 13

2.6. Natural Antioxidants 14

2.6.1. Sources of natural antioxidants and their preparation 14

2.6.2. The use of natural antioxidants in food products 16

2.6.3. Measuring antioxidant activity 18

2.7. The regulation of antioxidants in food 24

3 Materials and Methods 25

3.1. Structure of thesis experiments 25

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3.2. Raw material collection, transportation and sample preparation 26

3.3. Extraction process 28

3.3.1. Setting extraction parameters 28

3.3.2. Preparation of thyme crude extract 28

3.4. Analysis Methods 29

3.4.1. Evaluation of thyme antioxidant activity 29

3.4.1.1. Rancimat Method 29

3.4.1.2. Schaal oven test 30

3.4.2. Preservative effect of thyme extract on some food products 32

3.4.2.1. Chemical Analysis 32

3.4.2.2. Microbiological Analysis 34

3.5. Statistical Analysis 35

4 Results and Discussion 36

4.1. Effect of extraction parameters on thyme antioxidant activity and its extract yield

36

4.2. Evaluation of thyme antioxidant activity on oil and butter 41

4.2.1. The Rancimat Method 41

4.2.2. Schaal Oven Test Method 43

4.2.3. Effect of thyme crude extract concentration on its antioxidant activity

45

4.3. Preservative effect of thyme on oil, butter and meat 47

4.3.1. Free Fatty Acids 47

4.3.2. Peroxide Value 49

4.3.3. Acid Value 51

4.3.4. Total Viable Count 52

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4.3.5. Aerobic Mold and Yeast Count 54

4.3.6. Enterobacteriaceae Count 56

5 Suggesting Technology for the production of thyme crude extract 59

5.1. Production of thyme crude extract 59

5.2. Material and Energy balance on major unit operations 61

5.3. Economic Evaluation of the Plant 70

5.4. Plant Location 74

6 Conclusions and Recommendation 75

6.1. Conclusions 75

6.2. Recommendation 77

References 78

Appendices 84

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List of Tables

Table No. Title Page

4.1 Effect of extraction parameters on antioxidant activity of thyme 37

4.2 Effect of extraction parameters on the yield of thyme crude extract 39

4.3 Antioxidant activity of thyme evaluated by Rancimat on soybean oil

41

4.4 Antioxidant property of butter collected from different locations as determined by Rancimat

43

4.5 Peroxide value of thyme extract treated with soybean oil for evaluating thyme antioxidant activity using Schaal Oven Test

44

4.6 Correlation of thyme extract concentration and its antioxidant activity

46

4.7 Free fatty acid value of both soybean oil and butter treated by thyme crude extract

48

4.8 Peroxide value of soybean oil and butter of thyme crude extract treatments

50

4.9 Acid Value of soybean oil and butter treated by thyme crude extract 51

5.1 Plant capacity and production program 70

5.2 Purchased Equipment costs 70

5.3 Estimation of Direct and Indirect costs 71

5.4 Total production cost estimation 72

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List of Figures

Figure No. Title Page

2.1 Major antioxidants among thyme extract constituents 8

3.1 Experimental framework of the thesis 25

3.2 Rancimat apparatus for antioxidant activity evaluation 27

4.1 Aerobic plate count of butter and meat 53

4.2 Aerobic Mold and Yeast Count of butter and meat 55

4.3 Enterobacteriaceae count of both butter and meat 56

5.1 Basic steps for the production of thyme crude extract 59

5.2 Equipment layout of thyme crude extract production plant 60

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List of Abbreviations

AArefence Antioxidant Activity of reference

AAt Antioxidant Activity of thyme

ANOVA Analysis of Variance

AOAC Association of Official Analytical Chemists

AV Acid Value

BHA Butylated hydoxylanisole

BHT Butylated hydroxytoluene

Cfu Colony Forming Units

DPPH 2, 2-diphenyl-1-picrylhydrazyl

Dwb dry weigh basis

FFA

IT

Free Fatty Acids

Induction Time

JMP John’s Macintosh Project

mEq Milli Equivalent

OSI Oil Stability Index

PCA Plate Count Agar

PDA Potato Dextrose Agar

PF Protection Factor

PV Peroxide Value

TVC Total Viable Count

VRBA Violet Red Bile Agar

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Abstract

The study was conducted with the objective of evaluating antioxidant activity of Thymus

Schimperi and its preservative effect on oil, butter and meat. Thyme was prepared by

investigating the effect of ethanol concentration (0-97%), extraction time (180-240 min) and

extraction temperature (20-40℃) on its antioxidant activity and extract yield. Based on ANOVA

analysis, extraction parameters have significant effect (P<0.05) on thyme antioxidant activity

and its extract yield. The best levels of extraction parameters for higher antioxidant activity and

extract yield were distilled water (0% ethanol concentration) for 210 min at 20℃. Antioxidant

activity of thyme was evaluated by adding 0.1% thyme crude extract to refined soybean oil and

butter. For comparison, 0.05% α-Tocopherol was added as positive control and none thyme

extract was used as negative control. The results were expressed as induction time determined by

Rancimat (hr) and Schaal Oven Test (day) methods. Induction time of thyme was 3.25±0.02 hr

and six day when determined in soybean oil. But, α-Tocopherol has 4.98±0.10 hr and seven day

induction time. Thyme has 5.28±0.08 hr induction time when evaluated in butter. The

preservative effect of thyme was also studied by performing free fatty acid, Peroxide value, Acid

value (on oil and butter); Aerobic plate count, Aerobic Mold & Yeast count and

Enterobacteriaceae count on butter and meat. In the study, all samples were treated with 0, 0.1%

and 0.2% levels of thyme extract and analysis were performed in weekly basis. Compared to

thyme extract treated samples of oil and butter, control sample showed higher lipid oxidation

rate in each storage weeks. Highest microbial load was obtained in controlled samples of butter

and meat. Samples containing 0.2% thyme extract have lower count of total viable microbes,

mold & yeast and enterobacteriaceae. Based on the results, samples with 0.2% thyme extract

significantly (P<0.05) improved both the oxidative and microbial stability. Hence, Ethiopian

thyme has antioxidant activity and preservative effect as evaluated on soybean oil, butter and

meat. The preliminary techno-economic feasibility study also showed good profit margin and

financial feasibility for crude thyme extract production.

Keywords: antioxidant activity, induction time, thyme extract, thymus schimperi, preservative

effect, protection factor.

1

Chapter One

Introduction

1.1. Background Food products are now often sold in areas of far distant from their production sites and need

extended safety and storage stability. Many foods are perishable by nature and require protection

from spoilage during their preparation, storage and distribution to give them longer shelf-life.

The major cause of lipid-based food products deterioration is rancidity. Significant changes can

occur in product odor, taste, color, texture, nutritive value. Progressing oxidation results in

complete spoilage of foods. Use of antioxidants can postpone problems caused by rancidity thus

they are frequently used to retard oxidation processes in the food industry (Allen and Hamilton,

1994).

Antioxidants are an increasingly important feature in food processing. Their traditional role

involves, as their name suggests, inhibiting the development of oxidative rancidity in foods, like

meat, dairy products and fried foods. During recent years the most widely used definition of

antioxidants was proposed by Halliwell and Guteridge (2007): “A substance that, when present

at a low concentration compared with that of an oxidizable substrate, inhibits oxidation of the

substrate”.

In recent years there is a much focus on replacing synthetic food additives which might have

adverse effects with those of plant-based natural ones (Paradiso et al., 2008; Descalzo and

Sancho, 2008). Currently, the uses of natural antioxidants are becoming very popular in food and

preventive medicine due to the claims that they are safer and have disease–preventing and health

promoting attributes. It is well known that plants are the richest source of bioactive

phytochemicals and antioxidant nutrients (Elless et al., 2000). Spices and herbs provide foods

with flavors and food-preserving power, including antiseptic and antioxidant activity. The

increasing uses of herbal products demand extra attention with particular focus on their safety,

effectiveness and drug interactions. Over the last few decades, a substantial body of scientific

evidence is available demonstrating wide range of pharmacological and nutraceutical activities

of medicinal herbs (Burt, 2004; Celiktas et al., 2007; Edris, 2007). These include antioxidant,

antimicrobial, anticancer, anti-inflammatory activities.

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The essential oils and herbs-derived extracts are gaining much recognition as a potential source

of natural and safer antioxidants and bio-actives (Burt et al., 2003; Burt, 2004). Essential oils of

some spices and herbs such as sage, oregano, thyme, and Satureja etc. have shown their

antioxidant potential (Ruberto and Baratta, 2000; Rota et al., 2004; Rota et al., 2008) and thus

can be used as natural antioxidants for the protection of fats/oils and related products (Burt,

2004; Sacchetti et al., 2005; Bozin et al., 2006). Many authors, in fact, have reported

antimicrobial, antifungal, antioxidant and radical-scavenging properties of spices and essential

oils and, in some cases; a direct food-related application has been tested. Literatures outline

different approaches within this trend and both the biological screening of new essential oils and

the evaluation of new properties of already marketed oils have been done (Fullas, 2003).

The antioxidant activities of herbs and spices extracted with solvents, such as methanol or

acetone, have been evaluated in various test systems, including hydrophilic and lipophilic

systems. So far, many investigations on the antioxidant activities of methanol extracts of

rosemary, sage, and thyme have been carried out in hydrophilic and lipophilic test systems. In

2006, Bozin B. et al found that ethanolic extract or essential oil of thyme has a significant rate of

antifungal and antimicrobial activities with strongly inhibited lipid peroxidation and high -OH

radical scavenging.

Numerous studies have been conducted on the antibacterial and antioxidant activity of herbs and

vegetable extracts and subsequent effect on the shelf life of different food products. It is of a

great interest to consumers and nutritionists to quantify the antioxidant of various foods because

antioxidants are clearly important to human life. There are many different methods for

determining antioxidant function which rely on different generators of free radicals, acting by

different mechanisms. In the literature, antioxidant properties are denoted as antioxidant capacity

(Jan and Michael, 2001).

Thymus is an aromatic plant belonging to the Lamiaceae family, used for medicinal and spice

purposes almost everywhere in the world. Many pharmacological in vitro experiments carried

out during the last decades revealed well defined pharmacological activities of both, the thyme

essential oil and its extracts. The non-medicinal use of thyme is worthy of attention, because

thyme is used in the food and aroma industries; it is widely used as culinary ingredient and it

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serves as a preservative for foods especially because of its antioxidant effect. The major phenolic

components in thyme extracts, especially thymol and carvacrol, present higher antioxidant

activity than the well-known BHT (butylated hydroxytoluene) and α-tocopherol antioxidants

(Lee et al., 2005).

The antioxidative property of thyme is important in both the medicinal and non-medicinal

context. Several papers show that the essential oils and extracts of thyme exhibit antioxidative

property. The phenolic monoterpenes in thyme, thymol and carvacrol, are the primary

compounds which contribute to the characteristic aroma of its essential oil. They are also known

to inhibit lipid peroxidation. Thymus schimperi is rich in medicinally important constituents,

thymol and carvacrol (Dagne et al., 1998).

Even though, our country has considerably abundant of Lamiaceae family herb, wild growing

species of thyme (T. Schimperi), its antioxidant potential has not yet well studied on the shelf-

stability of fat-based food products. Therefore, the purpose of the research work was to evaluate

the antioxidant capacity of thyme extract using lipid oxidation inhibition system and determine

its preservative effect on butter, oil and meat food products.

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1.2. Statement of the problem

The quality of food products are becoming deleterious after short storage by microbiological and

chemical changes which are the major factors affecting food sensory characteristics, safety,

quality and cost. Lipid oxidation is one of the most undesirable change that affect the quality of

foodstuff during storage due to deterioration of polyunsaturated fatty acids (Pazos et al., 2008).

Vegetable oils and fats are recognized as important components of our diet. Particularly,

oxidative deterioration of oils and fats is a great concern in the shelf stability of foods and

resulted in decreasing of safety and nutritional quality. Due to these changes, consumers do not

accept oxidized products and industries suffer from economic losses. The oil industry has to pay

special attention in this context, as oils, fats and fatty foods suffer stability problems (Wu and

Nawar, 1986). In order to overcome the stability problems of oils and fats, synthetic antioxidants,

such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ter-butyl

hydroquinone (TBHQ) have been used as food additives. But recent reports reveal that these

compounds may be implicated in many health risks, including cancer and carcinogenesis (Hou,

2003; Prior, 2004).

Due to these safety concerns, there is an increasing trend among food scientists to replace these

synthetic antioxidants with natural ones, which, in general, supposed to be safer. Investigating

for cheap and abundant sources of natural antioxidants is attracting worldwide interest since the

trends of consumers and many health-related industries these days tend to shift preferences to

natural sources. The use of spices, herbs as antioxidants in perishable foods is becoming of

increasing importance in the food industry.

Health protection and economic reasons have required investigation areas aimed at enhancing the

oxidation and microbiological stability of foods such as oil, butter and meat. Preservation of

butter, oil and meat is the main application area of natural antioxidant to prevent the rate of

oxidative rancidity. For instance, butters of various locations have different selling price.

Knowing the quality of butter regarding its antioxidant content was one among the aim of this

study.

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Some plant extracts have been known as antimicrobials as well as antioxidants in food systems.

Thyme is the one among the potential herbs for extracting natural antioxidants. Lee et al. (2005)

reported that the major phenolic components in thyme extracts, especially thymol and carvacrol,

present higher antioxidant activity than the well-known BHT and α-tocopherol antioxidants. Our

country has abundant wild thyme (Thymus Schimperi) to extract natural antioxidant and

antimicrobial from this potential herb. However, it has not yet cultivated and exploited properly

for these purposes. Thus, the research was aimed at extraction of thyme crude extract and

evaluating its antioxidant activity antimicrobial effect in preservation of soybean oil, butter and

meat.

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1.3. Objectives

General objective

The general objective of this thesis work was to evaluate the total antioxidant activity of thyme

(Thymus Schimperi) and study its preservative effect on oil, butter and meat to prevent the

challenge of lipid oxidation.

Specific Objectives

The specific objectives of this study were to:

Study the effect of extraction parameters on thyme antioxidant activity and its extract

yield

Evaluate the total antioxidant activity of T. Schimperi on soybean oil and butter

Examine the antioxidant content of butter samples collected from different locations

Study the preservative effect of thyme on butter, meat and oil food products

Conduct techno- economic feasibility analysis of the present work for thyme extraction

process

1.4. Scope of the study

The study was extended in collection of T. Schimperi species of thyme from North Shewa Zone;

Tarmaber. Drying and grinding it to appropriate size. Determinations of thyme crude extract

antioxidant activity by measuring induction period using oxidation inhibition system.

Application of the thyme extract on specified foods such as butter, meat and oil at different

concentration and study the thyme extract preservative effect. Chemical and microbiological

analyses were performed. Finally, the techno-economic feasibility study was done for crude

thyme extract production.

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Chapter Two

Literature Review

2.1. Distribution of thyme in Ethiopia Thyme is mainly a temperate taxon and is uncommon in the African tropics. Ethiopia has

considerably abundant Lamiaceae family plants growing at different regions and possesses a

variety of the wild growing species of this family. Many species belonging to different genera

of the family Lamiaceae have been reported to found in different parts of the country. The two

species, T. Schimperi Ronniger and T. serrulatus Hochst.ex indigenous to Ethiopia (Demsew et

al., 1994; Asfaw et al., 2000) while T. vulgaris is a species, native to southern Europe. Thymus

Schimperi is comparatively widespread in Central, Eastern and Northern Ethiopia. It is locally

known as Tosign. Bale, Shewa, Gonder and Wollo are the major growing areas in Ethiopia

(Demsew, 1993; Asfaw et al., 2000). Wild thyme of T. Schimperi is harvested and dried by

people living close to the town of Dinsho and near Menz, put in plastic bags and sold to

travelers on buses.

2.2. Chemical Composition and use of thyme The principal components of thyme are thymol and carvacrol (up to 64% of oil), along with

linalool, p-cymol, cymene, hymene, α- pinene, apigenin, luteolin, and 6-hydroxyluteolin

glycosides, as well as di-, tri- and tetramethoxylated flavones, all substituted in the 6-position

(for example 5,4-dihydroxy-6,7-dimethoxyflavone, 5,4-dihydroxy-6,7,3-trimethoxyflavone) and

its 8-methoxylated derivative 5,6,4-trihydroxy-7,8,3-trimethoxyflavone. Thymol and γ-

terpinene are found to be the major constituents of T. vulgaris. Investigation of the chemical

compositions of the two thymus species in Ethiopia (T. schimperi R. and T. serrulatus Hochst.ex

Benth) was limited except for the volatile oil constituents by Dagne et al. (1998) for essential oil

constituents. It was found that oil obtained from T. Schimperi grown in Addis Ababa was rich in

carvacrol (66.2%), γ-terpinene (13.2%) while thymol (50%), γ-terpinene (12.1%) carvacrol

(10.1% and p-cymene (10.0%) were the major constituents in the same species from Dinshu.

The main uses of thyme in culinary and food processing are defined by the following properties

of thyme components: (i) Aroma and flavour, (ii) antioxidant and (iii) antimicrobial activities.

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Thyme is prepared as infusion to treat spasmodic cough, laryngitis, bronchitis and urinary

infections. It is also used as a decongestant, as a cholagoge, to reduce flatulence and to fight

parasites. External uses of thyme include preparations to wash skin wounds or infections

(Asfaw et al., 2000). Researches conducted in the Ethiopian traditional medicine shows that the

plant has many medicinal applications. Some of the reported applications are for the treatment

of gonorrhea, cough, inflammation, spasm, thrombosis, urinary retention, mental illness, eye

disease, toothache, stomach problems, leprosy, lung TB, acne and ascaris (Peter, 2004).

