NUTRITIONAL AND BIOCHEMICAL POTENTIAL OF ...

NUTRITIONAL AND BIOCHEMICAL POTENTIAL OF

FENUGREEK SUPPLEMENTED FLOUR

By

BAHZAD AFZAL

97-ag-1418

M.Sc. (Hons.) Food Technology

A thesis submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

IN

FOOD TECHNOLOGY

NATIONAL INSTITUTE OF FOOD SCIENCE & TECHNOLOGY

FACULTY OF FOOD, NUTRITION & HOME SCIENCES

UNIVERSITY OF AGRICULTURE, FAISALABAD

PAKISTAN

2015

i

DECLARATION

I hereby declare that the contents of the thesis, studies on “Nutritional and biochemical

potential of fenugreek supplemented flour” are the product of my own research and no part

has been copied from any published source (expect the references, standard mathematical or

genetic models/equations/formulas/protocols etc.). I, further, declare that this work has not

been submitted for the award of any other diploma/degree. The university may take action if

the information provided found inaccurate at any stage. (In case of any evasion the scholar will

be proceeded against as per HEC plagiarism policy).

Bahzad Afzal

97-ag-1418

ii

The Controller of Examinations,

University of Agriculture,

Faisalabad.

We, the Supervisory Committee, certify that the contents and form of this thesis submitted by

Bahzad Afzal, Reg. # 97-ag-1418 have been found satisfactory, and recommend that it be

processed for evaluation by the External Examiner(s) for the award of degree.

SUPERVISORY COMMITTEE:

Chairman:

(Dr. Imran Pasha)

Member:

(Prof. Dr. Tahir Zahoor)

Member:

(Prof. Dr. Haq Nawaz)

iii

DEDICATED To

Holy Prophet Muhammad

ملسو هيلع هللا ىلص

&

MY PARENTS

for their love, endless support and encouragement

iv

ACKNOWLEDGEMENTS

To esteem the Highness of Almighty Allah, I feel myself inept as my words have lost their expressions, knowledge is lacking and diction is too short to express gratitude in the rightful manner to the blessings and support of Allah Almighty whose help had flourished my ambitions and helped me to attain goals. Quivering hands feel mortified to hunt for words of praise for Holy Prophet Muhammad (P.B.U.H.) for enlightening our lives with the faith in Allah, selecting course of contented conscience, converging all His kindness and mercy upon him. Allah Almighty had been so helpful in His blessings by giving me a prospect to toil under the esteem supervision of Dr. Imran Pasha, Associate Professor, National Institute of Food Science and Technology, University of Agriculture, Faisalabad. I have no words to express my gratitude for his diligent cooperation, scrupulous support and cheering perspective during the entire degree program. I deem it my utmost pleasure in expressing my gratitude with the insightful benedictions to Prof. Dr. Tahir Zahoor, National Institute of Food Science and Technology, University of Agriculture, Faisalabad. His sympathetic attitude, parental guidance, scholarly suggestions and criticism indeed are incalculable wealth for me. Abstemious and stanch appreciation to Prof. Dr. Haq Nawaz, Institute of Animal Feed and Nutrition, University of Agriculture, Faisalabad, for his advices and valued suggestions throughout the research project. I feel much honor to express my deepest sense of gratitude and indebtedness to honorable Prof. Dr. Masood Sadiq Butt, Dean/Director General, National Institute of Food Science & Technology, Faculty of Food. Nutrition & Home Sciences, University of Agriculture, Faisalabad from the core of my heart for his marvelous guidance, encouraging behavior, scholastic and sympathetic attitude, masterly advice and indefatigable assistance at all time during the entire study program. I want to express my great appreciation and sincerest gratitude to all my fellows at the National Institute of Food Science & Technology, UAF for their dexterous, dynamic, untiring help, friendly behavior and moral support during my whole study. I am indeed thankful to all my sweet juniors for their earnest support throughout the course of my studies. Here I would be very selfish if I do not express my sincere and special gratitude for my loving father Muhammad Afzal (Late), my dearest mother who has always wished to see me glittering high on the skies of success. Their endless efforts and best wishes sustained me at all stages of my life & encouraged me for achieving high ideas of life and whose hands always remain raised in prayer for my success. I heartily thankful to my loving Brothers, Sisters, Bhabi and my sweet Fahad, Abdullah & Ayesha for their inspiring encouragement and moral support.

Bahzad Afzal

v

LIST OF CONTENTS

Acknowledgements iv

List of Tables ix

List of Figures xiii

Abstract xiv

1. INTRODUCTION 1

2. REVIEW OF LITERATURE 7

2.1 Functional foods and human health 8

2.2 Plants and their bioactive components 9

2.3 Fenugreek: an introduction 11

2.4 Phytochemistry of fenugreek 13

2.5 Fenugreek and flour rheological properties 14

2.6 Perspectives of product development 16

2.7 Fenugreek and antioxidant potential 19

2.8 Fenugreek and hyperglycemia 23

2.9 Fenugreek and hypercholesterolemia 27

2.10 Potential health risk 31

3. MATERIAL AND METHODS 32

3.1. Procurement of raw material 32

3.2 Preparation of raw material 32

3.3 Analysis of raw material 32

3.3.1. Proximate analysis 32

3.3.1.1 Moisture Content 32

3.3.1.2 Ash Content 32

3.3.1.3 Crude protein 33

3.3.1.4 Crude fat 33

3.3.1.5 Crude fiber 33

3.3.1.6 Nitrogen free extract (NFE) 33

3.3.2 Mineral profile 33

3.4 Rheological properties 33

3.5 Development of composite flour 34

3.6 Analyses of composite flour 34

3.6.1 Proximate analysis 34

3.6.2 Mineral profile 34

3.7 Rheological properties 35

3.8 Polyphenols extraction 35

3.9 Antioxidant assay 35

3.9.1 Total phenolic content 35

3.9.2 Total flavonoids 35

3.10 Antioxidant activity 36

3.10.1 Free radical scavenging activity (DPPH assay) 36

3.10.2 ß-carotene and linoleic acid assay 36

3.10.3 Ferric reducing antioxidant power (FRAP assay) 36

3.11 Preparation of bread 36

vi

3.12 Physicochemical characterization of bread 37

3.13 Antioxidant assay of bread 37

3.14 Antioxidant activity of bread 37

3.15 Sensory evaluation of bread 37

3.16 Selection of best treatments 38

3.17 Efficacy trial 38

3.17.1 Feed & water intakes 39

3.17.2 Serum lipid profile analysis 40

3.17.3 Serum glucose and insulin levels 40

3.17.4 Liver function tests 40

3.17.5 Renal function tests 40

3.17.6 Hematological analysis 40

3.18 Statistical analysis 40

4. RESULTS AND DISCUSSION 41

4.1 Proximate analysis of raw material 41

4.2 Mineral content of raw material 43

4.3 Proximate analysis of supplemented flour 45

4.3.1 Moisture content 45

4.3.2 Ash content 46

4.3.3 Crude protein 49

4.3.4 Crude fat 50

4.3.5 Crude fiber 51

4.3.6 Nitrogen free extract (NFE) 51

4.4 Mineral content of supplemented flour 52

4.4.1 Sodium (Na) 53

4.4.2 Potassium (K) 53

4.4.3 Iron (Fe) 54

4.4.4 Calcium (Ca) 57

4.4.5 Copper (Cu) 58

4.4.6 Zinc (Zn) 59

4.4.7 Manganese (Mn) 59

4.5 Rheological study 60

4.5.1 Farinographic studies 61

4.5.1.1 Water absorption 61

4.5.1.2 Arrival time 62

4.5.1.3 Dough development time (DDT) 63

4.5.1.4 Departure time 64

4.5.1.5 Dough stability 64

4.5.1.6 Mixing tolerance index 65

4.5.2 Mixographic studies 66 4.5.2.1 Mixing time 66

4.5.2.2 Peak height percentage 69

4.6 Antioxidant assay of supplemented flour 72

4.6.1 Total phenolic content (TPC) 72


4.7 Antioxidant activity 74

vii

4.7.1 Free radical scavenging activity (DPPH Assay) 74

4.7.2 β‐carotene and linoleic acid assay 78


4.8 Preparation of bread 79

4.9 Proximate analysis of bread 80

4.9.1 Moisture content 80

4.9.2 Ash content 81

4.9.3 Crude protein 81

4.9.4 Crude fat 84

4.9.5 Crude fiber 85

4.9.6 Nitrogen free extract (NFE) 86

4.10 Mineral content of bread 87

4.10.1 Sodium (Na) 87

4.10.2 Potassium (K) 88

4.10.3 Iron (Fe) 88

4.10.4 Calcium (Ca) 91

4.10.4 Copper (Cu) 92

4.10.6 Zinc (Zn) 93

4.10.7 Manganese (Mn) 93

4.11 Antioxidant assay of bread 94

4.11.1 Total Phenolic Content (TPC) 95


4.12 Antioxidant activity of bread 96

4.12.1 Free radical scavenging activity (DPPH Assay) 96

4.12.2 β-carotene and linoleic acid assay 97


4.13 Color of bread 98

4.14 Texture of bread 102

4.14.1 Texture profile of bread supplemented with leaves powder 102

4.14.2 Texture profile of bread supplemented with seeds powder 105

4.15 Sensory evaluation of Bread 110

4.15.1 Volume of bread 110

4.15.2 Aroma of bread 111

4.15.3 Taste of bread 115

4.15.4 Crust color of bread 115

4.15.5 Character of bread crust 119

4.15.6 Texture of bread 119

4.15.7 Crumb color for bread 122

4.15.8 Grain of bread 125

4.15.9 Symmetry of bread form 126

4.15.10 Evenness of bake of bread 126

4.15.11 Overall acceptability of bread 128

4.16 Efficacy study 134

4.16.1. Feed intake 134

4.16.2. Water intake 137

4.16.3. Body weight 139

viii

4.16.4 Serum profile analysis 140

4.16.4.1 Glucose 140

4.16.4.2 Cholesterol 144

4.16.4.3 Insulin 147

4.16.4.4 High density lipoprotein (HDL) 149

4.16.4.5 Low density lipoprotein (LDL) 150

4.16.4.6 Triglycerides 150

4.16.5 Liver functions tests 155

4.16.5.1 Aspartate aminotransferase (AST) 156

4.16.5.2 Alanine transaminase (ALT) 156

4.16.5.3 Alkaline phosphatase (ALP) 156

4.16.6 Renal function tests 161

4.16.6.1 Urea 161

4.16.6.2 Creatinine 161

4.16.7 Hematological analysis 164

4.16.7.1 Red blood cell (RBC) 164

4.16.7.2 White blood cells count (WBCs) 164

4.16.7. Platelets counts (PLC) 164

5. SUMMARY 168

ECONOMIC PERSPECTIVES 175

CONCULUSIONS 176

RECOMMENDATIONS 177

LIMITATIONS AND FUTURE VISTAS 178

LITERATURE CITED 179

APPENDICES 210

ix

LIST OF TABLES

Table # Title Page #

3.1 Treatments used in the study plan 34

3.2 Diet plan used for efficacy trial 39

4.1 Proximate analysis (%) of raw material 44

4.2 Mineral content (mg/100g) of raw material 44

4.3 Mean squares for proximate analysis of different treatments

supplemented with fenugreek leaves powder 47

4.4 Proximate analysis (%) of different treatments supplemented with

fenugreek leaves powder 47

4.5 Mean squares for proximate analysis of different treatments

supplemented with fenugreek seeds powder 48

4.6 Proximate analysis (%) for different treatments supplemented with

fenugreek seeds powder 48

4.7 Mean squares for mineral content of different treatments supplemented

with fenugreek leaves powder 55

4.8 Mineral content (mg/100g) of different treatments supplemented with


4.9 Mean squares for mineral content of different treatments supplemented

with fenugreek seeds powder 56

4.10 Mineral content (mg/100g) of different treatments supplemented with


4.11 Mean squares for Farinographic parameters of different treatments


4.12 Farinographic parameters of different treatments supplemented with


4.13 Mean squares for Farinographic parameters of different treatments

supplemented with fenugreek seeds powder

68

4.14 Farinographic parameters of different treatments supplemented with


4.15 Mean squares for Mixographic parameters of different treatments


4.16 Mixographic parameters of different treatments supplemented with


4.17 Mean squares for Mixographic parameters of different treatments


x

4.18 Mixographic parameters of different treatments supplemented with


4.19 Mean squares for antioxidants in different treatments supplemented with


4.20 Antioxidant in different treatments supplemented with fenugreek leaves

powder 76

4.21 Mean squares for antioxidants in different treatments supplemented with


4.22 Antioxidants in different treatments supplemented with fenugreek seeds

powder 77

4.23 Means squares for proximate analysis of bread supplemented with


4.24 Proximate analysis (%) of bread supplemented with fenugreek leaves

powder 82

4.25 Mean squares for proximate analysis of bread supplemented with


4.26 Proximate analysis (%) of bread supplemented with fenugreek seeds

powder 83

4.27 Mean squares for mineral content of bread supplemented with fenugreek

leaves powder 89

4.28 Mineral content (mg/100g) of bread supplemented with fenugreek leaves

powder 89

4.29 Mean squares for mineral content of bread supplemented with fenugreek

seeds powder 90

4.30 Mineral content (mg/100g) of bread supplemented with fenugreek seeds

powder 90

4.31 Mean squares for antioxidants in bread supplemented with fenugreek

leaves powder 99

4.32 Antioxidants in bread supplemented with fenugreek leaves powder 99

4.33 Mean squares for antioxidants in bread supplemented with fenugreek

seeds powder 100

4.34 Antioxidants in bread supplemented with fenugreek seeds powder 100

4.35 Mean squares for color of bread supplemented with fenugreek leaves

powder 103

4.36 Mean values for color of bread supplemented with fenugreek leaves

powder 103

4.37 Mean squares for color of bread supplemented with fenugreek seeds

powder 104

xi

4.38 Mean values for color of bread supplemented with fenugreek seeds

powder 104

4.39 Mean squares for bread texture supplemented with fenugreek leaves

powder 106

4.40 Mean value for bread texture supplemented with fenugreek leaves

powder 106

4.41 Mean squares for bread texture supplemented with fenugreek seeds

powder 107

4.42 Mean value for bread texture supplemented with fenugreek seeds powder 107

4.43 Mean squares for sensory parameters of bread supplemented with


4.44 Mean squares for sensory parameters of bread supplemented with


4.45 Effect of treatments and storage on volume of bread supplemented with


4.46 Effect of treatments and storage on volume of bread supplemented with


4.47 Effect of treatments and storage on aroma of bread supplemented with


4.48 Effect of treatments and storage on aroma of bread supplemented with


4.49 Effect of treatments and storage on taste of bread supplemented with


4.50 Effect of treatments and storage on taste of bread supplemented with


4.51 Effect of treatments and storage on crust color of bread supplemented


4.52 Effect of treatments and storage on crust color of bread supplemented


4.53 Effect of treatments and storage on character of bread crust supplemented


4.54 Effect of treatments and storage on character of bread crust supplemented


4.55 Effect of treatments and storage on texture of bread supplemented with


4.56 Effect of treatments and storage on texture of bread supplemented with


4.57 Effect of treatments and storage on crumb color of bread supplemented


xii

4.58 Effect of treatments and storage on crumb color of bread supplemented


4.59 Effect of treatments and storage on grain of bread supplemented with


4.60 Effect of treatments and storage on grain of bread supplemented with


4.61 Effect of treatments and storage on symmetry of bread form


4.62 Effect of treatments and storage on symmetry of bread form


4.63 Effect of treatments and storage on evenness of bake of bread


4.64 Effect of treatments and storage on evenness of bake of bread


4.65 Effect of treatments and storage on overall acceptability of bread


4.66 Effect of treatments and storage on overall acceptability of bread


4.67 Effect of diets and time interval on feed, water intake & body weight of

rats in different studies 135

4.68 Effect of supplemented diets on glucose (mg/dL) 145

4.69 Effect of supplemented diets on cholesterol (mg/dL) 148

4.70 Effect of supplemented diets on Insulin (µU/mL) 151

4.71 Effect of supplemented diets on HDL (mg/dL) 152

4.72 Effect of supplemented diets on LDL (mg/dL) 153

4.73 Effect of supplemented diets on triglycerides (mg/dL) 157

4.74 Effect of supplemented diets on serum AST (IU/L) 159

4.75 Effect of supplemented diets on serum ALT (IU/L) 159

4.76 Effect of supplemented diets on serum ALP (IU/L) 160

4.77 Effect of supplemented diets on Urea (mg/dL) 160

4.78 Effect of supplemented diets on creatinine (mg/dL) 163

4.79 Effect of supplemented diets on red blood cell indices (cells/pL) 163

4.80 Effect of supplemented diets on white blood cell Indices (cells/nL) 165

4.81 Effect of supplemented diets on Platelets count 165

xiii

LIST OF FIGURES

Fig. # Title Page #

4.1 Feed intake of normal rats (study I) 136

4.2 Feed intake of hyperglycemic rats (study II) 136

4.3 Feed intake of hypercholesterolemic rats (study III) 136

4.4 Water intake of normal rats (study I) 138

4.5 Water intake of hyperglycemic rats (study II) 138

4.6 Water intake of hypercholesterolemic rats (study III) 138

4.7 Body weight of normal rats (study I) 141

4.8 Body weight of hyperglycemic rats (study II) 141

4.9 Body weight of hypercholesterolemic rats (study III) 141

4.10 Percent reduction in glucose as compared to control 145

4.11 Percent reduction in cholesterol as compared to control 148

4.12 Percent increase in Insulin as compared to control 151

4.13 Percent increase in HDL as compared to control 152

4.14 Percent decrease in LDL as compared to control 153

4.15 Percent decrease in triglycerides as compared to control 157

xiv

ABSTRACT

Globally, lifestyle related health concerns are amongst the serious challenges to mankind.

These disorders are attributed to sedentary living habits and poor dietary practices. In

this context, diet based regimen is gaining importance to alleviate these issues. Fenugreek

(Trigonella foenum-graecum) is well recognized for imparting flavor to various dietary

edibles. Alongside, it contains fair proportions of nutrients helpful in preventing several

metabolic syndromes. Purposely, the present research was designed to elucidate nutritional

and antioxidant potential of fenugreek leaves and seeds followed by their incorporation

in bread formula. Moreover, hypoglycemic and hypocholesterolemic perspectives were

also explored. Based on the current study outcomes, fenugreek leaves and seeds have

appeared as rich sources of protein, fiber and minerals that offer immense opportunities for the

development of various value added products to ensure improved nutritional value and health.

The leaves and seeds powders were incorporated into wheat flour @ 5, 10 and 15% to made

composite flour. Chemical and mineral analysis of composite flours explicated that

supplementation enhanced protein and mineral content. The leaves and seeds flour showed

potent antioxidant potential attributed to numerous nutraceutics. The improvement in

rheological characteristics of flour like water absorption, dough development time and dough

stability were also observed with the increment in supplementation levels. In product

development phase, higher acceptability was noticed in bread containing fenugreek

leaves powder up to 5% and seeds powder by 10%. The phytochemical analysis of the

resultant bread revealed TPC as 198.00±9.10 & 341.00±16.02 mg GAE/100g and total

flavonoids 2.47±0.08 & 2.68±0.10 mg CE/g, respectively for leaves & seeds powder.

Likewise, antioxidant activity of the prepared bread in terms of DPPH scavenging activity

was up to 37.00±1.59 & 49.00±2.20%, β-carotene & Linoleic acid assay as 31.00±1.42 &

40.00±1.88% and FRAP 201.00±9.24 & 401.00±18.44 µmol Fe2+/g, respectively.

Furthermore, efficacy trials revealed that diets containing 5% leaves powder or 10%

seeds powder were effective in ameliorating hyperglycemia and hypercholesterolemia in

rat models. In this connection, glucose reduction was up to 6.78 & 10.67% in

hyperglycemic rats fed on leaves & seeds powder enriched diets as compared to control.

Furthermore, insulin level decreased significantly in control group whilst, inclining trend

was observed in groups fed on leaves and seeds enriched diets. Accordingly, elevation

by 2.96 & 4.01% in insulin level of groups was observed in groups relying on leaves and

seeds powder accordingly. In hypercholesterolemic rats, elevated level of cholesterol was

markedly reduced as a function of fenugreek leaves and seeds powder i.e. 6.32 & 12.03%,

respectively. Similarly, fenugreek leaves and seeds supplemented diets showed declining

trend in LDL by 4.56-12.25 & 4.52-11.34%, respectively. It is therefore inferred that apart

from flavor and fragrance, fenugreek leaves and seeds have a lot to offer in terms of health

improvement. Conclusively, fenugreek based dietary approach is recommended for boosting

health and suppressing metabolic ailments.

1

Chapter 1

INTRODUCTION

The recent era has witnessed the coinage of lifestyle related malfunctions due to poor dietary

habits, altering lifestyles and elevated consumption of refined and processed foods. The

increased metabolic dysfunctions have identified themselves as a great threat for healthy life.

In this context, the prevention of these maladies has captured a vital status to improve the

healthy living standards. Researchers have proved the preventive and therapeutic effects of

various foods and food ingredients. Likewise, the use of natural remedies for prevention of

lifestyle related disorders has gained paramount importance amongst consumers. Recently

number of researchers has explored the facts of natural foods to curtail metabolic ailments. In

this regard, herbs and spices are in limelight having greater potential in research areas to

develop certain products that improve the health status alongside providing basic nutrition.

Moreover, the use of phytochemicals in routine products is of immense consideration to uplift

the protective role of food products against numerous malfunctions. However, the utilization

of functional ingredients in daily foods is a preventive approach against various disorders (Liu

et al., 2007; Sethi et al., 2008). Similarly, these ingredients are helpful in reducing the risk of

such ailments due to their pharmacological perspectives (Misra and Khurana, 2008).

Fenugreek (Trigonella foenum-graecum) locally known as methi, a member of legume family

originated from Asia and Southeastern Europe but presently it is ubiquitous in Pakistan, India,

Egypt and many other countries of the world (Betty, 2008). Fenugreek has a typical fragrance

and grown everywhere in Pakistan. However, due to large cultivation in Kasur district, it is

also called as Kasuri Methi (Erum et al., 2011). The fresh leaves of fenugreek are being used

as vegetable in the diets which provides β-carotene, fiber, calcium and zinc (Jani et al., 2009).

In addition fenugreek seeds possess little bit sweetish and pleasant bitter taste. It has central

hard yellowish embryo and white semi-transparent endosperm which contains a carbohydrate

and gum like consistency namely galactomannans (Betty, 2008). Fenugreek seeds contains

25.2-30.1% protein, 7.2-9.3% lipids, 20.1-25.3% insoluble fiber, 20.4-30.2% galactomannan

and 5.3-7.3% saponins, volatile oils, free amino acids, mucilaginous fiber and flavonoids (Raju

and Bird, 2006). The fenugreek seeds also comprises alkaloids of pyridine-type, generally

2

trigonelline (0.21-0.35%), choline (0.51%), carpaine and gentianine; flavonoids apigenins,

orientin, quercetin, rutin, free amino acids; such as hydroxyl-isoleucine (0.08%), arginine,

histidine and lysine, some minerals like iron and calcium, glycosides that yield steroids on

hydrolysis i.e. tigogenin, yamogenin and neotigogenin (Mehrafarin et al., 2010).

Globally, fenugreek leaves and seeds powder are being used in food formulations, as a

medicinal herb, as a coffee alternative, insect repellent in grain storages and in fragrance

manufacturing industries (Ahari et al., 2009). In Sudan and Egypt, the seeds are used in making

beverages and in some countries the roasted seeds are used as a coffee substitute, probably

because of the alkaloid trigonelline content, which is a basic constituent of the coffee seeds.

Whilst in Egypt, the seeds of fenugreek are added to bread as a supplement of wheat and maize

(Hidvegi et al., 1984). In India and Pakistan, it is utilized as green leafy vegetable and has rich

nutritional profile i.e. iron, calcium, carotenoids and other water soluble vitamins (Sharma et

al., 1996).

The fenugreek leaves and seeds are widely utilized in cooking and have a maple smell and

flavor which make them a unique spice in foods, beverages and confections. The sprouted

seeds eaten raw in salads along with fresh green leaves or cooked into curries, soups, bread

and many other recipes (Turner and Frey, 2005). The seeds are also being used in number of

dishes, tea, garnishing desserts (Chhibba et al., 2007), pickles & supplements (Srinivasan,

2006; Liu et al., 2012) and specialty products like nans and breads (Sharma et al., 1996).

Indians used methi for preparation of number of products i.e. raita, ladoo, basin methi

particularly in Rajistan (Mathur and Choudhry, 2009). Flour containing fenugreek seeds

powder has good attribute for bakery products and wheat flour have been supplemented with

fenugreek seeds for achieving better results in developing number of food menus (Hooda and

Jood, 2004).

Owing to economic benefits and enhancement in nutritional profile, use of composite flour is

gaining importance day by day in baking science. Many regions of the world use composite

flours in product development. The basic theme behind composite flour is its enriched profile

regarding nutrition and health benefits (Dasappa et al., 2003). Intakes of some legumes and

other similar plants can be boosted up by composite flour technology. The benefit of producing

cereal legume composite foods may be considered as two fold: (1) There is an overall increase

3

in the protein content of composite food compared to when only cereal forms the base, and (2)

combined use of legumes and cereals can provide balanced amino acids, which can be used to

treat malnutrition (Ribotta et al., 2005: Gomez et al., 2008).

In previous investigation the development and evaluation of composite flour for missi

roti/chapatti was carried out to develop nutritious flours from various food commodities like

wheat flour, chickpea, soybean and fenugreek leaves powder. The supplementation of

fenugreek powder increased the nutritional quality of flour particularly in terms of mineral

content (calcium and iron) and fiber. All blended flours were found to have good sensory

quality characteristics of products as control and could be well stored in polyethylene bags or

tin boxes for the period of three months without any deterioration of quality (Kadam et al.,

2012). Fenugreek can be added upto 10% in wheat flour for preparation of biscuits with

appealing characteristics and it also augmented protein quantity & quality, calcium, iron

content and fiber (Hooda and Jood, 2004).

For preparation of baked goods, rheological properties of the dough are of vital importance.

Wheat, the main ingredient in most baked foods, contains viscoelastic gluten, which confers

specific rheological properties to the dough, and this in turn influences the final quality of the

baked product. Therefore, it would be expected that during preparation of composite flours and

dough, partial replacement of wheat with another ingredient resulted with devoid of gluten that

would influence rheological properties of the resultant dough (Abdel‐Kader, 2000). For

preparation of bakery products rheological properties of flour paly important role for end

product quality. So, the supplementation of fenugreek and other legumes in wheat flour

increased the protein content that affects the rheological parameters (Mahmoud, 2013). The

rheological, organoleptic and physiological characteristics of fenugreek-wheat supplemented

mixes have displayed promising outcomes with respect to increase in protein and fat content

(Hooda and Jood, 2005).

Wheat flour supplemented with 8-10% fenugreek seeds powder has been utilized for preparing

baked items like bread, muffins, pizza and cakes with suitable sensory attributes (Srinivasan,

2005). Enclosure of raw, steeped or germinated fenugreek produces good sensory and

physicochemical profile in developed bakery products unless used at 15% level (Hooda and

Jood, 2005). The physical characteristics of composite baked goods influence their sensory

4

quality and acceptability by consumers. Of interest characteristics are crust & crumb color,

loaf volume, loaf height, biscuit width, and spread factor. Generally, with incorporation of a

legume, the composite baked product tends to become darker as a result of Maillard reaction

due to the relatively higher levels of lysine. Composite bread also tend to have lower volume

& height, denser and are more compact in structure due to the reduction in levels of gluten.

With current emphasis on healthy bread with low glycemic index, high protein and increased

dietary fiber, the use of composite flours in baked goods is to be favored. Therefore, given the

inherent nutritional and therapeutic advantages of fenugreek, it could find useful application

in the baking industry. Many studies have reported on the possibility of replacing (although

partially) wheat flour with those obtained from local crops for the purpose of making bread

and other baked products (Mepba et al., 2007; Ade-Omowaye et al., 2008; Olaoye and

Onilude, 2008), however, consumer acceptability of these products is still under study.

Fenugreek, a rich source of phytochemicals is being used as medicinal plant. It contains lysine

& L-tryptophan rich proteins, mucilaginous fiber and other important chemical constituents

like coumarin, saponins, sapogenins, fenugreekine, phytic acid, nicotinic acid, scopoletin &

trigonelline, which are supposed to be for many of its beneficial health effects. Different

components of the seeds have various beneficial functions like associated with treatment or

protection from of chronic diseases such as cardiac complications, hypertension, diabetes,

cancer and other medical complications. Phenolic compounds, for instance, through their

antioxidant activity, are hypothesized to have the ability to reduce the risk of developing certain

cancers by potentially protecting body cells against oxidative damage caused by reactive

oxygen species. On the other hand, many of these phytochemicals together with other seeds

components are regarded as antinutritional factors (Mehrafarin et al., 2010). Therefore, with

regard to these phytochemicals or antinutritional factors, the objective of producing composite

baked goods may either be to retain these factors in order to exert potential health benefits or

to use the processing to eliminate or inactivate them so as to reduce their antinutritional effects.

However, it appears that with regard to composite baked goods, not much research has been

conducted to determine the effect of compositing and processing on levels of these

phytochemicals.

5

During baking, there was a decrease in free phenolics and an increase inbound phenolics,

whereas antioxidant activities remained relatively stable. Hence fenugreek has rich profile of

polyphenolic compounds (Rayyan et al., 2010) possessing numerous favorable activities, like

antioxidant effect (Ravikumar and Anuradha, 1999; Bukhari et al., 2008), cancer precautionary

activity (Raju et al., 2004), anti-diabetic activity and cholesterol lowering effect (Sowmya and

Rajyalakshmi, 1999; Broca et al., 2000; Meghwal and Goswami, 2012). Ample utilization of

fenugreek declines the blood cholesterol level and improves the fat profile of blood in rats

(Kamal-Eldin et al., 2000). This possesses excellent antioxidant capability because free radical

produced in body are easily scavenged by it and hence cancer is prevented (Ozsoy et al., 2009).

These components excellently reduce peroxides in serum and avoid hydrogen peroxide attack

at cell by initiating peroxisomes which also saves breakdown of blood cells (Kaviarasan et al.,

2004).

Diabetes is proliferating attributed to economic and social factors particularly in developing

countries. According to survey by 2030 approx. 300 million people will become victim of this

disease. Presently, Pakistan occupies 6th position which is drastic and can be at 5th until 2030

(Wild et al., 2004). Although drugs are being used for treatment of this disease but they are

more problematic than therapy (Zakir et al., 2008). Fenugreek has both hypoglycemic and

hypocholesterolemic effects. Moreover, fenugreek ingestion found non-toxic effects in both

rat and human studies (Vijayakumar et al., 2005). Consumption of wheat-fenugreek bread

found composite effect for diabetics reduction and it does not disturb the sensory

characteristics but controls insulin sensitivity especially in type II diabetics. Hence its addition

to bread is vital for controlling glycemic index (Losso et al., 2009). Fenugreek also improves

blood lipid profile like LDL and HDL in a way that it lowers LDL and boosts up HDL along

with enhancing the body antioxidant status (Stancu and Sima, 2001). Its beneficial influences

may be owing to sapogenins, which rise biliary cholesterol secretion, in turn reducing serum

cholesterol levels (Stark and Madar, 1993).

From nutritional and health point of view, fenugreek seeds and leaves are good source of

minerals and phytochemicals. Their incorporation in diet through appropriate processing

procedures could bring numerous benefits to the society with special reference to micronutrient

malnutrition prevailing in the country as well as chronic ailments and oxidative stress mediated

6

dysfunctions in response to nutrition transition towards hypercaloric diets that are dominating

over the globe particularly among the young urbanities. Besides, the research is of applied

nature and the provision of fenugreek based designer foods has the ability to enhance the

nutritional and non-nutritive aspects of the already existing leavened bread. Furthermore,

nutrification of exogenous antioxidants in designer foods would not only improve health status

but also enhance endogenous antioxidant capacity such as superoxide dismutase, catalase,

glutathione, etc. These foods not only ensure long-term positive impacts on health status but

also give you mind satisfaction for having a food with holistic nature. All these benefits would

surely boost up your immune defense, not only curtailing free radical mechanism and related

oxidative stress in the body but also giving a positive concept of “eat healthy, live healthy” to

prevent the disease onset at very initial stages by suggesting food modifications and

diversification, rather depending on medication with negative health effects at longer run.

In addition, despite of few researches on its medical value, fenugreek has not been well studied

with respect to nutritional composition, phytochemicals concentration, processing

characteristics and value added products development. Particularly in Pakistan, the historical

and traditional utilization of fenugreek has not been supported and improved by academic

researches regarding its agro-climatic preference, productivity, disease resistance, variety

distribution, storage & handling conditions, chemical & nutritional composition, processing

quality, therapeutic & functional values, product diversification and process technology

developments. Hence, the impact of fenugreek could be substantial for pharmaceutical and

food industry because of its two fold helpful influence on hyperglycemia and

hypercholesterolemia. Therefore, this study was conducted at the expectation that it contributes

its part to pursuit the stated problems and to enhance the awareness and exploitation of the

functional and nutritional values of fenugreek. The objectives of the study were;

1. To evaluate nutritional and biochemical worth of Pakistani fenugreek.

2. To produce functional bakery product with the addition of fenugreek leaves and seeds

powder, separately.

3. To assess nutraceutical potential of composite flour of fenugreek leaves and seeds

powder through model feed trial.

7

Chapter 2

REVIEW OF LITERATURE

Functional foods and legumes have been recognized as health promoters and therapeutics that

playing important role in today busy life (Bouchenak and Lamri-Senhadji, 2013). These

entities are prime source of protein, fibers, mineral like Fe, Zn, Ca, Mg, K & have low Na and

cholesterol. As compared to dietary guidelines, nutrient composition confers ideal properties

to these foods (Trinidad et al., 2010). Mostly found phytochemicals in legume family are like

phytoestrogens, phytohemagglutinins (lectins), enzyme inhibitors, oligosaccharides, phenolic

compounds and saponins that modify metabolism of human after their consumption

(Bouchenak and Lamri-Senhadji, 2013). Legumes have various physiological effects that can

be studied by using any member of this family, thus investigation of legumes that are consumed

less may uncover new sources. Hence, therapeutic effects and disease prevention can be carried

out by using fenugreek. Owing to its medicinal importance, the chemical composition has been

explored which reveal that it is an excellent source of L-tryptophan, mucilaginous fiber, lysine,

nicotinic acid, phytic acid, sapogenins and other constituents (Mullaicharam and GeetaliDeori,

2013). Considering the facts, the current research was intended to explore the nutritional and

nutraceutical role of locally grown fenugreek with special reference against lifestyle related

disorders. The literature regarding different features of the current work has been piled under

the following headings.

2.1 Functional foods and human health

2.2 Plants and their bioactive components

2.3 Fenugreek: an introduction

2.4 Phytochemistry of fenugreek

2.5 Fenugreek and flour rheological properties

2.6 Perspectives of product development

2.7 Fenugreek and antioxidant potential

2.8 Fenugreek and hyperglycemia

2.9 Fenugreek and hypercholesterolemia

2.10 Potential health risk

8

2.1 Functional foods and human health

Food supplies all necessary nutrients to our body and provides energy, supports metabolism

and helps in growth and maintains body’s vigor. Dietary fiber and designer food have been

tailored to achieve health benefits because these reduces the risk of numerous diseases (Kaur

and Das, 2011).

In next decades functional foods will be the major one including fruits, vegetables, fortified

foods and whole grains (Jones and Varady, 2007; Schwager et al., 2008). Dairy products, soya

products, eggs, fats and oils have been developed to maintain health and obtain other benefits

(Watson et al., 2006). Canadian health define that functional foods are similar to that of

traditional foods having defined health benefits (Shahidi, 2009). According to definition,

functional food is part of human diet and is demonstrated to provide health benefits that

decrease the risk of chronic diseases beyond those provided by adequate nutrition. The

functional foods include (i) usual foods with naturally occurring bioactive substances (ii) foods

supplemented with bioactive substances (iii) derived food ingredients introduced in

conventional foods. Functional foods should have novel prospective, rather than a food

product. It should also be mentioned that functional foods are not medicines such as pills but

are consumed as part of normal diet (Grajek et al., 2005).

Functional foods are different than medicinal foods but are used in specific conditions. These

foods are increasing in trend and food sector is rapidly developing new products to promote

health among consumers (Mollet and Lacroix, 2007). Many unique functional foods have been

developed by combining food with herbal medicines. In some countries traditional herbal

products are widely used as medicine in dietary supplements, daily foods and functional foods,

for replenishment and health promotion purposes. The concept is connected with immune

potentiation, the improvement of system circulation, disease prevention and control of aging

(Shi et al., 2010).

Good organoleptic qualities can be conferred to food consumers as they accept functional foods

over the traditional foods in the market (Klahorst, 2006). The development of functional foods

requires a multidimensional approach in order to meet consumer needs under the existing food

regulations. The nutritionists, food chemists, food technologists, biochemists, toxicologists and

clinicians must work together to produce a product that can claim the appropriate health

9

benefits. Epidemiological investigation may provide evidence-based scientific information,

which needs to be studied experimentally for biological responses, using appropriate

biomarkers. Further, these will need to be tested in clinical trials for establishing their health

benefits or risk-reducing effects. Foods such as cereals, pulses, nuts, vegetables, fruits,

beverages and spices have been widely studied. Genetic manipulations to increase the content

of active ingredients may also be useful for enhancing bio-potency (e.g. omega 3 eggs, golden

rice). Technological innovations can improve the product (e.g. soy fermented sauces, pre- and

probiotics) and it is also possible to add bioactive compounds to a traditional food (for instance

guar gum, fenugreek powder or bran). In such situations, it is necessary to keep in mind the

bioavailability of both the active and other physiologically relevant ingredients (Fogliano and

Vitaglione, 2005). Health related properties of functional foods may affect consumer choice &

acceptability and they always think about health benefits while buying such foods (Sanlier and

Seren Karakus, 2010). Promotional and educational technologies are important in creating

consumer awareness and studies on consumption behavior of functional foods indicated that

people awareness about the functionality of such foods increased their demand (Rosemen and

Kurzynske, 2006).

Depending upon consumption pattern and functionality functional foods should be

distinguished from conventional foods (Doyon and Labrecque, 2008). People who are health

conscious will become more inclined towards functional and nutraceutical foods. So, health

concern is a driving force that determines the purchase of such foods (Worsley and Lea, 2008).

In similar way Shahzad and Khattak (2011) performed market analysis of functional foods in

Pakistan and five different points were selected to know about consumer response. The results

of this survey illustrated that male consumers were more attracted towards functional qualities

than females who were fastidious about nutritional values. Wheat based products are important

for introducing nutraceutical components to masses in Pakistan where staple food is wheat

(Jacob and Leelavathi, 2007) and fenugreek i.e. rich in nutritional profile can be added to wheat

flour for conferring health benefits.

2.2 Plants and their bioactive compounds

Initially people consumed food plants for their health benefits but owing to medicinal

performances various components have been extracted to use them as cure for different

10

diseases (Vinatoru, 2001). During Roman and Greek times many drugs were formulated for

treating numerous ailments like Theophrastus, Hippocrates, Dioscorides and Celsus did a lot

of work in this regard (Paulsen, 2010). Romanian pharmacopeia was introduced that indicated

many medicinal herbs and an institute was established in Roman Empire (Vinatoru, 2001).

Bioactive compounds are very important but they were discovered later as a result of scientific

advancement and these compounds are produced as metabolites of secondary stage (Bernhoft,

2010).

The production process of the secondary metabolites depends on stage of life and specific need

of the plant. Like aroma synthesis in floral plants to attract different insects for pollination and

fertilization, production of toxic chemicals or compounds to safe guard against pathogens as

well as hinder the growth of neighboring plants (Dudareva and Pichersky, 2000). Some of

these secondary metabolites consider as role in biological systems that considered as bioactive.

So we can define bioactive compounds in plants as: secondary plant metabolites producing

pharmacological or toxicological effects in human and animals (Bernhoft, 2010). According

to Croteau et al. (2000) the bioactive compounds available in plants have three main categories:

(a) terpenes and terpenoids (b) alkaloids and (c) phenolic compounds. These bioactive

ingredients are diverse in nature like phenolic and carotenoids showing considerable

antioxidative activity (Dahan et al., 2007; Henson et al., 2008; Shahidi, 2009).

General composition of an herb or a spice contains sugar, proteins, essential oil, fiber, vitamins,

minerals and pigments (Viuda-Martos et al., 2007). Biomolecules enclosed in an herb are like

phenolic acids, flavonoids, cumarins and sterols (Uhl, 2000). Phenolic acids are the

predominant one for most of their health augmenting properties, but it depends on type and

quantity of a phenolic component. The exact configuration of phenolic can be determined by

various parameters such as the specific portion of the plant utilized, its vegetative state,

harvesting practices and environmental surroundings etc. (Viuda-Martos et al., 2010). One of

the major renowned compound enclosed in herbs and spices and accountable for most of the

purposeful properties of designer foods are phenolic composites in any of their forms. It could

be in the form of a simple phenols& flavonoids i.e. flavones, flavanols, flavanones and

anthocyanins etc. Many scientific and systematic studies have pointed out the functional

aspects of phenolic compounds and more precisely, flavonoids. Such pharmacological

11

potentials include their antioxidant (Bozin et al., 2008; Lin et al., 2009), antiviral (Tait et al.,

2006), antibacterial (Babajide et al., 2008) and anti-inflammatory (Lin et al., 2008)

capabilities. They also possess promising vigor of cardioprotection (Gamelin et al., 2004),

anticarcinogenic potential (Pergola et al., 2006; Russo et al., 2006) and inhibition of platelet

aggregation (Arct and Pytkowska, 2008). These compounds also cure cardiovascular diseases

and mostly these compounds are present in vegetables, fruits, flaxseeds oil and soy etc. Potent

antioxidants are phenolic and flavonoids which are mostly found in nuts and are affective

against inflammation, oxidation and cancer. Moreover, sulfurous compounds are also present

in onion, fruits and some cherries and their cardio-protective action can be utilized (Kris-

Etherton et al., 2002).

In short, various bioactive compounds emerge to have favorable health effects. Much of the

systematic research requires to be performed before science-based dietary recommendations

to start. Regardless of this, there is an adequate evidence to have a practical perspective. So

various food commodities especially fenugreek has been studied using model feed trial.

2.3 Fenugreek: an introduction

Fenugreek is an annual leguminous herb, have common name as methi in Pakistan is

extensively cultivated in various regions of Asia, Middle East and European countries

including India, Bangladesh, Egypt, Turkey, Morocco, China, Argentina, France and Spain are

major fenugreek producing countries. Fenugreek is also useful leguminous crop for inclusion

into short-duration rotation, for livestock feed, for fixing nitrogen in soil and increasing its

fertility etc. (Amin et al., 2005; Thomas et al., 2011). Fenugreek possess strong spicy flavor &

also well-known for its appetizing appeal and being utilized for dietary preparations (Brar et

al., 2013). Most utilized parts of fenugreek are leaves and seeds i.e. particularly popular in

savory recipes, snacks and also have medicinal importance (Khosla et al., 1995).

