Omer Mukhtar Tararprr.hec.gov.pk/jspui/bitstream/123456789/34/1/145S.pdf · To my paranymphs Taj...

Development, Characterization and Shelf Life Optimization of

a Prototype Nutrient Dense Food Bar

By

Omer Mukhtar Tarar M.Sc. (Hons.) Food Technology

A Thesis Submitted in Partial Fulfil lment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

IN

FOOD TECHNOLOGY

NATIONAL INSTITUTE OF FOOD SCIENCE & TECHNOLOGY

UNIVERSITY OF AGRICULTURE,

FAISALABAD,

PAKISTAN

2009

To The Controller of Examinations,

University of Agriculture,

Faisalabad-Pakistan

“We, the Supervisory Committee, certify that the contents and

form of thesis submitted by OMER MUKHTAR TARAR , 92-ag-

1273 , have been found satisfactory and recommend that it be

processed for evaluation, by the External Examiner(s) for the award

of degree”.

Supervisory Committee

1. Chairman ___________________________ (Prof. Dr. Salim-ur-Rehman) 2. Member ___________________________ (Dr. Tahir Zahoor) 3. Member ___________________________ (Dr. Amer Jamil) 4. Special Member ___________________________ (Dr. Khalid Jamil)

Dedicated to

My Beloved Parents

ACKNOWLEDGEMENTS All praises and thanks are for Almightily Allah who is the entire source of all

knowledge and wisdom endowed to mankind.

The work described in this thesis was made possible by contributions from

various individuals and institutions to whom I am indebted. I thank all those

who, in different ways, have walked beside me along the way; offering support

and encouragement, challenging my thinking, and teaching me to consider

alternative views.

Prof. Dr Salim-ur-Rehman, thank you for believing in me; for your

encouragement through kind emails and phone calls across the miles at the

start of work; the support and your guidance; the way you provided

constructive guidance in the manuscript writing.

Prof. Dr. Faqir Muhammad Anjum, special thanks are due for your kind

guidance I received whilst my stay at NIFSAT for this degree.

Dr Javaid Aziz Awan, I value your comments. I began to really get to know you

during this period. I don’t take it for granted that you always made time to

discuss the work and give valuable comments on the work I presented you in

spite of your busy schedule.

Dr. Tahir Zahoor, a cooperative and affectionate personality, special thanks

are extended to you for the care, impetus and encouragements.

Dr. Khalid Jamil and Dr. Aamir Jamil, thank you for your mentoring and

support through it all. Your sound advice and contributions have gone a long

way in making all this possible.

To Dr Nuzhat Huma for your kind affection extended during my stay at

Faisalabad.

Dr. Tanzeel Haider Usmani, it was your sincere backup which make me able to

complete this work without any worry. Mrs Askari Begum, your telephonic

encouragement across a long distance is big moral booster for me. It acted as

source of illumination, to achieve the high goals.

To authorities at Higher Education Commission (HEC); who provided an

opportunity to me believe in myself; financial support for PhD studies through

HEC scholarship program make me able to achieve this mile stone which

could otherwise was almost impossible; thank you very much. I thank PCSIR

for granting me study leave and providing hurdles free financial support

throughout my studies.

To Dr. Anjum Murtaza, Semee Mumtaz and Ghulam Mueen-ud-Din who in

different ways have been supportive right from start of my study till thesis

writing; special thanks to you trio.

My heartfelt thanks also go to the following for their support:

To Dr Essa Khan and Aftab Ahmad, for organizing the parts of my

experimental work. Dr Shahzad Hussain, Dr Ali Asghar and Aneela Hamid;

occasional chit chat helped to improve my study. To my paranymphs Taj Din,

Tariq Ismail, Haider, Ghulam Mustafa and Ilyas; for assisting during the

course of experimental work. To Manzoor Ahmad, for helping out with the

official matters at Karachi.

To all my friends, especially Akif Nadeem, Aleem Sarwar and Imran Saeed.

Thank you for your prayers, encouragement and support.

Mr Iqbal Qureshi; you patronized me in true sense of affection and kindness

right from joining the university till completion of the work. I am forever

grateful.

Dr. Mumtaz Akhtar Cheema and Prof. Dr. Zafar Iqbal Qureshi your act as

elder brothers provided me strength to face all the hardships encountered.

To late Dr. Ishtiaq Tarar, your sad demise is irrecoverable loss for me. Your

imprint on my soul is ever lasting. It provides me the courage to work hard

and stand firm on principles.

To my brother Saad Tarar, I am deeply obliged for sincere wishes offered by

you during my study.

Thanks are due to Mr. and Mrs. Maj. Rashid Ahmad Tarar, Col. Inam, Maj.

Ahtisham and Saira for your well wishes and prayers.

To my children Hamna, Rida and Muhammad who deeply felt my absence

during long week days. I also missed you my children!

To my better half Mrs. Sadia Tarar for devoting his energies to make me able

to work worries free; for sacrificing while taking care of all of the family in

splendid manner; for understanding when I could not be able to join my

family on important events. Thank you for all, what you did for me.

Last but not least, warm thanks to my parents; for support, moral strength

and material welfare throughout my life and for being there for me during this

time. Your love, encouragement and prayers made a big difference.

Omer Mukhtar Tarar

TABLES OF CONTENTS Chapters Title Page

1 INTRODUCTION 01 2 REVIEW OF LITERATURE 06 2.1. Development of a food bar 06 2.2 Selection of grains for food bars 09 2.2.1. Complementation of cereals 09

2.2.1.1. A candidate for complementation - Indian vetch (Lathyrus sativus L.) 09

2.2.2. Antinutritional factors in legumes 10 2.2.2.1. Trypsin inhibitors 11 2.2.2.2. Tannin 11 2.2.2.3. Phytates 11 2.2.2.4. β-ODAP 12 2.2.3. Antinutritional factors reducing strategies 14 2.2.3.1. Soaking 14 2.2.3.2. Autoclaving/ Cooking 15 2.2.3.3. Controlled fermentation 16 2.2.3.4. Natural fermentation 17 2.2.3.5. Germination 18 2.2.4. Bioavailability of minerals 19 2.2.5. Protein quality evaluation 23

2.2.5.1. Protein Digestibility Corrected Amino Acid Score (PDCAAS) 23

2.2.5.2. Biological efficacy studies 24 2.2.5.3. In-vitro digestibility 29 2.4 Storage changes in foods 30 2.5. Extending shelf life 30 2.5.1. Packaging materials 31 2.5.2. Use of antioxidants 31 2.5.2.1. Pomegranate (Punica granatum L.) extract 33 2.5.2.2. Amla (Emblica officinalis) fruit extract 35 2.6. Optimization of shelf life 36 2.6.1. Response surface methodology (RSM) 36 3 MATERIALS AND METHODS 38 3.1. Materials 38

3.2. Processing of Indian vetch (Lathyrus sativus L.) and chickpea (Cicer aritenum L.) 38

3.2.1. Soaking 38 3.2.2. Autoclaving 39

3.2.3. Germination 39 3.2.4. Controlled fermentation 39 3.2.5. Natural fermentation 40 3.3. Anti-nutritional factors in processed legumes 40 3.3.1. Estimation of tannin contents 40 3.3.1.1. Preparation of Folin-Denis reagent 40 3.3.1.2. Preparation of carbonate solution 40 3.3.1.3. Preparation of standard tannic acid solution 40 3.3.1.4. Preparation of working solution 40 3.3.1.5. Procedure 41 3.3.2. Estimation of total phenols 41 3.3.2.1. Preparation of standard solution 41 3.3.2.2. Procedure 41 3.3.3. Trypsin inhibitor activity 42 3.3.3.1. Preparation of 0.1 M phosphate buffer (pH 7.6) 42 3.3.3.2. Preparation of 0.1 M phosphate buffer (pH 7.0) 42 3.3.3.3. Casein 2% solution 42 3.3.3.4. Trypsin solution (5mg/ml) 42 3.3.3.5. Trichloroacetic acid (TCA) 5% solution 42 3.3.3.6. Procedure 42 3.3.3.7. Protein determination 43 3.3.3.8. Preparation of alkaline CuSO4 solution 43 3.3.3.8. Procedure 43 3.4. Phytate content 43 3.3.4.1. Preparation of phytate reference solution 43 3.3.4.2. Preparation of ferric solution 43 3.3.4.3. Preparation of 2, 2- bipyridine solution 43 3.3.4.4. Procedure 44 3.3.5. Determination of β-ODAP in Indian vetch 44 3.3.5.1. Preparation of OPA reagent 44 3.3.5.2. Procedure 44 3.4. Mineral profile 45 3.4.1. Preparation of sample 45

3.4.2. Estimation of mineral contents (Cu, Zn, Mn, Mg and Fe) 45

3.4.3. Estimation of sodium (Na) and potassium (K) contents 45

3.4.4. Estimation of phosphorous (P) content 45 3.4.4.1. Preparation of 2.5% ammonium molybdate 45

3.4.4.2. Preparation of aminonaphthol sulphonic acid 45 3.4.4.3. Preparation of phosphorus standard solution: 46 3.4.4.4. Procedure 46 3.4.5. Minerals extractability 46 3.4.5.1. Procedures 46 3.5. Proximate analysis 46 3.5.1. Moisture 46 3.5.1.1. Procedures 47 3.5.5. Total ash 47 3.5.5.1. Procedure 47 3.5.2. Crude protein 47 3.5.2.1. Procedure 47 3.5.3. Crude fat 48 3.5.3.1. Procedure 48 3.5.4. Crude fibre 48 3.5.4.1. Procedure 48 3.5.6. Nitrogen Free Extract (NFE) 49 3.6. Development of food bar 49 3.6.1. Product preparation through baking 50 3.7. Biological evaluation of protein meals 50 3.7.1. Preparation of meals 50 3.7.2. Biological assay 51 3.7.3. Nutritional indices 52 3.7.4. Amino acid analysis 52

3.7.5. Protein digestibility-corrected amino acid score method (PDCAAS) 53

3.8. Prototype selection 54 3.8.1. Sensory evaluation 54 3.8.2. In-vitro protein digestibility (IVPD) 54 3.8.2.1. Preparation of pepsin solution 54 3.8.2.2. Procedure 54 3.8.3. In-vitro starch digestibility (IVSD) 54 3.8.3.1. Preparation of porcine pancreatic amylase solution 54 3.8.3.2. Procedure 55 3.8.4. Proximate analysis. 55 3.8.5. Gross energy value 55 3.9. Extraction of antioxidant extract 55 3.10. Screening of antioxidant extract 55 3.10.1. Estimation of total phenols 56

3.10.2. Determination of antioxidant activity 56 3.10.2.1. Procedure 56 3.10.3. Radical scavenging activity 56 3.10.3.1. Procedure 56 3.11. Shelf life testing of bars 57 3.12. Experimental design for shelf life optimization 57 3.13. Determination of dependent variables 58 3.13.1. Sensory testing 58 3.13.2. Moisture content 58 3.13.3. Color measurement 58 3.13.4. Hue angles 59 3.13.3.2. Chroma values 59 3.13.4. Product slurry pH 59 3.13.5. Free fatty acid determination 59 3.13.5.1. Procedure 59 3.13.6. Peroxide value determination 59 3.13.6.1. Procedure 59 3.14. STATISTICAL ANALYSIS 60 4 RESULTS AND DISCUSSION 61 4.1. Effect of processing techniques on the nutritional

quality of legumes 62

4.1.1. Objective 62 4.1.2. Results 62 4.1.2.1. Moisture 62 4.1.2.2. Ash 62 4.1.2.3. Crude fat 65 4.1.2.4. Crude protein 65 4.1.2.5. Crude fiber 65 4.1.2.6. Nitrogen free extract (NFE) 69 4.1.3. Discussion 69 4.1.4. Conclusion 76 4.2. Effect of processing on antinutritional factors of legumes 77 4.2.1. Objective 77 4.2.2. Results 77 4.2.2.1. Tannin content 77 4.2.2.2. Total phenols 77 4.2.2.3. Trypsin inhibitor activity (TIA) 80 4.2.2.4. Phytic acid 82 4.2.2.5. β-ODAP 82 4.2.3. Discussion 85 4.2.4. Conclusion 93 4.3. Effect of processing on HCL-extractability of

minerals in legume seeds 94

4.3.1. Objective 94

4.3.2. Results 94 4.3.2.1. Copper 94 4.3.2.2. Manganese 96 4.3.2.3. Magnesium 96 4.3.2.4. Sodium 99 4.3.2.5. Potassium 99 4.3.2.6. Zinc 102 4.3.2.7. Iron 102 4.3.2.8. Phosphorous 105 4.3.3. Discussion 107 4.3.4. Conclusion 109 4.4. Biological evaluation of protein meals for nutrient

dense food bar making 110

4.4.1. Objective 110 4.4.2. Results 110 4.4.2.1. Feed /protein intake 110 4.4.2.2. Weight gain 110 4.4.2.3. Feed efficiency 112 4.4.2.4. Feed utilization (FU) 112 4.4.2.5. Protein utilization (PU) 112 4.4.2.6. Protein efficiency ratio (PER) 112 4.4.2.7. Net protein retention (NPR) 115 4.4.2.8. Corrected protein efficiency ratio (C-PER) 115 4.4.2.9. Relative protein efficiency ratio (RPER) and

Relative net protein ratio (RNPR) 115

4.4.3. Discussion 117 4.4.4. Conclusion 119 4.5. Protein Digestibility-Corrected Amino Acid Score

(PDCAAS) method 120

4.5.1. Objective 120 4.5.2. Results 120 4.5.3. Discussion 121 4.5.3.1. Role of amino acids 121 4.5.3.2. In vivo assay for true protein digestibility 128 4.5.3.3. Protein digestibility-corrected amino acid score

method (PDCAAS) 129

4.5.4. Conclusion 130 4.6. Selection of a prototype nutrient dense food bar 131 4.6.1. Objectives 131 4.6.2. Results 131 4.6.2.1. Proximate analysis 131 4.6.2.2. Calorific values 131 4.6.2.3. In vitro protein digestibility (IVPD) 131 4.6.2.4. In vitro starch digestibility (IVSD) 133 4.6.2.5. Sensory characteristics 133 4.6.3. Discussion 136 4.6.4. Conclusion 140 4.7. Antioxidant extract from agriculture waste materials 141 4.7.1. Objectives 141 4.7.2. Results 141

4.7.2.1. Extraction of antioxidants from food processing wastes

141

4.7.2.2. Total phenols 141 4.7.2.3. Antioxidant activity 144 4.7.2.4. Radical scavenging activity 144 4.7.3. Discussion 144 4.7.3.1. Extraction of antioxidants from food processing

wastes 144

4.7.3.2. Screening of natural antioxidant extract 152 4.7.4. Conclusion 155 4.8. Optimization of antioxidant extract levels for a shelf

stable bar using response surface methodology (RSM) 156

4.8.1. Objective 156 4.8.2. Results 156 4.8.2.1. Free fatty acids content 156 4.8.2.2. Peroxide value 161 4.8.2.3. Appearance 166 4.8.2.4. Taste 166 4.8.2.5. Flavor 172 4.8.2.6. Texture 177 4.8.2.7. Overall acceptability 177 4.8.2.8. Moisture content 177 4.8.2.9. Chroma Value 184 4.8.2.10. Hue Angle 184 4.8.3. Discussion 187 4.8.4. Conclusion 1925 SUMMARY 193 6 LITERATURE CITED 201 APPENDICES 237

LIST OF TABLES

Table Title Page 3.1 Experimental treatments for food bar formulation 49 3.2 Product Formulation 50 3.3 Experimental treatments for protein meal formulation 51

3.4 Essential amino acid requirements for pre-school children, 2–5 y 53

3.5 Levels of independent variables 57 3.6 Design showing independent variable combination levels 58

4.1 Mean sum of squares of the effect of processing treatments on moisture content of chickpea and Indian vetch

63

4.2 Effect of processing treatments on moisture content of chickpea and Indian vetch 63

4.3 Mean sum of squares of the effect of processing treatments on ash content of chickpea and Indian vetch 64

4.4 Effect of processing treatments on ash content of chickpea and Indian vetch 64

4.5 Mean sum of squares of the effect of processing treatments on fat content of chickpea and Indian vetch 66

4.6 Effect of processing treatments on fat content of chickpea and Indian vetch 66

4.7 Mean sum of squares of the effect of processing treatments on protein content of chickpea and Indian vetch

67

4.8 Effect of processing treatments on protein content of chickpea and Indian vetch 67

4.9 Mean sum of squares of the effect of processing treatments on crude fiber content of chickpea and Indian vetch

68

4.10 Effect of processing treatments on crude fiber content of chickpea and Indian vetch 68

4.11 Mean sum of squares of the effect of processing treatments on nitrogen fee extract (NFE) of chickpea and Indian vetch

70

4.12 Effect of processing treatments on nitrogen fee extract (NFE) of chickpea and Indian vetch 70

4.13 Mean sum of squares of the effect of processing treatments on tannin content of Indian vetch and chickpea 78

4.14 Effect of processing treatments on tannin content of Indian vetch and chickpea 78

4.15 Mean sum of squares of the effect of processing treatments on total phenol content of Indian vetch and chickpea

79

4.16 Effect of processing treatments on total phenol content of 79

Indian vetch and chickpea

4.17 Mean sum of squares of the effect of processing treatments on trypsin inhibitor activity of Indian vetch and chickpea

81

4.18 Effect of processing treatments on trypsin inhibitor activity of Indian etch and chickpea 81

4.19 Mean sum of squares of the effect of processing treatments on phytic acid content of Indian vetch and chickpea

83

4.20 Effect of processing treatments on phytic acid content of Indian vetch and chickpea 83

4.21 Mean sum of squares of the effect of processing treatments on β-ODAP content of Indian vetch 84

4.22 Effect of processing treatments on β-ODAP content of Indian vetch 84

4.23 Mean sum of squares for effect of processing treatments on HCl-extractability of copper in Indian vetch and chickpea

95

4.24 Effect of processing treatments on HCl-extractability of copper in Indian vetch and chickpea 95

4.25 Mean sum of squares for effect of processing treatments on HCl-extractability of manganese in Indian vetch and chickpea

97

4.26 Effect of processing treatments on HCl-extractability of manganese in Indian vetch and chickpea 97

4.27 Mean sum of squares for effect of processing treatments on HCl-extractability of magnesium in Indian vetch and chickpea

98

4.28 Effect of processing treatments on HCl-extractability of magnesium in Indian vetch and chickpea 98

4.29 Mean sum of squares for effect of processing treatments on HCl-extractability of sodium in Indian vetch and chickpea

