Yemsrach Yishak

Addis Addis Ababa A baba University Addis Addis Abab A baba a Institute Institute of Technology (AAiT) School of Chemical and a nd Bi B io Engin E nginee ee ring ring

Byproducts Utiliz Utili zation from Wheat Milling Mill ing Industries Industries for Development of Value Added Products

A Thesis Submitted to the School of Chemical and Bio Engineering of Addis Ababa Institute of Technology, in Partial Fulfillment of the requirements for the Degree of Master of Science in Chemical Engineering (Food Engineering Stream)

By: Yemsrach Yishak 

Advisor: Dr. Eng. Shimelis Admassu (Associate Professor)

Addis Ababa, Ethi Et hiop opia ia March, 2014 i

Addis Ababa Unive Unive rsity Addis Ababa Institute of Technology (AAiT) School Of Chemical Of  Chemical and Bio-Engineering Foo Foo d Engineer Enginee ring ing S tream

Byproducts Utilization from Wheat Milling Industries for Development of Value Added Products

A Thesis Submi Sub mitted tted to the School of Graduate Studies of Addis Ababa Institute Institute of Technology, in Partial Fulfillment of the Requirements for the Degree of Master of Science in Chemical Engineering (Food Engineering Stream) By: Yemsrach Yishak

Approved by the Examining Examining Board

Signatures

Ato Taye Zewdu Zewdu (Chair man, Department’s Graduate Committee)

Dr. Eng. Shimelis Admassu (Associate professor) (Advisor)

Ato Adamu Zegeye Zegeye (Internal Examiner) Dr. Ashagrie Zewdu (External Examiner)

i

Addis Ababa Unive Unive rsity Addis Ababa Institute of Technology (AAiT) School Of Chemical Of  Chemical and Bio-Engineering Foo Foo d Engineer Enginee ring ing S tream

Byproducts Utilization from Wheat Milling Industries for Development of Value Added Products

A Thesis Submi Sub mitted tted to the School of Graduate Studies of Addis Ababa Institute Institute of Technology, in Partial Fulfillment of the Requirements for the Degree of Master of Science in Chemical Engineering (Food Engineering Stream) By: Yemsrach Yishak

Approved by the Examining Examining Board

Signatures

Ato Taye Zewdu Zewdu (Chair man, Department’s Graduate Committee)

Dr. Eng. Shimelis Admassu (Associate professor) (Advisor)

Ato Adamu Zegeye Zegeye (Internal Examiner) Dr. Ashagrie Zewdu (External Examiner)

i

Acknowledgments First and for most of all I would love to thank Almighty God for all the blessings he has given me; enabling me to accomplish this thesis work, for all the special people surrounded me. Apart from my effort the success of this thesis depends mainly on the encouragement, guidelines, assistance and support of many others. I take this opportunity to express my gratitude, for the people who have been instrument in the successful achievement of this thesis. First, I would love to express my deepest appreciation to my thesis advisor Dr. Eng. Shimelis Admassu starting from choosing the right topic and guided me through. Your advice, hard work, inspirational conversations, the ability to look, wonder and dare to see beyond the limits makes you exceptional. Thanks for all the encouragement, guidance and assistance; and of course love to express my sincere gratitude to Mr. Bekele Mekuria, for assisting me and fill me me with wit h new new ideas and clues during dur ing my my preliminary preliminar y work. work. I genuinely acknowledge Prof. Kibret Mequanint; your efforts were backbones to the whole thesis thesis work. work. Th T hank you for the financ financ ia l support, willingness, willingness, a nd all the positive feedbacks. I love to acknowledge Ato Hintsa for continuously trying to work with devotion, without your effort and miracle job on Supper critical fluid extractor machine my thesis work would ha ve been totally tota lly different. Most importantly of all, I express my deep sense of gratitude to my most affectionate and  beloved fian fia ncé a nd family for all the spectra you ran for me. Tha Tha nks for being there for me for each never last seem journey. You taught me to never surrender for battles in my life, and keep on reminding me that God is already there. I just want to say thank you for your love, undying encouragement, wisdom, advice, and for being there in every aspect of my life. Meri my little sister extra thanks to you dear, you really mean something to me. I honestly could not have done this without your support. And last but not least, my friends Habtish and Dera I’m grateful from the bottom of my hypothalamus, thanks for assisting me when I was in need of support; Dr. Ashagre, PHD students Alexa and Engida in Science Faculty without your guidance all would have been impossible. Finally, I would love to thank Sirgute and Tigist for all the secretarial works you help me out.

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Abstract The main byproducts of wheat milling industries, wheat germ and bran, have been known as an outstanding sources of protein, dietary fiber, trace minerals, antioxidants, phytochemicals and allied micronutrients. This research was conducted to evaluate utilization of wheat germ (WG) and wheat bran (WB) for the development of value added cookies and tea substitute  products; resp resp ectively. Supercrit ical fluid ex tractor tract or (SFE) was used used t o ext ract oil from wheat  germ and the defatted wheat germ germ flour (DWGF) (DWGF) was used as a supplement f or wheat flour at 10%, 15% and 20% blending ratios (BR 1 , BR2 &BR3 ) and baking temperatures tem peratures of 150, 180 and 210 o C (T 1 , T 2  &T 3 ).Wheat  ).Wheat bran (WB), the t he other ot her byproduct of wheat milling indust ries, used to made tea substitute by milling, screening, and heating before utilized as final product. Chemical composition of raw materials, physico-chemical and rheological characteristics of  flours were investigated prior to cookies preparation. Proximate analysis analysis for wheat flour (WF)(0.83,11.92,0.52,0.45,9.33)

and

defatted

wheat

germ

flour

(DWGF)

(4.72,

12.98,1.01,5.17,28.11); for ash, moisture, fat, fiber and protein were resulted respectively; as a result nutrient dense DWGF used used as a substitute for development of cookies . It It was found that protein, fiber, ash and minerals (Ca, K, P, and Mg) contents in the blends increased  significantly  significantly (P<0.05) with an increase increase in DWGF substitution. The effect of BR2 and T 2 on  proximate composition composition resulted with (protein (14.29), (14.29), fiber (2.84), and ash (1.31)) resulted better together with sensory quality evaluation. Rheological and functional properties of  flours, physical properties propert ies for cookies and organoleptic properties for both products were analyzed. Total phenolic content (TPC) and antioxidant activity were determined by using  Folin-Ciocalteu  Folin-Ciocalteu and DPPH scavenging activity activi ty assays respectively. Extraction Ex traction procedure went out using methanol at three different temperatures (40, 60 and 80) 0 C. Higher total  phenolic  phenolic content ranged from 1.037 to t o 3.68mg of gallic gallic acid equivalent (GAE)/gm of dried extract obtained at 60 0 C using methanol. While antioxidant activity with lower half maximal inhibitory concentration (IC 50 activ ity for 50 ) (mg/ml) v alue of (1.4, 1.75, 2.13) scavenging activity ascorbic acid, methanol solvent extract of wheat bran and by absolute methanol respectively.  Finally,  Finally, cook ies baked bak ed at 180oC using blend ratio 15 % resulted better sensory qualification and wheat bran extracted using solvent methanol at 60 oC showed potential antioxidant activity activit y and TPC. Keywords: defatted wheat germ, cookies, antioxidant, folin-Ciocalteu assay, DPPH scavengi cavengi ng capacity capaci ty assay assay

iii

Table of contents

Chapter

Title

Page

Title page

i

Acknowledgements

ii

Abstract

iii

Table o f contents

iv

List of tables

v

List of figures

viii

List of abbreviations 1

2

ix

Introduction

1

1.1

Background

1

1.2

Statement of the problem

3

1.3

Objectives

4

1.4

Limitation of the study

4

1.5

Significance of the study

5 6

Literature Review

2.1

Overview on cereals

6

2.1.1 Wheat consumption and production in Ethiopia

7

2.1.2 Uses and varities of wheat

8

2.1.3 Morphology and composition of wheat

11

2.2

Effect of milling process

12

2.3

Phytochemicals and antioxidants

14

2.4

Raw materials for developed products

17

2.4.1 Wheat bran

17

2.4.2 Wheat germ

19

Process descript ion

21

2.5.1 Process descript ion for wheat flow

21

2.5

iv

3

2.5.2 Process descript ion for biscuit production

25

2.6

Sensory quality attributes

31

2.7

Concluding Remarks

32

Materials and Me thods

3.1

Raw material collection, transportation, preparation and storage

34

3.2

Frame work of the research experiment

35

3.3

Methods of processing

36

3.3.1 Preparation of defatted wheat germ flour

36

3.3.2 Blend formulation and cookies production

37

Methods of ana lysis

39

3.4.1 Analysis of pro ximate composition of flours and cookies

39

3.4

3.5

3.6 3.7 4

34

3.4.2Determination of rheology property of flours

44

3.4.3 Determination of funct ional properties of flours

44

3.4.4 Deter mination of physical properties of cookies

44

Analysis of antioxidant activity and total phenolics

45

3.5.1 Sample extraction

45

3.5.2 Determination of total phenolic content

45

3.5.3 Deter mination of free radical sca venging activity

46

Sensory quality evaluatio n

48

Experimental des ign and statistica l data analysis

48

Result and Discussion

49

4.1

Proximate chemical composition of flours and cookies

49

4.2

Effect of Blend ratio and baking temperature on Proximate composition of cookies

50

4.2.1 Effect of blend ratio and baking temperature on moisture content

50

4.2.2 Effect of ble nd ratio and baking te mperature on crude  protein

51

4.2.3 Effect of blend ratio and baking temperature on crude fiber

52

4.2.4 Effect of blend ratio and bak ing te mperature on ash

52

4.3 Rheological property of flours

53

4.3.1 Water absorptio n

53

4.3.2 Dough development time

55 v

4.4

4.5

4.6

4.7 5

6

4.3.3 Dough stability

56

4.3.4 Farinograph quality number (FQN)

56

Functional properties of flours

56

4.4.1 Bulk density

56

4.4.2 Water absorptio n capacity

57

4.4.3 Oil absorption capacity

57

Physical properties of cookies

58

4.5.1 Effect of blend ratio and bak ing te mperature on weight of cookies

58

4.5.2 Effect of blend ratio and baking te mperature on dia meter of cookies

59

4.5.3 Effect of blend ratio and bak ing te mperature on cookies height

59

4.5.4 Effect of blend proportion and temperature on spread ratio

59

Total phenolic content and antioxidant activity of bran

60

4.6.1 Total phenolic content of wheat bran

60

4.6.2 Antioxidant content of wheat bra n

61

Sensory quality evaluation of products

62

Process Technology

64

5.1

Production process for cookies and tea substitute

64

5.2

Suggested cookies manufacturing plant

65

Conclusion and Recomme ndation

67

6.1

Conclusion

67

6.2

Recommendation

68

References

69

Appendices

76

Appendix I

Score card for the sensory quality eva luation using nine point hedonic scales

76

for cookies Appendix II

Score card for the sensory quality evaluation using nine point hedonic scales for tea substitute

77

Appendix III

Data obtained for bran extraction and tests

78

Appendix IV

Pictorial representations for actual frame work

80

vi

List of tables Table

Title

Page

2.1

Wheat composition and the milling process effect on nutrient composition

12

2.2

Fatty acid composition of wheat ger m oil

19

3.1

percentage composition of composite flour for cook ies

36

4.1

Proximate composition and minera l of flours

47

4.2

Effect of blend ratio & baking temperature on proximate composition

51

4.3

Mineral composition of biscuit at different blend proportion

53

4.4

Functional properties of flours

58

4.5

Physical Properties of cookies

58

4.6

Effect of blend ratio and temperature on diameter of cookie

59

4.12 Effect of blend ratio and temperature on cookie height

59

4.13 Effect of blend proportion and temperature on spread ratio

60

5.1

66

Legend for suggested cookies manufactur ing plant

vii

List of figures Figure

Title

Page

2.1

Production of key crops from 2010-2013

2.2

Schematic diagram of wheat

11

2.3

Free radicals and disease

16

2.4

Process steps in wheat milling

23

2.4

Schematic representation of idealized phase diagram

25

2.5

Parameters in Pharinograph representation

28

2.6

Wheat production trend in Ethiopia

31

3.1

Wheat grain and its byproducts (from Hora Complex PLC.)

33

3.2

Frame work of the research exper iment

34

3.3

Oil obtained from defatted wheat germ flour (DWGF) using supercritical fluid extractor (SFE) before and after separat ion via separatory funnel

5

35

3.4

Simplified diagram for Preparation of DWGF

36

3.5

Blended flours, defatted wheat germ flour and control flour

37

3.6

Extraction method for antioxidant activities and phenolics analysis

45

4.1

Farinograph values of control flour/ WF

54

4.2

Farinograph value for BR 1

54

4.3

Farinograph measure ment for BR 2

55

4.4

Farinograph value for BR 3

55

4.6

Free radical scave nging methanolic extract of wheat bra n and control

62

4.7

Sensory quality evaluatio n for products

62

5.1

Flow chart diagram for deloped products

64

5.2

Equipment layouts for cookies production

65

viii

List of Abbreviations

AACC

American Association of Cereal Che mists

AOAC

Association of Analytical Chemist

BR1

Blend ratio 10% substitute of wheat flour

BR2

Blend ratio 15% substitute of wheat flour

BR3

Blend ratio 20% substitute of wheat flour

DPPH

2,2-diphenyl,1-picrylhydrazyl

DWGF

defatted wheat germ flour 

EGTE

Ethiopia Grain Trade Enterprise

EHNRI

Ethiopian Health and Nutrition Research Institute

Eq.

Equation

FAO

Food and Agricultural Organization

FQN

Farinograph quality number

FU

Farinograph Unit

IC 50

Half maximal inhibitory concentration

IFIC

Internat ional food information council foundation

Mmt

million metric tones

OAC

Oil absorption capacity

ODC

Ozone depleting che micals

SD

Standard deviation

TPC

total phe nolic content

t/ha

Ton per hectare

VOC

Volatile organic compounds

WAC

Water absorption capacities

WF

Wheat flour

WHO

World Health Organization

ix

CHAPTER ONE

Introduction 1.1 Background Cereal crops are staple foods that provide essential nutrients to numerous populations of the world. Cereals are dominant in the food sector beca use they are a versatile and reliable so urce of food. They are easy to store and may be used to produce a myriad of food products. Cereals processing thus forms a large and important part of the food production chain. It also  plays a lesser, but no less important role in the non-food sector. It is for these reasons that ways of improving cereal processing technology and practice need to be addressed on a continual basis. Practically every meal produced today contains cereals in some form, while the range of non- food applications (Galvin, 2001). Wheat is a farinaceous grass, known botanically as triticum spp., is one of the most consumed cereal grains worldwide and makes up a substantial part of the human diet. It provides more nourishment (calories & proteins) for humans than any other single food crops. According to Statista 2013/2014, the global production volume of wheat amounted approximately 710 million metric tons, which has shown a 7.7% increment from the previous year. It is the second most important food crop in the developing world after rice. In sub-Saharan Africa, 14 countries produce wheat; Ethiopia and South Africa are the two major producers. Along with Teff, wheat and maize represent the three most important cereal crops in Ethiopia. Wheat is one of the various cereal crops largely grown in highlands of Ethiopia.

It is

 produced largely in the southeast, central and northwest parts of Ethiopia (Karin & Leo, 2013). Cereal processing industry may be described as any industry that takes a cereal or a cereal  product as its raw mater ial. The wheat-based industry is a multi-billion dollar market; hence wheat is one of the top three cerea ls crop in the world. The milling process o f wheat produces large amount of wheat bran and germ as a byproduct. During milling, the endosperm is  broken down into fine particles (white flour) while bran and germ are removed. Wheat is a significant agricultural and dietary commodity worldwide with known antioxidant properties concentrated mostly in the bran. Wheat germ, being a byproduct of the flour milling industry, is reported to be one of the most potential and excellent sources of much-needed vitamins, minerals, dietary fiber, calories, proteins, and some functional micro-compositions at a relative low cost (Yiqiang et al ., 2001 and Shao & LiYu, 2011).