Green fenugreek leaves (fresh or dried) are one of the earliest herb (Thomas et al., 2011), being

used as green leafy vegetable in the diets. Fresh leaves considered as good source of nutrients

and for better retention of these nutrients leaves should be stored in refrigerator or oven dried,

blanching time not more than five minutes and cooking should be carried out in pressure cooker

(Yadav et al., 2010). Fenugreek seeds are also useful and most important part of fenugreek

12

plant (Altuntaş et al., 2005). The ground powder of seeds is being used in different products

like vegetable dishes, pickles, spice powder and as condiments (Jani et al., 2009).

Knowing the chemical constituents of a plant is important in order to determine specific health

effects. During a study of observing different varieties of fenugreek genotypes, it was

discovered that it can vary in chemical constituents (fiber, saponins, amino acids, protein and

fatty acid contents), morphology, growth habit and seeds production ability (Acharya et al.,

2006). The valuable properties of fenugreek are credited to its ingredients diversity i.e. amino

acids, nitrogen compounds, steroids, polyphenolic substances and volatile constituents etc.

(Mehrafarin et al., 2010). The fresh leaves of fenugreek comprises 2.37% ash, 85.14%

moisture, 4.32% protein, 0.66% fat, 1.59% crude fiber, 91.05% carbohydrate and minerals

(mg/100g), 618.41 calcium, 111.13 Iron, 3.44 zinc and 1.77 manganese (Mahmoud et al.,

2012).

Fenugreek seeds and leaves

Fenugreek seeds contain 3.0-3.9% ash, 3.2-5.35 moisture, 25.2-30.1% protein, 7.2-9.3% lipids,

20.1-25.3% insoluble fiber, 20.4-30.2% galactomannan and 5.3-7.3% saponins, volatile oils,

free amino acid, mucilaginous fiber and flavonoids (Raju and Bird, 2006). It also comprises

diosgenin, a steroid sapogenin i.e. starting compound for more than 60% of the total steroid

production by the pharmaceutical industry. Other sapogenins available in fenugreek are;

gitogenin, yamogenin, neotigogens & tigogenin and seeds also contain alkaloids like

gentianine, trigonelline and carpaine compounds (Mullaicharam and GeetaliDeori, 2013). The

composition of seeds, cotyledons and husk revealed that endospermic portion contributes to

maximum quantity of saponins (3.92 g/100g) and protein (39.6 g/100g) content. Conversely,

13

husk had rich profile of polyphenols 103.8 mg GAE/g and total dietary fiber 77.4g/100g,

including insoluble fiber 31.87 g/100g and soluble dietary fiber 45.18 g/100g (Naidu et al.,

2011). The fatty acid profile of fenugreek seeds showed mainly linolenic, linoleic, palmitic

and oleic acids. It has 44.64% total carbohydrates with 14.90% of galactose and mannans

moieties that is a soluble fiber (Schryver, 2002).

2.4 Phytochemistry of fenugreek

Phytochemistry has been gaining importance from last few decades and herbal products are

becoming important part of life. Plant based functional foods are gaining popularity across the

world due to an array of evidences for their safer therapeutic applications. The health claims

associated with the consumption of plants are due to their rich phytochemistry (Tapsell et al.,

2006). Phytochemicals like dietary fibers, antioxidant, Ω-3-fatty acids, vitamins, plant sterols

and flavonoids are helpful in maintaining the health of an individual thus reducing the risk of

various maladies (Ramaa et al., 2006).

Fenugreek; renowned from ancient times as folk medicine to manage various diseases and is

one of the oldest medicinal plant. In different parts of Asia, the young tender plants used as

"pot herbs" and dried seeds as spice & herbal medicine. In other parts of the world its leaves

are being used for their cooling properties and seeds for their tonic, carminative and

aphrodisiac effects as well as stimulating effect in digestive process. From the older times of

Greek and Latin history, it is consider as effective against diabetes and hypercholesterolemia.

It also reported as curative agent against ulcer, possess anti-bacterial, anti-fertility, anti-

helminthic and anti-nociceptive effects due to presence of saponin and galactomannan

(Chauhan et al., 2011). Different types of phenolic compounds present in fenugreek seeds that

leads to its beneficial health effects (Rayyan et al., 2010). Phytochemical analysis of fenugreek

seeds depicted that it contains different type of saponins, alkaloids, flavonoids and

carbohydrates (Chauhan et al., 2011), saponins found 4 to 8% and alkaloids 1% approximately

(Ambasta and Ramchandran, 1986).

The seeds to fenugreek separated into endosperm and husk by Naidu et al. (2011) who

determine their proximate composition and found protein content (43.8 g/100) & saponin

(4.63 g/100 g) in endosperm. Total polyphenolic contents found higher (103.8 mg/g GAE) in

14

husk with total dietary fiber (77.1 g/100 g), insoluble dietary fiber (31.9 g/100 g) and soluble

dietary fiber (45.2 g/100g), respectively. Three flavonoids (apigenin-7-O-rutinoside,

kaempferol 3-O-glycoside and naringenin) identified in fenugreek ethyl acetate extract by

using LC-MS/MS apparatus. The concentration of naringenin was found to be highest i.e. 7.23

mg/g of dry extract (Belguith-Hadriche et al., 2010). The study of neutraceutical properties of

trigonelline i.e. major alkaloid in fenugreek was carried out to evaluate its effect against

diseases like central nervous system and diabetes. Trigonelline has hypoglycemic,

hypolipidemic, neuroprotective, antimigraine, memory-improving, sedative, anti-tumor,

antibacterial and antiviral activities and it also has ability to reduce platelet aggregation and

diabetic auditory neuropathy. It play its role by affecting β-cell regeneration, insulin secretion,

altered the activities of glucose metabolism related enzymes, reactive oxygen species, axonal

extension, and neuron excitability (Zhou et al., 2012).

There are number of steroidal sapogenins available in fenugreek seeds with diosgenin found

in the oily embryo. In stem found alkaloids like trigocoumarin, trigonelline, trimethylcoumarin

and nicotinic acid. The leaves of fenugreek contain seven saponins, known as graecunins and

these compounds are glycosides of diosgenin. The seeds contain 0.1% to 0.9% diosgenin and

can be commercially extracted. It also contains saponin fenugrin B. and different coumarin

compounds as well as a number of alkaloids like gentianine, trigonelline and carpaine. Three

minor steroidal sapogenins present in seedsi.e. sarsapogenin, smilagenin & yuccagenin and on

compressing it also yield as much as 8% of a fixed, foul-smelling oil (Snehlata and Payal,

2012). Fenugreek oil has many beneficial roles and different genotypes of fenugreek that are

rich source of saponins and fixed oil are important for pharmaceutical industries. Arivalagan

et al. (2013) analyzed different genotypes of fenugreek for steroidal saponin and fixed oil

content. They found sponin and fixed oil content varied from 0.92 to 1.68g and 3.25 to 6.88g

with corresponding mean value of 1.34 g and 5.19 g/100 g, respectively. The steroidal saponin

and diosgenin can be useful therapeutics in nutrition world.

2.5 Fenugreek and flour rheological properties

Rheological instrumentation and evaluations have turned out to be indispensable means in

analytical laboratories for characterization of constituent materials and final products, to

monitor process conditions and predict product performance and consumer acceptance. The

15

division of science which is related to the pour and twist of substances is known as rheology.

Information about the rheological and mechanical characteristics of different food systems has

significance in the design of flow processes for the control of quality, in forecasting storage

stability and designing texture (Herh et al., 2000).

Rheological characterization of flour dough is compulsory for triumphant manufacture of

bakery items as it has influence on mechanical handling and quality characteristics of the

finished products (Amjid et al., 2013). The appropriateness of wheat flour for baking various

products as breads, cakes, biscuits and chapatties depends primarily on particular rheological

dough properties like water absorption, dough stability, strength, extensibility, elasticity etc.

Dough rheology characterization is an imperative factor in the assessment of bread wheat

quality that indicates dough handling properties and the tendency of the dough to contract

(Pedersen et al., 2004). Different methods including farinograph, amlyograph, mixograph and

extensograph are used for characterization of the rheological properties of flour.

The pragmatic rheological quantifications are recurrently performed using farinogarphic tools

(Razmi-Rad et al., 2007). The results obtained from the graph used as factors in preparation of

product to predict the amount of water to be added for the formulation of dough, to check the

influence of constituents on assimilation characters, to appraise flour mixing requirements and

to test out flour consistency (Meintjes, 2004). The mixograph provides an indication of the

mixing requirements of flour (Manu and Rao, 2008). Soft wheat flours are evaluated through

physical dough testing instruments on the grounds of supposition that the rheological

characteristics of soft wheat flour are contrary to firm wheat flours (Meintjes, 2004). The

mixographic results from experiment are convenient in finding out the gluten potency and

distinctiveness of flour for bead making therefore wheat breeders use results from mixograph

in order to evaluate early generation lines for gluten strength of dough (Pedersen et al., 2004).

Water absorption of flour determined by the mixograph frequently acts as bake amalgamation

in bread baking analysis. The technological experiments have demonstrated remarkable

changes in quality considerations such as the mixograph peak time and mixograph tolerance

index. The quality values of these two parameters ranged from awfully underprivileged to

superfluous strapping, for bread making, the loaf volume and the bread value point also

displayed an ample array of worth (Grausgruber et al., 2000).

16

The supplementation of different vegetables protein in wheat flour increased dough water

absorption due to high water absorbing capacity of these proteins and their ability to compete

with other constituents of dough for water absorption. According to different other researchers,

the high farinograph water absorption values is due to high water absorption capacity of protein

available in flour (El-Soukkary, 2001; Doxastakis et al., 2002). The amount of water addition

during bakery product development is very important for distribution materials in dough, their

hydration and development of gluten protein network (Amjid et al., 2013).

Replacement of whole wheat flour with dehydrated leaves (dill-DDL, fenugreek leaves DFL)

at 0, 5, 7.5 and 10% increased the water absorption (68.5 to 70.2%), dough development time

(3.5 to 5.9 min) and mixing tolerance index values (78 to 98 BU) (Sudha et al., 2013). When

increased the supplementation level of fenugreek in wheat flour, dough water absorption

increased from 65.10% (control) to 68.5% in the composite flour with 5% fenugreek powder.

When supplementation level increased to 10%, water absorption of dough decreased by

66.37% but remained higher than control and further decreased up to 61.2% when

supplementation level reached 20% (Hooda and Jood, 2003). Different levels of fenugreek

seeds flour increased most of the rheological properties. The water absorption observed 52%

in control that increased up to 62-70% when supplementation of fenugreek powder increased

in wheat flour (Sulieman et al., 2000). The rheological properties of supplemented flour with

different grain blends (chick pea, soya bean, barley and fenugreek seeds powder) was studied.

The increasing percentage of multigrain resulted in increased water absorption & decreased

dough stability, extensibility, extensograph resistance to extension, amylograph peak viscosity

and overall quality score of NIP from 38 to 53 for the maximum score of 60 (Indrani et al.,

2011).

2.6 Perspectives of product development

The emergence of new value added foods with the addition of various functional ingredients

are gaining popularity nowadays due to increasing consumer awareness about healthy diets.

As a result competition among such food producers increasing day by day to develop new

products that can meet consumer demand (Annunziata and Vecchio, 2013). Nowadays,

supplemented flours are being prepared by blending root, tuber and legume flours with cereals

at a predetermined ratio. These are then used to prepare various food products, including

17

fermented flat breads, biscuits, and tortillas. An important motivation for the production of

supplemented based foods is to improve nutritional quality. Cereal legume composite foods

serve as a good example of this. Legumes are protein rich relative to cereals and are also

generally better sources of required amino acids, particularly lysine. In comparison, cereals,

although lysine-deficient, are relatively better sources of sulfur-containing amino acids such

as methionine. The benefit of producing cereal legume composite foods may be considered as

twofold: (1) There is an overall increase in the protein content of the composite food compared

to when only the cereal forms the base, and (2) legumes contributed lysine and methionine by

cereals result in balanced amino acids. The combination of flours affects nutritional quality as

well as functional, sensory, and phytochemical characteristics of the final products. Various

factors play a role, including preprocessing steps followed in the preparation of the flours, the

ratio of cereal to legume flours used, as well as the procedures used during the preparation of

the end product. In this part of study discusses the potential effects of compositing flours and

subsequent baking on quality parameters such as nutritional, phytochemicals and sensory

properties.

Fenugreek is known since older times, having nourishing value and being used in different

food products throughout the world (Meghwal and Goswami, 2012). Armenians use the

fenugreek seeds with garlic paste and chili pepper in a spice called chemen, Yemenite Jews

use them in a seasoning called zhug, and in the United States, seeds are used in bean soups,

chutneys, spice blends, icing and meat seasoning (Uhl, 2000). In Greece, the seeds are boiled

and eaten with honey, and in Africa they are soaked and used as legume. The dry seeds are

also roasted and used as a coffee substitute (Pruthi, 2001). It also used as seasoning in foods

and the vegetative parts are used as a green leafy vegetable (Youssef et al., 2009). Nowadays,

it has been extensively used in the food industry because of its high protein content, dietary

fiber stabilizing and emulsifying properties (Meghwal and Goswami, 2012). Enclosure of

soaked, germinated and raw flour of fenugreek in wheat flour amplified the amount of protein

(10.46, 10.37 and 10.09%), lysine (2.16, 2.21 and 2.26 g/100 g protein), total calcium (58.2,

56.1, 57.8 mg/100 g), total Fe (7.40, 7.26 and 7.36 mg/100 g) and dietary fiber (12.68, 11.28

and 10.22%) with 10% substitution (Ibrahium and Hegazy, 2009). The addition of fenugreek

powder in wheat flour improved protein (16.30%) and fat (2.9%) content of blended flours but

18

decreased gluten content. The overall acceptability scores of biscuits, bread, macroni and

noodles were found acceptable by addition of powder upto 10, 15, and 20% levels in wheat

flour (Hooda and Jood, 2003).

In this context flour added with 8-10% dietary fiber from fenugreek has been utilized in baked

products such as pizza, biscuits, muffins, bread and cakes (Roberts, 2011). It has appreciable

amount of protein, fiber, fat & minerals and highly nutritional biscuits can be made by its

supplementation in wheat flour (Hussein et al., 2011). The incorporation of fenugreek powder

in wheat flour increased the protein, lysine, dietary fiber, total calcium and total iron content.

The biscuits prepared from this flour with acceptable sensory attributes with shelf life upto one

month in polypropylene bags (Hooda and Jood, 2005).

The parathas prepared with optimum levels of leaves, i.e. either 25% of normal dill/fenugreek

leaves or 7.5% of dehydrated dill/fenugreek leaves were evaluated for proximate composition

compounds. The ash content of parathas incorporated with either of the leaves was higher than

the control paratha. The increase in ash content of parathas mainly due to high mineral content

found in fenugreek leaves powder (Sudha et al., 2013). The incorporation of fenugreek powder

in wheat flour increased the water absorption of wheat flour without effecting the loaf volume

up to the 3% level of substitution, and gave a satisfactory loaf volume up to the 7.5% level.

Bread prepared with addition of fenugreek powder contained more protein, dietary fiber and

lysine than the control. Bread containing up to 3% fenugreek was considered as good as the

control in terms of acceptability and inclusion of fenugreek flour up to 7.5% gave a product of

acceptable quality as judged by sensory evaluation studies (Chauhan and Sharma, 2000).

The blending of fenugreek powder in wheat flour from 5 to 20% level increased protein, fat,

lysine, fiber and minerals content of bread. The bread prepared with 15% supplementation

exhibited acceptable baking and sensory attributes with protein (13.06%), dietary fiber

(14.73%), total lysine (2.89 g/100g protein) and appreciably amount of mineral. The wheat

flour bread contained iron (7.78 mg/100g), calcium (58.70 mg/100g) and zinc (3.51 mg/100

g), while in supplemented bread mineral content found increased i.e. iron (7.97 mg/100g),

calcium (59.76 mg/100g) and Zinc (3.84 gm/100g). However, at this incorporation level

observed acceptable loaf volume with mean values 470.50 ml, loaf weight 153.75 g and other

sensory quality attributes like crumb color score 5.66, appearance 6.17, texture 6.25, flavor

19

5.60, taste 5.00 and overall acceptability 5.73 (Hooda and Jood, 2005). The replacement of

fenugreek gum with wheat flour @ 0, 5 and 10% (w/w) was carried out to measure the bread

production features of the flour. Bread comprising fenugreek gum @ 5 and 10% presented

texture (468 g) and volumes (905 cc) similar to control treatment of bread (Roberts et al.,

2012).

Appreciable variations in chemical components existed among the different fenugreek-wheat

supplemented flour samples when compared with wheat flour. The moisture, ash, protein and

crude fiber content increased in the supplemented flours compared to the control wheat flour.

The protein content increased from 15-30% and mean weight & volume of the bread also

increased with increased level of supplementation with fenugreek flour (Sulieman et al., 2000).

The bread formulations were prepared with fenugreek-wheat blended flour to record their

nutritional, consumer acceptability and blood glucose lowering property. The bread containing

fenugreek had higher protein (21.57%) & fat content (2.02%) and there was no momentous

difference for color, texture, firmness and flavor between control and supplemented bread

(Losso et al., 2009).

2.7 Fenugreek and antioxidant potential

Antioxidants has play important role in body such as protective role against free radicals that

naturally produced or associated with diseased like diabetes mellitus, acute respiratory distress

syndrome, cardiovascular diseases, inflammatory diseases and cancer (Guerrero et al., 2007;

Toppo et al., 2009). Fruits and vegetables are rice source of various nutrients like dietary fiber

and natural product content such as phenolic compounds (antioxidant agent) that correlated

with their beneficial health effects (Al-Musayeib et al., 2011). Oxidative damage occurred in

body at cellular or subcellular level may be the major cause of various diseased like

carcinogenesis, coronary vascular disease, diabetes, inflammatory disease and aging. Reactive

oxygen species are very harmful to body cells at both membrane and genetic levels. These

increased lipid peroxidation in cellular membranes, generating lipid peroxides that cause

extensive damage to membranes and membrane mediated chromosomal damage. Fenugreek

has rich source of nutrients and it can reduce the increasing lipid peroxidation and alterations

in the content of circulating antioxidant molecules, such as β-carotene, glutathione and α-

tocopherol, in alloxan-diabetic rats (Ravikumar and Anuradha, 1999).

20

Fenugreek is categorized as high in phenolic content among vegetables group using ethanol as

solvent and observed very high antioxidant activity (Kaur and Kapoor, 2002). The total

polyphenol content of extracts obtained from extraction of dried fenugreek leaves with

methanol, ethanol, and isopropanol solvents was 48, 44, 28 mg/100 g Gallic acid equivalents,

respectively (Naidu et al., 2012). The total phenolic content of fenugreek observed in methanol

and ethanol extracts were reported as 575±0.002 and 685±0.002 (mg GAE/100g), respectively

(Bukhari et al., 2008). In an earlier study conducted by Naidu et al. (2011) who has observed

total phenolic content 85.8 mg/g in fenugreek seeds extract that could be responsible for its

antioxidant activity. Fenugreek extract had polyphenols (9.47±0.10 mg GAE/g dry seeds) that

plays a role as antioxidant principle. Caffeic acid (164.550 μg), gallic acid (170.335μg), ellagic

acid (184.879 μg) and quercetin (215.814 μg)/g on dry weight fenugreek seeds basis were

identified by HPLC analysis (Dua et al., 2013). The estimated phenolic content found 22±1.5

μg/mg GAE in extracted fenugreek sample (Kumar et al., 2013). Different fenugreek samples

were observed for total phenolic and found 139.2 mg GAE/100g followed by 130.0 mg

GAE/100g and 127.8 mg GAE/100g (Ali et al., 2015). The study conducted by Premanath et

al. (2011) observed 4.9 mg/g polyphenols in ethanol extract of fenugreek leaves powder. In

another research work mean phenolic content found to be 52.8 mg/g GAE in fenugreek leaves

supplemented chicken patties (Devatkal et al., 2012). The maximum content of 48 mg/g GA

equivalent of total phenolic in fenugreek leaves extract determined by Naidu et al. (2011) and

also reported that antioxidant activity is may be related with polyphenolic content present in

fenugreek.

Different flavonoids like tricin, vitexin, quercetin, naringenin and tricin-7-O-b-

Dglucopyranoside identified in fenugreek (Shang et al., 1998). The ethanol extract of

fenugreek leaves powder found flavonoids 0.47 mg/g reported by Premanath et al. (2011).

Significant antioxidant activity was observed in fenugreek seeds extract due to presence of

different flavonoids (Dixit et al., 2005). Methanol, ethanol, hexane, ethyl acetate and acetone

were used for extraction and observed flavonoid content in the range of 607±3.6, 653±4.3,

208±4.2, 251±3.3 and 416±2.7 QE µg/g of fenugreek (Bukhari et al., 2008). Flavonoid content

observed in fenugreek by Kumar et al. (2013) who found total flavonoids 16.6±1.2 (μg

QE/mg). Ishtiaque et al. (2013) observed flavonoid content 5.80 mg QE/g in fenugreek seeds

and supplementation in wheat flour resulted in increased flavonoid content. The study of

21

antioxidant properties of fenugreek seeds exhibited that significant antioxidant activity in seeds

may be due to presence of polyphenols and flavonoids (Dixit et al., 2005).

The extracts of fenugreek leaves exhibited antioxidant activity in DPPH assay ranged from 41

to 47%. Extracts from dried fenugreek leaves depicted free radical scavenging activity ranging

from 18 to 56% and the activity increased with increasing concentration of various solvents

used, methanol extract of fenugreek leaves exhibited 42% activity followed by ethanol and

isopropanol extracts. Further, extract of fenugreek leaves obtained with aqueous methanol,

exhibited higher free radical scavenging activity (56%). So antioxidant activities of the extract

mainly dependent on the composition of phenolic compounds available in fenugreek leaves

(Naidu et al., 2012).

The determination of fenugreek seedsantioxidant activity was carried out to estimate its ability

to scavenge DPPH and observed result i.e.89.91 ±3.09 (% inhibition per DPPH) (Belguith-

Hadriche et al., 2010). Hydro-alcoholic extract of fenugreek seeds were subjected for in vitro

antioxidant activity by different methods viz, 1-diphenylpicryl-hydrazyl radical (DPPH),

hydroxyl, and ABTS radical cation assay. The extract exhibited potent DPPH and ABTS

radical scavenging activity with IC50 values of 350μg/ml, and 962.5μg/ml, respectively. The

seeds of fenugreek showed significant total antioxidant capacity with IC50 value of 192 μg/ml

and hydroxyl radical with IC50 value of 587.5μg/ml, respectively. Based on the results it can

be concluded that hydro-alcoholic extract of fenugreek may have potential antioxidant effects

against several oxidants (Priya et al., 2011). The study carried out by Saeed et al. (2013) who

observed Kasuri fenugreek (methanol + water extract) have DPPH radical scavenging activity

(19.01%), followed by Kasuri fenugreek water extract (16.49%). The DPPH radical

scavenging activity Lahori fenugreek methanol + water extract and water extract were 18.36

and 14.59%, respectively at same concentration i.e. 1 mg/ml. The determination of DPPH

assay of fenugreek leaves and seeds depicted that free radical scavenging activity of seeds was

better than that of leaves as seen by a lower E.C.50 value obtained for seeds as compared to a

higher E.C.50 value for the leaves. However, the seeds were found to have a better antioxidant

potential than the leaves in vivo (Jha and Srivastava, 2012).

The antioxidant activity of fenugreek leaves and seeds may be due to presence of β-carotene.

In a study, antioxidant activity of fenugreek different fractions were measured by bleaching of

22

β-carotene. Different fenugreek fractions like fenugreek husk, endosperm and seeds extracts

exhibited 73%, 6% and 22% activity when compared with the corresponding values of 95%

for BHA (Naidu et al., 2011). The ethanol extract of fenugreek inhibited β‐carotene oxidation

suggesting that the antioxidant activity could be related to the high levels of phenolic

compounds. The antioxidant activity of 70% ethanol extract of fenugreek by β‐carotene

bleaching method was carried out. Fenugreek extract was employed in the range of 25-400

mg/mL and by adding more quantity of the extract, absorbance was decreased and the reason

behind was inhibition of bleaching of the color β‐carotene. The extract depicted inhibition at

concentration of 0.202 mg/mL (Subhashini et al., 2011).

Higher content of β‐carotene (22.5mg/100g) observed in low humidity air dried fenugreek

leaves compared to radiofrequency dryer dried sample (6.2 mg/100g; 76.2 mg/100g) and hot

air dried fenugreek leaves (6.0 mg/100g; 148.1 mg/100g) (Naidu et al., 2012). The

supplementation of fenugreek seeds in the diet of rats prevented enzymatic leakage, alter lipid

peroxidation and enhanced antioxidant potential (Thirunavukkarasu et al., 2003). So both

fenugreek leaves and seeds can be a good source of bioactive compounds like the addition of

fenugreek leaves powder in rats diet @ of 1g/kg of body weight decreased lipid peroxidation

and improved significant system (Annida and Prince, 2004). No bioactive components were

detected in paratha prepared with wheat flour. However, β-carotene content found slightly

higher when 25% of normal fenugreek leaves were incorporated in wheat flour (Sudha et al.,

2013).

A study was carried out by Khole et al. (2014) to separate and characterize bioactive molecules

from germinated fenugreek seeds. Ethyl acetate, water and n-butanol were used for extraction

purpose and the results for FRAP assay were obtained as 1.27±0.02, 131.027±11.05 and

0.14±0.007 (mM AEAC), respectively. In a meta-analysis, fenugreek seeds were evaluated for

FRAP assay. For this purpose, fenugreek seeds powder extracted systematically, at ambient

temperature selecting solvents of varying polarity such as methanol, dichloromethane, hexane

and water. The results of the study gave a range for various solvents in the range of

0.135±0.055 to 77.352±0.627 TE mg/g (Kenny et al., 2013).

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2.8 Fenugreek and Hyperglycemia

Despite the heterogeneity, hyperglycemia is the most common metabolic disorder in diabetic

patients (Steppan et al., 2001). At the molecular level, loss of pancreatic β-cells plays

significant role in the development of insulin deficiency and progression of diabetes mellitus

(Lapshina et al., 2006; Lee et al., 2004). Drug therapies often lose their effectiveness and

consensus is being sought to explore the new natural compounds for the treatment of diabetes

and its complications (Wolff, 1993; Lapshina et al., 2006). Diet diversification is important in

the module of diet-based therapies and slight modification in the daily diet possibly prevents

the onset of diabetes mellitus (Butt et al., 2007). For this purpose, in different parts of the world

utilization of different natural compounds is gaining wide popularity. These natural

compounds act as micronutrients and in the existing era, extensive research work has being

carried out to explicate their therapeutic mechanisms (van Dam et al., 2002; Hu et al., 2007;

Steyn et al., 2008).

The use of natural products for treatment of various diseases has been proved since thousands

year back and these are playing primary role in health care of poor and developing countries

of the world (King et al., 1998). Fenugreek is being commonly used in India and other parts

of the world as a condiment, rich source of dietary fiber that can play important role against

diabetes. The seeds contain 44.4% dietary fiber (13.3% soluble and 32% insoluble), with gum

composed of mannose and galactose that are related to reduce cholesterolemia and glycaemia.

Its hypoglycemic result has been particularly accepted in animals and humans (Roberts, 2011).

The earlier research work of Xue et al.(2007) and Mowl et al.(2009) proved that there was

decrease in glucose level when diet supplemented with fenugreek leaves. The anti-diabetic also

proved in rats when group of animals provided with fenugreek seeds rich diet (Annida and

Prince, 2004). The enrichment of diet with fenugreek seeds fraction to diabetic and non-

diabetic rats decrease the glucose level. The decreased absorption of glucose, intestinal

disaccharidase activity and increased gastrointestinal motility observed with the addition of

seeds fraction. The antidiabetic action of fenugreek is facilitated by slowing down digestion &

absorption of carbohydrate and improvement of peripheral insulin action (Hannan et al., 2007).

The findings of Madar and Shomer (1990) & Sauvaire et al. (1998) exhibited that anti-

hyperglycemic effects of fenugreek are attributed, at least partly, by reducing intestinal glucose

24

absorption. The findings also indicated that the seeds fraction increased sucrose content of the

stomach in non-diabetic and type 2 diabetic rats at 30-60 min supports the thought that

fenugreek also reduces gastric emptying (Nahar et al., 2000).

Literature suggested that the application of fenugreek can control blood sugar level in both

type 1 and type 2 diabetics. It possess free amino acid, 4-hydroxyisoleucine occurred only in

fenugreek, which acquire hypoglycemic properties through increased secretion of insulin

under diabetic situations and enhanced sensitivity to insulin (Haeri et al., 2012). Through in

vitro experiment it was found that the amino acid 4-hydroxyisoleucine available in fenugreek

seeds increased glucose-induced insulin release in experimental rats and human pancreatic islet

cells, it was experienced that 4-hydroxyisoleucine extracted from fenugreek seeds has insulin

tropic activity (Yadav et al., 2010; Akbari et al., 2012).

The amino acid was found to attack pancreatic beta cells while the levels of glucagon and

somatostatin were not affected. In human experimentation, through fenugreek addition, there

was reduction in the area under the plasma glucose curve and increment in the number of

insulin receptors (Smith, 2003). In a study it was concluded that inclusion of fenugreek powder

in diet showed reduction in sugar level in urine and blood with associated betterment in glucose

tolerance and diabetic complications in type II diabetic patients (Mitra and Bhattacharya,

2006). In Pakistan a study has been conducted to evaluate hypoglycemic activity of fenugreek

and concluded that it’s hypoglycemic activity by retarding glucose uptake and increasing its

usage (Zia et al., 2001).

The mean fasting blood glucose in diabetic untreated rats group (control positive) after

induction of 21 days of diabetes was 280±8.33 mg/dl while in the normal healthy group the

value was 76±2.59 mg/dl. When comparison of values between positive control group and the

group which was treated with fenugreek extract was carried out, it was observed that treated

group exhibited lower mean fasting blood glucose i.e. 141.83±9.04 mg/dl. In similar way mean

values of serum insulin observed in control positive group was 4.17±0.17 μU/ml and in normal

healthy group it was 10.53±0.66 μU/ml. The comparison of positive control group and

fenugreek extract treated group illustrated significantly higher mean serum insulin 7.27±0.6

μU/ml in treated group (El-Soud et al., 2007).

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The addition of fenugreek @ 5% in diet of experimental diabetic rats significantly reduced

fasting blood glucose by 33% (360 to 244.0 mg/dl) as compared to diabetic control exhibiting

its hypoglycemic influence during diabetes (Shetty and Salimath, 2009). A bread incorporating

fenugreek was tested for its consumer taste acceptability and effect on blood glucose level. The

trial was carried out on eight diet controlled diabetic patients that were served two slices (56

g) added with 5% fenugreek. The bread supplemented with fenugreek was indistinguishable

from the whole wheat bread and its maintained fenugreek’s functional property of reducing

insulin resistance (Losso et al., 2009). Previously, Shakib and Gabrial (2010) evaluated

postprandial blood glucose level in healthy subjects and determine their glycemic index values

after feeding with bread supplemented with barely, fenugreek or ginger. The all supplemented

bread reduced postprandial blood glucose responses than did the control (wheat flour bread),

suggesting that soluble dietary fiber available in these supplemented products had an impact

on glucose tolerance with glycemic indices ranging from 38 (fenugreek) to 52 (Barley wheat

bread). Fenugreek supplemented bread having crude fiber 6.25 g/100g so it can be replaced

with white wheat flour bread since it provides easily digestible and healthy carbohydrate based

diet that can help to maintain postprandial blood glucose level within normal range.

According to another research it was found that fenugreek antidiabetic effect is due to presence

of high percentage of soluble fiber, galactomannan, which acts to decline the gastric emptying

time and in this way cause delay in the glucose absorption from the small intestine (Akbari et

al., 2012). As suggested through animal trails that fenugreek is effective against diabetes by

aiding insulin secretion, because typically these patients are unable to produce insulin or less

than normal required. Through several animal experiments, the mechanism was deduced and

it showed that galactomannan gum blocks the intestinal absorption of glucose. Water soluble

fibers enhance the viscosity of food inside the intestine and inhibit glucose absorption (Smith,

2003).

The scientific study of Khalil(2004) was focused on clarifying the role of fenugreek seeds

aqueous extract in its therapeutic dose on beta cells number, blood glucose and plasma insulin

levels in diabetic rats. For this purpose rats were divided into groups, control, diabetic and

diabetic rats fed with fenugreek seeds aqueous extract (0.1 mg/kg body weight). During

induction of diabetes, insulin level was reduced (12.5±0.5 to 7.5±0.8 μU/dL). By feeding

26

fenugreek extract based diet, inslin level was elevated to higher value in diabetic group (10±1

μU/dL). Another study was conducted to evaluate the comparative effect of various fenugreek

extracts and antidiabetic drug. The findings of the study suggested that fenugreek extract

produce compatible hypoglycemic activity to the antidiabetic drug. Oral administration of

fenugreek extract at various dose level (0.1, 0.25, 0.5 g/kg body weight) for 14 days notably

reduced serum glucose, triglyceride, total cholesterol, AST, uric acid, creatinine and ALT

levels, whereas it enhanced serum insulin in diabetic rats (Eidi et al., 2007).

A study was carried out by Gad et al.(2006) who assessed the effect of administration of

fenugreek extracts for 21 days on the blood glucose and serum insulin of diabetic rats. During

period of diabetic induction, serum insulin level was reduced from 1.106±0.269 to 0.216±0.005

μg/L. when fenugreek based diet was fed to the diabetic models, serum insulin level rose to

0.326±0.077 μg/L. In similar way Rajarajeswari et al. (2012) investigated the antidiabetic

activity of fenugreek in alloxan induced diabetic rats. The effect observed was compared with

a known antidiabetic agent glibenclamide. In active pancreatic damage induced by alloxan

(i.p.75mg/kg b.w.), fenugreek seeds aqueous extract (1300 mg/kg b.w.) and ethanol extract

(1g/kg b.w) administration significantly reduced the elevated blood glucose and increased

serum insulin level.

The treatment of normal and alloxan-diabetic rabbits for one month was carried out with

isolated trigonelline from fenugreek, standard trigonelline, and ethanol fenugreek extract that

considerably lower blood glucose. At the end of experiment blood glucose was reduced

27.05%, 27.53% & 32.61% and isolated trigonelline depicted a momentous hypoglycemic

effect in normal and diabetic animals but more effective in diabetic group. It reduces 22.34%

blood glucose in non-diabetic and 27.53% in the diabetic animals (Al-Khateeb et al., 2012).

When supplementation of experimental rats was carried out with fenugreek leaves powder in

streptozotocin (STZ) induced diabetic rats with moderate hyperglycemia that resulted in

decreased blood glucose and increased plasma insulin level. One of the mechanistic approach

is that STZ incompletely destructs pancreatic β-cells even though the rats became permanently

diabetic. The β–cells of islets of Langerhans get stimulated and glucose level increased when

experimental rat’s diet supplemented with fenugreek leaves powder that increased plasma

insulin level. The process of glycolysis increased and gluconeogenesis decreased leads to

27

control glucose level when supplementation of leaves increased in the diet. This is possible as

it controls the activities of the key enzymes of glycolysis (Devi et al., 2012). The increase in

insulin level resulted in glucose decreased that exhibited reverse correlation. Blood glucose

level significantly increased and decrease in insulin level observed in positive control group

when compared to the rest groups. The addition of fenugreek extract in rats diet depicted

significant effect against blood glucose and insulin level. The level of glucose decreased from

258.41mg/dl (positive control) to 167.57 mg/dl and insulin level improved 5.81 IU/ml to 7.89

IU/ml (El-Dakak et al., 2013).

2.9 Fenugreek and hypercholesterolemia

Hypercholesterolemia is very serious medical and social problem, greatly correlated with

cardiovascular diseases, hence leading to higher mortality rate (Moosa et al., 2006). Through

various research works it has suggested that cholesterol reducing effect of fenugreek correlated

to the presence of flavonoids like naringenin. The addition of germinated fenugreek seeds

powder was carried out in diet resulted in reduction of LDL and total cholesterol level of

hypercholesterolemic adults. The hypocholesterolemic effect of fenugreek occurred through

various mechanisms like increased in excretion of fecal bile acids and neutral sterols with

reduction in level of cholesterol stores in liver. Its addition in diet stimulates the production of

bile in liver and the process of cholesterol conversion into bile salts or high fiber available

potentially reduces absorptive mucosal surface glucose diffusion rate, cholesterol and drug

absorption. The soluble fiber also increases the viscosity of the digest & unstirred layer

thickness in small intestine or inhibit the uptake of cholesterol and bile acids. For fermentation

in large bowel by microorganisms, soluble fiber is consider as an excellent source. The

resultant product of fermentation are volatile fatty acids that enter into the blood stream and

appear to decrease the synthesis of hepatic cholesterol. The seeds of the fenugreek contains

trigonelline and diosgenin in the form glycosides that can combine with cholesterol to make

complexes in the intestine and reduced its absorption. Another reason for reducing glucose

effect could be based on amino acid pattern present in fenugreek proteins (Sowmya and

Rajyalakshmi, 1999).

A study was carried out by Al‐Habori and Raman (1998) reported that the crude saponins

extract was the most effective in reducing hypercholesterolemia. The lipid profile is extracted

28

in bile and then reabsorbed from the intestine. Therefore, it can be suggested that saponins may

bind bile salts which are necessary for absorption of these substances or bind directly in the

intestine and prevent reabsorption (Madar and Shomer, 1990). This may be due to the fact that

saponins form insoluble complex with lipid profile (Rao and Gurfinkel, 2000). Saponins and

crude fiber extracted from fenugreek seeds significantly resulted in reduction of cholesterol

and triglyceride (El-Hussary, 1993). In a study of Narender et al. (2006)who reported that

amino acid, 4-hydroxy isoleucine isolated from fenugreek seeds significantly decreased total

cholesterol (22%), plasma triglyceride (33%) and free fatty acids (14%), accompanied by an

increase in High Density Lipoprotein (HDL) by 39% in the dyslipidemic hamster model. There

was a significant decreased in total lipid, triglyceride and LDL concentration on long term

application of fenugreek seeds in all the supplemented groups. However, long term

administration of fenugreek seeds resulted with significant increase on HDL. Low density

lipoprotein and high density lipoprotein worked as antagonist, decreased in LDL and increased

HDL which represents protection against atherosclerosis and coronary heart disease (Oraby et

al., 2008).

Hypocholestrolemic potential of fenugreek is thought to be attributed due to certain steroidal

saponins such as, diosgenin, tigogenin, yamogenin and neotigogenin etc. These

phytochemicals perform their action by inhibition of cholesterol synthesis and absorption.

Clinical studies on hypocholetrolemic potential of fenugreek revealed a statistically

remarkable reduction in human serum total cholesterol, triglycerides and LDL cholesterol

(Smith, 2003). Another hypothesis attributed the hypocholesterolemic potential of fenugreek

to the fiber-rich gum portion of the seeds. Gum slow down the rate of hepatic synthesis of

cholesterol and also influences intestine walls to secrete various enzymes and hormones which

ultimately affect the biosynthesis of cholesterol in liver (Yadav et al., 2010).

The higher content of high density lipoproteins (HDL) are very important in body due to

relation with reduced risk of heart diseases (Young et al., 2004). The high level of HDL

expedites cholesterol transport from serum to liver, where it catabolized and finally excreted

from the body. The ratio of LDL and HDL is consider as important risk factor of atherosclerosis

and this ratio can be decreased with supplementation of fenugreek seeds powder and ethyl

acetate extract in diet. The low level of serum HDL and high level of LDL are correlated with

29

increased atherosclerosis risk (Korhonen et al., 1996). The increasing level of HDL and

decreasing LDL level in experimental rats depicted the antiatherogenic property of the ethyl

acetate extract of fenugreek. The possible mechanism of hypocholesterolemic property may

be due to dietary cholesterol absorption inhibition in intestine or its production by the liver

(Bursill and Roach, 2006) or stimulation of the biliary secretion of cholesterol and excretion

through feces (Krzeminski et al., 2003).

The supplementation of ethanolic extract of fenugreek seeds was carried out with two separate

doses @ 30 or 50g in diets of hypercholesterolemic rats. The seeds contains saponins have

hypocholesterolemic property that interact with bile salts in the digestive tract (Stark and

Madar, 1993). The addition of fenugreek extract in diet of diabetic rats lowered significantly

serum triglycerides and cholesterol level. The findings also supported by the research work of

other scientist who have reported that fenugreek added diet feeding to diabetic rats resulted in

reducing total cholesterol (Khosla et al., 1995). A study was carried out with healthy

individuals fed with 2.5g fenugreek twice in day for three months resulted in no change in

blood lipids and blood sugar level (fasting and postprandial). However, when same dose was

given to coronary artery disease patients and non-insulin dependent patients for same period,

it significantly reduced blood lipid (total cholesterol and triglycerides) without affecting the

HDL-cholesterol (Bordia et al., 1997).

Similarly, Prasanna (2000) proved hypolipidemic role of fenugreek by given it dose of 25 and

50g twice in a day before meal to hypercholesterolemic patients. The hypolipidemic role of

fenugreek soluble dietary fiber fraction could be the result of retardation of carbohydrate and

fat absorption due to the presence of bioactive fiber in the agent (Hannan et al., 2003). The

study of Pipelzadeh et al. (2003) confirmed that serum total cholesterol decreased by feeding

rats with fenugreek diets. Treatment with 100 mg/kg fenugreek seeds powder reduced total

cholesterol 162.9 to 120 mg/dl while dose of 500 mg/kg also significantly reduced the total

cholesterol level and other serum lipids. The results concerned LDL-cholesterol and

triglycerides decreased by feeding rats with fenugreek diets and increased the serum HDL

cholesterol as compared with non-treated control group. In another study found

supplementation of fenugreek leaves in diet decreased triglycerides in diabetic rats (Annida

and Prince, 2004).

30

The fenugreek dose of 25g orally two times in a day for period of three and six weeks to

hypercholesterolemic individuals produces significant reduction of serum total cholesterol

(Moosa et al., 2006). In an experiment mean serum total cholesterol values in normal healthy

group was found 98.5±2.1 mg/dl that observed significantly increased in positive control group

(140.33±3.2 mg/dl). When fenugreek extract was added in diet, it significantly reduced the

serum total cholesterol to 107.83±2.2 mg/dl (El-Soud et al., 2007). Fenugreek extract was

added in diet of rats and observed lower blood glucose, glycated haemoglobin, total

cholesterol, triglycerides and higher HDL compared with diabetic rats (Xue et al., 2007).

The results of Elmnan et al.(2012) showed that fenugreek seeds had a significant decrease on

plasma total, triglyceride and LDL. However, a numerical increase was observed in HDL

(35.17 to 36.56) with increasing levels of fenugreek seeds. Long term administration fenugreek

seeds had a significant decrease on plasma total lipid (377.46 to 313.44), triglyceride (118.34

to 76.72), LDL (82.33 to 62.33) and a significant increase was obtained on HDL in the last

week. Studies have reported that diabetic state of an individual resulted by reduced insulin

secretion that could be responsible for serum high triglycerides level, as the insulin stimulated

the synthesis of adipose tissue by agency of lipoprotein lipase. The quantity and quality of

protein in the diets also have a direct influence on the levels of cholesterol like plant protein

found to decrease cholesterol level. The fenugreek is rich source of plant protein (26%), so

hypocholesterolemic potential can also be attributed to this component. Another

phytochemical, diosgenin decreased the elevated level of cholesterol, LDL and increased HDL

of serum in cholesterol fed rats. Diosgenin also inhibited cholesterol absorption and suppressed

its uptake in serum and liver as well as its accumulation in the liver (Akbari et al., 2012).