100

4.30 Effect of processing treatments on HCl-extractability of sodium in Indian vetch and chickpea 100

4.31 Mean sum of squares for effect of processing treatments on HCl-extractability of potassium in Indian vetch and chickpea

101

4.32 Effect of processing treatments on HCl-extractability of potassium in Indian vetch and chickpea 101

4.33 Mean sum of squares for effect of processing treatments on HCl-extractability of zinc in Indian vetch and chickpea 103

4.34 Effect of processing treatments on HCl-extractability of zinc in Indian vetch and chickpea 103

4.35 Mean sum of squares for effect of processing treatments 104

on HCl-extractability of iron in Indian vetch and chickpea

4.36 Effect of processing treatments on HCl-extractability of iron in Indian vetch and chickpea 104

4.37 Mean sum of squares for effect of processing treatments on HCl-extractability of phosphorous in Indian vetch and chickpea

106

4.38 Effect of processing treatments on HCl-extractability of phosphorous in Indian vetch and chickpea 106

4.39 Mean sum of squares for feed intake, protein intake and weight gain values in rats 111

4.40 Feed intake, protein intake and weight gain values in rats 111

4.41 Mean sum of squares for feed efficiency, feed utilization and protein utilization values in rats 113

4.42 Feed efficiency, feed utilization and protein utilization values in rats 113

4.43 Mean sum of squares for protein efficiency ratio (PER) and net protein retention (NPR) in rats 114

4.44 Protein efficiency ratio (PER) and net protein retention (NPR) in rats 114

4.45 Mean sum of squares for corrected protein efficiency ratio (C-PER), relative protein efficiency ratio (RPER) and relative net protein retention (RNPR) in rats

116

4.46 Corrected protein efficiency ratio (C-PER), Relative protein efficiency ratio (RPER) and Relative net protein retention (RNPR) in rats

116

4.47 Mean sum of squares for essential amino acid contents in Indian vetch (Lathyrus sativus) and chickpea (Cicer arietinum) flours incorporated food bar meals

122

4.48 Essential amino acid contents (mg/g) for Indian vetch (Lathyrus sativus) and chickpea (Cicer arietinum) flours incorporated food bar meals

123

4.49

Mean sum of squares for true protein digestibility (TPD) and Protein Digestibility-Corrected Amino Acid Score (PDCAAS) for Indian vetch (Lathyrus sativus) and chickpea (Cicer arietinum) flours incorporated food bar meals

124

4.50

True protein digestibility (%) and Protein Digestibility-Corrected Amino Acid Score (PDCAAS) in rats for given meals for Indian vetch (Lathyrus sativus) and chickpea (Cicer arietinum) flours incorporated food bar meals

124

4.51 Mean sum of squares for proximate composition of Indian vetch (Lathyrus sativus) and chickpea (Cicer arietinum) flours incorporated food bars

132

4.52 Mean proximate composition and energy values for Indian vetch (Lathyrus sativus) and chickpea (Cicer arietinum) flours incorporated food bars

132

4.53 Mean sum of squares for In-vitro digestibilities of Indian vetch (Lathyrus sativus) and chickpea (Cicer arietinum) flours incorporated food bars

134

4.54 In-vitrodigestibilities of Indian vetch (Lathyrus sativus) and chickpea (Cicer arietinum) flours incorporated food bars

134

4.55 Mean sum of squares for sensory studies of Indian vetch (Lathyrus sativus) and chickpea (Cicer arietinum) flours incorporated food bars

135

4.56 Mean values for sensory scores of Indian vetch (Lathyrus sativus) and chickpea (Cicer arietinum) flours incorporated food bars

135

4.57 Mean sum of squares for extraction rate of food processing waste materials under influence of different solvents

142

4.58 Extraction rate (%) of food processing waste materials under influence of different solvents 142

4.59 Mean sum of squares for polyphenols in various solvent extracts from food processing waste materials 143

4.60 Total polyphenols content (mg/g) in various solvent extracts from food processing waste materials 143

4.61 Mean sum of squares for DPPH quenching activity of various solvent extracts from food processing waste materials

145

4.62 Mean values for DPPH quenching activity (%) of various solvent extracts from food processing waste materials 146

4.63 Mean sum of squares for antioxidant activity of various solvent extracts from food processing waste materials 148

4.64 Mean values for antioxidant activity (%) of various solvent extracts from food processing waste materials 149

4.65 Coefficients of variables in models for free fatty acids in bars during storage and their respective R2 157

4.66 Analysis of variance for the full regression of model 157

4.67 Coefficients of variables in models for peroxide value of bars during storage and their respective R2 162


4.69 Coefficients of variables in models for appearance of bars during storage and their respective R2 167


4.71 Coefficients of variables in models for taste of bars during storage and their respective R2 168


4.73 Coefficients of variables in models for flavor of bars during storage and their respective R2 173


4.75 Coefficients of variables in models for texture of bars during storage and their respective R2 178


4.77 Coefficients of variables in models for overall acceptability of bars during storage and their respective R2

179


4.79 Coefficients of variables in models for moisture of bars during storage and their respective R2 183


4.81 Coefficients of variables in models for chroma value of bars during storage and their respective R2 185


4.83 Coefficients of variables in models for hue angle value of bars during storage and their respective R2 186


LIST OF FIGURES

Figure Title Page

4.1 DPPH scavenging capacity of various solvent extracts from food processing waste materials 147

4.2 Antioxidant activity of various solvent extracts from food processing waste materials 150

4.3 Effect of independent variables (X1, X2) on free fatty acids in nutrient dense food bars during storage 158



4.6 Effect of independent variables (X1, X2) on peroxide value of nutrient dense food bars during storage 163



4.9 Effect of independent variables (X1, X2) on taste of nutrient dense food bars during storage 169



4.12 Effect of independent variables (X1, X2) on flavor of nutrient dense food bars during storage 174



4.15 Effect of independent variables (X1, X2) on overall acceptability of nutrient dense food bars during storage 180



ABSTRACT The Indian vetch (Lathyrus sativus L.) and chickpea (Cicer aritenum) were selected for nutritional profile augmentation of food bars. Various processing techniques were used to reduce antinutritional factors in these legumes. The prepared bars were characterized for their nutritional quality attributes. Different antioxidant extracts were screened for their antioxidant activity. Response Surface Methodology (RSM) was used to optimize their levels in nutrient dense food bar during four months storage. The natural fermentation decreased crude protein level of legumes during processing, however other main constituents i.e. crude fat, total ash and crude fiber were least affected. The antinutritional factors i.e. trypsin inhibitors, tannins, polyphenols, phytates and β-ODAP (in Indian vetch only) were reduced effectively by fermentation and germination processes. The HCl-extractability (an index of bioavailability) of minerals in processed and raw Indian vetch and chickpea flours varied considerably in all cases. The processing treatments inserted positive influence over this. As part of criteria for selection of best treatment for nutrient dense food bar making, in-vitro protein and starch digestibilities, sensory characteristics, proximate composition and calorific value were analysed. In-vitro protein digestibility (IVPAD) and in-vitro starch digestibility (IVSD) for nutrient dense food bars were improved by the incorporation of processed Indian vetch and chickpea flours. All of the sensory attributes especially flavor, taste and overall acceptability deteriorated on the incorporation of natural fermented flour in food bars, whereas controlled fermented flour containing bars were preferred. The proximate composition data showed that bars with Indian vetch flour contributed high portion of crude protein and fat, whereas chickpea bars contributed marginally less. This investigation showed that these food bars are calorific dense as well by the virtue of their rich nutrient levels. The processed flours were thereafter blended with other protein sources to produce balanced protein meal. The protein quality of these diets was assessed by implying in-vivo rat assays. The values for relative protein efficiency ratio (RPER) and relative net protein ratio (RNPR) in close proximity to each other for processed meals acted as an indicator for good protein quality of these meals. The food bar meals showed significant variation among samples for isoleucine and sulfur containing amino acids i.e. methionine and cysteine. High contents of these amino acids were studied in meals carrying chickpea flours. When this amino acid profile was compared with amino acid requirement criteria for 2-5 years old children, it matched well. High TPD was achieved by food bars carrying controlled fermented Indian vetch and chickpea flour meals, followed closely by germinated flour possessing meals. The computed PDCAAS value was 1 for aforementioned meals. Food processing waste materials were extracted using different solvents and ethanol 80% solution in water was found effective in extracting all materials. Higher values of total polyphenol content were achieved by pomegranate peel among all solvent extracts, whereas DPPH scavenging and β-carotene bleaching capacities was also much better in pomegranate peel extracts than extracts from other materials. Then food bars were prepared taking into account the best formulation, incorporating Emblica officinalis extract, Punica granatum peel extract and citric acid at different levels for shelf stability of nutrient dense food bar. The second-order polynomial models were fitted well for independent variables on storage data at various intervals. It was observed that the optimized levels of Emblica officinalis (1.05%), Punica granatum extract (1.86%) and citric acid (0.059%) in food bar formulation generated through using Response Surface Methodology, resulted in sensory acceptable and shelf stable nutrient dense food bars.

Chapter-1

Introduction

Snacks are the food taken between two meals. In general, most of the snacks available

in market are devoid of balanced nutrients e.g. potato chips, extruded products,

chocolates etc. These are unhealthy offerings for the consumers especially school

going children. The food bars are snack foods of good sensory and nutritional

characteristics due to their high content of proteins, lipids and carbohydrates (Estevez

et al., 2000). Increasing demand from consumers for nutritious snacks, have prompted

the food industry to develop food bars that combine convenience and nutrition (Izzo

and Niness, 2001). The desired characters primarily for developing a bar are improved

nutritional value (Steinkraus, 1996), favorable texture, sensory quality (Trugo et al.,

1993) and partial or complete elimination of antinutritional factors (Khalifa and

Zinay, 1994).

There is a variety of food bars available in the market such as energy bars, nutrition

bars, low carbohydrate bars, granola bars etc. These all offer specific benefits for the

target consumers. These are ready to use, thus lure the school going children, office

workers and athletes to maintain their energy level and can even be supplied to the

target populations in the situation of an emergency. A nutrient dense food bar is a

food article carrying all the major nutrients in good balance. It contains some

processed cereals including wheat, oat, rice, barley as the main ingredient and others

include nuts, fruit chunks, chocolate chips or coatings.

Wheat is one of the popular cereals that supply the basic nutritional and energy

requirements of the population. However, its protein quality is poor. Lysine is the first

limiting essential amino acid in wheat flour; threonine and tryptophan are also in

meager amounts (Schaafsma, 2005). Thus, the food products prepared from wheat are

not of high nutrition value. The quality of a product with a big portion of wheat

constituents can be improved by fortifying with suitable edible proteinaceous

materials such as legumes, relatively rich in lysine, threonine and tryptophan. With

the great ability to fix nitrogen from the atmosphere and a much higher efficiency to

produce protein per unit land area, legumes can go a long way towards meeting

protein needs of an increasing world population (Hernandez et al, 1995). On the other

hand, legumes possess poor digestibility and contain antinutritional factors such as

phytates, tannins (Yadav and Khetarpaul, 1994), amylase inhibitors (Lajolo et al.,

1991), hemagglutinins (Bressani, 1993). These antinutritional factors are of great

concern. Tannins have been reported to crosslink with proteins and polysaccharides

(Barry, 1989) and form complexes with digestive enzymes such as trypsin, lipase, α-

amylase (Longstaff and McNab, 1991), thus lowering their biological effectiveness.

The trypsin inhibitors can inhibit the proteolytic activity of the digestible enzyme

trypsin and lead to reduced availability of amino acids (Liener and Kakade, 1980).

Phytic acid (inositol hexaphosphate, IP6) is considered to be a major cause of

impaired absorption of several essential minerals especially divalent cations such as

calcium, magnesium, zinc and iron, turning them into insoluble forms, which become

unavailable to the living beings (Reddy and Pierson, 1994).

In Pakistani diet, chickpea is one of the popular legumes. Its supplementation to

wheat flour at different levels has extensively been investigated (Khan et al., 1976).

The chickpea protein is rich in lysine and threonine contents, however, like other

legumes, it has low digestibility and carries other negative factors such as tannins,

trypsin inhibitors, phytates and hemagglutinins (Bressani, 1993).

Indian vetch (Lathyrus sativus L.) on the other hand is one of the cheapest legumes

rather least investigated source having high protein value. It also contains high lysine

content. However, in addition to other antinutritional factors, the presence of the

neurotoxin, β-N-oxalyl- α, β-diaminopropionic acid (β -ODAP), is a cause of public

health concern and a barrier to the utility of this legume.

The elimination of antinutritional and toxic factors of legumes makes these a good

candidate for supplementation to wheat flour (Lodhi et al., 2003). The use of

indigenous food-processing technologies such as fermentation (Sarkar et al., 1997)

and germination (Donangelo et al., 1995) can be used as a strategy to overcome

problem of antinutritional factors in legumes. Fermentation is one of the oldest and

most economical methods of processing and preserving foods. The fermentation of

legumes incurs desirable changes, such as improvement in sensory characteristics,

protein quality, starch digestibility and contents of some minerals and vitamins

(Vidal-Valverde et al., 1993; Tabera et al., 1995). Germination also improves the

digestibility of legume proteins (Khalil and Mansour, 1995). The legumes processing

thus exert a positive impact through separation or partitioning of minerals from their

food matrices or through the destruction of inhibitors. It also helps to form beneficial

complexes between food components and minerals, thus enhances mineral availability

(Watzke, 1998). The blending of processed legumes with cereal meals in right

proportions can provide a balanced mixture of amino acids in a diet. The primary

source of legume protein for supplementation can be in various forms such as flour,

concentrates, isolates, or textured vegetable protein (Wondimu and Malleshi, 1996).

The quality evaluation of protein dense foods is important for the selection of

particular protein source or assessing the effectiveness of processing methods

employed in the preparation of these foods (Sogi et al., 2005). The protein quality

evaluation methods include growth and nitrogen balance studies that deal with

digestibility of the proteins and their availability (Ekanayake et al., 2000). Several

bioassay methods have been used to determine protein quality of foods (Rozan et al.,

1997). Net protein ratio (NPR) and relative NPR (RNPR) methods were considered to

be improvements over the protein efficiency ratio (PER) method for measuring the

protein quality of foods (Sarwar and McDonough, 1990). The protein digestibility-

corrected amino acid score method, based on the actual amino acid profile of a protein

in comparison with the amino acid requirement of humans, is considered to be a

suitable routine method for evaluating the protein quality of vegetable protein

products (FAO/WHO, 1990). All these methods have the specific utility in

determination of biological value of a particular food.

Shelf stability is the most critical factor in the success of a food product. Lipid

oxidation has been recognized since long as a major problem in the storage of foods.

Molecular oxygen has the ability to interact with organic molecules. A wide variety of

organic molecules are susceptible to such chemical attacks. These lipids are very

much prone to this attack and experience oxidative damage. The quality parameters of

a food are affected by the oxidation of lipid portion. The oxidation of fatty material

results in deterioration of flavor, nutritional and food safety qualities (Kanner and

Rosenthal, 1992). In rancid food, new volatile odorous compounds are formed

through aroma changes. The flavor modifications are caused by hydroxyl acids,

condensation reaction between oxidation products and proteins results in the color

darkening, and a new texture might be attributed to the oxidative induction of protein

cross links.

Usually synthetic antioxidants such as butylhydroxyanisole (BHA), butylhydrotoluene

(BHT) and tert-butyl hydroquinone (TBHQ) are used to decelerate these processes.

The latest research indicates that these antioxidants pose health risks for human

beings on their consumption (Martinez-Tome et al., 2001). Thus, it is need of the hour

that the natural alternatives of synthetic antioxidants should be explored. The food

processing waste materials i.e. almond skin, pomegranate peel, onion scales and

peanut skin carry appreciable amount of natural antioxidants and have the potential to

be recognized as cheap source of these compounds. Amla (Emblica officinalis) fruit

extract is another good source of high antioxidant activity. In order to optimize the

levels of variables i.e. natural antioxidant, the experiment should be designed using

the suitable statistical tools. Response Surface Methodology (RSM) is reported to be

an effective measure for optimizing a process when the independent variables e.g.

antioxidant combinations, are hypothesized to possess a sovereign or cumulative

effect on the desired responses (Martınez et al., 2004).

To supply the public with nutritious food alternatives of junks, the nutrient dense food

bars could be a good candidate. To achieve the nutrients dense status of these bars,

cheap underutilized food sources with good nutritional value should be explored. The

means and methods should be devised to reduce the load of antinutritional factors or

toxic constituents from these food materials. Moreover, natural constituents should

extensively be investigated to enhance the shelf stability of such products. Therefore,

present project was planned on the basis of above mentioned lines to explicit the

following objectives:

• To investigate the effect of processing techniques including fermentation and

germination on the nutritional quality of legumes (chickpea and Indian vetch).

• To evaluate protein quality of legume flour supplemented food bars by the

protein efficacy studies.

• To assess suitability of wheat meal supplementation with chickpea or Indian

vetch flours for development of nutrients dense prototype food bars by

applying physico-chemical and sensory tests.

• To screen out the natural antioxidant extracts from food processing waste

materials for their activity.

• To establish a set of optimum antioxidant extract combinations using

Response Surface Methodology (RSM) for obtaining bars with better sensory

acceptability and shelf stability.

Chapter-2

Review of

Literature

To support the study, literature has been reviewed under the following headings: -

2.1. Development of food bar

2.2. Selection of grains for food bars

2.3. Protein quality evaluation

2.4. Storage changes in foods

2.5. Extending shelf life of food products

2.6. Optimization of shelf life

2.1. Development of a food bar

Food bars are snack foods of good sensory characteristics due to their constituents,

contributing rich contents of protein, lipids and carbohydrates. The popular varieties

of food bars include nutrition, energy, nutraceutical and diet bars. These have huge

growth in market potential around the globe; for instance only in the United States, a

$1.6 billion cereal bar market exists. One of the reasons for this growth is growing

consumer’s awareness of the health benefits of grains (Palazzolo, 2003).

The snacks with good nutritional value could play an important role in the physical

and mental development of children and teenagers since they show a great preference

for them. Chemical analysis reveals that cereal bars are marginally better than

favourite traditional snacks on the basis of their sugar, fat, salt and fiber contents

(Boustani and Mitchell, 1990).