1

In general from wheat milling industries release a byproduct of (25-40) % and these by products utilized for animal feed, bioethanol production, succ inic acid production, like a  blend for baked products as nutritional improvement, for cosmetics, meat substitute, neutraceutical/ phar mace utical products and for many more others. A value add ition, any step in the production process that improves the product for the customer and results in a higher net worth of the last product. Using by-products from wheat milling industries for value addition is accustomed in the developed countries like U.S.A for instance defatted wheat germ helps meet today’s demands for full flavor grain-based foods that are rich in protein and fiber (Dotty, 2012). Oil inside the wheat germ extracted using different mechanisms such as the common method organic solvent extraction (Hexane, Methanol, Chloroform-methanol, etc) which recovers about 90% of the oil, by mechanical pressing, which recovers about 50% (Singh and Rice 1979) or by using super critical extraction methods (85%). The extracted wheat germ oil from the former two mechanisms resulted in having lower free fatty acid and α-tocopherol content; in other word oil obtained by supper critical extraction can overcome these negative factors; in fact, the oils are solvent-free and do not need the traditional refining processes, and extraction yields are similar to those usually need to be refined (Panfali et. al, 2003). Above all, recent research demonstrates that wheat grain contains significant level of natural antioxidants, mostly concentrated at the outer part. Wheat is an important agricultural commodity and a primary food ingredient worldwide and contains considerable beneficial nutritional components. Wheat and wheat-based food ingredients rich in natural antioxidants can ideally serve as the basis for development of functional foods designed to improve the health of millions of consumers (Tomas et. al., 2014). Tea/ coffee substitutes are non-coffee  products, usually without caffeine,  that are used to imitate coffee. This substitutes can be used for younger children, medical, economic and religious reason, or simply because coffee is not readily available. Coffee and tea substitutes made from wheat and barley have been produced for a cent ury; however, limited research has gone into the antioxidant benefits from roasted wheat and coffee beverages. As the benefits of wheat antioxidants become better known, the wheat and coffee beverage markets may emerge as well. According to researches by naturopathic clinic caffeine stimulates c entral and sympathetic nervous systems, resulting in an elevation of the stress hormones released by  pituitary, adrenal a nd hypothalamus glands. These hormones c an ca use short term spikes in

2

our blood pressure by raising both systolic and diastolic pressures. Release of stress hormones causes our body to enter a state similar to a fight or flight response, causing blood to be redirected from our stomach and digestive system and potentially causing indigestion (Doty, 2012).

1.2. Statement of the problem Wheat milling industries process and finally ground wheat kernel in to flour by separating the wheat grain in to its constituents endosperm, bran and germ. The end product flour mainly contains endosperm where as bran and germ removed as byproduct from wheat milling industries. Wheat bran removed from being part of final flour with aim to produce flour with a white rather than a brown color, and eliminated the fiber. Neither of these objectives is necessarily desirable from the nutritional point of view. Similarly, for the reason that wheat germ, if left in flour, has an adverse effect on the functional properties of dough, and reduces the shelf life of final flour hence it’s rich in polyunsaturated fats (which have a tendency to oxidize and beco me ra ncid on storage). Consequently wheat germ removed during processing improved the storage qualities of flour and milled as part of mill feed and the final flour/ white flour so ld without enrichment process. Abroad, countries like United States, Far East and others developed countries managed to utilize their wheat milling industries byproducts beyond meeting the nutritional needs of their customers. Wheat germ used as a resource for value addition purpose after extracting the oil. When wheat germ defatted; it becomes ideal ingredient for grain based products; hence it is high in protein, fiber and is virt ually fat free. Processing and finally blending with wheat flour to get better functional qualities included improved stability, nutritional values and flavor of processed foods besides making consumer goods of all kinds. The basic problems in developing countries like ours unlike the developed ones; instead of maximizing (using available) resources in our hand, lose it as if we couldn’t gain any importance from it. Hence , the number of wheat milling industries are increasing year after year, as that of consumption and production of wheat; then utilizing the (25- 40) % of the total would be nice than waste it. Wheat milling industries byproducts, wheat germ and bran, were collected to feed animals therefore underutilized. Hence, aim of this thesis was utilization of byproducts from wheat milling industries at industrial level (produced cookies from blend of wheat germ after defatted and a non caffeine tea substitute from wheat bran) help to attempt the shortage of

3

wheat by developing composite products and insures food security in the country.

By

creating awareness in consumer’s mind; commercialization and promoting healthier products should be given attention.

1.3 Objectives of the study  General o bjectives

The general objective of this research was to utilize byproducts from wheat milling industries for de velopment of value added products.  Specific objectives

The specific objectives of this research were to:  Assess the proximate composition of raw materials: wheat germ flour, defatted wheat germ

flour, wheat flour and identify if defatted wheat germ flour was able to supplement wheat flour to develop last product cookies.  Evaluate mineral contents of wheat flour and defatted wheat germ flour and the composite

cookies developed.  Evaluate rheological properties o f control and blended flours.  Deter mine the functiona l property of b lended flours and control.  Determine the effect of baking temperature and blend ratios on the proximate composition,

 physical as well as organoleptic property of cookies  Evaluate the antioxidant property of wheat bran

1.4 Limitation of the study

Even though the research has reached its aspire, there were some avoidable limitations. The first one is scarcity of byproducts that are used as raw materials for the development of the value added products i.e wheat germ and wheat bran separately. Because most industries here avoid those byproducts together for animal feed; but Hora food complex was willing to open the accurate pipes in the middle of processing the ker nel to wheat flour. The seco nd limit was means of knowing the amount & which specific amino acids present in the defatted wheat germ flour however there wasn't means of knowing all the amino acids present inside the wheat germ protein content from nitrogen was calculated.

1.5 Significance of the study This research studied the importance of utilization of cheap byproducts (wheat bran and germ) from wheat milling industries. Hence, the final refined white flour resulted in most of

4

the industries in Ethiopia; do not pass through enrichment process unlike the developed countries. This as a result leaves the society poor in nutrition, and the industries less  profitable; instead of utilizing these byproducts either to maximize resource (by blending with defatted wheat germ flour) or bran as raw material for tea substitute. Utilizing wheat germ in baked products will not only explore its functional and neutraceutical role but also contribute towards value addition in wheat milling sectors so that consumers benefit nutritionally. This indirectly encourages cosmetic industry sectors to make use of oil from that of defatted wheat germ instead of importing expensive goods from abroad. Finally a new idea and practices to develop a non caffeine tea substitute from antioxidant rich wheat bran. Overall outcome of the research will raise profit, creates awareness and give alternatives for  processors especially contribute its part to achieve food security.

5

CHAPTER TWO

Literature Review 2.1. Over view on cereals A cereal is a grass cultivated for the edible components of its grain, composed of endosperm, germ and bran. Cereal grains are grown in greater quantities and provides more food energy worldwide than any other type of crop, they are therefore called staple crops. World cereal  product ion would fractionally decline from the 2013 peak.

According to FAO, 2014

estimated wheat production is 721.12 million metric tons achieving a new record from 715.13 million tons last year represent an increase of 5.98 million tones of wheat production around the globe. According to USDA report, Ethiopia is the second largest wheat producing country in Africa next to South Africa. Hence, among major grain crops grown in the country are teff, wheat, maize, barley (categorized as primarily cool weather crops) and maize, sorghum, and millet (categorized as warm weather grain crops). It ranks fourth after teff, maize and sorghum in area coverage and third in total production. Wheat is mainly grown in the central and south eastern highlands during the main (Meher) rainy season (June to September) and harvested in October-November. Arsi, Bale, and parts of Shoa are considered the wheat growing belt. In Ethiopia there has been a substantial growth in yield and production of cereals since 2010. In 2013/14, the yields are estimated to be 2.2 MT/ha. However, by international standards such yields are considered to be low (Carlos and Doyle, 2009). 2010/11

34,834,826

2011/12

34,976,894

15,852,869

29,163,336

17,816,522

2012/13 37,652,411

0

28,556,817

17,033,465

25,000,000

49,861,254

60,694,130

34,347,061

50,000,000

75,000,000

61,583,175 100,000,000

Metric tons of production Teff

Barley

Wheat

Maize

Figure2.1  Production of key crops from 2010-2013.

6

Sorghum

39,598,973

39,512,942

39,598,973 125,000,000

150,000,000

2.1.1 Wheat Consumpti on and Production in Ethiopia In Ethiopia, Wheat ranks fourth in terms of area production and yield among food crops. Production of wheat increased from 2.2 (000 t) in 2004/2005 (CSA, 1998) to 2.8 (000 t) in 2010/2011 (CSA, 2000) an increase o f 31 percent. However, the s hare of wheat in total cerea l area decreased (-12.4 percent) over the same period, mainly due to a shift in cropping  patterns towards sorghum. Wheat yield in Ethiopia is also lagging behind other major  producers in Africa: average yield was 1.68 t/ ha during t he same period, about 32 percent and 39 percent below Kenyan and South African averages, respectively (FAOSTAT). The apparent low productivity can be attributed to several factors, including slow progress in developing wheat cultivars with durable resistance to diseases, and depleted soil fertility (Demeke, 2013). Commercial imports of wheat have increased in the last couple of years, which is likely the result of the government’s efforts to stabilize wheat prices following a significant increase in domestic food prices. Ethiopia remains one of the largest recipients of food aid in Africa, receiving around 27% of the global food aid given to sub-Saharan Africa. In May 2012/13, Ethiopian Grain Trade Enterprise (EGTE) imported 322,415 MT of wheat, primarily from India, 26 percent of which was from the US and from food aid too (Demeke, 2013). There are around 216 flour mills in Ethiopia, with a total production capacity of about 4.2 million tons of wheat flour a year. Almost a third of these mills are located in Addis Ababa. Mills are able to obtain wheat through two channels, namely subsidized wheat from the EGTE and from domestic production o n the open market, whose price is higher than imports. The state-owned EGTE controls all commercial wheat imports and makes wheat available to millers at a subsidized price; this accounts for roughly a quarter of the wheat market and the rest of the market is supplied from domestic production, whose price is not controlled and whose price is higher than imported wheat (Abu, 2014). It accounts for about 11% of the national calorie intake. The largest volume of the main season production of wheat originates from Oromia (55 per cent), Amhara (29 per cent) and the Southern Nations, Nationalities, and Peoples Region, SNNPR (9 per ce nt) (CSA, 2010).

7

2.1.2 Use s and vari ties of wheat  Uses of wheat

In Ethiopia, wheat grain is used in the preparation of a range of products such as: the traditional pancake ( “injera”), bread (“dabo”), local beer (“tella”), porridge and several others local food items (i.e., "dabokolo","ge nfo", "kinche”). Besides, wheat straw is commonly used as a roof thatching material, and as a feed for animals. Wheat contributes approximately 200 kcal/day in urban areas and about 310 kcal in rural areas. Globally, there is no doubt that the number of peop le who rely on wheat for a substantial part of their diet amounts to several billions. According to P. kumar et al   journal wheat, as  produced b y nature, contains se veral medicinal virtues and re levant to human being discussed as follow: Every part of the whole wheat grain supplies elements needed by the human body. Wheat  bran is used as a s upplemental source of dietary fiber for preventing colon diseases ( including cancer), preventing gastric ca ncer, helps constipation by speeding up the colon and increas ing stool output and bowel frequenc y. Treating irritable bowel syndrome, reducing the risk of  breast cancer a nd gallbladder disease, and type 2 diabetes The germ forms only 3% of the weight of a wheat grain; nonetheless, contains about 25% of the protein, lecithin, vitamins and minerals. Its oil is highly rich unrefined oil, richest sources of vitamin E, A and D, has a shelf life nearly 6-8 months. This oil widely used for external application, as it helps a great deal in getting rid of skin irritation including skin dryness and cracking, improves the circulation of blood and helps to repair the skin cells destroyed by the scorching heat of sun, has exceptional nourishing qualities, as a result; increasingly finding its way in the making of skin care products. Wheat germ oil is known for its antioxidant  properties, a good source of fatty acids that are ver y vital for the healthy growth of the body and this explains the reason why it is added to other carrier oils. It keeps away the symptoms of dermatitis, thereby preventing the skin from being victimized by various kinds of  problems. Wheat grass therapy can be effectively used for skin diseases and ulcerated wounds as by retarding bacterial action, it promotes cell activity and normal re-growth. By drinking wheat grass juice regularly, an unfavorable environment is created for bacterial growth. Poultice of wheat grass juice can be applied on the infected area, as it is an able sterilizer. Externally, wheat flour is useful as a dusting powder over inflamed surface as in burns, scalds and

8

various itching and burning eruptions, Whole wheat flour, mixed with vinegar, boiled and applied outwardly removes freckles. The young stem of wheat used in the treatment of biliousness and intoxication. The ash is used to remove skin blemishes. The fruit is antipyretic and sedative. The light grain is antihydrotic. It is used in the treatment of night sweats and spontaneous sweating. The seed is said to contain sex hormones and has been used in China to promote female fertility. The seed sprouts are antibilious, antivinous and constructive. They are used in the treatment of malaise, sore throat, thirst, abdominal coldness and spasmic pain, constipation and cough. The plant has anticancer properties also. The straw has many uses, as a biomass for fuel, for thatching, as mulch in the garden. A fiber obtained from the stems is used for making paper. The stems are cut into usable pieces and soaked in clear water for 24 hours. They are then cooked for 2 hours in lye or soda ash and then beaten in a ball mill for 1½ hours in a ball mill. The fibers make a green-tan paper. The starch from the seed is used for laundering, sizing textiles, etc. Antioxidants in wheat bran exist in the forms of vitamins (tocopherols  –   vitamin E), minerals (selenium), phenolic acids (ferulic acid, vanillic acid), tocotrienols, phytic acid, phytosterols, flavonoids, and carotenoids (lutein) (El-Sayed et al., 2008). Coffee and tea contain abundant levels of antioxidants as do wheat and barley kernels. Coffee and tea also naturally contain caffeine. Coffee and tea substitutes made from wheat and barley have been produced for a century; however, limited research has gone into the antioxidant benefits from roasted wheat and coffee beverages.  Species and varieties of wheat

Today wheat is one of the world’s most important grains, as it covers more of the earth’s surface than any other grain crop. Wheat is a cereal grain of the genus Triticum within the grass family Poaceae. Botanically, there are more than 30, 000 wheat varieties, categorized into six major classes according to planting and harvesting dates as well as hardness, color and shape of the kernels(Kelly, 2009): Wheat varieties included were (i) Hard or soft, which relates to the hard ness of the kernel. (ii) Red or white, which relates to the presence or absence of a red pigment in the outer layers of the wheat kernel. (iii) Winter or spring wheat varieties that are categorized as such depending on whe n the wheat is planted. Ethiopian farmers traditionally grow several varietal mixtures (even less productive cultivars and wild relatives) in the same field that might have advantage to add variety to their diet,

9

reduce the risk of pests and diseases or unusual environmental conditions, and also preserve cultivars and ge netic diversity (Bekele, 1984; Ja in, 2000). Ethiopian wheat includes tetraploid and hexaploid species. Tetraploid wheats are indigenous, whereas hexaploid wheats are  probably a recent introduction (Bechere, 2000). In wheat breeding histor y Ethiopian tetraploid wheat landraces were often used as sources of earliness, disease and pest resistance, nutritional quality, resistance to drought and other stresses, adaptation to low soil fertility and other characteristics useful in low-input agriculture (Worede, 1997). Durum wheat is of the species Triticum durum  distinctly different from common wheat in that it produces very hard kernels and has yellow pigments throughout the endosperm rather than in the outer layers. It is typically used to produce pasta products, while common wheat is used, for example, in breads, cakes, cookies, and crackers (Korolchuk   et. al ., 2006). The species according to Ministry of agriculture and rural de velopment, (2009) can be categorized as:  Hexaploid species

Common wheat or Bread wheat (T. aestivum) is hexaploid species that is the most widely cultivated one in the world.  Spelt (T. spelta) is another hexaploid species cultivated in limited quantities. Spelt is sometimes considered a subspecies of the closely related species  common wheat (T. aestivum), in which case its botanical name is considered to be Triticum aestivum subsp.  spelta. Among the differe nt varieties of bread wheat in Ethiopia that

have been

currently developed to satisfy the growing production demands are: Tura, Sirbo, Bobicho, Tossa, Sofumar, Digalu, Senkegna, Dinknes h, Alidoro, Menze, Meraro,

Warkaye,

Dereselgne, Dashen, Mitike, Kubsa, Wabe, Galema, Megala, Abola, Tuse, Simba, Katar, Shina, Wetera, Gasay, Sulla, Meraro, Warkaye, Jiru, Senke gna, Millennium.  Tetraploid Species

Durum (T. durum) –   The only tetraploid form of wheat widely used today, and the second

most widely cultivated wheat. It has very narrow adaptation and lower yield potential as compared to bread wheat includes: Hitosa, Denbi, Werer (Mamouri I), Tate, Flakit, Obsa, Ejersa, Bakalcha, Kokate, Malefia, Oda, Ilani, Megenagna, Quami, Mettaya, Ude, Selam, Ginchi, Robe, Laste , Asasa, Arsi-Robe, Mosobo . Emmer (T. dicoccum) is also tetraploid species originated in the Near East., cultivated in

ancient times but no longer in widespread use worldwide. Indeed, it is one of the first cereals ever do mesticated a nd was part of the early agriculture of the Fertile Crescent.

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 Diploid Species

Einkorn (T. monococcum) is diploid species with wild and cultivated variants. Domesticated

at the same time as emmer wheat, but never reached the same importance. Triticale (X-Triticosecale)

This one is a man-made crop developed by crossing wheat (Triticum turgidum or Triticum aestivum) with Rye (Secale cereale). In Ethiopia triticale is only a recent introduction: Dilfekar, Logaw Shibo, Minet, Snan.