Ethyl acetate extract of fenugreek seeds was investigated by Belguith-Hadriche et al. (2013)

who observed reduced level of total cholesterol, low density lipoprotein (LDL), triglycerides

and increased level of high density lipoprotein (HDL) when compared with those rats that have

cholesterol-rich diet. In similar study El-Dakak et al. (2013) observed decreased serum

glucose, total cholesterol, triacylglycerol, urea, uric acid, creatinine, AST, ALT and ALP

levels, whereas increased serum insulin level with administration of aqueous extract of

Lepidium sativum L., lupin and fenugreek or their mixture in streptozotocin-induced diabetic

rats. Serum total cholesterol level increased in STZ-diabetic rats and daily administration of

31

fenugreek for 30 days succeeded to reduce cholesterol 48.22% when compared with diabetic

control rats (Marzouk et al., 2013).

2.10 Potential health risk

Food safety is an important issue and is crucial nowadays because people are very much

conscious for their health. Therefore, one should be aware of the fact that how much, in what

way, at what time, and in which condition, fenugreek should be used. Although fenugreek has

traditionally been considered safe and well tolerated, some side effects have been associated

with its use. Caution in using fenugreek is warranted in patients known to be allergic to it

because of possible cross reactivity (Patil et al., 1997). Fenugreek possesses estrogen content

that could stimulate the uterus hence avoided during pregnancy. Fenugreek is an insulin

substitute but it may interfere with insulin therapy taken by diabetics. Furthermore, there are

some chances that high mucilage presence in fenugreek could coat the stomach and reduce the

absorption of set medications thereby fenugreek consumption need to reduce in some cases

(Mullaicharam and GeetaliDeori, 2013).

Further studies are still required to explicit bystander effects of fenugreek on the overall health

of humans. However, based on the available data, it appears that fenugreek has wide variety of

health-promoting activities. It is important to increase awareness of the public, researchers,

doctors and nutritionists as to the unique properties of this vegetable that are most beneficial

for maintaining good health and preventing disease.

32

Chapter 3

MATERIAL AND METHODS

3.1 Procurement of raw material

The present research work was carried out at National Institute of Food Science and

Technology (NIFSAT), University of Agriculture, Faisalabad, Pakistan. For the purpose,

wheat variety Lasani 2011 and fenugreek (Qasoori methi) fresh leaves and seeds were procured

from Ayub Agricultural Research Institute (AARI), Faisalabad.

3.2 Preparation of raw material

The wheat was cleaned by sieving followed by tempering. Afterwards, milling was carried out

by using Quadrumate Senior mill to obtain straight grade flour. Likewise, fenugreek leaves

and seeds were cleaned by screening and washing, followed by drying and grinding separately

to get fine powder. The resultant powder were stored in air tight containers prior to analysis

(Kadam et al., 2012).

3.3 Analysis of raw material

3.3.1. Proximate analysis

Proximate composition of wheat flour, fenugreek leaves and seeds powder was estimated by

determining moisture, ash, crude protein, crude fat, crude fiber and nitrogen free extract (NFE)

according to the prescribed methods given in AACC (2000).

3.3.1.1 Moisture Content

Moisture content of wheat flour, fenugreek leaves and seeds were determined by drying the

samples in Air Forced Draft Oven (Memmert Germany) at 105±5oC till constant weight by

following the procedure described in AACC (2000) Method No. 14-15A.

3.3.1.2 Ash Content

The ash content was estimated by using Muffle Furnace (MF-1/02, PCSIR, Pakistan). After

charring, incineration of the samples was carried out at 550oC till greyish white residues

(AACC, 2000; Method No. 08-01).

33

3.3.1.3 Crude protein

Crude protein content were determined using Kjeltech Apparatus (Model: D-40599, Behr

Labor Technik, Gmbh-Germany) following the procedure given in AACC (2000) Method No.

46-30.

3.3.1.4 Crude fat

Soxhlet system (Model: H-2 1045 Extraction Unit, Hoganas, Sweden) was used to estimate

the crude fat content in raw material using hexane as solvent by following the guidelines given

in AACC (2000) Method No. 30-25.

3.3.1.5 Crude fiber

The raw material were extracted for fat and hence the fat free samples were utilized for

determination of crude fiber. For the purpose, the samples were treated with 1.25% H2SO4 for

20 minutes followed by washing and digestion with 1.25% NaOH solution using Fiber tech

apparatus (Labconco Cooperation, Kansas, USA) as per directions illustrated in Method 32-10

(AACC, 2000).

3.3.1.6 Nitrogen free extract (NFE)

NFE was calculated following the expression described below:

NFE (%) = 100 – (moisture + ash + crude protein + crude fat + crude fiber)

3.3.2 Mineral profile

Mineral like Na and K of wheat, fenugreek leaves and seeds powders were determined by using

Flame Photometer (Sherwood Scientific Ltd., Cambridge, Model 410) whilst Fe, Ca, Cu, Zn

and Mn were analyzed using Atomic Absorption Spectrophotometer (Varian AA240,

Australia) following the procedures described in AACC (2000).

3.4 Rheological properties

The rheological properties of wheat flour including water absorption, dough development

time, dough stability, mixing tolerance & softening of dough were explored using Farinograph-

34

E (Brabender D-4100; Germany) while mixing properties of dough like mixing time and peak

height percentage were assessed using Mixograph (National NSI-33R) following the methods

(54-21 & 22-10) described in AACC (2000).

3.5 Development of composite flour

Composite flours containing various levels of wheat flour, fenugreek leaves and seeds powder

were developed as per treatment plan illustrated in Table 3.1.

Table 3.1 Treatments used in the study plan

Treatment

Composite flour

Wheat (%) Fenugreek leaves

powder (%)

Fenugreek seeds

powder (%)

T0 100 - -

T1 95 5 -

T2 90 10 -

T3 85 15 -

T4 95 - 5

T5 90 - 10

T6 85 - 15

3.6 Analyses of composite flour

3.6.1. Proximate Analysis

The proximate composition (moisture, ash, crude protein, crude fat, crude fiber and nitrogen

free extract) of composite flour was ascertained according to the prescribed methods of AACC

(2000).

3.6.2 Mineral profile

Amongst the minerals in composite flour, Na and K contents were determined using Flame

Photometer whilst Fe, Ca, Cu, Zn and Mn were analyzed using Atomic Absorption

Spectrophotometer (Varian AA240, Australia) as per guidelines described by AAAC (2000).

35

3.7 Rheological properties

The prepared composite flours were subjected to evaluation of rheological properties like water

absorption, dough development time, dough stability, mixing tolerance index, softening of

dough and mixing properties of composite flours following the respective procedures detailed

in AACC (2000).

3.8. Polyphenols extraction

The polyphenols from fenugreek leaves &seeds and composite flour were extracted by

following the protocol of Rusak et al.(2008), using ethanol at constant temperature of 60°C for

48 hours. Resultant extracts were filtered using vacuum filtration assembly and solvents

were recovered by Rotary Evaporator (EYELA, N-N series, Japan) at a temperature of

40°C. The yield of respective sample was calculated and resultant extracts were stored at

4°C until used, afterwards the extracts were subjected to different assays as described below;

3.9 Antioxidant assay

3.9.1 Total phenolic content

Total phenolics of resultant extracts were estimated spectrophotometricaly by following Folin-

Ciocalteau method (Singleton et al., 1999). The extract (I mL) was mixed with 0.5 mL of Folin-

Ciocalteau reagent along with 7.5 mL of distilled water and allowed to stand for 20 min.

Following resting period, 1.5 mL of sodium bicarbonate solution (7%) was added to the

mixture. Then stay for 20 min by placing sample tubes in water bath (40oC), take 0.5 ml from

this solution and add 2 mL of distilled water. After that absorbance was measured at 755 nm

using a UV/vis Spectrophotometer (CECIL, CE7200) against control. Total polyphenols were

calculated and expressed as Gallic acid equivalent (mg Gallic acid/100 g).

3.9.2 Total flavonoids

Total flavonoids were determined by following the procedure of Zhishen et al.(1999). Take

two mL of aqueous extract in a 10 mL volumetric flask, then add 5 mL of distilled water

followed by 0.3 mL of 5% NaNO2 and after 5 min 0.6 mL of 10% AlCl3 was added. After

another 5 min stay 2 mL of 1 M NaOH was added and volume was made upto 10ml with

distilled water. Take one ml from this solution after mixing this solution, add 9 ml distilled

36

water and measured absorbance by using spectrophotometer at 510 nm. Total flavonoids were

expressed as Catechin equivalents per dry matter.

3.10 Antioxidant activity

3.10.1 Free radical scavenging activity (DPPH assay)

The DPPH radical scavenging activity of all extracts was measured according to the procedure

described by Oktay et al.(2003). Purposely, 1 mL of DPPH was added to each extract (4 mL)

and incubated at room temperature for 30 min. The absorbance was noted at 520 nm using

Spectrophotometer. Percent inhibition was calculated using the following formula;

Reduction of absorbance =AB − AA

AB× 100

AB = absorbance of blank sample (t = 0 min)

AA = absorbance of tested extract solution (t = 30 min)

3.10.2 ß-carotene and linoleic acid assay

Total antioxidant activity of the extracts was monitored by using assay based on coupled

oxidation of β-carotene and linoleic acid (Adegoke et al., 1998). Briefly, 2 mg of -carotene

was dissolved in 20 mL chloroform along with 40 mg linoleic acid and 400 mg Tween 20.

After removing chloroform, 3 mL of the prepared emulsion was added in 0.10 mL sample

and placed in a water bath for 120 min. Oxidation of ß-carotene was determined

spectrophotometricaly at 470 nm.

3.10.3 Ferric reducing antioxidant power (FRAP assay)

The ferric reducing ability of all the extracts was measured according to the procedures of

Benzie and Strain(1996). Purposely, extract (0.5 mL) was mixed with phosphate buffer (1.25

mL, 0.2 M, pH 6.6) and potassium ferricyanide (1.25 mL, 1 %). After incubation, 10 % TCA

(1.25 mL) along with 0.1 % ferric chloride were added in the mixture and then left at room

temperature for 10 min. Sample absorbance was measured with spectrophotometer at 700 nm.

3.11 Preparation of bread

For the purpose, respective flour, sugar, oil, yeast and water were used to make the bread

(AACC, 2000; Method No. 10-09). After preparation, the bread were cooled and packed in air

tight polythene packages before further analyses.

37

3.12 Physicochemical characterization of bread

Proximate composition (moisture, ash, crude fat, crude fiber, crude protein and nitrogen free

extract) of bread were determined (AACC, 2000). Color and texture of the product were

ascertained by using colorimeter and texture analyzer, respectively, adopting the procedures

described by Amir et al.(2013). The product color, L* (lightness), a* (–a greenness; +a

redness), and b* (–b blueness; +b yellowness) were measured using CIE-Lab Color Meter

(CIELAB Color Tech-PCM, USA). The data thus obtained was used to calculate Chroma (C*)

and hue angle according to method illustrated by Rodriguez-Garcia et al. (2012).

For texture analysis, the triple beam snap (three-point break) technique of Texture Analyzer

(TA-HDi, Stable Microsystems, UK) was used for measuring the texture of prepared bread. A

crosshead speed of 10 mm/min with a load cell of 50 kg was used. The force required to break

individual product was noted and average value was calculated according to protocol described

by Lara et al. (2011) and Rodriguez‐García et al.(2012).

3.13 Antioxidant assay of bread

Antioxidant assays like total phenolic content & flavonoids were performed for the developed

product by using procedures described by Singleton et al. (1999)and Zhishen et al. (1999),

respectively.

3.14 Antioxidant activity of bread

Free radical scavenging ability (Oktay et al., 2003), ß-carotene and linoleic acid assay

(Adegoke et al., 1998) and FRAP assay (Benzie and Strain, 1996) were also determined for

prepared bread.

3.15 Sensory evaluation of bread

The assessment of bread was carried out for sensory characteristics i.e. volume, color of crust,

symmetry of form, evenness of bake, character of crust and internal characteristics like grain,

color of crumb, aroma, taste and texture by panel of six judges (Lawless and Heymann, 1999).

The bread was examined for sensory evaluation using nine point hedonic scale system ranged

Chroma (C*) = [a*2 + b*2]1/2

Hue angle (h) = tan-1 (b*/a*)

38

from extremely liking to disliking (9 = like extremely; 1 = dislike extremely) following the

guidelines of Meilgaard et al. (2007). The detail is given in Appendix-I. Panelists were

provided separate booths equipped with white fluorescent light. To remove any biased from

the experiment, samples were presented them randomly and requested to assign scores for

selected characteristics.

3.16 Selection of best treatments

On the basis of antioxidant assay, rheological characteristics and overall acceptability of

prepared bread, two best treatments one each from leaves and seeds powder were selected for

efficacy study.

3.17 Efficacy trial

The selected best treatments were subjected to in vivo trials using rodent experimental model.

For the purpose, Sprague Dawley rats procured from National Institute of Health (NIH)

Islamabad were housed in the Animal Room of National Institute of Food Science and

Technology, University of Agriculture, Faisalabad. The rats were acclimatized by feeding on

basal diet for a period of one week. The environmental conditions were maintained throughout

the trial like temperature (23±2ºC) and relative humidity (55±5%) along with 12 hour light-

dark period. For efficacy trials, the study was carried out in three categories involving normal,

hyperglycemic and hypercholesterolemic. At the start of the study, some randomly selected

rats from each group were sacrificed to get baseline values. In each study, three groups were

formed having five rats in each group. During eight week trial, control and selected composite

flour diets (Appendix II) were given to all three groups to evaluate their restorative potential.

Feed & water intakes as well as body weight were measured throughout the experimental

period. At the end of the study, the overnight fasted rats were sacrificed & blood and sera were

collected to trail selected parameters including serum lipid profile, glucose and insulin levels.

For serum collection, blood samples were subjected to centrifugation using centrifuge machine

@ 4000 rpm for 6 min. The respective sera samples were examined for various biochemical

assays by using Microlab 300, Merck, Germany.

39

Table 3.2 Diet plan used for efficacy trial

Normal rats Hyperglycemic rats Hypercholesterolemic rats

D0 D1 D2 D0 D1 D2 D0 D1 D2

D0 = Control diet

D1 = Fenugreek seeds powder supplemented diet

D2 = Fenugreek leaves powder supplemented diet

Category I: Normal rats

In this study, rats were divided in to three homogeneous groups fed on normal diet along with

provision of respective functional diets.

Following similar approach, two other studies were also conducted to find out the impact of

fenugreek diets against respective disorders i.e. high sucrose and high cholesterol to check its

significance.

Category II: Hyperglycemic rats

In category II, high sucrose diet containing 40% sucrose was given to induce hyperglycemia

in rats and determined its effect on serum glucose and insulin levels. Besides, effect of

functional diets on the induced syndrome was measured in each group at the termination of the

trial.

Category III: Hypercholesterolemic rats

In category III, high cholesterol diet i.e. 1.5 % of cholesterol was given to the normal rats to

induce hypercholesterolemia. Periodic examination of rats was carried out to assess the

induction of hypercholesterolemia. The prepared functional diets were provided to the rats

concurrently to synchronize their effect on the respective group.

3.17.1 Feed & water intakes

Feed intake was measured daily by subtracting the spilled diet from the total diet during the

whole trial (Wolf and Weisbrode, 2003). The water intake of each group was also recorded

daily by monitoring the differences in the graduated bottles. Change in body weight of

experimental groups was measured weekly throughout the study period to monitor any

suppressing effect of functional diets.

40

3.17.2 Serum lipid profile analysis

Serum lipid profile of rats including cholesterol, high density lipoproteins (HDL), low density

lipoproteins (LDL) and triglycerides were determined. Serum cholesterol level was measured

by using CHOD-PAP method following the protocol of Rifai et al. (1999). High density

lipoprotein (HDL) was assessed by HDL Cholesterol Precipitant method (Alshatwi et al.,

2010). Whilst, triglycerides in sera samples were measured by liquid triglycerides (GPO-PAP)

method as outlined by Allain et al. (1974).

3.17.3 Serum glucose and insulin levels

From each study, collected sera were evaluated for glucose concentration by GOD-PAP

method as described by Ribes et al. (1986), whereas insulin level was assessed by following

the method of Temple et al.(1992).

3.17.4 Liver function tests

Liver function tests including aspartate aminotransferase (AST), alanine aminotransferase

(ALT) and alkaline phosphatase (ALP) were assessed. Levels of AST and ALT were measured

by the dinitrophenylhydrazene (DNPH) method using Sigma Kits 59-50 and 58-50,

respectively and ALP by Alkaline Phosphates–DGKC method (Basuny et al., 2009).

3.17.5 Renal function tests

The serum samples were also analyzed for urea by GLDH-method and creatinine by Jaffe-

method using commercial kits (Jacobs et al., 1996; Thomas et al., 1998) to assess the renal

functionality of different groups.

3.17.6 Hematological analysis

Red blood cell (RBC) and white blood cell (WBC) indices and Platelets count were assessed

by following the respective methods described by Fischbach (1996).

3.18 Statistical analysis

The collected data from the current study was subjected to statistical analysis by applying

completely randomized design (CRD) using Cohort version 6.1 (Costat-2003) according to

guidelines of Steel et al. (1997).

41

Chapter 4

RESULTS AND DISCUSSION

The prime objective of this study was to investigate and endorse the nutritional as well as

nutraceutical profile of fenugreek leaves & seeds powder by making wheat-fenugreek

supplemented flours; afterwards evaluated their suitability for production of bread. The

nutraceutical potential was examined against hyperglycemia and hypercholesterolemia as it

was hypothesized that these are effective against such disorders. Accordingly, fenugreek leaves

and seeds powder were tested in experimental modeling rats and applying statistical design.

After conducting comprehensive analytical experiments, the results of this detailed study were

segregated in three main sections to allow systematic representation of data, namely;

nutritional profile, antioxidant properties & product development and efficacy studies.

4.1. Proximate analysis of raw material

Proximate composition is a key factor for assessing the quality of raw material. Wheat flour

was evaluated for proximate composition by analyzing various parameters; including

13.5±0.25, 0.41±0.06, 10.5±0.13, 1.13±0.04 and 0.31±0.04% of moisture, ash, crude protein,

crude fat, crude fiber, respectively, while nitrogen free extract (NFE) was found to be

74.15±2.42%. Fenugreek leaves powder (dry weight basis) was subjected to different quality

assessment and revealed moisture, ash, crude protein, crude fat, crude fiber and NFE as

9.38±0.47, 6.37±0.32, 4.30±0.22, 0.88±0.07, 1.98±0.10 and 77.09±3.85%, respectively.

Proximate composition of the fenugreek seeds has been illustrated as moisture (10.65±0.53%),

ash (4.14±0.21%), crude protein (22.86±1.14%), crude fat (6.98±0.35%), crude fiber

(7.90±0.43%) and NFE (47.47±2.37%) (Table 4.1).

Findings of present research regarding proximate analysis are in harmony with the results of

studies conducted earlier, however some variations observed in the present study may due to

environmental factors like growing conditions, soil and genetic makeup. Khan et al. (2009)

used different spring wheat varieties grown in Pakistan for proximate analysis and found

12.92±0.22 to 13.42±0.88% moisture, 10.23±0.54 to 11.60±0.53% crude protein and ash

content 0.41±0.04 to 0.55±0.03%. Moisture content in different wheat varieties flour ranged

from 11.78 to 12.09%, crude protein 11.71 to 12.05%, crude fat 1.29 to 1.40%, crude fiber

42

0.41 to 0.44%, whereas ash content in the range of 0.48 to 0.57% (Mueen-ud-Din et al., 2009).

In an earlier study it was revealed that hard wheat flour depicted 10.84% protein, 1.68% fat,

0.5% fiber and 1.84% ash content whereas soft wheat flour had 9.61% protein, 1.15% fat, 0.4%

fiber and 1.06 % ash content (Kasaye and Jha, 2015).

The study of Mahmoud et al. (2012) narrated that fenugreek leaves contained (on dry weight

basis) 14.14% dry matter, 4.32% protein, 0.66% fat, 91.06% carbohydrate and 387.46 kcal/g

energy. The dehydrated fenugreek leaves had moisture content 9.38% i.e. evident since

dehydration removes all the moisture content present in samples (Sudha et al., 2013) and an

important contribution for the texture of the leaves is the moisture content (George, 2003). The

ash content of methi leaves and kasuri methi were found to be 13.36% and 9.84%, respectively,

and the respective moisture content were 6.07% and 5.22% which have an influence on the

shelf stability of the samples. Likewise, fat content were estimated to be 5.82% and 4.12% for

methi leaves and kasuri methi. Crude fiber was obtained in appreciable amounts in both

samples and the respective values were found to be 2.6% and 2.4%, respectively (Pasricha and

Gupta, 2014). The seeds of fenugreek have moisture content i.e. (8.30%), ash (2.92%), crude

protein (24.60%), crude fat (6.11%), crude fiber (7.72%) and nitrogen free extract (50.35%)

(Mahfouz et al., 2012). In another study moisture content were found to be 7.49% and other

components such as crude protein (25.40%), crude fat (7.90%), ash (3.38%) and crude fiber

(50.00%) were observed in fenugreek seeds whereas soluble and insoluble dietary fiber were

found to be 21.7 and 26.8%, respectively (Meghwal and Goswami, 2012). The proximate

analysis showed that fenugreek seeds contains 6.57% moisture, 4.03% total ash, 26.78%

proteins, 6.35% lipids, 6.75% crude fiber and 49.52% carbohydrates (Aljawofi, 2014).

Similarly, Saeed et al. (2013) determined the chemical analysis of fenugreek seeds and found

7.50 ± 0.7% moisture, 3.10 ± 0.3% ash, 6.47 ± 0.5% fat, 7.60± 0.7% fiber and 22.50 ± 1.5%

protein, while in another study by Rasool et al. (2013) who observed 12% moisture content,

26% protein, 8% crude fat and 3% ash content in fenugreek seeds. Findings of the present

study are also correlated with the research investigation of Kasaye and Jha (2015) who found

that fenugreek flour had crude protein (29.89%), crude fat (7.91%) crude fiber (11.34%),

carbohydrates (51.55%) and ash content (2.94%) on dry weight basis.

43

4.2 Mineral content of raw material

Mineral profile of current study has been depicted in Table 4.2. It was found that wheat flour

contains sodium, potassium, iron, calcium, copper, zinc and manganese as 3.12±0.15,

130.00±5.50, 3.50±0.12, 22.00±0.96, 0.13±0.02, 0.19±0.03 and 0.70±0.08 mg/100g,

respectively. The mineral composition of fenugreek leaves powder indicated 58.71±2.93

sodium, 481.91±24.09 potassium, 6.72±0.33 iron, 589.00±29.45 calcium, 0.15±0.02 copper,

0.90±0.09 zinc and 0.75±0.08 mg/100g manganese. The mineral profile of fenugreek seeds

powder illustrated that it contains potassium in the highest quantity of 296.41±14.82 mg/100g

followed by calcium (160.00±8.02 mg/100g), sodium (23.69±2.18 mg/100g), iron (19.60±1.98

mg/100g), zinc (2.10±0.19 mg/100g), manganese (1.20±0.06 mg/100g) and copper (0.75±0.08

mg/100g).

In the present research, mineral content remained in the ranges described in literature. Mineral

content of different cereal and legumes are influenced by genetic as well as non-genetic factors

like soil, climatic conditions and use of fertilizers etc. (Wiesler et al., 2003). The determination

of mineral content of different fenugreek genotypes grown in various locations was carried out

and observed variations among different varieties (Pathak and Agrawal, 2014). Different

Pakistani spring wheat varieties contain 2.23±0.06 to 2.90±0.13 iron, 22.94±0.61 to

26.15±1.71 calcium, 0.15±0.01 to 0.25±0.00 copper and zinc 0.90±0.06 to 2.10±0.10 mg/100g

(Khan et al., 2009). The fresh leaves of fenugreek comprise of 618.41 calcium, 111.13 iron,

3.44 zinc and 1.77 mg/100g manganese (Mahmoud et al., 2012). In another research potassium

was found in highest quantity of 517.23±13.01 followed by phosphorus 293.4±6.1, calcium

229.6±9.7, magnesium 157.01±5.26, sodium 73.5±1.2, iron 21.8±0.52, zinc 1.9±0.7,

manganese 1.3±0.2 and copper 0.8±0.1 mg/100g in fenugreek seeds (Tanveer, 2014).

Likewise, mineral profile of fenugreek seeds depicted that it contains K, Mg, Ca, Zn, Mn, Cu

and Fe in the range of 603.0±15.0, 42.0±5.0, 75.0±9.0, 2.4±0.2, 0.9±0.1, 0.9±0.1 and 25.8±1.2

mg/100g (Al-Jasass and Al-Jasser, 2012) i.e. in harmony with current study findings. In

another study fenugreek seeds has been observed as good source of mineral i.e. iron 11.51,

calcium 168.88, zinc 4.43 and magnesium 153.10 mg/100g (Kasaye and Jha, 2015).

44

Table 4.1 Proximate analysis (%) of raw material

Parameter Wheat flour Fenugreek leaves

powder

Fenugreek seeds

powder

Moisture 13.5±0.25 9.38±0.47 10.65±0.53

Ash 0.41±0.05 6.37±0.32 4.14±0.21

Crude Protein 10.5±0.13 4.30±0.22 22.86±1.14

Crude Fat 1.13±0.04 0.88±0.07 6.98±0.35

Crude Fiber 0.31±0.04 1.98±0.10 7.90±0.43

NFE 74.15±2.42 77.09±3.85 47.47±2.37

Values expressed are means ± standard deviation

Table 4.2 Mineral content (mg/100g) of raw material

Mineral Wheat flour Fenugreek leaves

powder

Fenugreek seeds

powder

Sodium 3.12±0.15 58.71±4.93 23.69±2.18

Potassium 130.00±5.50 481.91±24.09 296.41±14.82

Iron 3.50±0.12 6.72±0.33 19.60±1.98

Calcium 22.00±0.96 589.00±29.45 160.00±8.02

Copper 0.13±0.02 0.15±0.02 0.75±0.08

Zinc 0.19±0.03 0.90±0.09 2.10±0.19

Manganese 0.70±0.08 0.75±0.08 1.20±0.06


45

4.3 Proximate analysis of supplemented flour

The determination of food composition is a fundamental to theoretical and applied

investigations in food science, and is often the basis of establishing the nutritional value

and overall acceptance from consumer stand point. Different supplemented (treatments)

flour prepared by the addition of fenugreek leaves and seeds powder were analyzed for

different chemical characteristics and the results are described here-in-after.

4.3.1 Moisture content

Mean squares for moisture content showed that different treatments significantly affected with

the addition of fenugreek leaves powder (Table 4.3). The mean moisture content (Table 4.4)

in different treatments ranged from 12.88±0.20 to 13.51±0.17%. The highest value was

observed in T0 (13.51±0.17%) followed by T1 (13.29±0.15%), T2 (13.09±0.09%) and T3

(12.88±0.20%). The present outcome shows that the moisture content among different

treatments have declining trend with increasing the supplementation of fenugreek leaves

powder.

The statistical results regarding moisture content in different treatments (Table 4.5) exhibited

that it was significantly affected with supplementation of fenugreek seeds powder. Mean

values for moisture content has been given in Table 4.6. The maximum moisture content was

observed in T0 (13.51±0.17%) followed by T4 (13.33±0.06%), T5 (13.21±0.07%) and T6

(13.08±0.12%). The findings of current study revealed that the moisture content of wheat flour

decreased with the addition of fenugreek seeds powder.

Moisture content of the products play a significant role in storage stability and shelf life. At

low moisture content the quality of the baked products is less deteriorated due to low

respiration and microbial activities. For better keeping quality of flour, moisture content is key

factor like flour with high moisture content deteriorates rapidly as compared to flour obtained

from dry and sound grains (Pomeranz, 1988). The result of present study revealed that moisture

content decreased with increasing the supplementation level because drying process reduced

the moisture content of fenugreek leaves and seeds powder. Variation in the moisture content

also may be attributed due to differences in climatic conditions persisted during harvest and

storage (Randhawa et al., 2002). Findings of current study is comparable with findings of

Tharise et al. (2014) who found significant differences in the moisture content of different

46

composite flour and significantly lower when compared with control (wheat flour). The

moisture content of composite flour in current study varied from 12.88 to 13.51% compared

to reported values of 11 to 15% depending upon hygroscopic nature and storage conditions of

flour. The level of moisture content observed in composite flour was within the recommended

moisture levels of 14% for safe storage. To avoid chemical changes and microbial activities in

food products moisture content should be below 14% during storage (Shahzadi et al., 2005).

4.3.2 Ash content

Mean squares (Table 4.3) for ash content in different treatments indicated that ash content

varied significantly with supplementation of fenugreek leaves powder. Mean values (Table

4.4) for ash content in different treatments ranged from 0.41±0.05 to 1.30±0.15%. The values

of ash content in different treatments reported as T0; 0.41±0.05%, T1; 0.71±0.09%, T2;

1.01±0.12% and T3; 1.30±0.15%. Maximum ash content were found in T3 while minimum in

T0 that depicted ash content increased with increasing the supplementation of fenugreek leaves

powder.

Mean squares (Table 4.5) for ash content revealed highly significant difference among

treatments with supplementation of fenugreek seeds powder. Mean values of ash content falls

in the range of 0.41±0.03 to 0.97±0.10% (Table 4.6). The highest value for ash was exhibited

in T6 (0.97±0.10%) followed by T5 (0.78±0.06%), T4 (0.60±0.05%) and T0 (0.41±0.03%). The

findings of research work illustrated that the ash content in wheat flour tend to be increase with

increasing the supplementation level of fenugreek seeds powder.

The ash content of flour represents the inorganic residues left after the organic matter present

in flour brunt and it is an important parameter in the milling industry (Wei, 2002). The higher

ash content of different treatments reported in the current study attributed due to higher ash

content of fenugreek leaves and seeds powder as compared to wheat flour. The ash content

increased from 0.85 to 2.5% when wheat flour make composite with chickpea flour observed

by Hefnawy et al. (2012). Similar findings were observed by Tharise et al. (2014) who reported

ash content of composite flours ranged from 0.75 to 1.12% i.e. significantly higher than found

in wheat flour.

47

Table 4.3 Mean squares for proximate analysis of different treatments supplemented

with fenugreek leaves powder

SOV df Moisture Ash Crude

Protein

Crude

Fat

Crude

Fiber NFE

Treatment 3 0.21705** 0.44305** 0.48847** 0.00071NS 0.03450** 0.11561*

Error 8 0.00512 0.00077 0.00333 0.00048 0.00033 0.01848

Total 11

**= Highly significant

* = Significant

NS= Non Significant

Table 4.4 Proximate analysis (%) of different treatments supplemented with

fenugreek leaves powder

Trt. Moisture Ash Crude Protein Crude Fat Crude Fiber NFE

T0 13.51±0.17a 0.41±0.05d 10.50±0.06a 1.13±0.03a 0.31±0.02d 74.15±0.19d

T1 13.29±0.15ab 0.71±0.09c 10.18±0.05b 1.12±0.02ab 0.39±0.02c 74.31±0.10c

T2 13.09±0.09ab 1.01±0.12b 9.85±0.04c 1.11±0.07ab 0.48±0.01b 74.48±0.12b

T3 12.88±0.20b 1.30±0.15a 9.57±0.06d 1.09±0.08b 0.56±0.02a 74.60±0.14a


T0 Wheat Flour (Control)

T1 5% fenugreek leaves powder supplemented wheat flour



48

Table 4.5 Mean squares for proximate analysis of different treatments supplemented

with fenugreek seeds powder


Protein

Crude

Fat

Crude

Fiber NFE

Treatment 3 0.09611** 0.17558** 1.88099** 0.43514** 0.72207** 8.97403**

Error 8 0.00418 0.00046 0.00380 0.00045 0.00108 0.00787

Total 11


* = Significant

Table 4.6 Proximate analysis (%) of different treatments supplemented with

fenugreek seeds powder

Trt. Moisture Ash Crude

Protein Crude Fat Crude Fiber NFE

T0 13.51±0.10a 0.41±0.03c 10.50±0.16b 1.13±0.03b 0.31±0.02d 74.15±0.19a

T4 13.33±0.06a 0.60±0.05d 11.13±0.15ab 1.42±0.08ab 0.68±0.04c 72.82±0.09ab

T5 13.21±0.07b 0.78±0.06b 11.74±0.18ab 1.72±0.10ab 1.07±0.11b 71.48±0.10bc

T6 13.08±0.12b 0.97±0.10a 12.35±0.26a 2.01±0.15a 1.45±0.09a 70.15±0.10c



T4 5% fenugreek seeds powder supplemented wheat flour



49

4.3.3 Crude protein

The analysis of variance (Table 4.3) for crude protein with supplementation of fenugreek

leaves powder shows highly significant difference among treatments. Mean values for crude

protein content (Table 4.4) for different treatments are; T0 (10.50±0.06%), T1 (10.18±0.05%),

T2 (9.85±0.04%) and T3 (9.57±0.06), while maximum amount of crude protein observed in T0

(control). From the present results it is confirmed that crude protein content in wheat flour was

declined by increasing the level of fenugreek leaves powder.

Mean squares (Table 4.5) for crude protein content in different treatments depicted that protein

content significantly differ with supplementation of fenugreek seeds powder. Mean values

(Table 4.6) regarding protein content in different treatments varied from 10.50±0.16 to

12.35±0.26%. The highest protein content were observed in T6 (12.35±0.26%) followed by T5

(11.74±0.18%), T4 (11.13±0.15%) and T0 (10.50±0.16%). The results of present research

described that crude protein content in wheat flour has increasing trend with increasing the

supplementation level of fenugreek seeds powder.

Protein content of flour is an important quality parameter and quality of protein is

dependent on the genetic makeup of the crop that relates to dough strength, elasticity and

extensibility (Khan et al., 2009). Protein deficiency is a major health problem of developing

countries like Pakistan because the diet is based on mainly cereals. The supplementation of

other sources like protein rich vegetables in regular diet has being getting popular to combat

this deficiency problem. The supplementation of high protein source like fenugreek with wheat

flour increased protein quality by making balanced amino acid profile (Ibrahium and Hegazy,

2009). In current study protein content of treatments supplemented with fenugreek leaves

powder found decreasing trend with increasing the supplementation level. The reason of low

protein content of such treatments is due to low protein content in fenugreek leaves powder.

Similar findings of low protein (4.32%) was observed by Mahmoud et al. (2012) in fenugreek

leaves powder. On the other hand treatments supplemented with fenugreek seeds powder found

higher protein content with increasing supplementation level. The higher protein content

observed in treatments because of high protein content available in fenugreek seeds. Raju and

Bird (2006) also observed high protein content (25.2-30.1%) in fenugreek seeds powder. The

quality of proteins mainly genetically controlled but quantity varies with climatic condition

50

and soil during growing stages (Bushuk, 1997). Supplementation of wheat flour with fenugreek

seeds powder @ 5 to 20% levels showed high protein content in the range of 13.5-16.3% i.e.

due to high protein content of fenugreek seeds (Hooda and Jood, 2004). The results are also in

line with Hussein et al. (2011) who found fenugreek seeds powder as better protein source and

increasing trend found by its supplementation in corn flour.

4.3.4 Crude fat

Mean squares of crude fat content for different treatments indicated that the values has non-

significant change with supplementation of fenugreek leaves powder (Table 4.3). The mean

crude fat (Table 4.4) values ranged from 1.09±0.08 to 1.13±0.03% and in different treatments

found as T0 (1.13±0.03%), T1 (1.12±0.02%), T2 (1.11±0.07%) and T3 (1.09±0.08%). Maximum

level of crude fat content were present in T0 (control) and minimum level in T3 that indicated

a decreasing trend with the addition of fenugreek leaves powder for this trait.

The statistical analysis (Table 4.5) for crude fat in different treatments depicted that fat content

increased in different treatment with supplementation of fenugreek seeds powder. The mean

values (Table 4.6) ranged from 1.13±0.032 to 2.01±0.15% with maximum fat content observed

in T6 (2.01±0.15%) followed by T5 (1.72±0.10%), T4 (1.42±0.08%) while minimum in T0

(1.13±0.03%). The increasing trend was observed by increasing levels of fenugreek seeds

powder for this trait.

Fats are available in smaller quantity in cereals but have significant role in product quality and

texture due to their ability to make complex with proteins and starch (Sramkova et al., 2009).

The highest fat content (2.01%) were recorded for maximum fenugreek seeds powder level in

composite flour while lowest (1.09%) in treatment having highest (15%) of fenugreek leaves

powder. The initial fat content of the raw material effected the fat content of the respective

composite flour. The present findings of wheat-fenugreek seeds supplemented flour are in

harmony with results of Hooda and Jood (2004) who observed increased fat content of wheat

flour with supplementation of fenugreek. The increasing trend of fat content was also

experienced by Hussein et al. (2011) with increasing fenugreek supplementation in corn flour.

51

4.3.5 Crude fiber

Mean squares (Table 4.3) for crude fiber content of different treatments indicated that the

variation in the fiber content was highly significant with supplementation of fenugreek leaves

powder. Mean values regarding crude fiber content has been represented in Table 4.4 which

exhibited that results ranged from 0.31±0.02% to 0.56±0.02% for this trait. The findings of the

present study revealed that crude fiber content in wheat flour significantly increased with

incorporation of fenugreek leaves powder.

The analysis of variance (Table 4.5) for crude fiber content in different treatments revealed

that differences are significant with supplementation of fenugreek seeds powder. Mean values

of fiber content given in Table 4.6 ranged from 0.31±0.02 to 1.45±0.09%. The maximum crude

fiber content observed in T6 (1.45±0.09%) followed by T5 (1.07±0.11%), T4 (0.68±0.04%) and

T0 (0.31±0.02%), respectively. It is obvious from current study that there was increasing trend

of fiber in wheat flour with addition of fenugreek seeds powder.

Fenugreek leaves and seeds powder has high fiber content as compared to wheat flour that

resulted in higher fiber content in different supplemented treatments. Fenugreek leaves has

crude fiber content 1.59% (Mahmoud et al., 2012) and seeds flour 6.88-9.42% (Hooda and

Jood, 2004), that supported current study findings of increased fiber content in different

treatments supplemented with fenugreek powder at different percentages when compared with

control. Similar findings were observed by Okoye and Mazi (2011) & Pasha et al. (2013) who

observed higher crude fiber content in kidney bean and pumpkin composite flours as compared

to wheat flour.

4.3.6 Nitrogen free extract (NFE)

The present outcomes depicted that NFE in wheat flour was significantly increased with the

addition of fenugreek leaves powder. The mean squares (Table 4.3) for NFE in different

treatments supplemented with fenugreek leaves powder portrayed that the difference in NFE

was significant for this trait. Mean values for NFE have been unveiled in Table 4.4 indicated

that it ranged from 74.15±0.19 to 74.60±0.012%. The values of NFE in different treatments

were found to be T0; 74.15±0.19, T1; 74.31±0.10, T2; 74.48±0.12 and T3 74.60±0.14%.

52

Mean squares for NFE given in Table 4.5 portrayed that supplementation of fenugreek seeds

powder significantly effected different treatments. The mean values (Table 4.6) of NFE in

different treatments varied from 74.15±0.19 to 70.15±0.10% that shows a decreasing trend of

this trait with increasing supplementation level of fenugreek seeds powder. The highest value

was observed in T0 (74.15±0.19%) followed by T4 (72.82±0.09%), T5 (71.48±0.10%) and T6

(70.15±0.10%).

Appreciable variations in chemical components existed among different fenugreek-wheat

supplemented flour samples as compared to control (wheat flour). The moisture, crude protein

and fat observed with decreasing trend in fenugreek leaves supplemented flours while ash,

crude fiber and NFE found increased. The flour supplemented with fenugreek seeds powder

have more ash, protein, fat, fiber content while moisture and NFE with decreasing trend when

compared with control.

4.4 Mineral content of supplemented flour

Malnutrition of micronutrients now effected more than 40% population of world and rapidly

increasing in the developing countries like Pakistan. Now a days, deficiencies like iodine and

iron are the major concern to nutritionist and healthcare bodies with other mineral deficiencies,

including calcium, zinc, magnesium and selenium. The impact of malnutrition in these nations

is clearly visible in societal areas like rapidly increasing rate of morbidity and mortality,

reduces mental abilities and educational attainment of children, lower labor productivity,

deteriorates community development efforts and affect the quality of life. Keeping in view

current scenario of malnutrition, consumers are adopting behavior and preference of foods that

not only provide basic or traditional nutrients as well as compounds beneficial for health. So,

the current food systems of world should be designed and changed in such a ways that ensure

continuous and balanced nutrition supplies with affordability (Sramkova et al., 2009).

The current estimation of micronutrients illustrated variation among different treatments under

study. Momentous differences observed in sodium, potassium, iron, calcium and zinc whereas

copper and manganese content found non-momentous among different treatments

supplemented with fenugreek leaves powder. The results for fenugreek seeds powder

supplementation exhibited significant difference among treatments for under studied mineral.

Wheat flour contained calcium; 22.00±1.10 mg/100g, iron; 3.50±0.10 mg/100g, potassium;

53

130.00±6.50 mg/100g and zinc; 0.19±0.03 mg/100g (Table 4.2), i.e. comparatively less than

fenugreek leaves and seeds powders. Therefore, supplementation of wheat flour with

fenugreek powder has significant effect with respect to mineral content.

4.4.1 Sodium (Na)

The statistical results (Table 4.7) for sodium content depicted that different treatment differed

significantly among each other with supplementation of fenugreek leaves powder. Means for

sodium content ranged from 3.12±0.15 (T0) to 11.46±0.15 mg/100g (T3) as shown in Table

4.8. Momentous increase was observed in sodium content with increasing percentage of

fenugreek leaves powder in wheat flour.

Mean squares given in Table 4.9 indicated significant difference among treatments for sodium

content with the addition of fenugreek seeds powder. Means for different treatments (Table

4.10) exhibited that maximum sodium content were observed in T6 (11.98±0.59 mg/100g)

followed by T5 (9.03±0.45 mg/100g), T4 (6.07±0.29 mg/100g) whereas, control (T0) found

minimum value (3.12±0.15 mg/100g). The Na content revealed increasing trend by increasing

the fenugreek seeds powder in wheat flour.

Sodium is essential for normal tissue activity and major cation i.e. accounts for 90% of all

cations available in plasma. It has fundamental importance in maintaining the osmotic pressure

of intercellular fluid. The individual physiological properties of Na include its effect on the

swelling capacity of protein colloids, maintenance of the normal activity of the cardiac muscle,

participation in the processes of nerve excitation and transmission of the nerve impulse

(McDonald et al., 2002). In the present study we found increasing trend of sodium content

with increasing the supplementation of fenugreek leaves and seeds powder in wheat flour.

Fenugreek leaves and seeds has higher sodium content than wheat flour as observed by

Srinivasan (2006) i.e. 76mg/100g and 19mg/100g, respectively. Likewise, Tanveer (2014) &

Pasricha and Gupta (2014) also observed the higher sodium content of fenugreek than wheat

flour that are in agreement with our findings.