Antimicrobial activity

Thyme essence, especially the phenolic components thymol and carvacrol, show antibacterial

activity against gram-positive and gram-negative bacteria, due to their effects on the bacterial

membrane. Since thymol and carvacrol are eliminated through the respiratory tract, these

compounds have respiratory antiseptic action. Because of its antibacterial activity, thyme is also

useful as an antiseptic for the urinary tract, mouth and skin wounds. Thyme water extract

showed significant in vitro inhibitory effects on the growth of Helicobacter pylori and its

powerful urease activity (Fullas, 2003).

Antioxidant activity

The Thymol and carvacrol, present in thyme essence, as well as the flavonoids and other

polyphenols are considered to be involved in the antioxidant activity.

OH OH

Thymol Carvacrol

Figure 1: Major antioxidants among thyme extract constituents (source: Dorman and Hitunen, 2004)

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Rosmarinic acid, hydroxycinnamic derivatives and flavonoid compounds showed important in

vitro antioxidant activity by inhibiting iron-induced superoxide anion formation and lipid

peroxidation in microsomal and mitochondrial systems. Furthermore, the thymol present in the

essential oil showed in vitro antioxidant activity by neutralizing the DPPH (diphenyl-

picrylhydrazyl) radical (Awraris Derbe, 2009).

2.3. The development of oxidative rancidity in foods

Fats, oils and lipid-based foods deteriorate through several degradation reactions both on heating

and on long term storage. The main deterioration processes are oxidation reactions and the

decomposition of oxidation products which result in decreased nutritional value and sensory

quality. The off-flavours that develop during lipid oxidation normally act as a warning that a

food is no longer edible, although this does not apply to polyunsaturated lipid supplements taken

in capsule form. Understanding of the mechanisms by which lipids deteriorate developed rapidly

during the twentieth century. Autoxidation reactions commonly show an induction period, which

is a period during which very little change occurs in the lipids. After the end of the induction

period, oxidative deterioration of the lipids occurs much more rapidly. Off-flavours become most

noticeable after the end of the induction period. Lipid oxidation is one of the major causes of

quality deterioration in muscle foods following storage at refrigerated or frozen temperatures.

Often seen in later stages of storage, quality losses are manifested through a variety of

mechanisms (Allen and Hamilton, 1994).

The two major components involved in lipid oxidation are unsaturated fatty acids and oxygen. In

this process, oxygen from the atmosphere is added to certain fatty acids, creating unstable

intermediates that eventually break down to form unpleasant flavor and aroma compounds.

Although enzymatic and photogenic oxidation may play a role, the most common and important

process by which unsaturated fatty acids and oxygen interact is a free radical mechanism

characterized by three main phases:

Initiation: Initiator (In) + RH → InH + R•

Propagation: R• + O2→ ROO•

ROO• + RH → R• + ROOH

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Termination: 2ROO• → O 2 + ROOR

ROO• + R• → ROOR

Initiation occurs as hydrogen is abstracted from an unsaturated fatty acid, resulting in a lipid free

radical, which in turn reacts with molecular oxygen to form a lipid peroxyl radical. While

irradiation can directly abstract this hydrogen from lipids, initiation is frequently attributed in

most foods, including muscle foods, to reaction of the fatty acids with active oxygen species. The

propagation phase of oxidation is fostered by lipid–lipid interactions, whereby the lipid peroxyl

radical abstracts hydrogen from an adjacent molecule, resulting in a lipid hydroperoxide and a

new lipid free radical. Interactions of this type continue 10 to 100 times before two free radicals

combine to terminate the process. Additional magnification of lipid oxidation, however, occurs

through branching reactions (also known as secondary initiation). The radicals produced will

then proceed to abstract hydrogen from unsaturated fatty acids (Michael, 2001).

A. Initiation The direct reaction of a lipid molecule with a molecule of oxygen is highly improbable because

the lipid molecule is in a singlet electronic state and the oxygen molecule has a triplet ground

state. To avoid this spin restriction, oxygen can be activated by any of the following three

initiation mechanisms:

formation of singlet oxygen;

formation of partially reduced or activated oxygen species such as hydrogen peroxide,

superoxide anion, or hydroxyl radical; and/or

formation of active oxygen–iron complexes (ferryl iron or ferric–oxygen–ferrous

complex).

In addition, the oxidation of fatty acids may occur either directly or indirectly through the action

of enzyme systems, of which three major groups are involved: microsomal enzymes,

peroxidases, and dioxygenases, such as lipoxygenase or cyclooxygenase. Therefore, activated

oxygen species are likely to be present in the food item even before it is harvested, not just

produced during processing and storage.

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B. Propagation Propagation reactions form the basis of the chain reaction process and in general include the

following:

Radical coupling with oxygen: R• + O2 → ROO•

Atom or group transfer: ROO• + RH → ROOH + R•

Fragmentation: ROO• → R• + O2

Rearrangement and Cyclization

Conditions that determine the chain propagation length include initiation rate, structures of

aggregates (increasing with increasing structure of the aggregates), temperature, presence of

antioxidants, and chain branching. Chain branching involves the breakdown of fatty acid

hydroperoxides to the lipid peroxyl or alkoxyl radical. Given the bond dissociation energies of

LOO–H (about 90kcal/mol) and LO–OH (about 44kcal/mol), spontaneous decomposition is

unlikely at refrigerated or freezing temperatures. Instead, breakdown of hydroperoxides would

be dominated by one-electron transfers from metal ions during low temperature storage. The

major contributors to decomposition of lipid hydroperoxides in food and biological systems

would be heme and non-heme iron, with reactions involving the ferrous ion occurring much

more quickly than those involving ferric ion.

C. Termination To break the repeating sequence of propagating steps, two types of termination reactions are

encountered: radical–radical coupling and radical–radical disproportionation, a process in which

two stable products are formed from two radicals by an atom or group transfer process. In both

cases, non-radical products are formed. However, the termination reactions are not always

efficient. Secondary and primary peroxyl radicals, on the other hand, terminate efficiently by a

mechanism in which the tetroxide decomposes to give molecular oxygen, an alcohol, and a

carbonyl compound.

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The retardation of these oxidation processes is important for the food producer and, indeed, for

all persons involved in the entire food chain from the factory to the consumer. Oxidation may be

inhibited by various methods including prevention of oxygen access, use of lower temperature,

inactivation of enzymes catalyzing oxidation, reduction of oxygen pressure, and the use of

suitable packaging. Another method of protection against oxidation is to use specific additives

which inhibit oxidation. These are correctly called oxidation inhibitors, but nowadays are mostly

called antioxidants.

2.4. Antioxidants and their effects in food preservation Antioxidants in food may be defined as any substance which is capable of delaying, retarding or

preventing the development of rancidity in food or other flavour deterioration due to oxidation.

Antioxidants delay the development of off-flavours by extending the induction time. Addition of

antioxidants after the end of this period tends to be ineffective in retarding rancidity

development. The induction time (IT) is very sensitive to small concentrations of components

that shorten it; the pro-oxidants, or lengthen; antioxidants. Metal ions are the most important pro-

oxidants in foods, whereas antioxidants include compounds that act by radical scavenging, metal

chelating or other mechanisms (Michael, 2001).

Antioxidants can inhibit or retard oxidation in two ways: either by scavenging free radicals, in

which case the compound is described as a primary antioxidant, or by a mechanism that does not

involve direct scavenging of free radicals, in which case the compound is a secondary

antioxidant. Primary antioxidants include phenolic compounds such as vitamin E (α-tocopherol).

These components are consumed during the induction period. Secondary antioxidants operate by

a variety of mechanisms including binding of metal ions, scavenging oxygen, converting

hydroperoxides to non-radical species, absorbing UV radiation or deactivating singlet oxygen.

Normally, secondary antioxidants only show antioxidant activity when a second minor

component is present. This can be seen in the case of sequestering agents such as citric acid

which are effective only in the presence of metal ions, and reducing agents such as ascorbic acid

which are effective in the presence of tocopherols or other primary antioxidants (Michael, 2001).

13

The most important mechanism of antioxidants is their reaction with lipid free radicals, forming

inactive products. Additives with this mechanism are antioxidants in the proper sense. Usually,

they react with peroxy or alkoxy free radicals, formed by decomposition of lipid hydroperoxides.

Other inhibitors stabilize lipid hydroperoxides, preventing their decomposition into free radicals.

Some substances called synergists demonstrate no antioxidant activity in themselves, but they

may increase the activity of true antioxidants (Allen and Hamilton, 1994).

2.5. Antioxidants and health

Hence, food scientists often equate antioxidants with “inhibitors of lipid peroxidation and

consequent food deterioration.” By contrast, in the human gastrointestinal tract as well as within

the body tissues, oxidative damage to proteins and DNA is just as important as damage to lipids.

Indeed, oxidative DNA damage may be a major risk factor for the development of cancer, so that

dietary antioxidants able to decrease such damage in vivo would be expected to have an

anticancer effect (Halliwell and Gutteridge, 2007).

In the last few decades, several epidemiological studies have shown that a dietary intake of foods

rich in natural antioxidants correlates with reduced risk of coronary heart disease; particularly, a

negative association between consumption of polyphenol-rich foods and cardiovascular diseases

has been demonstrated. This association has been partially explained on the basis of the fact that

polyphenols interrupt lipid peroxidation induced by reactive oxygen species. A large body of

studies has shown that oxidative modification of the low-density fraction of lipoprotein is

implicated in the initiation of arteriosclerosis. More recently, alternative mechanisms have been

proposed for the activity of antioxidants in cardiovascular disease, which are different from the

simple shielding of low density lipoprotein from reactive oxygen species, induced damage.

Several polyphenols recognized for their antioxidant properties might significantly affect cellular

response to different stimuli, including cytokines and growth factors (Lester and Enrique, 2002).

Dietary consumption of polyphenols is associated with a lower risk of degenerative diseases. In

particular, protection of serum lipids from oxidation, which is a major step in the development of

arteriosclerosis, has been demonstrated. More recently, new avenues have been explored in the

capacity of polyphenols to interact with the expression of the human genetic potential. The

understanding of the interaction between this heterogeneous class of compounds and cellular

14

responses, due either to their ability to interplay in the cellular antioxidant network or directly to

affect gene expression has increased (Virgilli et al., 2001).

Foods rich in antioxidants may afford a degree of protection against free radical damage not only

in foods, but also in the human body, protecting against cardiovascular diseases, damage of

nucleic acids, and other deteriorative processes. The absorption of tocopherols and carotenoids

into the blood stream is well known, but much less has been published on the fate of other

antioxidants and their reaction products. Some antioxidants may not be absorbed in the intestinal

tract at all, even when they are active in foods (Lester and Enrique, 2002).

2.6. Natural antioxidants Antioxidants were first used before World War II for food preservation. These early antioxidants

were natural substances. They were, however, soon replaced by synthetic substances, which were

cheaper, of more consistent purity, and possessed more uniform antioxidant properties. Much

interest has developed during the last few decades in naturally occurring antioxidants because of

the adverse attention received by synthetic antioxidants and because of the world wide trend to

avoid or minimize the use of artificial food additives. The increased use of various synthetic food

additives was then challenged by consumer groups. Consumers wished to have these additives

replaced by natural materials, which were considered to be more acceptable as dietary

components. Industrial producers have tried to comply with consumers’ wishes, and have moved

to increased use of natural antioxidants. Most natural antioxidants are common food components,

and have been used in the diet for many thousands of years so that humans have adapted to their

consumption (Jan and Michael, 2001).

2.6.1. Sources of natural antioxidants and their preparation Herbs, spices and teas are one of the most important targets in the investigation of natural

antioxidants from safety the point of view. In the evaluation of spices as natural antioxidant,

some investigations have been carried out using the whole spice. Rosemary and sage were the

most effective antioxidants in lard and both spices were found to have a low redox potential in

sausages indicating antioxidative activity. However, in an oil-in-water emulsion, clove was the

most effective spice. In general, the stabilization factors obtained for the spices in the emulsions

15

were several times greater than those in lard, indicating a higher efficiency against oxidation in

the emulsion (Jan and Michael, 2001).

Thyme comes originally from the regions around the Mediterranean and is used as cough

medicine. The common English word ‘thyme’ covers both the genus and the species most widely

used, Thymus vulgaris L. (common thyme, garden thyme). Thymus vulgaris is native to southern

Europe, from Spain to Italy. It is commonly cultivated there as well as in most mild-temperate

and subtropical climates, which include southern and central Europe (Peter, 2004).

From the aromatic and medicinal points of view, thyme is indeed the most important herb and is

widely used as a flavouring agent, a culinary herb and as herbal medicine. The commercial

products that are obtained from thyme include essential oils, oleoresins, fresh and dried herbs.

The list of thyme applications includes almost all foods: beverages, cheese, fish, meat, salad

dressings, sauces, vegetables, egg dishes, poultry, soups and honey. Usually, owing to its sensory

characteristics, thyme is not suitable for sweet products (Peter, 2004).

There is a big difference between the preparation of synthetic antioxidants and natural

antioxidants for application in food products and processing. Synthetic antioxidants are produced

as pure substances of constant composition, and are applied as such or in well defined mixtures

with other pure substances. Application is thus relatively easy, requiring no substantial

modifications of the recipe and processing conditions. On the contrary, natural antioxidants are

available from raw materials of variable composition. Both the content of active substances

(usually a mixture of several compounds) and the content of various other compounds, either

inactive or possessing negligible activities, depend on the plant variety, agro-technology,

climatic conditions, degree of ripeness, and many other factors. Their composition should be

determined in every batch, and if necessary, the procedure of their preparation or application,

and the amount added to food products should be adapted according to analytical results. Some

nature-identical antioxidants, such as α-tocopherol or β-carotene, are available on the market in a

pure form or in defined solutions so that they can be added very easily in the amount desired.

Solutions of these compounds are prepared in the industry in order to improve the solubility of

the preparation in the food to be stabilized (Jan and Michael, 2001).

16

Many other food components possessing antioxidant activities are used in their natural form,

such as spices. The preliminary processing of such food components may be drying (in case of

leaves or stems), milling of dried material (such as seeds), or some other mechanical treatment.

Several ground spices (added in the amount of 5%) were found to be active in sunflower oil,

especially, sage, sumac and thyme. An alternative is to prepare a paste from soft, water-

containing substances, and to add it as a part of the recipe. These preparations are added to the

mass of components before processing, or they may be applied on the surface of food products

as it is exposed to heat and oxygen more than the inner layers. Rosemary oleoresin extract is

found to be efficient on application on the surface of muscle tissue from rainbow trout.

Sometimes, these ingredients are added after thermal processing, such as roasting, just to

prevent the destruction of antioxidants during the processing. The content of active

antioxidants in natural materials is usually rather low so that large additions would be

necessary to obtain a significant improvement in stability against oxidation. However, such

large additions could have a negative effect on the flavour or functional properties of the

product. It is often useful to prepare more concentrated materials. The easiest way is to remove

water by a suitable drying procedure and the next optimal procedure is extraction. The choice

of solvent is of crucial importance (Jan and Michael, 2001).

2.6.2. The use of natural antioxidants in food products Meat Control of oxidation in meat systems can occur in the raw or cooked meat system. In the raw

meat system, factors affecting the levels of endogenous antioxidants have been receiving a

great deal of attention in the literature in the 1990s. Most of the activity was focused on the

application of exogenous antioxidants during processing. Exogenous factors affecting the

oxidative stability of meats include the level of processing, cooking technique/time, pre- and

post-cooking storage time and temperature, packaging system, and/or use of antioxidants (Jan

and Michael, 2001).

Since about 1990, increasing the levels of tocopherols and carotenoids in muscle tissue via

dietary supplementation (endogenous antioxidants) has shown strong promise for increasing

the oxidative stability of muscle foods. Adding antioxidants during processing has been the

more traditional technique (exogenous antioxidants) used to control lipid oxidation in meats.

17

Some of the added compounds are found at low levels in meats, i.e. ascorbic acid and

carnosine, while others are derived from plants, i.e. phenolics/polyphenolics (Descalzo et al.,

2008).

Dairy product Milk is a very interesting and very perishable food system with a shelf-life affected by many

factors including microbial load, processing factors (such as agitation, temperature of

processing and/or storage prior to processing, exposure to light) (Jan and Michael, 2001).

The susceptibility of butter to oxidative reactions has been investigated. In 1986, Emmons

and his coworkers showed that butter held frozen (-18℃) and in the dark showed no evidence

of oxidation after 1 year of storage, but there was some loss of butter quality after 14 weeks

storage in the dark at 5°C. However, when (Luby et al, 1986) stored butter in light, either in

fluorescent in the cold (5°C) or daylight at 22°C, evidence of lipid oxidation (cholesterol oxide

production) was found. Their data showed that both singlet and free radical oxidation was

occurring.

Other researchers have looked at the potential for natural antioxidants to prevent lipid

oxidation in butter. In 1996, Zegarska and other researchers showed that an ethanolic extract of

rosemary increased the stability of butter against oxidation and that the effect was

concentration dependent. This study also evaluated the effectiveness of the rosemary extract in

inhibition of copper-catalyzed oxidation and found evidence that the extract was able to chelate

metals.

According to Farag et al. (1990) thyme and cumin essential oils could prevent oxidation in

butter stored at room temperature, and at 200ppm the essential oils were more effective than

BHT in inhibiting lipid oxidation in the butter. The researchers felt the preservative effect of

the essential oils from thyme and cumin was due to the phenols found in the oils. The phenolic

hydroxy group would be able to donate hydrogen to lipid.