2.1.3 Morphology and composition of wheat Wheat grains are generally oval shaped, although different types of wheat have grains that range from almost spherical to long, narrow and flattened shapes. The grain is usually  between 5 and 9 mm in length, weighs between 35 and 50 mg and has a crease down one s ide where it was originally connected to the wheat flower. The wheat grain contains 2-3% germ, 13-17% bran and 80-85% mealy endosperm (all constituents converted to a dry matter basis) (Zuzana et a l, 2009). The wheat kernel consists of three fractions, the endosperm, bran, and germ, which are compositionally and morphologically very different. Thus, products will have different coarseness, textures, and color depending on the portion of the wheat kernel being used. Refined wheat flour is formed primarily from the endosperm of the wheat kernel. The endosperm comprises approximately 82% of the wheat kernel. The functio n of the endosper m is to provide energy for the embryonic plant during germination of the wheat kernel. The endosperm contains approximately starch and 10-14% prote in (Korolchuk et a l., 2005). Compared to the bran and germ, the endosperm contains low amounts of fiber, lipid, vitamins, minerals, protein, pigments and other phytonutrients. This helps give the refined wheat flour its consistent, fine, starchy texture and off-white color compared to whole-grain wheat flour. The bran consists of several cell layers and contains a significant amount of fiber. The bran includes the aleurone layer, which separates the endosperm from the bran layers. The aleurone layer is rich in proteins, vitamins and phytonutrients. The germ is rich in lipids, fiber, vitamins, minerals and phytonutrients. Thus, refined wheat flour, which is made  primarily of endosperm is mainly starch a nd has limited amounts of fiber, proteins, lipids, vitamins, minera ls and ot her phytonutrients (Korolchuk et al., 2005).

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Figure 2.2 Schematic diagram of wheat (Sur get and Barron, 2005).

The bran (outer layers of wheat grain) is made up of several layers, which protect the main  part of the grain. In order to protect the grain and endosperm material, the bran comprises water-insoluble fiber. Chemical composition of wheat bran fiber is complex; it contains, essentially, cellulose and pentosans, polymers based on xylose and arabinose, which are tightly bound to proteins. These substances are typical polymers present in the cell walls of wheat and layers of cells such as aleurone layer. Proteins and carbohydrates each represent 16% of total dry matter of bran. The mineral content is rather high (7.2%). The two external layers of the grain (pericarp and seed coat) are made up of dead empty cells. The cells of the inner bran layer- aleurone layer are filled with living protoplasts. There are large differences  between the levels of certa in amino acids in the a leurone layer a nd t hose in flour. Glutamine and proline levels are only about one half, while arginine is treble and alanine, asparagine, glycine, histidine and lysine are double those in wheat flour (Cornell 2003).

2.2 Effect of milling process The consumption of white flour and bread have historically been associated with prosperity and the development of sophisticated roller mills in Austro-Hungary during the second part of the 19th century allowed the production of higher volumes of whiter flour than it was  possible to produce by traditional milling based on grinding betwee n stones a nd s ieving. The  bleached flour obtained at the end of the product is not rich in nutrient; however the by products obtained are excess ively prosperous to be left for animal feed (Jones, 2007). Generally, cereal grains are subjected to different processes to prepare them for human consumption. These processes significantly affect their chemical composition and consequently their nutritional value. Flour processing decreases the levels of naturally

12

occurring, non -bioavailable nutrients in flour. Iron a nd folic ac id are a mong the vitamins and minerals lost when bran and ger m are separated from endosperm (Victor, 2011). Table 2.1 Wheat composition and the milling process eff ect on nutrient composition Items

Wheat grain

Bran

Flour

Germ

Dietary fiber (g)

12

42.8

2.7

13.2

Protein (g)

15.4

15.5

10.3

23.1

Tryptophan (mg)

195

282

127

317

Threonine (mg)

433

500

281

968

Isoleucine (mg)

541

486

357

847

Leucine (mg)

1038

928

710

1571

Lysine (mg)

404

600

228

1468

Methionine (mg)

230

234

183

456

Cystine (mg)

404

371

219

458

Phenylalanine (mg)

724

595

520

928

Tyrosine (mg)

441

436

312

704

Valine (mg)

679

726

415

1198

Arginine (mg)

702

1087

417

1867

Histidine (mg)

330

430

230

643

Alanine (mg)

555

765

332

1477

Aspartic acid

808

1130

435

2070

Gluta mic acid (mg)

4946

2874

3479

3995

Glycine (mg)

621

898

371

1424

Proline (mg)

1680

882

1198

1231

Serine (mg)

663

684

516

1102

Thiamin (mg)

0.5

0.5

0.1

1.9

 Niacin (mg)

5.7

13.6

1.3

6.8

Vitamin B6 (mg)

0.3

1.3

-

1.3

Folate ( µg)

43

79

26

281

Amino acids

Vitamins

Source: Gramene 2009 Therefore, milling removes the fibrous layers of the grain; thus, refined cereals do not have the same nutritional and health benefits as the grain or by-products. Without the bran and

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germ, approximately 45% of the grain proteins are lost, along with 80% of fiber, 50 -85% of vitamins, 20-80% of minerals, and up to 99.8% of phytochemicals. In addition, important losses of amino acids (35-55%) occur during refining. Some fiber, vitamins, and minerals may be added back into refined cereal products through fortification or enrichment programs, which compensates for losses due to refining, but it is impossible to restore the  phytochemicals lost during processing (Rosell, 2007).

2.3 Phytochemicals and anti oxidants  Phytochemicals

Two hundred and ten thousand phytochemicals present in plants have been isolated and characterized according to the Dictionary of Natural Products (Hampden Data Services Ltd., 2008) but a large percentage of phytochemicals remain unknown. ‘Phytochemical’ refers to every naturally occurring chemical substance present in plants, especially those that are  biologically active (Zielinski and Kozlowska, 2000). Wheat grains are sources of phytochemicals with potential health benefits. Phytochemicals together with many other micronutrients are often found in the germ and the bran of wheat. Though, wheat bran and germ layers removed during milling contain 75% of the phytonutrients (Slavin, 2003; Jo nes et al., 2004) in the wheat kernel. Wheat grain, particularly, its bran fraction contains several classes of phytochemicals. Among them, phenolic acids, polyphenols (flavonoids and lignans), (both phenolic acids and flavonoids are major class of phytochemicals containing one or more aromatic ring and one or more hydroxyl group), carotenoid (another group of phytochemicals contributing to the pigments and are thought to provide health benefits in decreasing the risk of disease, particularly certain cancers and eye diseases), tocopherol/tocotrienols (used for treating diabetis, desease of brain and nervous system, to avoid complication in late pregnancy, to prevent aging etc.), and  phytostero ls/phytosterols (a group of p hytochemicals known for cholesterol reduct ion in human have been characterized and linked to many bioactivities related to human health). Most of these  phytochemicals have s hown strong a ntioxidant activities in both pure and mixed forms a lso have  been implicated to play a protective role against chronic d iseases such as cancer, cardio- vascular diseases, a nd diabetes (Lia ng, 2007).

Antioxidants Antioxidants are substances that may protect the cells against the effects of free radicals or compounds that detoxify reactive oxygen species (ROS) and prevent their damage through

14

multi mechanisms. Oxidation reactions can produce free radicals and these radicals are responsible to vast variety of human diseases including atherosclerosis, arthritis, ischemia diabetic mellitus, hypertension, aging, and cancer. Synthetic antioxidants have been in use as food additives for a long time, but reports on their involvement in chronic diseases have restricted their use in foods. It is established that consumption of antioxidant substances has  been linked to the reduct ion in the incidence of oxidative-stress related diseases. The use of currently available synthetic antioxidants like butylated hydroxy anisole (BHA), butylated hydroxyl toluene (BHT) has been limited due to their toxicity and side effects. They are suspected of being responsible for liver damage and carcinogenesis in laboratory animals. Hence strong restrictions have been placed on their application and therefore research for the determination of the natural oxidants source is important (Tapan et. al., 2013 and Magdy et al., 2014). They play many important roles such as free radical scavenger, reducing agent and antioxidant defense enzyme system activator. Rice and wheat are two very commonly consumed cereal grain that contain several antioxidative compounds and are shown to be  beneficial for a wide range o f medical conditions (Bishwajit et. al., 2013). Antioxidants terminate ROS attacks and appear to be of primary importance in the prevention of these diseases and health problems. It has been widely accepted that diet can significantly alter the overall health and quality of life. Development of functional foods rich in  bioa vailable antioxidants may play an important role in this regard. The k ey for developing functional foods is to provide a sufficient amount of the bioavailable safe active components, the functional additives/nutraceuticals, in the finished functional food products (Liangli, 2007). Antioxidant activity is an important biological property of many phytochemicals that protects living organisms from oxidative damage thereby preventing various deleterious events and diseases in plants and animals including human beings. Phenolic compounds possess antioxidant activity and these are aromatic secondary metabolites of phenylalanine, and, to a lesser extent, tyrosine that constitute one of the most diverse family of compounds found in  plants. Simple phenols, phenylpropanoids, flavonoids, tannins (proanthocyanidins and others), and lignins are among numerous categories of plant phenolics. Cereals have been known to contain phenolic acids, phytoestrogens, and small quantities of flavonoids. The  phenolic acids in cereals are benzoic and cinnamic acid derivatives; the latter being most

15

common. Cereals are also a major source of dietary lignans with potent antioxidant activity (Liangli, 2007). Phenolic acids exist as free, esterified and insoluble-bound forms. One of the advantages of  bound p hytochemicals is the ir ab ility to survive d igestion in the upper gut, allowing t hem to reach the colon and, therefore, exert health benefits. Flavonoids and phenolic acids are examples of antioxidants, which are important ingredients of many foods, and keenly sought in many ‘health foods’. They are thought to help protect against diseases like cancer, cardiovascular disorders, neurodegenerative diseases and ageing by mopping up potentially damaging free radicals that are re leased in the body (Asli et al., 2010). Postum is a powdered roasted-grain beverage once popular as a coffee substitute.  The caffeine-free beverage was created by Postum Cereal Co mpany founder 1895 and marketed as a healthful alternative to coffee. The Postum Cereal Company eventually became General Foods, which was bought by Kraft Foods. Post was a student of John Harvey Kellogg,  who  believed that caffeine was unhealthy. The "instant" drink mix version was de veloped in 1912, replacing the original brewed beverage Postum is made from wheat bran,  wheat,  molasses, and maltodextrin from corn (Pendergrast and Mark, 2010). The tea substitute made in this research paper only shared the wheat bran part from postum neither used the molasses nor corn instead wheat bra n stimulated by natural non caffeine herb /additives like mint, Cinnamon, funnel seed used as extra flavor for the drink when steeped in hot cup water. The main characteristic of an antioxidant is its ability to trap free radicals. Highly reactive free radicals and oxygen species are present in biological systems from a wide variety of sources. These free radicals may oxidize nucleic acids, proteins, lipids or DNA and can initiate de generative disease (Aruna et al., 2014).

16

Figure 2.3 Free radicals and disease.

Source: Pearso n Education I nc. Publishing as Be njamin Cummings (2005).

2.4 Raw materi als for developed products 2.4.1Wheat bran Bran, an outer layer of most cereal grains, is nutrient dense as it contains proteins, omega 3 and omega 6 fatty acids and antioxidants. Cereal bran is an excellent source of dietary fiber; for addition to food, it offers all the nutritional and neutrace utical benefits. It contributes a  pleasing sweet, nutty flavor when added as a flavor e nhancer in baked products a nd pasta. It is obtained from screened grains of wheat (Muhannad, 2010). Wheat bran is a major by-product o f the wheat milling industry used in value added products; the majority of it is used as animal feed and therefore underutilized. Wheat bran is known to contain many phytochemicals with numerous health benefits. wheat bran extracts have greater bioactivity than endosperm wheat extracts, suggesting that this major by-product of

17

the flour milling industry is highly nutritious, and contributes to the known health benefits of wheat for humans. Wheat extracts have shown high antioxidant activity, binding free radicals to promote healthy aging, and reduce risk of cardiovascular disease, diabetes and obesity, as well as some forms of cancer. Wheat bran had the highest level of antioxidant activity which was found by bioassay guided fractionation to be attributed to the unsaturated fatty acids; linolenic, linoleic and ole ic acids (Kelly, 2009). Wheat bran contains strong antioxidant activity. It may therefore provide protection against aging, cardiovascular disease, cancer, diabetes and obesity. The amino acid tryptophan was the prominent cause of the antioxidant activity observed in durum wheat bran. Wheat bran has been reported to contain 75% of the phytochemicals present in wheat, but the bioactivity and chemical identity of these phytochemicals is largely unknown (Kelly Marie, 2009). The antioxidant activity of wheat is derived mainly from the bran layers, with compounds found in the endosperm playing a minor role. Many studies have assessed wheat antioxidant activity with speculation on bioactives. A study of antioxidant activity from six milling fractions including head shorts, tail shorts, low-quality flour, low-grade flour, bran and control flour, showed that bran possessed the greatest antioxidant activity compared to all these samples. This is further supported by Adom et al. (2005), who found antioxidant activity of a bran/germ fraction, from milled fractions of different wheat varieties, had 13 – 27 fold increase in antioxidant activity compared to the endosperm in the hydrophilic assay (Kelly Marie, 2009). Antioxidants in wheat exist in the forms of vitamins (tocopherols  –   vitamin E), minerals (selenium), phenolic acids (ferulic acid, vanillic acid), tocotrienols, phytic acid, phytosterols, flavonoids, a nd carotenoids (lutein). About 36 Wheat species have widely differing quantities of antioxidants. Antioxidant content in modern white wheat varieties has tested to be lower than antioxidant content in modern red wheat varieties. Coffee and tea contain abundant levels of antioxidants as do wheat and barley kernels. Coffee and tea also naturally contain caffeine. Coffee and tea substitutes made from wheat and barley have been produced for a century; however, limited research has gone into the antioxidant benefits from roasted wheat, or from it’s  by products. As the benefits of wheat and barley antioxidants become better known, the wheat and co ffee be verage markets may e merge as we ll. (Neil et. al, 2012) Recovery of antioxidant compounds from plant materials is typically accomplished through different extraction techniques taking into account their chemistry and uneven distribution in the plant matrix. For example, soluble phenolics are present in higher concentrations in the

18

outer tissues (epidermal and sub-epidermal layers) of fruits and grains than in the inner tissues (mesocarp and pulp). Solvent extraction is most frequently used technique for isolation of plant antioxidant compounds. However, the extract yields and resulting antioxidant activities of the plant materials are strongly dependent on the nature of extracting solvent, due to the presence of different antioxidant compounds of varied chemical characteristics and polarities that may or may not be soluble in a particular solvent. Polar solvents are frequently employed for the recovery of polyphenols from a plant matrix. The most suitable of these solvents are (hot or cold) aqueous mixtures containing ethanol, methanol, acetone, and ethyl acetate (Neil et al., 2012).

2.4.2 Wheat germ Wheat germ, a part of the wheat kernel removed as by-product of the wheat milling industry, is considered as a natural source of highly concentrated nutrients (Shao and LiYa, 2011). The wheat germ is a unique source of concentrated nutrients, highly valued as food supplement. While the oil is widely appreciated for its pharmaceutica,l nutritional and cosmetic value, the defatted germ meal is a promising source of high-quality vegetable proteins. The germ is only a very small part of the kernel, approximately 3 percent in total. Wheat germ is very high (around 28 percent pro teins) (Finely, 1989; Bruce, 1997). The amount of nutrients that are contained within wheat germ seems endless. It contains more potassium and iron than any other food source. Also found in great quantities are riboflavin, calcium, zinc, magnesium and vitamins A, B1 and B3 . Vitamins B1 and B3 are very important to maintain energy levels and maintain healthy muscles, organs, hair and skin. Another important vitamin found in wheat germ is vitamin E; which is a very important antioxidant. It is helpful in preventing the body's aging process and also to prevent heart disease, helps to prevent blood clots and is needed to strengt hen the body’s immune system. Wheat germ has been found to be very beneficial in order to keep the body in tip top condition. It is used by athletes in their diet to improve cardiovascular function and improve endurance levels (Sabate, 1993; Spiller, 1997). Body builders will also add wheat germ to their diets in order to bulk up and maintain the nutritional levels they need to perform (Neli et al., 2007). Wheat germ also contains some relatively functional phytochemicals such as flavonoids, sterols, octacosanols, and glutathione. It provides three times as much protein, seven times as much fat, 15 times as much sugar and six times as much mineral content than wheat flour.