4.4.2 Potassium (K)

Mean squares (Table 4.7) for potassium content in different treatments revealed that the

potassium (K) content significantly differed with the addition of fenugreek leaves powder in

54

wheat flour. Mean values (Table 4.8) elaborated that K content ranged from 130.00±6.50 to

182.79±9.14 mg/100g. The maximum K content were observed in T3 (182.79±9.14 mg/100g)

followed by T2 (165.19±8.26 mg/100g), T1 (147.60±7.38 mg/100g) whilst the lowest value

was exhibited in T0 (130.00±6.50 mg/100g).

Statistical analysis depicted in Table 4.9 indicated that potassium content of different

treatments affected significantly with the addition of fenugreek seeds powder. Means for

potassium content (Table 4.10) showed the highest value (192.76±9.63 mg/100g) for T6 trailed

by T5 (171.84±8.59 mg/100g) and T4 (150.92±7.55 mg/100g). The minimum content were

observed in T0 (130.00±6.50 mg/100g) that revealed potassium content increased with

increasing the percentage of fenugreek seeds powder in wheat flour.

Potassium is located intra-cellular in body, participates in maintaining the acid base balance,

osmotic pressure and also in the metabolic processes taking place in the cells (McDonald et

al., 2002). Potassium ions, in conjunction with Na participate in producing the resting potential

and active potential in nerve and muscle formations. It is required in the reactions involving

phosphorylation of creatinine required for the proper activity of muscles (Murray et al., 2003).

Fenugreek is a rich source of potassium as reported earlier by Al-Jasass and Al-Jasser (2012)

& Tanveer (2014) who found supplementation with wheat flour increased the potassium

content of treatments as observed in the current study. In an earlier study conducted by Pasricha

and Gupta (2014) who also observed high potassium content of Kasuri methi (fenugreek) seeds

and leaves.

4.4.3 Iron (Fe)

It is obvious from the statistical analysis (Table 4.7) that treatments have non-significant

difference among each other for iron with the addition of fenugreek leaves powder. It was

observed that Fe content ranged from 3.50±0.10 to 3.98±0.14 mg/100g among different

treatments (Table 4.8). This parameter gradually increased but non-significant variation exist

with the addition of fenugreek leaves powder.

55

Table 4.7 Mean squares for mineral content of different treatments supplemented with fenugreek leaves powder

SOV df Na K Fe Ca Cu Zn Mn

Treatment 3 38.5772** 1548.10** 0.12536NS 4018.99** 0.00179NS 0.00678** 0.00400NS

Error 8 0.1570 62.12 0.03575 12.96 0.00073 0.00037 0.00183

Total 11


* = Significant

NS= Non Significant

Table 4.8 Mineral content (mg/100g) of different treatments supplemented with fenugreek leaves powder

Trt. Na K Fe Ca Cu Zn Mn

T0 3.12±0.15d 130.00±6.50d 3.50±0.10 22.00±1.10d 0.13±0.02 0.19±0.03c 0.67±0.05

T1 5.90±0.12c 147.60±7.38c 3.66±0.18 50.35±2.52c 0.14±0.03 0.23±0.01bc 0.71±0.04

T2 8.68±0.13b 165.19±8.26b 3.82±0.19 78.70±3.93b 0.16±0.03 0.26±0.03ab 0.73±0.04

T3 11.46±0.15a 182.79±9.14a 3.98±0.14 107.05±5.36a 0.18±0.02 0.30±0.02a 0.75±0.07






56

Table 4.9 Mean squares for mineral content of different treatments supplemented with fenugreek seeds powder


Treatment 3 43.6306** 2188.02** 3.22943** 238.142** 0.00523** 0.04807** 0.00703*

Error 8 0.1680 66.47 0.05825 2.765 0.00053 0.00091 0.00137

Total 11


* = Significant

Table 4.10 Mineral content (mg/100g) of different treatments supplemented with fenugreek seeds powder


T0 3.12±0.15d 130.00±6.50c 3.50±0.17d 22.00±1.10d 0.13±0.02c 0.19±0.02c 0.67±0.08b

T4 6.07±0.29c 150.92±7.55bc 4.31±0.22c 28.90±1.44c 0.16±0.02bc 0.29±0.02b 0.73±0.02ab

T5 9.03±0.45b 171.84±8.59ab 5.11±0.26b 35.80±1.79b 0.19±0.03ab 0.38±0.04b 0.75±0.03ab

T6 11.98±0.59a 192.76±9.63a 5.92±0.29a 42.71±2.13a 0.22±0.05a 0.48±0.05a 0.78±0.06a






57

Mean squares (Table 4.9) showed that iron content of different treatments significantly affected

with the addition of fenugreek seeds powder. Mean values (Table 4.10) showed that maximum

content 5.92±0.29 mg/100g was observed in T6 followed by 5.11±0.26 in T5, 4.31±0.22 in T4

and minimum 3.50±0.17 mg/100g in T0. Addition of fenugreek powder significantly increased

the iron content of different treatments.

Iron compounds are present in all cells of the body and fulfill oxidative functions and other

biochemical reactions. Although the major portion of total body Fe is in hemoglobin,

myoglobin and storage but a small portion is also found in a number of enzymes (Soetan et al.,

2010). The iron deficiency commonly prevailed in females and to combat this problem like

iron supplemented capsules are not much effective because of absorption factor. Fresh

fenugreek leaves are rich source of iron (111 mg/100) reported by Mahmoud et al. (2012) as

well as fenugreek seeds i.e. 25.8 mg/100g (Al-Jasass and Al-Jasser, 2012). Therefore,

fenugreek and its supplemented products can be substantial source for iron deficiency problem

due to its better bioavailability. Similarly, Hooda and Jood (2004) found higher iron content

(9.4 to 9.6 mg/100g) of wheat flour when supplemented with up to 20% fenugreek powder

which is in agreement with the results of the current study. The high iron content was also

observed in kasuri methi (fenugreek) seeds (0.318 mg/g) and methi leaves (0.293 mg/g) by

Pasricha and Gupta (2014).

4.4.4 Calcium (Ca)

The analysis of variance (Table 4.7) for Ca content in different treatments illustrated significant

difference with the addition of leaves powder in wheat flour. Mean values regarding Ca content

(Table 4.8) ranged from 22.00±1.10 to 107.05±5.36 mg/100g and the maximum value was

observed in T3 (107.05±5.36 mg/100g) followed by T2 (78.70±3.93 mg/100g), T1 (50.35±2.52

mg/100g) and T0 (22.00±1.10 mg/100g). The results exhibited that Ca content in different

treatments increased with increasing supplementation of fenugreek leaves powder.

Mean squares indicated that Ca content varied significantly with the addition of fenugreek

seeds powder in different treatments (Table 4.9). Mean values for Ca content (Table 4.10)

indicated that it increased with the addition of fenugreek seeds powder as highest found in T6

(42.71±2.13) mg/100g, while lowest 22.00±1.10 mg/100g in T0.

58

Calcium is active in ionic form in tissues and body and principal agent in the formation of

skeletal bones. It also involves in muscle contraction, nerve sensitization, blood clotting and

lactation (Pravina et al., 2013) and deficiency may cause different diseases like osteoporosis

(Soetan et al., 2010). In present investigation we found increasing trend of calcium content of

supplemented flours with increasing the supplementation level of fenugreek i.e. due to high

calcium content. The calcium content of fenugreek leaves reported by Mahmoud et al. (2012)

i.e. 618.41 mg/100 and in seeds 168.88 mg/100g by Kasaye and Jha (2015). Likewise, Hooda

and Jood (2004) found calcium content in the range of 62.2-63.4 mg/100g by supplementation

of wheat flour with 5-20% of fenugreek seeds powder. The high calcium content of kasuri

methi seeds (11.364 mg/g) and methi leaves (10.988 mg/g) reported by Pasricha and Gupta

(2014), that in harmony with current findings.

4.4.5 Copper (Cu)

Mean squares showed non-substantial effect of treatments on copper with the addition of

fenugreek leaves powder (Table 4.7). In current study, Cu content ranged from 0.13±0.02 to

0.18±0.02 mg/100g with highest value was explicated in T3 (0.18±0.02 mg/100g) followed by

T2 (0.16±0.03 mg/100g) and T1 (0.14±0.03 mg/100g) whilst the lowest was exhibited in T0

(0.13±0.02 mg/100g) (Table 4.8).

The analysis of variance (Table 4.9) for copper content of different treatments elucidated that

there was significant effect of fenugreek seeds powder supplementation in wheat flour. Mean

values (Table 4.10) for copper content observed in the range of 0.13±0.02 to 0.22±0.05

mg/100g that revealed copper content increased with the addition of fenugreek seeds powder.

Copper is an essential part of many proteins, required for blood formation, catalyzes the

incorporation of Fe into the structure of the heme and assists maturation of the erythrocytes. It

also a constituent of many enzymes or essential for their activity (Soetan et al., 2010). The

high copper content of treatments found in current study resulted due to high content of

respective mineral in fenugreek as reported earlier by Pasricha and Gupta (2014). The results

regarding copper content of current investigation are corroborated with the work of Al-Jasass

and Al-Jasser (2012) & Tanveer (2014) who reported high values for this attribute when

compared with wheat. Thus, higher content of copper in fenugreek resulted in higher content

of supplemented flours as observed in current research work.

59

4.4.6 Zinc (Zn)

Mean squares (Table 4.7) for zinc content in different treatments described that zinc content

significantly affected with the addition of fenugreek leaves powder. Mean values (Table 4.8)

regarding zinc content has been given in Table 4.8 varied from 0.19±0.03 to 0.30±0.02

mg/100g. The highest zinc content were observed in T3 (0.30±0.02 mg/100g) followed by T2

(0.26±0.03 mg/100g), T1 (0.23±0.01 mg/100g) and T0 (0.19±0.03 mg/100g). The findings of

present study indicated that zinc content increased in treatments with increasing the fenugreek

leaves powder.

The analysis of variance for zinc content of different treatments has been presented in Table

4.9. It is apparent from data that zinc content has significantly affected with the addition of

fenugreek seeds powder. Zinc content ranged from 0.19±0.02 to 0.48±0.05 mg/100g among

different treatments (Table 4.10) and results revealed that zinc content increased with

increasing level of fenugreek seeds powder.

Zinc is an important micronutrient that can affect growth, development, reproductive function,

bone and blood formation, metabolism of nucleic acids, proteins and carbohydrates. It is

present in plasma in two forms, either firmly bound or weakly bound. Zinc globulin complexes

has role in enzymatic functions and it can act in conjunction with enzymes, of which it is an

essential component or activator (Soetan et al., 2010). The results of current investigation are

in harmony with findings of Hooda and Jood (2004) who found zinc content in the range of

4.8-5.0 mg/100g by supplementation of wheat flour with 5-20% of fenugreek seeds powder.

The zinc content found in kasuri methi seeds (0.032 mg/g) and methi leaves (0.0496 mg/g),

reported by Pasricha and Gupta (2014).

4.4.7 Manganese (Mn)

Statistical results regarding manganese (Mn) content in different treatments showed non-

significant difference with supplementation of fenugreek leaves powder (Table 4.7). Mean

values (Table 4.8) depicted that Mn content ranged from 0.67±0.05 to 0.75±0.07 mg/100g with

highest Mn content found in treatment that have 15% fenugreek leaves powder (T3).

Mean squares (Table 4.9) showed that Mn content of different treatments significantly affected

with the addition of fenugreek seeds powder. It was observed that Mn content (Table 4.10)

60

varied from 0.67±0.08 to 0.78±0.06 mg/100g and the highest content observed in T6

(0.78±0.06 mg/100g) followed by T5 (0.75±0.03 mg/100g), T4 (0.73±0.02a mg/100g) and T0

(0.67±0.08 mg/100g). It is obvious from current findings that increasing fenugreek seeds

powder resulted in increased Mn content in different treatments.

Manganese is actively participates in redox process, tissues respiration, bone formation &

affects growth, reproduction, blood formation and the function of endocrine organs. It also

function biochemically either as a cofactor that activating a large number of enzymes or as an

integral part of certain metallo-enzymes. About one third of the body required manganese

provided through cereal based products & remaining major through vegetables and beverages

(Davidsson et al., 1988; Finley et al., 1994), excess of this mineral can restrict iron absorption

(Rossander-Hulten et al., 1991; Finley, 1999). The current investigation found higher

manganese content of fenugreek-wheat supplemented flour i.e. due to higher content found in

fenugreek, also observed earlier 1.71 mg/100g by Mahmoud et al. (2012); 0.9 mg/100g (Al-

Jasass and Al-Jasser, 2012) and 1.3 mg/100g by Tanveer (2014). Similarly, Pasricha and Gupta

(2014) observed manganese content of kasuri methi seeds (0.027 mg/g) and methi leaves

(0.0364 mg/g). Thus, supplementation of fenugreek increased manganese content of wheat

flour as described in current study.

4.5 Rheological study

Rheology deals with the study of flow behavior of fluids and provide information regarding

rheological and mechanical characteristics of different food systems that has significance in

the design of flow processes for quality control, in forecasting storage stability and designing

textural parameters (Herh et al., 2000). Dough rheology characterization is an imperative factor

in the assessment of bread wheat quality & indicates dough handling properties and the

tendency of the dough to contract (Pedersen et al., 2004). Different methods including

farinograph, amlyograph, mixograph and extensograph are being used for characterization of

the rheological properties of flour (Mani et al., 1992). In current investigation incorporation

effect of different percentages of fenugreek leaves and seeds powder in straight grade flour has

been assessed through farinograph and mixograph to evaluate the rheology of dough.

61

4.5.1 Farinographic studies

For the preparation of baked goods, knowledge about rheological properties of the dough is

considered as prime importance. Wheat, the main ingredient in most baked foods, contains

viscoelastic gluten, which confers specific rheological properties to the dough, and this in turn

influences the final quality of the baked product. Therefore, it would be expected that during

preparation of composite flour and dough, partial replacement of wheat with another

component that may be devoid of gluten would influence rheological properties of the resultant

dough. Various rheological parameters are of interest in this regard, including water

absorption, dough strength, dough development time and mixing tolerance. Generally, the

major effect on dough rheological properties arises from dilution of the gluten content on

partially replacing the wheat component. As would be expected, the rheological properties of

the resultant composite dough are dependent on the relative hydrophobicity or hydrophilicity

of food components such as proteins endogenous to the composite flours. Whereas

incorporation of legumes in composite flour tends to bring about changes in rheological

properties like increase in the water absorption and dough development time (Preedy et al.,

2011).

4.5.1.1 Water absorption

Mean squares depicting the fenugreek leaves powder supplementation effect on water

absorption of dough prepared from different treatments has been given in Table 4.11. The

results showed significant difference among treatments with incorporation of fenugreek leaves

powder. The mean values given in Table 4.12 varied from 60.60±0.60 to 63.10±0.93%.

Maximum water absorption was found in T3 (63.10±0.93%) followed by T2 (62.90±1.05%),

T1 (61.39±1.01%) and T0 (60.60±0.60%). The results exhibited that water absorption increased

with increasing supplementation level of fenugreek leaves powder.

Mean squares (Table 4.13) elucidated significant difference among treatments with

supplementation of fenugreek seeds powder. The mean values (Table 4.14) for this trait

explained that water absorption varied from 60.60±0.60 to 62.17±1.14%. Maximum water

(62.17±1.14) was absorbed in flour have 5% fenugreek seeds powder and absorption increased

at certain level (10%) then decreased but remained higher than control.

62

Water absorption is expressed as a percentage and it is one of the most important parameter

commonly used and widely accepted in farinograph measurements. It is also considered as one

of the most important physical factor affecting the farinograms. Higher water absorption during

dough formation reflects the quality of the finished baked products. The findings of the present

investigation are corroborated with the results of Sudha et al. (2013) who replaced wheat flour

with dehydrated fenugreek leaves at 0, 5, 7.5 and 10 %, resulted in water absorption increased

from 68.5 to 70.2 % of dough. When supplementation level of fenugreek in wheat flour was

increased, dough water absorption also increased from 65.10% (control) to 68.5% in the

composite flour with 5% fenugreek powder. When supplementation level increased to 10%,

water absorption of dough decreased by 66.37% but remained higher than control and further

decreased up to 61.2% when supplementation level reached 20%. This is might be due to the

fenugreek interfering with gluten formation that makes the dough cohesive, less elastic and

offered less resistance of farinograph mixing blades (Hooda and Jood, 2003).

Different levels of fenugreek seeds flour increased most of the rheological properties. The

water absorption observed 52% in control that increased up to 62-70% when supplementation

of fenugreek powder increased in wheat flour (Sulieman et al., 2000). Likewise, farinograph

water absorption increased by the addition of Mungbean powder in wheat (Pasha et al., 2011),

the incorporation of legume flour in wheat flour increased water absorption capacity and this

might be due the presence of high protein and fiber (Hefnawy et al., 2012). Enhanced protein

content results due to an increase in pentosans, especially ribose and deoxyribose, which has a

higher water holding capability (Shahzadi et al., 2005). Generally, fiber provides better

properties of water absorption and this is mainly due to more number of OH groups present in

fiber structure that through hydrogen bonding allows a stronger interaction of water (Rosell et

al., 2001). The present findings are also in corroboration with the values reported earlier by

Indrani et al. (2011), Metwal et al. (2011) and Kasaye and Jha (2015) who reported dough

water absorption increased 55.8 to 63.9% with blending up to 10 percent while found decreased

water absorption 61.8% when blending increased up to 15% for fenugreek.

4.5.1.2 Arrival time

The mean squares (Table 4.11) for arrival time exhibited significant effect by the addition of

fenugreek leaves powder in wheat flour. The mean values (Table 4.12) demonstrated that

63

control treatment showed maximum arrival time i.e. 3.56±0.17 min, while the addition of

different levels of fenugreek leaves powder in wheat flour gradually decreased the arrival time.

Mean squares for arrival time (Table 4.13) showed substantial effect among treatments with

the addition of fenugreek seeds powder. The mean values range from 2.58±0.10 to 3.56±0.17

min and the results depicted that control treatment have maximum arrival time that decreased

with increasing the supplementation level of fenugreek seeds powder (Table 4.14).

Arrival time of wheat-fenugreek supplemented flour decreased when compared with wheat

flour (Sulieman et al., 2000). The findings of current study are also in accordance with Saleh

et al. (2012) who made different blends of wheat flour with soybean and chickpea flour and

found arrival time decreased from 1.5 (control) to 0.5 min with soybean supplementation.

4.5.1.3 Dough development time (DDT)

Mean squares for dough development time (DDT) depicted highly significant effect with

supplementation of different levels of fenugreek leaves powder in wheat flour (Table 4.11).

Means regarding DDT (Table 4.12) with addition of fenugreek leaves powder has been

revealed that maximum time was taken by the flour have 15% addition of fenugreek leaves

powder (T3) as 7.50±0.16 min while minimum time 6.17±0.15 min taken by T0.

The statistical analysis (Table 4.13) for DDT of different treatments exhibited that it was

significantly affected with the addition of fenugreek seeds powder. The dough development

time varied from 6.17±0.15 to 8.40±0.10 min in flour have different levels of fenugreek seeds

powder (Table 4.14). The mean values depicted that dough development time increased with

increased supplementation level of fenugreek seeds powder in wheat flour.

Results of dough development time resemble the values observed by Sudha et al. (2013) who

replaced wheat flour with dehydrated fenugreek leaves at 0, 5, 7.5 and 10 % level resulted in

dough development time (DDT) increased from 3.5 to 5.9 min. The incorporation of debittered

and defatted fenugreek seeds powder in wheat flour resulted in increased DDT and this increase

might be the reason of delayed gluten network development because of the fiber content of

fenugreek (Metwal et al., 2011). In general, the relations between non-wheat based protein,

gluten and fiber leads to delay hydration and gluten development that could be the reason of

increased dough development time (Dhinda et al., 2012). The DDT increased from 1.5 min

(control) to 3.5, 7.0 and 9.0 min for 10%, 20% and 30% supplementation levels of fenugreek

64

(Chauhan and Sharma, 2000). Findings of the present study are also correlated with the study

of Hooda and Jood (2003) & Hefnawy et al. (2012) who found increased DDT with addition

of fenugreek in wheat flour. Similarly, Kasaye and Jha (2015) also observed increased dough

development time from 4.2 to 7.5 min with increased supplementation of fenugreek in wheat

flour.

4.5.1.4 Departure time

Analysis of variance reported significant effect by the incorporation of fenugreek leaves in

different treatments for departure time (Table 4.11). The mean values ranged from 15.27±1.02

to 15.65±1.06 min that shows gradually increasing trend with increasing the percentage of

fenugreek leaves powder (Table 4.12). The maximum time taken by T3 (15.65±1.06 min)

followed by T2 (15.48±1.01 min), T1 (15.42±1.09 min) while minimum in T0 (15.27±1.02

min).

Mean squares (Table 4.13) for departure time illustrated that there was significant difference

among different treatments with supplementation of fenugreek seeds powder. The mean values

(Table 4.14) representing increasing trend in departure time as maximum time (16.37±1.01

min) was taken by the dough prepared from flour have 15% fenugreek seeds powder (T6). Thus

the outcomes of the parameter reveals that flour with more quantity of seeds powder will take

longer to leave the 500BU line.

Findings of the current study in harmony with outcomes of Sulieman et al. (2000) who

observed different levels of fenugreek seeds flour increased most of the rheological properties

like water absorption, mixing time, dough stability, departure time and resistance to extension.

The findings of Kasaye and Jha (2015) also have same trend of increasing departure time when

supplementation of fenugreek powder increased in wheat flour.

4.5.1.5 Dough stability

Data available in Table 4.11 revealed that the incorporation of fenugreek leaves powder in

wheat flour has a significant impact on stability time of dough. Mean values (Table 4.12) for

treatments have different levels of fenugreek leaves powder fall in range of 11.71±0.21 to

12.81±0.23 min correspondingly with maximum time taken by flour having 15% addition of

fenugreek leaves powder (T3).

65

The statistical analysis for dough stability given in Table 4.13 shows similar increasing trend

for seeds powder supplementation like fenugreek leaves powder. Flour have 15% fenugreek

seeds powder (T6) had maximum dough stability i.e. 13.78±0.11 min (Table 4.14) followed by

T5 (13.30±0.10 min), T4 (12.61±0.12 min) and T0 (11.71±0.21 min). The current observation

concluded that supplementation of fenugreek powder in wheat flour increased dough stability

when increased supplementation of both fenugreek leaves and seeds powder.

When supplementation of wheat flour was carried out with fenugreek powder, dough stability

increased with increasing the level of supplementation (Hooda and Jood, 2003) that

strengthened the results of present exploration. In another study when fenugreek powder was

incorporated in wheat flour @ 1.5 to 9%, the stability of dough increased up to 4 to 5% due to

slow hydration properties of fenugreek (Chauhan and Sharma, 2000). It is evident that addition

of fenugreek powder in wheat flour increased dough stability (Sulieman et al., 2000; Metwal

et al., 2011). The results of Kasaye and Jha (2015) are also in accordance with the current

findings, who observed increased dough stability from 5.9 to 10.5 min with blending of

fenugreek in wheat flour.

4.5.1.6 Mixing tolerance index

Mean squares for the mixing tolerance index (MTI) exhibited highly significant effect among

treatments with supplementation of fenugreek leaves powder (Table 4.11). The mean values

for MTI of different wheat flour with fenugreek leaves powder at varying level has been

depicted in Table 4.12. Maximum value was observed as 26.00±2.00 BU in control (T0)

treatment while minimum value (18.45±1.12 BU) was found in flour have 15% of fenugreek

leaves powder (T3) added to it.

The statistical analysis (Table 4.13) exhibited that MTI substantially affected with the addition

of fenugreek seeds powder in different treatments. The mean values of MTI for the flour having

different levels of fenugreek seeds powder were in range of 16.46±1.01 to 26.00±2.00 BU

(Table 4.14). There was decline in the MTI value by the incorporation of fenugreek seeds and

leaves powder to wheat flour.

The results of current investigation in accordance with findings of Sulieman et al. (2000) who

observed mixing tolerance index decreased with supplementation of fenugreek powder in

wheat flour. The possible reason is that dough become more stable with supplementation of

66

fenugreek that offered some resistance to weakening of dough when mixing carried out.

Similar outcomes of fiber substitutions in dough were observed by other research workers

(Haridas and Rao, 1991; Chauhan and Sharma, 2000). The study conducted by Hooda and Jood

(2003) also strengthen our results who observed mixing tolerance index decreased from 65 BU

to 25 BU with increasing level of fenugreek substitution in wheat flour.

4.5.2 Mixographic studies

Mixograph measures the dough physical properties and used by wheat breeders to check the

quality of the wheat flour. It is really a useful tool for quality evaluation of wheat where sample

size is limited because this method requires small size of sample. This helps the breeders to

call out the undesirable material in early generations in order to save time, labor and investment

in the subsequent generation. Mixograph determine attributes like water absorption, over

mixing tolerance and viscoelastic strength of dough (Atwell, 2003). These attributes mostly

are functions of flour protein quality and quantity as well as other ingredients present in dough

like shortening (Bello et al., 1991). The ratio of water and flour used during current

mixographic studies was based on initially determine moisture and protein content of flour.

Mixing of water and flour was carried out in mixograph bowl. The mixing time and peak height

attributes observed through mixogram curve used to conclude strength of the dough.

4.5.2.1 Mixing time

Mean squares for the mixing time demonstrated significant effect by incorporating different

levels of fenugreek leaves powder in wheat flour (Table 4.15). The mean values (Table 4.16)

depicted that addition of fenugreek leaves powder in wheat flour lowered its mixing time from

5.73±0.05 min to 4.70±0.20 min. Maximum mixing time was found in wheat flour without any

fenugreek acting as control (T0) while minimum value was observed in flour have 15%

fenugreek leaves powder (T3).

Mean squares (Table 4.17) illustrated that supplementation of fenugreek seeds powder in

different treatments significantly affect mixing time. The mean values for different levels of

fenugreek seeds powder has shown that mixing time reduced from 5.73±0.05 min to 4.80±0.10

min (Table 4.18). There was a decreasing trend for this trait with increasing the percentage of

fenugreek seeds powder in different treatments.

67

Table 4.11 Mean squares for Farinographic parameters of different treatments supplemented with fenugreek leaves

powder

SOV df Water

absorption Arrival time

Dough

development

time

Dough

stability

Mixing

tolerance

index

Departure

time

Treatments 3 10.1927** 0.30414** 0.95436** 0.66681** 39.7193** 0.07264*

Error 8 0.0126 0.00048 0.00105 0.01013 2.0003 0.00019

Total 11

**= Highly Significant

*= Significant

Table 4.12 Farinographic parameters of different treatments supplemented with fenugreek leaves powder

Treatments

Water

absorption

(%)

Arrival time

(min)

Dough

development

time (min)

Dough stability

(min)

Mixing

tolerance

index(BU)

Departure time

(min)

T0 60.60±0.60d 3.56±0.17a 6.17±0.15d 11.71±0.21d 26.00±2.00a 15.27±1.02d

T1 61.39±1.01c 3.26±0.06b 6.80±0.14c 12.61±0.16c 24.00±2.01a 15.42±1.09c

T2 62.90±1.05b 2.98±0.11c 7.11±0.12b 12.50±0.20b 19.33±1.21b 15.48±1.01b

T3 63.10±0.93a 2.84±0.13d 7.50±0.16a 12.81±0.23a 18.45±1.12b 15.65±1.06a






68

Table 4.13 Mean squares for Farinographic parameters of different treatments supplemented with fenugreek seeds

powder

SOV df Water

absorption Arrival time

Dough

development

time

Dough

stability

Mixing

tolerance

index

Departure

time

Treatments 3 1.72460** 0.56627** 3.01639** 2.42460** 57.7019** 0.74968*

Error 8 0.01022 0.00028 0.00667 0.00265 1.0002 0.00025

Total 11

**= Highly Significant

*= Significant

Table 4.14 Farinographic parameters of different treatments supplemented with fenugreek seeds powder

Treatments

Water

absorption

(%)

Arrival

time

(min)

Dough

development time

(min)

Dough

stability

(min)

Mixing

tolerance

index(BU)

Departure

time

(min)

T0 60.60±0.60c 3.56±0.17a 6.17±0.15d 11.71±0.21d 26.00±2.00a 15.27±1.02d

T4 62.17±1.14a 2.88±0.16b 7.80±0.10c 12.61±0.12c 18.19±1.02b 15.49±1.02c

T5 61.52±1.01b 2.72±0.11c 8.13±0.15b 13.30±0.10b 17.38±1.10b 16.02±0.91b

T6 60.63±1.32c 2.58±0.10d 8.40±0.10a 13.78±0.11a 16.46±1.01b 16.37±1.01a






69

Mixing time is an important criteria affected by quality of the flour. Similar to present data

mixing time decreased as level of legume (Matri) flour increased in wheat flour (Lodhi and

Huma, 2003). The results of current research are also comparable with the earlier findings of

Pasha et al. (2013) who observed mixing time decreased from 3.67 to 2.77 min as level of

pumpkin flour supplementation increased in wheat flour.

4.5.2.2 Peak height percentage

Analysis of variance regarding the peak height for different treatments with varying percentage

of fenugreek leaves powder has been given in Table 4.15. The mean values (Table 4.16) ranged

from 53.00±3.00 to 58.00±2.01% in wheat flour have different levels of fenugreek leaves

powder (Table 4.16). The incorporation of fenugreek leaves powder lowered the peak height

as measured by the mixograph and a decreasing trend was observed while maximum peak

height found in control sample.

Mean squares depicted non-momentous difference among different treatments have fenugreek

seeds powder (Table 4.17). The addition of fenugreek seeds powder in wheat flour resulted in

decreasing trend at T4 (55.00±1.01 %) then gradually increased but remained lower than

control (T0) up to 15% supplementation level (Table 4.18).

Peak height gives some indication of the strength and absorption of the flour. However, the

mixogram is not as sensitive to absorption as the farinograph, so the peak height percentage

gives only an indication of the value. Results of mixographic value resemble the values

determined by Lodhi and Huma (2003) & Pasha et al. (2013) who observed mixograph peak

height decreased from 59.30 to 55.50 BU as level of supplementation with matri and pumpkin

flour increased in wheat flour. Mixograph peak height for wheat flour was 45.4% and found

50 to 70% lower in flour supplemented with freeze and oven dried, pre-cooked fiber (Gajula

et al., 2009).

70

Table 4.15 Mean squares for Mixographic parameters of different treatments

supplemented with fenugreek leaves powder

SOV df Mixing time Peak height

Treatments 3 0.63019* 14.7500*

Error 8 0.01090 3.7500

Total 11

**=Highly Significant

*= Significant

Table 4.16 Mixographic parameters of different treatments supplemented with


Treatments Mixing time (min) Peak height (%)

T0 5.73±0.05a 58.00±2.01a

T1 5.30±0.01b 56.00±1.03ab

T2 4.89±0.06c 54.00±1.02b

T3 4.70±0.20c 53.00±3.00c






71

Table 4.17 Mean squares for Mixographic parameters of different treatments


SOV df Mixing time Peak height

Treatments 3 0.45409** 5.41667NS

Error 8 0.00347 2.33333

Total 11

**=Highly Significant

NS= Non-significant

Table 4.18 Mixographic parameters of different treatments supplemented with


Treatments Mixing time (min) Peak height (%)

T0 5.73±0.05a 58.00±2.01

T4 5.39±0.01ab 55.00±1.01

T5 5.21±0.02b 55.67±0.57

T6 4.80±0.10c 57.00±2.04






72

4.6 Antioxidant assay of supplemented flour

Reactive oxygen species (ROS) produced in our body through different ways, either from inner

body mechanisms, exposure to environmental pollutants, UV radiations, toxic chemicals and

cigarette smoke or stress generating current life style, results in weakened body defense system

(Valko et al., 2006). Under normal circumstances, our healthy natural defense system can

neutralize these ROS but over production creates problem for our body. Antioxidants are the

compounds that can scavenge the ROS like free radicals with the help of their redox properties

(Middleton et al., 2000). The continuous availability of antioxidants through various dietary

supplements can safeguard our body against harmful effects of free radicals and can increase

the antioxidant capacity. Fenugreek has antioxidant potential and found to be effective

antioxidant in vivo and vitro to deal with oxidation stresses (Mariod et al., 2009). In the current

study we have investigated the antioxidant potential of fenugreek leaves and seeds powder

supplemented wheat flour.

4.6.1 Total phenolic content (TPC)

Statistical results (Table 4.19) showed that highly significant difference was observed for total

phenolic content (TPC) among different treatments with the addition of fenugreek leaves

powder. It is obvious from the results given in Table 4.20 that total phenolic content in different

treatments ranged from 117.00±5.26 (T0) to 390.00±19.11 mg GAE/100g (T3). Increasing

trend was observed for TPC with increased percentage of fenugreek leaves powder.

Mean squares (Table 4.21) depicted that highly significant variations were observed for TPC

between treatments by adding fenugreek seeds powder. The mean values given in Table 4.22

revealed that TPC in different treatments as 117.00±5.26 (T0), 305.00±14.03 (T4),

410.00±19.27 (T5) and 450.00±22.05 mg GAE/100g (T6). The results showed that total

phenolic content increased with increasing the percentage of fenugreek seeds powder in wheat

flour.

In current study wheat-fenugreek supplemented flour extracts depicted momentous antioxidant

properties, depending upon the percentage of fenugreek in wheat flour. Wheat flour has low

phenolic content as compared to fenugreek, as observed in an earlier study total phenolic

content of refined wheat flour found in the ranged of 1.16 to 1.55 mg FAE/g. The organic

73

solvent extracts of hard and soft whole wheat flour were used to determine TPC and found

values as 0.353 and 0.478 mg FAE/g, in the same way 0.137 and 0.161 mg FAE/g values

observed in hard and soft wheat flours (Liyana-Pathirana and Shahidi, 2006). In another

research work, TPC was determined in different wheat varieties flour and observed values in

the range of 1.46 to 2.26 mg GAE/g (Liu et al., 2010). Fenugreek seeds contain different

phenolic content like quercetin and naringenin that could be effective against free radicals. The

hunting activities of these phenolic compounds are credited to active hydrogen (H) donating

capability of the hydroxyl (OH) substitutions (Bors et al., 1996).

The research work of Kaur and Kapoor (2002) categorized fenugreek among high phenolic

content vegetables group using ethanol as extracting solvent and observed very high

antioxidant activity. The total polyphenol content of extracts obtained from extraction of dried

fenugreek greens with methanol, ethanol, and isopropanol solvents was 48, 44, 28 mg

GAE/100g, respectively (Naidu et al., 2012). Similar finding were observed by Bukhari et al.

(2008) who did an experiment to find total phenolic content of fenugreek methanol and ethanol

extracts and reported results as 575±0.002 and 685±0.002 mg GAE/100g, respectively. In

another study conducted by Naidu et al. (2011) who has observed total phenolic content

(85.8 mg/g) in fenugreek seeds extract that could be responsible for its antioxidant activity.

Fenugreek extract had polyphenols (9.47 mg GAE/g) that plays a role as antioxidant (Dua et

al., 2013). The results were also in harmony with another research work of Kumar et al. (2013)

who determined phenolic content and found 22±1.5 μg/mg GAE in extracted fenugreek

sample. The study of different fenugreek samples was carried out to observed total phenolic

content and found 139.2 mg GAE/100g followed by 130.0 mg GAE/100g and 127.8 mg

GAE/100g, respectively (Ali et al., 2015).


Mean squares regarding the effect of fenugreek leaves powder supplementation in different

treatments on total flavonoids content has been given in Table 4.19. The results explicated that

fenugreek addition has a significant effect on different treatments. The mean flavonoids values

given in Table 4.20 depicted that highest content (2.97±0.14 mg CE/g) were reported in T3

trailed by T2 (2.80±0.07 mg CE/g), T1 (2.60±0.15 mg CE/g) and T0 (2.38±0.08 mg CE/g).

74

Analysis of variance for total flavonoids (Table 4.21) explicated momentous difference in

values as a function of fenugreek seeds powder. Means for total flavonoids (Table 4.22) for

different treatments; T0 (2.38±0.08 mg CE/g), T4 (2.57±0.10 mg CE/g), T5 (2.79±0.09 mg

CE/g) and T6 (3.01±0.14 mg CE/g), showing increasing trend with increased percentage of

fenugreek seeds powder.

Flavonoids are diverse group of polyphenolic components with exceptional strength to act as

free radical scavenger, anti-inflammatory and antibacterial agent. Different flavonoids like

tricin, vitexin, quercetin, naringenin and tricin-7-O-b-Dglucopyranoside were identified in

fenugreek (Shang et al., 1998). Significant antioxidant activity was observed in fenugreek

seeds extract due to presence of different flavonoids (Dixit et al., 2005). Methanol, ethanol,

hexane, ethyl acetate and acetone were used for extraction and observed flavonoid content in

the range of 607±3.6, 653±4.3, 208±4.2, 251±3.3 and 416±2.7 QE µg/g of fenugreek (Bukhari

et al., 2008). To verify its worth, a study was designed to determine flavonoid content present

in fenugreek by Kumar et al. (2013) who observed total flavonoids 16.6±1.2 μg QE/mg.

Likewise, Ishtiaque et al. (2013) also found flavonoid content 5.80 mg QE/g in fenugreek seeds

and supplementation in wheat flour resulted in an increased flavonoid content.

4.7 Antioxidant activity

4.7.1 Free radical scavenging activity (DPPH Assay)

Mean squares regarding DPPH scavenging activity of different treatments has been presented

in Table 4.19. A significant difference was observed as a function of fenugreek leaves powder

supplementation in wheat flour. Mean values given in Table 4.20 indicated that this attribute

increased with increase in supplementation level i.e. T3 (47.00±2.30%) followed by T2

(45.00±2.11%), T1 (41.00±1.88%) and T0 (35.00±1.57%).

From mean squares of DPPH scavenging activity (Table 4.21), a highly significant relation

was observed for the effect of fenugreek seeds powder supplementation. Means for different

treatments (Table 4.22) found in the range from 35.00±1.57% (T0) to 58.00±2.84% (T6).

Results showed that the highest value was recorded in 15% supplementation while the lowest

for control that exhibited increasing trend for this trait with increased level of fenugreek seeds

powder.

75

The DPPH values of different wheat flour were found in the range of 6.48 to 8.57 μmol TE/g

(Liu et al., 2010). Different factors like genetic, environmental conditions, processing and

extraction solvent & procedure affect antioxidant level in wheat flour (Mpofu et al., 2007). In

current study ethanol extracts has showed significant and concentration dependent DPPH

scavenging activity indicating the antioxidant potential of fenugreek as reported earlier by

Bukhari et al. (2008). They observed DPPH scavenging activity of ethanol and methanol

extracts of fenugreek seeds are correlated to the total polyphenol content of the extracts. High

polyphenol content in the fenugreek extract seems to be the major free radical scavenger in

current investigation. Extracts from dried fenugreek leaves depicted free radical scavenging

activity ranging from 18 to 56% and the activity increased with the increasing concentration

of the various solvents used, methanol extract of fenugreek leaves exhibited 42% activity

followed by ethanol and isopropanol extracts. Further, extract of fenugreek leaves obtained

with aqueous methanol, exhibited higher (56%) free radical scavenging activity. So antioxidant

activities of the extract mainly dependent on the composition of phenolic compounds available

in fenugreek leaves (Naidu et al., 2012).

The data acquired from present study is in harmony with results of Belguith-Hadriche et al.

(2010) who determined fenugreek seeds antioxidant activity by estimating its ability to

scavenge DPPH and observed result i.e. 89.91±3.09%. In another study conducted by Saeed et

al. (2013) who observed Kasuri fenugreek (methanol + water extract) have DPPH radical

scavenging activity (19.01%), followed by Kasuri fenugreek water extract (16.49%). The

DPPH radical scavenging activity of Lahori fenugreek methanol + water extract and water

extract were 18.36 and 14.59%, respectively at same concentration i.e. 1 mg/ml. The

determination of DPPH assay of fenugreek leaves and seeds depicted that free radical

scavenging activity of seeds was better than that of leaves as seen by a lower E.C.50 value

obtained for seeds as compared to a higher E.C.50 value for the leaves. However, the seeds

were found to have a better antioxidant potential than the leaves in vivo (Jha and Srivastava,

2012).

76

Table 4.19 Mean squares for antioxidants in different treatments supplemented with


SOV df TPC Flavonoids DPPH

β-carotene &

Linoleic acid

assay

FRAP

Treatment 3 41408.3** 0.19410** 83.9767** 20.0000* 20494.8**

Error 8 179.7 0.01373 3.9517 2.8683 224.5

Total 11


* = Significant

Table 4.20 Antioxidants in different treatments supplemented with fenugreek leaves

powder

Trt.

TPC

(mg GAE/

100g)

Flavonoids

(mg CE/g)

DPPH

(%)

β-carotene &

Linoleic assay

(%)

FRAP

(µmol Fe2+/g)

T0 117.00±5.26d 2.38±0.08c 35.00±1.57c 33.00±1.48b 222.00±9.99c

T1 226.00±10.39c 2.60±0.15bc 41.00±1.88b 35.00±1.61ab 257.00±11.82c

T2 314.00±14.75b 2.80±0.07ab 45.00±2.11ab 37.00±1.73ab 351.00±16.49b

T3 390.00±19.11a 2.97±0.14a 47.00±2.30a 39.00±1.91a 401.00±19.94a






77

Table 4.21 Mean squares for antioxidants in different treatments supplemented with



β-carotene &

Linoleic acid

assay

FRAP

Treatment 3 66431.6** 0.19410** 292.658** 168.000** 46531.2**

Error 8 270.5 0.01373 5.452 4.209 388.8

Total 11


* = Significant

Table 4.22 Antioxidants in different treatments supplemented with fenugreek seeds

powder

Trt

TPC

(mg GAE/

100g)

Flavonoids

(mg CE/g)

DPPH

(%)

β-carotene &

Linoleic acid

assay (%)

FRAP

(µmol Fe2+/g)

T0 117.00±5.26c 2.38±0.08c 35.00±1.57c 33.00±1.48c 222.00±9.99c

T4 305.00±14.03b 2.57±0.10bc 49.00±2.25b 43.00±1.97b 421.00±19.36b

T5 410.00±19.27a 2.79±0.09ab 53.00±2.49ab 45.00±2.11b 465.00±21.85ab

T6 450.00±22.05a 3.01±0.14a 58.00±2.84a 51.00±2.49a 501.00±24.54a






78

4.7.2 β‐carotene and linoleic acid assay

Mean squares for this trait (Table 4.19) showed significant effect of fenugreek leaves powder

addition in different treatments. The mean values (Table 4.20) illustrated that maximum value

(39.00±1.91%) was recorded in T3 followed by T2 (37.00±1.73%), T1 (35.00±1.61%) while

lowest in T0 (33.00±1.48%). The findings of the current research illustrated that this trait

increased with increasing percentage of fenugreek leaves powder.

The analysis of variance for β‐carotene (Table 4.21) revealed significant effect of fenugreek

seeds powder supplementation in wheat flour. The mean values (Table 4.22) illustrated that

values ranged from 33.00±1.48% (T0) to 51.00±2.49% (T6). It is obvious from results that this

assay increased with increasing the supplementation level of fenugreek seeds powder.