18

Edible oil Lipids in foods of vegetable origin are usually more unsaturated than lipids of foods of animal

origin, therefore, the initiation rate of oxidation reactions is higher and natural antioxidants,

originally present in foods are more rapidly consumed than in lard or tallow and other animal

fats. The stabilization of products of vegetable origin against autoxidation is thus less efficient

than the stabilization of animal products. Protection factors of comparable antioxidants are

several times higher in lard than in edible oils (Jan and Michael, 2001).

Edible oils become rancid on storage, the type of rancid off-flavor depending on their fatty acid

composition (for example, it may become painty and fishy in oils containing linolenic acid,

such as rapeseed oil) and the presence of minor components (for example, in flavor-reverted

soybean oil). Edible oil producers try to prolong the shelf life of edible oils by different

techniques, including the addition of antioxidants. The presence of natural antioxidants should

always be taken into account, when appropriate levels of added antioxidants are considered

(Mariassvoya, 2006).

The antioxidative effect of an ethanol extract from savory (Satureja hortensis L.) in sunflower

oil was investigated during high temperature treatment (at 180°C). The extract improved the

oxidative stability of sunflower oil even after 50 hrs at 180 °C and inhibited the oxidative

processes more than the thermal processes under these conditions. Main components of the

extract are thymol and carvacrol, the former being more active than the latter (Beddows et al.,

2000).

2.6.3. Measuring antioxidant activity Antioxidants are used in a wide variety of food products, and their activity may vary depending

on the temperature, food composition, food structure and availability of oxygen. Temperatures

at which antioxidant activity may be required range from 180–200°C for frying oils, to about

5°C for products such as margarine or mayonnaise that are stored in the fridge. Besides the

processing and storage temperatures to which these products are exposed, the accompanying

constituents including water, proteins, carbohydrates, vitamins, minerals and other food

components vary, and the physical structure of the food also varies. This can cause big changes

in the activity of the antioxidant in different food systems. It is commonly observed that a non-

polar antioxidant such as α-tocopherol is relatively ineffective in oil but is strongly effective in

19

an oil-in-water emulsion. In contrast, a polar antioxidant such as ascorbic acid or trolox (a

water-soluble derivative of α-tocopherol) is more effective in oil than in an emulsion (Michael,

2001).

Normally, a more rapid measurement of antioxidant activity is required than would be obtained

by making the food product, storing it at ambient temperature and then measuring the oxidative

state of the food. Consequently, there are three decisions to be made:

The model food system used for antioxidant activity test

Most assessments of antioxidant activity have been performed in oil. This commonly gives

sensible predictions for the activity in oil or water-in-oil emulsions such as margarine, but the

data may be misleading for oil-in-water emulsions. Some information may be gained by the

use of a radical-scavenging test in an organic solvent.

Method of accelerating oxidation process

The most common methods of accelerating oxidation are to raise the temperature and to

increase the supply of oxygen. The combination of these effects can reduce the oxidative

stability by a large amount. Other factors affecting the oxidation rate include the content of

metal ions in the test sample, the oxidative state of the test sample before the addition of

antioxidant and exposure to UV light.

Monitoring method for oxidation process

In principle, one could consider measuring the loss of lipid starting material, i.e. fatty acids or

triglycerides, or the formation of oxidation products as a method of monitoring oxidative

deterioration or antioxidant activity. In practice, the formation of oxidation products is a much

more sensitive method of monitoring oxidation. However, the assessment of antioxidant

activity by monitoring the formation of oxidation products is not a simple task. Since a

complex mixture of oxidation products is formed and the relative amounts of these products

depend on a variety of variables including temperature, metal ion content, and other

components present such as water, deciding which components to monitor is an important

decision. Monitoring antioxidant activity under frying conditions may well require other

products to be monitored than if the activity is to be assessed under ambient conditions. Thus,

20

hexanal formation can be used to monitor oxidative deterioration in ambient stored products,

but not in used frying oils.

Antioxidant activity of a given compound is assessed as either resistance to oxidation of lipids

in the presence of that particular compound or free-radical scavenging capacity. Therefore,

most of the methods described and used to assess antioxidant activity follow oxidation and the

stages of oxidation of unsaturated lipid substrates. Many techniques have been developed to

determine the antioxidant efficacy of the compounds of interest, but all of these have to be

employed and interpreted carefully. In 1993, Frankel has listed the following parameters such

as Substrate, condition, analysis, concentration and calculation that are fairly important in

choosing methods to evaluate antioxidants.

An updated review by (Antolovich et al., 2002) discusses the methods of determining

antioxidant activity extensively. The methods used in measuring antioxidant activity may be

categorized into three groups, which directly or indirectly measure the rate or extent of the

following:

Decay of substrate, probing compound, or oxygen consumption

Formation of oxidation products by the oxidizing substrate

Formation or decay of probing free radicals for DPPH (radical scavenging methods)

The first two methods measure antioxidant activity as an inhibitory effect exerted by the test

compound on the extent or rate of consumption of reactants or the formation of oxidation

products. The antioxidant activity (AA) of a compound or a component mixture that is a

function of many parameters of the assay method employed may be defined using the

following mathematical expressions:

AA = f (time or rate; temperature; substrate; concentration of antioxidant; concentration of

other substances; partitioning behavior).

For a fixed set of assay conditions, AA could be defined independent of the test method. It

should be noted here that there are no standard units for reporting the antioxidant activity

because such activity (assay, capacity, efficiency, effectiveness, etc.) is independent of the test

procedure.

21

A. Radical scavenging method Radical scavenging is the main mechanism by which antioxidants act in foods. Several

methods have been developed in which the antioxidant activity is assessed by the scavenging

of synthetic radicals in polar organic solvents, e.g. methanol, at room temperature. Those used

include 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2'-azinobis (3-ethylbenzthiazoline-

sulphonic acid) (ABTS) radicals.

In the DPPH test, the scavenging of DPPH radicals is followed by monitoring the decrease in

absorbance at 515 nm which occurs due to reduction by the antioxidant (AH) or reaction with a

radical species (R·)

DPPH· + AH → DPPH–H + A·

DPPH· + R· → DPPH–R

Fast reaction of DPPH radicals occurs with some phenols e.g. α-tocopherol, but slow

secondary reactions may cause a progressive decrease in absorbance, so that the steady state

may not be reached for several hours. Most papers in which the DPPH method has been used

report the scavenging after 15 or 30 min reaction time. The data is commonly reported as

EC50, which is the concentration of antioxidant required for 50% scavenging of DPPH radicals

in the specified time period.

The ABTS radical cation is more reactive than the DPPH radical, and reaction of the ABTS

radical cation with an antioxidant is taken as complete within 1 min. The method of generation

of the radical cation has changed several times since the method was first described. The most

recent method describes the use of potassium per sulphate to oxidize ABTS to the radical

cation. The radical scavenging activity assessed by the ABTS method has been expressed as

the TEAC (trolox equivalent antioxidant capacity) value in most papers employing this

method. These methods may be useful for screening antioxidants, but antioxidant effectiveness

in foods must always be studied by other methods because their activity in foods is dependent

on a variety of factors including polarity, solubility, and metal-chelating activity (Antolovich et

al., 2002).

22

B. Inhibitory effect of antioxidant on lipid oxidation Some methods can be applied for assessing the current state of an oil or food sample. In order

to be applied in assessment of antioxidant effectiveness, an experiment must be designed in

which the antioxidant is incorporated into the food and the food is stored under controlled

conditions. The principles of these methods are described below.

I. Sensory analysis For the food industry, the detection of oxidative off-flavours by taste or smell is the main

method of deciding when a lipid-containing food is no longer fit for consumption.

Consequently, any antioxidant used in the food will ultimately be evaluated by its potential for

extending the time before this off-flavour can be detected. The ability of individuals to describe

the nature of the aroma is useful, and the sensitivity of a trained panel to oxidative off-flavours

may allow detection of oxidative deterioration. The main problems with sensory evaluation are

that different individuals vary in their sensitivity to these off-flavors, and their performance

may vary depending on their state of health and other variables. Trained panelists are much

more reliable than untrained panelists, but the reproducibility of sensory analysis is normally

worse than that of chemical or instrumental methods (Jan and Michael, 2001).

II. Peroxide Value (PV) The PV is still the most common chemical method of measuring oxidative deterioration of oils

and fats. Although hydroperoxides decompose to a mixture of volatile and non-volatile

products and they also react further to endoperoxides and other products, the PV measurement

is a useful method of monitoring oxidative deterioration of oils and fats, although it should

normally be combined with a method of monitoring secondary oxidation products to provide a

fuller picture of the progress of oxidation. The traditional method of determining PV involves a

titration of the oil and fat containing potassium iodide in a chloroform–acetic acid mixture. The

hydroperoxides oxidize the iodide to iodine, which is determined by titration with sodium

thiosulphate. In order to avoid the use of chloroform, the AOCS has developed an alternative

method which uses iso-octane as solvent, although the method is limited to PV less than 70meq

kg-1, as described in the AOCS guidelines (AOCS, 1989).

23

III. Thiobarbutric Acid Value (TBA)

Malonaldehyde may be formed from polyunsaturated fatty acids with at least three double

bonds. The concentration of this product may be assessed by reaction with thiobarbituric acid

which reacts with malonaldehyde to form red condensation products that absorb at 532–535nm

with molar absorptive of 27.5 absorbance units/µmol. However, the reaction is not specific, and

reaction with a wide variety of other products may contribute to the absorbance. Saturated

aldehydes normally absorb at lower wavelengths after reaction with TBA. The TBA test has

recently been reviewed. Several food components including proteins, Maillard browning

products and sugar degradation products affect the determination. In order to emphasize the lack

of specificity, the values obtained in the test are commonly described as TBARS (TBA reactive

substances) (Guillen-Sans and Guzman-Chozas, 1998).

C. Predictive methods

Oil stability Index (OSI) is predictive method in which samples are continuously monitored

during accelerated oxidation conditions. The OSI is an automated development of the AOM

(active oxygen method). In the AOM, the time for an oil to reach a PV of 100 meq kg-1 during

oxidation at 97.8°C, with an air flow of 2.33 ml per tube per second is determined. Instruments

for determining the OSI are the Rancimat, manufactured by Metrohm, Basel or the Oxidative

Stability Instrument, manufactured by Omnion, Rockland, USA. These instruments depend on

the increase in electrical conductivity, when effluent from oxidizing oils is passed through

water. The samples, assessed by the OSI methods, are held at 100°C, 110°C, 120°C, 130°C, or

140°C. The temperature may be adjusted to allow the oxidation time to fall within the range of

4–15 h. The sample size is 2.5 g or 5 g depending on the instrument used. Volatile carboxylic

acids are generated in the oxidizing oil and these cause the increase in electrical conductivity

(Jan and Michael, 2001).

Rancimat test is an accelerated method to assess oxidative stability of fats and oils. In this test,

the sample is subjected to an accelerated oxidative process (by heat in presence of oxygen),

where short-chain volatile acids are produced. The acids formed are measured by conductivity.

Mariassyova (2006) studied the antioxidant potential of natural antioxidant concentrates with

high contents of flavonoids, carotenoids, and phenolic acids using this method. The assay has

24

been used to evaluate the antioxidant activity of several food products, including herbs and

spices (Beddows et al., 2000).

Among these antioxidant activity measuring methods, the research was focused to use some of

them based on the availability of chemicals and equipments required for the study. The

antioxidant activity of thyme was studied using Inhibitory effect of antioxidant on lipid

oxidation such as predictive and peroxide value method. In other hand, these methods are called

Rancimat and Schaal Oven test methods which are mentioned a lot in this study.

2.7. The regulation of antioxidants in food

Since food is essential to life and can be improperly prepared or handled, it can threaten life. The

purveyor of food therefore has a duty to provide safe and wholesome products to every customer.

Given the fundamental importance of food, it is appropriate for any government to define and

enforce this ethical obligation and thereby protect what many would consider the right of every

individual to safe and wholesome food. From the legal point of view, antioxidants are substances

which prolong the shelf-life of foodstuffs by protecting them against deterioration caused by

oxidation, such as fat rancidity, color changes and loss of nutrient value. Antioxidants are

extensively tested for the absence of carcinogenity and other toxic effects in themselves, in their

oxidized forms, and in their reaction products with food constituents, for their effectiveness at

low concentrations, and for the absence of the ability to impart an unpleasant flavor to the food

in which they are used (Jan and Michael, 2001).

Antioxidants should satisfy several requirements before being accepted for incorporation into

food products. The use of antioxidants in food products is governed by regulatory laws of the

individual country or by internal standards. Even though many natural and synthetic compounds

have antioxidant properties, only a few of them have been accepted as generally recognized as

safe substances for use in food products by international bodies such as the Joint FAO Expert

Committee on Food Additives and the European Community’s Scientific Committee for Food.

Antioxidants can be added directly to vegetable oils, melted animal fats or other fat-containing or

polyphenol-containing systems. Food products can also be sprayed with, or dipped in solutions

or suspensions of, antioxidants, or they can be packed in films containing antioxidants.

25

Chapter Three Materials and Methods

3.1. Structure of Thesis Experiment The research was conducted following this general flow sheet which contain the major unit

Operations and activities performed during the study.

Fig. 3.1: Experimental framework of the thesis

Raw Material (thyme) Preparation Harvesting

Cleaning Drying and Grinding

Extraction Ethanol solvent Extraction Temperature Extraction Time

Thyme Antioxidant Activity Evaluation

Schaal Oven Test

Rancimat Method

Butter

Soybean Oil

Preservative Effect of thyme

Chemical Analysis

Microbiological Analysis

Meat

Free Fatty Acids Peroxide Value Acid Value

Total Viable Count Yeast &Mold Count Enterobacteriaceae

Count

Solvent

Thyme Extract Yield

26

3.2. Raw material Collection, Transportation and Storage Thyme (Thymus Schimperi R.) was obtained from Tarmaber areas of North Shewa zone, 180km

away from Addis Ababa, where this thyme variety is found. Both leave and flower part of thyme

were manually collected and dried with natural sun drying system in protective and shaded way.

The dried thyme was packed in polyethylene plastic bags and taken to Bahir Dar University,

Technology Institute, Food Chemistry & Analysis Laboratory for further study. The thyme was

mill by a small scale commercial laboratory grinder to pass a sieve size of 500µm and retained

on 420µm to get uniformly sized thyme powder. The moisture content of dried thyme was

determined before extraction. It has a moisture content of 12.8% in wet weight basis.

Thyme Antioxidant evaluation Equipment: 743 Rancimat

The Rancimat (Model 743, Metrohm, Switzerland) is a modern, PC-controlled analytical

instrument for the comfortable determination of the oxidation stability of oils and fats. The

unique temperature extrapolation allows a rough estimation of the storage stability of a product.

The Rancimat has two independent heating blocks that allow up to eight samples which has been

analyzed at one or two temperatures. Each measuring position can be started individually. As

soon as the measurement has been completed the measuring position is immediately ready for a

new sample, which means that the instrument can be used to its full capacity. The conductivity

cell is incorporated in the measuring vessel cover. When the cover is placed in position the cell is

immersed in the distilled water. At the same time electrical contact is made to the electronics in

the instrument to measure conductivity versus induction time data. All its functions are

controlled by the PC connected with RS-232 cable. The air flow used for the measurement is

aspirated through a filter that prevents particles entering to instrument. The molecular sieve

removes water vapor from the aspirated air; as water contributes to the hydrolytic decomposition

of the fat molecules, it could interfere with the measurement. The amount of air that passes

through the sample is automatically controlled by the rotation of rate of the built-up in pump

according to the method setting. During the measurement a stream of air is passed through the oil

or fat sample contained in a sealed and heated reaction vessel. This treatment results in oxidation

of the oil or fat molecules in the sample, with peroxides initially being formed as the primary

oxidation products.

27

After some time the fatty acids are completely destroyed; the secondary oxidation products

formed include low-molecular organic acids in addition to other volatile organic compounds.

These are transported in the stream of air to a second vessel containing distilled water. The

conductivity in this vessel is recorded continuously. The organic acids can be detected by the

increase in conductivity. The time that elapses until these secondary reaction products appear is

known as the induction time, induction period or Oil Stability Index (OSI).

Fig 3.2: Rancimat apparatus for antioxidant activity evaluation

28

3.3. Extraction Process

3.3.1. Setting Extraction Parameters A 23 Full-Factorial Experiment Design with a central point was used to identify the relationship

existing between the dependent responses (antioxidant activity and yield) and independent

process variables as well as to determine conditions that optimized the extraction process. The

three independent variables or factors studied were: extraction solvent (ethanol) concentration of

(97 and 0%), extraction temperature (20 and 40℃) and extraction time (3 and 4 hours) for actual

variable levels. For each factor, an experimental range was adjusted based on the results of

literature data and on the performance of preliminary experiment trials.

These three factors: extraction solvent concentration of ethanol, extraction temperature and

extraction time were selected as independent variables, because of their influence on antioxidant

properties of phenolic extracts in plant materials (Wettasinghe and Shahidi, 1999).

In this study, the particle size was controlled as constant by passing mill thyme through 500µm

and retaining on 420µm sieve openings. The 420-500µm sieve size is optimal for extraction,

while smaller particles may become slimy during extraction and create difficulty during filtration

(Sukhdev et al., 2008).

3.3.2. Preparation of thyme extract Samples of about 10g of the dried, mill and sieved thyme were extracted with 100mL of solvent.

The extraction process was performed using a magnetic stirrer with hot plate. After extraction,

the samples were filtered using 125 mm diameter filter paper (Whatman Ltd., England). The

solvent ethanol was separated from extracts using a rotary evaporator (Buchi Rota-vapor R-124

fitted with Buchi water bath B-480, Switzerland) under vacuum at 45℃ and then weighed to

measure thyme extraction yield. The concentrated thyme extract was stored at -18℃ till its

antioxidant activity was determined. Whereas, aqueous extract of thyme was further freeze dried

for antioxidant activity evaluation.