19

Wheat germ protein has been classed effectively with superior animal proteins and is rich in amino acids, especially the essential amino acids lysine, methionine, and threonine, in which many cereals are deficient (Shivani and Sudha, 2011). Wheat germ oil is used in products such as foods, biological insect control agents, pharmaceuticals and cosmetic formulations (Alessa ndra et al., 2009). It is a good source of healthy fatty acids that help lower cholesterol, lower inflammation, and supports a healthy nervous system which can lower anxiety levels and improve mood (Pinna and Peter, 2009). Table 2.2 Fatty acid composition of wheat germ oil Fatty acid

%

Palmitic acid

17.4

Stearic acid

0.9

Oleic acid

17.1

Linoleic acid

56.1

Linolenic ac id

6.9

Arachidonic acid

0.2

Eicose noic acid

1.4

Source: Asuman Kan, (2012). Stabilizing wheat germ by defatting increased the protein content to 38% and also increased the soluble fiber from 2.07 to 3.01% and insoluble fiber increased from 14.4 to 24.49% (Bansal and Sudha 2011). Defatted wheat germ is the ideal ingredient for grain based food  products. Natural and nutritious, it enhances the flavor and texture of hundreds of applications. Wheat germ is processed to retain the natural nutritional and flavor characteristics of fresh, high quality wheat germ. The functional qualities of each include improving the stability texture, nutritional value and flavor of processed foods and consumer goods of all kinds. Each is high in protein and fiber and is virtually fat free. The product is granular and possesses a toa sty mocha- like flavor (Garuda, 2011). According to IFIC food and health Survey of 2013 defatted wheat germ helps meet today’s demands for full flavor grain-based foods that are rich in protein and fiber. This multidimensional ingredient offers 26%+ protein, 15%+ fiber, Low fat, 12-month shelf life and a multitude of vitamins a nd minerals. Defatted Wheat Germ improves nutrition, enhances flavor and enriches the texture for your end product.

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2.5 Process description 2.5.1 Process description for wheat flow A modern milling operation involves much more than grinding wheat to a powder. Three general operations are usually involved: cleaning, tempering, and milling. Cleaning removes unwanted material; tempering softens the grain, making it easier to separate and grind; and milling involves grinding the wheat and isolating wheat co mponents of a specific size. i.

Cleaning

Wheat unloaded from a truck, into a mill elevator contains a sizable percentage of non wheat kernel components, termed “dockage.”  Dockage consists of other types of seeds, underdeveloped or unsound wheat kernels, insects, stems, stones, and other debris commonly found in a wheat field. Before milling, this debris must be removed, and this is accomplished in the wheat cleaning section of the mill. Although numerous machines exist to clean wheat, they are all classified based on separation by size, shape, density, and magnetism. Different mills vary greatly with respect to the order of the operations in a cleaning process. Usually, one of the first separations removes a ny ferrous metal in the wheat using magnetic separators. Removing metal early in the process helps avoid damage to equipment farther downstream. A milling separator may be next, to remove sticks, stones, stems, and other plant debris. Lighter, less-dense components in the wheat are removed here via aspiration. Air circulates upward through the grain as it is fed into the separator, and lighter material is drawn away from the wheat kernels. The wheat then falls onto a sieve, which allows the wheat to pass through but retains stones and larger seeds. Another sieve follows which retains the wheat and allows smaller seeds to pass through. A disk separator, which separates wheat from other grains of equal density, is also likely to  be included in the cleaning process. This mac hine separates grains based o n shape. Pockets in rotating disks accept seeds of certain lengths and reject those o f other sizes. Generally there is more than one disk separator. One will accept wheat and another will reject wheat to remove  both lar ger and smaller grains. Dirt or mold adhering to wheat kernels is largely re moved using a scourer. This machine uses a screen or an abrasive surface to remove any material adhering to the kernel. Materials such as small stones similar in size to a wheat kernel are separated based on density in a gravity table or dry stoner. The gravity table is an oscillating inclined plane. Denser material such as stones moves down the table faster than lighter

21

material. The dry stoner removes the dense material with aspiration sufficient to raise the grain and allow the stones to drop out. ii.

Tempering :

is the addition of predetermined amounts of water to wheat during specific holding periods. It toughens the bran, making it easier to separate from the endosperm and germ. It also softens the endosperm, allowing it to break apart with less force. Tempering involves adjusting the moisture level of the wheat. For soft wheat, optimal tempering brings the grain to 13.5 –  15.0% moisture and takes 6 – 10 hr. For hard wheat, the final moisture is 15.5 – 16.5%, and tempering times are 12 – 18 hr. Incoming wheat is generally lower in moisture content than this; hence, water is usually added and the grain is allowed to equilibrate for a period of time. This time varies considerably based on the hardness of the wheat. Conditioning of wheat refers to the application of heat in the tempering process to increase the rate of penetration of moisture into the kernels. Temperatures lower than 50°C are e mployed during conditioning to ensure that the functionality of the flour components, especially the gluten, is maintained. iii.

Milling:

At this point, the wheat is ready for milling and starts through the various systems in the mill. The first machine in almost every mill is the roller mill. Two rolls, one rotating clockwise and the other counterclockwise, are separated by a small distance called the “gap.” One of the rolls usually rotates faster than the other one. Consequently, at the nip, due to the rotation of the rolls the wheat e xperiences a s hearing action as well as a cr ushing action. The first roller mills are employed in the break system. This is the part of the milling operation designed to remove the endosperm from the bran and germ. Rolls in this process have spiral grooves called “corrugations” cut parallel to the long axis of each roll. Generally there are about five roller mills or five “breaks” in the system. The germ is removed in the first two breaks, as is much of the bran. The germ is pliable and tends to flatten when it goes through the rollers. Bran particles are usually in the form of low-density small flakes. These  properties allow millers to separate the germ and bran fractions from the endosperm fraction. After each break, a set of sieves and/or purifiers (aspirators) separates the ground material by size and density.

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iv.

Sieving :

Small particles are channeled into the flour, and large particles are either removed (as is the case with the germ and bran) or sent to the next break (as occurs for large endosperm pieces). Once the endosperm is isolated, the large particles that result (called middlings) are reduced in the reduction system to a particle size distribution consistent with flour. This means they must be able to pass through a 136-μm  opening. The rollers in the reduction system are smooth and are operated at low differentials, providing a crushing action that yields the fine  particles of flour (although a small amount of shear is s till important). A lar ge percentage o f the particles compos ing the final flour come off the reduction rolls. Flour from the break and reduction rolls may be combined in many ways to create different types of flour, but it is usually sifted again in the flour dressing system and passes through sieves meets the particle size standard for flour. Larger particles are recirculated back to the appropriate point in the grinding process. The flour may be further treated with chlorine or a  bleaching agent depe nding on t he requirements of the customer. In the mill feed s ystem, the germ and bran are separated from each other, and adhering endosperm is removed. The coarse bran from the early breaks is termed “bran”  and composes about 11% of the total  products from the mill. The finer branny material from the later steps is called shorts; it represents about 15% of the total. Germ is generally recovered at the rate of about 2 – 3.0% of the total wheat depending on the type of equipment used. These special products or ingredients for human consumption are usually sold as animal feed in our country. The steps were shown in Fig 2.4 Process steps in wheat milling.

23

Figure 2.4 Process steps in wheat milling.

Source: School of Biological Sciences, U niversity of Bristol, England (2007)

24

2.5.2 Process description for biscuit producti on Defatting process by supercritical fluid extraction

There are several methods for oil extraction that all have their advantages and disadvantages. Mechanical expression (pressing) and organic solvent extraction are both being used for commerc ial extraction of wheat germ oil (WGO). Solvent extraction is by far the most widely used method to extract oil (Woerfel, 1995). In recent years supercritical fluid extraction (SFE) has received increased attention as an important alternative to conventional methods.This is due to regulatory and environmental  pressures on hydrocarbon a nd o zone-dep leting e missions. SCF-based processes have helped to eliminate the use of hexane and methylene chloride as solvents. With increasing scrutiny of solvent residues in pharmaceuticals, medical products, and neutraceuticals, and with stricter regulations on volatile organic compounds (VOC) and ozone depleting compounds (ODC) emissions, the use o f SCFs is rapidly proliferating in all industrial sectors. Supercritical fluids have adjustable extraction characteristics due to their density, which can  be controlled b y changes in pressure or temperature. In addition, other properties such as low viscosity, high diffusivity and low surface tension enhance the solute mass transfer from inside a solid matrix. SCFs are advantageously applied to increasing product performance to levels that cannot be achieved by traditional processing technologies, and such applications for SCFs offer the potential for both technical and economic success (Sultana,  et.al,  2007, Reverchon et. al., and Lang et.al., 2001). Supercritical carbon dioxide (SC-CO2 ), being nontoxic, nonflammable, inexpensive and easily separable from the extracts, has been the most frequently used extractant in the food and pharmaceutical industries. Furthermore, the low critical temperature of carbon dioxide allows extraction of thermolabile compounds without degradation (Alessandra et al., 2009). It is an efficient extraction method, which is non-explosive and non-toxic, leaving non-solvent residues. The o ils extracted with this met hod do not need the traditional refining processes. In addition, SFE is a mild process which can avoid fatty acid oxidation and protein in defatted wheat germ denaturation. Therefore SFE has received increased attention as a promising alternative to conventional extraction met hods over the last decades (S hao and LiYu, 2011). A supercritical fluid is the phase of a material at critical temperature and critical pressure of the material. Critical temperature is the temperature at which a gas cannot become liquid as

25

long as there is no extra press ure; and, critical pressure is the minimum amount of pressure to liquefy a gas at its critical temperature. Supercritical values for these features take place  between liquids and gases. The formation o f a supercritical fluid is the result of a dynamic equilibrium. When a material is heated to its specific critical temperature in a closed system, at constant pressure, a dynamic equilibrium is generated. This equilibrium includes the same number of molecules coming o ut of liquid p hase to gas phase by gaining e nergy and going in to liquid phase from gas phase by losing energy. At this particular point, the phase curve  between liquid and gas phases disappears and supercritical material appears (Mustafah and Andrew, 2013). There is another characteristic point in the phase diagram; the critical point (CP) is obtained at critical temperature (Tc) and critical pressure (Pc). After the CP, no matter how much  pressure or temperature is increased, the material cannot transform from gas to liquid or from liquid to gas phase. This form is the supercritical fluid form. Increasing temperature cannot result in turning to gas, and increasing pressure cannot result in turning to liquid at this point. In the phase diagram, the field above Tc and Pc values is defined as the supercritical region. (Mustafah and Andrew, 2013).

Figure 2.4 Schematic representation of idea lized phase diagram.

Source: (http://cnx.org/content/m46150/1.2/)

26

According to thermodynamic research laboratories of university of Illinois’ SCFE is advantageous: 1.

SCFs have solvating powers similar to liquid organic solvents, but with higher diffusivities, diffusivities, lower viscosity, and and lower su s urface tension.

2.

Since the solvating power can be adjusted by changing the pressure or temperature separation of analytes from solvent is fast an a nd easy.

3.

By adding modifiers to a SCF (like methanol to CO2) its polarity can be changed for ha ving more more se lectiv ect ivee separation power.

4.

In industria industria l processes involving food or ph p harmaceu armace uticals, tica ls, one does not not have to worry worr y about solvent solvent residuals res iduals as you would if a "typica l" orga orga nic solvent solvent were used.

5.

Candidate SCFs are generally cheap, simple and are safe. Disposal costs are much less and and in industria industria l processes, t he fluids ca n be simple to recycl recyc le

6.

SCF technology requires sensitive process control, which is a challenge. In addition, the phase transitions of the mixture of solutes and solvents have to be measured or  predicted  predicted quite quite accurately. accurate ly. Generally the phase tran tra nsiti sit ion in the critical critica l region is rather complex and difficult to measure and predict. Advantages of Using Carbon dioxide is the most commonly utilized SCF in SFE machine. It is chemically stable, has re la tive tive ly low toxicity, is not flamm fla mmable, able, is inex ine xpensive and produ prod uces zero su s urface tension. Furthermore, it has a mild critical temperature required for extraction of thermolab thermolab ile compounds compounds a nd is separated separated easily eas ily from fro m the samp samplle.

Farinograph Farinograph analysis an alysis The Farinograph is an apparatus which is commonly used to measure the rheological  properties  properties of dough (Inn, et al., 2007). It measures measures (as torque) torque) and records the res istan sta nce to mixing of dough as it is formed from flour and water (AACC, 2000). Viscoelastic properties of wheat dough are the result of the presence of a three dimensional net work of gluten  proteins. The Visco-e lasti ast ic properties enable dough to retain reta in gas which is esse ntial tia l for  product  product ion of baked products with wit h a light textur t exture. e. Rheolo Rheolo gical properties prope rties such as e lasti ast icity cit y, viscosity and extensibility are important in the prediction of the processing parameters of dough and quality of end product (Hruskova, 2001). Farinograph results include absorption, arrival arrival time, t ime, stability time, peak time, t ime, depart departu ure time, t ime, and mixing mixing tolerance index.

27

 Absorption (%): is the amount of water required to center the farinograph curve on the

500 brabender units (BU) line. This relates to the amount of water needed for a flour to  be optimally processed process ed into into e nd products.  Peak Time (minute) - indicates dough development time, beginning the moment water is

added until the dough reaches maximum consistency. This gives an indication of optimum mixing time under standardized conditions. It is expressed in minutes.  Arrival Time (minute) - is the time when the top of the curve touches the 500-BU line.

This indicates the rate of flour hydration (the rate at which the water is taken up by the flour). Arrival time is expressed in minute.  Departure Time (minute) - is the time when the top of the curve leaves the 500-BU line.

This indicates the time when the dough is beginning to break down and is an indication of dough consistency during processing. Departure time is expressed in minutes.  Stability Time (minute) - is the difference in time between arrival time and departure

time. This indicates the time the dough maintains maximum consistency and is a good indication of dough strength.  Mixing Tolerance Index (MTI) is the difference in BU value at the top of the curve at

 peak time t ime an a nd the value at the top of o f the t he c urve 5 minutes minutes after the peak. This indica indica tes the degree of softening during mixing. Mixing tolerance index is expressed in Brabender units(BU). units(BU). Weak gluten flour has a lower water absorption absorptio n a nd shorter stability stabil ity time time th t ha n strong gluten flour.

28

Figure Figure 2.5 Parameters in in Pharinograph Phar inograph representat ion from fr om manual in Kali Ka lity ty Factory Factor y (1923).

Preparati Preparation on of value adde adde d cookies The demand for food and agricultural products is changing in unprecedented ways. The nature and extent of the changing structure of agri-food demand offer extraordinary opportunities for diversification and value addition in agriculture, particularly in developing countries. The prospects for continued growth in demand for value-added food and agricultural products constitute an incentive for increased attention to agro industries development within the context of economic growth, food security and poverty-fighting strategies. Agro-industries, here understood as a component of the manufacturing sector where value is added to agricultural raw materials through processing and handling operations are known to be efficient engines of growth and development. With their forward and backward linkages, agro-industries have high multiplier effects in terms of job creation and value addition (Carlos and Doyle, 2009). According to New Brunswick Value-added Food Sector Strategy 2012-2016, any step in the  product  product ion process that improves improves the product for the customer and results in a higher net worth called value addition. Value-added food sector encompasses companies producing agriculture and seafood-based products, beverages and other food made from both local and imported resources. The sector includes live, fresh, frozen, packaged, processed and  preserved  preserved food products whose va lue lue and pro fitabil fitability ity has bee n increased increased by making them more appealing and valuable to the buyer. In Ethiopia, the food-processing sector is by far the largest manufacturing industry and accounts for 39% of the gross value of production in large and medium size manufacturing in

29

2009/2010 which expected to arouse even more by now. The gross value of production (GVP) equals 16,220 million Birr (900 million USD), of which small-scale manufacturers achieve a GVP of 308 million Birr in food processing excluding grain milling, and the grain millers produce a GVP of 1,113 million Birr. The largest sectors are sugar, bakery, and grain milling, which together cover about 47% of the total GVP (Soethoudt et. al., al., 2013). Increasing

awareness

of

consumers

regarding

health

and

nutrition

has

led

to

experimentations for modification and development of bakery products to value added health foods. This may become a boon for further development of bakery products using low cost, nutritious ingredients. Among these bakery products cookies/biscuits are popular and well accepted as snack food. ‘Cookie’  ‘Cookie’  is chemically leavened product, also known as ‘biscuit’ (Uma Uma Ballolli Ba llolli,, 2010).

Cookies are textural and flavorful wonders, they are easy to make and usually require no special equipment. Cookie recipes run the gamut from chic and classic to simple, homespun, and familiar. Cookies are versatile: They can be huge or miniature, chewy or crisp, filled or frosted or plain, sweet or savory. They can be round, square or rectangular or take the shape of animal, vegetable, or mineral. They can be kitschy, chic or both at the same time. Many welcome welcome variation ariatio ns (John, 2002). The The ingredients of cookies are flour, sh s hortening orte ning,, eggs, salt, sa lt, leavening age nts, additives, flavors, liquids, and various other enriching ingredients. Each one of these ingredients has its own role and function in the preparation of the product (Sumnu and Sahin, 2008). The importa importan nce/ functio n of ingredie ingredie nts used in cookies making are re vealed be low: ow : Fat Fat  is added for flavor and controls how chewy or crunchy the cookie is. More fat is a

chewier cooki cook ie, less fat is a crunchier crunc hier cookie. O ptions for fat are butter, margar ine, ine, shortening, or oil. Since shortening melts at a higher temperature, it is the best choice if one wants to keep spreading to a minimum. Shortening, butter, and margarine are all fats. But not all fats are created equal when it comes to baking cookies. Fats are used in cookies to: tenderize and soften the texture, ad moistness and richness, increase the keeping quality, add flavor, flavor, ass ist in leavening when when used as a cr eaming agent (ob viou us ly!) and tenderizer, tende rizer, while wh ile contro contro lling how muc muc h the cooki cook ie Sugar is a sweete ner (obvio spreads. Using white sugar s ugar will result in a crispi crisp ie r cooki cook ie, while brow n sug su gar will w ill help help retain moisture, making cookies chewier. Adding sugar increases the spread of a cookie, so cookies with less sugar will be puffier.