Food bars can be produced through various techniques. Extrusion is one of such

technology. Extruded product of a peanut/sorghum flour mixture was utilized as a

wafer subassembly of a snack bar or a meal bar. Food filler i.e. chocolate, peanut

butter paste etc., were added to enhance the nutritional quality and consumer appeal

of the final product (Anderson and Jones, 1999). The development of food bars could

be carried out through blending the grains, nuts and other ingredients along some

binding material including gums, liquid glucose and sucrose etc. The mixture is then

shaped into a bar by passing through a roller (Al-Hooti et al., 1997) or baked in a

baking oven at moderate heat i.e. below 150°C (Brisske et al., 2004).

The examination of sensory aspects of food bar snacks presents a picture of relative

importance to consumer perception and degree of liking. Chewy, nutty and chocolate

bars are liked the most due to high levels of filling flavor, chewy and crunchy textures

and sweetness. Majority of consumers ranked taste as the most important

characteristic influencing their purchase intention, followed by textural features, price

and appearance (Bower and Whitten, 2000). Most of consumers preferred cereal bars

which contain chocolate but these were not the healthier than whole grain plain cereal

bars (Boustani and Mitchell, 1990). However, the cereal bars with peanut and walnut

have shown to be snack foods of good sensory characteristics and high calorific value.

Protein content is higher in the bars with mesquite cotyledon and thermal processing

has no effect on their chemical composition. However, on the basis of sensory

characteristics, bars with mesquite cotyledon treated by microwave have shown a

higher acceptability (Escobar et al., 2000).

Three different cereal bars were prepared with different amount of oat, wheat germ,

and puffed amaranth. During accelerated storage (37°C for 15 days), water activity

and moisture content reached to low levels of 0.48 and 5.9%, respectively, in one of

the bars. Peroxide content increased gradually up to 12 and 17 meq/kg at 15 days

(Escobar et al., 1994). In another study, four types of bars were elaborated with 6%

microwaves treated or toasted mesquite cotyledon, and were carrying 18% peanut or

walnut. The peroxides values (4.9-13.8 meq/kg of oil) for bars stored for 90 days at

room temperature indicated that bars remained shelf stable during storage (Escobar et

al., 1998).

The walnuts can successfully be used in the manufacture of snack bars as these offer

good nutritional and sensory quality and remain stable in storage. Six snack-type bars

were manufactured, to contain oat and wheat germ and two different walnut levels,

agglutinated with natural sweeteners and fats. Two bars additionally contained toasted

amaranth with brown sugar cover and wheat extrudate, while two others, contained

puffed instead of toasted amaranth. The drying time for the cereal and walnut based

bars was 45 min at 120°C. All bars presented a good fiber supply and the bar

containing only oat, wheat germ and walnut, had the high protein content. Sensory

evaluation revealed that the bars with 18% walnut level got greatest preference.

During storage, the moisture and water activity decreased in all the bars. Peroxides

remained within the acceptable ranges; acceptability based on sensory evaluation was

preferable in the bar with toasted amaranth (Estévez et al., 1995).

In other study, granola bars were developed by using common black and red beans

(Phaseolus vulgaris). On appearance basis, the red bean granola bars were more

acceptable than the black bean products (Maurer et al., 2005). Five confectioned

energy bars containing papaya, hazelnut, almond, apple and orange chunks showed

good microbiological and sensory quality. The energy supply was 520 Kcal/100g;

thus ingest of two prototypes cover between 25-30% of recommended intake for the

most frequently practiced sports. The shelf life studies of the product stored in

ambient conditions (20-25°C and 55-60% relative humidity) showed that the product

without additives maintained its quality up to 60 days. Whereas, neither rancid odor

nor rancid flavor was detected in the product with added preservative and antioxidant

and it stayed without quality modifications up to at least 120 days (de Penna et al.,

1992).

Two varieties of chocolate coated soy-based candy bars carrying almonds and nuts

were developed for sportsmen who need a higher protein intake. The ingredients used

were isolated soy protein, texturized soy flour, milk solids, cocoa powder, toasted oat,

nuts, almonds, authorized flavors, preservatives and antioxidants. The nutritional

composition of both bar varieties averaged at 12.4% proteins, 9% lipids and 58.7%

carbohydrates, and the mean calorific value was 375.2 kcal/100 g (de Penna et al.,

1993).

Packaging material plays a crucial role in keeping the food bars shelf stable. The

double layered polypropylene package film can effectively protect the quality of

energy bars having fruit chunks (de Penna et al., 1992). The quality of soy-based

candy bars packed in aluminum foil can be maintained without significant changes

during 30 days storage for the nut candy, and at least for 60 days for the almond

candy bar (de Penna et al., 1993). Similarly, the packaging materials maintained the

cereal bars in stable condition i.e. moisture (7.4-11.2%), water activity (0.50-0.65) for

extended period. The bars remained acceptable from sensory point of view as well

(Escobar et al., 1998).

2.2. Selection of grains for food bars

Among the food grains, cereals constitute a major portion of human diet. Although,

cereal grains are rich in carbohydrates but these are not a good contributor of quality

protein as these lack in much needed essential amino acids (Horn and Schwartz,

1961). On the other hand, legumes represent a major dietary source for a large section

of the population of developing countries and contribute significantly towards protein,

mineral and vitamin needs of people (Bressani, 1993).

2.2.1. Complementation of cereals

The supplementation of wheat flour with inexpensive staples, such as pulses, helps to

improve the nutritional quality of wheat products (Sharma et al., 1999). Combining

cereals and legumes has proven to improve overall nutritional quality of diet by

countering the nutritional lacking of each other (Bressani, 1987). There are many

pulse candidates available for complementation of cereals; Indian vetch and chickpea

are two of them.

2.2.1.1. A candidate for complementation - Indian vetch (Lathyrus sativus L.)

Indian vetch or grass pea (Lathyrus sativus L.) is an annual leguminous crop

cultivated in North America, temperate parts of South America, Eurasia, and East

Africa for animal or human consumption. Common names for Indian vetch include

grass pea or chickling pea in the UK and North America; shan li dou in China;

khesari or batura in India; guaya in Ethiopia and matri in Pakistan (Smartt, 1990). It

is a useful legume having many advantages among other food crops. It has the

multiple tolerances to drought and water logging and shows resistance to insects and

pests and fixes nitrogen at good rate and possesses high grain yield with high protein

content (Spencer, 1989; Campbell et al., 1994). Its human consumption is still

confined to the countries of developing world, facing the water scarcity (Praveen et

al., 1994) but over the past few decades it has also been cultivated in many parts of

Asia (Haque et al., 1996), Europe (Roldan et al., 1994), Africa (Getahum et al., 1999)

and Latin America (Mera et al., 2000). In Italy, it has now bean extensively cultivated

by organic farmers due to its hardy nature. In Polish register, it has been reintroduced

as an agricultural crop in 1997 (Milczak et al., 2001). Indian vetch has recently been a

popular crop in the famine hit areas of world i.e. Ethopia and Afghanistan (Praveen et

al., 1994). It is a cheap legume and usually its splits are used as adulterant into other

legumes. Gram (Cicer arietinum) flour adulteration with vetch flour is known which

is used to produce certain savory products popular in subcontinent such as pakora

(Rehman et al., 2007). In Nepal the dried grains are split either in a stone grinder on a

home scale or milled to make dal which is consumed with rice. The grains are also

ground and made into flour for use in a pancake-like preparation of badi or pakora.

Vetch is one of the richest sources of protein among legumes and carries 25-31%

protein. Mixing grass pea with cereals is a way of improving the amino acids profile

of the final product, as they are complementary in their amino acid compositions

(Cheftel et al., 1996). However, the legume fortification to cereals is preferred in

unleavened products because non-glutinous legume protein portion may exert a

volume depressing effect on leavened products when used at relatively high levels,

necessary to reach the desired amount of fortification (Akobundu et al., 1988). Indian

vetch, due to its good quality protein has been recognized as a genuine candidate for

protein quality improvement of cereal foods through cereal-legume complementation

(Rehman et al., 2007). Its usage potential has been explored in different food products

such as biscuits (Rehman et al., 1997), bread (Lodhi et al., 2003), ice cream (Rehman

et al., 2004), protein rich milk powder (Rehman et al., 2006a), chapatti (Rehman et

al., 2006b), doughnuts (Rehman et al., 2007) and milk blend (Rehman et al., 2007).

2.2.2. Antinutritional factors in legumes

Although, legumes have been proven as a cheap source of protein, the nutritional

quality of a legume could be affected by a few of antinutritional factors i.e. proteolytic

inhibitors, phytates, compounds causing favism, polyphenols, phytohemagglutinins,

lathyrogens, cyanogenetic compounds and saponins (Gupta, 1987).

2.2.2.1. Trypsin inhibitors

Trypsin inhibitors (TI) are low molecular weight proteins formed by joining identical

small size peptide chains i.e. upto 8 disulfide bonds. (Mueller and Weder, 1990).

Generally, these are not carrying carbohydrate moieties. The trypsin inhibitors from

different sources have diverse structures, molecular weights, and chemical

compositions; however, the amino acid sequence of the active sites is similar (Liener,

1994). They inhibit the activity of the digestive enzyme trypsin and result in stunted

growth through reduced availability of amino acids. Their presence in the diet leads to

the formation of the irreversible complexes with trypsin enzyme which reduces its

level in intestine and a decrease in the protein digestibility of a diet. Under this

situation, the secretory activity of the pancreas increases in the subject, which could

cause pancreatic hypertrophy and hyperplasia. Since pancreatic enzymes such as

trypsin and chymotrypsin are particularly rich in the sulfur-containing amino acids

especially cysteine, pancreatic hypertrophy and/or hyperplasia diverts these amino

acids from the synthesis of body tissue protein to the synthesis of these enzymes. This

loss in sulfur-containing amino acids in legume protein which is already deficient in

these amino acids results in the stunted growth in living being (Liener and Kakade,

1980).

2.2.2.2. Tannin

Tannins have been reported to occur in appreciable amount in legumes. The seed coat

color of legumes has largely been associated with tannin content (Nozolillo and de

Bezeda, 1984). These have the tendency to form stable or weak complexes with

proteins, carbohydrates and vitamins. These can cross link with protein by reacting

with lysine, threonine and tyrosine making them unavailable during digestion (Yu et

al., 1996). The degree of polymerization of these polyphenolic compounds plays an

important role in protein digestibility and availability of vitamins and minerals

(Suschetet, 1975).

2.2.2.3. Phytates

Phytates are the principal storage form of phosphorus and are particularly abundant in

cereals and legumes (Reddy et al., 1989). These chelate divalent cations such as

calcium, magnesium, zinc and iron, thereby also reducing their bioavailability

(Sandberg, 2002). These can inhibit the activity of several enzymes (Knuckles et al.,

1989) and can also reduce the digestibility of legume proteins by forming the

complexes with them.

2.2.2.4. β-ODAP

The presence of the neurotoxin, β-ODAP (β-N-oxalyl-L- α, β-diaminopropionic acid)

also known as BOAA (β-N-oxalyl-amino-L-alanine) is a major hazard to the

utilization of Indian vetch (Kuo et al., 1998). This toxin can cause irreversible

paralysis on consumption in large quantities for prolonged periods such as over three

months (Spencer et al., 1993). In laboratory animals (chicks, rodents, and other

species), the neurolathyrism is mostly represented by convulsions. However, these do

not appear in humans (Roy and Spencer, 1989). The primates feeding on Indian vetch

(Lathyrus sativus) for extended periods may develop central motor pathway and hind

limb problems which are similar to those observed in the human case (Spencer et al.,

1986). Moreover, heavy consumption of grass pea for several weeks or months may

cause lathyrism with prodromal symptoms of muscle cramping associated with a

sensation of heaviness and weakness of the legs. These initial symptoms of the

disease are probably reversible. However, consuming grass pea for long causes severe

and irreversible spastic paraparesis arising from the selective involvement of the

central motor pathway (Haque et al., 1996). Horses are quite susceptible to

neurolathyrism and sometimes die on seed consumption for long (Bellido, 1994;

Hanbury et al., 2000).

However the effects of feeding L. sativus are species-specific. Sheep, cattle and other

ruminants, can withstand high levels of it (70% grass pea with 0.09% β-ODAP). It is

suggested that β-ODAP may be broken down in rumen (Hanbury et al., 2000).

Similarly the calves fed on L. sativus up to 30% of daily diet for prolonged periods

showed no signs of toxicity like ill health, or changes in behavior or vigor (Dhiman et

al., 1983). However, some pathological changes were noted in liver and kidney cells

of ruminants (Liu et al., 1989; Chen et al., 1992).

In a study on patients suffering from neurolathyrism in Bangladesh, it was observed

that 12% (all male) complained of bone pain and showed symptoms of osteolathyrism

i.e. skeletal deformities. On X-ray examination, a failure of fusion in both vertebral

and iliac epiphyses was found in two patients. As the patients were of 30 and 37 years

age, such failure was considered a clear evidence of osteolathyrism. All patients were

used to consume the green parts of L. sativus as well as foods made from the seeds of

the same vetch plants containing β-ODAP (Haque et al., 1997).

Two hundred patients with chronic neurolathyrism were examined 25-35 years after

the appearance of signs and symptoms of intoxication of the L. sativus. Their daily

food intake, in a German forced labor camp during World War II, consisted of 400 g

L. sativus cooked in water plus 200 g bread baked of barley and straw. Apart from the

classic signs of neurolathyrism, in some cases, experimental osteolathyrism was

observed. Its symptoms included absence of ossification centers of the iliac crests,

ischial tuberosities and vertebrae as well as bowing of the femoral shaft with

thickening (Cohn and Streifler, 1983).

An exploratory study was conducted in the rural Ethiopia to identify the role of type

of grass pea (L. sativus) diet in the susceptibility to neurolathyrism. Five-hundred

subjects were included in the study. The gravy (Shiro) grass pea preparation was

consumed by 91.6% of the study population, boiled (Nifiro) by 86%, and roasted

(Kollo) by 56.4%. Almost, half (48%) of the cases consumed grass pea for > 4 months

compared to 8% of controls. There was a significant association between the risk for

neurolathyrism and the consumption of boiled and roasted forms of grass pea. There

was no risk of paralysis associated with consumption of the gravy form of grass pea

(Getahun et al., 2002).

2.2.3. Antinutritional factors reducing strategies

The removal of undesirable components is essential to improve the nutritional quality

of legumes. In this way, these could effectively be utilized to their full potential as

human food. It is widely accepted that simple and inexpensive traditional processing

techniques are effective methods of achieving desirable changes in the composition of

seeds. Soaking, cooking, fermentation and germination may improve the quality of

legumes due to the removal of some antinutritional factors. In many instances, usage

of only one method may not impart the desired removal of antinutritional compounds

and a combination of two or more methods is required.

2.2.3.1. Soaking

A soaking procedure usually is a prior step to legume cooking in which aroma and

palatability is enhanced. It could be one of the processes to remove soluble

antinutritional factors, which can be eliminated with discarded soaking solution.

Moreover, some metabolic reactions can also take place during soaking, which may

affect the content of some antinutritional compounds (Ibrahim et al., 2002). This

process may affect antinutritional factors in legumes in the variable manner. The

soaking of soybeans in water at 22°C for 24 hrs had no effect on the trypsin inhibitor

activity (TIA) value (Liu and Markakis, 1987). In many cultivars of P. vulgaris, 98-

99% of TIA was retained when the seeds were soaked in water for 18 hrs (Deshpande

and Cheryan, 1983). Navy and red kidney beans soaked for 16 hrs in water at room

temperature, showed insignificant decrease in TIA in both cultivars (Dhurandhar and

Chang, 1990). Trugo et al. (1990) reported the retention of TIA when black beans

were soaked in water for 16 hrs. Whereas a 58-66% decrease was observed in TIA

after soaking the lentil seeds for 24 hrs in distilled water (Batra et al., 1986). Also, on

soaking faba beans in distilled water and 0.07% sodium bicarbonate solutions, a

decrease in TIA took place. However, soaking of faba beans in 0.1% citric acid

solution showed no effect on TIA. It could probably be attributed to the stability of

the inhibitor in acidic pH (Fernandez et al., 1993). However, high reduction in phytic

acid content can be achieved at low pH (5.5) (Ford et al., 1978). An increase in the

tannin content was noted after soaking faba beans in distilled water, 0.1% citric acid,

and 0.07% bicarbonate solutions. Soaking the seeds in 0.07% sodium bicarbonate

solution did not reduced the phytic acid (PA) content as effectively as water (27% vs

23%), whereas 0.1% citric acid solution was more efficient than water alone (37% vs

23%) (Fernandez et al., 1993).

Moreover, β-ODAP is a water-soluble amino acid so it can leach from seed by

soaking in water (Akalu et al., 1998). Steeping grass pea in a large volume of cold

water for 3 min leached out approximately 30% of β-ODAP, with greater losses when

hot water was employed (Tekele-Haimanot et al., 1993). Similarly, soaking dehusked

seed in hot water for several hours and boiling the seed in water removed 70–80% of

the neurotoxin (Mohan et al., 1966). Moslehuddin et al. (1987) found that washing

the vetch seed partially removed β-ODAP.

As far as studies on comparative reduction of antinutritional factors are concerned,

soaking reduced the contents of phytate, polyphenols and trypsin inhibitors in various

media to a significant extent within four lines of L. sativus. The losses were higher in

freshly boiled water, alkaline and tamarind solutions than after soaking in drinking

water. The highest losses (65-70%) were observed for β-ODAP in boiled water,

followed by trypsin inhibitors (42-48%) and polyphenols (30-37%) (Srivastava and

Khokhar, 1999).

2.2.3.2. Autoclaving/ Cooking

It has been found that the thermostability of antinutritional factors in legumes varies

not only with legume source but also with the different conditions used during

processing, such as pH, humidity, time, temperature, and pressure (Weder and Link,

1993; Vidal-Valverde et al., 1994; Urbano et al., 1995; Frias et al., 2000). It was

noted that autoclaving/cooking treatment of legumes reduced the antinutritional

factors but at variable rate.

It was noted that cooking for 60 min at 100°C was sufficient to inactivate over 90% of

TIA in P. vulgaris (Trugo et al., 1990) and eliminated completely on heating soaked

red gram seeds in boiling water for 5 min (Mulimani and Paramjyothi, 1993). TIA of

pre-soaked faba beans decreased significantly after they were cooked for 35 min

(Fernandez et al., 1993). Moreover, application of wet and/or heat processing reduced

the trypsin inhibitor levels in faba beans down to 42 to 56% of the original. It was

observed in all cases that as the duration and temperature of treatments were

increased, it resulted in further reduction of TI (Anderson et al., 1994). Cooking of

pre-soaked winged beans in boiling water for 30 min was sufficient to inactivate

trypsin inhibitor (TI) (Kadam and Smithard, 1987). About 30-40% of polyphenols can

be removed from Phaseolus vulgaris by cooking and discarding the cooking water

solution (Bressani and Elias, 1980).