29

3.4. Analysis Methods 3.4.1. Evaluation of Thyme Antioxidant activity

The best combination of extraction parameters like extraction temperature, time and solvent

concentration for maximum thyme extract antioxidant activity and yield were taken for further

thyme antioxidant activity evaluation and its preservative effect study. The Antioxidant activity

of thyme extract was determined by Rancimat and Schaal Oven test method to get induction

period.

3.4.1.1. Rancimat Method The Rancimat method is an automated version of the active oxygen method for the determination

of induction time the so called stability time of food products. In this method, the highly volatile

organic acids produced by oxidation are absorbed in distilled water and used to indicate the

induction time.

Antioxidant activity of thyme was evaluated taking a real food model system (substrate) for lipid

oxidation analysis occurs in lipid foods. Refined soybean oil and butter were taken as model food

substrates. Favorable conditions for substrate oxidation were provided to accelerate rate of lipid

oxidation process in a controlled environment of Rancimat. The induction period for the

formation of oxidative products of oxidizing substrate were measured for antioxidant activity

evaluation.

Samples of thyme extracts were added to 5.0g refined soybean oil and butter at concentration of

0.1% (w/w). For comparison, vitamin E (α- tocopherol) was added to the oil and butter at 0.05%

(w/w) concentration. At the same time the induction time of samples without thyme extract was

determined as negative control to calculate the protection factor. The maximum level of synthetic

antioxidants concentration allowed to be added in food is 0.02% for the safety reasons. In the

case of natural antioxidants, higher concentrations (0.05–0.2%) are necessary because of their

lower activities and presumed lower toxicity (Frankel, 2007). The concentration of 0.1% was

studied as it is most often used in the research as a model substance representing natural

antioxidant.

Three parallel treatments are filled into the reaction vessels and introduced in the heating blocks.

The treatments were kept at stable temperature (130℃) and continuous air stream of 20L/hr

pumped through the samples. The induction time was detected by conductivity measurements

30

and recorded by computer. Antioxidant activity of thyme extract was expressed as a protection

factor. The protection factor (PF) was calculated as:

𝑃𝑃𝑃𝑃 =𝐼𝐼𝐼𝐼𝑆𝑆𝐼𝐼𝐼𝐼𝑂𝑂

According to (Altolovich et al., 2002), Antioxidant activity of thyme extract was calculated by

measuring induction time as independent variable.

𝐴𝐴𝐴𝐴𝑡𝑡 =(𝐼𝐼𝐼𝐼𝑆𝑆 − 𝐼𝐼𝐼𝐼𝑂𝑂)([𝐴𝐴𝐴𝐴])𝐼𝐼𝐼𝐼𝑂𝑂

Rearranged as:

𝐴𝐴𝐴𝐴𝑡𝑡 =[𝑃𝑃𝑃𝑃 − 1]

[𝐴𝐴𝐴𝐴]

Where: ITs = the induction time of the sample (oil/butter + extract) [hr]

ITo = the induction time of control soybean oil [hr]

AAt = antioxidant activity of thyme extract

[AH] = concentration of thyme extract added to the oil or butter

Based on the calculation result, the protection factor can be interpreted in three ways:

PF=1 or if ITs = ITo, the thyme extract does not have antioxidant activity

PF<1, the thyme extract shows pro-oxidant activity

PF>1, the thyme extract shows antioxidant activity

3.4.1.2. Schaal Oven Test The Schaal oven test was used to determine the antioxidant activity of thyme extract by

measuring the induction time in order to measure the oxidative stability of oil. In the Schaal oven

test, 40g of samples of refined soybean oil supplemented by 0.1% thyme extract were put in

50ml bottle and placed in a drying Oven at 60℃. For comparison, both positive (α-Tocopherol at

0.05%) and negative (without thyme extract) treatments were prepared and stored in the same

condition. For each treatment, the time required to reach at the targeted peroxide value of 20mEq

31

O2/kg soybean oil (the point at which soybean oil has poor quality) has taken as Induction Time

to evaluate thyme antioxidant activity.

The Peroxide Value was determined based on (AOAC, 2000) using official method 965.33 until

it reaches 20mEq active oxygen/Kg soybean oil. Five gram sample was accurately weighed (to

the nearest 0.001 g) into each two 250-ml glass-stoppered Erlenmeyer flasks. Following 30ml of

acetic acid-chloroform, 0.5 ml saturated KI (Potassium iodide) solution was added and allowed

for 1 min with occasional shaking. Samples were slowly titrated with 0.1 N sodium thiosulfate

solution, with vigorous shaking until yellow color was almost gone. The titration was continued

by adding 0.5 ml 1% starch solution shaking vigorously to release all iodine from chloroform

layer, until blue color just disappeared. The volume of titrant was recorded. In parallel, blank

sample was Prepared (omitting only the oil) and titrated. The volume of titrant for blank sample

was also recorded. Then, the peroxide value (PV) of the samples was calculated using the

formula:

PV = (S−B)W

N × 1000

Where:

PV= mEq peroxide per kg of sample

S = volume of titrant (ml) for sample

B = volume of titrant (ml) for blank

N = normality of Na2S2O3 solution (mEq/ml)

1000 = conversion of units (g/kg)

W = sample mass (g)

32

3.4.2. Evaluation of thyme antioxidant activity in butter Initially, the antioxidant activity of all butters collected from Tarmaber, Sheno, Bahir Dar, Rut &

Tsega Milk cows breeding dairy production & processing plc (Hirut dairy PLC) and Lame dairy

PLC, were determined by Rancimat without adding thyme extract. After that the antioxidant

activity of crude thyme extract was evaluated on butter bought from Lame Dairy PLC at the

concentration of 0.1% using Rancimat method as mentioned earlier in 3.4.1.1.

3.4.3. Preservative effect of Thyme on some food products Thyme extract was added to each test samples of meat, butter and oil at three different

concentration levels; 0, 0.1and 0.2% and the samples were stored for 7, 14 and 22 days.

A total of eighteen butter samples with 40g weigh, were separately stored for microbial and

chemical analysis in both microbiology and food chemistry & analysis laboratories at 4℃

refrigeration temperature. For each analysis three butter samples were treated as blank (without

thyme extract), the other six samples were treated with 0.1% and 0.2% crude thyme extract.

For microbiological analysis of meat, three treatments were prepared with 0, 0.1 and 0.2% crude

thyme extract. Each treatment has about100g of meat and stored at 4℃ refrigeration temperature.

In every week, about 25g sample was taken from each treatment for total viable count, mold and

yeast and pathogenic microbial count.

Preservative effect of thyme crude extract was also studied on soybean oil. Nine soybean oil

samples were prepared. Each has a weight of 40g and treated with 0, 0.1 & 0.2% crude thyme

extract. The samples were stored at room temperature in dark place for three consecutive weeks.

The rancidity parameters (free fatty acid, peroxide value and acid value) were evaluated for each

treated samples per each storage week.

3.4.3.1. Chemical Analysis method Butter of Lame dairy plc and refined soybean oil were used to study the preservative effect of

thyme by chemical analysis. Oxidative rancidity parameters in fat and oil were considered.

Among rancidity parameters such as peroxide value, free fatty acid (FFA) and acid value, only

Peroxide value has direct relation and is good indicator of fat and oil oxidative rancidity. FFA

and acid value indirectly show susceptibility of butter and oils for rancidity. These rancidity

33

parameters were determined in weekly basis following the recommended methods of AOAC.

Butter and oil are selected due to their highly use in food and easily can undergo rancidity.

Determination of Free Fatty Acids (Acid Value)

Free fatty Acid and Acid value of soybean oil and butter were determined according to (AOAC,

2000) Official method of 972.28. About five gram of melted oil and butter samples were placed

in 250ml Erlenmeyer flasks and 100ml of neutralized ethanol and 2ml of phenolphthalein

indicator were added. The mixture was vigorously shake and titrated with standard 0.1N NaOH

base until the endpoint reached when slight pink color can persist for 30 seconds. This was done

in duplicate and the volume the titrant was recorded for acid value/free fatty acid calculation.

Similarly, blank was prepared without adding oil or butter and the amount of titrant it took was

recorded. For each samples the free fatty acid was calculated with the formula:

%FFA (as oleic acid) =V × N × 28.2

W

Where,

%FFA = percent free fatty acid expressed as oleic acid

V = Volume of NaOH titrant (ml)

N = Normality of NaOH titrant (mol/ 1000 ml) and

W = Sample weight (g)

Determination of Peroxide Value

The Peroxide Value of butter and oil was determined using official method 965.33 based on

(AOAC, 2000) as mentioned in 3.4.1.2.

34

3.4.3.2. Microbiological analysis Aerobic Plat Count Microbiological analysis of butter and meat was done in terms of Total Viable Count (TVC)

following (NMKL, 2006). It involved addition of 225mL peptone saline to each stomacher bag

containing 25gram samples (0, 0.1 & 0.2% thyme crude extract treated butter and meat),

followed by homogenization for 1.5 minute using stomacher. Plate Count Agar (PCA) medium

was prepared, poured to each Petri dish, and allowed to solidify and inoculated with serial

diluted sample. Each Petri dish incubated at 30oC for 72 h before colonies count. Microbial

numbers on decimally diluted plates were converted into CFU cm-1 in a standard manner.

Aerobic Mold and Yeast Count The number of viable aerobic mould and yeast per gram or mililiter of product conducted

following (NMKL, 2005). A 25gram of each butter and meat samples (0, 0.1 & 0.2% thyme

crude extract treated) was added in 225ml peptone water and homogenized at 260 rpm for 1.5

minute. Each homogenate was mixed with a specified (18-20ml solidified Potato Dextrose Agar

(PDA)) agar medium. It was assumed that each viable mould/ yeast could multiply under these

conditions and give rise to a colony. Six consecutive serial dilutions were performed for each

sample taking 1ml from mixed homogenates to 9ml peptone water (labeled as 10-1). In the same

way, 1ml was added to the next 9ml peptone water and labeled 10-2. Similar procedure was

followed until 10-6 serial dilution was obtained. From each food sample 100μl was inoculated on

appropriately marked duplicate petri-dishes. Incubation was done for specific conditions of time

and temperature (at 30℃ for 48hrs).

Enterobacteriaceae count Enterobacteriaceae count of both meat and butter was performed according to (NMKL, 2005)

with little modification. A 25gram of each butter and meat sample (control, 0.1 & 0.2% thyme

crude extract treated) was added in 225ml peptone water and homogenized at 260 rpm for 1.5

minute. Each homogenate was mixed with a specified (18-20ml solidified Violet Red Bile Agar

(VRBA)) agar medium.

Six consecutive serial dilutions were performed for each sample 1ml from mixed homogenates to

9ml peptone water (labeled as 10-1). In the same way, 1ml was added to the next 9ml peptone

35

water and labeled10-2. Similar procedure was followed until 10-6 serial dilution was obtained.

From each food sample 200μl was inoculated on appropriately marked duplicate petri-dishes.

Incubation was done under anaerobic jar for specific conditions of time and temperature (at 35℃

for 48hrs).

3.5. Experimental Design and Statistical Analysis

A 23 Full-Factorial Experiment Design with a central point was used to study the effect of

extraction parameters on thyme antioxidant activity and its extract yield as clearly stated under

3.3.1. Data obtained from the experiment were analyzed using Analysis of Variance (One way

ANOVA) method in duplicate to compare the mean value with standard deviation at significant

level of (P<0.05) of treatments by JMP statistical Analysis software version 5.0 and Design

Expert Software Version 7.0.0.

36

Chapter Four

Results and Discussion

4.1. Effect of extraction parameters on thyme antioxidant activity and its

extract yield

In this study ethanol and water were selected as extraction solvent because they are safer and less

toxic as compared to acetone, methanol and other organic solvent. As materials and methods

part, these three extraction factors: ethanol solvent concentration, extraction temperature and

time were selected as independent variables, because of their influence on antioxidant properties

of phenolic extracts in plant materials (Wettasinghe and Shahidi, 1999). For each factor, an

experimental range was adjusted based on the results of literature data and on the performance of

preliminary experiment trials.

Antioxidant activity of ethanol and distilled water extract of thyme was determined. In Table 4.1,

the results of the Rancimat analysis were given as induction time, protection factor and

antioxidant activity of the thyme with respect to extraction parameters: solvent concentration,

extraction temperature and time that affect thyme antioxidant activity and its extract yield. The

particle size of mill thyme was taken constant since 420-500µm sieve size is optimal for thyme

extraction (Sukhdev et al., 2008). The experimental results showed a different trend in the

antioxidant activity of the ethanol and distilled water thyme extract using the Rancimat assay.

The induction time of thyme crude extract widely ranged from 2.62±0.16hr to 4.09±0.39hr. The

result of one-way ANOVA showed that the distilled water extract exhibited significantly higher

total antioxidant activity (P<0.05) than that of ethanol thyme crude extract. It could be concluded

that the different polarity of the extracts might contain different antioxidant constituents that

demonstrated a varying reactivity in the model food substrates used in this work.

Results in Table 4.1 also showed that the effects of ethanol concentration on antioxidant

capacities of thyme. In this study ethanol and water were selected as extraction solvent since they

are safer and less toxic as compared to acetone, methanol and other organic solvent. The result

showed that the extraction solvent had significant effect (p<0.05) on thyme antioxidant activity.

Kwon et al. (2003) noted that the ethanol concentration had critical role in the extraction of

37

soluble components from different natural products. Even though the extraction yield of thyme

extract was increased with increasing ethanol concentration, its antioxidant activity was greatly

decreased. Thyme antioxidant activity determination by Rancimat method shows that thyme

extract obtained by distilled water resulted in higher antioxidant activity. This provides green

extraction technology for further study.

Table 4.1: Effect of extraction parameters on thyme antioxidant activity

S. No.

Extraction parameters

Induction Time (hr)

Protection Factor

(PF)= ITsIT0

Antioxidant activity

AAt =[PF − 1]

[AH] Ethanol

conc.(%)

Temp(℃)

Time(hr)

1 97 40 4.0 2.65±0.08d 1.38±0.041

d 3.8±0.410

d

2 0 20 3.0 3.92±0.25a 2.04±0.130

a 10.4±1.40

a

3 0 40 3.0 3.22±0.04b 1.68±0.021

b 6.8±0.21

b

4 0 40 4.0 3.09±0.18bc

1.61±0.093bc

6.1±0.92bc

5 0 20 4.0 4.09±0.39

a 2.24±0.203

a 12.4±2.03

a

6 97 20 4.0 2.87±0.09cd

1.50±0.047cd

5.0±0.47cd

7 97 20 3.0 2.62±0.16d 1.40±0.085

d 4.0±0.85

d

8 97 40 3.0 2.86±0.22cd

1.49±0.120cd

4.9±1.20cd

9 48.5 30 3.5 3.04±0.10bc

1.58±0.052bc

5.8±0.52bc

Means within the same column followed by the same letters are not significant difference at p< 0.05, by student’s t-test. All values are mean ± standard deviation. ITs, is Induction Time of thyme antioxidant and IT0, Induction Time of control and AAt, is antioxidant activity of thyme

Following the general principle of solvent extraction, “like dissolves like”, which means that

solvents only extracts those phytochemicals which have similar polarity with the solvents (Zhang

et al., 2007), the results of this study show that there was no single ethanol concentration could

give the highest value for crude thyme extract antioxidant activity examined by Rancimat

method. This implies that thyme contained diverse phenolic compounds that can dissolve in

different polarity of solvents. Based on Table 4.1, the highest thyme antioxidant activity was

achieved at 0% ethanol concentration or pure distilled water solvent. This circumstance could be

38

due to 100% ethanol was unable to extract polar phenolic compounds that had high antioxidant

capacity (Chirinos et al., 2007). Previous studies stated that antioxidant capacities of phenolic

compounds were associated with the availability of the phenolic compound acting as hydrogen-

donating radical scavengers (Karadeniz et al., 2005). Thus, it could be expected that there was a

high availability of phenolic compounds which can act as hydrogen-donating radical scavengers

in thyme extract obtained by distilled water.

The effects of extraction temperature on antioxidant activity (induction time) of thyme was also

shown in Table 4.1. Extraction temperature was found to be the most significant factor affecting

antioxidant activity of thyme at P < 0.05 level. Induction time was decreased with increased

thyme extraction temperature.

Based on the above result, antioxidant activity of thyme crude extract was increased

proportionally with the decreasing of extraction temperature, reaching maximum values at 20℃

(room temperature). As the extraction temperature increases, phenolic compounds of thyme

extract could be degraded and resulted in the loss of its antioxidant activity. According to Chan

et al. (2009); Liyana-Pathirana and Shahidi (2005), the loss in antioxidant capacities of plant

extracts at high extraction temperature was likely due to degradation of phenolic compounds

which were previously mobilized at low temperature. Similarly, Mueller-Harvey (2001) reported

that some phenolic compounds decomposed rapidly under high temperature and thus caused a

reduction in the antioxidant capacity of plant sample. Thus, it has been shown that the phenolic

compounds which were extracted under high temperature had lower antioxidant capacity as

compared to those which were extracted under low temperature. Based on Table 4.1, the highest

value of induction time was achieved at extraction temperature of 20℃.

Table 4.1 also shows the effects of extraction time on thyme antioxidant activity expressed as

induction time. Overall, extraction time had no significant effect (p>0.05) on antioxidant activity

of thyme. In general, the maximum thyme antioxidant activity was achieved at extraction time of

210 min (3.5 hour). After this point, thyme antioxidant capacity was decreased. It was believed

that prolonged extraction time would lead to exposure of more oxygen and thus increase the

chances for occurrence of oxidation on phenolic compounds (Naczk and Shahidi, 2004; Chirinos

et al., 2007). As stated by Naczk and Shahidi, prolonged extraction would increase the chance

for occurrence of oxidation of phenolic compounds.