30

Flour is a stabilizer and thickener and controls how much the cookie rises. It holds the cookie

together, providing it with its structure. If too little flour used cookie won’t keep its shape but if one use too much you’ll end up with a thick tasteless cookie. Also, dif ferent types of flour result in different cookie textures. All-purpose flour is the standard flour used most often. Dough may have a ratio of 1:6 or higher and might be used for cook ies or pastry dough. Rising agent  or leavener most commonly used is either baking soda or baking powder. If

 baking soda used, recipe must include another acidic ingredient, like sour cream, lemon juice, or buttermilk. On the other hand, baking powder has its own built in acid. Baking soda increases browning and spreading, resulting in a flatter cookie. Baking powder will give a  puffier cookie. Binding agents  are the liquid in the recipe that hold the cookie together. Examples of binding

agents are eggs, milk, honey, and fruit juice. Cookies with more eggs will rise more and spread less. Salt, Spices, Flavorings, and Extracts / additives Salt is used to bring out flavor of many

foods, including sweet cookies while Spices, flavorings, and extracts add flavor to the cookies. Usually these are added in sma ll amounts.

2.6 Sensory quality attributes Human accepts food on the basis of certain characteristics that he/she defines and perceives with his/her senses. These attributes are described in terms of sensations and sometimes referred as qualitative or sensory qualities. They include perception of appearance factors such as color, size, shape and physical aspects; kinesthetic factors such as texture, viscosity, consistency, finger feel and mouth feel; and flavor factors or sensations combining odor and taste. Human judges are used to measure sensory characteristics of food. Sensory analysis is too commonly often overlooked as a requirement before product launched. Similarly in this research work sensory data such as appearance, color, smell, taste, texture aroma and mouth feel are obtained through subjective eva luation by panelists.  Color and other aspects of appearance influence: Color is a quality factor that greatly

influences the appearance of a product. There are five functions that should be considered in understanding human reactions to color in foods are (perception, motivation, emotion, learning and thinking). The human eye has remarkably fine qualitative discrimination for color, but it is not quantitative instrument. Appearance refers to the size, shape together with defects and color are appearance factors that

31

greatly influence initial consumer impressions for this work the quality of the cookies and tea – substitute in terms of shape, size, color, form and thickness.  Taste, aroma and flavor: Flavor as attribute of foods and beverages is defined as the

sum of the perceptions resulting from stimulation of the sense ends that are grouped together at the entrance of the alimentary and respiratory tract. When food is consumed, the interaction of taste, odor and textural feeling provides overall sensation best defined as Flavor. Flavor results from compounds that are divided in to two broad classes: Those responsible for taste and those responsible for odors, the later often designated as aroma which provides both sensation. In simple term taste is the sensation perceived when a small portion of cookies/ tea-substitute is taken by mouth in terms of saltiness, sweetness, bitterness, and sourness caused by soluble s ubstances in the mouth.  Texture as property of foodstuff apprehended both by the eye and muscle senses in the

mouth embracing roughness, smoothness, chew ab ility, stickiness, and so fort h.  Overall acceptability is the sum of all the quality parameters and liking and disliking of

the products sensed by the consumer.

2.7 Concluding remarks This chapter demonstrates byproducts from wheat milling industries (wheat germ and wheat  bran) could be used as a means of resource for production of cookies and tea substitutes. According to data found from FAO and CSA, wheat production in Ethiopia is mounting. Hence wheat production in 1997(<1,000,000 tons) till last year a nd become 4,039,113 tons in 2013 though the growth of production was not monotonous as can be seen in the figure. 4500000 4000000    )   s   n   o    t    (

3500000

  n   o    i    t   c   u    d   o   r   p

2500000

3000000 2000000 1500000 1000000 500000 0    7    9    9    1

   8    9    9    1

   9    9    9    1

   0    0    0    2

   1    0    0    2

   2    0    0    2

   3    0    0    2

   4    0    0    2

   5    0    0    2

   6    0    0    2

   7    0    0    2

   8    0    0    2

   9    0    0    2

   0    1    0    2

   1    1    0    2

   2    1    0    2

   3    1    0    2

Figure 2.6 Wheat production trend in Ethiopia.

Source: Graph developed on data of CSA and FAO (1997-2013)Moreover; according to USDA foreign agricultural service report, 2013; in 2012, there were around 216 large flour

32

mills in Ethiopia with a total of 4.2 million tons of milling capacity of flour per year; which is expected to increase in number by now. The wheat consumption trend in Ethiopia is gradually increasing in urban areas due to high population growth (about 2.6 percent a year), migration of people to urban areas, and changes in life styles. Amount of byproducts from milling industries is about (25-35) %; which can result more than 11 million quintals of  byproducts per year. The above facts show byproducts of wheat milling industries can be used as means o f resource without short supply throughout a year. Milling industries in developed countries; use a source of enrichment for the final refined white flour either directly by adding mineral and vitamins to the last product or by using different wheat byproduct flours as a source of enrichment. This is done because they are fully aware of wheat flour alone is nutritionally incompetent for daily use. Hence, developing countries like Ethiopia show more scarcity in wheat and deficient it’s better to use byproducts for human use rather than as animal feed. In view of the fact that defatted wheat germ flour can be used as a supplement for the newly developed cookie; due to its high quality nutrition content in protein, fiber, minerals. Similarly, from the studies wheat bran showed higher amount of antioxidant activity. A conclusion from studies establishes basic ideas and possibilities, which can be favorable to support the development of wheat milling industries to use their own byproducts as a resource. Apart from utilizing byproducts as a resource, minimizing agricultural waste for  product ion of new products to human better usage is something.

33

CHAPTER THREE

Materials and Methods 3.1. Raw materi als collection, transportation and storage The basic raw material used to make a defatted wheat germ cookies and tea substitute were wheat grain byproducts directly harvested from Hora’s land production. It was collected from one of the modern Midroc’s company named Hora Food Complex found in Alemgena. Wheat  bran, white wheat flour refined/ extracted at 76% and wheat germ samples were obtained separately during production from this compa ny.

Each flour samples that were taken

carefully from the appropriate pipes were kept with a zipped, high-quality hygienic food grade polypropylene plastic bags at room temperature where as the germ and bran kept in ice  box during transportation and sta yed in freezer (0 – 5 o C) till laboratory analysis done. Carbon dioxide was purchased from Moha Soft Drinks Industry, Gotera. Analytical grade chemicals and reagents; 2, 2-diphenyl-1-picrylhydrazyl (DPPH), Folin Ciocalteau reagent were purchased from Sigma-Aldrich Co. (St. Louis, Missouri,  U.S.A). Methanol, filter paper, safety equipments, vitamin C, sodium carbonate was bought from Wise PLC. Other ingredients such as salt, sugar, aluminum and plastics-bags with seal,  baking powder, egg, butter, vanilla, oil and spices were obtained from well trusted markets like Shoa and Abadir super market.

A) Wheat grain

B) Wheat germ

C) Wheat bran

Figure 3.1 Wheat grain and its byproducts (from Hora Co mplex PLC.).

34

3.2 Frame work of the research experi ment

Flours from wheat milling Industries

Wheat flour

Wheat bran

Extraction using

Wheat germ

Proximate com osition Anal sis

Defatting Wheat germ flour process

methanol

Blending flours

 by SFE

(WF & DWGF) Deter mining total

Defatted wheat germ

 phenolic content by FolinCiocalteau assay and

flour BR 2

BR 1

BR 3

WF

antioxidant by DPPH

Tea substitute

Sensory quality

-

Mixing Kneading Cutting Baking

-

Cooling

-

Packing

Farinogram Analysis and Functional property analysis (OAC, WAC, BD)

evaluation Proximate composition

Cookies Produced

Analysis of physical properties (diameter, height, and weight)

Figure 3.2 Frame work of the research experiment.

35

3.3 Methods of processing 3.3.1 Preparation of defatted wheat germ flour Raw wheat germ carefully selected and cleaned to remove contaminants. Accord ing to the method described by Zhu and Zhou, (2005), the enzymes in the raw wheat germ were inactivated by heating for 30 minutes at 105°C. Wheat germ samples were loaded into the extraction vessel carbon dioxide from a cylinder was passed through a chiller kept at 2°C and  pumped into the extractor by a high pressure pump. Raw wheat germ was defatted by using supercritical fluid extractor machine that is found in AAiT’s Laboratory.  According to methods by Alessandra et. al , 2009 and Shao and LiYa, 2011 the machine operated to defat wheat germ at pressure of 300 bar; temperature of 40°C; CO 2  flow rate of 20kg/h. Then the defatted wheat germ flour milled by coffee machine the flour passed through a 250µm sieve diameter. F inally the flour stored in freezer (0-40 C); after extraction, the oil was collected separators while water and volatile components were recovered. The extracted oil obtained was together with iced form water separated using separator y funnel. The oil collected looks like as shown below:

(a)

(b)

Figure 3.3 Oil obtained by defatting wheat germ flour (WGF) by supercritical fluid extractor

(SFE) before and after separation by using separator y funnel. (a) Before separation by separator y funnel

(b) after separation by separatory funnel

The extracted oil from the wheat germ is not pure oil form instead (icy form) latter melted in to water and the oils float above. Separatory funnel is used in order to separate the oil from the water part as shown in figure.

36

Oil Raw wheat germ

Heating at

SFE operated at T=40 o C & P= 300 Barr

T= 1050 C

DWGF

Stored at Freezer

Milling

(1-50C)

Sieving

Figure 3.4 Simplified diagram for Preparation of DWGF.

3.3.2 Blend formulation and cookies producti on Blend formulation The basis for blend formulation of blended flour was done by taking into consideration some important facts about the characteristics of DWGF results obtained after making farinograph analysis and based on previous studies. The choice of these blend ratios were based on studies of Muhammad, 2006 and Sahar, 2012 modified by using result obtained from farinograph. That blends of wheat flour and DFWG flours containing 0%, 10%, 15%, and 20% DFWG flour, on a replacement basis, were taken. After blending, the mixture was  packed in poly propylene plastic ba gs and s tored at room temperature t ill further analysis is done.

10%DWGF

15% DWGF

20%DWGF

37

100% DWGF

Control (100%Wheat flour)

Figure 3.5 Blended flours, defatted wheat germ flour and control flour.

Table 3.1 Percenta ge composition of blended flour for cook ies Flour blend

Wheat flour (%)

DWGF (%)

100

-

BR-1

90

10

BR-2

85

15

BR-3

80

20

BR-0/control/WF

Cookies production Cookies were prepared according to the procedure described by (Taha et al., 2006) with some modifications on type and amount of spices added. The basic ingredients used were 110 g of flour blend, 29g shortening, 34 g of sugar, 13 g of beaten whole egg, and 1.1 g of baking  powder, 1.5 g of salt, 1.2 g vanilla, 2 g cinnamon, 1g c love, 0.6 g ginger a nd 5.3gm water. First, dry ingredients were weighed and mixed thoroughly in a bowl by hand for 3 – 5 min. The shortening, sugar and egg creamed together was added to the mixed dry ingredients and rubbed in until uniform. The dough was sheeted using Lasagna sheeting machine with a uniform thickness (5 mm) and cut out using a round cutter of diameter 45mm. The cut out dough pieces were baked on lightly greased pans at baking temperatures 150, 180 a nd 210 o C for 10-12 minutes in a baking oven. The prepared cookies were cooled to room temperature and packed in polyet hylene bags.

38

3.4 Methods of analysis 3.4.1 Analysis of proximate composition Proximate chemical composition analysis such as moisture content, total ash, crude protein, crude fat, and crude fiber of raw materials and finished products were carried out according to AOAC (2000) official methods 925.09, 923.03, 920.87, 450.1 and 962.09 respec tively. Determination of moisture content

Moisture content was determined according to AOAC, (2000) using the official method (925.09). Empty dishes and their lids (made of porcelain) was dried using drying oven for 1 hour at 100 o C and cooled for 30 minute in the desiccators (with granular silica gel). A clean dried and covered dish was weighed and about 5gm of the sample was transferred to the dish (W1 ). The dish then placed in the oven at 100 o C for 5hrs and cooled in desiccators and reweighed (W2 ). Then, the moisture content on wet basis estimated by the formula:

  

Moisture content in percent (%) =

Eq. (3.1)

Where: W1 = weight of fresh sample (g), W2  = weight of dry sample (g) Determination of total ash

The ash content was determined by using AOAC, (2000) the official method (923.03). About 2.5g of sa mple was added into each dish. The dis hes were placed on a hot plate under a fumehood and the temperature was s lowly increased until smoking ceases a nd the samples become thoroughly charred. The dishes (c rucibles) were placed inside the muffle furnace at 550o C for 6 hr, and removed from the muffle and then placed in desiccators for 1hr to cool. Finally weight of total ash was calculated by difference and expressed as percentage using the formula:

Total Ash (%) =

     

Eq. (3.2)

Where: W1  = Weight in grams of the crucible with the sample W2  = Weight in grams of the crucible plus ash W = Weight in grams of empty crucible Determination of crude protein

Crude protein was determined by Kjeldahl method according to (AOAC, 2000) using the official method (920.87). About 1.0 gm of fresh samples were taken in a Tecator tube and 6ml of acid mixture (5parts of concentrated orthophosphric acid and 100 parts of

39

concentrated sulfuric acid) was added, mixed, thoroughly and 3.5ml of 30% hydrogen  peroxide was added step by step. As soon as the violent reaction had ceased, the tubes were shaken for a few minutes and placed back into the rack. A 3.00g of the catalyst mixture (ground 0.5g of selenium metal with 100g of potassium sulfate) was added into each tube, and allowed to stand for about 10min. before digestion. When the temperature of the digester reached 370o C, the tubes were lowered into the digester. The digestion was continued until a clear solution was obtained for about 1h. The tubes in the rack was transferred into the fume hood for cooling, 15ml of deionized water was added, and shaken to avoid precipitation of sulfate in the solution. Then, 250ml conical flask containing 25ml of the boric acid-indictor solution was placed under the condenser of the distiller with its tips immersed into the solution. The digested and diluted solution was transferred into the sample compartment of the distiller. The tubes were rinsed with two portions of about 5ml de-ionized water and the rinses were added into the solution. A 25ml of 40% sodium hydroxide solution was added into the compartment and washed with a small amount of water, stopped and the steam switched on. About 100ml solution of the sample was distilled, and then the receiver was lowered so that the tip of the condenser is above the surface of the distiller. The distillation was continued until a total volume of 150ml is collected. The tip was rinsed with a few milliliter of water before the receiver was removed. Finally, the distillate is titrated with standardized 0.1N sulphuric acid to a reddis h color. The percent total nitro gen and crude prote in were calculated us ing equation (3.3).

Nitrogen (%) =

  

Eq. (3.3)

Where : N = Normality of standard sulfuric acid (0.1N). T = Volume in ml of standard sulfuric acid solution used in the titration for the test material. B = Volume in ml of standard sulfuric acid solution used in the titration for the blank determination. W = sample weight on dry matter basis and 14.007 is the molecular weight of nitrogen. Crude protein content percent per weight = Total N itrogen * universal conversion factor  N.B: The % o f nitrogen is converted to % of protein by using appropriate conversion factor i.e., (6.25 for biscuits, 6.31 for bran and 5.7 used for flour) according to Jones, (1941).

40

Determination of crude fat

Crude fat was determined based on the Sohxlet extraction method of AOAC (2000) using official method 920.39. A 250 ml quick fit round bottom flask was washed and dried in an oven (Gallenkamp, model OV 880, England) at 105°C for 25 minutes and allowed to cool to room temperature before it was weighed. A clean and dried muslin thimble containing about 5 g of dried sample and covered with fat free cotton at the bottom and top was placed in the extraction chamber. 2.0g of the samples were weighed into the thimble. This was inserted into the extraction column with the condenser connected. 200ml of the extracting solvent (petroleum ether, boiling point 40-60°C) was poured into the round bottom flask and fitted into the extraction unit. The flask was then heated with the aid of electro-thermal heater at 60°C for 8 hrs. Losses of solvent due to heating were checked with the aid of the condenser so that it cooled and refluxed the evaporated solvent. After extraction, the thimble was removed and the solvent salvaged by distillation. The flask containing the fat and residual solvent was placed on a water bath to evaporate the solvent followed by a further drying in an oven (Gallenkamp, model: OV 880, England, 1974) at 105°C for 30 minutes to completely evaporate t he solve nt. It was t hen cooled in desiccators and weighed. The flask conta ining the extracted fat was dried on a steam bath at 98ºC to a constant mass. The fat obtained was expressed as a percentage of the initial weight of the sample using the formula.