The discoloration of β‐carotene has been broadly used to determine different plants extract

antioxidant potential because it is very susceptible to free radical mediated oxidation of linoleic

acid. The formation of linoleic acid free radical occurred with the abstraction of a hydrogen

atom from one of its diallylic methylene groups that attacks the highly unsatured β‐carotene

molecules. As β‐carotene molecules by process of oxidation loses their double bonds resulted

in loss of yellow color of compound. Likewise, antioxidant activity of fenugreek fractions as

measured by the bleaching of β-carotene used different fenugreek fractions like fenugreek

husk, endosperm and seeds extracts that exhibited 73%, 6% and 22% activity when compared

with the corresponding values of 95% for BHA (Naidu et al., 2011). Current study results are

in agreement with explorations of Subhashini et al. (2011) who determine the antioxidant

activity of 70% ethanol extract of fenugreek by β‐carotene bleaching method. The ethanol

extract of fenugreek inhibited β‐carotene oxidation suggesting that the antioxidant activity

could be related to the high levels of phenolic compounds. The extract was employed in the

range of 25-400 mg/mL and by adding more quantity of the extract, absorbance was decreased

and the reason behind was inhibition of bleaching of the color β‐carotene. Fenugreek extract

depicted inhibition at concentration of 0.202 mg/mL.


Data regarding mean squares for FRAP assay (Table 4.19) indicated a significant effect of

fenugreek leaves powder supplementation in different treatments. Means for the given

79

parameter (Table 4.20) illustrated that T3 exhibited maximum reducing power (401.00±19.94

µmol Fe2+/g) followed by T2 (351.00±16.49 µmol Fe2+/g), T1 (257.00±11.82 µmol Fe2+/g) and

T0 (222.00±9.99 µmol Fe2+/g). The observation of present study revealed that reducing powder

of treatments increased with increased percentage of fenugreek leaves powder.

Analysis of variance for FRAP (Table 4.21) explicated momentous difference in values as a

function of fenugreek seeds powder addition in wheat flour. The mean values for FRAP (Table

4.22) ranged from 222.00±9.99 (T0) to 501.00±24.54 µmol Fe2+/g (T6) that indicates increasing

trend with increasing percentage of fenugreek seeds powder.

Reducing power is correlated with antioxidant activity and may be consider as significant

reflection of the antioxidant activity (Arabshahi-Delouee and Urooj, 2007). The results of the

present investigation for reducing power demonstrated the electron donor characteristic of

fenugreek, thus by neutralizing free radicals to form stable products. A study was carried out

by Khole et al. (2014) to separate and characterize bioactive molecules from germinated

fenugreek seeds. Ethyl acetate, water and n-butanol were used for extraction purpose and the

results for FRAP assay were obtained as 1.27±0.02, 131.027±11.05 and 0.14±0.007 mM

AEAC, respectively. Results of current investigation are comparable with earlier findings in

which meta-analysis was carried out and fenugreek seeds evaluated for FRAP assay. For this

purpose, fenugreek seeds powder extracted systematically, at ambient temperature selecting

solvents of varying polarity such as methanol, dichloromethane, hexane and water. The results

of the study gave a range for various solvents such as 0.135±0.05 to 77.352±0.62 TE mg/g

(Kenny et al., 2013). Thus, the high reducing power of fenugreek than wheat flour support the

findings of current investigation that increasing the percentage of fenugreek in wheat flour

increased reducing power.

4.8 Preparation of bread

In developing countries like Pakistan, demand of baked products like bread has being

increasing from the last few decades and to couple with need of ever-growing urban

population, the composite flour/bread technology could be very useful. The ingredients used

in composite flours depend on the availability of raw materials in the country concerned.

Pakistan is endowed with an enormous biodiversity of plant resources, which could be

exploited for this purpose. Unfortunately, many of these abundant resources remain largely

80

under-utilized like many indigenous crops that required low-input to cultivate but these can be

play their role through agricultural diversification and as a unique opportunity to fulfill food

requirements of increasing population as well as nutritional insecurity. With the current

emphasis on healthy bread with a low glycemic index, high protein and increased dietary fiber,

the use of composite flour in baked goods is to be favored. Therefore, given the inherent

nutritional and therapeutic advantages of these crops, they could find useful application in the

baking industry. Many studies have reported on the possibility of partially replacing wheat

flour with those obtained from local crops for the purpose of making bread and other baked

products (Mepba et al., 2007; Ade-Omowaye et al., 2008; Olaoye and Onilude, 2008), however

consumer acceptability of these products is still under study.

4.9 Proximate analysis of bread

4.9.1 Moisture content

The analysis of variance (Table 4.23) of moisture content exhibited significant difference with

the addition of fenugreek leaves powder in bread. The mean moisture content ranged from

34.46±1.09 to 34.68±1.21% (Table 4.24) with highest moisture content were observed in T3

(34.68±1.21%) while lowest in T0 (34.46±1.09%). The results for moisture described

increasing trend with the addition of fenugreek leaves powder.

Mean squares (Table 4.25) for moisture content of bread supplemented with fenugreek seeds

powder indicated that it was significantly affected by the incorporation levels. The mean

moisture content of bread varied from 34.46±1.06 to 34.63±10.17% (Table 4.26). The results

depicted increasing trend for moisture with increased supplementation T0 (34.46±1.09%), T4

(34.49±1.06%), T5 (34.56±1.35%) and T6 (34.63±1.17%), respectively.

The moisture content of bread prepared from different fenugreek leaves and seeds powder

supplemented flour has increased with increasing the supplementation level. In an earlier study

the moisture content of the composite bread increased with soybean flour substitution by a

range of 32.0 to 37.00% due to increased fiber and protein content. In a similar way increased

moisture content observed that associated with increase in fiber content (Akhtar et al., 2008;

Elleuch et al., 2011; Haruna et al., 2011). The results of present study also in harmony with

earlier study in which bread was prepared and chemical analysis revealed that moisture content

81

increased from 30.09 to 35.54% when supplementation of fenugreek increased from 5 to 15%

in wheat flour i.e. due to high crude fiber content of fenugreek (Kasaye and Jha, 2015).

4.9.2 Ash content

The statistical analysis (Table 4.23) for ash content showed significant difference among

different treatments with the addition of fenugreek leaves powder. The mean values (Table

4.24) depicted that lowest ash content were observed in T0 (1.24±0.02%), while highest in T3

(2.24±0.08). The results has revealed that the incorporation of fenugreek in bread resulted in

increased ash content i.e. @5% (1.74±0.03%) @10% (1.98±0.06%) and @15% (2.24±0.08%).

The analysis of variance (Table 4.25) for ash content of bread significantly differed with

supplementation of fenugreek seeds in different treatments. The mean values for ash content

in bread found in a wide range as T0 (1.24±0.02%), T4 (1.39±0.07%), T5 (1.55±0.05%) and T6

(1.72±0.06%) that shows increasing trend with addition of fenugreek seeds powder (Table

4.26).

In current study high ash content observed in supplemented bread i.e. mainly due to high

mineral content found in fenugreek leaves and seeds powder. The results are in harmony with

findings of Sudha et al. (2013) who prepared parathas with optimum levels of fenugreek

leaves, i.e. either 25% of normal dill/fenugreek leaves or 7.5% of dehydrated dill/fenugreek

leaves and evaluated their proximate composition. The ash content of parathas incorporated

with leaves were higher than the control paratha. The result for ash content also in accordance

with findings of Kasaye and Jha (2015) who reported ash content of bread increased from 1.85

to 2.97% when fenugreek blending increased up to 15% in wheat flour.

4.9.3 Crude protein

Mean squares in Table 4.23 indicated that crude protein content significantly affected by the

addition of fenugreek leaves powder. Means for the effect of fenugreek leaves powder (Table

4.24) depicted that the highest crude protein (11.12±0.55%) were recorded in T0 followed by

T1 (10.85±1.08%) while the lowest in T3 (10.29±1.07%). There was a decreasing trend for

protein content with the addition of fenugreek leaves powder.

82

Table 4.23 Mean squares for proximate analysis of bread supplemented with fenugreek leaves powder

SOV df Moisture Ash Crude Protein Crude Fat Crude Fiber NFE

Treatment 3 0.02523* 0.32094** 0.38463** 0.00228NS 0.01588** 0.01796NS

Error 8 0.00502 0.00042 0.00625 0.00033 0.00033 0.02133

Total 11

** = Highly significant

* = Significant

NS= Non-significant

Table 4.24 Proximate analysis (%) of bread supplemented with fenugreek leaves powder

Treatment Moisture Ash Crude Protein Crude Fat Crude Fiber NFE

T0 34.46±1.09a 1.24±0.02d 11.12±0.55a 5.97±0.15 1.27±0.01d 45.49±1.42

T1 34.54±0.91b 1.74±0.03c 10.85±1.08b 5.94±0.23 1.33±0.01c 45.53±1.17

T2 34.61±1.75ab 1.98±0.06b 10.57±0.90c 5.93±0.16 1.38±0.02b 45.60±1.19

T3 34.68±1.21ab 2.24±0.08a 10.29±1.07d 5.90±0.10 1.44±0.02a 45.67±1.88






83

Table 4.25 Mean squares for proximate analysis of bread supplemented with fenugreek seeds powder


Protein Crude Fat Crude Fiber NFE

Treatment 3 0.01994* 0.05873** 1.57943* 0.39865* 0.57634** 7.25433**

Error 8 0.00418 0.00051 0.28559 0.08222 0.00813 0.44778

Total 11


* = Significant

Table 4.26 Proximate analysis (%) of bread supplemented with fenugreek seeds powder

Treatment Moisture Ash Crude Protein Crude Fat Crude Fiber NFE

T0 34.46±1.09a 1.24±0.02d 11.12±0.55c 5.97±0.15c 1.27±0.01d 45.49±1.42a

T4 34.49±1.06b 1.39±0.07c 11.74±0.99b 6.25±0.23b 1.65±0.08c 44.35±2.08b

T5 34.56±1.35bc 1.55±0.05b 12.32±1.20ab 6.54±0.44ab 1.99±0.11b 43.04±2.13c

T6 34.63±1.17c 1.72±0.06a 12.79±1.06a 6.81±0.34a 2.29±0.11a 41.90±1.84d






84

The analysis of variance (Table 4.25) for crude protein content of bread made with fenugreek

seeds powder indicated that it was affected by variation in the levels of substitution. Mean

values for crude protein content (Table 4.26) revealed increasing trend as T0 (11.12±0.55%),

T4 (11.74±0.99%), T5 (12.32±1.20%) and T6 (12.79±1.06%).

When Indian parathas prepared either with 25% of normal dill/fenugreek leaves or 7.5% of

dehydrated dill/fenugreek leaves found protein content in range of 8.3 to 9.5% that are lower

than control. These findings are in accordance with our study that with increased

supplementation level of fenugreek leaves resulted in decreased protein content of bread i.e.

due to low protein content in fenugreek leaves as compared to wheat. On the other hand legume

seeds such as fenugreek is valuable source of protein and can be consider as supplement with

wheat flour for bread production (Kasaye and Jha, 2015). The high protein content determined

in bread prepared with different wheat-fenugreek blended flour and improvement in protein

content is due to high content available in fenugreek seeds. The findings of Hooda and Jood

(2003) & Eissa et al. (2007) also confirmed current study results who prepared biscuits with

different supplementation level of fenugreek seeds powder in wheat flour resulted in higher

protein content in supplemented biscuits when compared with control. Similar findings

observed by Chauhan and Sharma (2000) who determine protein content of bread and observed

increased protein content in bread prepared with flour supplemented with fenugreek seeds

powder. When bread was prepared with 5 to 20% supplementation of fenugreek seeds powder,

increased protein content observed when compared with control (Hooda and Jood, 2005). High

protein content (13.66%) observed in 15% blended wheat-fenugreek bread as compared to

control bread (Kasaye and Jha, 2015).

4.9.4 Crude fat

The analysis of variance (Table 4.23) for crude fat content of bread supplemented with

fenugreek leaves powder represented that there was non-significant difference among different

treatments. The mean crude fat content (Table 4.24) in bread prepared with different levels of

substitution was observed as T1 (5.94±0.23%), T2 (5.93±0.16%) and T3 (5.90±0.10%). The

highest fat content was observed in T0 (5.97±0.15%) having no supplementation and lowest in

T3 (5.90±0.10%) with 15% supplementation level. These results indicated that the replacement

of wheat flour with fenugreek leaves powder in bread resulted a gradual decrease in fat content.

85

The statistical analysis (Table 4.25) for crude fat content of bread supplemented with fenugreek

seeds powder referred that it was affected significantly with level of supplementation. Mean

crude fat content (Table 4.26) in T0 (control) was found to be 5.97±0.15%, whereas, the

incorporation of seeds powder in the bread resulted a progressive increase in fat content i.e. T4

(6.25±0.23%), T5 (6.54±0.44%) and T6 (6.81±0.34%).

The results of current study illustrated that fat content decreased with increasing the fenugreek

leaves powder in wheat flour i.e. due to low fat content of fenugreek leaves. In an earlier study

parathas (Indian bread) with optimum levels of leaves, i.e. either 25 % of normal dill/fenugreek

leaves or 7.5% of dehydrated dill/fenugreek leaves were prepared and observed fat content

8.9–9.2 % that are lower than control one (Sudha et al., 2013). However, fenugreek seeds has

higher fat content than wheat flour resulted in higher fat content in seeds powder supplemented

bread. Similar results were reported by Ibrahium and Hegazy (2009) that incorporation of

fenugreek flour in biscuits formula increased fat content. In similar way Mahmoud (2013)

determined chemical composition of non-fortified and fortified bread with two levels (5 and

10%) of fenugreek seeds and found higher fat content in fortified bread as compared to control.

The current study results are also comparable with findings of Kasaye and Jha (2015) who

observed high fat content in wheat-fenugreek blended flour bread i.e. 2.11% as compared to

wheat flour bread.

4.9.5 Crude fiber

Mean squares in Table 4.23 for crude fiber content of bread supplemented with fenugreek

leaves powder showed that it was significantly different among treatments. The mean crude

fiber content available in Table 4.24 revealed that incorporation of fenugreek leaves powder

in bread resulted an increase in fiber content i.e. @ 5% (1.33±0.01%), @ 10% (1.38±0.02%)

and @ 15% (1.44±0.02%), while lowest found in control (1.27±0.01%).

The analysis of variance (Table 4.25) for crude fiber content of bread supplemented with

fenugreek seeds powder indicated that there was a significant difference among treatments.

The mean fiber content (Table 4.26) were observed in the range of 1.27±0.01% (T0) to

2.29±0.11% (T6). The bread have different levels of seeds powder resulted an increase in fiber

content i.e. T4 (1.65±0.08%), T5 (1.99±0.11%) and T6 (2.29±0.11%). These results indicated

that fenugreek can be used as a good source of fiber in bread.

86

The crude fiber content of the supplemented bread increased with progressive inclusion of

fenugreek powder in wheat flour. The health benefits of high fiber diets is getting popular in

now a days busy life and people are becoming more conscious for healthy diets like whole

grain bread. Supplementation has been consider as important toll to improve fiber availability

and fiber fortified baked products are available for consumers since years (Eastwood and

Kritchevsky, 2005). The fiber content of Indian parathas found higher enriched either with

25% of normal dill/fenugreek leaves or 7.5% of dehydrated dill/fenugreek leaves when

compared to the control paratha (Sudha et al., 2013). Similar findings were observed when

bread was prepared with 5 to 20% supplementation of fenugreek powder in wheat flour

resulted in higher fiber content (Hooda and Jood, 2005). Likewise, bread prepared from wheat-

fenugreek blended flour found increased fiber content from 0.65 to 3.08% as supplementation

of fenugreek powder increased (Kasaye and Jha, 2015) that supports the findings of current

study.

4.9.6 Nitrogen free extract (NFE)

Mean squares (Table 4.23) for NFE in bread prepared with fenugreek leaves powder stated

that the difference in NFE was non-significant. Mean values has been given Table 4.24

revealed that NFE content in bread supplemented with different levels of fenugreek leaves

powder ranged from 45.49±1.42 to 45.67±1.88%. The highest value observed in T3

(45.67±1.88%) followed by T2 (45.60±1.19%) and T1 (45.53±1.17%) while lowest value was

observed in T0 (45.49±1.42%).

The analysis of variance (Table 4.25) for NFE in bread prepared with fenugreek seeds powder

specified that the differences in NFE were highly significant. Mean values for NFE have been

illustrated in Table 4.26 revealed a decreasing trend as T0 (45.49±1.42%), T4 (44.35±2.08%),

T5 (43.04±2.13%) and T6 (41.90±1.845%).

The chemical composition of non-fortified and fortified bread with two levels (5 and 10%) of

fenugreek seeds was determined. It was observed that fortified bread with fenugreek seeds

powder found increased total protein, fat, ash and fiber content, while moisture content and

carbohydrates decreased than that of the non-fortified bread (Mahmoud, 2013). The moisture,

ash, protein, fat and fiber content increased with increasing supplementation level of wheat-

fenugreek blended bread while carbohydrates decreased. Bread prepared from wheat flour has

87

NFE 65.59% as compared to bread prepared with supplementation of fenugreek in wheat flour.

NFE content decreased from 65.59 to 55.72% when supplementation increased up to 15% in

wheat flour (Kasaye and Jha, 2015). So, the results of the current study are in line with earlier

research work of different scientist who also observed with increasing fenugreek seeds powder

supplementation increased the nutritional profile of bread.

4.10 Mineral content of bread

4.10.1 Sodium (Na)

Mean squares (Table 4.27) for sodium content in bread supplemented with fenugreek leaves

powder showed significant difference among different treatments. The mean values for sodium

content ranged from 4.10±0.15 (T0) to 12.28±0.57 mg/100gm (T3) as given in Table 4.28. The

results showed that sodium content increased with increasing the percentage of fenugreek

leaves powder.

Analysis of variance (Table 4.29) for sodium content in bread indicated significant effect with

the addition of fenugreek seeds powder in different treatments. Means for different treatments

(Table 4.30) explained that maximum sodium was (12.83±0.59 mg/100g) found in T6 followed

by T5 (10.05±0.45 mg/100g), T4 (7.02±0.30 mg/100g) whereas, control (T0) showed minimum

value (4.10±0.15 mg/100g). The Na content exhibited increasing trend by increasing the

fenugreek seeds powder in prepared bread.

The high sodium content of bread prepared from different wheat-fenugreek composite flour

resulted due to high sodium content present in fenugreek. Salt plays an important functional

roles during bread making process like it not only rises the dough osmotic pressure as well as

partially neutralizes electrostatic repulsion between gluten proteins. The combined role of

these function have significant effect on quality of the bread. Due to gluten protein

strengthening effect, gas bubbles expand to their maximum size without rupture. Bread

prepared without addition of salt resulted with large bubbles, attached with collapsed bubbles

due to their poor gluten strength, leads to poor loaf volume. The mixing time of dough and

mixing tolerance index increased through salt and gluten proteins interaction as well as it also

improves sensory characteristics of bread (Heidolph et al., 2011).

88

4.10.2 Potassium (K)

Statistical analysis depicted in (Table 4.27) for potassium content (K) in different treatments

revealed that in prepared bread potassium (K) content significantly differed with the addition

of fenugreek leaves powder. Mean values (Table 4.28) illustrated that K content ranged from

124.00±6.20 (T0) to 177.18±8.86 mg/100g (T3). The highest K content during analysis were

observed in T3 (177.18±8.86 mg/100g) followed by T2 (159.46±7.97 mg/100g), T1

(141.73±7.09 mg/100g) whilst the lowest value was exhibited in T0 (124.00±6.20 mg/100g).

Mean squares given in Table 4.29 indicated that potassium content (K) of bread prepared from

different treatments affected significantly with the addition of fenugreek seeds powder. Means

for potassium content (Table 4.30) exhibited highest value (187.30±9.36 mg/100g) for T6

trailed by T5 (166.20±8.31 mg/100g) and T4 (145.10±7.25 mg/100g). The minimum content

was observed in T0 (124.00±6.20 mg/100g) that revealed that potassium content increased with

increased supplementation of fenugreek seeds powder in bread.

In the present research work, we found higher potassium content in bread prepared from

different treatments due to high potassium content in fenugreek. It is a good source of

potassium as reported by Naidu et al. (2012) who determined 4.7 mg/100g of fenugreek leaves.

Bread prepared with white flour with the addition of three different percentage of sodium

chloride and equal mixture of sodium chloride with potassium chloride (NaCl/KCl). The

0.75% level of sodium chloride and 1.0% level of KCl/NaCl were not significantly differ in

flavor when compared with control bread and made acceptable by panel of judges (Wyatt and

Ronan, 1982). Potassium plays an important role in controlling blood pressure. So keeping this

in view a study was conducted in which two bread were prepared, one with 30% replacement

of sodium with potassium salts and second with soy flour replaced 10% of the wheat flour.

The sensory results were acceptable and found alike control bread (Braschi et al., 2009).

4.10.3 Iron (Fe)

It is obvious from the statistical analysis (Table 4.27) that iron (Fe) in prepared bread have non-

momentous difference among each other with the addition of fenugreek leaves powder. It was

observed that the Fe content ranged from 3.44±0.17 (T0) to 3.86±0.19 mg/100gm (T3) among

different treatments (Table 4.28). Although, addition of fenugreek leaves powder gradually

increased the iron content but non-significant among different treatments.

89

Table 4.27 Mean squares for mineral content of bread supplemented with fenugreek leaves powder


Treatment 3 38.5587** 1571.23** 0.09361NS 3997.19** 0.00065NS 0.00438* 0.00018NS

Error 8 0.1541 57.68 0.03339 12.23 0.00081 0.00104 0.00086

Total 11


NS= Non Significant

Table 4.28 Mineral content (mg/100g) of bread supplemented with fenugreek leaves powder


T0 4.10±0.15d 124.00±6.20c 3.44±0.17 19.96±1.00d 0.11±0.01 0.17±0.01b 0.66±0.03

T1 6.81±0.29c 141.73±7.09bc 3.59±0.18 48.23±2.41c 0.12±0.01 0.21±0.03ab 0.69±0.04

T2 9.54±0.43b 159.46±7.97ab 3.71±0.18 76.50±3.82b 0.14±0.05 0.22±0.03ab 0.70±0.09

T3 12.28±0.57a 177.18±8.86a 3.86±0.19 104.78±5.24a 0.16±0.01 0.26±0.05a 0.71±0.08






90

Table 4.29 Mean squares for mineral content of bread supplemented with fenugreek seeds powder


Treatment 3 43.7094** 2226.19** 3.11267** 233.518** 0.00419** 0.04627** 0.00210NS

Error 8 0.01680 61.96 0.05490 2.427 0.00033 0.00100 0.00131

Total 11


NS= Non Significant

Table 4.30 Mineral content (mg/100g) of bread supplemented with fenugreek seeds powder







T0 4.10±0.15d 124.00±6.20c 3.44±0.17d 19.96±1.00d 0.11±0.01c 0.17±0.01c 0.66±0.03

T4 7.02±0.30c 145.10±7.25c 4.23±0.21c 26.79±1.34c 0.14±0.01bc 0.28±0.04b 0.71±0.05

T5 10.05±0.45b 166.20±8.31b 5.01±0.25b 33.62±1.68b 0.17±0.02ab 0.36±0.03b 0.73±0.07

T6 12.83±0.59a 187.30±9.36a 5.81±0.29a 40.46±2.02a 0.19±0.02a 0.46±0.02a 0.74±0.09

91

Mean squares (Table 4.29) showed that iron content of bread prepared from different

treatments significantly affected by the addition of fenugreek seeds powder. Mean values

(Table 4.30) showed that maximum value (5.81±0.29 mg/100g) was recorded in T6 followed

by (5.01±0.25) in T5, (4.23±0.21) in T4 and minimum (3.44±0.17 mg/100g) in T0 (control).

This parameter gradually increased with increasing the supplementation of fenugreek seeds

powder.

The concentration of iron increased in bread when supplemented with 5-15% of fenugreek

powder when compared with control bread (Fretzdorff and Brummer, 1992). The

supplementation of fenugreek leaves powder (100g/meal) in standard cereal meal, significantly

increased the iron content (3.24 mg to 9.12 mg). This could be credited to the high iron content

of fenugreek leaves (Jonnalagadda and Seshadri, 1994). The results of current study also

confirmed by Hooda and Jood (2005), who determined low iron content in control biscuits and

it significantly increased with supplementation of fenugreek powder in wheat flour due to high

iron content of fenugreek powder. The addition of fenugreek seeds powder increased iron

content in biscuits as compared to wheat flour biscuits (Ibrahium and Hegazy, 2009). Similar

higher iron content (11.95mg/100g) of biscuits observed by Mahmoud et al. (2012). In another

study the iron content found in range of 7.79 to 8.03 mg/100g of bread when prepared with

wheat-fenugreek blended flour as compared to wheat flour (Kasaye and Jha, 2015).

4.10.4 Calcium (Ca)

Mean squares (Table 4.27) for calcium (Ca) content in bread prepared from different

treatments indicated that these content differed significantly with supplementation of

fenugreek leaves powder. Mean values illustrated that Ca content in bread ranged from

19.96±1.00 to 104.78±5.24 mg/100g. The maximum value was observed in T3 i.e. 104.78±5.24

mg/100g followed by T2 (76.50±3.82 mg/100g), T1 (48.23±2.41 mg/100g) and T0 (19.96±1.00

mg/100g) (Table 4.28).

Analysis of variance indicated that calcium content varied significantly with the addition of

fenugreek seeds powder in bread prepared from different treatments (Table 4.29). Mean

calcium content (Table 4.30) in bread indicated that it increased with the addition of fenugreek

seeds powder with highest value found in T6 i.e. 40.46±2.02 mg/100g and lowest 19.96±1.00

mg/100g in T0.

92

The calcium content of bread increased by fenugreek addition in wheat flour @ 5 to 15% as

compared to wheat flour bread (Fretzdorff and Brummer, 1992). The value of calcium content

was 58.70 mg/100g in control bread and it increased to 59.76 mg/100g when supplementation

of fenugreek powder was carried out in wheat flour (Hooda and Jood, 2005). The earlier

findings also strengthen the results of present investigation that mineral content of bread

increased when prepared with wheat-fenugreek blended flour as compared to wheat flour. The

calcium content determined in bread varied from 64.13 to 78.04 mg/100g when supplemented

with various percentage of fenugreek powder. In the same way when biscuit prepared with

wheat-fenugreek blended flour and analyzed for mineral profile found highest calcium

(70.71±0.39 mg/100g) when wheat supplemented with 15% fenugreek flour while the lowest

(56.82±0.30 mg/100g) for control (Kasaye and Jha, 2015).

4.10.5 Copper (Cu)

The analysis of variance (Table 4.27) for copper content in different treatment bread showed

non-momentous difference among each other with the addition of fenugreek leaves powder.

The mean Cu values (Table 4.28) ranged from 0.11±0.01 to 0.16±0.01 mg/100g with highest

Cu content was explicated in T3 (0.16±0.01 mg/100g) followed by T2 (0.14±0.05 mg/100g),

T1 (0.12±0.01 mg/100g) whilst the lowest was exhibited in T0 (0.11±0.01 mg/100g).

Mean squares (Table 4.29) for copper content in bread prepared from different treatments

explained that there was significant effect of fenugreek seeds powder supplementation. Mean

values related to copper content have been depicted in Table 4.30. The copper content ranged

from 0.11±0.01 to 0.19±0.02 mg/100g that exhibited copper content increased with the

addition of fenugreek seeds powder.

The mineral content of wheat flour increased with supplementation of fenugreek powder.

Likewise, biscuits prepared with supplemented flour exhibited higher copper content that

might be credited to higher mineral content of fenugreek powder (Hooda and Jood, 2005). It

was observed in the current study that supplementation of fenugreek powder to wheat flour

significantly increased copper content of bread that also in harmony with the findings of

Kasaye and Jha (2015).

93

4.10.6 Zinc (Zn)

The statistical analysis for zinc content in different treatments has been presented in Table 4.27

which indicated that the zinc content differed significantly with the addition of fenugreek

leaves powder. Mean values of zinc content varied from 0.17±0.01 to 0.26±0.05 mg/100g. The

highest zinc content was observed in T3 (0.26±0.05 mg/100g) followed by T2 (0.22±0.03

mg/100g), T1 (0.21±0.03 mg/100g) and T0 (0.17±0.01 mg/100g) (Table 4.28).

Mean squares for zinc content of different treatment bread has been given in Table 4.29. It is

obvious from the results that zinc content significantly affected by the addition of fenugreek

seeds powder. Zinc content ranged from 0.17±0.01 to 0.46±0.02 mg/100g among different

treatments (Table 4.30) that revealed that zinc content of bread increased with increasing

supplementation level of fenugreek powder.

It was apparent that the supplementation of fenugreek powders to wheat flour significantly

increased zinc content in bread and current exploration is supported by Hooda and Jood (2005)

who observed zinc content of bread increased when wheat flour supplemented with fenugreek

powder and result found in the range of 3.51 mg/100g to 3.84 gm/100g. In another study similar

increased of Fe, Zn and Ca content were observed in fenugreek supplemented biscuits

(Ibrahium and Hegazy, 2009). The zinc content of bread increased from 2.15±0.00 to

2.44±0.07 mg/100g when prepared with wheat-fenugreek blended flour as compared to wheat

flour. In the same study when biscuit prepared with wheat-fenugreek blended flours and

analyzed for mineral profile found zinc content in the range of 1.65±0.00 to 2.01±0.05 mg/100g

(Kasaye and Jha, 2015).

4.10.7 Manganese (Mn)

Statistical results given in Table 4.27 illustrated that manganese content in different treatments

showed non-significant results with supplementation of fenugreek leaves powder. Mean values

(Table 4.28) exhibited that Mn content ranged from 0.66±0.03 to 0.71±0.08 mg/100g with

highest Mn content found in treatment having 15% fenugreek leaves powder (T3).

Mean squares (Table 4.29) showed that Mn content of different treatment bread significantly

affected by the supplementation of fenugreek seeds powder. The mean values for Mn content

varied from 0.66±0.03 to 0.74±0.09 mg/100g and the highest content observed in T6

94

(0.74±0.09 mg/100g) followed by T5 (0.73±0.07 mg/100g), T4 (0.71±0.05 mg/100g) and T0

(0.66±0.03 mg/100g). The result exhibited that Mn content gradually increased with increasing

the percentage of fenugreek seeds powder.

The higher Mn content in fenugreek-wheat supplemented bread were observed when compared

with the control bread. The results are in harmony with observations of Mahmoud et al. (2012)

who determined higher mineral content like Mn in the range of 0.34 to 0.48 mg/100g with 10%

supplementation in biscuits as compared to control. Fenugreek has higher mineral content as

compared to wheat flour and our results are also in agreement with outcomes of Singh et al.

(2013) who found manganese content in range of 1.79 to 1.35 mg/100g, with an average of

1.53mg/100g in fenugreek seeds.

4.11 Antioxidant assay of bread

Phytochemicals are non-nutritive plant chemicals that are associated with protective or disease

preventing role against different chronic diseases. On the other hand, many of these

phytochemicals together with other seeds components are regarded as anti-nutritional factors.

Therefore, with regard to these phytochemicals or anti-nutritional factors, the objective of

producing composite baked goods may either be to retain them in order to exert potential health

benefits or to use the processing to eliminate or inactivate them so as to reduce their anti-

nutritional effects. However, it appears that with regard to composite baked goods, not much

research has been conducted to determine the effect of the compositing and processing on

levels of these phytochemicals (Anton et al., 2008).

The phenols content of bread prepared with wheat flour in current study were lower as

compared to respective supplemented flour. The baking process degraded or damaged the

active compounds responsible for antioxidant activity of flour and during other process like

dough mixing and kneading losses are also observed for antioxidants. In bread antioxidant

activity can be modified by active oxidative enzymes available in different ingredients used

during production or oxidized by ambient oxygen (Holtekjolen et al., 2008; Leenhardt et al.,

2006).

95

4.11.1 Total Phenolic Content (TPC)

The mean squares in (Table 4.31) found momentous effect on TPC with supplementation of

fenugreek leaves powder among different treatments. Means (Table 4.32) related to TPC

showed momentous variations among treatments i.e. T0, T1, T2 & T3; 99.00±4.45, 198.00±9.10,

213.00±10.01 and 315.00±15.43 mg GAE/100g, respectively. The results showed that there is

significant increase in TPC value with increasing the supplementation of fenugreek leaves

powder.

The bread supplemented with fenugreek seeds powder exhibited a significant difference in

TPC among different treatments (Table 4.33). Means from Table 4.34 indicated highest TPC

(413.00±19.82 mg GAE/100g) in T6 followed by T5 (341.00±16.02 mg GAE/100g) and T4

(238.00±10.94 mg GAE/100g). The increasing trend was observed for this trait with increasing

the supplementation of fenugreek seeds.

According to findings of current investigation TPCs found lower in wheat flour bread when

compared with fenugreek-wheat supplemented bread. The study conducted by Premanath et

al. (2011) observed 4.9 mg/g polyphenols in ethanol extract of fenugreek leaves powder. In

another research work mean phenolic content found to be 52.8 mg/g GAE in fenugreek leaves

supplemented chicken patties (Devatkal et al., 2012). The maximum content of 48 mg/g GA

equivalent of total phenolic in fenugreek leaves extract determined by Naidu et al. (2011) and

also reported the antioxidant activity may be related with polyphenol content present in

fenugreek. However, low total phenolic content i.e. 0.52 and 1.01 mg FAE/g determined in

refined and whole wheat flour bread (Preedy et al., 2011) that in agreement with current study

findings. The total phenolic content found in bread prepared with refined and whole wheat

flour as 0.87 and 1.58 mg FAE/g, respectively (Yu and Nanguet, 2013). During baking

decreased of 67% and 72% of total phenolic content observed in refined and whole wheat flour

bread due to loss of heat liable phenolic acid like vitamin C (Han and Koh, 2011).


Analysis of variance for total flavonoids (Table 4.31) explicated momentous difference in

values as a function of fenugreek leaves powder supplementation among different treatments.

Means for total flavonoids (Table 4.32) for different treatments; T0 (2.13±0.09 mg CE/g), T1

96

(2.47±0.08 mg CE/g), T2 (2.65±0.11 mg CE/g) and T3 (2.84±0.13 mg CE/g), showing

maximum results for treatment with 15% supplementation.

Mean squares for total flavonoids (Table 4.33) showed significant effect among treatments

with supplementation of fenugreek seeds powder. As examined from mean values (Table 4.34),

maximum flavonoids value (2.91±0.12 mg CE/g) was recorded in 15% fenugreek seeds

powder supplemented treatment (T6) while minimum (2.13±0.19 mg CE/g) in T0.

The shielding impact of fenugreek against chronic diseases has been accredited to the

antioxidant potential of its flavonoid content like alkaloids, flavonoids and saponins (Kumar

et al., 2013). The ethanol extract of fenugreek leaves powder found flavonoids 0.47 mg/g as

reported by Premanath et al. (2011). The study of antioxidant properties of fenugreek seeds

exhibited that significant antioxidant activity in seeds may be due to presence of polyphenols

and flavonoids (Dixit et al., 2005). The content of total flavonoids in wheat-fenugreek

supplemented bread were found higher when compared with control bread because fenugreek

powder is a better source of flavonoid compounds than wheat flour. However, wheat-fenugreek

flour bread having flavonoids content lower than supplemented flour. The possible reason of

loss during processing conditions like baking, there might be loss of flavonoid compounds due

to thermal process that leads to reduce antioxidant activities (Dietrych-Szostak and Oleszek,

1999). Therefore, it is recommended that to keep such losses minimum conditions of product

processing should be optimized keeping in view nature of the product.

4.12 Antioxidant activity of bread

4.12.1 Free radical scavenging activity (DPPH Assay)

Mean squares regarding DPPH scavenging activity of different treatments has been presented

in Table 4.31. A significant difference was observed as a function of fenugreek leaves powder

addition in different treatments. Means shown in Table 4.32 indicated that free radical

scavenging activities of different treatments ranged from 31.00±1.27 to 43.00±1.97%. In

addition, the highest scavenging activity was found in T3 (43.00±1.97%) that revealed that

activity increased with increasing the supplementation level.

Analysis of variance for DPPH scavenging activity (Table 4.33) explicated momentous

difference in values as a function of fenugreek seeds powder among different treatments.

Means for DPPH scavenging activity (Table 4.34) for the different treatments; T0

97

(31.00±1.24%), T4 (45.00±1.93%), T5 (49.00±2.20%) and T6 (51.00±2.39%), showing

increasing trend with increased percentage of fenugreek seeds powder.

The antioxidant strength of bread prepared from different treatments was estimated by using

the free radical scavenging (DPPH) activity. The DPPH value for bread prepared from wheat

flour found lower than fenugreek-wheat supplemented bread. The bread prepared from refined

and whole wheat flour having DPPH values in the range of 2.83 to 3.90 μmol TE/g and 2.79

to 4.05 μmol TE/g (Yu and Nanguet, 2013). However, average DPPH value was observed

64.2% in fenugreek leaves supplemented chicken patties due to higher activity of fenugreek

(Devatkal et al., 2012). During the process of bread production the scavenging activity found

decreased i.e. in harmony with the early investigation in which average scavenging ability

observed decreasing trend of 30 to 32%, respectively. The loss of this activity may be due to

loss of phenolic compounds, because high temperature required during baking leads to

destruction of these compounds (Han and Koh, 2011).

4.12.2 β-carotene and linoleic acid assay

Mean squares in Table 4.31 indicated β-carotene values were significantly affected by

fenugreek leaves powder supplementation in bread. Means for this trait (Table 4.32) exposed

that the highest β-carotene values 35.00±1.68% was recorded in T3 followed by T2

(33.00±1.45%) and T1 (31.00±1.42%) while the lowest 29.00±1.18% in T0.

Mean squares presented in Table 4.33 indicated significant effect of fenugreek seeds powder

addition in different treatments. Means for the effect of β-carotene values (Table 4.34)

illustrated that maximum value for this trait was found in T6 (43.00±2.10%), while minimum

in T0 (29.00±1.30%). The results of present study exhibited that β-carotene values increased

with increasing the supplementation of fenugreek in prepared bread.

In a previous study, higher content of ß-carotene (22.5mg/100 g) observed in low humidity air

dried fenugreek leaves compared to radiofrequency dryer dried sample (6.2 mg/100 g; 76.2

mg/100 g) and hot air dried fenugreek leaves (6.0 mg/100 g; 148.1 mg/100 g) (Naidu et al.,

2012). The supplementation of fenugreek seeds in the diet of rats prevented enzymatic leakage,

alter lipid peroxidation and enhanced antioxidant potential (Thirunavukkarasu et al., 2003). So

fenugreek leaves and seeds can be a potential source of bioactive compounds and addition of

fenugreek leaves powder in rats diet @ of 1g/kg of body weight lowered lipid peroxidation

98

and improved significant system (Annida and Prince, 2004). No bioactive components were

detected in paratha prepared with wheat flour. However, β-carotene content found slightly

higher when 25% of normal fenugreek leaves were incorporated in wheat flour (Sudha et al.,

2013).


Data regarding mean squares for FRAP assay (Table 4.31) indicated a significant effect of

fenugreek leaves powder supplementation. Means for the given parameter (Table 4.32)

illustrated that T6 exhibited maximum reducing power (356.00±17.08 µmol Fe2+/g) followed

by T2 (254.00±11.93 µmol Fe2+/g) and T1 (201.00±9.24 µmol Fe2+/g) while minimum in T0

(167.00±7.51 µmol Fe2+/g), that indicated increasing trend with increasing supplementation

level.

Mean squares for FRAP assay showed significant effect of fenugreek seeds powder addition

in bread prepared from different treatments (Table 4.33). As indicated from mean values (Table

4.34), highest reducing power (450.00±21.60 µmol Fe2+/g) was recorded in 15% seeds powder

supplementation while lowest (167.00±7.01 µmol Fe2+/g) in wheat flour (T0).

The findings of the present study revealed that reducing power increased with increasing the

fenugreek supplementation in bread. Fenugreek has more reducing power when compared with

wheat flour as discuss earlier. In a meta-analysis, fenugreek seeds were evaluated for FRAP

assay. For this purpose, fenugreek seeds powder extracted systematically, at ambient

temperature selecting solvents of varying polarity such as methanol, dichloromethane, hexane

and water. The results of the study gave a range for various solvents in the range of

0.135±0.055 to 77.352±0.627 TE mg/g (Kenny et al., 2013).

4.13 Color of bread

Color tonality includes L*, a*and b*, Chroma and Hue angle values. Usually, L* value

indicates lightness of the tested substance, while b* gives indication of yellowness whereas a*

indicates greenness and redness of the final product. Chroma inform about color intensity,

while tan inverse of a/b is called as hue angle. Mean squares regarding attributes of color

tonality indicated that supplementation of fenugreek leaves and seeds powder significantly

affected these traits (Table 4.35, 4.37).

99

Table 4.31 Mean squares for antioxidants in bread supplemented with fenugreek

leaves powder

Source df TPC Flavonoids DPPH

β-carotene &

Linoleic acid

assay

FRAP

Treatment 3 23442.4** 0.27722** 74.9957** 20.0000** 20420.5**

Error 8 110.3 0.01167 2.7506 2.0945 144.2

Total 11


Table 4.32 Antioxidants in bread supplemented with fenugreek leaves powder

Trt.

TPC

(mg GAE/

100g)

Flavonoids

(mg CE/g )

DPPH

(%)

β-carotene &

Linoleic acid

assay (%)

FRAP

(µmol Fe2+/g)

T0 99.00±4.45c 2.13±0.09c 31.00±1.27c 29.00±1.18c 167.00±7.51d

T1 198.00±9.10b 2.47±0.08b 37.00±1.59b 31.00±1.42bc 201.00±9.24c

T2 213.00±10.01b 2.65±0.11ab 39.00±1.71ab 33.00±1.45ab 254.00±11.93b

T3 315.00±15.43a 2.84±0.13a 43.00±1.97a 35.00±1.68a 356.00±17.08a






100

Table 4.33 Mean squares for antioxidants in bread supplemented with fenugreek

seeds powder


β-carotene &

Linoleic acid

assay

FRAP

Treatment 3 55723.6** 0.32412** 244.040** 108.750** 48330.0**

Error 8 197.4 0.01112 3.976 3.143 290.7

Total 11


Table 4.34 Antioxidants in bread supplemented with fenugreek seeds powder

Trt.

TPC

(mg GAE/

100g)

Flavonoids

(mg CE/g )

DPPH

(%)

β-carotene &

Linoleic acid

assay (%)

FRAP

(µmol Fe2+/g)

T0 99.00±4.45d 2.13±0.19c 31.00±1.24c 29.00±1.30c 167.00±7.01c

T4 238.00±10.94c 2.50±0.10b 45.00±1.93b 37.00±1.70b 398.00±17.51b

T5 341.00±16.02b 2.68±0.10ab 49.00±2.20ab 40.00±1.88ab 401.00±18.44b

T6 413.00±19.82a 2.91±0.12a 51.00±2.39a 43.00±2.10a 450.00±21.60a






101

Means regarding L* values of bread containing fenugreek leaves powder are presented in

Table (4.36). Progressive increase in leaves powder gave lower L* values; maximum

(87.96±0.13) was recorded in T0 (control), while minimum value of 43.01±0.08 was observed

in T3 (15% fenugreek leaves powder). Likewise, fenugreek seeds powder addition decreased

L* from 87.96±0.13 to 60.16±0.05 (Table 4.38).