39

The effects of independent variables on the yield of thyme extract were also studied. Yield of

thyme extract was determined by mass and component balance done before and after drying at

the temperature of 105℃ in oven dryer. Table 4.2 shows that ethanol concentration has direct

relation with the yield of thyme extract; it increases with increasing ethanol concentration. Based

on the result of one-way ANOVA analysis, It has significant (p<0.05) effect on thyme extract

yield. The highest recovery of thyme extract was exhibited at 48.5 and 97% ethanol

concentration. The extraction yield ranges from 10.6% to 18.13% of plant material.

Table 4.2: Effect of extraction parameters on the yield of thyme extract determined with dry weight basis (dwb)

S. No. Extraction parameters Yield of thyme extract (% dwb) Ethanol conc. (%)

Temp.(0C)

Time(hr)

1 97 40 4.0 17.17±1.46a

2 0 20 3.0 12.47±1.50bc

3 0 40 3.0 10.60±1.21

c

4 0 40 4.0 11.23±0.60bc

5 0 20 4.0 13.33±1.25

b

6 97 20 4.0 18.13±0.98a

7 97 20 3.0 18.13±1.32a

8 97 40 3.0 16.40±1.40a

9 48.5 30 3.5 18.12±1.06a

Means within the same column followed by the same letters are not significant difference at p<0.05, by student’s t-

test. All values are mean ± standard deviation

The other critical parameter affecting thyme extract yield is temperature. It has a significant

effect at P<0.05 level and greatly influence the extraction process. Based on the result, higher

thyme extract yield was obtained at lower extraction temperature; particularly at room

temperature using 48 & 97% ethanol. Thyme extract yield was decreased as the extraction

temperature increase to 40℃ depending on the concentration of the solvent.

40

Extraction time has no significant (P>0.05) effect on the yield of thyme extract as shown in the

above Table 4.2. The impact of this factor could not be seen clearly on the extract yield at fixed

levels of the other parameters.

No significant association was found between the extraction yield and antioxidant activity of

crude thyme extract. Higher yield was obtained at solvent concentration of 97 and 48.5 % of

ethanol however; distilled water alone gives superior antioxidant activity of thyme. This implies

that water soluble components of thyme exhibit better antioxidant activity as evaluated using

Rancimat method and they may not soluble enough in ethanol solvent. The antioxidant activity

of thyme was not clearly shown in Schaal Oven Test method as compared to Rancimat due to

immiscibility of thyme extract and soybean oil and butter (model food substrates to evaluate

thyme antioxidant).

Interaction effects of extraction parameters (ethanol concentration, extraction temperature and

time) were analyzed using Design expert software version 7.0.0. In evaluation of thyme

antioxidant activity in terms of induction time; inhibition time of soybean oil and butter to

oxidative rancidity, there were significant interaction effects between ethanol concentration and

extraction temperature on antioxidant of thyme extract at P< 0.05 level. It was also observed that

the interaction between the extraction temperature and extraction time was significantly related

to antioxidant activity of crude thyme extract at P<0.05 level. However, all extraction parameters

did not have interaction effect on yield of crude thyme extract. Extraction time and ethanol

concentration have no interaction effect on both antioxidant and yield of thyme extract.

Extraction Parameter optimization was required for further analysis of thyme antioxidant activity

and its extract yield. Optimal extraction conditions for the extraction yield and cumulative

antioxidant activity of thyme were determined by statistical analysis of experiment data with

Design Expert 7.0.0 software and techno-economic considerations. Based on the analysis of

results, the software recommended that zero percent ethanol concentration (distilled water), at

room temperature (20℃) for 3.5hrs extraction temperature and time respectively as best

extraction condition to obtained maximum antioxidants of thyme and yield of its extract.

41

4.2. Evaluation of thyme Antioxidant Activity 4.2.1. Rancimat Method

Table 4.3 contains thyme antioxidant activity of three sample treatments of soybean oil as

evaluated by Rancimat method. Thyme extract treatment is the main task of this study that has

oil and 0.1% thyme extract. The other two, positive and negative control were used for

comparison of thyme antioxidant activity. Positive control is a treatment of oil with α-tocopherol

as a reference antioxidant whereas, the negative control contain oil alone. All three treatments

were put under the same experimental conditions in Rancimat. Antioxidant activity of the

negative treatment cannot be determined based on the formula mentioned in Table 4.1 above,

since the denominator becomes zero.

Table 4.3: antioxidant activity of thyme evaluated by Rancimat on soybean oil

Treatments Induction Time (hr) Protection Factor Antioxidant

activity

Thyme extract 3.25±0.02b 1.69±0.010

b 6.9±0.10

b

Positive control 4.98±0.10a 2.59±0.052

a 15.9±0.50

a

Negative control 1.92±0.08c 1.00±0.042

c ∗

Means within the same column followed by the same letters are not significant difference at p<0.05, by student’s t-test. All values are mean ± standard deviation. ∗ Antioxidant activity was undefined for soybean oil sample without thyme extract

Based on the result mentioned in Table 4.3, the induction time was automatically determined by

computer connected to Rancimat Equipment for each treatment. Crude thyme extract shows an

antioxidant activity (P<0.05) since the protection factor of thyme extract treatment is greater than

one. The higher induction period of the soybean oil with the thyme extract added, compared to

the control, the better the antioxidant activity of thyme. It was effective in maintaining stability

of the oil for extended time when it was exposed to accelerated condition in Rancimat. As

shown in the Table 4.3, thyme extract has low antioxidant activity than Vitamin E (α-tocopherol)

applied at 0.05% concentration as positive treatment. This may happen due to the low purity and

solubility of thyme crude extract used in the treatments.

42

Antioxidant activities of thyme largely depend on the composition and concentration of the

extracts as well as on the conditions of the test system and influenced by many factors.

Therefore, it was necessary in this study to perform more than one type of antioxidant activity

measurements to take into account the various mechanisms of the antioxidant action.

Dagne et al. (1998) found that essential oil obtained from Thymus Schimperi grown in Addis

Ababa was rich in carvarol (66.2%) and thymol (50%) which are responsible constituents of

thyme for its antioxidant activity. Similarly, Awraris Derbe (2009) stated that thymol and

carvacrol, present in thyme essence, as well as the flavonoids and other polyphenols are

considered to be involved in the antioxidant activity. He was also reported as thyme extract is

recommended to formulate cosmetic products aimed at the protection of skin and hair integrity

against oxidative processes.

Evaluation of thyme antioxidant activity in Butter

Data of Induction time and protection factor were presented in Table 4.4 for butter samples

collected from different locations. These butter samples were collected from Tarmaber, Sheno,

Bahir Dar, Hirut Dairy PLC and Lame Dairy PLC. All butter samples were treated under the

same way during transportation, storage and evaluation. This data table do not have antioxidant

activity column since all samples were determined without addition of thyme extract. In this

case, the study was intended examine thyme feed cows can provide butter enriched in

antioxidants and see the selling price of these butter in the market. On market, some butter have

higher selling price. This study wants to consider the perception regarding to the antioxidant

content of the butter.

The cumulative antioxidant activity of thyme was also evaluated in butter, collected from

Tarmaber, Sheno, Bahir Dar, Hirut dairy PLC and Lame dairy PLC to examine the quality of

butter related to its antioxidant content.

43

Table 4.4: Antioxidant content of butters collected from different locations as determined by Rancimat

Locations of Butter samples taken

Induction Time (hr) Protection Factor

Tarmaber 4.66±0.08a 1.12±0.020

a

Sheno 3.78±0.14b 0.96±0.035

b

Hirut Dairy PLC 4.22±0.37ab

1.07±0.043ab

Lame Dairy PLC 3.96±0.11

b 1.00±0.028

b

Bahir Dar 2.24±0.28c 0.57±0.071

c

Means within the same column followed by the same letters are not significant difference at p<0.05, by student’s

t-test. All values are mean ± standard deviation.

The results presented in Table 4.4 shows; these butter samples collected from different locations

have different antioxidant content. Both Tarmaber and Hirut Dairy PLC butters exhibit higher

antioxidant activity since they were obtained from cows eating thyme together with their feeds.

Butter of Tarmaber has higher induction time and protection values (4.66±0.08 hr and 1.12±0.02

respectively) than butter of other locations and implies better antioxidant activity. It also has

unique yellow color. This indicates that butters enrich by caroteniods, can have antioxidant.

Whereas, butters taken from Sheno and Bahir Dar did not have antioxidant activity as compared

to that of Tarmaber and Hirut dairy PLC butters indicated in the above table. Commonly, butter

sellers give higher price for Sheno butter and sell it to customers. But, based on this study, Sheno

butter was not as such quality butter enriched by antioxidant as compared to Tarmaber and Hirut

dairy PLC butters which have higher antioxidant content.

In 2009 report, Assefe Berhane stated the antioxidant and preservative effect of thyme in his

technology transfer through stronger feed capability to increase the capacity of Hirut Dairy PLC.

The PLC is more concentrated on feed input to produce butter.

4.2.2. Schaal Oven method

Table 4.5 contains the raw data of peroxide value soybean oil to evaluate thyme antioxidant

activity using Schaal Oven test method. All treatments were stored at 60℃ till the targeted

peroxide value was reached to determine the induction time for thyme antioxidant activity

44

evaluation. The deterioration indexes employed to analyze experimental results from storage

experiments are the peroxide value. In this case, the induction time is storage time which

required for each treatment to reach the peroxide value of 20mEq O2/kg soybean oil where the

oil has poor quality and assumed to be rancid.

Directly, Table 4.5 shows the effect of the addition of thyme extract on the peroxide value of

soybean oil. As an overall observation, the addition of thyme antioxidant decreased the peroxide

value of the oil compared to the control. Initially, the peroxide value the oil was 4.00±0.00 mEq

oxygen/kg of soybean oil. From the results obtained, the peroxide value increased with storage

time. For soybean oil, without the addition of thyme antioxidant, the peroxide value significantly

increased from 4.00±0.00 to 58.33±0.71 within seven day of storage time. The peroxide value of

soybean oil contain in g th y me antiox id ant was lower than the oil withou t it. In th is case, α-

Tocopherol was found to be the most effective antioxidant since there was a slightly increment in

peroxide value under the seven storage time.

Table 4.5: Peroxide value of soybean oil treatments for evaluating antioxidant activity of thyme using Schaal Oven test.

Storage time (day) Treatments

Control 0.1% thyme extract 0.05% Vitamin E (α-Tocopherol)

0 4.00±0.00e 4.25±0.35

f 4.00±0.00

f

2 8.50±0.71d 7.00±0.00

e 6.40±0.56

e

4 11.00±0.82d 8.10±0.71

d 7.60±0.56

d

5 19.67±0.47c 13.50±0.71

c 12.25±0.35

c

6 30.30±1.70b 18.33±0.71

b 16.40±0.56

b

7 58.33±0.71a 28.70±0.35

a 19.86±0.42

a

Means within the same column of each treatment followed by the same letters are not significant difference at

p>0.05, by student’s t-test. All values are mean ± standard deviation.

Based on the results mentioned above in Table 4.5, each treatment has taken different induction

time to reach at the targeted peroxide value of 20mEq O2/kg soybean oil. Induction time required

for reaching the targeted peroxide value of control and thyme treatments were five and six days

45

respectively. Whereas, reference antioxidant, needs seven days. Thyme has also a protection

factor of 1.20 when its induction time was converted. It exhibits relative antioxidant activity. As

mentioned in Rancimat method above this antioxidant activity of thyme comes from is thymol

and carvacrol constituents.

Antioxidant activity of thyme was also evaluated using Schaal Oven Test method. Thyme

showed significantly higher antioxidant activity (P<0.05) than the negative control. Reference

antioxidant (α-tocopherol) exhibited stronger antioxidant activity (P<0.05) than both negative

control and thyme treatments. Even though this method of antioxidant activity determination

shows as thyme exhibits antioxidant activity, it is not well comparable with Rancimat method.

This behavior can be attributed due to the very low solubility of thyme extract in the oil. It has

been previously found that antioxidant’s protecting efficacy increased when the continuous

airflow facilitates emulsification (Velazco et al., 2000).

4.2.3. Effect of thyme extract concentration on its antioxidant activity Data of induction time, Protection factor and antioxidant activity were presented in Table 4.6

when 0, 0.05, 0.1 and 0.2 % of thyme extract was added in the two model food substrates

(soybean oil and Butter). That was done to examine the effect of thyme extract concentration

added to food samples on antioxidant activity of thyme. The concentration levels were

designated based on (Frankel, 2007) range value. As he stated, the maximum level of synthetic

antioxidants concentration allowed to be added in food is 0.02% for the safety reasons. But, in

the case of natural antioxidants, higher concentrations (0.05–0.2%) are necessary because of

their lower activities and presumed lower toxicity. Further addition of thyme extract

concentration above 0.2%, changes the color, flavor, even the viscosity of food samples.

For this study, α-tocopherol was used as a reference antioxidant (positive control) in both

soybean oil and butter for comparison. Samples of both soybean oil and butter without thyme

antioxidant (0% thyme concentration) were prepared separately as negative control, and used to

compare and calculate the protection factor values.

46

Table 4.6: Correlation of thyme extract concentration and its antioxidant activity

Model food substrates

Thyme extract conc. (%) Induction time (hr)

Protection Factor

Antioxidant activity

Refined Soybean oil

0 1.92±0.08d 1.00±0.042

d ∗

0.05 2.70±0.06dc

1.41±0.045dc

4.10±0.59c

0.1 3.22±0.03bc

1.68±0.048bc

6.8±0.43bc

0.2 4.28±0.07

b 2.23±0.036

b 12.5±0.18

b

0.05 (α-tocopherol) 4.98±0.10a 2.59±0.052

a 15.9±1.08

a

Butter (Lame dairy PLC)

0 3.78±0.10e 1.00±0.026

e ∗

0.05 4.16±0.16d 1.10±0.045

d 2.0±0.59

d

0.1 5.28±0.08c 1.40±0.021

c 4.0±0.21

b

0.2 6.02±0.10b 1.59±0.026

b 2.95±0.13

c

0.05 (α-tocopherol) 7.12±0.06a 1.88±0.015

a 8.8±0.30

a

Means within the same column of food substrate followed by the same letters are not significant difference at

p>0.05, by student’s t-test. All values are mean ± standard deviation. ∗Antioxidant activity was undefined for

soybean oil sample without thyme extract

Antioxidant activity of the negative treatment cannot be determined based on the formula

mentioned in Table 4.1 above, since the denominator becomes zero. According to PF values

presented in Table 4.6, antioxidant activity of thyme increases with increasing its concentration

added in both soybean oil and butter.

The value of induction time ranged from 1.92±0.05 to 4.28±0.07 and 3.78±0.10-6.02±0.10 hr on

soybean oil and butter respectively. Based on the results presented in Table 4.6, thyme extract

concentration has significant (P<0.05) effect on its antioxidant activity as it was evaluated in

refined soybean oil and pure butter using Rancimat method.

Furthermore, the data showed that the antioxidant activity of the thyme extract increases when

the extract concentration added to the test samples was increased. Based on induction time data,

thyme extract concentration and its antioxidant activity have strong positive correlation

(R2=0.997). Thyme extract with a concentration of 0.2% shows a higher protection factor in both

47

oil and butter implies that it has better antioxidant activity. At this concentration crude extract of

thyme has almost equivalent antioxidant activity with 0.05% vitamin E (α- tocopherol)

concentration which was taken as reference antioxidant. As observed from the result thyme

antioxidant is more effective on soybean oil than butter food substrate.

4.3. Preservative effect of thyme crude extract The preservative effect of thyme crude extract was studied on pure butter, refined soybean oil

and meat using both chemical and microbiological analysis. The levels thyme extract

concentration for all analysis were given depends on the findings of Frankel (2007) as mentioned

in 4.2.3. Storage weeks were set based on microbiological results of preliminary runs. The

microbial count of the control samples of butter and meat become too difficult to count and

enumerate for analysis.

Acid Value, Free fatty acid and peroxide value of both refined soybean oil and pure butter were

studied to examine the preservative effect of thyme crude extract in chemical analysis at its

different concentration. These rancidity parameters were performed for three consecutive storage

weeks for each food samples.

4.3.1. Free Fatty Acids (FFA)

Table 4.7 contains mean and standard deviation of FFA data of the sample of two food substrates

treated by 0, 0.1 and 0.2% thyme extract. Formation of free fatty acids might be an important

measure of rancidity in oil and butter. The Free fatty acids of both refined soybean oil and butter

treated with different concentration of thyme extract were determined using titration method for

three consecutive weeks. In Table 4.7 the results of free fatty acid analysis are given and derived

from the titration of thyme extract treated soybean oil and butter with standardized sodium

hydroxide titrant. The results were expressed by the percentage of oleic acid value.

Table 4.7 shows the changes in free fatty acid values. Initially, FFA value of the control

treatment of soybean oil (oil without thyme extract) was found to be 0.296±0.02. After one

week, the FFA value was promoted to 0.340±0.002. After the completion of three week storage

time, FFA value of the control treatment of soybean oil was increased to 0.423±0.02. This

change of FFA content was significant according to statistical analysis. Soybean oil treated by

0.1% thyme extract, has the FFA value of 0.231±0.01 and 0.353±0.02 at the first and third week

48

analysis respectively. Whereas, 0.2% treated refined soybean oil have the value of 0.198±0.04

and 0.284±0.21 during the 1st and 3rd week storage time. The result of one-way ANOVA analysis

showed that thyme crude extract treated soybean oil has significantly lower free fatty acid value

(P<0.05).