Crude fat, % by weight =



Eq. (3.4)

Where: W1 = weight of the extraction flask ( g), W2= weight of extraction flask plus t he dried crude fat (g), and W= weight of samples ( g) Determination of crude fiber

Crude fiber was conducted using the method of AOAC, (2000) official method (962.09). About 1.6g of fresh sample was placed into a 600ml beaker, 200ml of 1.25% sulfuric acid was added, and boiled gently exactly for 30 minutes placing a watch glass over the mouth of the beaker. During boiling, the level of the sample solution was kept constant with hot distilled water. After 30 minute boiling, 20ml of 28% KOH was added and boiled gently for further 30 minute, with occasional stirring. The solution was poured from beaker into sintered glass crucible and then the vacuum pump was turned on. The wall of the beaker was rinsed

41

with hot distilled water several times; washing were transferred to crucible, and filtered. The residue in the crucible was first washed with hot distilled water and filtered and then it was washed with 1% H2 SO 4 and filtered. Secondly the residue was washed with hot distilled water and filtered; and again washed with 1% NaOH and filtered. Finally the residue was washed with water- free acetone. The crucible with its content dried for 2 hr in an electric drying oven at 130 0 C and cooled for 30 min in the desiccators (with granular silica gel), and then weighed. The crucible was transferred to a small muffle furnace and incinerated for 30 min at 5500 C. The crucible was cooled in the desiccators a nd weighed.

 ) =      

Eq. (3.5)

Where: W1 = dried weight of crucible W2 = Weight of crucible after ashing W3 = dried weight of sample M = % moisture o f the sample Determination of total carbohydrates Total carbohydrates of the samples including crude fiber were determined by subtraction:

               

 (3.6)

Energy calculation (kcal/100gm)

Energy content was obtained by multiplying the mean values of crude protein, crude fat and total carbohydrate by the Atwater factors of 3.91, 9, 4.12 respectively, taking the sum of the  products and expressing the result in kilocalor ies per 100 g sa mple as reported by Ede m et al ., (1990) and Onyeike et al., (1995). The formula for calculating energy is shown below.

              

Eq. (3.7)

Minerals analysis Calcium and Magnesium

Calcium and Magnesium were determined using AOAC (1998) official method (985.35). They were quantifying by atomic absorption spectrophotometric method. About 25 ml composite aliquot was placed in previously cleaned evaporating dish. Then the aliquot was dried in oven at 100 0 C for overnight. After completion of drying it was heated on hot plate until smoking cease. Next the dish was placed in furnace at 525 0 C to obtain ash of white and free from carbon for 4 hrs. Then dish was removed from the furnace and cooled. Following this H2 O and 2ml of HNO 3 was added, dried on hot plate and the dish was returned to 525 0c

42

furnace for 2hrs. After t hat the as h was dissolved in 5ml 1N HNO 3 and warmed on steam bath for 3 min to aid in solution. Next add solution to 50ml volumetric flask with 1 N HNO 3 and repeated with 2 additional portion of 1N HNO 3 . Lanthanum Chloride (LaCl) solution was added to final dilution of each standard then solution was tested to make 0.1% (W/V) La. Blanks were prepared to represent all reagents and glassware. Then calibration curve was  prepared (concentration Vs absorbance) to determine each mineral using wave length and flame specified in the table. Finally determine concentration of each mineral from its calibration c urve, and calculate the values using the following relation:

        

Eq. (3.8)

Where : W = Weight of t he sa mple (g), V = Volume of the extract (ml), A = Co ncentration (µg/ml) of sample solution and B = Concentration (µg/ml) of blank solution Phosphorous determination

Phosphorous was determined by the colorimetric method using ammonium molybdate (AOAC, 1984) using the official method (965.17). It was converted to phosphomolybdate, which was reduced to a blue molybdenum compound. A sample solution was obtained from mineral analysis. About 1 ml of the clear extract was taken from the sample solution and diluted to 100ml with deionized water in a 100 ml volumetric flask. A 5ml (duiplicate) of the sample dilution was added into test tubes. A 0.5 ml of molybdate and a 0.2ml aminonaphthosulphonic acid was added into the test tube (sample solution) and mixed thoroughly step by step. A 0.2 ml amino naphtholsulphonic acid was added into the test tube repeatedly each time until the solution become clear. The solution was allowed to stand for 10min. The absorbance (reading A) of the solution was measured at 660 nm against distilled water. Simultaneously with sample phosphorous, standard and blank analysis were carried out. Standard and blank solutions were prepared as above but 5ml of working standard (reading As) and 5ml of de-ionized water (reading AB) in place of the sample dilution were used respectively. A standard curve was made from absorbance versus concentration and the slope was used for calculation. First AB subtracted from all other readings

       

Eq. (3.9)

Where : A = reading of the sample solution; AB = read ing of the blank solution; WF = weight of fresh sample.

43

Potassium

Potassium was determined using AOAC (1998) official method (969.03) by using flame absorption photometric. About 4 g sample was added into crucible and char on over flame. Then it was placed in cold furnace at temperature of 525 o C and ashed for 2 hrs. Then 15 ml of dilute HNO3 was added to crucible and it was filtered into 100 ml volumetric flask through acid- washed quantitative paper. Then residue was washed with H 2 O. Next it was diluted for direct readout as follows: about 1 ml aliquot was placed in 25 ml volumetric flask and dilute to volume with H 2 O. At the same time blank solution was prepared by diluting 2 ml HNO 3  to 100 ml with H 2 O. Finally read the blank, standards, and samples at 767 nm until results were reproduced; record % T or absorption for each and Convert % absorption to absorbance (A). At last standard curve was plotted A against concentration. Finally read unknown concentrations fro m the c urve and determine the values by the following relation:

      

Eq. (3.10)

Determination of rheol ogy property of flours A 300g of each blended and control flours were prepared and placed into the corresponding farinograp h mixing bowl. Water from a burette was added to the flour and mixed to form dough. As the dough was mixed, the farinograph recorded a curve on graph. The amount of water added (absorption) affects the position of the curve on the graph paper. Less water increases dough consistency and moves the curve upward. The curve is centered on the 500Brabender unit (BU) line ±20 BU by adding the appropriate amount of water and is run until the curve leaves the 500-BU line.

Functi onal properties of flour Water and oil absorption capaci ty Water and oil absorption capacity of flour was determined with the method reported by Anderson et al .,(1969) as cited by Sukhcharn et al.,  (2008). Five gram flour of each sample was weighed into a centrifuge tube and 30 ml of distilled water or oil was then added and mixed thoroughly. This was allowed to stand for 30 min and centrifuged at 3,000 rpm for 15 min. The supernatant was then decanted and the sample weighed again. The amount of water or oil retained in the sample was recorded as weight gain and was taken as water or oil absorbed. The results were expressed as weight of water absorbed in grams per gram dry matter of the sample.

Bulk density

44

The bulk density of the composite flour was analyzed according to the method stated by Oladele and Aina (2007) in which a mass of 50 g of the sample was put in to a 100 ml measuring cylinder. The cylinder was tapped cont inuous ly until a cons tant volume was obtained. The bulk density was then calculated as weight of the grounded flour (g) divided by its volume (ml).

Determination of physical properties of cookies For the determination of diameter (width), thickness and spread factor, AACC (1995) methods were followed. Diameter

To determine the diameter (D), six cookies were  placed edge to edge. The total diameter of the six cookies was measured in cm by using a ruler. The cookies were rotated a t an angle o f 90 for duplicate reading. This was repeated once more and average diameter was taken in centimeter. Thickness

To determine the thickness (height), six cookies were placed on top of one another. The total height was measured in millimeters with the help of ruler. This process was repeated twice to get an average value and results were taken in mm.

Spread ratio Spread ratio was determined by dividing the diameter to height of cook ies.

3.5 Analysis of antioxidant activity and total phenolics 3.5.1 Sample extraction Method used by (David, 2006, Bushra et al., 2009 and Barinderjit et al., 2012) with some modification used for extraction. Ten grams of grounded fine bran weighed using an aluminum foil and transferred in to a beaker. 40 ml methanol was added in a beaker. The  beakers were capped, p laced in water bath at (40, 60 and 80 0 C) for 20 min and were shaken twice, while it’s  inside the water bath. Finally, incubator shaker used to extract effectively. Then the solvent layer from each test tube was separated by centrifugation at 5000rpm for 14 min. The residue was then extracted with two additional 20 ml portions of solvent as described above. And the re-dissolved sample by the respective solvents used then passed through what man No. 4 paper. The combined extracts were put below 50 0 C in thermostat oven. The separated solvent super natant with the bioactive compounds in it was transferred to clean, previously weighed and labeled test tube. Beaker cleansed, dried, weighted and made

45

ready. Weight differences were calculated for each samples resulted as shown below. All samples were placed in refrigerator prior to testing.

Grounded

10gm measured

40 ml methanol

Cupped

wheat bran

using

added

water bath at T= 40, 60 and

Al

foil

and

placed

in

80o C for t=20 min.

Vortex the test tube 2X during incubation

Incubator Shaker Centrifuged

Solvent supernatant

evaporator/ o ven at

transferred in clean,

o

T≤ 50 C to remove solvent

weighted and labeled test

Centrifuged with ω=2000rpm,

tube using Watman filter

t= 15 min.

Weighted to measure the yield of sample (mass of dry extract= by mass

Solvent supernatant

Residue

Mixed

with

V=20 ml of Methanol,

difference)

and Vortexed

Prepare stock solution and place in refrigerator prior to use

Testing for determination TPC and DPPH

Figure 3.6: Extraction method for antioxidant activities and phenolics ana lysis.

3.5.2 Determination of total phenolic content Phenolic compounds concentration of methanolic extracts was estimated by using slightly modified procedure by (Singleton and Rossi, 1965) as illustrated below. After extraction of the bioactive chemicals, a stock solution of 10mg/ml extract in methanol (10:1) prepared.

46

Then 1ml stock solution taken and diluted by 1ml met hanol to have 2 ml total volume, but the concentration is diluted by half i.e. 5mg/ml. 1ml of Fo lin-C iocalteu a nd 1ml of 7% of sodium carborbonate added. The samples were vortexed for 3 min before sodium carbonate added. Finally, 7ml water added to the sample then vortexed for the last time before absorption read. Incubated for 90 minutes and spectrophotometer read at an absorbance of 725nm model (Perkin Elmer Lamda 950 UV/Vis/NIR). First Gallic acid calibration curve standard is required, so absorption for the gallic acid done in place of extract till R 2 ≥0.98 achieved. All  phenolic compounds carried out in triplicate. Total phenolic content was expressed as mg gallic acid equivalents (GAE)/100g weight. The total content of phenolics in wheat bran extracts in gallic acid eq uivalent was calculated by the following formula:

   

Eq. (3.11)

Where TPC is the total content of phenolic compounds, mg/g fresh material, in GAE; C is the concentration of gallic acid established from the calibration curve (Absorbance = 0.0134 gallic acid /g –   0.0144, R 2  = 0.9918); V the volume of extract (L); m is the weight of extract the concentration of gallic acid established from the calibration curve.

3.5.3 Determination of free radical scavenging activity The effect of methanolic extracts on the DPPH radical was estimated according to Kirby and Schmidt (1997). A 0.004% solution of DPPH radical solution in methanol was prepared and then 4 ml of this solution was mixed with 1 ml of various concentrations (2 –  14 mg/ml) of the extracts in methanol. Finally, the samples were incubated for 30 min in the dark at room temperature. Scavenging capacity was read spectrophotometrically model (Perkin Elmer Lamda 950 UV/Vis/NIR) by monitoring the decrease in absorbance at 517 nm. The maximum absorption was first verified by scanning freshly prepared DPPH from 200 to 800 nm using the scan mode of the spectrophotometer. Ascorbic acid was used as a standard and mixture without extract was used as the control. Inhibition of free radical DPPH in percent (I%) was t hen calc ulated:



      

Eq. (3.12)

Where A0  is the absorbance of the control and A 1  is the absorbance of the sample.

3.6 Sensory quality evaluation The sensory quality evaluation for coded samples (cookies and tea substitutes) done by using descriptive sensory analysis via ten trained panelists. It was conducted in Addis Ababa

47

institute of Technology (AAiT) and in quiet, daylight, room temperature separated house at different sessions. Descriptive tests can be qualitative or quantitative and involve detection and description of both qualitative and quantitative sensory attributes. This has successfully  been used to obtain detailed descriptions of sensory attributes like aroma, flavor, texture and others attributes of the cookies and tea substitute. Samples were evaluated for a number of attributes by trained panelists (Lea et al.1998). Factors like health status, allergies, availability, personality, verbal creativity, concentration, motivation, smoker, sensitivity, medications were considered when selecting sensory panelists. For ease of evaluation, the panelists were handed a scored sheets with 9-point hedonic scale (a balanced bipolar scale around neutral at the center with four positive and four negative categories on each side). The categories are labeled with phrases representing various degrees of affect and those labels are arranged success ively to suggest a single continuum of likes and dislikes (Peryam & Pilgrim, 1957). The panelists were instructed to rate the products using a 9 point hedonic scale with 1 = dislike extremely, 5 = neither like nor dislike, 9 = like extremely was used for attributes according to Amerine et al., (1965); Then coded samples of cookies and tea substitute were presented to panelists together with water for mouth wash within each taste interval.

3.7 Experi mental design and statistical data analysis The data obtained from each experiment were analyzed by using JMP statis tical analysis software version 7.0; using complete ra ndomized design (CRD). Significance was accepted at 0.05 level of probability (p<0.05). Mean separation was performed by “Each Pair Student’s t test” for multiple comparisons of means. All of experiments were performed in triplicates and duplicates. For defatted wheat germ enriched biscuits a factor of two; (Temperat ure a nd blend ratio at level of three attained while for the tea substitute extraction Temperature). Data analysis output of some properties and proximate compositions were listed in result and discussion. The effect of replacing wheat flour by DWGF on the acceptability of the product developed was evaluated by comparing them to a control and measuring the least significant difference (LSD) at 5% according to method described by Mc Clave et al (1991). For wheat  bran sample analysis, average value was taken using Excel, 2007.

48

CHAPTER FOUR Results and Discussion 4.1 Proximate chemical composition Proximate chemical composition for raw materials including wheat germ, defatted wheat germ, wheat flour, wheat bran and cookies were performed.

Proximate chemical composition of raw materi als Proximate analysis is crucial as one part of quality parameter starting from raw material  processing throughout the development process up to final state of product obtained in almost every food product development, production/ process/. The proximate composition of foods is used to determine the functional property, amount of nutrition value, and over all acceptability of the final food product. Proximate composition analysis was made for flours and cookies, which was made from different blend ratio of composite flours and baking temperature. It offers vital clues about the overall composition and nutritional status intended for edibility purpose. The results of  proximate analysis of raw materials WF, WGF, DWGF, and WB flour used for making cookies and preparation of a tea substitute respectively are presented in tables 4.1, 4.2 and 4.3. Table 4.1 Proximate composition of flours Flour Types Proximate Composition

WF

WGF

DWGF

Ash (%)

0.84±0.06c 

3.90±0.11

4.72±0.04a

Moisture (%)

11.92±.0.06c 12.08±0.03

Crude Fat (%)

0.52±0.03

9.91±0.03 a 

1.01±0.04

3.25±0.13

Crude Fiber (%)

0.45±0.07c 

1.19±0.03

5.18±0.08a 

11.91±0.85 a

Crude Protein (%)

9.33±0.26

18.41±0.04

28.12±0.32 a  14.17±0.28 c

Total Carbohydrates (%)

77.39±0.17a 

54.51±0.18 c  53.17±0.19 c  67.43±.1.8

Samples

Bran

4.8±0.18 a

12.99±0.04 a  10.38±0.06

Minerals (mg/100 gm) Mg

P

Ca

K

WF

35±0.14

192.35±0.64

27±0.42

171±0.28

DWGF

45.95±0.35a

392±0.42a

46.6±0.92a

1044.9±0.14a

All a-c values are means of duplicate ± SD on dry weight basis Means followed by different superscript within the same row differ significantly (P < 0.05). Where WF is wheat flour; WGF is wheat germ flour and DWGF is defatted wheat germ flour.

49

From the table 4.1 above, in proximate evaluat ion of the three types of flours, DWGF has as h (4.72%), crude fiber (5.18%) and crude protein (28.12%) has a huge difference from WGF with ash (3.9%), crude fiber (1.19%) and crude protein (18.41%) and WF with ash (0.84%), crude fiber (0.45%) and cr ude protein (9.33%). Similarly in Table 4.2 DWGF resulted having considerable amount of minerals than that of WF; resulting WF has the lowest nutritional content. Consequently from table 4.1 the total a mount of minerals a nd proximate composition obtained from DWGF is much higher than that of WF; thus DWGF can be used for supplementation as substitute of WF for upgrading nutritional content of the flour and its  product. The data obtained were in agreement with the findings of various investigators Sahar, (2012). The variation in moisture content value may be caused by due effect of conditioning and storage conditions. A better yield of protein, ash and fiber in DWGF might be due to wheat germ by nature is most nutrient rich part of the kernel. Brans of cereals have been used mainly as source of dietary fiber in cereal foods due to  physiological a nd metabolical effects. Both insoluble and soluble fibers have many positive effects on health and can help prevent d iseases.