A complete destruction of TI after cooking pre-soaked chickpeas for 35 min was

noted (Nestares et al. 1993). Similarly, chickpea seeds were cooked for 40 min and

complete elimination of TI was observed (Savage and Thompson, 1993). Overnight

soaking whole beans, chickpeas, lentils, and pea seeds, followed by boiling for 2 hrs

almost completely abolished TIA (Weder and Link, 1993). This complete inactivation

action of trypsin inhibitor, potentially improves legume protein digestibility. TI

isolated from faba bean seeds were inactivated after autoclaving. However heat

treatments improved legume protein digestibility which is attributed to the removal of

the heat-labile antinutritional constituents (Kozlowska et al., 1990).

It has been observed that ordinary cooking and pressure cooking of pre-soaked seeds

were more effective in reducing the level of the contents of phytate, polyphenols,

trypsin and amylase inhibitors than soaking within different lines of L. sativus

(Srivastava and Khokhar, 1999). The α-form of ODAP is considered to be less toxic.

The heating procedure isomerizes β-form to the α-form under the influence of time

temperature dependent process. This conversion was noted in some common Indian

cooking preparations not more than 40%. However, significant proportion of the toxin

(about 60%) remains as the toxic β-form after cooking (Padmajaprasad et al., 1997).

2.2.3.3. Controlled fermentation

Fermentation is one of the oldest and most economical methods of processing and

preserving foods. There are many ways to produce foodstuffs by fermenting legumes.

Whole or ground seeds, either raw or cooked, can act as substrate for fermentation.

The fermented legumes are popular due to improved sensory characteristics, protein

quality, starch digestibility and contents of some minerals and vitamins, as well as

partial or complete elimination of antinutritional factors (Paredes-Loapez and Harry,

1988; Vidal-Valverde et al., 1993; Tabera et al., 1995)

The presence of natural flora on legume flours may interfere during the controlled

fermentation, so it has always been essential to autoclave the legume extract. The use

of a mixed culture of 5% of L. casei and 5% L. plantarum inoculated in MRS broth

decreased fermentation time in autoclaved chickpea extract. Its addition in

logarithmic phase to the extract, increased the lactic acid production and decreased

the pH value in 6 hrs which was less time than obtained with each of lactobacillus

strain (de Leon et al., 2000). Also the PA content can be reduced (26–39%) by

fermentation in different legumes. It can be reduced by more than 50% in pigeonpea.

The fermentation carried out in pearl millet (Pennisetum typhoideum) at 30°C for 48

hrs with Lactobacillus plantarum and Rhodotorula isolated from naturally fermented

pearl millet and Lactobacillus acidophilus, Candida utilis and natural microflora had

completely eliminated the phytic acid and amylase inhibitors, especially in the

soaked, debranned and germinated samples. Moreover, the polyphenols were altered

non-significantly in general, however, fermentation with Lactobacillus plantarum +

Rhodotorula and natural microflora caused a significant increase in polyphenols

(Sharma and Kapoor, 1996). However, fermentation has been proved more effective

in destroying enzyme inhibitors (amylase inhibitors by 69-71%; trypsin inhibitors by

65-66%) than either phytates or polyphenols within four different lines of L. sativus

(Srivastava and Khokhar , 1999).

2.2.3.4. Natural fermentation

The microorganisms isolated from cowpeas and chickpeas were Lactobacillus casei,

L. leichmanii, L. plantarum, Pediococcus pentosaceus and P. acidilactici after natural

fermentation for 4 days at 25°C. Lactobacillus helveticus was found in chickpeas

only. This makes the fermentation chiefly lactic acid. Pathogenic organisms were not

found in the system (Zamora and Fields, 1979).

The lentil (Lens culinaris var. vulgaris) flour was naturally fermented for 4 days at

different temperatures (28°C, 35°C and 42°C) and concentrations (79 g/l, 150 g/1 and

221 g/1). It caused a decrease in TIA and a rather sharp decline in the tannin/catechin

ratio. The minimum initial lentil concentration and temperature used (79 g/1, 28°C)

proved most effective in this regard. The TIA was more affected by temperature than

by concentration, and a high reduction (62.5%) was observed at 42°C and 79 g/l

concentration (Tabera et al., 1995). The content of total inositol phosphates in lentil

showed a maximum reduction of 63% at 72 hrs under the natural fermentation

conditions of 42°C and 79 g/l broth concentration (Cuadrado et al., 1996).

The fermentation process possesses the ability to inactivate β-ODAP in fermented

dough to a great extent. The neurolathyrism risk increased due to consumption of

boiled and raw unripe green grass pea, but it was not observed to be associated with

the consumption of fermented pancake. (Getahun et al., 2002).

2.2.3.5. Germination

Germination has been an effective treatment to remove antinutritional factors in

legumes e.g. phytates. These are the mobilizing secondary metabolic compounds

which are thought to function as reserve nutrients (Reddy et al., 1978). The phytic

acid serves as an important reserve of phosphate generated by the action of phytase

during seed germination for the developing seedling. However, this conversion

depends upon the type of beans and germinating conditions. In lentils, germination

reduces phytic acid content at a higher rate than soaking or cooking. It can reduce the

phytic acid content of chickpea and pigeon pea seeds by over 60% and that of mung

bean, urd bean, and soybean by about 40% (Chitra et al., 1996).

A marked reduction in TIA after 24 hrs of germination was observed in lentil and

thereafter the rate decreased, which indicated that these compounds might be utilized

in the first stage of germination as a source of energy (El-Mahdy et al., 1981).

However, Batra et al. (1986) found that germination for 3 days decreased TIA only

slightly, while germination for a prolonged duration reduced it substantially (21-

54%). As far as affect of germination on tannins is concerned, germination could

make tannins move in the same way as that soaking and cooking do, and the fact that

these compounds are heat and light sensitive make them more prone to destruction.

As the germination period increased to six days, TIA and the PA contents decreased

to a large extend, while amounts of tannins and catechins increased in two lentil

cultivars (Vidal-Valverde et al., 1994). Moreover, germination was considered as an

effective treatment for destroying trypsin enzyme inhibitors than either phytates or

polyphenols in L. sativus (Srivastava and Khokhar , 1999).

2.2.4. Bioavailability of minerals

Being a good source of minerals, legumes fulfill dietary requirements of human in

adequate manner among different food groups (Fennema, 2000). Legume plants get

minerals from their soil environment and deposit these to their seeds. Roots utilize

specific and/or selective transport proteins to obtain minerals that are essential for

plant growth and development including calcium (Ca), phosphorus (P), iron (Fe), zinc

(Zn), copper (Cu), manganese (Mn), magnesium (Mg), potassium (K) and sodium

(Na) (Grusak and Dellapenna, 1999). These minerals collectively contribute towards

ash fraction of the seed. These take part in many metabolic activities including

photosynthesis, respiration, chlorophyll synthesis, cell division and various responses

to biotic stress. The concentrations of any given mineral in legume seeds vary

depending on genotype and environmental constraints (Wood and Grusak, 2007).

A number of studies have been carried out for the evaluation of processing impact on

the fate of minerals. In general, mineral contents seem not to be affected during food

processing. However, the loss of minerals on soaking and cooking may be attributed

to their leaching out into discarded water (Saikia et al., 1999; Duhan et al., 2002)

which influences bioavailability of minerals during processing treatments. The

minerals have a multiple and complex type of interactions within the food matrix.

Processing usually exerts a positive impact through separation or partitioning of

minerals, or through the destruction of inhibitors or the beneficial complex formation

between food components and metal ions, thereby enhancing their availability

(Watzke, 1998).

The bioavailability of a nutrient can be subdivided in three constituent phases i.e.

availability in the intestinal lumen for absorption, absorption and/or retention in the

body and finally utilization by the body. The food processing can only influence the

first phase by determining the amount of minerals (content in raw materials minus

losses) and the speciation of the metal ion in the product and in the intestinal lumen.

However, other phases depend on the homeostatic regulation mechanisms and

individual physiological needs of the body (Watzke, 1998). As the minerals are

extractable in 0.03N HCl, the concentration of HCl found in human stomach, is an

index of its in-vitro bioavailability from foods.

The determination of HCl-extractability of minerals in processed legumes showed a

great improvement in this attributes. However, in soaked seeds in the absence of

further treatments, the HCl-extractability of minerals was recorded at par with

unprocessed seeds of chickpea (Cicer arietinurn) and blackgram (Vigna rnungo).

Comparatively, poor extractability of minerals when soaked in water might be due to

leaching out of some minerals into discarded soaking water under the influence of

concentration gradient (Kim and Zemel, 1986; Nolan and Duffin, 1987). Hence, these

minerals from legume seeds may be lost when water used during cooking was

discarded. Haytowitz and Matthews (1983) reported that cooking in boiling water

caused great losses of potassium (24%), copper (15%) and iron (8%). Reduction in

iron content of the soaked grains as compared to raw ones was also noted (Lestienne

et al., 2005).

The soaking and cooking treatments are found as effective techniques and caused

improvement in the availability of both major and trace minerals in white bean

(ElMaki et al., 2007). In unprocessed rice bean seeds, the HCl-extractability of

calcium, iron and phosphorus was 70.2, 78.0 and 33.4%, respectively whereas the

soaking the seeds for 12 hrs enhanced the HCl-extractability of calcium, iron and

phosphorous by 4, 8 and 13%, respectively, over the control values (Saharan et al.,

2001).

Autoclaving is a very effective method of cooking to increase the HCl-extractability

of all the minerals. However, draining of water after cooking is the source of nutrient

loss (Adebooye and Singh, 2007). Similar results were obtained for Bengal gram,

broad beans, cowpea, field beans, green gram, horse gram, lentil and French bean

after cooking (Khatoon and Prakash, 2004). Attia et al. (1994) reported mineral

nutrient losses after cooking of chickpea because cooking water was drained and

discarded before chemical analyses of the cooked seeds. Longe (1983) reported losses

of 31% copper and 22% magnesium from mature cowpeas after autoclaving. The loss

of potassium on cooking may be attributed to its leaching out into discarded water.

Sodium, zinc, copper, manganese and iron followed the same trend as observed for

potassium. Other major elements did not affect by cooking (Saikia et al., 1999 and

Duhan et al., 2002). The mineral losses of cooked foods in mass cooking were on an

average about 30-40% of those in raw or uncooked foods. Among various cooking

methods, loss of mineral was largest in squeezing after boiling (Kmura and Itokawa,

1990).

Except for soaking and dehulling, the remaining processing and cooking methods did

not lower the contents of total calcium, phosphorus and iron in pigeon pea seeds. HCl-

extractability of dietary essential minerals was enhanced significantly when the

pigeon pea seeds were processed and cooked (Duhan et al., 2002). With extended

cooking time HCl-extractable ash and phosphorus were enhanced. High temperature

such as pressure cooking showed the highest percentages of both extractable ash

(94.93%) and phosphorus (4.36%) in vegetable peas (Habiba, 2002).

Sprouting of seeds is a useful method in increasing the availability of minerals but

their raw consumption is sometimes unacceptable from sensory point of view.

Therefore, the consumption of cooked sprouts may be encouraged to enhance the

availability of food legumes and to reduce the incidence of several prevailing mineral

deficiencies particularly in developing countries. It has the most pronounced effect on

increasing the HCl-extractability of calcium, phosphorus, iron, zinc, copper and

manganese of primarily processed chickpea and black gram (Jood and Kapoor, 1997).

Further, sprouting followed by dehulling and soaking has been the best method for

improving the extractability of calcium, phosphorous and iron in seeds of rice bean

and faba bean. The husks of these legumes have significantly lower extractable levels

of these minerals (Saharan et al., 2001). HCl- extractability of calcium, iron and zinc

in kidney beans (Phaseolus vulgaris), sprouted for inclusion into a weaning food

formulation, increased by 55.2%, 54.7% and 53.0%, respectively (Mbithi et al.,

2001). Moreover, germination up to 48 hrs enhanced the HCl-extractability of zinc

and copper to a remarkable extent. In case of pigeon pea, germinated for 36 hrs

followed by pressure cooking, it increased up to 18–19% and 16-18% by 48 hrs

germination. Copper extractability enhanced to the maximum extent (3–4%) over the

control when the seeds of pigeon pea cultivars were germinated for 36–48 hrs (Duhan

et al., 2004). Significant improvements in HCl-extractability of all minerals were

observed after sprouting the seeds for 24 hrs. In rice bean seeds, maximum

enhancement was noticed in phosphorus extractability followed by iron and calcium

(El-Adawy, 2002). Fermentation also improves HCl-extractability of all minerals.

However, fermentation of the cooked seeds almost has no effect on contents of all

major and minor elements studied (Saharan et al., 2001). A fermented batter of

rice+black gram - 2:1 (idli) and 3:1 (dosa) – had higher bioaccessibility values for

zinc (71 and 50%, respectively), while iron bioaccessibility values were increased in

these cases of fermentation to an even greater extent, namely 277 and 127%,

respectively. Zinc and iron bioaccessibility was not improved by fermentation of the

combination of chickpea, green gram, black gram and rice (1:1:0.5:0.5; dhokla)

(Hemalatha et al., 2007).

Doli ki roti is a popular diet among an Indian Punjabi community and is prepared by

the natural fermentation of wheat flour dough kneaded with water containing a spice

mixture and leguminous stuffing. The reduction in phytic acid content by

fermentation was followed by a concomitant improvement in the HCl-extractabilities

of calcium (5-17%) and iron (15-38%). Further, it was improved at higher

fermentation temperature (40°C) for a longer period (24 hrs) (Bhatia et al., 2002).

The tendency of controlled fermentation to enhance HCl-extractability of minerals has

been reported. Fermentation of the rice and Bengal gram-whey blends did not change

their total mineral content but the HCI-extractability of calcium and iron was

enhanced considerably after whey incorporation and fermentation (Sharma and

Khetarpaul, 1994). However, a combination of germination and fermentation is a

potential process for decreasing the antinutrient levels and enhancing mineral

extractability. Processing reduced PA content by 60% with an increase in HCl-

extractable minerals of 47%. The phytate x Ca/Zn molar ratio decreased from 163 to

66.2, indicative of an increased zinc bioavailability (Sripriya et al., 1997).

2.2.5. Protein quality evaluation

Biological evaluation is the best tool for judging the quality of protein since a number

of factors affect the ultimate protein quality (Sogi et al., 2005). It includes growth and

nitrogen balance studies that deal with digestibility of the protein and availability of

amino acids (Ekanayake et al., 2000). Several bioassay methods, such as protein

efficiency ratio (PER), net protein ratio (NPR), true protein digestibility (TPD),

biological value (BV) and net protein utilization (NPU) have been used to determine

protein quality of foods (Rozan et al., 1997).

The NPR and relative NPR (RNPR) methods were considered to be improvements

over the PER method for measuring the protein quality of foods (Sarwar and

McDonough, 1990). The protein digestibility-corrected amino acid score method,

based on the actual amino acid profile of a protein in comparison with the amino acid

requirement of humans, was later recommended to be the most suitable routine

method for evaluating the protein quality of vegetable protein products (FAO/WHO,

1990).

2.2.5.1. Protein Digestibility Corrected Amino Acid Score (PDCAAS)

In 1991, the Food and Agricultural Organisation (FAO) and World Health

Organisation (WHO) recommended the Protein Digestibility-Corrected Amino Acid

Score (PDCAAS) as a more accurate method for evaluating protein quality. It

determines protein quality of foods intended for humans 2-5 years old. The amino

acid score (AAS) is calculated by dividing the amino acid content of a test food by

amino acid (AA) requirements for pre-school age children (FAO/WHO/UNU, 1985).

True protein digestibility (TPD) is determined by measuring nitrogen in food and

feces using the rat balance assay (McDonough et al., 1990). The PDCAAS is then

calculated by multiplying the lowest AAS value by TPD (Wu et al., 1995). The food

selection for a dietary plan based on its protein quality could be facilitated through

creation of table that contains the AAS, TPD values, and PDCAAS of foods

(Kizlansky and Lopez, 2006).

The PDCAAS score was calculated for 70 foods, taking the AA pattern for children >

1 year old and adults proposed by the U.S. National Academy of Sciences for the year

2002 as reference. For vegetable foods, the obtained amino acid score values and

PDCAAS were, respectively: vegetables 88.5%/73.4%, tubercles 89.44%/74.24%,

fresh fruits 75.6%/64.3%, dried fruits 65.6%/48.1%, legumes in general

89.2%/69.58%, chickpea and soybean 100%/78%, cereals and derivatives

68.8%/58.5% (Kizlansky and Lopez, 2006). The processing of food components

improves the PDCAAS value. The PDCAAS of weaning food with germinated

cowpea flour was higher (55.49%) than cooked cowpea flour (46.74%) (Jirapa et al.,

2001).

2.2.5.2. Biological efficacy studies

Biological evaluation on albino rats is the suitable technique to assess the nutritional

quality of diets. Poor biological utilization of legume proteins is well documented and

is known to be related to various factors such as the low content of sulfur containing

amino acids (Wu et al., 1995); the antinutritional compounds, which may modify

digestibility and alter release of amino acids (Aw and Swanson, 1985); the compact

structure of native legume proteins, which may resist proteolysis (Deshpande and

Nielsen, 1987) and increased endogenous nitrogen excretion (Deshpande and Nielsen,

1987; Mongeau et al., 1989; Marletta et al., 1992). Effects of antinutritional factors on

the protein quality can be assessed by animal feeding experiments (Ekanayake et al.,

2000).

The feed intake of the rats has been found strongly associated with their liking for

diet. The diet components also interfere with diet intake. The tannin present in raw

legumes (Nestares et al., 1993) decreases intake of food by precipitating salivary

proteins and thus interfering with swallowing (Mole, 1989). The significantly higher

intakes of the processed diets especially containing chickpea soaked in basic medium,

in comparison with raw chickpeas may have been due to reductions in antinutritional

factors during treatment (Nestares et al., 1993).