Table 4.7: Free fatty acid value of both of soybean oil and butter treated by thyme crude extract

Food Substrates Storage weeks Concentration of thyme crude extract (%)

0 0.1 0.2

Refined soybean oil

1st 0.296±0.02a 0.231±0.006

ab 0.198±0.041

b

2nd 0.340±0.002a 0.296±0.027

b 0.256±0.035

c

3rd 0.423±0.02a 0.353±0.021

b 0.284±0.21

b

Butter (Lame Dairy PLC )

1st 0.353±0.02a 0.31±0.04

a 0.296±0.02

a

2nd 0.423±0.04a 0.381±0.02

a 0.377±0.01

a

3rd 0.592±0.04a 0.458±0.01

b 0.403±0.01

b

Means within the same row of food substrate and column of thyme extract concentration followed by the same

letters are not significant difference at p<0.05, by student’s t-test. All values are mean ± standard deviation.

According to the result mentioned in Table 4.7, the order of free fatty acid value decreased with

0 > 0.1 > 0.2% concentration of thyme crude extract under specified week of storage time. In

each treatment, the value of free fatty acid increased with respect to the increasing of storage

time (weeks). Changes of FFA value in thyme extract containing soybean oil samples showed

the significant blockage of oxidation phenomenon as compared to control treatment of the oil.

In the case of butter, the control treatment (butter without thyme extract) has the value of free

fatty acids 0.353±0.02 to 0.592±0.04 at the 1st and 3rd week of storage time. Free fatty acid value

of 0.1% thyme crude extract treated butter increased from 0.31±0.04 to 0.453±0.01 and that of

0.2% treatment has a free fatty acid value of 0.296±0.02 and 0.403±0.01 at 1st and 3rd week of

butter storage time respectively. Even though, the value of free fatty acid decrease with

increasing thyme extract concentration across the storage week of butter, the output of one-way

49

ANOVA analysis exhibited that thyme extract has no significant effect (P>0.05). The

significance of thyme extract treatments has not been detected within 95% confident interval

fixed for this study. According to Farag and his coworkers (1990) thyme and cumin essential oils

could prevent oxidation in butter stored at room temperature, and at 200ppm the essential oils

were more effective than BHT in inhibiting lipid oxidation in the butter. The researchers felt the

preservative effect of the essential oils from thyme and cumin was due to the phenols found in

the oils since the phenolic hydroxy group would be able to donate hydrogen to lipid.

To sum this up, thyme crude extract has the potential of preserving both food substrates. Its

preservative effect depends on its concentration as shown in the result presented in Table 4.7. An

increasing of free fatty acid values of food substrates related to the decomposition their fats. It is

quite clear that the free fatty acid values were retarded with the addition of thyme crude extract

in each food substrates. These findings explored the strong antioxidant ability of thyme extract in

preservation of oil and butter.

4.3.2. Peroxide Value (PV)

The results are shown the amount of peroxide present in treated samples since peroxide value

measures the oxidative rancidity of fats by determining the lipid peroxides. The peroxide value

of thyme extract treated food products were evaluated using titration method for three week of

storage time. In Table 4.8 the results were derived by titrating food substrates with standardized

sodium thio-sulphate titrant in weekly basis.

Peroxide value is widely used measure of the primary oxidation indicating the amount of

peroxides formed in fats and oils during oxidation (Gulcan and Bedia, 2007). In the case of

soybean oil, changes in peroxide values are showed in Table 4.8. The peroxide value of control

treatments of soybean oil was 5.20±1.13. It was increased to 15.0±1.41 at the end of three week

storage time. The peroxide values of 0.1% and 0.2 % thyme extract treated oil samples were

changed from 4.50±0.71 to 6.50±0.71 and 5.00±1.41 to 6.00±0.00 at the 1st and 3rd week of

analysis respectively. These changes were significant indicating the noticeable phenomenon of

lipid oxidation. There were statistical differences (P<0.05) among control and thyme crude

extract treatments. One-way ANOVA analysis result, showed that refined soybean oil treated

with thyme crude extract (0.1 and 0.2%) has significantly lower peroxide value (P<0.05)

50

compared to untreated oil. This implies more peroxides were created in untreated soybean oil

samples than samples of thyme extract treated oil. According to the results mentioned in Table

4.8, the order of peroxide value increased 0.2 < 0.1 < 0% concentration of thyme crude extract in

specified week of storage time. In each treatment, the peroxide value increased weekly.

Table 4.8: Peroxide value of soybean oil and butter of thyme crude extract treatments

Food Substrates Storage weeks Concentration of thyme crude extract (%)

0 0.1 0.2

Refined Soybean oil

1st 5.20±1.13a 4.50±0.71a 5.00±1.41a

2nd 9.50±0.71a 7.00±1.41ab 5.50±0.71b

3rd 15.0±1.41a 6.50±0.71b 6.00±0.00b

Butter (Lame Dairy PLC)

1st 3.25±1.06a 1.50±0.71a 1.50±0.71a

2nd 15.0±1.41a 10.0±1.41b 9.50±0.71b

3rd 21.0±1.41a 15.0±1.41b 13.5±0.71b

Means within the same row of food substrate and column of thyme extract concentration followed by the same

letters are not significant difference at p<0.05, by student’s t- test. All values are mean ± standard deviation.

In butter, the control has higher peroxide values ranged from 3.25±1.06 to 21±1.41 at the 1st and

3rd week of storage time. Butter treated with 0.1% thyme extract has peroxide values of 1.5±0.71

and 15±1.41 during storage weeks. After the 2nd week of storage time, the result of one-way

ANOVA analysis exhibited that thyme extract has significant effect (P<0.05) as compared to the

control (untreated) butter sample. Both 0.1 and 0.2% thyme extract treated butter samples show

lower peroxide value (P<0.05). During the first week of sample storage time, treatments have not

showed a significance effect (P>0.05) even though the data observed in Table 4.8 looks like

different. The butter samples treated with 0.1 and 0.2% thyme extract concentration did not show

a significant difference (P>0.05) at the 2nd and 3rd week of storage time.

Generally, the preservative effect of thyme extract has been clearly exhibited in this study

conducted weekly as compared to the control food substrates. The results from peroxide value

measurement showed that the addition of thyme extract at 0.1 and 0.2% delayed effectively the

51

oxidation of both refined soybean oil and butter during three weeks of storage time. The thyme

extract has a potential to reduce the chance of peroxides formation in food products. The

oxidative rancidity of both refined soybean oil and pure butter was retarded with the addition of

thyme crude extract.

4.3.3. Acid Value (AV) Acid value of both refined soybean oil and butter treated with different concentration of thyme

extract were determined using titration method for three consecutive weeks. In Table 4.9 the

results of acid value analysis are given, they derived from the titration of both untreated and

thyme extract treated samples of butter and soybean oil with standardized sodium hydroxide

titrant. Acid value results are expressed as milligram KOH alcohol per gram of oil or butter.

Table 4.9: Acid value of soybean oil and butter treated by thyme crude extract

Food substrates Storage weeks Concentration of thyme extract (%)

0 0.1 0.2

Refined Soybean oil

1st 0.592±0.04a 0.462±01ab 0.395±0.08b

2nd 0.68±0.00a 0.592±0.04b 0.512±0.01c

3rd 0.874±0.04a 0.606±0.04b 0.592±0.04b

Butter (Lame Dairy plc)

1st 0.704±0.04a 0.62±0.08a 0.592±0.04a

2nd 0.846±0.08a 0.762±0.04a 0.714±0.03a

3rd 1.182±0.08a 0.916±0.02b 0.806±02b Means within the same row of food substrate and column of thyme extract concentration followed by the same letters are not significant difference at p<0.05, by student’s t-test. All values are mean ± standard deviation.

In the case of soybean oil, the acid values of blank soybean oil (without thyme extract) were

0.592±0.04 and 0.874±0.04 at the 1st and 3rd week of storage time at room temperature. At these

storage weeks, both 0.1 and 0.2% thyme extract treated soybean oil samples have acid values

ranged from 0.462±0.01-0.606±0.04 and 0.395±0.08-0.592±0.04 respectively. The differences in

mean acid value between different thyme extract treatments and blank soybean oil samples were

significant (p<0.05) from the beginning storage and during the subsequent storage weeks. The

52

results showed that there was a significant (p<0.05) increase in acid values in different

treatments during storage by different rates. The highest incremental rate was found in the

control sample of soybean oil. The concentrations of thyme crude extract at 0.1 and 0.2%

showed the highest significant effects on lipid oxidation by lowering acid values than control

sample till the end of cold storage.

Blank butter (0% thyme extract) has the acid values of 0.704±0.04 and 1.182±0.08 at the 1st and

3rd week of cold storage at 4℃ temperature. At these storage weeks, both 0.1 and 0.2% thyme

crude extract treated butter samples have acid values of 0.62±0.08; 0.916±0.02 and 0.592±0.04;

0.806±0.02 respectively. The result of one-way ANOVA analysis showed that thyme crude

extract added in butter samples has no significant (P<0.05) effect even though they have lower

acid value than blank sample. According to the result mentioned in Table 4.9, acid value

decreased as the concentration of thyme extract increased under specified week of cold storage

time. In each treatment, acid value also increased with increasing of cold storage weeks of butter.

4.3.4. Total Aerobic Viable Count

Total aerobic plate Count was enumerated for different treatments of butter and meat during the

cold storage as given in Fig. 4.1. The results of each treatment of both butter and meat were

expressed by the logarithm value of colony forming units obtained by direct count colonies on

each serial dilution per ml of sample inoculated to plate count agar medium in duplicate.

At all three weeks of butter cold storage, control sample showed the highest counts comparing to

other samples contained thyme extract with different concentrations. The value of its aerobic

plate count significantly increased during each cold storage week of butter. Both 0.1 and 0.2%

thyme extract treated butter samples showed lower value of aerobic plate count. It is quite clear

that thyme extract has antimicrobial effect in preservation of butter. But, the significant

difference of thyme extract treatments has not been seen clearly. This may happened due to the

closeness of thyme extract concentration levels taken for this study.

In the case of meat, the colony count values of control sample were higher than other thyme

extract treated samples during the first two weeks of meat cold storage time. But at the third

week of meat storage, it was very difficult to count colonies grown on plate count agar medium.

In meat, both concentration of thyme crude extracts (0.1 & 0.2%) significantly decreased the

53

value of aerobic plate count. In Fig. 4.1, it has been clearly seen that 0.2% thyme extract has

lower count value in each cold storage week of meat.

(a)

(b)

Fig.4.1: Aerobic plate count (a) butter (b) meat

Aerobic plate count of both butter and meat was gradually increased during cold storage for all

samples with different rates depending on the concentration of thyme extract. The incremental

0

1

2

3

4

5

6

7

1 2 3

control0.1% thyme extract0.2% thyme extract

log

(Cfu

/ml)

storage weeks

0123456789

1 2 3

control0.1% thyme extract0.2% thyme extract

log

(Cfu

/ml)

storage weeks

54

pattern of the colony count value can be arranged in an ascending order as follows: 0.2% thyme

extract, 0.1% thyme extract and finally control sample. In general, as the concentration of thyme

extract increase the colony count numbers decreased with the same storage week of both meat

and butter. Tabak and his coworker (1996) aqueous extracts of thyme significantly inhibited the

growth of Helicobacter pylori. Many reports show that plants leaves possess high antimicrobial

activity than other parts (Nwaogu et al., 2008). These strong antimicrobial activities are mostly

attributable to the presence of phenolic compounds such as thymol and carvacrol, and to

hydrocarbons like γ- terpinene and p-cymene (Lambert et al., 2000).

4.3.5. Aerobic Mold and Yeast Count It was done for both meat and butter samples treated by 0, 0.1 and 0.2% concentration of thyme

extract and cold stored at 4℃ for three consecutive weeks. The results are expressed as log

(cfu/ml) of both samples of meat and butter.

The values of aerobic mold and yeast count for control sample of butter were higher than thyme

crude extract treated samples in all three weeks of butter cold storage time. The values were

increased significantly during each week. Both 0.1 and 0.2% thyme crude extract treated butter

samples showed lower value of aerobic mold and yeast count. It is quite clear that thyme extract

has antimicrobial effect against the growth of both mold and yeast in cold stored butter. But, as

shown in Fig 4.2 (a), the significant difference between 0.1 & 0.2% thyme extract treatments

were not seen clearly. It may happen due to the closeness of thyme extract concentration levels

taken for this study.

Aerobic growth of mold and yeast was also studied in meat samples of thyme extract treatments.

Higher values of aerobic mold and yeast count were obtained in the control sample of meat

during the 1st and 2nd weeks of meat cold storage time. However, its count value at the 3rd week

of analysis was too difficult to count colonies grown on Potato Dextrose Agar medium. Both

concentration of thyme crude extracts (0.1 & 0.2%) significantly decreased the value of aerobic

mold and yeast growth count during the whole weeks of analysis. In Fig. 4.2 (b), it has been also

clearly seen that 0.2% thyme extract has lower count value in each cold storage week of meat.

55

(a)

(b)

Fig. 4.2: Aerobic mold and yeast count (a) butter (b) meat

In 2007, Omidbeygi and his coworkers evaluated the antifungal activity of three essential oils

(thyme, summer savory and clove) in culture medium and as a real system in tomato paste (in

vitro and in vivo). Results clearly showed that in vitro each essential oil had notable antifungal

activity. Thyme essential oil has the highest antifungal activity, followed by summer savory and

clove essential oils.

33.25

3.53.75

44.25

4.54.75

55.25

5.5

1 2 3

control0.1% thyme extract0.2% thyme extract

log

(Cfu

/ml)

storage weeks

4

4.5

5

5.5

6

6.5

7

1 2 3

control0.1% thyme extract0.2% thyme extract

log

(Cfu

/ml)

storage weeks

56

4.3.6. Enterobacteriaceae Count

It was counted for different treatments of butter and meat during the cold storage as given in Fig.

4.3. The results of each treatment of both butter and meat were expressed by the logarithm value

colony forming units per ml of sample inoculated to plate count agar medium.

(a)

(b)

Fig. 4.3: Enterobacteriaceae Count (a) butter (b) meat

2

2.5

3

3.5

4

4.5

1 2 3

control0.1% thyme extract0.2% thyme extract

log

(Cfu

/ml)

storage weeks

2

2.5

3

3.5

4

4.5

5

1 2 3

control0.1% thyme extract0.2% thyme extract

log

(Cfu

/ml)

Storage weeks

57

As shown in Fig. 4.3 (a), it could be observed that control sample of butter had the highest

counts of enterobacteriaceae at all three weeks of cold storage compared to other treatments. The

count of enterobacteriaceae of butter significantly increased during each weeks of cold storage at

4℃. Samples of butter treated by both 0.1 and 0.2% thyme crude extract showed lower value of

enterobacteriaceae. But, in Fig. 4.3 (a), the deviation between enterobacteriaceae counts of

thyme extract treatments on has not been clearly seen in this analysis.

The values for enterobacteriaceae count of control sample of meat were higher than other thyme

extract treated samples during the two weeks of meat cold storage time as shown in Fig.4.3 (b).

But, at the third week of meat storage, it was very difficult and too much to count colonies grown

on plate count agar medium. Both concentration of thyme crude extracts (0.1% and 0.2%)

significantly decreased the value of enterobacteriaceae count. In Fig. 4.3 (b), it has been clearly

seen that enterobacteriaceae count values were increased in each cold storage week of meat for

each fixed treatment.

In general, the values of enterobacteriaceae counts decreased as the concentration of thyme

extract increase with the same storage week of both meat and butter. From the same Fig. 4.3, it

could be easy to say that, thyme crude extract has preservative effect on both butter and meat

against the growth of enterobacteriaceae.

Numerous studies have been published on the antimicrobial activities of plant extracts against

different types of microbes, including foodborne pathogens (Beuchat, 1994; Kondo et al., 2004).

It has been reported that spices owe their antimicrobial properties mostly to the presence of

alkaloids, phenols, glycosides, steroids, essential oils, coumarins and tannins (Otung et al.,

1991). As reviewed by Lopez-Malo et al in 2006, some of antimicrobial components that have

been identified in spices and herbs are: eugenol from cloves, thymol from thyme and oregano,

carvacrol from oregano, vanillin from vanilla, allicin from garlic, cinnamic aldehyde from

cinnamon, allyl isothiocyanate from mustard.

There has been several more general studies about antimicrobial activity of thyme. One of them

is the work of Gutierrez et al. (2009), who evaluated essential oil of lemon balm, marjoram,

oregano and thyme, and applied them on food model media based on lettuce, milk and meat.

Minimum inhibitory concentrations were determined against Enterobacter spp., Listeria spp.,

58

Lactobacillus spp. and Pseudomonas spp. According to his findings, the average efficacy of

essential oils against Listeria spp. was in the following order: oregano>thyme> lemon balm,

while the efficacy order against the spoilage bacteria was: oregano>thyme> marjoram.

For successful applications of thyme in different food systems, potential interactions between

thyme extract and food components have to be determined. There is a number of examples where

some studies have shown that plant extracts are useful for reduction of pathogens in some food

product, while others reported very low antimicrobial activity or no effect when the same plant

extract were applied to other product. Thus, the application of plant extract requires the

evaluation of efficacy within food products or in model systems that closely mimic food

composition, because the efficacy of many antimicrobials may be reduced by certain food

components (Gutierrez et al., 2009).

59

Chapter Five

Suggested technology for the production of thyme crude extract

5.1. Production of Crude thyme extract

The production of crude thyme extract passes the following procedures in the laboratory and can

be industrially scaled-up. The preliminary operations that comprises of harvesting, cleaning,

drying, grinding, sieving, blending, extraction, filtration, freeze drying and finally packaging

and storage till further analysis. The appropriate sample preparation and careful processing are

the major procedures that affect the analysis of final product. Generally, the following flow sheet

clearly describes the process for production of crude thyme extract.