Increasing the supplementation of fenugreek leaves powder resulted a marked decrease in a*

(Table 4.36) from 2.92±0.11 in T0 (control) to -3.00±0.03 in T3 (15% fenugreek leaves

powder). The bread with varying level of fenugreek seeds powder (Table 4.38) resulted in

progressive increase in a* from 2.92±0.11 in T0 (control) to 5.10±0.03 in T6 (15% fenugreek

seeds powder).

It is evident from results (Table 4.38) that b* values decreased as a function of fenugreek leaves

powder i.e. 22.26±0.04 in T0 (control) to 22.02±0.02 in T3 (15% fenugreek leaves powder). In

contrary, progressive increase in fenugreek seeds powder concentrations gave momentous

increase; minimum value of 22.26±0.04 was recorded in T0 (control), while maximum

(27.41±0.04) was recorded in T6 (Table 4.38).

Means depicting the effect of treatments on Chroma (Table 4.36) exhibited that maximum value

(27.88±0.03) was recorded in T3 (15% fenugreek leaves powder), while minimum value

(22.45±0.05) was observed in T0 (control). Likewise, treatments containing fenugreek seeds

powder indicated that additional concentrations gave progressive increase in Chroma values.

Maximum value of 26.00±0.08 was recorded in T6 (15% fenugreek seeds powder), while

minimum value of 22.45±0.05 was observed in T0 (Table 4.38).

It is depicted from Table (4.36) that increasing the concentration of fenugreek leaves powder

resulted in obvious decrease of Hue angle; maximum Hue angle (82.52±0.06) was observed in

T0 (control), while minimum value of -82.25±0.07 in T3 (15% fenugreek leaves powder). The

addition of fenugreek seeds powder decrease Hue angle from 82.52±0.02 in T0 (control) to

79.47±0.08 in treatment having 15% fenugreek seeds powder (Table 4.38).

From consumers point of view color is very important sensory attribute that act as first appeal

to make choice of a product. However, development of color also indicate the completion of

product production process like golden brown color development at the end of baking process.

102

The end product color of products like bakery products depends of physical-chemical

properties of the raw materials and working conditions applied during baking (Zanoni et al.,

1995). In the present study color changes with the addition of fenugreek leaves and seeds

powder were found to be significant. The increase in color tonality was certainly due to

presence of coloring pigments (Cheikh-Rouhou et al., 2008). Color tonality in paratha was

studied by Sudha et al. (2013) who observed with increasing levels of dried fenugreek leaves

in parathas from 5 to 15%, the lightness value decreased, and the parathas had increasing green

color, which is due to the high green coloration of normal fenugreek leaves. Findings of

Hussein et al. (2011) also supported the current findings who observed color of final product

is dependent upon the raw materials used that ultimately affect tonality values. L values of all

legume supplemented flour found declining trend with compared with control. In another study

the color of fenugreek-wheat supplemented biscuits getting darker, redder (a-values) and with

higher browning index (BI) as compared to control samples. Also the results of a-values

observed higher in fenugreek-wheat flour biscuit as compared to control. Similar results were

also reported when germinated fenugreek powder added in wheat flour for product

development (Eissa et al., 2007).

In another study of Srivastava (2012) who observed L-value decrease from 68.00 to 57.30 with

15% supplementation of fenugreek seeds husk in muffins. On the other hand (+b) value

increased from 17.63 to 23.00 for said product. The study of Jyotsna et al. (2011) also

strengthen our results who experienced higher “b” values in vermicelli prepared with

fenugreek-wheat supplemented flour that could be due to the presence of carotene content in

fenugreek seeds.

4.14 Texture of bread

4.14.1 Texture profile of bread supplemented with leaves powder

It is evident from mean squares in Table 4.39 regarding texture of bread supplemented with

leaves powder that non-significant variations were observed for the effect of treatments on all

textural parameters like hardness, gumminess, cohesiveness, elasticity, chewiness and

springiness. The mean values of textural parameters like hardness, gumminess, cohesiveness,

springiness, chewiness and elasticity are presented in Table 4.40.

103

Table 4.35 Mean squares for color of bread supplemented with fenugreek leaves

powder

SOV Df L* value a* value b* value Chroma Hue angle

Treatment 3 1329.61** 24.0764** 9.69105** 21.9979** 20669.7**

Error 8 0.10 0.0035 0.00259 0.0023 0.01987

Total 11


Table 4.36 Mean values for color of bread supplemented with fenugreek leaves

powder

Trt. L* value a* value b* value Chroma Hue angle

T0 87.96±0.13a 2.92±0.11a 22.26±0.04c 22.45±0.05c 82.52±0.02a

T1 49.61±0.61b -2.01±0.02b 25.92±0.08a 23.40±0.04c -85.56±0.04c

T2 45.99±0.08c -2.99±0.01c 22.99±0.03b 26.02±0.04b -82.59±0.09b

T3 43.01±0.08d -3.00±0.03c 22.02±0.02d 27.88±0.03a -82.25±0.07b






104

Table 4.37 Mean squares for color of bread supplemented with fenugreek seeds

powder

SOV df L* value a* value b* value Chroma Hue angle

Treatment 3 454.732** 2.73505** 20.4063** 9.07592** 6.52997**

Error 8 0.006 0.00428 0.0020 0.00300 0.02433

Total 11


Table 4.38 Mean values for color of bread supplemented with fenugreek seeds powder

Trt. L* value a* value b* value Chroma Hue angle

T0 87.96±0.13a 2.92±0.11d 22.26±0.04c 22.45±0.05d 82.52±0.02a

T4 68.00±0.03b 4.01±0.04c 22.08±0.04d 22.71±0.02c 79.70±0.11b

T5 64.40±0.05c 4.72±0.03b 25.59±0.04b 23.18±0.03b 79.56±0.06b

T6 60.16±0.05d 5.10±0.03a 27.41±0.04a 26.00±0.08a 79.47±0.08b






105

It is evident from results that hardness of the bread increased non-significantly among

treatments from T0 to T3 with increasing the supplementation of fenugreek leaves powder. The

highest value of hardness was observed in T3 (3.63±0.05) while lowest was found in T0

(3.51±0.03). While treatments T1 and T2 having values 3.56±0.04 and 3.59±0.06, respectively.

As for as cohesiveness is concerned the mean Table 4.40 exhibited that cohesiveness of the

bread supplemented with leaves powder increased non-significantly in different treatments.

The highest value of cohesiveness was found in T3 (0.95±0.02) followed by T2 (0.93±0.05), T1

(0.92±0.03) and T0 (0.91±0.04).

Mean values for gumminess of bread supplemented with fenugreek leaves powder given in

Table 4.40 showed non-significant increasing trend. Highest value of gumminess was observed

in T3 (3.24±0.03) while lowest in T0 (3.19±0.03) that shows gumminess increased with

increasing percentage of fenugreek leaves powder in bread.

Springiness of fenugreek leaves powder supplemented bread increased down the treatments

from T0 to T3 as presented in Table 4.40. The mean value ranged between 0.98±0.03 (T0) to

1.05±0.01 (T3) and results exhibited that springiness increased non-significantly with the

addition fenugreek leaves powder.

Mean values for chewiness of bread supplemented with fenugreek leaves powder are available

in Table 4.40. It is obvious from the results that chewiness increased non-significantly among

treatments by the incorporation of fenugreek leaves powder. The lowest value for chewiness

was found in T0 (3.13±0.03) while highest in T3 (3.18±0.02). However, T1 and T2 showed

values 3.15±0.01 and 3.17±0.01, respectively.

The mean values regarding bread elasticity given in Table 4.40 indicated that the bread from

15% fenugreek leaves powder (T3) addition got the highest values (92.19±1.12) and elasticity

decreased as the supplementation level decreased. The mean value ranged from 89.79±1.00

(T0) to 92.19±1.12 (T3) described non-significant difference for elasticity among different

bread.

4.14.2 Texture profile of bread supplemented with seeds powder

The mean squares for the texture profile of bread supplemented with seeds powder have been

given in Table 4.41. The data exhibited non-significant behavior in treatment for all parameters

like hardness, cohesiveness, elasticity, gumminess, chewiness and springiness of bread.

106

Table 4.39 Mean squares for bread texture supplemented with fenugreek leaves

powder

SOV df Chewiness Cohesiveness Elasticity Gumminess Hardness Springiness

Trt. 3 0.00207NS 0.00072NS 3.13144NS 0.00140NS 0.00850NS 0.00322NS

Error 8 0.00053 0.00139 1.08482 0.00103 0.00244 0.00100

Total 11

NS= Non Significant

Table 4.40 Mean values for bread texture supplemented with fenugreek leaves

powder

Trt. Hardness Cohesiveness Gumminess Springiness Chewiness Elasticity

T0 3.51±0.03 0.91±0.04 3.19±0.03 0.98±0.03 3.13±0.03 89.79±1.00

T1 3.56±0.04 0.92±0.03 3.21±0.04 1.01±0.04 3.15±0.01 90.53±1.12

T2 3.59±0.06 0.93±0.05 3.22±0.01 1.03±0.03 3.17±0.01 91.23±0.89

T3 3.63±0.05 0.95±0.02 3.24±0.03 1.05±0.01 3.18±0.02 92.19±1.12






107

Table 4.41 Mean squares for bread texture supplemented with fenugreek seeds

powder

SOV df Chewiness Cohesiveness Elasticity Gumminess Hardness Springiness

Trt. 3 0.00643NS 0.00083NS 8.41461NS 0.00122NS 0.00260NS 0.00126NS

Error 8 0.00133 0.00101 3.13486 0.00093 0.00212 0.00169

Total 11

NS= Non Significant

Table 4.42 Mean values for bread texture supplemented with fenugreek seeds powder

Trt. Hardness Cohesiveness Gumminess Springiness Chewiness Elasticity

T0 3.51±0.03 0.91±0.04 3.19±0.03 0.98±0.03 3.13±0.03 89.79±1.00

T4 3.53±0.04 0.92±0.02 3.21±0.02 0.98±0.03 3.15±0.04 90.25±1.11

T5 3.55±0.05 0.93±0.03 3.23±0.03 1.00±0.03 3.19±0.03 91.41±2.52

T6 3.58±0.05 0.95±0.02 3.24±0.03 1.02±0.05 3.23±0.03 93.54±1.98






108

It is depicted that all textural parameters didn’t vary by the addition of seeds powder up to 15%

level. Mean values concerning bread hardness given in Table 4.42 exhibited that maximum

hardness was observed in T6 (3.58±0.05) followed by T5 (3.55±0.05), T4 (3.53±0.04) and T0

(3.51±0.03). It is also apparent from the findings that prepared bread possessed non-significant

differences for this trait.

The mean values for cohesiveness of bread portrayed in Table 4.42 illustrated that bread from

15% fenugreek seeds powder supplementation got the highest values for cohesiveness

(0.95±0.02). As we decrease the percentage of supplementation the value decreased, so

minimum cohesiveness value found in T0 (0.91±0.04). Dough cohesiveness is the property of

being cohesive and sticky holding particles in a homogeneous body together. If cohesiveness

is more, stickier the dough will be and hardness tendency will be more if cohesiveness

decreased.

Data regarding the means for gumminess of bread prepared with supplementation of fenugreek

seeds powder has been given in Table 4.42. The values ranged between 3.19±0.03 (T0) to

3.24±0.02 (T6) exhibited that gumminess of bread increased non-significantly with the addition

of fenugreek seeds powder.

The mean values (Table 4.42) for springiness of bread described increasing trend with various

levels of supplementation with fenugreek seeds powder. The maximum springiness was

observed in T6 (1.02±0.05) followed by T5 (1.00±0.03), T4 (0.98±0.03) whereas minimum was

observed in T0 (0.98±0.03).

Mean values of chewiness of bread prepared with supplementation of fenugreek seeds powder

given in Table 4.42. Results presented a non-significant increasing trend with increasing

percentage of supplementation, so maximum value was found in T6 (3.23±0.03). The values

found by the other treatments are T5 (3.19±0.03), T4 (3.15±0.04) and T0 (3.13±0.03).

The values regarding the means of elasticity of bread has been presented in Table 4.42 which

depicts that this trait of bread increased with increasing the percentage of fenugreek seeds

powder. The minimum value of elasticity has been observed against T0 and highest against T6

which was 89.79±1.00 and 93.54±1.98, respectively.

The gluten content of wheat flour play an important role for quality of baked products. It also

influence flour water absorption, viscosity, cohesion, elasticity, tolerance to kneading,

109

extensibility, ability to gas retention, resistance to deformation and dough strengthening

properties (Lazaridou et al., 2007; Wieser, 2007). In a study, texture of methi paratha before

and after radiation processing was determined using a texture analyzer. Parameters like

hardness and puncture force were determined. The shear force as measured using texture

analyzer significantly decreased from 829g for control to 520 and 497g force on addition of

increasing levels of dried fenugreek leaves. Lowering in shear force showed improvement in

the paratha quality, this indicates that addition of leaves in dehydrated form made the paratha

slightly harder (Indrani et al., 2011). The reduction in shear force may be due to the presence

of dietary fiber coming from leaves, thereby diluting the gluten protein which makes the

product more extensible and chewy. Addition of leaves in dehydrated form also had acceptable

quality (Sudha et al., 2013). Wheat flour was supplemented with legume flour (pigeon pea and

chick pea) @ 10 to 20% to prepared flat bread and then observe nutritive value, textural

attributes, and sensory acceptability of the product. The bread was found with low moisture

content and hard texture when compared with control bread (Sharma et al., 1995).

The harder texture was found in bread when prepared with supplementation of cassava flour as

compared to wheat flour bread. The findings are also in harmony with studies of Abdelghafor et

al. (2011) and Phattanakulkaewmorie et al. (2011) who observed with supplementation of

sorghum flour in bread resulted in harder texture. The hard texture of bread depend on moisture,

moisture migration and redistribution of moisture (Osella et al., 2005) and gluten-starch

interactions (Every et al., 1998). After baking of bread when it gets cool, starch retrogrades and

gel within inter granular spaces, provide rigidity that leads to bread hardness (Zobel and Kulp,

1996). Contrast finding was observed by Anton et al. (2008) who reported firmness and

cohesiveness of wheat bean composite tortillas decreased with increased substitution with bean

flour.

There was no statistically significant difference in color, firmness, texture and flavor intensity

between the fenugreek and wheat bread (Losso et al., 2009). Similarly, Shakib and Gabrial

(2010) evaluated different bread formulations containing wheat, barley and fenugreek. Two

type of fenugreek flour replacements was done i.e. 5% and 2.5% in basic bread recipe and

tested for their overall acceptability by a panel of 20 judges. Sensory evaluation of fenugreek

bread with 5% and 2.5% formulations showed good acceptability level for color (2.0±0.26 and

110

2.5±0.23), breakability (2.7±0.15 and 3.0±0.18), taste (2.4± 0.32 and 2.6±0.1) and chewability

(2.2±0.12 and 2.4±0.21).

4.15 Sensory Evaluation of bread

Bread can be defined as a fermented confectionary product prepared with mixture of

ingredients like wheat flour, sugar, water, salt and yeast passes through a production process

involving mixing, kneading, proofing, shaping and baking (Dewettinck et al., 2008). Bread is

an excellent source of numerous vitamins and minerals especially phosphorus and copper but

still it consider as nutritionally poor because wheat protein i.e. main ingredient is deficient in

some essential amino acids (Wrigley et al., 1988). Composite flour not only improve the

nutritional status of bread but also comply the need of vegetarian consumers who want to fulfill

their protein requirement from plant sources (Chen, 2009).

The sensory attributes of the product plays very important role in acceptability of the product

by consumers. The sensory evaluation of bread under study was carried out by experienced

panelist who provide valuable feedback from consumer point of view. The supplementation of

fenugreek leaves and seeds powder in different treatments resulted in change of sensory

characteristics of the bread.

4.15.1 Volume of bread

The analysis of variance showed that the fenugreek leaves powder supplementation had

significant effect on the volume of bread. The interaction of treatments and storage time did

not significantly influence the volume of the bread as compared to control (Table 4.43). The

mean score for bread volume prepared from different treatments varied from 6.19±0.46 (T3) to

7.13±0.21 (T0) that shows it was negatively related to the quantity of leaves powder added

which is depicted by the higher value (7.13±0.21) for volume of control and progressively

decreasing volume in the treatments. The time of storage also negatively influenced the volume

of the bread reduced and all treatments showed slightly reduction in the volume during storage

(Table 4.45).

It was statistically depicted that the addition of fenugreek seeds powder in bread and storage

time had significant effect on the volume of bread. The score for volume ranged from

6.55±0.07 to 7.13±0.10 for treatment and 6.49±0.07 to 7.22±0.07 for storage time as shown in

111

Table 4.46. The interaction of treatment and storage time also had significant effect on the

volume of bread which shows that the combination of these factors has to be taken into account

while considering volume of the bread prepared from addition of fenugreek seeds powder

(Table 4.44).

Bread volume has equal importance as that of crust color and softness because large volume

of bread looks bigger and attracts the consumers. The volume of bread can be achieved as

desired but its shrinkage always proved to be a problem and its retention greatly depends upon

bread texture quality. Fine cells of equal size during fermentation and proofing found to have

good texture, which ultimately affects the softness and volume. Studies has proved that volume

of bread is affected with the quality and quantity of flour protein as well as other factors like

time for proofing, baking time and baking temperature. Thus, protein has played major role in

quality of bread, so supplementation of wheat flour with legume flour certainly affects the end

product characteristics due to dilution in gluten protein (Sliwinski et al., 2004).

The supplementation of legume flours like chickpea and lentil affect the qualitative parameters

of baked products like bread volume. The other bakery product like pastries volume decreased

mainly due to decrease in the amount of gluten caused by the addition of materials from which

it is not possible to isolate gluten. By lowering the amount of gluten, the ability to keep ferment

gas during the rising of dough is also lowered and consequently it influences the lower volume

and lower porosity of pastries (Bojnanska et al., 2010). The results of another study also in

harmony with our findings that volume of the bread decreased as the level of legume flour

increased due to the dilution of the gluten structure by added protein (Hefnawy et al., 2012).

Significant difference regarding bread volume was also observed in fenugreek-wheat flour

supplemented bread. This might be due to physiochemical properties of the fenugreek protein

which present in the seeds (Rasool et al., 2013).

4.15.2 Aroma of bread

Aroma of bread prepared with fenugreek leaves powder supplemented flour was significantly

affected by both the quantity of leaves powder (treatment) and storage time (Table 4.43). The

mean values (Table 4.47) for aroma of bread among different treatments ranged between

6.10±0.10 to 6.96±0.06 and during storage period found in the range of 6.31±0.09 to

112

6.95±0.12. The values revealed that aroma was negatively influenced by increasing percentage

of the fenugreek leaves powder as well as the storage time. The combination of fenugreek

leaves powder addition and storage time did not bring about significant change in aroma of

bread.

The analysis of variance showed that the addition of fenugreek seeds powder and storage time

exhibited highly significant influence on the aroma of bread (Table 4.44). The mean score for

aroma of bread prepared by addition of fenugreek seeds powder was noted from 6.67±0.14 to

6.96±0.12 which shows the increase in amount of fenugreek seeds powder added is inversely

proportional to the aroma of bread. During storage of bread mean scores varied from 6.53±0.15

to 7.12±0.17 that shows decreasing trend with the passage of time (Table 4.48).

The combination of treatment and storage had no significant influence on the aroma of bread

and depicts that the treatment and storage independently influenced the aroma and their

combined effect was not noticeable (Table 4.44).

Aroma is an important sensory characteristic that plays an important role in creating consumer

appeal for a product. In an earlier study aroma of bread was non-significantly differed with

fenugreek seeds powder supplementation when compared with control bread. When fenugreek

was added in wheat flour it modified the sensory attributes like taste and mouth feel of the

product. There was a decreasing trend for odor and taste, probably due to fenugreek flavor

(Hooda and Jood, 2004).

The desirable aroma of fenugreek seeds came from polysaccharides (galactomannan), volatile

oils and alkaloids, such as choline and trigonelline, that is why the addition of 1% of fenugreek

seeds was more desirable to add in such cases (Rasool et al., 2013). Our findings are in support

of Kasaye and Jha (2015) who observed aroma of bread found score 4.46 with supplementation

of 15% fenugreek flour while 7.80 in control. The aroma of fenugreek flour supplemented

bread samples scored maximum 7.87 in 5:95 (bread with 5% fenugreek flour) followed by

10:90 (7.53) and lowest (5.00) being for 15:85 blend and 7.76 in the control bread.

113

Table 4.43 Mean squares for sensory parameters of bread supplemented with fenugreek leaves powder

SOV df Volume Aroma Taste Color of

Crust

Character

of Crust Texture

Color of

Crumb Grain

Symmetry

of form

Evenness

of bake

Overall

acceptability

Trt.(A) 3 1.85677* 1.49512* 2.75754* 0.45931* 1.29167* 1.36090* 10.1280* 0.30758NS 0.59486* 0.13944NS 0.81472NS

Time

(B) 3 0.91471* 1.00774* 1.00921* 0.47993* 0.59226* 1.16167* 0.5625* 0.80339NS 0.99819* 0.84482* 7.24632*

A x B 9 0.05548NS 0.16481NS 0.21112NS 0.00636NS 0.14567NS 0.01945NS 0.0039NS 0.00413NS 0.00242NS 0.00172NS 3.97683NS

Error 32 0.05359 0.07744 0.13592 0.08612 0.09333 0.18692 0.0962 0.24082 0.13853 0.18756 1.04254

Total 47


* = Significant

NS= Non Significant

114

Table 4.44 Mean squares for sensory parameters of bread supplemented with fenugreek seeds powder

SOV df Volume Aroma Taste Color of

Crust

Character

of Crust Texture

Color of

Crumb Grain

Symmetry

of form

Evenness

of bake

Overall

acceptability

Trt.(A) 3 0.79018* 0.19015 * 0.89237* 0.15695* 0.55982NS 0.45301* 1.02104* 0.09807NS 0.41142* 0.02221NS 2.20278NS

Time

(B) 3 1.21454* 0.86651* 1.30672* 0.49088* 0.85042* 0.86358* 0.81557* 0.91824NS 1.09509* 0.88959* 7.24632*

A x B 9 0.17625NS 0.00183NS 0.00389NS 0.00535NS 0.17721NS 0.00676NS 0.00132NS 0.00111NS 0.00251NS 0.00084NS 3.99425NS

Error 32 0.05084 0.03918 0.12012 0.02296 0.31712 0.02570 0.09586 0.20247 0.14734 0.12775 1.04264

Total 47


NS= Non Significant

115

4.15.3 Taste of bread

Taste of bread is considered the primary sensory attribute which determines the acceptability

of the bread by the consumer. The analysis of variance depicted that the effect of treatments

and storage time on the taste of bread was significant (Table 4.43). It is obvious from the results

given in Table (4.49) that mean score of taste for different treatments bread ranged from

6.06±0.11 to 7.14±0.15, while with the passage of time values also decreased. Significantly

the highest mean score for taste (7.14±0.15) was observed for T0 followed by T1 (6.36±0.12)

and T2 (6.22±0.07) and the lowest value (6.06±0.11) was found in T3. The combination of

fenugreek leaves powder addition and storage time did not bring about significant change in

taste of bread.

The analysis of variance illustrated that degree of fenugreek seeds powder supplementation

and the time for which the bread are kept, had significant effect on the taste of the bread. The

combination of treatment and storage period did not exhibited momentous significant effect on

the taste of the bread. (Table 4.44). Progressively decreasing values of taste were observed

both treatments & storage time. The mean score varied from 6.53±0.14 to 7.14±0.15 for

treatments and 6.43±0.10 to 7.17±0.13 during storage time (Table 4.50).

The finding of the current study are in harmony with finding of Hooda and Jood (2004) who

prepared bread with supplementation of fenugreek powder in wheat flour upto 15% level with

acceptable taste but when increased the supplementation level, taste of bread little bit bitter

because of distinct taste of fenugreek. However, supplementation upto 20% level resulted in

poorest taste of the product. In an earlier research work taste of wheat-fenugreek supplemented

bread found different when compared with control due to distinct taste of fenugreek (Rasool

et al., 2013).

4.15.4 Crust color of bread

It was statistically observed that addition of fenugreek leaves powder in bread prepared from

different treatments showed highly significant effect on the crust color (Table 4.43). The

supplementation of flour used to prepare bread with fenugreek leaves had negative effect on

their color and it was hampered with increasing quantity of fenugreek leaves powder. It is

obvious from the results given in Table 4.51 that bread the highest crust color score (7.20±0.15)

116

Table 4.45 Effect of treatments and storage on volume of bread supplemented with


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.39±0.04 7.21±0.13 7.02±0.16 6.90±0.12 7.13±0.21a

T1 7.06±o.22 6.92±0.27 6.72±0.24 6.55±0.32 6.81±0.22b

T2 6.99±0.20 6.81±0.30 6.61±0.28 6.47±0.31 6.72±0.22b

T3 6.85±0.13 6.13±0.22 5.94±0.32 5.82±0.24 6.19±0.46c

Mean 7.07±0.23a 6.77±0.46ab 6.57±0.45bc 6.44±0.44c


Table 4.46 Effect of treatments and storage on volume of bread supplemented with


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.39±0.06 7.21±0.13 7.02±0.11 6.90±0.08 7.13±0.10a

T4 7.29±0.04 7.14±0.08 6.95±0.05 6.78±0.12 7.04±0.08a

T5 7.19±0.12 7.06±0.10 6.86±0.07 6.68±0.10 6.95±0.09a

T6 7.02±0.09 6.86±0.14 6.74±0.09 5.57±0.04 6.55±0.07b

Mean 7.22±0.07a 7.07±0.12ab 6.89±0.08bc 6.49±0.07c


117

Table 4.47 Effect of treatments and storage on aroma of bread supplemented with


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.26±0.18 7.02±0.15 6.81±0.16 6.74±0.12 6.96±0.06a

T1 6.97±0.13 6.64±0.09 6.40±0.09 6.29±0.05 6.58±0.08b

T2 6.85±0.07 6.53±0.18 6.25±0.05 6.17±0.07 6.45±0.14b

T3 6.72±0.16 5.48±0.10 6.19±0.11 6.03±0.10 6.10±0.10c

Mean 6.95±0.12a 6.42±0.14b 6.41±0.12b 6.31±0.09b


Table 4.48 Effect of treatments and storage on aroma of bread supplemented with


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.26±0.08 7.02±0.15 6.81±0.12 6.74±0.09 6.96±0.12a

T4 7.11±0.18 6.86±0.20 6.57±0.16 6.51±0.14 6.76±0.17ab

T5 7.08±0.12 6.85±0.18 6.54±0.19 6.48±0.17 6.74±0.20b

T6 7.01±0.20 6.77±0.12 6.48±0.21 6.40±0.19 6.67±0.14b

Mean 7.12±0.17a 6.87±0.16b 6.60±0.20c 6.53±0.15c


118

was observed for T0 followed by T1 (6.10±0.17), T2 (5.28±0.20) and T3 (4.91±0.19). It is also

evident from results that the crust color score varied from 5.68±0.96 to 6.10±1.07 during

storage period. This attribute was also negatively associated with increasing duration of storage

and all treatments showed significant deterioration in the color of the bread crust. The

interaction of treatments and storage time did not significantly influence the crust control of

the bread as compared to control.

Color of bread is instrumental in determining the quality of bread. Mean squares depicted that

supplementation of fenugreek seeds powder and storage time had significant effect on the crust

color of bread (Table 4.44). The mean score for crust color of bread showed decreasing trends

with increasing quantity of fenugreek seeds powder & storage time and had values between

6.78±0.13 to 7.20±0.10 & 6.81±0.15 to 7.27±0.09 (Table 4.52). The interaction of the two

variables showed non-significant results and depicted no strong linkage between coexistence

of treatment and storage time with the crust color of bread (Table 4.44).

Color is an important criterion from consumer point of view for acceptability of the baked

product. Moreover, the color development occurs during later stage of baking and its indication

for completion of the baking process. Color of the baked product depends on physicochemical

properties of the dough (pH, water content, amino acid content and reducing sugars) and

processing conditions applied during baking process (relative humidity, temperature, air speed,

modes of heat transfer (Zanoni et al., 1995). When supplementation of fenugreek flour

increased in wheat flour, the color of bread crust changed to dull brown in seeds flour

supplemented bread and greenish in leaves supplemented flour. Similar findings were observed

when supplementation of wheat flour upto 15% with fenugreek powder was carried out

resulted in dark brown curst color (Chauhan and Sharma, 2000). In another study the crust

color scores for bread prepared with addition of raw, soaked and germinated fenugreek flour

were 5.66, 5.50 & 5.37 and 7.15 score found by control bread. The darker color was observed

in bread supplemented with germinated fenugreek flour and lighter color in raw and soaked

fenugreek flours supplemented bread (Hooda and Jood, 2004). It can be concluded that the

increased in protein content due to fenugreek seeds flour supplementation and green color of

fenugreek leaves probably the reason of dark crust color.

119

4.15.5 Character of bread crust

Analysis of variance depicted that the effects of both fenugreek leaves powder addition and

storage on character of crust of bread were highly significant. The interaction of treatment and

storage did not significantly impact the character of crust (Table 4.43). The mean score for this

trait in different treatments ranged from 6.71±0.13 to 7.43±0.14 (Table 4.53). Significantly the

highest score (7.43±0.14) was observed for control (T0) followed by T1 (6.85±0.10), T2

(6.78±0.07) and T3 (6.71±0.13). It is also evident from results that the score for character of

crust varied from 6.63±0.10 to 7.15±0.07 during storage period that exhibit decreasing trend

with increasing the supplementation level of fenugreek leaves powder.

It was statistically observed that the addition of fenugreek seeds powder and interaction of

these variables did not have significant effect on the character of crust (Table 4.44), while time

period as significant effect on this trait. It had uniform value with non-significant difference

among each other (Table 4.54).

4.15.6 Texture of bread

It was observed from the statistical analysis that the effect of fenugreek leaves powder

supplementation and storage time was significant. The crust texture of bread was decreased

momentously with increasing the supplementation level of fenugreek leaves powder as well as

the storage time. The interaction of treatment and time didn’t has considerable effect on the

texture as depicted by (Table 4.43). The maximum mean score of 6.97±0.12 was noticed in

(T0) while in other treatments it was 6.39±0.12 (T1), 6.31±0.07 (T2) and 6.22±0.13 (T3). The

mean score for texture was 6.16±0.14 to 6.85±0.08 during storage study of the prepared bread

(Table 4.55).

The analysis of variance showed that the addition of fenugreek seeds powder and storage time

exhibited significant influence on the texture of bread (Table 4.44). The mean score for texture

of breads prepared by addition of fenugreek seeds powder was noted from 6.51±0.13 to

6.97±0.12 which shows the increase in amount of fenugreek seeds powder added is inversely

proportional to the texture score of bread. During storage study of bread the score with

decreasing trend from 7.05±0.11 to 6.44±0.12 (Table 4.56). The combination of treatment and

storage had no significant influence on the texture of bread and depicts that the treatment and

120

Table 4.49 Effect of treatments and storage on taste of bread supplemented with


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.62±0.06 7.23±0.13 6.90±0.21 6.80±0.18 7.14±0.15a

T1 6.72±0.10 6.46±0.16 6.19±0.15 6.05±0.10 6.36±0.12b

T2 6.60±0.08 6.31±0.09 6.07±0.13 5.92±0.06 6.22±0.07bc

T3 6.15±0.12 5.92±0.07 6.65±0.16 5.50±0.14 6.06±0.11c

Mean 6.77±0.09a 6.48±0.12b 6.45±0.17b 6.07±0.12c


Table 4.50 Effect of treatments and storage on taste of bread supplemented with


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.62±0.06 7.23±0.15 6.90±0.15 6.80±0.13 7.14±0.15a

T4 7.12±0.12 6.82±0.13 6.52±0.08 6.38±0.07 6.71±0.10b

T5 7.02±0.08 6.67±0.10 6.40±0.05 6.30±0.16 6.60±0.15b

T6 6.91±0.15 6.62±0.16 6.36±0.13 6.24±0.11 6.53±0.14b

Mean 7.17±0.13a 6.84±0.14ab 6.54±0.12bc 6.43±0.10c


121

Table 4.51 Effect of treatments and storage on crust color of bread supplemented with


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.52±0.03 7.28±0.16 7.06±0.14 6.92±0.20 7.20±0.15a

T1 6.32±0.20 6.12±0.16 6.05±0.23 5.91±0.15 6.10±0.17ab

T2 5.47±0.16 5.29±0.23 5.24±0.21 5.11±0.24 5.28±0.20b

T3 5.09±0.20 4.93±0.21 4.88±0.19 4.76±0.20 4.91±0.19b

Mean 6.10±1.07a 5.90±1.04ab 5.81±0.98ab 5.68±0.96b


Table 4.52 Effect of treatments and storage on crust color of bread supplemented with


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.52±0.03 7.28±0.16 7.06±0.14 6.92±0.12 7.20±0.10a

T4 7.32±0.10 7.13±0.09 6.96±0.09 6.88±0.15 7.07±0.12ab

T5 7.23±0.12 7.07±0.13 7.00±0.07 6.83±0.17 7.03±0.15b

T6 7.01±0.15 6.81±0.15 6.66±0.12 6.62±0.14 6.78±0.13b

Mean 7.27±0.09a 7.07±0.12ab 6.92±0.10ab 6.81±0.15b


122

storage independently influenced the texture and their combined effect was not noticeable

(Table 4.44).

The baking conditions (temperature and time variables); the state of the bread components,

such as fibers, starch, protein (gluten) weather damaged or undamaged and the amount of

absorbed water during dough mixing, all contribute to the final texture of the bread (Serrem et

al., 2011). The bread characteristics like texture, crust color, thickness, and crumb color were

affected with fenugreek flour. However, there was no significant differences between control

sample and the sample supplemented with 5% fenugreek flour as regard to the sensory

attributes (Sulieman et al., 2000). There was decreasing trend observed in bread crust color

with supplementation of wheat flour with fenugreek powder. The color score of 4.87 was

observed with 20% supplementation of raw fenugreek powder while score of 4.71 and 4.60

was noticed with 20% supplementation level of soaked and germinated fenugreek seeds

powder (Hooda and Jood, 2004).

The replacement of fenugreek gum with wheat flour @ 0, 5 and 10% (w/w) was carried out to

measure the bread production features of the flour. Bread comprising fenugreek gum @ 5 and

10% presented texture and volume similar to control treatment of bread (Roberts et al., 2012).

Texture and overall acceptability of bread samples made from blends of fenugreek-wheat @

5:95 and 10:90 are within acceptable limits. No significant differences were noticed with

respect to control bread samples (Kasaye and Jha, 2015).

4.15.7 Crumb color for bread

Crumb color is one of the most important characters determining the consumer acceptability

of the bread. The scientific evaluation determined that addition of fenugreek leaves powder

and storage time both had significant effect on crumb color of bread (Table 4.43). The

combination of fenugreek leaves powder addition and storage time did not bring about

significant change in crumb color of bread. It is apparent from the results that bread prepared

from 100% wheat flour had highest score (7.09±0.09) followed by bread prepared from 95%

wheat flour (WF) and 5% fenugreek leaves powder (6.08±0.06). The lowest score (5.07±0.09)

given by panelist to bread prepared with supplementation of 15% fenugreek leaves powder

(Table 4.57). During storage study the score for this trait ranged from 5.66±0.05 to 6.14±0.06

and color becomes darker with increasing the supplementation of fenugreek leaves powder.

123

Table 4.53 Effect of treatments and storage on character of bread crust supplemented


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.52±0.06 7.34±0/18 7.98±0.17 6.87±0.11 7.43±0.14a

T1 7.12±0.07 6.92±0.08 6.74±0.12 6.62±0.15 6.85±0.10b

T2 7.00±0.06 6.85±0.10 6.69±0.08 6.57±0.07 6.78±0.07b

T3 6.96±0.09 6.81±0.14 6.63±0.16 6.45±0.14 6.71±0.13b

Mean 7.15±0.07a 6.98±0.13a 7.01±0.12a 6.63±0.10b


Table 4.54 Effect of treatments and storage on character of bread crust supplemented


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.52±0.06 7.34±0.10 7.98±0.05 6.87±0.08 7.43±0.07

T4 7.41±0.12 7.07±0.17 6.86±0.09 6.73±0.12 7.02±0.10

T5 7.42±0.14 7.08±0.13 6.85±0.07 6.75±0.10 7.03±0.12

T6 7.30±0.13 7.02±0.12 6.79±0.10 6.70±0.08 6.95±0.11

Mean 7.41±0.11a 7.13±0.11ab 7.12±0.09b 6.76±0.09b


124

Table 4.55 Effect of treatments and storage on texture of bread supplemented with


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.22±0.07 7.22±0.14 7.22±0.11 6.76±0.13 6.97±0.12a

T1 6.82±0.04 6.82±0.10 6.82±0.13 6.02±0.18 6.39±0.12b

T2 6.71±0.09 6.71±0.04 6.71±0.08 5.98±0.10 6.31±0.07bc

T3 6.64±0.11 6.64±0.08 6.64±0.16 5.88±0.15 6.22±0.13c

Mean 6.85±0.08a 6.85±0.07ab 6.85±0.14b 6.16±0.14c


Table 4.56 Effect of treatments and storage on texture of bread supplemented with


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.22±0.07 7.02±0.14 6.86±0.11 6.76±0.13 6.97±0.12a

T4 7.05±0.13 6.72±0.09 6.47±0.12 6.40±0.15 6.66±0.14b

T5 7.02±0.16 6.68±0.05 6.44±0.08 6.37±0.11 6.63±0.08b

T6 6.89±0.09 6.57±0.12 6.35±0.16 6.24±0.07 6.51±0.13b

Mean 7.05±0.11a 6.75±0.10b 6.53±0.14c 6.44±0.12c


125

The sensory evaluation of the bread showed that the degree of fenugreek seeds powder

supplementation and the time for which the bread are kept, had significant effect on the crumb

color of bread (Table 4.44). Progressively decreasing score for this trait were observed for both

treatments and storage time. The highest mean score was observed in T0 (7.09±0.07) followed

by T4 (6.84±0.13), T5 (6.74±0.16) and T6 (6.39±0.14). The mean score during storage ranged

between 6.50±0.13 to 7.09±0.12 represents decreasing trend with the passage of time (Table

4.58). The combination of treatment and storage period did not show significant effect on the

taste of the bread (Table 4.44).

Findings of Gouveia et al. (2007) supported the current findings as they suggested that color

of final product is dependent upon the raw materials used that ultimately affect tonality values.

The results of current study are also in agreement with findings of Sulieman et al. (2000) who

observed decreasing trend for crumb color score with increasing fenugreek flour

supplementation in wheat flour. The decrease in color score was also reported when

supplementation of wheat flour was carried out with different levels of fenugreek powder

(Chauhan and Sharma, 2000). The crumb color of fenugreek flour supplemented bread, scored

maximum points (7.97) for 5:95 followed by 10:90 (7.77) and lowest (5.04) being for 15: 85

blend as against 8.13 for the control sample (Kasaye and Jha, 2015).

4.15.8 Grain of Bread

It was statistically depicted that the effect of fenugreek leaves powder addition and interaction

of treatment & time on grain of bread was not significant, while time period had noticeable

effect on this trait of bread (Table 4.43). It is obvious from the results given in Table 4.59 that

bread crumb grain score in different treatments ranged from 6.94±0.06 to 7.32±0.08.

Significantly the highest crumb grain score (7.32±0.08) was observed for T0 followed by T1

(7.09±0.06), T2 (7.04±0.09) and the lowest (6.94±0.06) was found in T3. During storage period

of bread the mean score for crumb grain decreased from 7.41±0.07 to 6.81±0.08 with the

passage of time (Table 4.59).

Statistical analysis of the data from sensory evaluation depicted that supplementation of

fenugreek seeds powder, storage time and the interaction of these variables did not have

significant effect on the grain of bread (Table 4.44) and it had uniform value with non-

significant difference among each other (Table 4.60).

126

4.15.9 Symmetry of bread form

Statistical analysis showed that addition of fenugreek leaves powder among different

treatments and storage time significantly influence bread symmetry. The symmetry of bread

form was negatively affected with increase in the storage time as compared to control (Table

4.43). It is obvious from the results given in Table 4.61 that bread symmetry score in different

treatments ranged from 6.69±0.27 to 7.23±0.26. Significantly the highest bread symmetry

(7.23±0.26) was observed for treatment T0 followed by T1 (6.92±0.31), T2 (6.91±0.29) while

the lowest in T3 (6.69±0.27). It is also evident from results that the bread symmetry score varied

from 6.65±0.23 to 7.28±0.22 with the passage of time that shows decreasing trend in score

among different treatments.

The analysis of variance depicted that the symmetry of form of bread was significantly affected

by the addition of fenugreek seeds powder and time period (Table 4.44). The mean score (Table

4.62) for this trait ranged between 6.78±0.12 to 7.23±0.10 with supplementation of treatments

while with passage to time mean score decreased from 7.38±0.12 to 6.72±0.11. The combined

effect of seeds powder addition and storage time also did not have any significant influence on

the symmetry of the bread (Table 4.44).

The symmetry of form of bread is an important sensory characteristic from consumer point of

view. The finding of current study score in accordance with earlier findings that revealed

highest symmetry, bread texture, crumb color aroma and taste were obtained by using 1%

fenugreek in wheat flour. This may be due to the effect of the emulsion agent which present in

fenugreek seeds which lead to produce a desirable and uniform air bubbles in the dough and

final product (Rasool et al., 2013).

4.15.10 Evenness of bake of bread

Evenness of bake of bread is a critical factor from consumer point of view for acceptability

and its shelf life. It was statistically observed that the evenness of bake of bread supplemented

with fenugreek leaves powder was not significantly affected by supplementation but the effect

of storage time was significant. The interaction of treatment and storage time did not

significantly influence the evenness of bake (Table 4.43). The mean score for evenness of bake

portrait that highest score (7.06±0.10) was observed in T0 while the lowest in T3 (6.80±0.09).

127

Table 4.57 Effect of treatments and storage on crumb color of bread supplemented


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.41±0.08 7.18±0.12 6.96±0.06 6.82±0.11 7.09±0.09a

T1 6.35±0.04 6.17±0.09 5.93±0.09 5.86±0.07 6.08±0.06b

T2 5.51±0.06 5.38±0.13 5.14±0.14 5.08±0.05 5.28±0.07c

T3 5.28±0.10 5.15±0.05 4.94±0.11 4.89±0.04 5.07±0.09c

Mean 6.14±0.06a 5.97±0.08ab 5.74±0.09b 5.66±0.05b


Table 4.58 Effect of treatments and storage on crumb color of bread supplemented


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.41±0.04 7.18±0.09 6.96±0.06 6.82±0.10 7.09±0.07a

T4 7.18±0.11 6.92±0.14 6.69±0.11 6.56±0.16 6.84±0.13ab

T5 7.08±0.08 6.83±0.18 6.60±0.15 6.45±0.18 6.74±0.16b

T6 6.68±0.15 6.48±0.12 6.23±0.13 6.16±0.14 6.39±0.14c

Mean 7.09±0.12a 6.85±0.13ab 6.62±0.12bc 6.50±0.13c


128

It is also evident from results that mean score for evenness of bake varied from 6.67±0.13 to

7.26±.06 during storage period and got decreasing trend for this trait (Table 4.63).