Processing of legume seeds has tendency to affect the protein quality parameters. PER

of diets containing raw gram drastically reduces between 1.46 to 0.79 by cooking. It

significantly improved TPD from 64.57% to 77.87%. However, NPU increased from

36.69 to 38.44% only (Bhatti et al., 2000). The protein quality parameters for velvet

bean seed proteins were significantly improved by autoclaving when compared to

other processing techniques (Vadivel and Pugalenthi, 2007). Raw peas had low PER

(1.4), which increased significantly on cooking to 2.0 (Nagra and Bhatti, 2007). James

and Hove (1980) showed a similar increase in PER between 1.87 to 2.21. The NPU of

the diet containing raw peas was 41.6% and on cooking it significantly increased to

46.3% (Nagra and Bhatti, 2007).

The diets containing cooked gram and supplemented with different types of meat

significantly improved PER (1.02 to 1.45), TPD (78.13 to 86.58%) and NPU (38.61 to

44.4%) over non supplemented diets. Diets supplemented by meat at 20 percent level,

showed comparatively better results than other levels in case of PER, TPD and NPU

(Bhatti et al., 2000). The results obtained for PER, NPU and NPR for food

supplement prepared with a maize cultivar indicated that it contains good protein

value, especially when soybean flour was added (de Paula et al., 2004).

The germination, extraction (double extraction with 70% ethanol and water at

isoelectric point) and α-amylase treatments improved nutritional indices of chickpea

flours. The protein quality of germinated and α-amylase treated chickpea flours was

comparable with casein, while germinated plus α-amylase treated seeds appeared

nutritionally superior to casein. These treatments could be used successfully as a

source of concentrated high quality protein for baby food production. The corrected

amino acid indices gave better prediction of PER, BV, TD and NPU than actual

amino acid indices. Moreover, PER was highly correlated with corrected amino acid

score (Mansour, 1996).

The digestive and metabolic utilization of chickpea protein (Cicer arietinum L.) was

assessed for raw and processed chickpeas (dry-heated, soaked and soaked + cooked).

Food intake, calculated as a function of body weight was higher for processed than for

raw chickpeas. The digestibility of chickpea protein was not affected by soaking, but

subsequent cooking increased it. This effect may be related to reduced trypsin

inhibitor activity and tannin content. Nitrogen retention (nitrogen balance) was better

after soaking in basic medium without cooking. However, it was lower than expected

from the chemical analyses of the protein in the different diets (Nestares et al., 1996).

The rate of protein digestibility in the raw bean extract was lower than casein due to

the presence of hemagglutinins (Thompson et al., 1986). The TPD of peas increased

significantly on cooking from 74.7 to 79.8% whereas TPD of autoclaved peas

increased from 85 to 88% (Sarwar et al., 1975). The increase in digestibility on

cooking may be due to elimination of trypsin and chymotrypsin inhibitors.

Dry-heating under pressure significantly reduced protein apparent digestibility

coefficient (ADC) in comparison with the value found for raw chickpeas, despite the

fact that this processing reduced trypsin inhibitor activity (TIA) by 30% and reduced

tannins by 47% (Nestares et al., 1993). The low digestive utilization of chickpea

protein after dry-heating was due to denaturation of the protein molecules (Bender,

1978), which reduced the bioavailability of some amino acids (Kirk, 1984).

A significant increase in the body weight gain of rats due to cooking of the whole

seed has been reported (Shah, 1991). The body weight gain of the animals fed on

either raw whole meals of kalimattar and white faba bean was significantly lower than

that of controls given standard skimmed-milk powder as protein source. Compared to

those of rats fed on kalimattar and its cotyledons, dry body weights, weight gains and

feed to weight gain ratios were significantly lower from groups fed on white faba

bean meal, its cotyledons and hull fraction. However, performance indices (dry body

weight, weight gains and weight gain to feed ratios) obtained from rats fed on

kalimattar hull fraction was similar to those obtained from the standard protein

(Mortuza et al., 2000).

Reports are available on the nutritional quality and biological values of certain under-

utilized legumes like Bauhinia purpurea (Vijayakumari et al., 2007); Canavalia

ensiformis, Canavalia gladiata, Canavalia maritima, and Canavalia cathartica

(Bressani et al., 1987; Seena et al., 2005; Bhagya et al., 2006). Bressani et al., (1987)

observed a gain in weight of rats after feeding processed seeds (pressure cooking and

roasting). The rats fed with C. cathartica seed flours showed a slight gain in weight,

but it was vice versa on feeding a whole seed diet of C. ensiformis. Similarly, the

subject rats showed better growth when fed with processed whole seeds (roasting,

cooking, alkaline cooking and extrusion cooking) (Bressani and Sosa, 1990). No

significant difference was found between the NPU of raw cotyledon and processed

whole seed meal of C. gladiata (Ekanayake et al., 2000).

Through the rat balance (fecal) method the lowest TPD values (79–84) are obtained

for pinto beans, kidney beans and lentils, whereas intermediate values (89–92) are

obtained for chick peas, rolled oats, whole wheat cereal, and pea protein concentrate

in comparison to the highest values (94–100) obtained for casein and casein +

methionine (Sarwar et al., 1989).

There are contradictory reports regarding the effect of fermentation on protein quality

of diets. Fermentation did not improve the protein quality of whole white and whole

black bean-rice diets (Joseph and Swanson, 1994). Contrarily, Goyal (1991) showed

an improvement in digestibility for fermented rice-soy flour blend products. Boralkar

and Reddy (1985) and Goyal (1991) attributed the increase in protein digestibility to

an increase in proteinase activity in fermented soybeans and in fermented rice-soy

flour blended products. Khalifa and Zinay (1994) referred increase in the protein

nutritional value of sorghum to decrease in the tannin content of the globulin

fractions, following fermentation. The germination of the beans raised the TPD from

81 to 87% (Kannan et al., 2001). This increase is notable especially because it

brought the TPD of cooked germinated beans close to that of milk-based infant

formulas (Sarwar et al., 1989). Nielsen (1991) attributed the improvement in

digestibility upon germination to the modification of storage proteins. The

germination reportedly causes protein mobilization with the help of proteases, leading

to the formation of polypeptides, oligopeptides, and free amino acids, thus facilitating

an improvement in protein digestibility. Oyeleke et al. (1985) also found that

germination improves digestibility in legumes such as mung beans and garbanzo

beans. It can lead to lower levels of trypsin inhibitors, tannins, phytate (Gupta and

Sehgal, 1991), and lectins (Smets et al., 1985) and can decrease phyto-hemagglutinin

activity. However, there is no effect of germination of beans on PDCAAS. It may

likely be due to similarity in the amino acid contents of cooked beans and germinated

beans. Another reason is likely to be related to the stringent amino acid scores for

infants that were used in calculating the PDCAAS in the evaluation of the test

products (Kannan et al., 2001). The PDCAAS for cooked fermented beans duplicated

the score reported for wheat gluten (0.25-0.37) (Sarwar, 1987).

The TPD measured for cooked beans (80.94%) is consistent with the findings

reported for a similar bean product by Sarwar (1987) and is 18 and 8% higher than

that reported by Hernandez et al. (1995) and Sarwar and McDonough (1990),

respectively. The TPD values for cooked fermented beans and cooked fermented

cooked beans (both 80.3%) are similar to those reported for autoclaved pinto beans

(Sarwar et al., 1989), canned kidney beans, and canned lentils (Sarwar, 1987) and are

only somewhat higher than that reported for pinto beans (72-76%) (McDonough et

al., 1990).

In oat cultivars, lysine had been observed as the most limiting amino acid. The amino

acid score and PDCAAS were variable for cultivars. When compared with casein, all

other indices of protein quality were inferior for the oat cultivars. The oat cultivars

tended to be identical among them, except for apparent protein digestibility which

was significantly higher in the two cultivars. On an average, the PER values of the oat

cultivars were 82% of casein; the NPU was 88% of casein as determined in vivo and

49% by the PDCAAS method (Pedó et al., 1999).

In other study, raw and germinated faba bean and chickpea seed meals were

incorporated in essential amino acid-supplemented and energy-equalized diets as the

only sources of protein. Weight gains, gain/feed ratios, nitrogen retention and NPU

values of animals fed raw or germinated legumes were lower than those of rats given

lactalbumin as control diet, while faecal dry weights were higher. However, nitrogen

retention and NPU values of germinated faba bean diets fed rats were higher and

faecal dry weights were lower than those of non-germinated faba bean diets fed

animals (Rubio et al., 2002).Two formulations (generated by computer-assisted

successive approximation) were prepared with 45–50% maize, 35–40% cowpeas, and

either peanuts or soybeans to improve the amino acid balance and to raise the energy

level of a diet. Acceptable PERs of 2.1–2.4 were obtained for the diets when casein

was adjusted to 2.5. The TPD of these food formulations was (87.4–92.1%) close to

that of casein (96.4%). Similarly, NPR ranged from 3.0–3.3 which was slightly less

than NPR of the casein diet (3.5). The PDCAAS for the formulations ranged from

0.72–0.82 (Wilmot et al., 2001).

Three types of bars using different ratios of oat, wheat germ, peanut, toasted and

expanded amaranthus and wheat extrudate were evaluated for PER and NPR values.

These were found to contain good PER (2.59-2.57) and NPR (3.99-3.95) values

(Boustani and Mitchell, 1990). Six snack-type bars were prepared containing oat and

wheat germ, walnuts, toasted or puffed amaranth, wheat extrudate and natural

sweeteners and oil. The PER and NPR values of the bars did not differ significantly

showing values approximately 86% that of the casein value (Estévez et al., 1995).

2.2.5.3. In-vitro digestibility

The effect of different processing treatments on a particular diet could be predicted

well through in-vitro digestibility. The legumes containing minimum amount of

antinutritional factors had the highest protein digestibility (Preet and Punia, 2000).

Fermentation seems to enhance the nutritive value of legumes by increasing the levels

of essential nutrients and reducing the level of antinutrients in foods. It ultimately

contributes towards the improvement in the in-vitro digestibility of legumes (Granito

et al., 2005).

Bacillus subtilis is the dominant species involved in the soybean and African locust

bean fermentation. During fermentation for 48 hrs of autoclaved soybean, the quantity

of soluble and dialyzable matter increased from 22% and 6% up to 65% and 40%,

respectively. The protein and carbohydrate degradation during fermentation of

soybean with several Bacillus spp. was substantial during the first 18 hrs of

fermentation resulting in the release of high levels of low molecular weight

compounds i.e. peptides and oligosaccharides. The in-vitro digestibility was increased

from 29% up to 33–43% after Bacillus fermentation for 48 hrs (Kiers et al., 2000).

In-vitro protein digestibility was neither improved significantly by germination nor by

decortication, however it improved substantially by cooking (Ifendu et al., 1989). In

case of chickpea, the protein digestibility was not significantly improved by any of

such treatments. However, for horse gram and cowpea, improvement in protein

digestibility was observed after some of the different processing treatments. In-vitro

protein digestibility of doughnuts was found to increase (71.8 to 76.3 g/100 g) with

increasing level of vetch flour from 0 to 15 g/100 g in doughnuts. (Rehman et al.,

2007). Different treatments, except germination, caused a marked increase in the in-

vitro carbohydrate digestibility (El Faki et al., 1984).

2.4. Storage changes in foods

During storage, various factors are involved to affect the quality of food products.

After microbial spoilage, oxidation is the second most important cause of food

spoilage and leads to overt rancidity in fatty foods (Lindley, 1998). Lipid oxidation is

also most momentous of three chemical processes that lead to the undesired rancidity;

the other two processes are lipolysis and flavour reversion. Lipid oxidation (also

referred to as "oxidative rancidity" and "autoxidation of unsaturated fatty acids") is

irreversible and proceeds by a free radical chain mechanism that results in

hydroperoxide formation. A subsequent decomposition reaction produces a variety of

secondary oxidative products (Velasco et al., 2004). The initial hydroperoxide

formation is a three step mechanism involving initiation, propagation, and

termination. The reaction rates for autoxidative processes are influenced by the

presence of prooxidants, such as trace metals; antioxidants, either indigenous or

added; ultraviolet radiation; available oxygen levels; and temperature conditions

(Gordon, 1990). The free radical triplet oxygen is the primary mechanism for the

formation of volatile flavour compounds in edible oils oxidation due to

photosensitized singlet oxygen, initiated by chlorophyll, has also a significant role in

the initiation of lipid oxidation. As the degree of fatty acid unsaturation increases, the

rate of autoxidation proceeds more rapidly. Hydroperoxides are unstable, colourless,

odourless and flavourless. The real cause of the off-flavours and off-odours associated

with lipid oxidation are the products of the decomposition of the hydroperoxides, the

cleaving of the residues and/or the termination of the autoxidative process to form

aldehydes, alcohols, ketones, esters, lactones, aromatics, acids, and hydrocarbons

(Nawar, 1996).

2.5. Extending shelf life

Shelf life can be enhanced by proper handling, packaging and addition of certain

preservatives in food products. The role of some of these factors is discussed in the

following text.

2.5.1. Packaging materials

A storage study of deep-fat-fried banana chips was carried out for 8 weeks at ambient

temperature (27°C), using four types of packaging materials i.e. laminated aluminium

foil (LAF), oriented polypropylene (OPP), polypropylene (PP) and low-density

polyethylene (LDPE). The moisture content, water activity, TBARS and breaking

force values of all samples increased during 8 weeks storage. The colour also changed

during storage, showing high L and low a and b values. Samples packed in LAF had

the lowest moisture content, water activity, thiobarbituric acid reactive substances

(TBARS) and breaking force values. The most notable sensory change occurred

during storage was a decrease in crispness, however, the samples packed in LAF

gained higher scores among samples. While LDPE, gave the lowest scores for

crispness as well as product colour. There were no significant differences in rancid

odour among samples packed in OPP, PP and LDPE, however, the samples packed in

LAF differed significantly from other three samples as it was giving the lowest rancid

odour (Ammawath et al., 2002).

In another study, three packaging materials (tortilla-packing paper, low-density

polyethylene and high density polyethylene) were used to evaluate tortilla shelf-life at

two different temperatures (-10°C and 5°C). At -10°C, the quality of tortillas was not

affected significantly during 11 days of storage, and the influence of the packaging

material was negligible. The tortilla kept at 5°C in high or low-density polyethylenes,

had significantly better quality than the paper-packaged product. Textural changes

were best shown by the Instron puncture resistance test than by the cutting resistance

test. At 5°C, packaged tortillas had a microbiologically stable level for up to seven

days, while that at -10°C significantly improved over the storage period (Nieblas et

al., 1991).

2.5.2. Use of antioxidants

Antioxidants are used to preserve food quality and to increase the shelf life of lipids

and lipid containing products. Because of being responsible for liver damage as well

as carcinogenesis of synthetic antioxidants such as butylated hydroxytolune (BHT)

and butylated hydroxyanisole (BHA) (Grice, 1986; Wichi, 1988), naturally occurring

nutritive and non-nutritive natural antioxidants have gained popularity as a potent

antioxidant in lipid systems. Polyphenols are important class of phytochemicals that

act as antioxidants in food. Turmeric, betel leaves, clove, lemon-grass and G.

atriviridis extended the butter cake storage life to 4 weeks, while BHA was able to

extend the storage life to 3 weeks only. Turmeric and betel leaves had the best

antioxidative activities followed by clove and lemon grass, all performing better than

BHA or BHT. The Anisidin values of cakes containing G. atriviridis were similar to

control cakes but the peroxide values were low throughout the storage period,

indicating that G. atriviridis increased the rate of conversion of hydroperoxides to

aldehydes. Moreover, black pepper leaves appeared to be pro-oxidative (Lean and

Mohamed, 1999).

The polyphenols can also provide protection against some common health problems.

These protect cells and body against damage caused by free radicals (reactive atoms

that contribute to tissue damage in the body). For example, when low-density

lipoprotein (LDL) cholesterol is oxidized, it can become glued to arteries and cause

coronary heart disease. Furthermore, the polyphenols can also block the action of

enzymes that cancers need for growth and deactivate substances that promote the

growth of cancers (Adams et al., 2006). The polyphenol most strongly associated with

cancer prevention is epigallocatechin-3-gallate (Lin and Liang, 2000). Intake of

polyphenols has been inversely correlated to the incidence of several chronic diseases

such as several types of cancer and cardiovascular diseases (Block et al., 1992; Arts

and Hollman, 2005). Specifically, the intake of polyphenols has been shown to be

associated with an increased antioxidant potential in plasma and vascular protection

(Keen et al., 2005; Manach et al., 2005). Several other studies have been performed in

the assessment of absorption of polyphenols from beverages and dried extracts.

There are three methods widely employed in the evaluation of antioxidant activity;

namely 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method, static

headspace gas chromatography (HS-GC) and β-carotene bleaching test (BCBT). The

strengths and limitations of each method were illustrated by testing a number of

extracts, of differing polarity, from plants of the genus Sideritis, and two known

antioxidants (BHT and rosmarinic acid). The sample polarity was important for the

exhibited activity in the BCBT and HS-GC methods but not for the DPPH method.

The complex composition of the extracts and partition phenomena affected their

activity in each assay. The value of the BCBT method appears to be limited to less

polar samples. Although slow, the HS-GC method is preferable for assessing the

antioxidant inhibitory properties on the formation of unwanted secondary volatile

products. Being rapid, simple and independent of sample polarity, the DPPH method

proved very convenient for the quick screening of many samples for radical

scavenging activity (Koleva et al., 2002)

2.5.2.1. Pomegranate (Punica granatum L.) extract

Consumption of fruits and vegetables is negatively associated with the morbidity and

mortality of cardio- and cerebro-vascular diseases and certain types of cancers

(Johnsen et al., 2003; Rissanen et al., 2003; Temple and Gladwin, 2003). The

antioxidants contained in fruits and vegetables including ascorbic acid, carotenoids,

flavonoids, hydrolysable tannins, are supposed to play an important role in the

prevention of these diseases (Lampe, 1999; Knekt et al., 2002; Huxley and Neil,

2003). Evidences from animals and humans experiments also revealed that some

natural antioxidants other than ascorbic acid, carotenoids and vitamin E could be

absorbed significantly and act as potent antioxidants in-vivo (Scalbert and

Williamson, 2000; Cao et al., 2001; Pataki et al., 2002; Su et al., 2003).

The Pomegranate (Punica granatum L.) tree originated in the Middle East and India.