4.2 Effect of blend rati o and baking temperature on proximate composition of cookies In developed countries, wheat flour is generally fortified with vitamins B1 , B2 , niacin, with minerals: iron, calcium and folate. Vitamins A and D can also be added to flour (Fortification  basics, USAID). But in developing countries like Ethiopia mostly the final refined wheat flour, is nutritionally deficient wheat flour is consumed without being fortified this might be due to the cost needed to buy or import the required amount of minerals and vitamins to enrich the last refined white flour. However it’s possible to utilize the byproduct wheat germ to upgrade the nutritional content of white wheat flour with least cost as raw material. Effect of both baking temperature and blend ratio o n proximate o f baked cookies discussed below.

4.2.1 Effect of blend ratio and baking temperature on moisture content As it can be observed from table 4.2 the moisture content of the cookies was significantly affected by blend ratio, baking temperature, and their interaction (p< 0.05). The moisture content has a unit of g/100g. With rising baking temperature the moisture content of cookies  becomes smaller this makes the b isc uits to turn o ut to be cr ispier if kept for t he avera ge t ime required. Similarly, as the amount of blend ratio used for baking the cookies increased down

50

the column the amount of moisture content increased. This is caused b y the greater number of hydroxyl existed inside the fiber structure that allow more water interaction through hydrogen  bonding. Similar findings were obta ined b y Piergiovanni & Farris (2008) a nd Manoe la et al ., (2006). Hence an excellence cookie is baked when the cookies resulted crisp ier than hard to  be chewed besides the amounts of water inside cookies indirectly measure the shelf life. Table 2: Effect of blend ratio and baking temperature on proximate % Moisture c

%prote in

%fiber

%Ash c

BWFT1 BWFT2 BWFT3 BR1T1 BR1T2 BR1T3 BR2T1

7.14 ± 0.04 7.08 ± 0.04 7.01 ± 0.04 7.52 ± 0.04 c 7.28 ± 0.05   6.87 ± 0.07 a 7.64 ± 0.04  

9.85±0.04 9.35±0.08 9.17±0.06 c 13.83±0.09 c 13.35±0.10   c 12.87±0.06   14.43±0.06

1.84±0.04 1.79±0.06 1.71±0.05 2.23±0.08 c 2.17±0.08 c 2.08±0.10 a 2.93±0.13

 

     

0.81±0.06 c 0.79±0.09 c 0.74±0.04 1.28±0.03 1.24±0.06 1.19±0.11 1.38±0.05

BR2T2 BR2T3 BR3T1 BR3T2 BR3T3

7.49 ± 0.05 6.99 ± 0.06 a 7.69 ± 0.02   a 7.65 ± 0.06   a 7.47 ± 0.04  

14.29±0.08 14.19±0.04 a 15.88±0.18   a 16.78±0.11   a 15.08±0.12  

2.84±0.04 2.77±0.07 a 3.05±0.11   a 3.10±0.04   a 2.97±0.03  

1.31±0.06 1.23±0.07 a 1.55±0.06 a 1.51±0.09 a 1.47±0.07

a

All a-d values are means of duplicate ± SD on dry weight basis Means followed by different superscript within the same row differ significantly (P < 0.05).

Where BWFT1 = biscuit baked from wheat flour at T1  (150 o C), BWFT2 = biscuit baked from wheat flour at T2  (180 o C), BWFT3= biscuit baked from wheat flour a t T3  (210 o C), BR1T1=  biscuit baked from (90% wheat flour and 10 % defatted wheat germ flour) at 150 o C, BR1T2= biscuit baked from (90% wheat flour and 10 % defatted wheat germ flour) at 180 o C, BR1T3= biscuit baked from (90% wheat flour and 10 % defatted wheat germ flour) at 210 o C,BR2T1= biscuit baked from (85% wheat flour and 15 % defatted wheat germ flour) at 150 o C, BR2T2 =biscuit baked from (85% wheat flour and 15 % defatted wheat germ flour) at 180 o C, BR2T3= biscuit baked from (85% wheat flour and 15 % defatted wheat germ flour) at 210 o C, BR3T1= biscuit baked from (80% wheat flour and 20 % defatted wheat germ flour) at 150 o C, BR3T2= biscuit baked from (80% wheat flour and 20% defatted wheat germ flour) at 180 o C, BR3T3= biscuit baked from (80% wheat flour and 20% defatted wheat germ flour) at 210 o C.

4.2.2 Effect of blend ratio and baking temperature on crude protein content Proteins a lso b ind water o n a molecular basis owing to hydrogen bo nds within the solubilized  protein itself and therefore proteins also help to increase firmness of a product. Proteins in defatted wheat germ enhance the flavor in finished products Horizon milling, (2013). Baking temperature, blend ratio and their interaction affected the protein content of cookies. The average protein content was declined slightly with increasing in baking temperature along the row (not significant). This is either due to protein denaturation resulted due to the effect of high temperature, or Maillard reaction, a reaction by free amino groups of amino

51

acids and sugars. Similar result found by Gulen and Eris (2004) when studied the effect of heat stress on protein content. As can be seen from table 4.2 the value of protein raised with every increment of blend ratio down the column this is because the more amounts of amino acids are presence in each increment in blend ratio. The amount of water associated to proteins is closely related with its amino acids profile and increases with the number of charged residues, conformation, hydrophobicity, pH, temperature, ionic strength and protein concentration Damodaran, (1997).

4.2.3 Effect of blend ratio and baking temperature on crude fiber  Now a day a number of people in the world boost up co nsumption of dietary fiber intake b y accepting the fact it’s capable of reducing blood cholesterol levels, occurrence of colon cancer even best for weight loss hence it is the indigestible part of foods, determined from the residue remaining after extraction under specified conditions, it feels the belly full without leaving the individual obsessed. According to (FAO, 2003) daily intake of dietary fiber is 25gm/day. Thus it’s advantageous to use the byproduct DWGF with almost no cost. From table 4.2 baking t emperature of the cookies doesn’t have that much significant effect (P>0.05) on the crude fiber content of the cookies, however cookies were found significantly affected by blend ratio (p<0.05). As the blend ratio of the composite flour used increased the amount of crude fiber content also increased. Therefore supplementation of wheat flour with defatted wheat germ flour could be one a lternative to make our food prosperous nutritionally. Similar findings were obtained by (Mian et a l., 2009).

4.2.4 Effect of blend ratio and baking temperature on ash Ash is mineral content of foods, determined by combustion of the sample under defined conditions a nd weighing of the residue. The as h content of the cook ie was found significantly influenced by the blend ratio (p< 0.05) but not by baking temperature and their interaction. Increasing in the blend ratio of DWGF in the respective blend ratios similarly increased the amount of ash in the last product. This could be the result of higher amount of ash content in defatted wheat germ flour than wheat flour initially. Ash was found not significantly influenced by baking temperature (p > 0.05) similar finding with Biniyam, (2010).

52

Table 3: Mineral composition of biscuits at different blend proportions

Flours

Minerals Mg

P

Ca

K

WF

25.65±1.34 d 

179.15±1.20 d 

39.55±1.06 a 

130.1±0.85 d

BBR 1  

46.2±0.98 c 

207.85±0.35 c 

40.4±0.28 a 

213.8±0.42 c

BBR 2  

54.5±0.84 b 

216.7±0.57 b 

40.6±0.42 a 

235.0±0.28 b

BBR 3  

61.95±1.34 a 

232.5±0.84 a 

41.1±0.28 a 

278.7±0.84 a

All values are means of duplicate ± SD Means followed by different superscript within the same column differ significantly (P < 0.05).

According to (Fenema, 1996) Mineral elements, unlike vitamins and amino acids, cannot be destroyed by exposure to heat this could be the reason that baking temperature was not influence ash and mineral content of the cookie significantly. Therefore amount of minerals  presented in the cookies is s ignificantly affected (P<0.05) by blend ratio, this is basically true hence the defatted wheat germ flour has got magnificent amount of minerals than that of the control. Similar findings can be observed by (Mian et al., 2009).

4.3 Rheological property of flours Rheology is the science that studies the flow and deformations of solids and fluids under the influence of mechanical forces as a function of time. The rheological measures of a product in the manufacture stage can be useful in quality control. The microstructure of a product can also be correlated with its rheological behavior, allowing development of new materials (Gips y and Gustavo, 2004). In the food industry, rheology provides a scientific basis for subjective measurements such as mouth feel, spread ability and pour ability by using farinograph an instruments used to investigate the physical properties of dough. Rheological properties such as elasticity, viscosity and extensibility are important in the prediction of the processing parameters of dough and quality of end product (Hruskova, 2001). The measurements completed on the variation of the kneading torque by two different modalities, using an electronic brabender farinograph and an experimental plant with torques. Rheological properties of the different types of blends and the control were analyzed. Mixer temperature was set at 30 o C prior for all tests.

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Figure 4.1 Farinograph values of control flour/ WF.

Figure 4.2 Farinograph value for BR 1. 4.3.1 Water absorption

It is the amount of water required to center the farinograph curve on the 500-Brabender unit (BU) line. From this water absorption showed tendency of change in the physical characteristics of the dough found from wheat flour alone and blended with defatted wheat germ flour with the increase in the proportion of defatted wheat germ flour in the mixture.

54

The increase in absorption was mainly due to the increase in DWGF, which is higher in fiber content which cause high number of hydroxyl groups existing in the fiber molecules, responsible to allow more water interaction due hydrogen bonding similar findings with Abdullatif, (2009). The higher the blend proportion, the higher the water absorption of the flour, this might be due to the factor that affect flour water absorption are principally the flour moisture content, its protein level, a nd the a mount of damaged starch.

Figure 4.3 Farinograph result for BR 2.

Figure 4.4 Farinograph value for BR 3.

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4.3.2 Dough development time

Among all the samples wheat flour (control) has got the lowest value and the blend with higher proportion BR 3  took highest time to be developed. This showed the addition of more defatted wheat germ resulted higher amount of time the dough to be developed this again  basically due to increased a mount of moisture co ntent inside the water loving hydroxyl group which are present highly in the fiber part this as a result ended up the final dough development time to be higher as the more amount of defatted wheat germ blended. This finding is in agreement with Abdellatif, (2009).

4.3.3 Dough stabili ty The control needed less dough stability time, whereas the blended ones desired more time of stability. Though longer stability means easier handling for the baker and less possibility of over mixing, the dough with an increase in defatted wheat germ flour ratio resulted having difficulty to ac hieve stability with in short period of time as that of the control. This is mainly due to excellent amount of protein content present inside the defatted wheat germ flour. And this might be due the difference in protein content and quality of flours which is similar finding with (Holas and Tipples, 1978 and (Sudha et al., 2011).

4.3.4 Farinograph quality number (F QN) Wheat flour has got the lowest farinograph quality number while the blended ones have got uppermost farinograph quality number. As the level of the defatted wheat germ flour added increased the farinograph quality number to signifying that the flour had high water absorption capacity same finding as (Toufeili et al., 1999). Hence higher farinograph quality number obtained for higher a mount of proportion in the b lend.

4.4 Functional properties of flours Functional properties are those parameters that determine the application and use of food material for various food products. It is the characteristics of a substance that affect its  behavior and that of products to which it is added. Influence potential applications of a substance in the food industry, as a particular functional property may be espec ially use ful for the manufacture and stability of specific types of foods. Include a wide range of characteristics, such as water absorption capacity, bulk density, and oil absorption capacity. The wheat flour and blend of DWGF analyzed for their functional properties for the formulation of value added cookies.  The mean values f or bulk density, water a nd oil absorption capacities were shown in Table 4.9.

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4.4.1 Bulk density Bulk density, weight per unit volume of wheat flour and the blended composites flours,  presented in table 4.9. The highest bulk density was obtained b y BR 3 , BR 2 , BR 1  and finally WF. Having higher bulk density of composite flour exhibit better compactness and possible mixed effect caused by the interaction of the molecules of the DWGF and WF. The higher  bulk density ob served for the composite flour implies that a denser packaging material may  be required for this prod uct. Bulk de nsity gives infor mation on t he porosity of a product a nd can influence t he choice of packa ge and its design (Odedeji and Oyeleke, 2010).

4.4.2 Water absorption capacity The water absorpt ion capac ity is a function of water holding ability of the flour sample. Fro m the analysis reported in table 4.9. The highest WAC of DWGF could be attributed due the  presence of high protein, crude fiber and higher amounts of hydrophilic constituents in DWGF. Similar findings were obtained by Adeyemi and Beckley (1986) reported that water absorption capacities of flours correlate positively with the particle size of flours. Higher WAC of the composite flour may be attributed to their higher protein contents. Afoakwa, (1996), reported that proteins are mainly responsible for the bulk of water uptake in flours. Water absorption capacity is a critical function of protein in various food products like soups, gravies, doughs a nd baked products Sosulski et a l., (1976).

4.4.3 Oil absorption capacity Oil absorption capacity (OAC) is another vital functional property of flour hence it’s excellent in enhancing the mouth feel while preserve the flavor of food products. The removal of fat from the samples exposes the water binding sites on the side chain groups of  protein units previously blocked in a lipophilic environment t hereby leading to an increase in WAC value as in defatted flours as mentioned earlier. Similar observation has been reported  by Lin et al. (1974). Oil absorption increased in proportion to the protein contents of the flour. The mechanism of fat binding is not fully understood, but formation of lipid-protein complexes is markedly responsible for oil retention. As can be seen from table 4.9 the oil absorption capacity of flours increased from wheat flour through the blended ratios. The oil absorption of defatted wheat germ flour was higher than those of wheat flour. This implied DWGF may have more hydrophobic proteins flour; the more hydrophobic proteins demonstrate superior binding of lipids. Hence the major chemical

57

component affecting oil absorption capacity is protein, which is composed of both hydrophilic and hydrophobic parts. This on the other hand shows DWGF with the higher  blend ratio improved mouth feel a nd preserve t he flavor o f the value added cookies produced (Aremu et al., 2006). Table 4: Functional properties of flours

Flours

B.D

WAC

OAC

WF

0.625±.003

0.78±0.042

0.78±.04

BR 1

0.65±.028

0.87±.042

0.95±.021

BR 2

0.67±.008

1.07±0.049

1.19±.028

BR 3

0.68±.014

1.98±0.049

2.14±.042

All values are means of duplicate ± standard deviations Where B.D = bulk density, WAC=water absorption capacity & OAC= oil absorption capac ity

4.5 Physical properties of cookies The physical properties of cookies are one crucial feature that determines the consumer acceptability. Weights (gm), height (cm), diameter (cm) were measure and spread ratio was calculated as the diameter to height ratio. Table 5: Effect of blend ratio and baking temperature on physical properties of cookies Weight

diameter

height a

Spread ratio a

BWFT1 BWFT2 BWFT3 BR1T1 BR1T2 BR1T3

5.43±0.01 5.37±.007 5.30±0.01c  6.44±0.02c 6.29±0.02c 6.29±0.04

4.52±0.07   4.40±0.04 a  4.39±0.06 a  3.88±0.10 3.850±0.08 3.840±0.04

0.53±.001   0.52±0.04 a  0.51±0.01 a  0.522±0.01 0.51±0.07 0.50±.004

8.69±0.05 a 8.63±0.08 a 8.61±0.07 a 7.46±0.05 c 7.54±0.04 c 7.68±0.06 c

BR2T1 BR2T2 BR2T3 BR3T1 BR3T2 BR3T3

6.74±0.01 6.52±0.02 6.42±0.01 7.06±0.04a  6.87±0.06a  6.8±0.014a 

3.76±0.06 c  3.800±0.01 3.74±0.07 c  3.63±0.08 c  3.610±0.05 c 3.610±0.09 c

0.49±.0014 c  0.51±0.02 c  0.48±.001 c  0.48±.002 0.47±0.02 0.46±0.01

7.67±0.07b 7.65±0.09 c 7.65±0.03 c 7.72±0.07 7.85±0.06 7.84±0.08

All values are means of duplicate ± standard deviations Means followed by different superscript within the same column differ significantly (P < 0.05).

4.5.1 Effect of blend ratio and baking temperature on weight of cookies The method of Zoulias et. al., (2002) used for measuring the physical properties weight, diameter height and spread ratio of the value added cookies. And averages of the duplicate

58

measures of both blend ratio and baking temperature effect were analyzed. Physical analysis of cookies used as an essential tool for both consumers and manufacturers, hence spread of the biscuits should be according to specification. The effect of blend ratio and temperature showed a significance difference in the weight of cookies. As the blend ratio of the cookie increased the weight of the cookies also increased this could be majorly as a result of imbitions o f water due to the higher water absorption owing to high protein content in DWGF or could be higher b ulk de nsity of DWGF in each proportion increment that is similar finding with (Gernah et al., 2014). However, there is an adverse effect of temperature on weight loss of the cookies this might be due to the up taken of high amount of moisture content in every raise in temperature.