Water removed

Impurities

Undersized

Thyme crude extract

Fig. 5.1: Basic steps for the production of thyme crude extract

Harvesting Cleaning (washing)

Packaging & storage

Drying (Shade sun drying)

Sieving

Grinding

Filtration Water

Treatment Unit

Tape Water

Cooling Extraction

Residue

Freeze Drying

60

Equipment layout for thyme extract production plant

Fig.5.2: Equipment Layout of thyme crude extract production plant

61

5.2. Material and energy balance on major unit operations

Material balances are the basis of process design. A material balance taken over the complete

process will determine the quantities of raw materials required and products produced. Balances

over individual process units set the process stream flows and compositions. They are also useful

tools for the study of plant operation and trouble shooting. They can be used to check

performance against design; to extend the often limited data available from the plant

instrumentation; to check instrument calibrations; and to locate sources of material loss. On the

other hand, in process design, energy balances are made to determine the energy requirements of

the process: the heating, cooling and power required. In plant operation, an energy balance

(energy audit) on the plant will show the pattern of energy usage, and suggest areas for

conservation and savings (Coulson and Richardson, 2005).

In the case of this research, the need to conduct material and energy balances on major unit

operations was to scale up all the parameters used in the laboratory that resulted in the annual

production of the plant; in order to design the size of the equipment and / or for equipment

selection that helped in estimating purchased equipment cost. In addition to that they helped in

calculating the material, auxiliary and utility costs. Generally, they are needed to estimate

economic analysis; profitability and financial feasibility of the processing plant.

Data (Assumptions):

Based on the scale up experimental data, the plant will be assumed to have a capacity to

process 10Qtl (1000Kg) raw and cleaned thyme in batch process taking three days for

complete cycle.

The raw dried thyme is expected to have industrial quality fitting Quality Standard

Authority of Ethiopian (QSAE): maximum acceptable impurity is 5%.

62

Material Balance Material Balance on cleaning unit Impurities (5%) MI

Mt MC = 1,000Kg

Raw thyme Cleaned raw thyme

Where,

Mt –mass of harvested thyme

Mc – mass of cleaned thyme

Mi – mass of impurities

Therefore,

Mt = Mi + Mc ------------------------------------------------------------------------------ (5.1)

Mc = (0.95)Mt

Mt = 1,052.63 𝐾𝐾𝐾𝐾

And Mi = (0.05)Mt = 52.63 𝐾𝐾𝐾𝐾

Material Balance on Drying

Raw and cleaned thyme was exposed to drying in tray drier

Water Vapour (Mv)

Cleaned raw thyme (Mc) Dried thyme (Md)

m.c of 87.6% m.c of 12.8%

Where,

Md- mass of dried thyme

Mv- mass of water vapour removed during drying

Therefore,

Mc = 𝑀𝑀𝑣𝑣 + 𝑀𝑀𝑑𝑑 ------------------------------------------------------------------------------- (5.2)

1,000𝐾𝐾𝐾𝐾 = 𝑀𝑀𝑣𝑣 + 𝑀𝑀𝑑𝑑

Cleaning (washing)

Drying (Natural shade drying)

63

Solid balance on the drying process provides;

0.122 × 1,000 = 0.872 × 𝑀𝑀𝑑𝑑

𝑀𝑀𝑑𝑑 = 139.9 𝐾𝐾𝐾𝐾 dried thyme

And 𝑀𝑀𝑣𝑣 = Mc −𝑀𝑀𝑑𝑑

𝑀𝑀𝑣𝑣 = 1,000− 139.9

𝑀𝑀𝑣𝑣 = 860.1 𝐾𝐾𝐾𝐾 water could be removed during thyme drying.

Material Balance on Sieve fitted grinding unit

Only 5% loss is assumed to be occurred during grinding and sieving process.

Md = 139.9Kg

Undersized (5%) Mu

Ms

Where,

Mu-mass undersized (very fine thyme)

Ms-mass of mill thyme after sieving

Therefore,

𝑀𝑀𝑑𝑑 = 𝑀𝑀𝑢𝑢 + 𝑀𝑀𝑠𝑠 -------------------------------------------------------------------------- (5.3)

And, 𝑀𝑀𝑢𝑢 = (0.05)𝑀𝑀𝑑𝑑 𝑀𝑀𝑢𝑢 = 6.996 𝐾𝐾𝐾𝐾

𝑀𝑀𝑠𝑠 = 𝑀𝑀𝑑𝑑 − 𝑀𝑀𝑢𝑢 𝑀𝑀𝑠𝑠 = 132.92 𝐾𝐾𝐾𝐾 ground thyme is required for crude thyme extract production.

Grinding and Sieving

64

Overall Material balance on Extraction and Filtration Units

Distilled water in 10:1(v/w) ratio with ground thyme

Mw

Ms Recycled

m.c of 12.8%

Residue (m.c of 87.3%), Mr

Mf (m.c of 97.7%)

Where,

Mf- mass of filtered thyme extract Mr- mass of thyme residue after extraction

Mw-mass of distilled water Overall balance gives,

𝑀𝑀𝑠𝑠 + 𝑀𝑀𝑤𝑤 = 𝑀𝑀𝑓𝑓 + 𝑀𝑀𝑟𝑟 ----------------------------------------------------------------- (5.4)

𝑀𝑀𝑤𝑤 = 10 ×𝑀𝑀𝑠𝑠 𝐾𝐾𝑔𝑔𝑣𝑣𝑔𝑔𝑠𝑠 𝑀𝑀𝑤𝑤 = 10 × 132.92

𝑀𝑀𝑤𝑤 = 1,329.2 𝐾𝐾𝐾𝐾

Density of distilled water was assumed to be 1g/ml or 1Kg/L. While preparing thyme crude

extract, 10g of ground and sieved thyme was mixed with 100ml distilled water i.e., taking the

ratio of 1:10 (thyme to distilled water).

𝑀𝑀𝑓𝑓 +𝑀𝑀𝑟𝑟 = 𝑀𝑀𝑤𝑤 + 𝑀𝑀𝑠𝑠 and gives 𝑀𝑀𝑓𝑓 + 𝑀𝑀𝑟𝑟 = 1329.2 + 132.92

Then, 𝑀𝑀𝑓𝑓 +𝑀𝑀𝑟𝑟 = 1,462 -------------------------------------------------------------- (5.5)

Making solid balance on extraction unit helps to calculate the value of both Mf and Mr.

𝑀𝑀𝑠𝑠 × 0.872 = 𝑀𝑀𝑓𝑓 × (1 − 0.997 ×) + 𝑀𝑀𝑟𝑟 × (1 − 0.873)

132.92 × 0.872 = 0.023𝑀𝑀𝑓𝑓 + 0.127𝑀𝑀𝑟𝑟 ------------------------------------- (5.6)

From equation (5.5) 𝑀𝑀𝑓𝑓 = 1,462 −𝑀𝑀𝑟𝑟 and substituting it to equation (5.6) gives;

Extraction

Filtration

65

𝑀𝑀𝑟𝑟 = 791.1 𝐾𝐾𝐾𝐾 𝑎𝑎𝑎𝑎𝑑𝑑 𝑀𝑀𝑓𝑓 = 670.9 𝐾𝐾𝐾𝐾

Material Balance on Freeze Drying Unit

Aqueous extract of thyme was taken to freeze drying process to reduce at least 90% of its inlet

weight and produce freeze dried thyme crude extract.

Water/vapour (Mfv)

Mfd

Mf = 670.9Kg Freeze dried thyme crude extract

m.c of 97.7%

Where,

Mfy- mass of water/vapour removed from freeze dying of thyme extract Mfd- mass of freeze dried thyme extract

The amount of water contained in the coming aqueous thyme extract to freeze drier is;

𝑀𝑀𝑓𝑓 × 0.977 = 670.9 × .977

This gives 668.9 𝐾𝐾𝐾𝐾 of water has been contained in filtered thyme extract.

During freeze drying process, 90% of aqueous thyme extract weight coming to freeze drier has to be reduced.

Thus, the amount of water removed = 670.9 × (0.9)

= 603.8 𝐾𝐾𝐾𝐾 of water is removed during freeze drying.

The amount of freeze dried product; thyme crude extract can be calculated by,

𝑀𝑀𝑓𝑓 = 𝑀𝑀𝑓𝑓𝑣𝑣 +𝑀𝑀𝑓𝑓𝑑𝑑 ------------------------------------------------------------------------- (5.7)

670.9 = 603.8 + 𝑀𝑀𝑓𝑓𝑑𝑑

From this 𝑀𝑀𝑓𝑓𝑑𝑑 = 67.1 𝐾𝐾𝐾𝐾 thyme crude extract could be produce from 1000Kg raw and cleaned

thyme.

Freezing Drying

66

Energy Balance

Material quantities, as they pass through processing operations, can be described by material

balances. Such balances are statements on the conservation of mass. Similarly, energy quantities

can be described by energy balances, which are statements on the conservation of energy. The

increasing cost of energy has caused the industries to examine means of reducing energy

consumption in processing. Energy balances are used in the examination of the various stages of

crude thyme extract production, over the whole process and even extending over the total

production system from the raw thyme to the finished freeze dried thyme crude extract. All

calculated energy values are the quantity of energy required to run one batch process.

Energy Balance on water distillation unit

Heat Removed (Qout) Distilled water

Cold water (25℃)

Heat supplied (Qin)

In distillation process, feed tape water is heated and then evaporated to separate out dissolved

minerals. Based on the specification of distiller used for this study, the heater requires the power

(P) of 4kW means to boil five liter cold water in 8 minutes (t).

Thus, the amount heat supplied by electric heater becomes;

𝑄𝑄𝑔𝑔𝑎𝑎 = 𝑃𝑃𝑡𝑡--------------------------------------------------------------------------- (5.8)

𝑄𝑄𝑔𝑔𝑎𝑎 = 4000𝑗𝑗𝑗𝑗𝑢𝑢𝑗𝑗𝑔𝑔/ sec× 8𝑚𝑚𝑔𝑔𝑎𝑎 × 60𝑠𝑠𝑔𝑔𝑠𝑠/𝑚𝑚𝑔𝑔𝑎𝑎

𝑄𝑄𝑔𝑔𝑎𝑎 = 1920𝐾𝐾𝑔𝑔𝑗𝑗𝑗𝑗 𝐽𝐽𝑗𝑗𝑢𝑢𝑗𝑗𝑔𝑔s

Heater

Condenser

67

The quantity of heat gained by the water (Qw) in raising its temperature from 20℃ (room

temperature) to 100℃ is;

𝑄𝑄𝑤𝑤 = 𝑀𝑀𝐶𝐶𝑝𝑝∆𝐼𝐼------------------------------------------------------------------------- (5.9)

The mass of water is the product of its volume (5Lt) and density (1Kg/Lt) and gives 5Kg. The

specific heat capacity of water (Cp) is taken to be 4.187KJ/Kg. K.

𝑄𝑄𝑤𝑤 = 5 × 4.187(100− 20)

𝑄𝑄𝑤𝑤 = 1674.8𝐾𝐾𝑔𝑔𝑗𝑗𝑗𝑗 𝐽𝐽𝑗𝑗𝑢𝑢𝑗𝑗𝑔𝑔𝑠𝑠

The circulating cooling water is assumed to remove at least 85% of the heat gained by water and

condense the vapor coming from the heating unit.

𝑄𝑄𝑗𝑗𝑢𝑢𝑡𝑡 = 0.85(𝑄𝑄𝑤𝑤)

𝑄𝑄𝑗𝑗𝑢𝑢𝑡𝑡 = 0.85 × 1674.8 = 1423.6 𝐾𝐾𝑔𝑔𝑗𝑗𝑗𝑗 𝐽𝐽𝑗𝑗𝑢𝑢𝑗𝑗𝑔𝑔𝑠𝑠

The remaining 251.2 Kilo Joule heat energy is taken by distilled water.

Energy balance on Cooling and Freezing unit

Cooling and Freezing energy are required for filtered thyme extract prior to freeze drying

process. The ordinary refrigeration of thyme extract involves cooling only without any phase

change. The freezing of extract, on the other hand, involves three stages: cooling to the freezing

point (removing the sensible heat), freezing (removing the latent heat), and further cooling to the

desired subfreezing temperature (removing the sensible heat of frozen thyme extract).

After assuming that thyme extract is considered to be food, its thermal properties such as specific

heat and the latent heat of thyme extract are calculated with reasonable accuracy on the basis of

its water content alone. They are determined based on Siebel’s (1982) formula given by;

𝐶𝐶𝑝𝑝 , 𝑓𝑓𝑟𝑟𝑔𝑔𝑠𝑠ℎ = 3.35𝑎𝑎 + 0.48 ( 𝐾𝐾𝐽𝐽𝐾𝐾𝐾𝐾.℃

)------------------------------------------ (5.10)

𝐶𝐶𝑝𝑝 ,𝑓𝑓𝑟𝑟𝑗𝑗𝑓𝑓𝑔𝑔𝑎𝑎 = 1.26𝑎𝑎 + 0.84 ( 𝐾𝐾𝐽𝐽𝐾𝐾𝐾𝐾.℃

)---------------------------------------- (5.11)

Where, Cp, fresh and Cp, frozen are the specific heats of the thyme extract before and after

freezing, respectively; 𝑎𝑎 is the fraction of water content of the thyme extract.

The latent heat (h) of thyme extract during freezing is also depends on its water content and

given by;

ℎ = 334𝑎𝑎 (𝐾𝐾𝐽𝐽/𝐾𝐾𝐾𝐾)----------------------------------------------------------- (5.12)

68

After filtration, the thyme extract has a moisture content (𝑎𝑎) of 97.7%.

Thus, 𝐶𝐶𝑝𝑝 , 𝑓𝑓𝑟𝑟𝑔𝑔𝑠𝑠ℎ 𝑡𝑡ℎ𝑦𝑦𝑚𝑚𝑔𝑔 𝑔𝑔𝑒𝑒𝑡𝑡𝑟𝑟𝑎𝑎𝑠𝑠𝑡𝑡 = 3.35 × .977 + 0.48

= 3.75𝐾𝐾𝐽𝐽/𝐾𝐾𝐾𝐾.℃

And 𝐶𝐶𝑝𝑝 ,𝑓𝑓𝑟𝑟𝑗𝑗𝑓𝑓𝑔𝑔𝑎𝑎 𝑡𝑡ℎ𝑦𝑦𝑚𝑚𝑔𝑔 𝑔𝑔𝑒𝑒𝑡𝑡𝑟𝑟𝑎𝑎𝑠𝑠𝑡𝑡 = 1.26 × .977 + 0.84

= 2.07𝐾𝐾𝐽𝐽/𝐾𝐾𝐾𝐾.℃

Similarly, the latent heat of thyme extract (h) can be calculated as;

ℎ = 334 × 0.997 = 333𝐾𝐾𝐽𝐽/𝐾𝐾𝐾𝐾

As mentioned above, freezing of thyme extract needs both cooling and freezing energies.

The amount of heat removed as the thyme extract cooled from 20℃ to 0℃ is

𝑄𝑄𝑠𝑠𝑗𝑗𝑗𝑗𝑗𝑗𝑔𝑔𝑎𝑎𝐾𝐾 = 𝑀𝑀𝐶𝐶𝑝𝑝 ,𝑓𝑓𝑟𝑟𝑔𝑔𝑠𝑠ℎ × ∆𝐼𝐼𝑠𝑠𝑗𝑗𝑗𝑗𝑗𝑗𝑔𝑔𝑎𝑎𝐾𝐾 ---------------------------------------- (5.13)

= 670.9 × 3.75 × (20 − 0) = 50,317.5 𝐾𝐾𝐽𝐽

The amount of heat removed as the thyme extract frozen, is the sum of both latent heat remove at

0℃ and sensible heat from 0℃ to -18℃.

𝑄𝑄𝑓𝑓𝑟𝑟𝑔𝑔𝑔𝑔𝑓𝑓𝑔𝑔𝑎𝑎𝐾𝐾 = 𝑀𝑀𝐶𝐶𝑝𝑝 ,𝑓𝑓𝑟𝑟𝑗𝑗𝑓𝑓𝑔𝑔𝑎𝑎 × ∆𝐼𝐼𝑓𝑓𝑟𝑟𝑔𝑔𝑔𝑔𝑓𝑓𝑔𝑔𝑎𝑎𝐾𝐾 + 𝑀𝑀ℎ------------------- (5.14)

= (670.9×2.07×18) + (670.9×333)

= 248,407.4 𝐾𝐾𝐽𝐽

The total amount of heat removed when the thyme extract cooled and frozen before starting

freeze drying process becomes;

𝑄𝑄𝑡𝑡𝑗𝑗𝑡𝑡𝑎𝑎𝑗𝑗 = 𝑄𝑄𝑠𝑠𝑗𝑗𝑗𝑗𝑗𝑗𝑔𝑔𝑎𝑎𝐾𝐾 + 𝑄𝑄𝑓𝑓𝑟𝑟𝑔𝑔𝑔𝑔𝑓𝑓𝑔𝑔𝑎𝑎𝐾𝐾 -------------------------------------------------------- (5.15)

= 50,317.5 + 248,407.4

𝑄𝑄𝑡𝑡𝑗𝑗𝑡𝑡𝑎𝑎𝑗𝑗 = 298,725 𝐾𝐾𝐽𝐽

69

Energy of Freeze Drying

Freeze drying, is used to increase the stability and concentration of bioactive components of

thyme extract. The drying process needs certain amount of energy to remove water contained in

thyme extract at lower temperature and pressure. In general, since the thyme crude extract dried

has uniform and comparatively thin layer, and taking into account that the lyophilization process

is rather slow, temperature gradients within the thyme extract can be disregard in a first hand

approximation. Based on these assumptions, the total amount of heat to be supplied to thyme

extract was expressed according to the equation of Liu et al (2003).