It has been used for centuries in ancient cultures for its medicinal purposes. The

pomegranate fruit possess strong antioxidant and anti-inflammatory properties

(Seeram et al., 2005). It contains numerous antioxidant compounds including

anthocyanidins, catechins, proanthocyanidins, ellagitannins, gallotannins, ellagic and

gallic acids. While many pomegranate extracts are standardized simply to ellagic acid,

increasing research indicates that some of the most beneficial constituents of the

pomegranate may in fact be the ellagitannins. These ellagitannins can be found in

relatively abundant quantities in pomegranate juice. One family of ellagitannins,

known as punicalagins, has been shown to be especially important for supporting

healthy cell growth and immune function (Dinelli et al., 2006).

The polyphenolic constituents of pomegranate juice (0.2% to 1.0%) includes

anthocyanins, catechins, tannins and gallic and ellagic acid (Aviram et al., 2000). It

has been reported that the antibacterial action of pomegranate juice varied with fruit

variety and dependened on the contents of phenolic compounds, pigments and citric

acid (Kirilenko et al., 1978). Seeram et al. (2008) applied four tests of antioxidant

potency to polyphenol rich beverages. The pomegranate juice had the greatest

antioxidant potency composite index among the 12 beverages tested and was at least

20% greater than any of the other 11 beverages tested e.g. apple juice, acai juice,

black cherry juice, red wine, blueberry juice, cranberry juice, concord grape juice,

orange juice, black tea, green tea and white tea. Leaves and bark of this plant are

known to be a rich source of gallotannins and ellagitannins (Tanaka et al., 1986;

Nawwar et al., 1994).

Substantial amount of polyphenols such as ellagic tannins, ellagic acid and gallic acid

are present in pomegranate peel (Gil et al., 2000). The pomegranate peel yields

double the amount of antioxidants than the pulp, and have more potential as a health

supplement (Dinelli et al., 2006). Recent science has been focusing on the antioxidant

and cardio protective aspects of pomegranate. It has commonly been linked to

improve heart health, but other varied claims have been made including protecting

against prostate cancer and slowing cartilage loss in arthritis (Khalil, 2004).

2.5.2.2. Amla (Emblica officinalis) fruit extract

Amla (Emblica officinalis) fruit extract has many pharmacological activities. It

preserves against radiations (Scartezzini and Speroni, 2000), contains gastroprotective

effect (Al-Rehaily et al., 2002), possesses antidiabetic activity (Sabu and Kuttan,

2002) exhibits cytoprotective activity against heavy metals (Sai Ram et al., 2003) and

shows antivenom capacity (Alam and Gomes, 2003). The amla fruits exhibiting

antioxidant activity are of prime interest for food researchers for exploration of these

for shelf life enhancement of certain lipid rich foods. The E. officinalis fruit contains

0.40% (w/w) ascorbic acid and found that the ascorbic acid contributes approximately

45-70% of the total antioxidant activity exhibited by the fruit extract. It also shows a

great combination of vitamin C and tannins. Because of tannin, the ascorbic acid does

not oxidize even in dried fruit, thus it maintains its antiscurvy property. The fruit

extract of E. officinalis can effectively control hyperthyroidum and hyperglycemia.

Higher antioxidant activity has been attributed to the phenolic contents. It possesses

more free phenolics (126 mg/g) than the phenolics in bound form (3.0 mg/g). Gallic

acid and tannic acid are identified as the major antioxidant components in phenolic

fractions.

The antioxidant activity of phenolics increased when a lot of free hydroxyl groups

were present in the molecule (Kumeran and Karuakarun, 2006). The fruit extract of

amla contains tannins with significant antioxidant effect in-vivo (Ghosal et al., 1996)

and in-vitro (Bhattacharya et al., 2000; Bhattacharya et al., 2002). The tannins

responsible for this effect include low molecular weight hydrolysable gallotannins

comprising, gallic acid and ellagic acid. It had been noted that DPPH scavenging

activity of emblicannins A and B was 7.86 and 11.20 times more than that of ascorbic

acid and 1.25 and 1.78 times more than gallic acid. The concentration of E. officinalis

fruit pulp required for 15% inhibition of super oxide scavenging activity was 107

μg/ml in a method involving photoreduction of riboflavin and 232 μg/ml in the

xanthin-xanthin oxidase method and that for hydroxyl radical scavenging

(deoxyribone degradation method) was 3400 μg/ml. It has been reported that all the

antioxidant activity of E. officinalis was contributed by ascorbic acid (Khopde et al.,

2001).

Among the four fractions of E. officinalis fruit extracted with different solvents, ethyl

acetate partitioned fraction showed the strongest DPPH radical scavenging activity. It

was further subjected to separation and purification. Six phenolic compounds were

identified as geraniin, quercetin 3-β-D glycopyranoside, kaempferol 3-β-D

glycopyranoside, isocorilagin, quercetin and kaempferol. All the six compounds

showed antioxidative potential. Amongst, geraniin showed the highest antioxidant

activity (4.7 and 65.7 mM of IC50 values for DDPH and lipid peroxidation assay,

respectively) than other purified compounds (Liu et al., 2008).

The aqueous extract of the fruit of E. officinalis shows the ability to protect cellular

damage. It inhibited the β-radiation induced strand break formation in plasmid DNA.

It also inhibited the radiation induced lipid peroxidation in rat liver microsomes with

IC50 value less than 15 μg/ml. The extract has the ability to scavenge free radicals

such as DPPH and superoxides (Naik et al., 2005).

2.6. Optimization of shelf life

2.6.1. Response surface methodology (RSM)

Response surface methodology (RSM) is a useful statistical

procedure, consisting a set of techniques used in the empirical

study of relationships between one or more responses and a group

of input variables, to locate the region of lowest response values

where the lowest is considered to be the best (Cornell 1990). It

can be used to model or optimize any response affected by levels

of one or more quantitative factors (Dean and Voss 1999), such as

ingredients or process variables. Box et al. (1978) have described

some attractive features of RSM, including its wide applicability

and efficiency for optimization. It is more efficient than

traditional methods because it provides the minimum time and cost

required to determine the optimum product. RSM uses quantitative

data to determine and simultaneously solve the multivariate

equations that specify the optimum product for a specified set of

factors through mathematical models, while considering

interactions among test factors (Giovanni, 1983).

The Box-Behnken fractional factorial design is often preferred for product

optimization while evaluating sensory attributes, since interaction parameter estimates

are not completely confounded, and the design is considerably smaller than that of

other fractional factorial designs (Dean and Voss, 1999). Also, fractional factorial

designs are rotatable or nearly rotatable (Montgomery, 1991). However, since in

addition to the points generated by the composition, center points must be added to

the design for all model parameters to be estimable (Dean and Voss, 1999), the model

required that 15 treatments be tested by each panelist.

This method has been successfully applied to determine the

optimum formulation for various food products, while evaluating

sensory or physicochemical attributes (Martinez et al., 2004;

Singh et al. , 2004; Alizadeh et al., 2005). RSM was also used for

process optimization for blackberry jam (Acosta et al. , 2006),

bread (Demirekler et al., 2004), parotha (Indrani and Rao, 2001),

paneer (Nanda et al. , 2004), puri (Vatsala et al., 2001) and many

other food products.

Chapter-3

Materials &

Methods

The research project was undertaken in two phases. The first phase of research project

was carried out at Microbiology and Biotechnology Laboratory and second phase at

Food Analysis Laboratory, National Institute of Food Science and Technology,

University of Agriculture, Faisalabad. The details of materials used and analytical

methods employed during the study are given below.

3.1. Materials

Indian vetch (Lathyrus sativus L.) and chickpea (Cicer aritenum L.) seeds were

purchased from local grain market. Other ingredients such as wheat flour and farina,

whey protein isolate, fat, salt, lecithin, carboxy methyl cellulose and dry sugar were

procured from local market. Analytical grade chemicals were purchased from Merck

(KGaA, 64271, Darmstadt, Germany), Oxide (Basingstoke, Hampshire, England) and

Applichem (Ottoweg 10 b, D-64391, Darmstadt, Germany).

3.2. Processing of Indian vetch (Lathyrus sativus L.) and chickpea (Cicer aritenum L.)

3.2.1. Soaking

Indian vetch (Lathyrus sativus L.) and chickpea (Cicer aritenum L.) seeds were

soaked in distilled water for 9 hrs. The proportion of seed to soaking medium was 1:3

w/v. After this period extra soaking water was drained, and the soaked seeds were

weighed, dried at 60°C in an oven for 12 h (PCSIR Drying Oven, DO-1-30/02, PCSIR

Labs., Lahore, Pakistan), dehulled and finally ground in a mill. The samples were

preserved under low temperature (4°C) till analysis (Khalil et al., 2007).

3.2.2. Autoclaving

Raw ground Indian vetch and chickpea flours were dry heated in an autoclave (Sanyo

Lab Autoclave, MLS-3780-SV, Sanyo Electric Co. Ltd. Japan) at 121°C and 15 lb

pressure for 15 min and cooled to ambient temperature. The legume flours were

stored at low temperature (4°C) for analysis after removal of hull portion.

3.2.3. Germination

Germination of legume seeds was carried out as described by Jirapa et al. (2001).

Seeds were sterilized by soaking in 80% ethanol for 1 min. These were then soaked in

tap water at 1:3 seeds water ratio for 9 h at room temperature. The imbibed seeds

were germinated in plastic trays lined with wet towels. Seeds were also covered with

a wet towel to prevent rapid moisture loss. These were allowed to germinate in a dark

at room temperature with 3 times watering, for 36 h. The sprouts were washed and

dried at 60°C for 12 h in an electric oven (PCSIR Drying Oven, DO-1-30/02, PCSIR

Labs., Lahore, Pakistan). The dried sprouts were coarsely ground in a mill to facilitate

air classification of material. After hull separation, the material was again ground.

Finally, it was sieved using a 60 mesh screen to get fine particle size. The flour was

stored in polyethylene zip bags and stored in a refrigerator at 4°C until use.

3.2.4. Controlled fermentation

Legume seeds were fermented in controlled conditions according to procedure

described by Doblado et al. (2003) with certain modifications. Thermophilic lactic

culture (a mixed strain culture containing Lactobacillus helveticus and Streptococcus

thermophilus) was obtained from SACCO, Italy. These microorganisms are

commonly used for producing fermented milk products. MRS broth was inoculated

with starter and incubated for 24 h at 37°C. Then the broth carrying active culture was

centrifuged at 3000 rpm for 15 min and pellets were resuspended in saline buffer (7.2

pH) to obtain an approximate cell concentration of 1-5x108 cfu/ml.

The cracked seeds of Indian vetch and chickpea were ground to fine flour after

removing the hull. The flour was then sterilized by dry autoclaving for 15 min at

121°C and 15 lb pressure, and then mixed for 2 min with sterile water (40°C) using

sterile spatula to obtain slurries with dry legume content of 50%. Slurries were

individually inoculated with 5% of active culture and incubated at 37°C for 48 h in a

fermentation unit (Sanyo Orbital Shaker, Sanyo Electric Co. Ltd. Japan). The

fermented slurries were dried at 60°C for 12 h to get fermented flour.

3.2.5. Natural fermentation

The natural fermentation of legume flours was carried out as described by Martin-

Cabrejas et al. (2004). Suspensions of dehulled legume flours in sterilized tap water

were aseptically prepared and were allowed to ferment naturally (with only the

microorganisms on the seeds) at 37°C for 36 h without aeration, stirred at 150 rpm in

a fermentation unit (Sanyo Orbital Shaker, Sanyo Electric Co. Ltd. Japan). The

fermented slurries were dried at 60°C for 12 h to obtain natural fermented flour.

ANALYSES OF RAW MATERIALS

3.3. Antinutritional factors in processed legumes

3.3.1. Estimation of tannin contents

Tannin contents of flour were measured by Folin-Denis method (Schanderi, 1970).

3.3.1.1. Preparation of Folin-Denis reagent

Sodium tungstate (100 g) and phosphomolybdic acid (20 g) were dissolved in 750 ml

distilled water and later 50 ml phosphoric acid was added into the solution. Mixture

was refluxed for 2 h and volume was made to one liter with distilled water.

3.3.1.2. Preparation of carbonate solution

Sodium carbonate (350 g) was dissolved in one liter water at 70oC. Solution was

allowed to stand overnight and then it was filtered through glass-wool.

3.3.1.3. Preparation of standard tannic acid solution

Tannic acid (100 g) was dissolved in 100 ml distilled water.

3.3.1.4. Preparation of working solution

5 ml stock solution was diluted to 100 ml with distilled water. Each ml contained 50

µg of tannic acid.

3.3.1.5. Procedure

Ground sample (0.5 g) was taken in a 250 ml conical flask and 75 ml distilled water

was added to it. It was heated and boiled for 30 min and then centrifuged at 2000 rpm

for 20 min. The supernatant was collected in 100 ml volumetric flask and volume was

made up to the mark. In a 100 ml flask containing 75 ml water, 1 ml sample extract, 5

ml Folin-Denis reagent and 10 ml sodium carbonate solution were added and volume

was made up. Contents of the flask were shaken well and then absorbance was

measured at 700 nm after staying for 30 min. A blank was prepared with water instead

of sample and standard graph was produced by using 0-100 µg tannic acid.

3.3.2. Estimation of total phenols

Analysis for total phenols was carried out calorimetrically by using method described

by Malick and Singh (1980).

3.3.2.1. Preparation of standard solution

Standard solution was prepared by dissolving 100 mg catechol in 100 ml distilled

water. Then it was diluted 10 times for a working standard

3.3.2.2. Procedure

Sample (1 g) was taken and ground in 10 ml 80% ethanol in pestle and mortar. This

homogenate was centrifuged at 10,000 rpm for 20 min. Supernatant was saved and

residue was re-extracted with 80% ethanol, centrifuged and the supernatant was

saved. The supernatant was evaporated to dryness. The residue was dissolved in 5 ml

distilled water. Different quantities of samples (0.2-2 ml) were taken in the test tubes.

Volume of each tube was made to 3 ml with distilled water. Folin-Ciocalteu reagent

(0.5 ml) was added in each tube. After 3 min, 2 ml sodium carbonate (20%) solution

was added to each tube. The contents of the tubes were mixed thoroughly and the

tubes were placed in boiling water bath exactly for one minute, cooled and absorbance

was measured at 650 nm against a reagent blank. Standard curve was prepared by

using different concentrations of catechol.

3.3.3. Trypsin inhibitor activity

Trypsin inhibitor activities of legume samples were measured according to the

procedure of Roy and Rao (1971).

3.3.3.1. Preparation of 0.1 M phosphate buffer (pH 7.6)

Na2HPO4 (0.2M) 16 ml and NaH2PO4 (0.2M) 84 ml were added to 200 ml distilled

water and pH was adjusted to 7.6.

3.3.3.2. Preparation of 0.1 M phosphate buffer (pH 7.0)

50 ml of 0.1 M phosphate buffer was added to 100 ml distilled water and pH was

adjusted to 7.0

3.3.3.3. Casein 2% solution

A suspension of casein (2 g) with phosphate buffer (0.1 M, pH 7.6) was prepared and

dissolved by warming on steam bath for 10 min. The cold solution was diluted to 100

ml with phosphate buffer and stored in a refrigerator.

3.3.3.4. Trypsin solution (5mg/ml)

125 mg trypsin (20,000 F gross units/g) was dissolved in 20 ml phosphate buffer (0.1

M, pH 7.6)

3.3.3.5. Trichloroacetic acid (TCA) 5% solution

TCA (5 g) was dissolved in distilled water to make 100 ml.

3.3.3.6. Procedure

In flour sample (5 g), 25 ml of 0.05 phosphate buffer (pH 7.0) were added. The

mixture was shaken for 3 hours and centrifuged at 10000 rpm for 20 min. Different

sets of incubation mixture were prepared in test tubes in triplicate by adding trypsin

(0.5 ml), HCl (0.4 ml), casein solution (2 ml), TCA solution (6 ml). To test extract for

TIA, 1 ml extract was added in test tubes carrying incubation mixture and volume was

made up to 10 ml by adding phosphate buffer. Control tube was prepared by without

adding extract. These test tubes were incubated for 20 min at 37oC. A blank was

prepared parallel without incubation.

3.3.3.7. Protein determination

After incubation, the contents were centrifuged at 10000 rpm for 10 min. TCA soluble

proteins were determined in the supernatant by the method of Lowry et al. (1951).

3.3.3.8. Preparation of alkaline CuSO4 solution

Reagent A. 2% Na2CO3 in 0.1N NaOH solution.

Reagent B. 0.5% CuSO4 in 1% citrate solution

50 parts of solution A and 1 part of solution B were mixed just before use.

3.3.3.8. Procedure

To 0.5 ml of supernatant, 5 ml alkaline CuSO4 solution was added, mixed thoroughly

and allowed to stand for 10 min at room temperature. Later 0.5 ml folin phenol

reagent (double diluted the original reagent) was added and immediately mixed

properly. Water blank was also run side by side. The color intensity was read after 30

min at 520 nm. For preparing a standard curve, 0.1 ml to 0.5 ml of standard casein

solution was taken (400 μg/ml).

3.4. Phytate content

Phytate content in legume meals was determined by procedure elaborated by Haug

and Lantzsch (1983).

3.3.4.1. Preparation of phytate reference solution

Sodium phytate (0.15 g) was dissolved in 100 ml de-ionized water. Reference solution

was prepared by diluting the stock solution in the range of 1.2-11.7 ml stock solution

(1.2, 2.7, 4.2, 5.7, 7.2, 8.7, 10.2, 11.7 ml) in 100 ml volumetric flask and made the

volume with 0.2N HCl.

3.3.4.2. Preparation of ferric solution

Fe(NH4)2(SO4)2.6H2O (0.2 g) was dissolved in 100 ml 2N HCl and volume was made

to 1000 ml with de-ionized water.

3.3.4.3. Preparation of 2, 2- bipyridine solution

10 g 2,2- bipyridine and 10 ml thioglycolic acid were dissolved in de-ionized water

and made the volume 1000 ml.

3.3.4.4. Procedure

Sample (0.06) g was extracted in 10 ml 0.2N HCl solution in a test tube by shaking

for half an hour. Then 1 ml extract was taken in a test tube. 2 ml ferric solution was

added into the test tube and covered with a stopper. The test tube was fixed with clip

and then heated in a water bath for 30 min. Then test tube was cooled in ice water for

15 min and allowed to adjust at room temperature. 2,2- bipyridine solution (4 ml) was

added into the test tube and the contents were mixed. The absorbance was checked at

519 nm by spectrophotometer against de-ionized water after 30-60 sec. The method

was calibrated with reference solution as a substitute for the sample solution for each

set of analysis.

3.3.5. Determination of β-ODAP in Indian vetch

β-ODAP in Indian vetch was determined as per procedure described by Rao (1978).