4.5.2 Effect of blend ratio and baking temperature on diameter of cookies Effect of blend ratio on diameter has got a significant difference (P<0.05) of cookies. There was decrement in diameter and height as the blend ratio increased this might be due to an increment in fiber concentration in every blend formulation. The control cookie made of wheat flour had wider diameter than supplemented cookie. Cookies made of BR 3 were with the smaller diameter mainly due to the presence of more fiber. A similar decreased in diameter was also reported by Singh et al., (2008), and by Biniyam, (2009) wheat, quality  protein maize and carrot.

4.5.3 Effect of blend ratio and baking temper ature on cookies height

The effects of blend ratio on height of the cookies were similar to that of the diameter. As the supplementation of defatted wheat germ increased the height of the cookies resulted decreased. This might be due to the different flour quality of the blended flours (presence of high fiber) or the absence of ample amount of gluten inside the byproduct defatted wheat germ flour. When there was a raise in temperature the height of the cookie showed slight diminish this could be d ue to reduction by volume of cookies d ue high amount of moisture up taken by the raise in temperature.

4.5.4 Effect of blend ratio and temperature on spread ratio From the table above spread ratio decreased with an increase in blend ratio. Hence it’s an indication of the viscous property of dough and influenced by the recipe, ingredients,  procedures and conditions used in biscuit production (Dogan, 1998). McWatters (1978) reported that rapid partitioning of free water to hydrophilic sites during mixing increased

59

dough viscosity, thereby limiting cookie spread; or a decrease in spread factor and thickness was due to the increase in amount of protein, as addition of soy flour which could attribute to higher protein content of soy flour as reported by Mridula et. al ., (2007). These results were similar to those reported for cookies prepared from wheat – cowpea (McWatters et al., 2003) and wheat – soybean (Shrestha & Noomhorm, 2002) flour blends. However, it has been suggested that spread ratio is affected by the competition of ingredients for the available water, by flour or any other ingredient, which absorbs water during dough mixing, will decrease it (Fuhr, 1962).

4.6 Total phenolic content and antioxidant activity of bran 4.6.1Total phenolic content of wheat bran In preliminary determination of total phenolic content of wheat bran, standard curve for the gallic acid obtained by Excel, 2007 from table found in appendix III. Total phenolic content (TPC) was expressed as milligrams of gallic acid equivalent per gram (mg/g) of dry extract samples. Gallic acid, the major phenolic acid found in wheat, was used as a standard. Total  phenolic co ntent o f wheat bran using differe nt temperature range differed (1.037, 2.15, 3.58 and 3.68) mg of GAE /g for temperatures 40, 80, and 60

o

C using absolute and solvent

methanol for extraction. These results are in agreement with that observed by Vaher et al . (2010) who found that the bran layers have the highest content of total phenolics content. This may be due to the use of mixture alcohol and water present the advantage of depends mainly on the hydroxyl groups, the molecular size and the length of hydrocarbon. The higher total phenolic compounds were extracted by using organics solvent (alcohols) whose polarity is modified with water. These mixtures become ideal and selective to extract a great number of bioactive compounds of phenolic compounds. Whereas water given more amount of yield,  but only is not good to extract po lyphenols. Wa ter extracts o nly the water-soluble bioactive compounds; moreover much other residual substances and impurities are present in the aqueous extracts (Zohra, 2011). Therefore for the experiment, with increase in solvent extraction temperature, there was significant increase in total phenolic content in cereal brans. Maximum total phenolic contents were obtained at 60°C (3.68mg GAE g-l) while minimum at 40°C (1.037 mg GAE g- l). The difference in total phenolic content among each extraction may be due to the difference in heat labile nature of cerea l bra n (Dar and Savita, 2011).

60

As can be seen from the result from the table on appendix III; the effect of different temperatures during extraction result higher amount of TPC at 60 o C obtained 3.58 and 3.68mg/gm GAE using absolute and solvent methanol. According to Oufnac et al.  (2007) with rise in extraction temperature more phenolic compounds are released. Earlier research has shown that higher temperature during extraction has a tendency to increase antioxidant activity (Brand-Williams et al., 1995). Increased extraction temperature may breakdown or increase hydrolysis of the bonds of some bound phenolic compounds and causes them to  become extractable phenolic compounds. Similar fact was observed by Sun et al., (2007), while extracting total phenols from asparagus. And the aqueous methanol was better than the absolute one may be due to the fact that phenolics are often extracted in higher amounts in more polar solvents such as aqueous methanol as compared with absolute methanol similar results were obtained by Anwar et al., 2007), O nyeneho and Hettiarachchy, (1992), F ulcher et al.,  (1972) These results are in agreement with previous reports which also reported that antioxidants including phenolics are concentrated in the aleurone fraction of bran.

4.6.2 Antioxidant acti vity of wheat bran The DPPH method is common for determination of free radical scavenging activity of antioxidant. DPPH (2, 2- diphenyl-1- picrylhydrazyl) hence it’s a very stable organic free radical and presents the ability of accepting an electron or hydrogen radical. The capacity of wheat extract to scavenge the stable DPPH radical is shown in figure 4.5 where blue, red and green are percentage inhibition capacities for ascorbic acid, methanol solvent and absolute methanol with IC 50  value (1.43, 1.75, and 2.125) mg/ml respectively. IC 50  determined as the lowest concentration that will inhibit 50% of a process for ascorbic acid as control, followed  by extract by methanol solvent and absolute methanol respectively. This indicated extract b y solvent methanol has higher scave nging capacity t han absolute methanol. Similar findings found by Bushra, 2009 that extract yields and resulting antioxidant activities of the plant materials are strongly dependent on the nature of extracting solvent, due to the  presence of different antioxidant compounds of varied chemical c haracteristics and polarities that may or may not be soluble in a particular so lvent. Polar solvents are frequently employed for the recovery of polyphenols from a plant matrix. The most suitable of these solvents are (hot or cold) aqueous mixtures containing methanol, ethanol, acetone and ethyl acetate, and according to Liyangli, 2007each type of antioxidant compound is likely to exhibit different free radical scavenging properties depending on the nature and mechanism of the free

61

radicals used and their reactivities with different antioxidant compounds, a moderately polar extraction solvent such as 80% methanol may be more effective for extracting phenolic antioxidants and DPPH scavengers from wheat grain than absolute methanol. 110 100 90 80 70 Ascorbic

60 50

Me-ol & H2O

40

Me-ol

30 20 10 0 0

2

4

6

8

10

12

Figure 4.5 Free radical scavenging methanolic e xtract of wheat bran and control.

4.7 Sensory quality evaluation of products The last evaluation conducted by the panelist was the overall acceptability of cookies. Hence overall acceptability is the sum of all the quality parameters and loving the cookies, considered as basic for new product development. The overall acceptability of the cookie was influenced by both blend proportion and baking temperature. The uppermost score of  judgment on over all acceptability was observed for BR 2   at 1800 C while the least one was BR 3  at T1 . WFBT2 10 BBR3T3 8 6 BBR3T1 4 2 BBR3T2 0 BBR2T3

WFBT1

appea.

WFBT3

flavor BBR1T2 texture BBR1T1

BBR2T1

color

taste OAA

BBR1T3 BBR2T2

Figure 4.5 (a) Sensory evaluation for cookies

For the sensory evaluation cookies were selected primarily, followed by hedonic test. From this the overall acceptability of BR2 was more acceptable than all the others included control

62

(WF). This might be due to the enhanced flavor and enriched texture for end product imparted by the defatted wheat germ flour. This finding is not in agreement with (Sahar, 2012) this could be mainly due to culture d ifference a nd perception of acceptance. Appeara. 10 8 6 Overall A.

4

Aroma

2

a b

0

c d

Flavour

Taste

Figure 4.5 (b) Sensory quality evaluations for tea substitute. Where a= unflavored tea b= funneled tea c= mint tea d= cinnamon tea

For the sensory evaluation of tea substitute using a 9 point hedonic scale test, the flavored ones showed a better sensory quality than that of unflavored one. Especially result of tea substitute using additives funnel seed and cinnamon was exceptionally adored by the  pannalists.

63

CHAPTER FIVE

Process Technology 5.1 Production process for cookies and tea substitute

Byproducts from Wheat Milling Industries Wheat Germ

Wheat Bran

Cleaning

Cleaning

Enzyme inactivation

Drying

Milling

Defatting Wheat F lour Blending

Heating

Kneading Flavoring Forming Packaging  b) Tea-s ubstitute Molding/Cutting  b) Tea s ubstitute Baking

Cooling a) Cookie production Packaging Figure 5.1 Flow chart dia gram for deve loped products (a a nd b).

64

5.2 Suggested cookies manufacturing plant

Figure 5.2 Equipment layouts for cookies production

1. Wheat germ pretreat ment unit;

2. Supercrit ical fluid extractor -CO 2; 2-1 SFE- vesse l; 2- 2 Expans ion valve;

2-3 Co llection vesse l;

2-4 Gas pump;

3.Mill; 4.Sreen ing; 5.Blender; 6.Kneader ; 7.Sheeter; 8.Cutter; 9.Ba king oven; 10.Cooling; 11.End prod uct evaluation; 12. Pack ing and distribution

65

CHAPTER SIX Conclusions and Recommendations 6.1 Conclusions This study was primarily and mainly aimed to investigate the possibility of exploit underutilized byproducts (wheat germ and wheat bran) from wheat milling industries; with the intention of developing value added products (cookies and tea substitute) respectively. Secondly, it was attempted to determine appropriate blend ratio, temperature and their combination plus their impact on the (functional, physical and rheological property of flours (proximate and sensory quality)) of the newly formulated products; and tea substitute from wheat bra n demonstrated better antioxidant activity and phenolic content. From the findings, when wheat flour supplemented with defatted wheat germ flour with  protein (28.12), fiber (5.18) was more than adequate to be used as enrichment of wheat flour with protein (9.33), fiber (0.45) and much lesser amount of minerals (Mg, P, Ca, K) content than that of the defatted wheat germ flour. Hence, the Farinogram analysis demonstrated acceptable range of dough character existed till level of 15%. Finally, the 15% blended cookies baked at temperature of 180 o C result very good sensory acceptance, better nutritional

CHAPTER SIX Conclusions and Recommendations 6.1 Conclusions This study was primarily and mainly aimed to investigate the possibility of exploit underutilized byproducts (wheat germ and wheat bran) from wheat milling industries; with the intention of developing value added products (cookies and tea substitute) respectively. Secondly, it was attempted to determine appropriate blend ratio, temperature and their combination plus their impact on the (functional, physical and rheological property of flours (proximate and sensory quality)) of the newly formulated products; and tea substitute from wheat bra n demonstrated better antioxidant activity and phenolic content. From the findings, when wheat flour supplemented with defatted wheat germ flour with  protein (28.12), fiber (5.18) was more than adequate to be used as enrichment of wheat flour with protein (9.33), fiber (0.45) and much lesser amount of minerals (Mg, P, Ca, K) content than that of the defatted wheat germ flour. Hence, the Farinogram analysis demonstrated acceptable range of dough character existed till level of 15%. Finally, the 15% blended cookies baked at temperature of 180 o C result very good sensory acceptance, better nutritional content (protein=16.8, fiber=3) quite not destructively affected by temperature and blend ratio. The wheat bran extracted using absolute and solvent methanol for determination of total  phenolic content using Folin-C iocalteu assa y and antioxidant activity using DPPH scavenging assay at (40, 60 and 80) o C . Gallic acid and ascorbic acid were used as a control for both assays respectively. A higher total phenolic content (3.68mg/gm) of gallic acid equivalent was investigated at a temperature of 60 o C using solvent methanol. Percentage inhibition capacity for ascorbic acid, methanol solvent and absolute methanol with IC 50  value was (1.43, 1.75, and 2.125) mg/ml respectively. The tea substitute made from wheat bran flavored with funnel seed and cinnamon resulted better sensory acceptanc e.

67

6.2 Recommendations From the study result, partial substitution of wheat flour with defatted wheat germ flour for cookies making appeared to promising in nutrition point of view; similarly the tea substitute of variety additives were success too. The following recommendations are forwarded based on holistic assessment of the subject area for production of value added products from defatted wheat germ flour and wheat flour:  Establish benches mark for further research pertaining to work on the germ o il

 product ion and characteriza tion which is useful for making variety of products in different sectors; hence it owes high amount of tocopherol a nd unsaturated fatty acids  both of which are of great importance in human metabolism a nd cannot be synthesized by organism.  Hence defatted wheat germ flour resulted in much lesser amount of oil, which was

responsible for rancidity of the flour, long and durable researches should be done on determination of shelf life of flour. It can be used as nutrient supplement for the wheat flour alone which has smaller nutritional content; even more these by-products from wheat milling industries sold in much lesser price so anyone could buy it cheap and use it for home baking purpose; because this encourages the expansion of existing and development of value added flour, and a new type of cookies from inexpensive and available resources, besides it improves the health status of malnutrition vulnerable group of the society  Further studies on defatted wheat germ flour based value added products like as a

source for production of meat products by partial replacement of the meat should  be done.  Further studies on wheat bran alone for determination of all other antioxidant

activities; and other value added products like soft drink in United Kingdom requires further investigat ion.

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Appendices Appendix I: Scorecard for the sensory quality evaluation using nine point hedonic scales Nine Point Hedonic Scaling Scored-Card for Cookies Date .........................................

 Name....................................................... Instruction:   Taste the coded cookies samples. Fill your appropriate scores which best describes your feeling (according to the 9-point hedonic scale below). Please rinse your  palate by drinking water in between samples. 9. Point Hedonic Scaling 9.

Like e xt remely

6.

Like sl ightly

8.

Like very much

5.

Neither like nor d islike

4.

Dislike slightly

7.

Like moderately

Sample Code

3. 2.

Dislike moderate ly Dislike very much

1.

Dislike extremely

Attributes Appear ance

Col or

Fl avor

Texture

Taste

Over all accept.

BWFT1 BWFT2 BWFT3 BBR1T1 BBR1T2 BBR1T3 BBR2T1 BBR2T2 BBR2T3 BBR3T1 BBR3T2 BBR3T3

If you have additional Co mments/suggestions please do not hesitate to jot down: ...................................................................................................................................................... ...................................................................................................................................................... ...................................................................................................................................................... ...................................................................................................................................................... ...................................................................................................................................................... ................................................................................................... Thank you! 76

Appendix II Score card for t he sensory quality evaluation using nine point hedonic scales Nine Point Hedonic Scaling Scored-Card for tea substitute

Date .........................................

 Name....................................................... Instruction:  Taste the coded samples (a-d). Fill your appropriate scores which best describes

your feeling (according to the 9-point hedonic scale below). Please rinse your palate by drinking water in between samples. 9. Point Hedonic Scaling 9.

Like e xt remely

6.

Like sl ightly

8.

Like very much

5.

Neither like nor d islike

4.

Dislike slightly

7.

Like moderately

Sample Code

Appea rance

Aroma

3. 2. 1.

Attributes Mouth Flavor Feel

Dislike moderately Disl ike very much Dislike e xt remely

Overall acceptability

A B C D If you have additional Co mments/suggestions please do not hesitate to jot down: ...................................................................................................................................................... ...................................................................................................................................................... ...................................................................................................................................................... ...................................................................................................................................................... ...................................................................................................................................................... ...................................................................................................

Thank you! 77

Appendix III Data obtained for bran extraction and tests Standard Ga llic acid absorpt ion to produce standard c urve

Run

Test

GA

ME-ol

Vo l.T

FC

 Na2 CO3

tubes

5mg/m

(µlt)

(ml)

(ml)

(ml)

DD Water (ml)

Con.

Avg.

(µg/ml)

l 1

B11

0

100 µlt

0.1

1ml

1ml

7ml

0

2

B12

0

100

0.1

1

1

7

0

3

B13

0

100

0.1

1

1

7

0

4

GA 11

5

95

0.1

1

1

7

5

5

GA 12

5

95

0.1

1

1

7

5

6

GA 13

5

95

0.1

1

1

7

5

7

GA 21

15 µlt

85

0.1

1

1

7

15

8

GA 22

15 µlt

85

0.1

1

1

7

15

9

G23

15 µlt

85

0.1

1

1

7

15

10

GA 31

30 µlt

70

0.1

1

1

7

30

11

GA 32

30 µlt

70

0.1

1

1

7

30

12

GA 33

30 µlt

70

0.1

1

1

7

30

13

GA 41

50 µlt

50

0.1

1

1

7

50

14

GA 42

50 µlt

50

0.1

1

1

7

50

15

GA 43

50 µlt

50

0.1

1

1

7

50

16

GA 51

70 µlt

30

0.1

1

1

7

70

17

GA 52

70 µlt

30

0.1

1

1

7

70

18

GA 53

70 µlt

30

0.1

1

1

7

70

19

GA 61

100 µlt

0

0.1

1

1

7

100

20

GA 62

100 µlt

0

0.1

1

1

7

100

21

GA 63

100 µlt

0

0.1

1

1

7

100

Absorbance read for TPC of extracted samples Samples

TPC

Absorbance

Solvent methanol extracted at 60 o C

3.68

0.1824

78

0.07701

0..091

0.1182

0.1609

0.2154

0.2668

0.368