𝑄𝑄𝑡𝑡𝑗𝑗𝑡𝑡𝑎𝑎𝑗𝑗 = 𝑀𝑀𝑤𝑤𝑎𝑎𝑡𝑡𝑔𝑔𝑟𝑟 × ∆ℎ𝑠𝑠𝑢𝑢𝑠𝑠 + 𝑀𝑀𝑠𝑠𝑎𝑎𝑚𝑚𝑝𝑝𝑗𝑗𝑔𝑔 × 𝐶𝐶𝑝𝑝𝑠𝑠𝑎𝑎𝑚𝑚𝑝𝑝𝑗𝑗𝑔𝑔 (𝐼𝐼𝑠𝑠𝑎𝑎𝑚𝑚𝑝𝑝𝑗𝑗𝑔𝑔 − 𝐼𝐼0)-------------- (5.16)

Where; Qtotal is the total amount of heat to be supplied to the sample (J); Mwater: Mass of water

removed from the sample (kg); Δhsub: Latent heat of sublimation (J/kg); Msample: Sample dry

mass (kg); Cpsample: Sample specific heat (J.K-1.kg-1); Tsample: Sample temperature (K); To:

Arbitrary reference temperature (K).

Thus, 𝑄𝑄𝑡𝑡𝑗𝑗𝑡𝑡𝑎𝑎𝑗𝑗 = (603.8 × 2838 ) + 15.43 × 2.07 × (273− 238)

= 159,327.5𝐾𝐾𝐽𝐽

70

5.3. Economic Evaluation of the Plant

Plant Capacity and Production Programming

The plant is assumed to work for 300 days per annum (100 batch runs) and in a double shift of

16hr per day. Therefore, based on the market forecast and material balance, the plant has the

capacity of processing 10 ton of cleaned thyme for the production of 6,710 Kg thyme crude

extract per year is expected. The annual production program of the plant is formulated and

assumed to achieve 80% and 90% capacity utilization rate in the first and second year and full

capacity will be attained in the third year and onwards as shown in the table below.

Table 5.1: Plant capacity and production program

S. No. Description Production Program 1st year 2nd year 3rd year

1 Capacity Utilization (%) 80 90 100 2 Production Rate

(Kg per year) 5,376 6,048 6,720

Purchase Equipment Cost (PEC) Table 5.2: Purchased Equipment Cost S. No. Equipment Quantity Capacity Total Cost(Birr) 1 Raw material Silo 2 100 m3 389,390 2 Mill 1 140 Kg 46,300 3 Sieve machine 1 140 Kg 21,000 4 Distilled water Tank 1 450 Lt 13,400 5 Agitator fitted Extractor 1 500Lt 500,976 6 Water Pump 1 90Lt/hr 56,680 7 Filter Press 1 9.4 m2 diameter 77,900* 8 Freeze Dryer 1 6.71 kg 500,000 9 Water distiller 1 1.52 m2 diameter 71,135* Purchased Equipment Cost (PEC) 1,241,530 Birr Values for most equipment were taken from local company and personal contact. *Costs obtained from the internet (http://www.matche.com/EquipCost/Index.htm, retrieved on Jan, 18, 2012.

71

Total Capital investment Estimation

The ratio factors shown below are for estimating capital investment items based on delivered-

equipment cost of solid-liquid processing plant; according to Peters and Timmerhaus (1991) are

considered for the estimation of capital investment as shown in Table 5.3.

Table 5.3: Estimation of direct and indirect cost

Cost category Components Factors Cost (Birr)

i. Direct Plant Cost

Purchasing Equipment cost (PEC) PEC 1,241,530

Equipment installation 0.39 PEC 484,197

Piping 0.31 PEC 384,874

Instrumentation and control 0.13 PEC 161,400

Electrical 0.10 PEC 124,153

Building 0.29 PEC 360,044

Service facilities 0.55 PEC 682,842

Yard improvement 0.10 PEC 124,153

Land 0.06 PEC 74,492

Total Direct Plant Cost 3,637,685

ii. Indirect Plant Cost

Design and Engineering 0.25 PEC 310,383

Contractor’s fee 0.18 PEC 223,475

Contingency 0.10 PEC 124,153

Total Indirect Plant Cost 658,011

A. Fixed Capital Investment (FCI)

FCI = Total Direct cost + Total Indirect Cost

= 3,637,685 + 658,011

= 4,295,696 Birr

B. Working Capital

Working capital is an additional investment needed above the fixed capital to start up and

Operate the plant to the point in which income is earned.

Working Capital = 15% Fixed Capital

72

= 644,354 Birr Therefore,

Total Capital Investment = Fixed Capital + Working Capital

= 4,295,496 + 644,354

= 4,940,050 Birr

Estimation of Total Production Cost (TPC)

Three hundred working days per year and 16 hours per day are taken as assumption to estimate the production cost.

Before estimating the total production cost the operating labor and utilities required in the process must be evaluated. Total production cost estimation to produce 6,710 Kg thyme crude extract per year is provided in the following table.

Table 5.4: Total production cost estimation

Items percent Cost (Birr)

Direct

production

cost

Raw material 15Birr/kg 15,000

Operating labor 15% TPC 860,673.3

Direct supervisor and clerical labor 17.5 % operating labor 150,617.8

Utilities Electricity +water 3,183.4

Maintenance and repair 6 % FCI 257,741.8

Operating supplies 0.75% FCI 32,217.7

Fixed

charge

Depreciation 10% FCI 429,569.6

Local taxes 2.5% FCI 107,392.4

Insurance 0.7 % FCI 30,069.9

General

Expenses

Administration 3.5 % TPC 200,823.8

Distribution and selling 11% TPC 631,160.8

Research and development 5% TPC 286,891.1

Financing (interest) 3.5 % TCI 172,902

73

Total Production Cost (TPC) = Manufacturing Cost + General expenses

TPC = (Direct production cost + Fixed charge + plant overhead) + General expenses

TPC = (60%TPC + 10%FCI + 10%TPC) + (19.5%TPC + 3.5%TCI)

TPC − 0.895TPC = 0.1FCI + 0.035TCI

0.105TPC = 0.1 × 4,295,696 + 0.035 × 4,940,050

TPC = 5,737,822.3 Birr/year

The annual production rate of thyme crude extract is:

= 67.1Kgbatch

× 100batchyear

= 6,710 Kg thyme crude extract/year

Profitability evaluation

After estimating the total product cost, the attractiveness and profitability of proposed preliminary thyme crude extract production plant is evaluated in terms of rate of return on investment, payback time and present worth value.

Unit product cost =Total production cost

Annual production rate

= 5,737,822.36,710

= 855.1 Birr/kg thyme crude extract

The selling price of thyme crude extract with a minimum profit of 35% is 1,154.4 Birr per Kg of thyme crude extract.

Total Income =6,710Kg

year ×1,154.4 Birr

kg

= 7,745,958.2 Birr/year

i. Gross earning and Net earning

Gross earning (profit before tax) = Total Income− Total product Cost

= 7,745,958.2− 5,737,822.3

= 2,008,136Birr

Assuming that the income tax rate is 35% (the tax rate of Ethiopia);

74

Net annual earning = Gross earning − Income Tax

= 2,008,136 (1− 0.35)

= 1,305,288.3 Birr

ii. Return on Investment (ROI)

ROI = Net ProfitTotal capital investment

× 100

= 1,305,288.3/4,940,050 × 100 = 26.4%

iii. Payback Time

Payback Period is used to know when project’s initial investment will be fully recovered .The

investment cost and income statements are used to determine the pay-back period. The plant is

assumed to have depreciation of five years.

Payback period =FCI

Net profit + Depreciation

=4,295,696

1,305,288.3 + 859,139.2 = 1.98 years

Based on the preliminary economic evaluation, the suggested project has a return on investment

(ROI) of 26.4 % and payback period of 1.98 years. The evaluation shows that there is good profit

margin and the suggested project is financially feasible.

5.4. Plant Location To select proper locations and sites, certain key requirements or criteria should be set (i.e. raw

material proximity, Infrastructures, product market, human power) that would allow the

assessment of a number of potential locations. The remaining alternatives are subject to a more

in-depth qualitative and quantitative analysis of technical and financial criteria, including social,

environmental and economic aspects of location and site selection. For thyme crude extract

producing plant, based on proximity to market, availability of utilities such as water, electricity

and other infrastructures, ease of access and handling of human resource, Addis Ababa was

selected to locate the plant.

75

Chapter Six

Conclusions and Recommendation

6.1. Conclusions

Thyme is a potent source of polyphenols; thymol and carvacrol having both antioxidant and

antimicrobial properties. The aim of this study was to evaluate these properties of thyme applied

on certain foods like meat, oil and butter.

It is evident from the result of the present work and also the results of other researchers that

antioxidant of thyme crude extract depended on the extraction parameters (solvent concentration,

extraction temperature and extraction time), the kind of food substrate, concentration of the

extract being used in the substrate and the method of choice for antioxidant activity test. Higher

antioxidant activity and extract yield of thyme were found by distilled water extraction process at

20℃ extraction temperature for 3.5 hours.

The antioxidant activity of thyme has been well studied on refined soybean oil and butter using

both Rancimat and Schaal Oven Test methods at 0.1% concentration of thyme crude extract

added to these food model substrates. For comparison, 0.05% α-Tocopherol was added as

positive control and none thyme extract-negative control was also prepared and evaluated. The

results were expressed as Induction Time (IT) and Protection Factor (PF). Induction Time

(Protection Factor) of thyme and α-Tocopherol were 3.25±0.02 hrs (PF of 1.69) & six day (PF of

1.2) and 4.98±0.10 hr IT (PF of 2.59) & seven day (1.5PF) as determined by Rancimat and

Schaal Oven Test method in soybean oil respectively. Thyme also has 5.28±0.08 hrs IT (PF of

1.4) when evaluated in butter by Rancimat method. It may be concluded that thyme crude extract

could contain an effective antioxidant in stabilizing refined soybean oil and butter by both two

methods used for evaluation of antioxidant activity of thyme crude extract. The effectiveness of

the different treatments was strongly dependent on temperature and testing methods. Rancimat

method, exhibits more consistent and objective results of thyme antioxidant activity expressed as

induction time and protection factor than the Schaal Oven Test. At 0.2% thyme extract

concentration its antioxidant activity was also being comparable to natural reference antioxidant

Vitamin E (α-Tocopherol) used in this study.

76

The Preservative effect (chemical analysis) of thyme crude extract has been studied on refined

soybean oil and butter for three consecutive storage weeks. Free Fatty Acid value, Peroxide

value and Acid value of control treatments (without thyme extract) have increased for both butter

and oil during the three storage weeks. As the results show, both treatments of 0.1 and 0.2%

thyme extract significantly retard the values of these oxidative quality parameters of butter and

oil.

The results clearly indicated that supplementing 0.1% and 0.2% thyme crude extract has the

beneficial effect in controlling the microbial load (total aerobic plate count, total aerobic yeast &

mold and total count of Enterobacteriaceae) of both meat and butter during three weeks of

storage at 4℃. However, the control sample of both meat and butter showed higher microbial

load. Thyme crude extract applied at 0.2% has exhibited better capacity to delay the microbial

growth compared to other samples of both meat and butter.

In conclusion, this study provides an insight into understanding the behavior of added natural

antioxidants on soybean oil and butter oxidation. Based on the results, samples with 0.2% thyme

extract significantly (P<0.05) improved both the oxidative and microbial stability. Hence,

Ethiopian thyme has acceptable antioxidant activity and preservative effect as observed on thyme

extract treated food products.

77

6.2. Recommendation Extracts from natural materials are mixture of many components and the content of active

antioxidant is usually low so that large addition would be necessary to obtain significant

improvement against oxidation. However, such large addition may change the flavor and

functional properties of food products. Thus, active antioxidant content and composition of

thyme crude extract could be studied by high performance liquid chromatography (HPLC) and

Gas chromatography (GC).

Even though antioxidant activity cannot be measured directly rather indirectly by the effect of

the antioxidant in controlling the extent of oxidation in model food substrate, antioxidant activity

of thyme crude extract could also be determined using other antioxidant activity evaluation

methods like radical scavenging system.

In human food, its recommended that prior to thyme extraction it can be used in powder and leaf

form in preparing berbere, shiro, tea, butter, pizza and flour. Some of these applications of thyme

have already been used in the society. Due to antifungal property of thyme, it can be used in

preparing cosmetics for skin and hair.

These studies provide the basis for further research:

Develop purified natural antioxidant from this potential herb and others

Improve the quality of meat using thyme as animal feed.

Prepare agricultural pesticides and insecticides

Develop specific active packaging able to have antimicrobial property

Exploit new nutraceuticals safer for human health and more reliable for food industry

applications.

78

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Appendices Appendix 1: Raw data of induction time of thyme extract added refined soybean oil determined by PC-

controlled 743 Rancimat.

Control (without thyme extract)

97% Ethanol, 40℃, 3hr thyme crude extract

0% Ethanol, 40℃, 4hr thyme crude extract

1.88

0

5

10

15

20

25

30

35

40

45

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25

µS/cm

h

2.61

0

5

10

15

20

25

30

35

40

45

0.0 0.5 1.0 1.5 2.0 2.5 3.0

µS/cm

h

2.90

0

5

10

15

20

25

30

0.0 0.5 1.0 1.5 2.0 2.5 3.0

µS/cm

h

85

0% Ethanol, 40℃, 3hr thyme extract

0% Ethanol, 20℃, 3hr thyme extract

48.5% Ethanol, 30℃, 3.5hr thyme extract

3.18

0

5

10

15

20

25

30

35

40

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

µS/cm

h

3.82

0

10

20

30

40

50

60

70

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

µS/cm

h

3.06

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

µS/cm

h

86

Appendix 2: One-way ANOVA analysis of data output done by Design Expert Software 7.0.0.

Response1: Induction time

Sum of Mean F p-value Source Squares df Square Value Prob > F Model 2.05 6 0.34 63.48 0.0030 A-conc 1.28 1 1.28 238.26 0.0006 B-temp 0.30 1 0.30 55.18 0.0050 C-time 8.000E-004 1 8.000E-004 0.15 0.7253 AB 0.37 1 0.37 68.84 0.0037 AC 2.450E-003 1 2.450E-003 0.46 0.5479 BC 0.097 1 0.097 18.02 0.0239 Curvature 0.037 1 0.037 6.95 0.0779 Residual 0.016 3 5.372E-003 Lack of Fit 4.050E-003 1 4.050E-003 0.67 0.4987 Pure Error 0.012 2 6.033E-003 Cor Total 2.10 10 Summary of fit

R-Squared 0.9922

Adj R-Squared 0.9766

Pred R-Squared 0.8636

Adeq Precision 23.518

87

Response2: thyme extract Yield

Sum of Mean F p-value Source Squares df Square Value Prob > F Model 65.16 6 10.86 60.64 0.0032 A-conc 56.18 1 56.18 313.70 0.0004 B-temp 7.33 1 7.33 40.95 0.0077 C-time 1.33 1 1.33 7.42 0.0723 AB 9.800E-003 1 9.800E-003 0.055 0.8301 AC 9.800E-003 1 9.800E-003 0.055 0.8301 BC 0.30 1 0.30 1.66 0.2885 Curvature 27.74 1 27.74 154.91 0.0011 Residual 0.54 3 0.18 Lack of Fit 0.50 1 0.50 26.83 0.0353 Pure Error 0.037 2 0.019 Cor Total 93.44 10

Summary of fit R-Squared 0.9918 Adj R-Squared 0.9755 Pred R-Squared 0.6566 Adeq Precision 22.250 Optimization of thyme antioxidant extraction process; Report of Numerical optimazation Lower Upper Lower Upper Importance Name Goal Limit Limit Weight Weight conc minimize 0 97 1 1 3 temp minimize 20 40 1 1 3 time is in range 3 3.5 1 1 3 induction time maximize 2.62 4.09 1 1 3 yield maximize 10.6 18.27 1 1 3 Solutions: Number conc temp time induction time yield Desirability 1 0.08 20.00 3.50 3.97401 12.9042 0.725 Selected

2 7.61 20.00 3.32 3.82977 13.243 0.715 3 8.02 20.00 3.27 3.81097 13.246 0.712 4 6.41 20.00 3.09 3.78315 13.0949 0.700

88

Appendix 3: Human Power Requirement

No. Position No. of Person Monthly salary per person

Yearly salary

1 General Manager 1 6,500 78,000

2 Executive Secretary 1 2,500 30,000

3 Production & Technical manager

1 5,500 66,000

4 Production Head 1 3,500 42,000

5 Shift leaders 2 1,400 33,600

6 Packers 2 1,000 24,000

7 Electrician 1 2,500 30,000

8 Mechanic 1 2,500 30,000

9 Chemist 1 2,500 30,000

10 Sales Person 1 2,500 30,000

11 Accountant 1 2,000 24,000

12 Drivers 2 1,200 28,800

13 Guards 2 600 15,000

14 Cleaners 2 500 12,000

Total 21 473,400

89

Declaration

I, the undersigned, declare that this thesis is my original work and has not been presented for a

degree in any other University, and that all sources of materials used for the thesis have been

duly acknowledged.

Name: Gebrehana Ashine Hailemariam

Signature: _________________________

Place: Addis Ababa, Ethiopia

Date of submission: __________________________

This thesis has been submitted for examination with my approval as University advisor.

Name: Dr. Eng. Shimelis Admasu (Associate Prof.)

Signature: _____________________________