Earlier, the toxin was extracted from Indian vetch sample according to the method

described by Abegaz et al. (1997).

3.3.5.1. Preparation of OPA reagent

Mercaptoethanol (200 μl) and o-phthalaldehyde (100 mg) were dissolved in 100 ml

5M potassium tetraborate tetrahydrate.

3.3.5.2. Procedure

In order to screen the Indian vetch samples for their toxin level, 80 mg powdered

Indian vetch sample was suspended in 8 ml distilled water and subjected to 1 h of

extraction at 40-45°C to extract ODAP. Then, it was centrifuged at 4000 rpm, and a

0.1 ml aliquot of the supernatant was transferred to a 10 ml test tube for basic

hydrolysis using 3M KOH (0.2 ml) in a boiling water bath for 30 min, to convert

ODAP to L-2,3-diaminopropionic acid (DAP), that is, formed after cleavage of the -

COCOOH group. After neutralization of the hydrolysate with 0.2 ml 3M HCl, volume

was made up to 1 ml, then 2 ml OPA reagent was added, to form a colored adduct.

Absorbance of the colored adduct was measured at 476 nm against a reagent blank

after 30 min of standing time, after which the color was fully developed. To prepare

standard curve, stock solution of DAP (D-1502, Sigma Chemical Co., St. Louis, MO,

USA) (2.89 mM) was diluted to various concentrations and treated similarly as

sample with OPA to form color adduct and analyzed spectrophotometrically.

3.4. Mineral profile

3.4.1. Preparation of sample

Sample (0.5 g) was digested by the wet digestion method. It was first digested with 10

ml HNO3 at gentle temperature (60-70°C) for 20 min. Then the sample was digested

with HClO4, at high temperature (190°C) till the solution become clear. The digested

sample was transferred to 250 ml volumetric flask and volume was made with

distilled water and then filtered (Duhan et al., 2002).

3.4.2. Estimation of mineral contents (Cu, Zn, Mn, Mg and Fe)

The filtered sample solution was loaded to the atomic absorption spectrophotometer

(Varian AA 240, Victoria, Australia). The standard curve for each mineral was

prepared by running samples of known strength. The mineral contents of the samples

were estimated by using the respective standard curve prepared for each element

(AACC, 2000).

3.4.3. Estimation of sodium (Na) and potassium (K) contents

Sodium and potassium were determined by flame photometer (Sherwood Flame

Photometer 410, Sherwood Scientific Ltd. Cambridge, UK) according to AOAC

(1984). 3.4.4. Estimation of phosphorous (P) content

Ammonium vandate was used to determine phosphorus by aminonaphthol sulphonic

acid method (Kitson and Mellon, 1944).

3.4.4.1. Preparation of 2.5% ammonium molybdate

Ammonium molybdate (2.5 g) was dissolved in 3 ml conc. H2SO4 and volume was

made to 100 ml with distilled water.

3.4.4.2. Preparation of aminonaphthol sulphonic acid

Aminonaphthol sulphonic acid (0.5 g) was dissolved into 15% sodium bisulphate

solution (195 ml) with added 20% Na2SO3 (5 ml) and shaken to dissolve.

3.4.4.3. Preparation of phosphorus standard solution:

KH2PO4 (0.351 g) was dissolved in distilled water and volume was adjusted to 100

ml. Concentration of phosphorous achieved was 0.8 mg/ml.

3.4.4.4. Procedure

In the test tubes, ammonium molybdate solution (1 ml), amminonaphthol sulphonic

acid solutions (0.4 ml) and 0.1 ml sample or standard solutions at various dilutions

were added. Distilled water was added to make up the volume (10 ml). This solution

was shaken and allowed to stand for 5 min to develop color. Blank was used to set the

zero absorbance at 720 nm wavelength.

3.4.5. Minerals extractability

HCl-extractability of minerals (as an index of mineral bioavailability) was estimated

according to method prescribed by Duhan et al. (2002).

3.4.5.1. Procedures

The minerals in the legume samples were extracted with 0.03N HCl by shaking the

contents at 37°C for 3 h. The solution was then filtered through Whatman No. 42

filter paper. The clear extract obtained after filtration was oven-dried at 100°C and

digested following the procedure mentioned in 3.4.1. HCl-extractable minerals in the

digested samples were determined by the methods described for the estimation of

minerals in 3.4.2-4. Mineral extractability (%) was determined by applying Eq. 2.

Mineral extracted into 0.03N HCl Mineral extractability (%) = x l00….…(Eq. 1) Total minerals

3.5. Proximate analysis

3.5.1. Moisture

Moisture (%) in the meal components was determined by a gravimetric method

(AOAC, 2000).

3.5.1.1. Procedures

One gram of sample was pre-weighed (W1) in a beaker and placed in an oven (PCSIR

Drying Oven, DO-1-30/02, PCSIR Labs., Lahore, Pakistan) at 105°C for 24 h. The

sample was removed from the oven, cooled in a desiccator, and reweighed (W2).

Moisture percentage was calculated according to the formula:

Moisture (%) = (W1 – W2) / W1 x 100………………….(Eq. 2)

3.5.5. Total ash

Total ash content was determined as total inorganic matter by incineration of a sample

at 600°C (AOAC, 2000).

3.5.5.1. Procedure

Sample (1g) was weighed into a pre-weighed porcelain crucible and incinerated

overnight in a muffle furnace (MF-1-02, PCSIR Labs., Lahore, Pakistan) at 600°C.

The crucible was removed from the muffle furnace, cooled in desiccator and weighed.

Ash content was calculated according to the following formula:

Ash (%) = (ash weight / sample weight) x 100………………….(Eq. 3)

3.5.2. Crude protein

Crude protein was determined by the Kjeldahl method as described by AOAC (2000).

3.5.2.1. Procedure

Sample (700 mg) was placed in a Kjeldahl digestion tube. 5 g K2SO4 + 0.5 g CuSO4

and 25 ml conc. sulphuric acid were added to the sample. The sample was digested

for one h. 20 ml deionized water was added to the sample after allowing it to cool.

After adding 25 ml NaOH (40%), the sample was then distilled and the ammonia

liberated was collected in boric acid and titrated with 0.1N hydrochloric acid. A blank

was prepared and treated in the same manner except that the tube was free of sample.

Protein percentage was calculated according to the formula:

Crude protein (%) = (sample titre – blank titre) x 14 x 6.25 x 100 / sample

weight..(Eq. 4)

Where, 14 is molecular weight of nitrogen and 6.25 is the nitrogen factor.

3.5.3. Crude fat

Crude fat was estimated by employing solvent extraction using a Soxhlet extraction

unit (AOAC, 2000).

3.5.3.1. Procedure

One gram sample was weighed into an extraction thimble and covered with absorbent

cotton. 50 ml solvent (petroleum ether) was added to a pre-weighed cup. Both thimble

and cup were attached to the extraction unit (PCSIR Labs. Complex, Lahore,

Pakistan). The sample was subjected to extraction with solvent for 30 min followed

by rinsing for 1.5 h. The solvent was evaporated from the cup to the condensing

column. Extracted fat in the cup was placed in an oven at 110°C for 1 h and after

cooling the crude fat was calculated using following formula:

Crude fat (%) = (Extracted fat / Sample weight) x 100……………….(Eq. 5)

3.5.4. Crude fibre

Crude fibre in the samples was determined by the method described by AOAC

(2000).

3.5.4.1. Procedure

Defatted sample (1g) was placed in a glass crucible and attached to the extraction unit

(InKjel, D-40599, behr Labor-Technik GmbH, Dusseldorf, Germany). 150 ml boiling

1.25% sulphuric acid solution was added. The sample was digested for 30 min and

then the acid was drained out and the sample was washed with boiling distilled water.

After this, 1.25% sodium hydroxide solution (150 ml) was added. The sample was

digested for 30 min, thereafter, the alkali was drained out and the sample was washed

with boiling distilled water. Finally, the crucible was removed from the extraction unit

and oven dried at 110°C overnight. The sample was allowed to cool in a desiccator

and weighed (W1). The sample was then ashed at 550°C in a muffle furnace (MF-1-

02, PCSIR Labs., Lahore, Pakistan) for 2 h, cooled in a desiccator and reweighed

(W2). Extracted fibre was expressed as percentage of the original undefatted sample

and calculated according to the formula:

Digested sample (W1) – Ashed sample (W2) Crude fibre (%) = x 100……(Eq. 6)

Weight of sample

3.5.6. Nitrogen Free Extract (NFE)

Nitrogen free extract (carbohydrate) was calculated as following:

NFE (%) = 100 – (Crude protein + Crude fat + Total ash + Crude fibre) ………..(Eq.

7)

3.6. Development of food bar

A baking procedure for food bars preparation was adopted (Brisske et al., 2004).

Food bars were prepared by adding different proportion of meals. Treatments and

their respective meal quantities are given in Table 3.1.

Table 3.1. Experimental treatments for food bar formulation

S. Code Legume meals§

No.

meal*§ Raw Indian vetch

Controlled fermented

Indian vetch

Natural fermented Indian

vetch

Germi-nated Indian vetch

Raw chickpea

Controlled fermented chickpea

Natural fermented chickpea

Germi -nated

chickpea

1 BT0 44.00 - - - - - - -

2 BT1 33.00 11.00 - - - - - -

3 BT2 33.00 - 11.00 - - - - - -

4 BT3 33.00 - - 11.00 - - - - -

5 BT4 33.00 - - - 11.00 - - - -

6 BT5 33.00 - - - - 11.00 - - -

7 BT6 33.00 - - - - - 11.00 - -

8 BT7 33.00 - - - - - - 11.00 -

9 BT8 33.00 - - - - - - - 11.00 *Basic meal: Comprises of patent wheat flour, farina and rusk crumb mixed at the ratios of 2.25: 2.25: 12.0 respectively. § % quantity used in each formulation treatment

3.6.1. Product preparation through baking

Food bar formulation as described in Table 3.2 was adopted from Brisske et al.

(2004). Dry ingredients (basic meal, Indian vetch flour, chickpea flour, whey protein

isolate, salt and dry sugar) as per treatment were blended to give a uniform mixture.

Fat was melted to 112°C and mixed for 5 min with the lecithin using a mixer. Carboxy

methyl cellulose (CMC) was added in emulsified fat and mixed for 5 min. Sugar syrup

was added and mixed for another 5 min. Then blended dry ingredients were gradually

added until fully incorporated into the dough. Finally, water was incorporated and

the entire dough was mixed for 5 minutes. Immediately after mixing, 52.0 ± 0.02 g

dough was weighed into 7.6×4.4×3.2 cm loaf pans lined with aluminum sheet. The

cookie-like dough was compressed into the pans to form a firm mass of approximately

1.5 cm thick bar. The bars were baked at 150°C for 20 min in an oven (DO-1-30/02,

PCSIR Labs., Lahore, Pakistan) and removed from pans. After 5 min cooling, these

were packed in laminated aluminum pouches. Final bar weighed 50.0 ± 0.2 g.

Table 3.2. Product Formulation

Ingredient Quantity (%) Fat 13.5 CMC 2.20 Corn syrup 18.00 Sugar 12.30 Basic meal As per table 1 Legume meal As per table 1 Whey protein isolate (WPI 80) 7.00 Water 3.50 3.7. Biological evaluation of protein meals

3.7.1. Preparation of meals

Nine experimental bar meals were prepared by incorporating protein contributing

components from formulation i.e. basic meal, legume meal and whey protein isolate

at the level of 10% protein (Table 3.3) whereas, in control diet, the protein source was

casein. In protein free diet, the protein source was replaced completely by starch. The

fat level of test diet was analysed to be 9% to attain the same level for standard and

control (protein free) diets, refined corn oil (Rafhan Maize Industries, Faisalabad) was

added. Basic meal, legume meal and whey protein concentrate were blended to give a

uniform mixture. Thereafter, these were baked at 149°C for 20 min (PCSIR Drying

Oven, DO-1-30/02, PCSIR Labs., Lahore, Pakistan) to simulate the processing steps

to which the protein blends were about to be subjected during food bar preparation

(Brisske et al., 2004). Then other ingredients were added and pellets prepared by

adding little water.

Table 3.3. Experimental treatments for protein meal formulation

Components Overall quantities (%) Protein 10 Fat 9 Cellulose 5 Salt mixture* 5 Vitamin mixture* 2 Corn starch sufficient for 100 * composition given in appendix-I

3.7.2. Biological assay

Biological evaluation was done by measuring the protein quality of diets containing

protein blends as per treatments (Table 3.3). Albino rats of the Sprague–Dawley strain

were used. Weaning was done at 21 days of age. The rats were then put on stock diet

for 7 days prior to the experiment. They were arbitrarily divided into experimental

units of 3 rats each in such a way that the initial weight of the rats in each cage was

135 g; 2 experimental units were randomly allotted to each diet. One group was given

a casein diet, the second was fed on a protein-free diet, and the other eight groups

were fed on the experimental diets containing eight types of protein meal varying

from each other on legume protein source. The rats were fed the allotted diets ad

libitum for a period of 14 days. During this period, fresh and clean water was made

available at all times and room temperature was maintained at 24–27°C. The weight

of each replicate was recorded weekly. The fecal matter was separated from the

spilled food and collected for each group, dried overnight at 100°C, then weighed and

ground. The nitrogen content of the ground feces of test groups for the determination

of fecal protein (FP) was analyzed by the micro-Kjeldahl method. The feces from

protein free group were analyzed for estimation of metabolic fecal protein (MFP)

(Miller and Bender, 1955).

3.7.3. Nutritional indices

The amount of the feed given on dry weight basis was computed, based on moisture

content while moisture free refusal was obtained by drying overnight at 100°C. The

difference between diet given and refusals was taken as feed intake, which was further

used to calculate protein intake. Feed intake, protein intake and body weight gain

were used to compute the following nutritional indices:

Feed efficiency (FE) = Gain in body wt (g) /Feed intake (g)….……(Eq. 8)

Feed utilization (FU) = Feed intake (g) /Gain in body wt (g) ….…..(Eq. 9)

Protein efficiency ratio (PER) = Gain in body wt (g) /Protein intake (g) …(Eq. 10)

Corrected protein efficiency ratio (C-PER) = PER of casein (2.5) x PER of test

protein / Exp. PER of casein ………(Eq. 11)

Protein utilization (PU) = Protein intake (g) /Gain in body wt (g) …(Eq. 12)

Net protein ratio (NPR) = (Weight gain of test rat + Weight loss of non protein rat)/

Protein consumed by test rat ……………………………………..…(Eq. 13)

Relative PER (RPER) and relative NPR (RNPR) values calculated using the

following equations (Sarwar and Peace, 1994):

RPER = (PER of test diet/PER of control diet) x 100………………(Eq. 14)

RNPR = (NPR of test diet/NPR of control diet) x 100……………...(Eq. 15)

True protein digestibility value was calculated using the following equation (Sarwar

and Peace 1986):

True protein digestibility = TPD = [PI – (FP-MFP)] / PI x 100………(Eq. 16)

Where, PI = protein intake, FP = fecal protein and MFP = metabolic fecal protein.

3.7.4. Amino acid analysis

All the samples of selected food bars were subjected to determination of amino acids

composition using an amino acid analyzer according to the method of Schuster

(1988). The samples were hydrolyzed with 6N HCl under vacuum at 110ºC for 24 h.

The hydrolysates were dried in a rotary evaporator at 40°C under vacuum to remove

the excess acid (6N HCl). The dry residues were then dissolved in a known quantity

of citrate buffer (2.2 pH) and filtered to obtain a clean solution of the hydrolysate. An

aliquot of it was injected into the column (Shim-pack ISC-07/S1504 Na) of the HPLC

based amino acid analyzer (RF-10AXL, Shimadzu Corporation, Tokyo, Japan)

equipped with fluorescence detector (FLD-6A). Sodium hypochlorite and o-

phthalaldehyde solutions were used as reaction solutions.

3.7.5. Protein digestibility-corrected amino acid score method (PDCAAS)

Protein digestibility-corrected amino acid score was calculated in two steps. The first

step included the determination of the true protein digestibility for the casein and

protein meal diets. In the second step, determination of amino acid contents was done

to calculate the chemical score of the limiting amino acids in the various bar types.

mg of an essential amino acids in 1.0 g of test protein Amino acid score= ..(Eq. 17) mg of the same amino acids in 1.0 g of reference protein

Amino acid ratios for nine essential amino acids (His, Ile, Leu, Lys, Met + Cys, Phe +

Tyr, Thr and Val) were calculated using a human pattern of amino acid requirements

(FAO/WHO/UNU suggested pattern of amino acid requirements for pre-school

children, 2–5 y) as the reference proteins (Table 3.4).

Table 3.4. Essential amino acid requirements for pre-school children, 2–5 y

Essential amino acid (mg/g protein) Children1 (2-5 yrs)

Histidine 19

Isoleucine 28

Leucine 66

Lysine 58

Methionine and cysteine 25

Phenylalanine and tyrosine 63

Threonine 34

Tryptophan 11

Valine 35 1FAO/WHO/UNU, 1985.

The PDCAAS was calculated as follows (Sarwar and McDonough 1990):

PDCAAS = True protein digestibility x amino acid score ……………(Eq. 18)

3.8. Prototype selection

Sensory panel screened nine prototype bars based on sensory rating. Proximate

composition, calorific value, in-vitro digestibility of starch and protein were the other

factors for selection of best prototype.

3.8.1. Sensory evaluation

Food bars were evaluated for sensory characteristics such as color, flavor, taste,

texture and overall acceptability at room temperature in sensory evaluation laboratory

by a panel of twelve judges on 9-point Hedonic Scale (Land and Shepherd, 1988).

Score card for sensory evaluation is given in appendix-II.

3.8.2. In-vitro protein digestibility (IVPD)

In-vitro protein digestibility of flour was determined by pepsin digestibility method

(Mertz et al., 1984).

3.8.2.1. Preparation of pepsin solution

1.5 mg/ml solution of pepsin was prepared in 0.035M HCl with pH 2.0.

3.8.2.2. Procedure

Powdered material (200 mg) was suspended in 35 ml pepsin solution and incubated

for 2 h at 37°C with gentle shaking. This solution was centrifuged at 12,000g for 15

min at 40°C and the residue was suspended in 10 ml 0.035M HCl and recentrifuged.

Residue was collected and dried overnight at 40°C. Total nitrogen of the dried residue

was determined by micro-Kjeldahl method. A blank solution was run with each assay

without addition of enzyme solution.