The statistical analysis showed that supplementation with fenugreek seeds powder had non-

significant while storage time had significant effect on evenness of bake of bread (Table 4.44).

The mean score for evenness of bake of bread represented inverse relationship to increasing

quantity of fenugreek seeds powder and storage time. The highest mean score given by panelist

to treatment T0 (7.06±0.12) followed by T4 (7.01±0.14), T5 (7.02±0.09) and T6 (6.95±0.11).

The mean score observed during storage time ranged between 6.76±0.11 to 7.37±0.09 (Table

4.64). The interaction of the two variables showed non-significant results and depicted no

strong linkage between coexistence of treatment and storage volume with the evenness of bake

of bread (Table 4.44)

4.15.11 Overall acceptability of bread

The data obtained from the sensory evaluation indicates that the difference in the quantity of

fenugreek leaves powder addition was not significant while the storage time had highly

significant effect on it. Non-significant results were also obtained by employing the

combination of treatments and storage time (Table 4.43). It is evident from the mean results

given in Table (4.65) that bread overall acceptability in different treatments varied from

6.17±0.14 (T3) to 7.16±0.13 (T0). The decreasing trend was observed with increasing the

supplementation level of fenugreek leaves powder and same trend was observed 6.90±0.10 to

5.16±0.14 during storage of the bread.

The sensory evaluation illustrated that addition of fenugreek seeds powder into the bread did

not significantly influence the overall acceptability of the bread, whereas the storage time had

highly significant effect on this trait (Table 4.44). Mean score for overall acceptability among

different treatments with decreasing trend ranged between 6.75±0.15 to 7.16±0.13 (Table

4.66). The combined interaction of the treatment and storage time also had non-significant

effect on the overall acceptability of the bread prepared by supplementation with fenugreek

seeds powder (Table 4.44).

The current study sensory scores exhibited that there was decreasing score found for overall

acceptability score of wheat fenugreek supplemented bread when compared with control bread.

129

The overall acceptability score for control bread was 6.01 that decreased with increasing

supplementation level of fenugreek powder. However, bread contains up to 5% level of

fenugreek leaves and 10% seeds powder consider as more acceptable as control in terms of

acceptability. The results of the consumer acceptance studies obtained by Naidu et al. (2012)

who reported that 45% and 25% of the panelists gave ‘‘Like very Much’’ rating to rice

preparations containing dried and fresh fenugreek leaves, respectively, whereas 55% and 50%

have gave the rating ‘‘Like Moderately’’ to dried and fresh fenugreek leaves incorporated rice

preparations, respectively.

A small percentage of the population (25%) have rated the rice preparation with fresh

fenugreek leaves as ‘‘Like Slightly.’’ Since all the ratings come under the ‘‘Like’’ category,

both the samples were considered acceptable and rice prepared with dried fenugreek leaves

showed higher consumer preference. In another investigation overall acceptability score of

wheat-fenugreek supplemented baked products observed acceptable at 5% level of

substitution. Products like biscuits and bread prepared with raw, soaked and germinated

fenugreek supplemented wheat flour found acceptable at 15% (Hooda and Jood, 2004). The

sensory attributes like symmetry, texture, crumb color, aroma, taste and overall acceptably

were within range of consumer acceptance with fenugreek-wheat supplemented bread (Rasool

et al., 2013). In an earlier study appearance, texture and overall acceptability of bread samples

made from fenugreek-wheat blends of 5:95 and 10:90 are within acceptable limits. No

significant differences were noticed with respect to control bread samples (Kasaye and Jha,

2015).

From the present exploration, it is deduced that 5% fenugreek leaves and 10% seeds powder

showed better performance in bread preparation. Moreover, these treatments were also rich in

bioactive molecules hence selected for further use in efficacy studies.

130

Table 4.59 Effect of treatments and storage on grain of bread supplemented with


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.66±0.03 7.44±0.07 7.15±0.13 7.00±0.12 7.32±0.08

T1 7.40±0.05 7.14±0.12 6.98±0.10 6.84±0.05 7.09±0.06

T2 7.35±0.11 7.11±0.10 6.95±0.04 6.75±0.09 7.04±0.09

T3 7.22±0.04 7.04±0.08 6.86±0.07 6.63±0.10 6.94±0.06

Mean 7.41±0.07a 7.18±0.13ab 6.99±0.08ab 6.81±0.08b


Table 4.60 Effect of treatments and storage on grain of bread supplemented with


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.41±0.03 7.18±0.07 6.96±0.05 6.82±0.08 7.09±0.05

T4 6.35±0.06 6.17±0.12 5.93±0.10 5.86±0.12 6.08±0.08

T5 5.51±0.09 5.38±0.14 5.14±0.16 5.08±0.05 5.28±0.12

T6 5.28±0.05 5.15±0.10 4.94±0.15 4.89±0.10 5.07±0.09

Mean 6.14±0.06 5.97±0.11 5.74±0.14 5.66±0.09


131

Table 4.61 Effect of treatments and storage on symmetry of bread form supplemented


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.55±0.11 7.36±0.10 7.06±0.19 6.97±0.91 7.23±0.26a

T1 7.30±0.18 7.06±0.30 6.72±0.22 6.61±0.53 6.92±0.31ab

T2 7.27±0.32 7.02±0.27 6.72±0.08 6.62±0.43 6.91±0.29ab

T3 7.00±0.14 6.82±0.21 6.55±0.29 6.39±0.35 6.69±0.27b

Mean 7.28±0.22a 7.07±0.20ab 6.76±0.17bc 6.65±0.23c


Table 4.62 Effect of treatments and storage on symmetry of bread form supplemented


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.55±0.11 7.36±0.14 7.06±0.08 6.97±0.12 7.23±0.10a

T4 7.39±0.05 7.09±0.10 6.79±0.07 6.69±0.13 6.99±0.09ab

T5 7.43±0.14 7.13±0.11 6.83±0.15 6.74±0.10 7.03±0.13ab

T6 7.15±0.16 6.92±0.14 6.58±0.05 6.48±0.09 6.78±0.12b

Mean 7.38±0.12a 7.13±0.12ab 6.81±0.10b 6.72±0.11b


132

Table 4.63 Effect of treatments and storage on evenness of bake of bread


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.42±0.04 7.12±0.11 6.88±0.09 6.82±0.18 7.06±0.10

T1 7.28±0.05 7.02±0.17 6.81±0.12 6.68±0.11 6.95±0.12

T2 7.23±0.08 6.99±0.14 6.72±0.10 6.64±0.07 6.89±-.08

T3 7.10±0.06 6.92±0.12 6.64±0.08 6.54±0.14 6.80±0.09

Mean 7.26±.06a 7.02±0.15ab 6.76±0.14b 6.67±0.13b


Table 4.64 Effect of treatments and storage on evenness of bake of bread


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.42±0.07 7.12±0.11 6.88±0.09 6.82±0.16 7.06±0.12

T4 7.35±0.12 7.06±0.16 6.86±0.12 6.78±0.13 7.01±0.14

T5 7.39±0.08 7.09±0.13 6.85±0.07 6.75±0.08 7.02±0.09

T6 7.30±0.10 7.04±0.14 6.78±0.11 6.70±0.10 6.95±0.11

Mean 7.37±0.09a 7.08±0.13ab 6.84±0.10b 6.76±0.11b


133

Table 4.65 Effect of treatments and storage on overall acceptability of bread


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.46±0.15 7.22±0.17 7.07±0.14 6.89±0.10 7.16±0.13

T1 6.93±0.09 6.70±0.12 6.47±0.06 6.34±0.15 6.61±0.10

T2 6.70±0.14 6.47±0.08 6.25±0.10 6.13±0.12 6.39±0.11b

T3 6.50±0.05 6.25±0.13 6.03±0.08 5.89±0.17 6.17±0.14

Mean 6.90±0.10a 6.66±0.14a 6.45±0.09a 5.16±0.14b


Table 4.66 Effect of treatments and storage on overall acceptability of bread


Treatments

Time (Hours)

0 24 48 72 Mean

T0 7.46±0.15 7.22±0.17 7.07±0.14 6.89±0.10 7.16±0.13

T4 7.28±0.12 7.02±0.20 6.74±0.18 6.74±0.10 6.93±0.15

T5 7.23±0.18 6.97±0.22 6.74±0.12 6.74±0.15 6.89±0.14

T6 7.08±0.08 6.83±0.18 6.59±0.15 6.59±0.10 6.75±0.15

Mean 7.26±0.13a 7.01±0.16a 6.78±0.17a 6.78±0.13b


134

4.16 Efficacy study

In current research work efficacy study was carried out to explore the functional/nutraceutical

potential of fenugreek leaves and seeds powder against selected metabolic disorders using

experimental Sprague Dawley rats. The trial was conducted on rodents rather than humans due

to organized supervision, control diet & environmental conditions and convenient

management. In the instant investigation, efficacy trial was comprised of three studies (Study

I: normal rats, Study II: hyperglycemic rats, Study III: hypercholestrolemic rats) along with

simultaneous intake of supplemented diets (D0: normal diet, D1: 10% fenugreek seed powder

supplemented, D2: 5% fenugreek leaves powder supplemented). At the initiation of trial, some

rats were scarified to assess the baseline values, whilst, rest of the rats were killed at the

termination (56th day) of the study. Feed and water intakes were measured on daily bases

however, body weight was assessed weekly.

4.16.1. Feed intake

Mean squares (Table 4.67) explicated that the treatments and time intervals affected the feed

intake significantly in all studies. Mean values of normal rats indicated (Fig. 4.1) maximum

feed intake (36.21±2.98 g/rat/day) in D1 (seeds powder) trailed by D0 (control) and D2 (leaves

powder) as 34.80±2.82 and 33.49±2.64 g/rat/day. It was observed that feed intake was

minimum at the initiation of study and it was maximum at the termination of study i.e.

29.39±1.25 to 38.23±1.92 g/rat/day, correspondingly. The feed intake increased gradually with

the passage of time and at 1st week it was recorded as 29.28±0.73, 30.69±1.12 and 28.20±1.17

g/rat/day in D0, D1 and D2 groups that subsequently increased to 38.36±0.96, 40.01±1.39 and

36.25±1.47 g/rat/day, respectively at 8th week.

Similarly, in Study II, D0 showed 33.93±2.65 g/rat/day feed intake, whilst D1 and D2 exhibited

35.04±2.98 and 36.67±2.89 g/rat/day for respective trials. Time factor enhanced the feed

intake, at initiation of study it was 28.61±0.61, 29.39±1.02 & 30.90±1.26 g/rat/day (Fig. 4.2)

in D0, D1 and D2, respectively. However, an increasing trend at 8th week 36.74±1.46,

39.39±1.36 and 39.87±1.63 g/rat/day was observed. Likewise in study III, D0 exhibited

maximum (33.88±2.65 g/rat/day) feed consumption followed by D2 (33.32±2.71 g/rat/day)

whilst minimum (32.26±2.70 g/rat/day) was noticed in D1. During the time span, it was

increased from 27.92±0.761 to 36.52±0.44 g/rat/day at initiation to termination, respectively.

135

Table 4.67 Effect of diets and time intervals on feed, water intake & body weight of

rats in different studies

Studies SOV df Feed

intake

Water

intake

Body

Weight

Study 1

Intervals

(A) 8 118.354** 74.1181** 31473.4**

Diet (B) 2 83.692** 16.3865** 16949.2**

A x B 16 0.455NS 0.3132NS 419.3NS

Error 108 1.585 0.4708 303.9

Study II

Intervals

(A) 8 120.911** 67.7120** 19875.1**

Diet (B) 2 85.586** 27.4034** 16218.5**

A x B 16 0.497NS 0.2258NS 253.7NS

Error 108 1.487 0.3154 140.6

Study III

Intervals

(A) 8 110.367** 66.4694** 18849.0**

Diet (B) 2 30.525** 27.7632** 9684.5**

A x B 16 0.255NS 0.2288NS 273.1NS

Error 108 0.695 0.8094 210.5

** = Highly significant

* = Significant

NS = Non significant

136

Figure 4.1 Feed intake (g) of normal rats (study I)

Figure 4.2 Feed intake (g) of hyperglycemic rats (study II)

Figure 4.3 Feed intake (g) of hypercholesterolemic rats (study III)

D0 : Control diet

D1 : Fenugreek seeds powder @ 10%

D2 : Fenugreek leaves powder @ 5%

15

20

25

30

35

40

45

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9

D0 D1 D2

15

20

25

30

35

40

45


D0 D1 D2

15

20

25

30

35

40


D0 D1 D2

137

Similarly, in groups rely on D0, D1 and D2 (Fig. 4.3) elevation in feed consumption was

observed from 28.56±0.77 to 35.71±0.97, 27.08±0.48 to 36.02±0.64 and 28.13±0.80 to

36.85±1.05 g/rat/day at 1st and 8th week, respectively.

The results of present research are comparable with the findings of Anuradha and Ravikumar

(2001) in which rats were divided in five groups, amongst third group was diabetic and fed on

fenugreek rich diet. In this group feed intake increased from 83.8±2.3 to 98.2±5.2 (g/kg/day).

In another study non-momentous change in food intake was observed in rats groups with

fenugreek supplemented leaves diet (Annida and Prince, 2004). Similarly a team of another

researchers Kumar et al. (2005) investigated antidiabetic property of fenugreek seeds and

suggested that during research feed and drink intakes were calculated in control and diabetic

groups which were treated with fenugreek. In control treated with fenugreek feed intake was

12.8 ±0.2 g/24 hr and in diabetic treated with fenugreek it was 16.00±0.9 g/24 hr. Recently,

Mahmoud (2013) evaluated the effect of high protein diet containing fortified bread with 5 and

10% fenugreek seeds (FS) on the mean values of weight/body weight % of rats suffering from

diabetes. The mean value of feed intake in negative control group provided with normal protein

diet containing 250 g un-fortified bread was 16.67g, while it was 15.50g in the negative control

group fed on high protein diet containing the same type and amount of bread. The results of

present study comparable with findings of Geetha et al. (2011) who divide rats into groups and

fed with various levels of fenugreek based diets and observed feed intake reduce during first

week of the study but after that animals regained their appetite and feed intake increased as

study proceed.

4.16.2. Water intake

Mean squares for water intake in Table 4.67 showed momentous effect of treatments as well

as time intervals (weeks) during the course of study. Means for water intake in all modules

showed increasing trend with time intervals. In study I (Fig. 4.4) water intake at the beginning

was 19.54±0.39, 20.64±0.54 and 18.71±0.41 mL/rat/day, respectively for D0, D1 and D2 that

increased to 26.02±0.66, 27.50±0.91 and 26.40±0.92 mL/rat/day respectively at the end.

In study II (Fig. 4.5) the recorded values for water intake in D0, D1 and D2 were 19.98±0.32,

21.61±0.40 and 20.22±0.48 mL/rat/day, respectively that raised to 26.44±0.62, 28.25±0.78 and

26.54±0.53 mL/rat/day, respectively during the entire trial.

138

Figure 4.4 Water intake (mL) of normal rats (study I)

Figure 4.5 Water intake (mL) of hyperglycemic rats (study II)

Figure 4.6 Water intake (mL) of hypercholesterolemic rats (study III)

D0 : Control diet



15

18

21

24

27

30


D0 D1 D2

15

18

21

24

27

30


D0 D1 D2

15

18

21

24

27

30


D0 D1 D2

139

The mean water intake was 23.82±2.08, 25.33±2.22 & 24.22±2.07 mL/rat/day, respectively

with diets D0, D1 and D2. Similarly water intake for all diet groups was 20.60±0.88 mL/rat/day

at start of trail that increased to 27.08±1.36 mL/rat/day at the end to the trial. Rats fed on high

cholesterol diet (study III) showed highest water intake in D0 (27.47±1.21 mL/rat/day) at 8th

week followed by D1 (27.96±0.57 mL/rat/day) and D2 (25.97±0.76 mL/rat/day). The maximum

mean water intake (Fig. 4.6) was observed in rats fed with D2 (25.24±2.13 mL/rat/day)

followed by D0 (24.60±2.17 mL/rat/day) and D1 (23.87±2.02 mL/rat/day).

There was increasing trend of water intake observed in all groups under study. The thrust of

higher water intake can be a symptom of diabetes. Similar findings were also observed in a

study that reported that control rats drink 20 to 30 mL/rat/day of water, while fenugreek control

and diabetic groups consumed 27.6 and 83.4 mL/24hr, respectively (Kumar et al., 2005).

4.16.3. Body weight

Mean squares (Table 4.67) revealed that the body weight of rats in different studies varied

significantly with treatments and study weeks.

Body weight depicted (Fig. 4.7) that in the beginning (study I) the weight of different rat groups

D0, D1 and D2 were 224.24±13.44, 236.03±13.83 and 232.65±13.38 g/rat, respectively that

subsequently increased to 361.92±17.33 g/rat (D0), 403.31±3.63 g/rat (D1) and 352.88±25.86

g/rat (D2) at the termination. Mean values indicated highest weight gain in D1 followed by D0

and D2. Similarly, in study II (Fig. 4.8), maximum weight gain was observed in D1 from

231.90±13.62 to 344.14±18.70 g/rat, respectively for 1st and 8th week. The remaining groups,

exhibited increase in weight during entire trial from 237.85±3.64 to 331.07±11.99 (D2) and

227.56±4.95 to 313.89±16.34 (D0) g/rat.

In study III, gain in weight was more pronounced in D1 and during 1st week the recorded weight

for D0, D1 and D2 was 229.84±13.261, 222.97±14.611 & 233.24±3.763 g/rat that reached to

304.60±15.589, 332.75±14.515 & 326.76±6.30 g/rat, respectively at the final week (Fig 4.9).

Means for final body weight at the termination of study depicted substantial differences among

the treatments. In study 1, the highest weight was observed in D1 group (329.17±53.818 g/rat)

trailed by D0 (298.28±45.85 g/rat) and D2 (293.37±38.54 g/rat). Likewise in study II, the D1

(291.94±38.903 g/rat) group had more weight trailed by D2 (280.22±36.61 g/rat) and D0

(254.81±34.965 g/rat).

140

The results of current investigaton in harmony with scientific work of Anuradha and

Ravikumar (2001) in which rats were divided in five groups, amongst third and fifth groups

were diabetic fed on fenugreek rich diet and control fed on fenugreek, accordingly. In diabetic

group weight gain was from 167.4±2.1 to 195.3±3.1 g/rat/week, even intake of food was found

similar in groups. Likewise, Elmnan et al. (2012) reported fenugreek seeds found to increase

body weight and this weight gain may be due to the fact that, the seeds is considered as an

appetite stimulating agent (Blumenthal et al., 1998; Broca et al., 2000), thus increased feed

intake and a better use of nutrients available in diet lead to increase in bodyweight. The addition

of fenugreek leaves supplementation in diabetic rats diet depicted non substantial change in

food intake among different groups, while partially increased body weight observed in diabetic

rats that can be credited to its antidiabetic role (Devi et al., 2012). The findings of Petit et al.

(1995) depicted that steroid saponins present in fenugreek seeds stimulate food intake that

leads to increase in weight of rats. In another investigation non-significant increase in mice

body weight was observed when diets supplemented with fenugreek seeds (Choudhary et al.,

2001). Diabetic rats treated with fenugreek observed with weight gain, on the other hand

untreated diabetic rats group exhibited decrease in body weight (Xue et al., 2007). Similar

findings were observed by Abdalatif et al. (2012) who found slightly less weight gain in

diabetic animals when compared with non-diabetic ones. The addition of fenugreek in diet

resulted in gradual increase in diabetic as well as non-diabetic rats (Belguith-Hadriche et al.,

2013).

4.16.4 Serum profile analysis

4.16.4.1 Glucose

The statistical analysis showed that glucose level in different groups of rats was significantly

affected by diets in all studies except for study I (Table 4.68).

In study I mean glucose values in D0, D1 and D2 groups were 83.00±2.21, 79.27±2.92 and

78.18±2.54 mg/dL. In study II, D0 group illustrated highest glucose level 151.00±4.95 mg/dL,

that substantially decreased to 134.89±2.01 and 146.55±2.65 mg/dL in D1 and D2 groups,

respectively (Table 4.68). Likewise, high cholesterol fed rats (study III) had maximum glucose

141

Figure 4.7 Body weight (g) of normal rats (study I)

Figure 4.8 Body weight (g) of hyperglycemic rats (study II)

Figure 4.9 Body weight (g) of hypercholesterolemic rats (study III)

D0 : Control diet



150

200

250

300

350

400


D0 D1 D2

150

200

250

300

350


D0 D1 D2

150

200

250

300

350

400

450


D0 D1 D2

142

level 115.00±3.74 mg/dL in D0 group that reduced significantly in D1 (105.66±2.87 mg/dL)

followed by D2 (111.45±1.95 mg/dL).

Fig 4.10 depicted the percent reduction in glucose content; in study I treatment D1 showed

glucose reduction (4.50%) whereas D2 caused 3.15% reduction. Similarly, in study II, highest

decrease was observed in D1 (10.67%) trailed by D2 (6.78%). Similar diminishing trend was

observed during study III, the highest decline was noticed in D1 (8.12%) followed by D2

(5.45%). It is revealed that functional diet containing seeds powder (D1) performed better

against glucose related abnormalities than diets containing leaves powder (D2).

The earlier research work supported current research findings like Xue et al. (2007) and Mowl

et al. (2009) proved that there was decrease in glucose level when diet or rats supplemented

with fenugreek leaves. The anti-diabetic also proved in rats when group of animals were

provided with fenugreek seeds rich diet (Annida and Prince, 2004). The enrichment of diet

with fenugreek seeds fraction to diabetic and non-diabetic rats decrease the glucose level. The

decreased absorption of glucose, intestinal disaccharidase activity and increased

gastrointestinal motility observed with the addition of seeds fraction. The antidiabetic actions

of fenugreek facilitated by slowing down digestion of carbohydrate & absorption and

improvement of peripheral insulin action (Hannan et al., 2007). The findings of Madar and

Shomer (1990) & Sauvaire et al. (1998) exhibited that anti-hyperglycemic effects of fenugreek

are attributed, at least partly, by reducing intestinal glucose absorption. The findings also

indicated that the seeds fraction increased sucrose content of the stomach in non-diabetic and

type 2 diabetic rats at 30-60 min support the thought that fenugreek also reduces gastric

emptying (Nahar et al., 2000).

The mean fasting blood glucose in diabetic untreated rats group (control positive) after

induction of 21 days of diabetes was 280±8.33 mg/dl while in the normal healthy group the

value was 76±2.59 mg/dl. When comparison of values between positive control group and the

group which was treated with fenugreek extract was carried out, it was observed that treated

group exhibited lower mean fasting blood glucose i.e. 141.83±9.04 mg/dl. In similar way mean

values of serum insulin observed in control positive group was 4.17±0.17 μU/ml and normal

healthy group it was 10.53±0.66 μU/ml. The comparison of positive control group and


143

μU/ml in treated group (El-Soud et al., 2007). Fenugreek feeding at 5% significantly reduced

fasting blood glucose in diabetic rats by 33% (360 to 244.0 mg/dl) as compared to diabetic

control indicating its hypoglycemic influence during diabetes (Shetty and Salimath, 2009). A

bread incorporating fenugreek was tested for its taste acceptability and blood glucose lowering

effect. Eight diet controlled diabetic subjects were served two slices (56 g) for bread. The bread

supplemented with fenugreek was indistinguishable from the whole wheat bread and its

maintained fenugreek’s functional property of reducing insulin resistance (Losso et al., 2009).

Previously, Shakib and Gabrial (2010) evaluated postprandial blood glucose level in healthy

subjects and determine their glycemic index values after feeding with bread supplemented with

barely, fenugreek or ginger. The all supplemented bread reduced postprandial blood glucose

responses than did the control (wheat flour bread), suggesting that soluble dietary fiber

available in these supplemented products had an impact on glucose tolerance with glycemic

indices ranging from 38 (55 fenugreek) to 52 (Barley wheat bread). Fenugreek supplemented

bread have crude fiber 6.25 g/100g so it can be replaced with white wheat flour bread since it

provides easily digestible and healthy carbohydrate based diet that can help to maintain

postprandial blood glucose level within normal range.

When supplementation of experimental rats was carried out with fenugreek leaves powder in

streptozotocin (STZ) induced diabetic rats with moderate hyperglycemia that resulted in

decreased blood glucose and increased plasma insulin level. The mechanism of STZ that it

incompletely destruct pancreatic β-cells even though the rats became permanently diabetic.

The β–cells of islets of Langerhans get stimulated and glucose level increased when

experimental rats diet supplemented with fenugreek leaves powder that increased plasma

insulin level. The process of glycolysis increased and gluconeogenesis decreased leads to

control glucose level when supplementation of leaves increased in the diet. This is possible as

it controls the activities of the key enzymes of glycolysis (Devi et al., 2012). The increase in

insulin level resulted in glucose decreased that exhibited reverse correlation. Blood glucose

level significantly increased and decrease in insulin level observed in positive control group

when compared to the rest groups. The addition of fenugreek extract in rats diet depicted

significant effect against blood glucose and insulin level. The level of glucose decreased from

258.41mg/dl (positive control) to 167.57 mg/dl and insulin level improved 5.81 IU/ml to 7.89

IU/ml (El-Dakak et al., 2013). Serum glucose level was significantly elevated in STZ-diabetic

144

rats and the percentage increase was 195.81%. Fenugreek significantly decreased STZ-induced

hyperglycemia and the percentage reduction was 53.66% in comparison with the diabetic

control. Accordingly, there was a significant decrease in serum insulin concentrations in

diabetic rats, compared with normal control (23.13%), and administration of fenugreek

increase the insulin level and the percentage elevation was 14.62% as compared to the diabetic

control (Marzouk et al., 2013).

4.16.4.2 Cholesterol

The statistical analysis (Table 4.69) showed that diets imparted non-significant variations on

cholesterol in study I whereas, significant differences were observed in rest of the studies. In

study I, maximum cholesterol was observed in D0 (80.26±2.48 mg/dL) followed by D2

(79.63±1.08 mg/dL) and D1 (77.88±1.04 mg/dL) groups. Means for cholesterol in study II

indicated maximum value for D0 (100.05±2.19 mg/dL) that momentously reduced to

91.59±1.52 and 95.53±1.07 mg/dL in D1 and D2, respectively. Similarly in study III, high

cholesterol value 146.36±2.72 mg/dL was noticed in D0 followed by D2 137.17±4.82 mg/dL

and D1 127.87±5.68 mg/dL.

It is obvious from Fig 4.11 that D1 (diet containing seeds powder) caused maximum reduction

in cholesterol followed by D2 (diet containing leaves powder). In study I the treatments D1 and

D2 exhibited 2.96 and 2.03 % decline in cholesterol, respectively as compared to control.

Likewise, in study II highest reduction was exhibited by D1 (8.45%) trailed by D2 (4.52%).

Similarly, in study III maximum decrease (12.03%) was noticed in D1 whilst minimum

reduction was observed in D2 (6.32%) as compared to control.

The present results are supported by Moosa et al. (2006) who reported fenugreek dose of 25g

orally two times in a day for period of three and six weeks to hypercholesterolmic individuals

produces significant reduction of serum total cholesterol This study suggests that fenugreek

seeds powder would be considered as effective agent for lipid lowering purposes. The findings

of Pipelzadeh et al. (2003) supported current research results concerned serum total cholesterol

found decreased by adding fenugreek in diets of experimental rats. Treatment with 100 mg/kg

fenugreek seeds powder reduced total cholesterol 162.9 to 120 mg/dl while dose of 500 mg/kg

also significantly reduced the total cholesterol level and other serum lipids.

145

Table 4.68 Effect of supplemented diets on glucose (mg/dL)

Studies

Diets

F value

D0 D1 D2

Study I 83.00±2.21 79.27±2.92 80.01±2.54 3.26NS

Study II 151.00±4.95a 134.89±2.01b 146.55±2.65ab 6.43*

Study III 115.00±3.74a 105.66±2.87c 111.45±1.95b 14.13**


*= Significant

NS= Non Significant

D0 : Control diet



Study I : Normal rats

Study II : Hyperglycemic rats

Study III: Hypercholesterolemic rats

Figure 4.10 Percent reduction in glucose as compared to control

-4.50

-10.67

-8.12

-3.15

-6.78

-5.45

-12.00

-10.00

-8.00

-6.00

-4.00

-2.00

0.00

STUDY I STUDY II STUDY III

D1 Fenugreek seed powder supplemented diet

D2 Fenugreek leaves powder supplemented diet

146

In addition Prasanna (2000) proved hypolipidemic role of fenugreek by given it dose of 25 and

50g twice in a day before meal to hypercholesterolemic patients. Similar results were reported

by Belguith-Hadriche et al. (2010) who revealed that administration of fenugreek ethyl acetate

extract to rats fed on cholesterol-rich diet significantly lowered the plasma level of total

cholesterol. The lowering effect correlated to the presence of flavonoids in fenugreek

especially naringenin. Sowmya and Rajyalakshmi (1999) observed that addition of germinated

fenugreek seeds powder in diet resulted in reduction of LDL and total cholesterol levels of

hypercholesterolemic adults. The hypocholesterolemic effect of fenugreek occurred through

various mechanisms like increased in excretion of fecal bile acids and neutral sterols with

reduction in level of cholesterol stores in liver. Its addition in diet stimulates the production of

bile in liver and the process of cholesterol conversion into bile salts or high fiber available

potentially reduces absorptive mucosal surface glucose diffusion rate, cholesterol and drug

absorption. The soluble fiber also increases the viscosity of the digest & unstirred layer

thickness in small intestine or inhibit the uptake of cholesterol and bile acids. For fermentation

in large bowel by microorganisms, soluble fiber is consider as an excellent source. The

resultant product of fermentation are volatile fatty acids that enter into the blood stream and

appear to decrease the synthesis of hepatic cholesterol. The seeds of the fenugreek contains

trigonelline and diosgenin in the form glycosides that can combine with cholesterol to make

complexes in the intestine and reduced its absorption. Another reason for reducing glucose

effect could be based on amino acid pattern present in fenugreek proteins. Likewise, fenugreek

given in a dose of 2.5g twice daily for 3 months to healthy individuals did not affect the blood

lipids and blood sugar (fasting and postprandial). However, administered in the same daily

dose for the same duration to coronary artery disease patients also with non-insulin dependent

diabetes, fenugreek decreased significantly the blood lipid (total cholesterol and triglycerides)

without affecting the HDL-cholesterol (Bordia et al., 1997). The present results are on the line

with Stark and Madar (1993) who reported that feeding hypercholesterolemic rats with two

separate doses (30 or 50g) of ethanol extract from fenugreek seeds contained

hypocholesterolemic components which appear to be saponins that interact with bile salts in

the digestive tract.

The administration of fenugreek extract has significantly decreased serum triacylglycerol and

cholesterol in diabetic rats. The findings also supported by the research work of other scientist

147

who have reported that fenugreek added diet feeding to diabetic rats resulted in reducing total

cholesterol (Khosla et al., 1995). The hypolipidemic role of fenugreek soluble dietary fiber

fraction could be the result of retardation of carbohydrate and fat absorption due to the presence

of bioactive fiber in the agent (Hannan et al., 2003). In an experiment mean serum total

cholesterol value in normal healthy group was found 98.5±2.1 mg/dl that observed

significantly increased in positive control group (140.33±3.2 mg/dl). When fenugreek extract

was added in diet, it significantly reduced the serum total cholesterol to 107.83±2.2 mg/dl (El-

Soud et al., 2007). Ethyl acetate extract of fenugreek seeds was investigated by Belguith-

Hadriche et al. (2013) who observed reduced level of total cholesterol, low density lipoprotein

(LDL), triglycerides and increased level of high density lipoprotein (HDL) when compared

with those of rats that have cholesterol-rich diet. Serum total cholesterol level increased in

STZ-diabetic rats and daily administration of fenugreek for 30 days succeeded to reduce

cholesterol 48.22% when compared with diabetic control rats (Marzouk et al., 2013).

4.16.4.3 Insulin

The statistical analysis (Table 4.70) elucidated that diets imparted non-significant effect on

insulin in study I however, this trait was affected significantly in study II and III. Means for

insulin values in study I were 7.01±0.25, 7.16±0.16 and 7.09±0.20 µU/mL in D0, D1 and D2

groups, respectively. Nonetheless in study II, lowest insulin value was recorded in D0

(4.69±0.05 µU/mL) that significantly uplifted to 4.88±0.04 and 4.83±0.04 µU/mL in D1 and

D2 groups.

Similarly in study III, D0 exhibited lowest insulin level (6.32±0.09 µU/mL) that momentously

increased in D1 and D2 groups as 6.53±0.10 and 6.46±0.05 µU/mL, respectively (Table 4.70).

It is obvious from the Fig. 4.12 (Study I) that diets D1 and D2 resulted in 2.11 and 1.09%

increase in glucose while 4.01 and 2.96% increase in insulin was found with diets D1 and D2

in study II. Likewise in study III, highest elevation 3.25% was noticed in D1 followed by 2.22%

in D2.

The results obtained were in harmony with the findings of previous researchers. The scientific

study of Khalil (2004) focus on clarifying the role of fenugreek seeds aqueous extract in its

therapeutic dose on beta cells number, blood glucose and plasma insulin levels in diabetic rats.

148

Table 4.69 Effect of supplemented diets on cholesterol (mg/dL)

Studies

Diets

F value

D0 D1 D2

Study I 80.26±2.48 77.88±1.04 79.63±1.08 2.71NS

Study II 100.05±2.19a 91.59±1.52b 95.53±1.07c 54.8**

Study III 146.36±2.72a 127.87±5.68c 137.17±4.82b 20.4**


NS= Non Significant

D0 : Control diet






Figure 4.11 Percent reduction in cholesterol as compared to control

-2.96

-8.45

-12.03

-2.03

-4.52

-6.32

-14.00

-12.00

-10.00

-8.00

-6.00

-4.00

-2.00

0.00

Study I Study II Study III



149

For this purpose rats were divided into four groups, control, diabetic, diabetic rats and control

rats fed with fenugreek seeds aqueous extract (0.1 mg/kg body weight). During induction of

diabetes, insulin level was reduced (12.5±0.5 to 7.5±0.8 μU/dL). By feeding fenugreek extract

based diet, inslin level was elevated to higher value in diabetic group (10±1 μU/dL). Likewise,

Gad et al. (2006) assessed the effect of administration of fenugreek extracts for 21 days on the

blood glucose and serum insulin of diabetic rats. During period of diabetic induction, serum

insulin level was reduced from 1.106±0.269 to 0.216±0.005 μg/L. When fenugreek based diet

was fed to the diabetic models, serum insulin level rose to 0.326±0.077 μg/L.

Mean values of serum insulin observed in control positive group was 4.17±0.17 μU/ml and

normal healthy group it was 10.53±0.66 μU/ml. The comparison of positive control group and


μU/ml in treated group (El-Soud et al., 2007). When supplementation of experimental rats was

carried out with fenugreek leaves powder in streptozotocin (STZ) induced diabetic rats with

moderate hyperglycemia that resulted in decreased blood glucose and increased plasma insulin

level. The mechanism of STZ that it incompletely destruct pancreatic β-cells even though the

rats became permanently diabetic. The β–cells of islets of Langerhans get stimulated and

glucose level increased when experimental rats diet supplemented with fenugreek leaves

powder that increased plasma insulin level. The process of glycolysis increased and

gluconeogenesis decreased leads to control glucose level when supplementation of leaves

increased in the diet. This is possible as it controls the activities of the key enzymes of

glycolysis (Devi et al., 2012).

Serum glucose level was significantly elevated in STZ-diabetic rats and the percentage

increase was 195.81%. Fenugreek significantly decreased STZ-induced hyperglycemia and the

percentage reduction was 53.66% in comparison with the diabetic control. Accordingly, there

was a significant decrease in serum insulin concentrations in diabetic rats, compared with

normal control (23.13%), and administration of fenugreek increase the insulin level and the

percentage elevation was 14.62% as compared to the diabetic control (Marzouk et al., 2013).

4.16.4.4 High density lipoprotein (HDL)

It is indicated from the statistical analysis (Table 4.71) that diets imparted non-momentous

differences on HDL level in all three studies.

150

Means for HDL in study I (normal diet) showed the values 37.12±1.13, 37.62±1.02 and

37.49±1.04 mg/dL for D0, D1 and D2 groups, respectively. However, in study II lowest HDL

level (31.26±0.88 mg/dL) was observed in D0 that elevated non-significantly in D1 (32.28±1.05

mg/dL) and D2 (31.87±1.06 mg/dL). Mean HDL concentration for D0 group in study III was

28.96±0.85 mg/dL that increased non-substantially to 30.16±0.84 mg/dL in D1 and 29.54±1.08

mg/dL in D2.

It is evident from the Fig. 4.13 that in study I increase in HDL was 1.01 & 1.36% with diets

D2 and D1, similarly in study II & III, diets D1 and D2 led to non-substantial increase in HDL

as compared to control as 3.25 & 4.13% and 1.96 & 2.02%, correspondingly.

4.16.4.5 Low density lipoprotein (LDL)

The statistical analysis indicated significant effect of diets on LDL in all studies (Table 4.72).

Means of study I indicated maximum LDL value 31.12±1.06 mg/dL in D0 that diminished to

29.05±0.56 and 29.99±0.65 mg/dL in D1 and D2 groups, respectively. Whereas in study II,

LDL value 46.85±0.73 mg/dL in D0 group was significantly decreased to 42.31±0.63 mg/dL

(D1) and 44.71±0.87 mg/dL (D2). Likewise in study III, mean LDL values for D0, D1 and D2

differed momentously i.e. 59.63±1.49, 52.33±0.93 and 54.96±0.74 mg/dL, correspondingly.

Fig. 4.14 depicted percent decrease in LDL values among different groups of rats in various

studies. In study I, substantial decrease by 3.63 and 6.64% in D2 and D1 groups was recorded

as compared to control. Likewise, significant reduction in LDL was observed during Study II

i.e. 4.56% in D2 and 9.68% in D1. Likewise in study III, diet containing fenugreek leaves

powder (D2) reduced the LDL level by 7.83%. Whilst diet comprised of seeds powder (D1)

resulted 12.25% LDL reduction.

4.16.4.6 Triglycerides

The statistical analysis indicated significant effect of diets on triglycerides in study II and III,

whereas non-momentous effect observed in study I (Table 4.73). Means of study I indicated

maximum triglycerides value 79.63±5.32 mg/dL in D2 that decreased to 79.00±5.24 and

76.27±4.93 mg/dL in D1 and D2 groups, respectively. Whereas in study II, triglycerides value

96.00±3.31 mg/dL in D0 group was significantly decreased to 91.66±3.08 mg/dL (D1) and

86.51±3.76 mg/dL (D2). Similarly in study III, mean triglycerides values for D0, D1 and D2

differed momentously i.e. 105.00±5.09, 93.09±3.47 and 98.36±3.96 mg/dL, respectively.

151

Table 4.70 Effect of supplemented diets on Insulin (µU/mL)

Studies

Diets

F value

D0 D1 D2

Study I 7.01±0.25 7.16±0.16 7.09±0.20 0.65NS

Study II 4.69±0.05b 4.88±0.04a 4.83±0.04a 19.1**

Study III 6.32±0.09b 6.53±0.10a 6.46±0.05ab 7.50**


NS= Non Significant

D0 : Control diet






Figure 4.12 Percent increase in Insulin as compared to control

2.11

4.01

3.25

1.09

2.96

2.22

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50




152

Table 4.71 Effect of supplemented diets on HDL (mg/dL)

Studies

Diets

F value

D0 D1 D2

Study I 37.12±1.13 37.62±1.02 37.49±1.04 0.29NS

Study II 31.26±0.88 32.28±1.05 31.87±1.06 1.37NS

Study III 28.96±0.85 30.16±0.84 29.54±1.08 2.05NS

NS= Non Significant

D0 : Control diet






Figure 4.13 Percent increase in HDL as compared to control

1.36

3.25

4.13

1.01

1.96 2.02

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50




153

Table 4.72 Effect of supplemented diets on LDL (mg/dL)

Studies

Diets

F value

D0 D1 D2

Study I 31.12±1.06a 29.05±0.56b 29.99±0.65ab 8.52**

Study II 46.85±0.73a 42.31±0.63c 44.71±0.87b 45.4**

Study III 59.63±1.49a 52.33±0.93c 54.96±0.74b 56.3**


D0 : Control diet






Figure 4.14 Percent decrease in LDL as compared to control

-6.64

-9.68

-12.25

-3.63-4.56

-7.83

-14.00

-12.00

-10.00

-8.00

-6.00

-4.00

-2.00

0.00




154

It is apparent from the Fig.4.15 that in study I the maximum reduction 3.45% was observed in

D1 followed by 0.47% in D2. Similarly in study II, D1 exhibited the highest reduction 9.89%

whereas a decline in D2 4.52% was observed. In study III, diet containing seeds powder

exhibited a reduction of 11.34% while diet containing leaves powder showed a decline of

6.32%.

The findings of Pipelzadeh et al. (2003) supported current study results concerned cholesterol,

LDL & triglycerides observed decreasing trend by feeding experimental rats with fenugreek

supplemented diets and increased serum HDL cholesterol level as compared to non-treated

control group. Similar results were reported by Belguith-Hadriche et al. (2013) who observed

reduced level of low density lipoprotein (LDL), triglycerides and increased level of high

density lipoprotein (HDL) when compared with those of rats that have cholesterol-rich diet.

These lipid effects were correlated to occurrence of flavonoids in fenugreek, especially

naringenin i.e. major flavonoid compound present in fenugreek. The present results are

supported by Moosa et al. (2006) who reported fenugreek dose of 25g orally two times in a

day for period of three & six weeks to hypercholesterolmic individuals produces significant

reduction in triglyceride and LDL cholesterol. In another study found supplementation of

fenugreek leaves in diet decreased triglycerides in diabetic rats (Annida and Prince, 2004). The

higher content of high density lipoproteins (HDL) are very important in body due to relation

with reduced risk of heart diseases (Young et al., 2004). The high level of HDL expedites

cholesterol transport from serum to liver, where it catabolized and finally excreted from the

body. The ratio of LDL and HDL is consider as important risk factor of atherosclerosis and

this ratio can be decreased with supplementation of fenugreek seeds powder and ethyl acetate

extract in diet. The low level of serum HDL and high level of LDL are correlated with increased

atherosclerosis risk (Korhonen et al., 1996). The increasing level of HDL and decreasing LDL

level in experimental rats depicted the antiatherogenic property of the ethyl acetate extract of

fenugreek. The possible mechanism of hypocholesterolaemic property may be due to dietary

cholesterol absorption inhibition in intestine or its production by the liver (Bursill and Roach,

2006) or stimulation of the biliary secretion of cholesterol and excretion through feces

(Krzeminski et al., 2003).

155

The reduction in total lipid, triglyceride, LDL and increase of HDL in this study may be due

to crude fiber and saponins content in fenugreek. Al‐Habori and Raman (1998) reported that

the crude saponins extract was the most effective in reducing hypercholesterolemia. The lipid

profile is extracted in bile and then reabsorbed from the intestine. Therefore, it can be suggested

that saponins may bind bile salts which are necessary for absorption of these substances or

bind directly in the intestine and prevent reabsorption (Madar and Shomer, 1990). This may

be due to the fact that saponins form insoluble complex with lipid profile (Rao et al., 1996).

Saponins and crude fibre extracted from fenugreek seeds significantly resulted in reduction of

cholesterol and triglyceride (El-Hussary, 1993). Fenugreek seeds may reduce triglyceride by

decreasing non Esterified Fatty Acids (NEFA) which are always the major component for this

fraction of lipid profile (Reinila, 1981). The study of Narender et al. (2006) reported that, amino

acid, 4-hydroxy isoleucine isolated from fenugreek seeds significantly decreased total

cholesterol (22%), plasma triglyceride (33%) and free fatty acids (14%), accompanied by an

increase in HDL by 39% in the dyslipidemic hamster model. There was a significant decreased

in total lipid, triglyceride and LDL concentration on long term application of fenugreek seeds

in all the supplemented groups. However, long term administration of fenugreek seeds had a

significant increase on High Density Lipoprotein (HDL). Low density lipoprotein and high

density lipoprotein worked as antagonist, decreased in LDL and increased HDL which

represents protection against atherosclerosis and coronary heart disease (Murray et al., 2003).

Mean serum total cholesterol value in normal healthy group was found 98.5±2.1 mg/dl that

observed significantly increased in positive control group (140.33±3.2 mg/dl). When

fenugreek extract was added in diet, it significantly reduced the serum total cholesterol to

107.83±2.2 mg/dl (El-Soud et al., 2007). Daily administration of fenugreek for 30 days

succeeded to reduce total cholesterol and triglycerides significantly. Concerning triglycerides

it was 53.68% and for cholesterol it was 48.22% when compared with diabetic control rats

(Marzouk et al., 2013).

4.16.5 Liver functions tests

Liver function tests comprised of aspartate transaminase (AST), alanine transaminase (ALT)

and alkaline phosphatase (ALP) were carried out for safety reasons.

156

4.16.5.1 Aspartate aminotransferase (AST)

The statistical analysis given in Table 4.74 indicated that AST level was affected substantially

in all three studies. Mean AST values for D0, D1 and D2 groups in study I were 105.12±3.00,

100.33±2.26 and 101.27±2.44 IU/L, correspondingly. However, in study II, means for AST

indicated maximum value in D0 (134.56±4.31 IU/L) than that of D1 (121.52±3.31 IU/L) and

D2 (128.40±2.15 IU/L). Besides in study III, D0 group showed maximum AST level

(165.32±5.65 IU/L) that reduced substantially in D1 (146.75±5.33 IU/L) and D2 (156.01±4.510

IU/L).

4.16.5.2 Alanine transaminase (ALT)

It is obvious from the statistical analysis that diets substantially affected serum ALT level in

all studies except for study I (Table 4.75). In this case, mean ALT values for D0, D1 and D2

were 41.12±1.60, 38.52±1.40 and 39.41±1.93 IU/L, respectively. Nonetheless in study II, this

trait was higher in D0 (51.23±1.88 IU/L) that significantly lowered in D1 (45.16±1.69 IU/L)

and D2 (48.66±2.04 IU/L) groups. Likewise in study III, the higher ALT value 59.63±1.95

IU/L was in D0 group that significantly decreased to 53.40±0.96 and 56.68±1.60 IU/L in D1

and D2 groups, respectively.

4.16.5.3 Alkaline phosphatase (ALP)

It is deduced from the statistical analysis that ALP level was affected significantly by diets in

all studies (Table 4.76). Means for ALP in study I were 112.36±2.37, 104.91±3.43 and

107.20±2.45 IU/L in D0, D1 and D2 groups, respectively.

Nevertheless in study II, higher ALP value was noticed in D0 (185.12±2.64 IU/L) that

decreased significantly in D1 (165.77±3.46 IU/L) and D2 (175.64±3.48 IU/L) groups. In study

III, high cholesterol diet induced enhancement in the ALP level by 201.32±4.14 IU/L in D0

whereas ALP values were reduced in D1 and D2 groups by 174.65±4.71 and 188.46±4.09 IU/L,

respectively.

Serum enzymes like ALT and AST are used in the evaluation of hepatic disorders. The

increasing activity of these enzymes depicted active liver damage. Inflammatory hepatocellular

disorders result in extremely elevated transaminase levels (Hultcrantz et al., 1986).

157

Table 4.73 Effect of supplemented diets on triglycerides (mg/dL)

Studies

Diets

F value

D0 D1 D2

Study I 79.00±5.24 76.27±4.93 79.63±5.32 0.60NS

Study II 96.00±3.31a 86.51±3.76b 91.66±3.08ab 9.76**

Study III 105.00±5.09a 93.09±3.47b 98.36±3.96ab 9.92**


NS= Non Significant

D0 : Control diet






Figure 4.15 Percent decrease in triglycerides as compared to control

-3.45

-9.89

-11.34

-0.47

-4.52

-6.32

-12.00

-10.00

-8.00

-6.00

-4.00

-2.00

0.00




158

The study of Vozarova et al. (2002) reported that, high ALT value is an indicator of type 2

diabetes risk and proposed a potential role of liver in the pathogenesis of type 2 diabetes. In

similar way Kaviarasan et al. (2007) suggested that polyphenolic compounds present in

fenugreek seeds can be considered cytoprotective during ethanol induced liver damage. The

seeds extract addition in the diet had a positive effect on both lipid profile and qualitative &

quantitative properties of collagen in alcoholic liver disease. In diabetic rats, ALP activity was

found significantly increased by 72.31% to their normal levels. This protective role is due to

presence of bioactive phytochemicals in fenugreek seeds. (El-Dakak et al., 2013).

The increase in activity of ALP is mainly due to the leakage of these enzymes from liver

cytosol into the blood stream (Mansour et al., 2002). On the other hand, addition of fenugreek

extract in the diet of diabetic rats resulted in decreased ALT, ALP & AST activity and leads

towards normal values. The increased in values of serum urea, uric acid and creatinine were

observed in diabetic rats that may be due to disturbance in metabolic activity, reflected in high

activities of lipid peroxidation, xanthine oxidase and increased cholesterol and triacylglycerol

(Anwar and Meki, 2003). The study of Eidi et al. (2007) explained the effect of fenugreek

extract feeding for 14 days on the level of alanine aminotransferase (ALT) and aspartate

aminotransferase (AST) in normal and diabetic rats and found significant decreased in these

parameters. Similar significant changes observed with legumes diets on serum AST, ALT and

AP, compared with rats of the positive group. Serum AST, ALT and AP were the lowest in

rats fed on fenugreek seeds (Mahfouz et al., 2012).

The activities of liver function markers (AST, ALT, ALP) were significantly elevated in STZ-

diabetic rats by 14.33%, 42.73% and 91.96%, respectively when compared with the normal

controls rats. The rats administrated fenugreek for 30 days showed significant reduction in

these marker enzyme activities to almost normal levels. The percentage of decrease, when

compared to diabetic control rats was 38.30% for AST; 42.12% for ALT and 27.12% for ALP

(Marzouk et al., 2013).

A team of researchers Hamza et al. (2012) designed an experiment to explore preventive and

curatine perspective of fenugreek in type 2 diabetis rats fed on a fat rich diet. Fenugreek extract

was fed to the rats @ 2 mg/kg body weight for whole trial period. Rats were divided in three

groups i.e. control, high fat diet and fenugreek extract treated high fat diet group. Control group

159

Table 4.74 Effect of supplemented diets on serum AST (IU/L)

Studies

Diets

F value

D0 D1 D2

Study I 105.12±3.00a 100.33±2.26b 101.27±2.44ab 4.79*

Study II 134.56±4.31a 121.52±3.31c 128.40±2.15b 18.7**

Study III 165.32±5.65a 146.75±5.33c 156.01±4.51b 16.00**

* = Significant


Table 4.75 Effect of supplemented diets on serum ALT (IU/L)

Studies

Diets

F value

D0 D1 D2

Study I 41.12±1.60 38.52±1.40 39.41±1.93 3.16NS

Study II 51.23±1.88a 45.16±1.69b 48.66±2.04a 13.2**

Study III 59.63±1.95a 53.40±0.96c 56.68±1.60b 19.8**


NS= Non Significant

D0 : Control diet






160

Table 4.76 Effect of supplemented diets on serum ALP (IU/L)

Studies

Diets

F value

D0 D1 D2

Study I 112.36±2.37a 104.91±3.43b 107.20±2.45b 9.31**

Study II 185.12±2.64a 165.77±3.46c 175.64±3.48b 45.1**

Study III 201.32±4.14a 174.65±4.71c 188.46±4.09b 47.5**


Table 4.77 Effect of supplemented diets on Urea (mg/dL)

Studies

Diets

F value

D0 D1 D2

Study I 22.10±0.35 21.65±0.66 21.88±0.77 0.64NS

Study II 35.16±0.76a 33.58±0.75b 34.06±0.97ab 4.71*

Study III 29.63±0.63 28.41±1.72 28.67±1.94 0.86NS

*= Significant

NS= Non Significant

D0 : Control diet






161

provided a lower value of triglyceride (29.9±19.7 mg/dL), which was elevated by feeding on

high fat diet in second group (62.8±18.3 mg/dL). In third group a substantial decline in

triglyceride was found due to the presnce of fenugreek seeds extract (17.9±9.7 mg/dL). One of

the peers Kumar et al. (2014) investigated the effect of aqueous extract of fenugreek on serum

lipid levels in high fat diet (HFD)-induced obese rats to varify its potential to ameriolae

dislipidemia. By adding fenugreek seeds extract in different doses, the elevated level of LDL

reduced, restored the level of HDL and showed pronounced effect in reducing triglycerides.

4.16.6 Renal function tests

Renal functioning tests were carried out to determine the effect of fenugreek supplemented

diets against renal malfunctions.

4.16.6.1 Urea

The statistical analysis indicated significant effect of diet on urea in study II, whilst, non-

significant effect was observed for other two diets in study I & III (Table 4.77).

Means of study I indicated maximum serum urea value 22.10±0.35 mg/dL in D0 that

diminished to 21.65±0.66 and 21.88±0.77 mg/dL in D1 and D2 groups, respectively. Whereas

in study II, serum urea value 35.16±0.76 mg/dL in D0 group was significantly decreased to

33.58±0.75 mg/dL (D1) and 34.06±0.97 mg/dL (D2). Likewise in study III, mean serum urea

values for D0, D1 and D2 differed momentously i.e. 29.63±0.63, 28.41±1.72 and 28.67±1.94

mg/dL (Table 4.77).

4.16.6.2 Creatinine

The statistical analysis indicated non-substantial effect of diets on creatinine level in study I &

III whilst significant tendency for this trait was noticed in study II (Table 4.78).

In study I, D0 showed the highest creatinine value 0.81±0.02 mg/dL whereas, D1 and D2 groups

exhibited lower values as 0.78±0.02 and 0.79±0.02 mg/dL, respectively (Table 4.78).

Likewise, in study II, creatinine level in D0 was 1.09±0.03 mg/dL that differed momentously

in D1 and D2 as 1.04±0.01 and 1.06±0.03 mg/dL, respectively. Nevertheless in study III, a non-

significant decline was observed from 0.98±0.03 mg/dL in D0 to 0.94±0.02 mg/dL in D1.

162

The results of current study are in harmony with findings of Eidi et al., (2007) who studied

that effect of oral administration of fenugreek extract for two weeks on level of serum urea,

uric acid and creatinine in normal and diabetic rats. The feeding of extract significantly lower

the values of serum urea, uric acid and creatinine levels. Creatinine is a break down product of

creatinine phosphate and an indicator of kidney functioning. During healthy state, creatinine is

filtered by the kidney but during disease condition its level increased drastically. The elevated

blood creatinine is an indicator for impaired glomerulus filtration that is a first sign of kidney

diseases. Moreover, protein glycation in diabetes may lead to increased release of purine,

muscle wasting and main source of uric acid, as well as increased activity of xanthine oxidase

(Anwar and Meki, 2003).

Similarly, increased values of serum uric acid, urea nitrogen and creatinine in diabetic rats as

compared to non-diabetic. The increased uric acid level in diabetic rats may be due to metabolic

disturbance in diabetes reflected in high activities of xanthine oxidase, lipid peroxidation and

increased triglycerides and cholesterol (Madianov et al., 1999). Treating diabetic groups with

high protein diet containing bread fortified with fenugreek decreased the mean values of serum

uric acid, urea nitrogen and creatinine, as compared to the positive control groups. Suresh et

al. (2012) reported that in Southeast Asia, the water extract of fenugreek seeds is used in the

management of diabetes and is known to improve kidney function during diabetes.

The research work of Meera et al. (2009) reported that significant hepatoprotective effects

were obtained against liver damage by adding ethanol extract of fenugreek leaves in rats diets.

There was non-significant changes in serum creatinine, whereas serum urea was significantly

decreased in rats fed on fenugreek seeds (Mahfouz et al., 2012). The results of current

investigation are also in harmony with study of El-Dakak et al. (2013) who observed decreased

level of the urea, uric acid and creatinine with administration of aqueous extract of

Lepidiumsativum L., lupin and fenugreek or their mixture in streptozotocin-induced diabetic

rats. The positive control showed a significant increase in creatinine, urea, and uric acid

compared to negative control group. However, fenugreek extract treated rat group showed a

significant decrease in serum creatinine, urea, and uric acid compared to positive control group.

163

Table 4.78 Effect of supplemented diets on creatinine (mg/dL)

Studies

Diets

F value

D0 D1 D2

Study I 0.81±0.02 0.78±0.02 0.79±0.02 0.98NS

Study II 1.09±0.03 1.04±0.01 1.06±0.03 5.18*

Study III 0.98±0.03 0.94±0.02 0.95±0.03 2.44NS

* = Significant

NS= Non Significant

Table 4.79 Effect of supplemented diets on red blood cell indices (cells/pL)

Studies

Diets

F value

D0 D1 D2

Study I 7.02±0.28 7.23±0.36 7.11±0.40 0.43NS

Study II 6.01±0.34 6.23±0.48 6.09±0.36 0.36NS

Study III 5.32±0.38 5.54±0.45 5.43±0.54 0.27NS

NS= Non Significant

D0 : Control diet






164

4.16.7 Hematological analysis

4.16.7.1 Red blood cell (RBC)

The statistical analysis in Table 4.79 showed that all the diets non-significantly affected

the RBC content of rats in all the three studies.

In study I the maximum value was observed in the diet D1 as 7.23±0.36 cells/pL followed

by D2 and D0 as 7.11±0.40 & 7.02±0.28 cells/pL, correspondingly. Likewise in study II and

study III the maximum values for RBC was observed in D1 as 6.23±0.48 and 5.54±0.45

cells/pL, respectively while minimum RBC content were observed in control treatment in both

studies as 6.01±0.34 & 5.32±0.38 cells/pL, respectively (Table 4.79).

4.16.7.2 White blood cells count (WBCs)

The statistical analysis indicated that diets imparted non-significant differences on WBCs in

all the three studies (Table 4.80).

In study I the maximum value was observed in the diet D0 as 12.36±0.29 cells/nL

followed by D2 and D1 as 12.24±0.38 & 12.08±0.23 cells/nL, correspondingly. While in

study II maximum WBC content was observed in D0 as 13.29±0.54 cells/nL and minimum was

noticed in D2 12.85±0.49 cells/nL. Furthermore, in study III maximum level was observed in

Do while minimum was observed in D1 as 16.96±0.65 and 16.40±0.51 cells/nL, respectively.

4.16.7.3 Platelets count (PLC)

The statistical analysis given in Table 4.81 illustrated that all the diets non significantly

affected the platelets count of rats in all the three studies.

In study I the maximum value was observed in the diet D1 as 7.28±0.38 followed by D2

and D0 as 7.19±0.34 & 7.12±0.42, correspondingly. While in study II maximum platelets were

observed in D2 as 6.83±0.46 and minimum was noticed in D0 6.63±0.30. Furthermore, in study

III maximum level was observed in D2 while minimum was observed in D0 as 6.31±0.46 and

6.13±0.34, respectively (Table 4.81).

165

Table 4.80 Effect of supplemented diets on white blood cell Indices (cells/nL)

Studies

Diets

F value

D0 D1 D2

Study I 12.36±0.29 12.08±0.23 12.24±0.38 0.32NS

Study II 13.29±0.54 13.09±0.56 12.85±0.49 0.60NS

Study III 16.96±0.65 16.40±0.51 16.72±0.56 1.12NS

NS= Non Significant

Table 4.81 Effect of supplemented diets on Platelets count

Studies

Diets

F value

D0 D1 D2

Study I 7.12±0.42 7.28±0.38 7.19±0.34 0.21NS

Study II 6.63±0.30 6.72±0.39 6.83±0.46 0.35NS

Study III 6.13±0.34 6.23±0.39 6.31±0.46 0.26NS

NS= Non Significant

D0 : Control diet






166

The present result trend was in accordance with the outcomes of Khalil (2004) who explored

blood biochemical profile and its behavior under fenugreek administration in rats. Results

showed a non-momentous effect of fenugreek extract on red blood cells and white blood cells

as it changes in hyperglycemic and fenugreek treated hyperglycemic groups, correspondingly.

The protective effect of fenugreek polyphenolic fraction against oxidative damage to red blood

cells was investigated by Kaviarasan et al. (2004). They devised a trial to investigate and

validate the potency of fenugreek seeds polyphenols to protect RBC against hydrogen peroxide

(H2O2) induced oxidation in normal and diabetic models. In diabetic models, increased

vulnerability to oxidative damage and lipid peroxidation was found when compared with

normal subjects. In the study of Sindhu et al. (2012) effect of fenugreek extract various

hematological aspects was enumerated such as WBC count and RBC count in experimental

animals with induced arthritis. In control arthritic group, red blood cell were in lower range

i.e. 3.52±0.2 (103/mm3). Fenugreek extract was prepared in distilled water and fed @ 75

mg/kg/day through oral administration, that resulted in little rise in RBC content (4.52±0.03

103/mm3). In the same table data showed that the control +ve group was significantly

decreased RBCs 10/µL as compared with the control negative group. All supplemented

diets of anemic rats revealed an enhancement of red blood cells count as compared to

the control (+). The best results were obtained for diets supplemented with fenugreek

leaves and germinated fenugreek (7.6±0.8 and 7.1±0.9 10/µL) as compared with the

control negative group (Mahmoud et al., 2012).

The scientific research of Abdul-Rehman (2012) investigated the effect of fenugreek seeds on

blood characteristics such as WBC, RBC and thrombocytes etc. in experimental animals. The

animals were divided into four groups, control, fenugreek extract fed once a week, twice a

week and daily. Fenugreek seeds showed little improvement in blood picture in comparison

with control represented by the momentous increment in the RBCs (2.31±0.05 to 2.92±0.07

million/mm3 from 1st to 4th group. The findings also revealed non-significant and haphazard

effect of fenugreek seeds on white blood cell content as it 17379.33±272.75, 17358.67±263.05,

17582.00±234.61 and 17500.22±251.93 thousand/mm3 in 1st, 2nd, 3rd and 4th groups,

correspondingly. Fenugreek is an ancient therapeutic herb with documented use for the

prevention of arthritis and inflammation. A study was planned by Suresh et al. (2012) to

investigate the extent of fenugreek extract on blood parameters such as red blood cells and

167

white blood cells in albino rats. The RBC content in arthritic control was found to be

4.178±0.28 million/mm3, that showed a little increment 4.270±0.25 and 4.783±0.46

million/mm3 by feeding on fenugreek extract at different doses 200 and 400 mg/kg. The white

blood cell content in arthritic control was found to be 8,528±71.32 thousand/mm3, that showed

deduction upto 7,873±56.48 and 6,989±58.54 thousand/mm3 when fed on different doses of

fenugreek extract i.e. 200 and 400 mg/kg, correspondingly. The present outcomes were also in

harmony with the research of Abdelgawad et al. (2012) who determined the preventive

potential of fenugreek seeds @ 2 and 4% (w/w feed) on experimental models. The mean

difference in platelets count depicted non-momentous effect of fenugreek seeds on platelet

count in all groups i.e. fed on 2 and 4% diet.

168

Chapter 5

SUMMARY

The research work was carried out to evaluate the acceptability of fenugreek utilization in

baked products. The wheat was cleaned by sieving followed by tempering and milling to get

straight grade flour. Likewise, fenugreek leaves and seeds were cleaned by washing and

screening followed by drying and grinding to get fine powder. The resultant powder was added

in wheat flour with various percentages to get composite flours. The wheat flour, fenugreek

leaves and seeds powder was analyzed for their chemical composition, mineral profile and

wheat flour for rheological properties. The composite flours were also analyzed for their

chemical composition, mineral profile, antioxidant assay and rheological properties. The bread

prepared from composite flours was subjected to chemical analysis, mineral profile, color

tonality, texture analysis, antioxidant assay and sensory quality. On the basis of antioxidant

assay, rheological characteristics and overall acceptability of bread, two best treatments each

from leaves and seeds powder were selected for efficacy study.

Wheat flour was evaluated for chemical composition by analyzing various parameters;

including 13.5, 0.41, 10.5, 1.13 and 0.31% of moisture, ash, crude protein, fat, fiber,

respectively whilst, Nitrogen Free Extract (NFE) was found to be 74.15%. Fenugreek leaves

powder (dry weight basis) was subjected to different quality assessment and revealed moisture,

ash, crude protein, fat, fiber and NFE as 9.38, 6.37, 4.30, 0.88, 1.98 and 77.09%, respectively.

Chemical composition of the fenugreek seeds has been illustrated as moisture (10.65%), ash

(4.14%), crude protein (22.86%), fat (6.98%), fiber (7.90%) and NFE (47.47%). The mineral

profile of wheat flour contains sodium, potassium, iron, calcium, copper, zinc and manganese

was observed as 3.12, 130.00, 3.50, 22.00, 0.13, 0.19 and 0.70 mg/100g, respectively. The

fenugreek leaves powder mineral composition indicated 58.71 mg/100g sodium, 481.91

mg/100g potassium, 6.72 mg/100g iron, 589.00 mg/100g calcium, 0.15 mg/100g copper, 0.90

mg/100g zinc and 0.75 mg/100g manganese, respectively. The mineral profile of fenugreek

seeds powder exhibited that it contains potassium in the highest quantity of 296.41 mg/100g

followed by calcium (160.00 mg/100g), sodium (23.69±2.18 mg/100g), iron (19.60±1.98

mg/100g), zinc (2.10 mg/100g), manganese (1.20 mg/100g) and copper (0.75 mg/100g).

169

Different supplemented (treatments) flour prepared by the addition of fenugreek leaves and

seeds powder were also analyzed for different chemical characteristics. The mean values

ranged between 12.88 to 13.29, 0.71 to 1.30, 10.18 to 9.57, 1.12 to 1.13, 0.39 to 0.56 & 74.31

to 74.60% for moisture, ash, crude protein, crude fat, crude fiber and NFE, respectively in

different leaves powder supplemented treatments. In seeds supplemented treatments moisture

content were observed between (13.08 to 13.33%), ash (0.60 to 0.97%), crude protein (11.13

to 12.35%), fat (1.42 to 2.01%), fiber (0.68 to 1.45%) and NFE (70.15 to 72.82%), accordingly.

Momentous differences were observed in sodium, potassium, iron, calcium and zinc whereas,

copper and manganese content were found non-momentous among different treatments

supplemented with fenugreek leaves powder. The results for fenugreek seeds powder

supplementation exhibited significant difference among treatments for mineral. Mean values

for sodium, potassium, iron, calcium, copper, zinc & manganese content ranged from 5.90 to

11.46, 147.60 to 182.79, 3.66 to 3.98, 50.35 to 107.05, 0.14 to 0.18, 0.23 to 0.30 and 0.71 to

0.75 mg/100g in leaves powder supplemented treatments, whereas treatments with seeds

powder supplementation ranged from 6.07 to 11.98, 150.92 to 192.76, 4.31 to 5.92, 28.90 to

42.71, 0.16 to 0.22, 0.29 to 0.48 and 0.73 to 0.78 mg/100g, respectively.

The rheological characteristics were significantly affected with supplementation of

fenugreek leaves and seeds powder. Dough properties like water absorption increased from

60.60 to 63.10%, dough development time enhanced from 6.17 to 7.50 min, dough stability

also increased 11.71 to 12.81 min while mixing tolerance index decreased from 26.00 to 18.45

BU with leaves powder supplemented treatments. The dough properties of seeds powder

supplemented treatments had water absorption in range from 60.60 to 62.17%, dough

development time 6.17 to 8.40 min, dough stability 11.71 to 13.78 min and mixing tolerance

index 16.46 to 26.00 BU. Mixing time of dough decreased from 5.73 to 4.70 min in leaves

supplemented flours whereas in seeds supplemented flours 5.73 to 4.80 min.

The statistical analysis showed that addition of fenugreek in wheat flour imparted

significant differences on the antioxidant indices of treatments. Regarding leaves

supplemented treatments, highest TPC was found in T3 (390.00 mg GAE/100g) and

minimum in T0 (117.00 mg GAE/100g), in similar way increasing trend was found in total

flavonoids (2.38 to 2.97 mg CE/g) with increasing supplementation level. Likewise,

170

antioxidant activities enhanced with increasing the supplementation level with maximum

DPPH scavenging potential (47.00%), β-carotene assay (39.00%), FRAP (401.00 µmol Fe2+/g)

observed in 15% fenugreek leaves powder (T3). The mean values revealed that TPC in different

treatments were 117.00 (T0), 305.00 (T4), 410.00 (T5) and 450.00 mg GAE/100g (T6) while

total flavonoids 2.38 (T0) to 3.01 mg CE/g (T6), showed ascending order with increasing

fenugreek seeds powder supplementation in wheat flour. From mean squares of DPPH

scavenging activity a highly significant relation was observed for supplementation and mean

ranged from 35.00 (T0) to 58.00% (T6). The mean values for β-carotene assay illustrated that

it varied from 33.00% (T0) to 51.00% (T6), similar increasing trend observed for FRAP values

with maximum found in T6 (501.00 µmol Fe2+/g) and minimum in T0 (222.00 µmol Fe2+/g)

that indicates inclining trend with increasing percentage of fenugreek seeds powder.

In the product development phase, bread was prepared with varying level of fenugreek leaves

and seeds powder. Momentous variations in chemical composition of bread was observed like

moisture (34.46 to 34.68%), ash (1.24 to 2.24%), crude fiber (1.27 to 1.44%) and NFE (45.49

to 45.67%). The increase was observed by increasing leaves supplementation level while crude

protein and crude fat were decreased as supplementation level enhanced attributed to low

protein and fat content of fenugreek leaves. The mean moisture content of bread supplemented

with seeds powder varied from 34.46 to 34.63%, ash content found in a wide range as T0

(1.24%), T4 (1.39%), T5 (1.55%) and T6 (1.72%), crude protein revealed a decreasing trend as

T0 (11.12%), T4 (11.74), T5 (12.32%) and T6 (12.79%). The mean crude fat content in T0

(control) was found to be 5.97% whereas, the incorporation of seeds powder in the bread

resulted a progressive increase in fat content i.e. T4 (6.25%), T5 (6.54%) and T6 (6.81%). The

mean values for fiber content (Table 4.26) were observed in the range of 1.27% (T0) to 2.29%

(T6). Mean values for NFE illustrated decreasing trend as T0 (45.49%), T4 (44.35%), T5

(43.04%) and T6 (41.90%), respectively.

As both fenugreek leaves and seeds were found rich source of minerals hence its incorporation

in bread increased mineral content that leads to beneficial health effects. The mean values for

sodium content ranged from 4.10 to 12.28 and potassium 124.00 to 177.18 mg/100g. It was

observed that the iron content ranged from 3.44 to 3.86 and calcium content in bread ranged

from 19.96 to 104.78 mg/100g among different treatments. The highest copper value observed

in T3 (0.16 mg/100g) followed by T2 (0.14 mg/100g), T1 (0.12 mg/100g) whilst, the lowest

171

was exhibited in T0 (0.11 mg/100g). The zinc content varied from 0.17 to 0.26 mg/100g and

manganese content ranged from 0.66 to 0.71 mg/100g with highest content found in treatment

having 15% fenugreek leaves powder. Mean values for different seeds powder treatments

explained that maximum sodium was (12.83 mg/100g) in T6 followed by T5 (10.05 mg/100g),

T4 (7.02 mg/100g) whereas, control (T0) showed minimum value (4.10 mg/100g). The

potassium and iron content was found in the range of 124.00 to 187.30 mg/100g and 3.44 to

5.81 mg/100g, respectively. Mean values of calcium content increased by adding fenugreek

seeds powder with highest found in T6 (40.46 mg/100g) and lowest (19.96 mg/100g) in T0. The

copper content ranged from 0.11 to 0.19 mg/100g and zinc content varied from 0.17 to 0.46

mg/100g amongst different treatments. The highest manganese content observed in T6 (0.74

mg/100g) followed by T5 (0.73 mg/100g), T4 (0.71 mg/100g) and T0 (0.66 mg/100g).

Moreover, treatments had significant impact on antioxidant potential of the bread. The total

phenolic content showed momentous variations among leaves powder supplemented

treatments i.e. T0, T1, T2 & T3; 99.00, 198.00, 213.00 and 315.00 mg GAE/100g whereas,

means for total flavonoids; T0 (2.13 mg CE/g), T1 (2.47 mg CE/g), T2 (2.65 mg CE/g) and T3

(2.84 mg CE/g). The values of free radical scavenging activities ranged from 31.00 to 43.00%

that revealed enhanced antioxidant activity with increasing the supplementation level. The

highest β-carotene value 35.00% was recorded in T3 followed by T2 (33.00%) and T1 (31.00%)

whilst, the lowest 29.00% in control (T0). The recorded values for FRAP illustrated that T6

exhibited maximum reducing power (356.00 µmol Fe2+/g) followed by T2 (254.00 µmol

Fe2+/g) and T1 (201.00 µmol Fe2+/g) while minimum in T0 (167.00 µmol Fe2+/g). Means from

seeds powder supplemented treatments indicated highest TPC (413.00 mg GAE/100g) in T6

followed by T5 (341.00 mg GAE/100g) and T4 (238.00 mg GAE/100g). As examined from

mean values, total flavonoids varied from 2.13 to 2.91 mg CE/g that showed inclining trend

with increasing supplementation level. Means of DPPH scavenging activity for different

treatments observed as; T0 (31.00%), T4 (45.00%), T5 (49.00%) and T6 (51.00%). The

maximum β-carotene values found in T6 (43.00%) while minimum in T0 (29.00%), the mean

for FRAP values illustrated highest reducing power (450.00 µmol Fe2+/g) @ 15% seeds powder

supplementation while lowest (167.00 µmol Fe2+/g) in wheat flour (T0).

Mean squares regarding color tonality indicated the significant impact of supplementation on

treatments. Increasing concentration of leaves powder decreased color values except for b* and

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chroma. Likewise, additional increments of seeds powder decreased L* values & hue angle

while increasing trends of a* and b* and chroma. The addition of these powders non-

significantly influenced the textural characteristics i.e. chewiness, cohesiveness, elasticity,

gumminess, hardness and springiness of the bread.

Sensory evaluation of fenugreek leaves and seeds powder based bread explicated significant

effect of treatments and storage time on volume, aroma, taste and texture, while remaining

traits varied non-significantly. It was expounded from the means scores for overall

acceptability that bread containing 5% leaves powder and 10% seeds powder were considered

best with assigned score of 6.17 & 6.75 as compared to 7.16 (control). From the present

exploration, it is deduced that fenugreek leaves powder @ 5% and seeds powder @ 10% rated

better during sensory evaluation. Moreover, these treatments were also rich in bioactive

molecules hence selected for further use in efficacy studies.

Series of efficacy trials were conducted in normal (study I), hyperglycemic (study II) and

hypercholesterolemic (study III) rats modeling. The diets prepared from the selected treatments

i.e. 10% fenugreek seeds powder (D1) and 5% fenugreek leaves powders (D2) along with

control (D0) were fed to the respective groups of rats for a period of eight weeks. Water intake

and feed consumed on daily basis while body weights of individual rats in each group were

recorded weekly to determine the effect of respective diet on these parameters. The increase

of feed intake in different groups of rats explored as; normal rates (29.39 to 38.23 g/rat/day),

hyperglycemic rats (29.63 to 38.66 g/rat/day) and hypercholesterolemic rats (27.92±0.76 to

36.52±0.44 g/rat/day) fed to different groups. There was a progressive increasing trend in water

consumption by increasing fenugreek leaves and seeds powder in diets. Results regarding body

weight concluded that rats fed on 10% fenugreek seeds powder based diet (D1) observed with

maximum weight gain. The mean weight of rats in each group observed as; normal rats (230.97

to 372.64 g/rat), hyperglycemic rats (232.44 to 329.70 g/rat) and hypercholesterolemic rats

(228.68 to 321.37 g/rat) from start of experiment to termination.

It is worth mentioning that fenugreek leaves and seeds powder increased insulin level, reduced

blood glucose, cholesterol, LDL and triglycerides whilst, HDL improved non-significantly. In

study II and III, diets (D0, D1 & D2) showed significant effect on glucose whilst, non-

momentous effect was observed in study I. In study II, glucose reduction was 10.67% (D1)

173

followed by 6.78% (D2), while in study III maximum reduction was observed with D1 (8.12%)

followed by D2 (5.45%) when compared with D0 (control). The effect of diets was observed

momentous on cholesterol level in study II & III while non-momentous in study I.

Experimental diets reduced the elevated level of cholesterol by 2.96% (D1), 2.03% (D2) in

study I, in study II reduction was 8.45% (D1) & 4.52% (D2), while in study III level reduced

to 12.03% (D1) & 6.32% (D2), respectively. Insulin level found decreased in control group as

compared to increasing tendency of fenugreek leaves and seeds powder, however, insulin

secretion was significantly lowered in diabetic as compared to normal rats. There was an

increase of 4.01 and 2.96% for insulin level in groups of rat fed with fenugreek seeds and

leaves powder in study II, in similar way insulin secretion was more in D1 (3.25%), D2 (2.22%)

in study III as compared to control.

Both experimental diets improved the HDL level non-significantly in all studies while

significant reduction was observed in LDL and triglycerides. The addition of fenugreek leaves

(D2) seeds (D1) powder decreased LDL level by 3.63 & 6.64% in study I, 4.56 & 9.68% in

study II whereas, 7.83 & 12.25% recorded in study III when compared with control diet.

However, decrease of triglycerides in different groups of rats concluded that D1 showed

maximum reduction of 9.89 & 11.34% followed by D2 (4.52 & 6.32%) in study I & II with

reference to control.

In study I, II and III, diets depicted significant affect on liver function tests i.e. AST, ALT and

ALP. The AST value of rats reduced by 4.56 & 3.66% (study I), 9.69 & 4.58% (study II) and

11.23 & 5.63% (study III) when fed with experimental diets D1 and D2 as compared to control

diet (D0). Maximum reduction in ALT value was observed with D1 i.e. 6.32, 9.89 & 10.45%

in all studies, while decreased values for ALP in study I when fed with experimental diets was

4.59% (D2) & 4.59% (D1), in study II 5.12% (D2) & 10.45% (D1) and in study III 6.39% (D2)

& 13.25% (D1), respectively when compared with control.

Statistical analysis revealed non-significant effect of diets on red blood cells, white blood cells

and platelets count. In study I, red blood cells increased 1.23 to 3.01% & 1.36 to 3.69% (study

II) and 2.01 to 4.12% (study III) when fed with experimental diets D2 and D1. White blood

cells increased non-significantly from 1.01 to 2.13 (study I), 1.52 to 3.34% (study II) and 1.39

to 3.31% (study III) as a function of experimental diets as compared to control. Likewise in

174

study I, platelets count was increased from 1.01 to 2.22%, study II (1.36 to 2.96%) and study

III (1.63 to 3.01%), respectively. Conclusively, rich phytochemistry of fenugreek leaves and

seeds powder accredited for hypoglycemic and hypocholesterolemic effects.

The present investigation suggests that different chemical, rheological, antioxidant and product

sensory characteristics are variable among different treatments with supplementation of

fenugreek leaves and seeds powder. Feed intake and gain in body weight exhibited increasing

trend and significant reduction in glucose, cholesterol, LDL and triglycerides was observed

due to the addition of fenugreek leaves and seeds powder in wheat flour. The incorporation

of fenugreek do not add much to price, being an agricultural country, Pakistan should focus on

these neglected commodities that would not only solve the problems regarding mono-cropping

system but also ensure optimal health benefits to the society. Their micronutrient and

antioxidant potential prevent the society from escalating health care cost in managing various

diseases. According to World Bank report, cost of micronutrient malnutrition is approx. 5%

Gross National Product (GNP). On the other hand, interventional strategies cost up to 0.3% of

the total GNP. Therefore, interventions are considered cheaper over health care cost (Darnton-

Hill et al., 2005). Moreover, addition of fenugreek leaves and seeds cost only 50 and 200

Pakistani rupees per kilogram, respectively i.e. almost equal or comparable to any other

commodity incorporation. Furthermore, direct supplementation of fenugreek seeds and leaves

powder is much cheaper for low income countries as it saves labor cost and operational capital

regarding extraction & purification or encapsulation apart from hectic procedures.

175

ECONOMIC PERSPECTIVES

Pakistan is facing issues regarding poverty, unemployment, population growth (6th most

populous country), time constraints faced by working women in food preparation & reliance

on monotonous staples, difficulty in losing excess body fat due to closely spaced pregnancies

and lack of information & education to make prudent food choices. In addition, unequal

distribution or over-abundance of food with poor nutritional quality, high cost of nutritious

food relative to unhealthy snacks, education barriers particularly among females, inadequate

supply of safe water & sanitation facilities and street foods prepared under unhygienic

conditions are considered as the markers of societal failure. Under such prevailing

circumstances, it is important to go for some healthy dietary interventions that benefit the

society over long run and in a better way. Hence, it is the time to shift old paradigm of green

revolution; ensuring food security but no focus on nutritional value and human health with new

paradigm of ‘green consumerism’; utilizing plant moieties in functional food development thus

linking agriculture with nutritional & health security. Nowadays, food commodities with

multiple benefits such as fenugreek plant parts not only give food diversification but also

prevent various diseases. Furthermore, due to high poverty rate, dietary shift towards neglected

and diverse indigenous grains is necessitated to ensure ample provision of micronutrients and

phytochemicals to combat micronutrient deficiencies and lifestyle related disorders. In this

context, partial replacement or supplementation of fenugreek seeds and leaves in wheat flour

to form designer foods could be of nutritional and nutraceutical worth, promote underutilized

resources, reduce expensive wheat imports & save foreign exchange, encourage multigrain

concept and promote export of designer products as convenience food.

176

CONCLUSIONS

Fenugreek seeds are rich source of protein and has good antioxidant potential as

compared to fenugreek leaves, later is an excellent source of minerals as well.

Improved rheological properties of wheat-fenugreek composite flour have been

observed for economical and quality baked products.

Fenugreek leaves and seeds powder can be added up to 5 and 10% level in bread

formulation without affecting overall quality.

Hyperglycemia and hypercholesterolemia problems can be managed with the

utilization of fenugreek.

The prepared bread found with nutritionally rice profile, good acceptability and had

therapeutic value, thus be considered for commercialization.

With constant increase in consumer demand of bread and other bakery products in

many developing countries and to couple with ever-growing urban population, the

composite flour/bread technology could be very useful.

177

RECOMMENDATIONS

• Amongst diverse healthy options, use of fenugreek seeds and leaves need to promote

in daily cuisines for a health start owing to its micronutrient enhanced profile as well

as potent antioxidant activity

• Nutritionists should encourage the incorporation of fenugreek based designer products

with special reference to fenugreek seeds to mitigate hyperlipidemia and

hyperglycemia in addition to combat prevailing micronutrient malnutrition especially

calcium, iron and zinc

• To avoid the food extract based consumers controversies, inclusion of whole fenugreek

seeds and leaves could serve as a motivation, lead to tackle health menace in the society

besides guarantee public satisfaction regarding natural means by avoiding the use of

toxic solvent residues in case of extracts

• Besides, the education and adoption of healthy processing procedures such as

fermentation to reduce antinutrients like phytic acid ultimately improving

micronutrient bioavailability, at home scale as well as industrial level could

acknowledge the general public to get optimal benefits using prudent food

technological aspects

• Last but the most important is the use of indigenous crops especially minor grains and

cereals to bring economic stability, attraction amongst the eastern cuisines as well as

provision of platform for allied stakeholders

178

LIMITATIONS AND FUTURE VISTAS

• From clinical nutrition perspectives, it is important to experiment using human subjects

besides working on animals to better relate the outcomes and to improve the antioxidant

dose & modes of processing in accordance to the needs of the consumers

• Additionally, anti-oncogenic, anti-bacterial, anti-helmintic, anti-cholinergic and ulcer

& wound healing facets of fenugreek plant parts need to explore via efficacy trials

• To optimize the antioxidant effective dosage for healthy as well as diseased subjects,

there is a need to adopt novel extraction modes, nutrient sensitive processing

procedures and encapsulation technologies for maximum mass transfer, retention and

targeted delivery of nutraceutics, respectively

• Furthermore, concept of excipient foods is now finding their way into novel researches

where excipient ingredients serve as bioavailability enhancers in combination with

particular nutraceutic in designer foods

• For detailed knowledge, nutrikinetics study of an active moiety could tell us the

complete fate of a particular nutraceutic hence facilitate in targeting a specific body

organ where its retention is somewhat prolonged

• From microbiological aspect, food safety is the highlighted concept nowadays thereby

it is important to have knowledge about the potential of fenugreek antioxidants against

food spoilage agents

• Besides optimizing cooking losses and antioxidant retention, it is also important to

optimize cost and shelf life using different blends by applying response surface

methodology

• Consumer awareness programs regarding diet-health linkages ought to be launched

among the masses to motivate prudent food choices and to highlight the therapeutic

worth of designer breads over conventional edibles for healthy living and variety in

dietary patterns.

179

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APPENDIX I

Sensory Evaluation of Bread

Name of Judge ______________________

External Characteristics Treatments

Parameters T0 T1 T2 T3 T4 T5 T6

Volume

Color of Crust

Form Symmetry

Evenness of bake

Crust Character

Internal Characteristics Treatments

Parameters T0 T1 T2 T3 T4 T5 T6

Grain

Color of crumb

Aroma

Taste

Texture

Signature: ___________________ Date: ___________________

INSTRUCTIONS

Chew a sample and score for bread using the following 9-point Hedonic Scale:

Extremely poor 1 Very poor 2

Poor 3

Below fair above poor 4

Fair 5

Below good above fair 6

Good 7

Very good 8

Excellent 9

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APPENDIX II

Experimental Diets

Ingredients (%) D0 D1 D2

Wheat Flour 82 72 77

Corn Oil 10 10 10

Vitamin Mixture 3 3 3

Mineral Mixture 1 1 1

Casein 4 4 4

Fenugreek seeds powder - 10 -

Fenugreek leaves powder - - 5

Sucrose* 40 - -

Cholic acid** 1.5 - -

* Added in diet for first week of trial to induced hyperglycemia in respective group

** Added in diet for first week of trial to induced hypercholesterolemia in respective group

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