3.8.3. In-vitro starch digestibility (IVSD)

3.8.3.1. Preparation of porcine pancreatic amylase solution

Porcine pancreatic amylase (EC 3.2.1.1, 790 units/mg protein; catalog no. A6255,

Sigma) to give final concentration 0.4 mg/ml was used.

3.8.3.2. Procedure

In-vitro starch digestibility (IVSD) was assayed by employing porcine pancreatic

amylase (Singh et al., 1982). One unit of amylase liberated 1 mg of maltose from

starch in 3 min at pH 6.9 at 20°C. In brief, 50 mg food bar sample was incubated with

0.5 ml pancreatic amylase solution at 20°C for 2 h. After incubation, 2 ml 3,5-

dinitrosalicyclic acid reagent was added, and the mixture was boiled for 5 min. After

cooling, the absorbance of the filtered solution was measured at 550 nm with maltose

used as the standard. The values of starch digestibility were expressed as milligrams

of maltose released per gram of dry sample (Chau and Cheung, 1997).

3.8.4. Proximate analysis.

Proximate composition such as moisture, ash, crude protein, crude fat, crude fiber and

NFE of prototype bar samples was determined and expressed on dry matter basis

(AOAC, 1990).

3.8.5. Gross energy value

Using standard factors of 23.6, 39.5 and 17.2 kJ/g for protein, lipid and carbohydrate

respectively, the energy contents were summed to give total or gross energy of the bar

samples (Livesey, 1990).

3.9. Extraction of antioxidant extract

Selected food processing waste materials (almond skin, pomegranate peel, onion

scales and peanut skin) were dried in a hot air oven at 50°C. The dried plant materials

were ground separately and kept in air tight containers at 4°C until further use. A

weighed portion (25 g) of each dried sample was extracted with 100 ml of 80% (v/v)

ethanol, distilled water, ethanol and acetone for 6 h in a mechanical shaker. The

extracts were filtered and filtrates from ethanol and acetone extracts were evaporated

at 40°C under vacuum to dryness in a rotary evaporator. The dried extract obtained

was again diluted to 100 ml using distilled water and stored in an airtight container at

-21°C until further use.

3.10. Screening of antioxidant extract

The extracts were screened for their antioxidant activity through different methods as

mentioned below.

3.10.1. Estimation of total phenols

Analysis for total phenols was carried out by Folin-Ciocalteu reagent colorimetrically

as described by Malick and Singh (1980). The procedure was same as given in 3.3.2.

3.10.2. Determination of antioxidant activity

Antioxidant activity of the samples was examined by using a β-carotene/linoleic acid

system (Matthaus, 2002).

3.10.2.1. Procedure

…………Eq. 19

…Eq. 20

1 ml β -carotene solution (1 mg/ml in chloroform), 40 μL linoeic acid and 400 μl

Tween 80 (water soluble vitamin E) were transferred to a round bottom flask.

Chloroform in the samples was evaporated off under nitrogen stream. Then, 100 ml

distilled water was added slowly to the residue and the mixture was agitated

vigorously to give a stable emulsion. To an aliquot of 4.5 ml of this emulsion, 500 μl

(50-400 μg/ml) of appropriately diluted samples in a 10 ml test tube was added. The

tube was placed in water bath at 50°C and the absorbance was measured after 120 min

at 470 nm. A blank sample was prepared by adding 500 μl distilled water to the

control reaction mixture, and the absorbance was measured immediately after

preparation at 470 nm (Matthäus, 2002).

Antioxidant activity was calculated in different ways. Antioxidant activity (AA) was

calculated as percent inhibition relative to control using following equation (Al-

Saikhan et al., 1995)

Rcontrol - Rsample AA= X 100

Rcontrol

Where, Rcontrol and Rsample were the bleaching rates of β-carotene in reactant mix

without antioxidant and with sample extract, respectively.

3.10.3. Radical scavenging activity

Radical-scavenging activity of extracts was assayed according to 1,1-diphenyl-2-

picrylhydrazyl (DPPH) method described by Singh et al. (2002).

3.10.3.1. Procedure

Different concentrations (50 and 100 μl equivalent to 50 and 100 ppm) of extracts and

Gallic acid (25 and 50 ppm) and BHA (25 and 50 ppm) were taken in different test

tubes. The volume was adjusted to 100 μl by adding methanol (MeOH). 0.9 ml 0.1

mM methanolic solution of DPPH (3.94 mg DPPH/100ml MeOH) was added to these

tubes and shaken vigorously. The tubes were allowed to stand at ambient temperature

for 20 min. The control was prepared as above without any extract. Changes in the

absorbance of the samples were measured. MeOH was used for the correction of

baseline at 515 nm.

Radical scavenging activity was expressed as the inhibition percentage and was

calculated using the following formula:

Radical scavenging activity (%) = (control OD - sample OD/control OD) x 100

3.11. Shelf life testing of bars

The highest ranked bar formulation was selected for shelf life studies. Various

combinations of antioxidants treatments were suggested for such studies (Table 3.4).

Prepared prototypes were stored at 32°C for storage experiment for 120 days.

3.12. Experimental design for shelf life optimization

Response Surface Methodology (RSM) was used to relate storage responses to

independent variables i.e. Emblica officinalis extract, best performing extract from

food processing wastes and citric acid. The maximum level of each variable was

selected by earlier trials performed to validate the decisions regarding the ingredients’

levels, evaluating bar’s sensory evaluation. Coded levels and actual values of the

factors are given in Table 3.5.

Table 3.5. Levels of independent variables Independent variables (%) -1.0 0.0 1.0

Emblica officinalis extract 0 0.75 1.5

Food processing waste extract 0 1 2

Citric acid 0 0.03 0.06

Fifteen bar treatments were generated using a Box-Behnken design with 3 variables and 3

levels for each variable. 13 different formulations were produced in total and the central point

was evaluated in triplicate. The experimental design in actual and coded values is presented in

Table 3.6. Runs 13 to 15 correspond to centre point replicates.

Table 3.6. Design showing independent variable combination levels S t d

O r d e

r

R u n

O r d e

r

B l o c k

s

Coded Uncoded

Emblica officinalis

extract

Punica granatum

peel extract

Citric acid

Emblica officinalis

extract (%)

Punica granatum

peel extract

(%)

Citric acid (%)

6 1 1 + 0 - 1 . 5 0 1 . 0 0 0 . 0 0

1 2 2 1 0 + + 0 . 7 5 2 . 0 0 0 . 0 6

5 3 1 - 0 - 0 . 0 0 1 . 0 0 0 . 0 0

1 4 1 - - 0 0 . 0 0 0 . 0 0 0 . 0 3

4 5 1 + + 0 1 . 5 0 2 . 0 0 0 . 0 3

1 4 6 1 0 0 0 0 . 7 5 1 . 0 0 0 . 0 3

1 5 7 1 0 0 0 0 . 7 5 1 . 0 0 0 . 0 3

9 8 1 0 - - 0 . 7 5 0 . 0 0 0 . 0 0

1 0 9 1 0 + - 0 . 7 5 2 . 0 0 0 . 0 0

7 1 0 1 - 0 + 0 . 0 0 1 . 0 0 0 . 0 6

1 3 1 1 1 0 0 0 0 . 7 5 1 . 0 0 0 . 0 3

1 1 1 2 1 0 - + 0 . 7 5 0 . 0 0 0 . 0 6

3 1 3 1 - + 0 0 . 0 0 2 . 0 0 0 . 0 3

2 1 4 1 + - 0 1 . 5 0 0 . 0 0 0 . 0 3

8 1 5 1 + 0 + 1 . 5 0 1 . 0 0 0 . 0 6

3.13. Determinat ion of dependent variables

3 .13.1 . Sensory tes t ing

The attributes of appearance, taste, flavor, texture (in mouth), and overall

acceptability were evaluated by the panel using 9-point Hedonic scale as described in

3.8.1.

3.13.2. Moisture content

Moisture changes in food bars during storage were determined by the

standard air-oven method and results were calculated on dry basis (AOAC,

1990).

3.13.3. Color measurement

The color of food bars at different storage intervals was measured according to

method described by Rocha and Morais (2003) with a hand held tristimulus

reflectance color meter (PCM/PSM model, Color-Tec, 28 Center Street, Clinton, NJ,

USA).

Three replicates of bars were used for each storage time. The color was recorded

using a CIE–L*a*b* uniform color space (–Lab), where L* indicates lightness, a*

indicates chromaticity on a green (-) to red (+) axis, and b* chromaticity on a blue (-)

to yellow (+) axis. Numerical values of a* and b* were converted into hue angle and

chroma value (Francis, 1980). The L* value is a useful indicator of darkening during

storage resulting from oxidative reactions.

3.13.3.1. Hue angles

…………Eq. 21

The H° is an angle in a color wheel of 360°, with 0°, 90°, 180° and 270° representing

the hues red-purple, yellow, respectively. It was derived as the arctangent of the ratio

of CIE a* to CIE b* expressed as degrees (Nielsen, 1998).

3.13.3.2. Chroma values

The chroma value is the intensity or purity of the hue. It was calculated as the square

root of the sum of the squared values of both CIE a* and CIE b* (Nielsen, 1998).

3.13.4. Product slurry pH

Slurry of 5 g sample was prepared in 4 ml distilled deionized water. Product slurry pH

was determined with a pH meter standardized with a buffer at pH 7.01.

3.13.5. Free fatty acid determination

Free fatty acids were determined by AOAC procedure (2000).

3.13.5.1. Procedure

Extracted oil (5.0 g) was weighed into a 125 ml Erlenmeyer flask. 50 ml denatured

alcohol (95%) and 3 drops of phenolphthalein indicator were added in the flask. The

suspension was titrated against 0.1N NaOH until permanent faint pink color. The free

fatty acids are calculated as oleic acid using the following equation:

1 ml 0.1N NaOH = 0.028 g oleic acid

3.13.6. Peroxide value determination

Food bar samples were analyzed for peroxide values using the procedure described in

AOAC (2000) with certain modifications.

3.13.6.1. Procedure

Extracted oil (5.0 g) was weighed and 30 ml (3:2) HOAc and CHCl3 solution were

added and mixed until dissolved. 0.5 ml saturated KI solution was added and the

mixture was shaken for l min. 30 ml distilled water and 0.5 ml 1% starch solution

were added before titration to simplify the final determination. The sample was then

titrated against 0.01N Na2S2O3 until the blue color disappeared. Peroxide values were

determined using the equation:

PV = S x N x 1000 x M-1

Where, S is the volume of Na2S2O3 used for titration (ml), N is the normality of

Na2S2O3 solution, M is the mass of the bar sample (g), and PV is the mequiv

peroxide/kg sample. (AOAC, 2000)

3.14. STATISTICAL ANALYSIS

Results were statistically processed using analysis of variance technique. The

difference in means was evaluated by the Duncan Multiple Range test and Bartlett’s

test (Steel et al., 1997). The design of experiment and regression analysis was

performed for Response Surface Methodology (Montgomery, 2001) using

STATISTICA ver. 6.0 (StatSoft Inc. Tulse, Oklahama, USA). The data collected

during shelf life study was subjected to shelf life optimization by Minitab (ver. 14)

program (Minitab Inc. Quality Plaza, 1829 Pine Hall Rd. State College. PA. 16801.

United States).

Chapter-4

Results

&

Discussion

The results have been discussed according to the following phases of study:-

1. Effect of processing techniques on the nutritional quality of legumes.

a. Proximate composition of legumes

b. Antinutritional factors in legumes

2. Effect of processing on minerals bioavailability in legume seeds

3. Evaluation of protein quality of legume flour supplemented food bar meals by

the protein efficacy studies.

a. Biological evaluation studies of legume flour supplemented food bar

meals

b. Protein Digestibility-Corrected Amino Acid Score (PDCAAS) method

4. Assessment of suitability of chickpea or Indian vetch flours supplementation

for development of nutrient dense prototype food bars by applying physico-

chemical and sensory tests.

5. Screening out the natural antioxidant extracts from food processing waste

material for their activity.

6. Establishing a set of optimum antioxidant extract combinations for obtaining

bars with better sensory acceptability and shelf stability using Response

Surface Methodology (RSM).

4.1. EFFECT OF PROCESSING TECHNIQUES ON THE NUTRITIONAL

QUALITY OF LEGUMES

4.1.1. Objective

Legumes are considered as the suitable candidate for complementation to cereals.

These are proven as a rich source of major food components especially protein. The

processing of legume flours through different techniques such as soaking,

autoclaving, fermentation and germination, may alter these constituents. Present study

was conducted to evaluate the impact of processing methods on major nutritional

components of raw or processed chickpea and Indian vetch.

4.1.2. Results

4.1.2.1. Moisture

Moisture level determination is an integral part of the proximate composition

analysis of the foods. Moisture content in legume flour determines its stability. Mean

sum of squares and means regarding moisture content in raw and processed flours of

Indian vetch and chickpea have been presented in Tables 4.1 and 4.2, respectively.

Results show highly significant impact (P<0.05) of processing treatments on moisture

level.

Data revealed that the highest level of moisture was present in raw Indian vetch and

chickpea flour samples (7.46+0.10 and 6.42+0.08%, respectively). Whereas in the

controlled fermented Indian vetch and chickpea flour samples, minimum moisture

(5.00+0.27 and 4.73+0.17%) was observed, respectively. Natural fermented Indian

vetch flour contained the moisture level at par to the controlled fermented flour.

However, in chickpea, both fermented legume samples differed significantly (P<0.05)

from each other for their moisture contents.

4.1.2.2. Ash

Effect of processing treatments on ash content of chickpea and Indian vetch is

presented in Tables 4.4. Mean sum of squares for ash content are given in Table 4.3.

The statistical analysis of ash content in raw and processed legumes shows

significant difference (P<0.05) among mean values. In Indian vetch flour, ash

content after

Table 4.1: Mean sum of squares of the effect of processing treatments on

moisture content of chickpea and Indian vetch

SOV df Indian vetch

(Lathyrus sativus)

Chickpea

(Cicer arietinum)

Processing treatments 5 2.39** 1.00**

Error 12 0.118 0.059

Total 17

** =Highly significant (P<0.01)

Table 4.2: Effect of processing treatments on moisture content of chickpea and Indian vetch

Processing treatments Indian vetch‡*

(Lathyrus sativus)

Chickpea‡*

(Cicer arietinum)

Raw 7.46+0.10a 6.42+0.08a

Soaking 6.51+0.25b 5.76+0.10b

Autoclaving 6.75+0.17b 5.55+0.14bc

Controlled fermentation 5.00+0.27c 4.73+0.17d

Natural fermentation 5.45+0.09c 5.33+0.10bc

Germination 6.27+0.24b 5.12+0.20cd

‡ g/100g, estimated in dehulled flour + SE (Standard Error)

*Any two means not sharing a letter in a column differ significantly at P<0.05 (LSD) (n=3)

Table 4.3: Mean sum of squares of the effect of processing treatments on ash content of chickpea and Indian vetch

SOV df Indian vetch

(Lathyrus sativus)

Chickpea

(Cicer arietinum)


Error 12 0.005 0.007

Total 17


Table 4.4: Effect of processing treatments on ash content of chickpea and Indian vetch


(Lathyrus sativus)

Chickpea‡*

(Cicer arietinum)

Raw 1.38+0.01a 1.43+0.08a

Soaking 1.02+0.04b 1.12+0.03b

Autoclaving 1.23+0.02ab 1.36+0.03a

Controlled fermentation 1.35+0.06ab 1.41+0.03a

Natural fermentation 1.28+0.02ab 1.41+0.01a

Germination 1.35+0.06ab 1.29+0.06a

‡ g/100g on dry wt basis, estimated in dehulled flour + SE (Standard Error)


various processing treatments matched to each other and their ash values ranged

between raw and soaking treatments (1.38 and 1.02 g/100g, respectively). Ash

content of processed chickpea samples did not vary to a considerable extent from

raw flour, with exception of soaking treatment which deviated from this trend.

4.1.2.3. Crude fat

Mean values for crude fat in raw and processed chickpea and Indian vetch samples

are given in Table 4.6. Statistical analysis of data is shown in Table 4.5. Maximum

fat level was observed in the unprocessed Indian vetch flour (1.08±0.01 g/100g).

Soaking and autoclaving caused non significant change (1.05±0.01 and 1.02±0.03

g/100g, respectively). Change in the fat content by controlled fermentation was

significant (P<0.05) but at par to variation caused by germination. Natural

fermentation depicted higher decrease in fat level (19%). Similar kind of change

was observed in fat values of raw and processed chickpea flours. The higher

reduction in fat content (13%) was noted in natural fermented chickpea flour

samples. However this decrease was 6% percentile points lower than Indian vetch

case.

4.1.2.4. Crude protein

The data for crude protein in raw and processed Indian vetch and chickpea flour are

given in Table 4.8. This data underwent the statistical analysis and results are

depicted in Table 4.7. Crude protein content of unprocessed Indian vetch was the

highest among all samples (29.68±0.68 g/100g). Processing of Indian vetch caused

slight reduction in crude protein level. Natural fermented Indian vetch contained the

lowest quantity of crude protein (27.75±0.27 g/100g) among other processing

treatments. The results for germinated Indian vetch flour were at par to natural

fermented flour. However, no change in crude protein content was observed for raw

and processed chickpea flours.

4.1.2.5. Crude fiber

The data regarding effect of processing treatments on crude fiber of chickpea and

Indian vetch is shown in Tables 4.10. Mean sum of squares for crude fiber are given

in Table 4.9. The crude fiber content analyzed in unprocessed Indian vetch and

chickpea were 0.98±0.05 and 1.09 ±0.02 g/100g, respectively. Processing caused no

Table 4.5: Mean sum of squares of the effect of processing treatments on crude

fat content of chickpea and Indian vetch

SOV df Indian vetch

(Lathyrus sativus)

Chickpea

(Cicer arietinum)

Processing treatments 5 0.016** 0.010*

Error 12 0.002 0.002

Total 17


* =Significant (P<0.05)

Table 4.6: Effect of processing treatments on crude fat content of chickpea and Indian vetch


(Lathyrus sativus)

Chickpea‡*

(Cicer arietinum)

Raw 1.08+0.01a 4.21+0.01a

Soaking 1.05+0.01a 4.14+0.01abc

Autoclaving 1.02+0.03ab 4.17+0.03ab

Controlled fermentation 0.96+0.03bc 4.06+0.04bc

Natural fermentation 0.88+0.05c 3.94+0.04c

Germination 1.03+0.01ab 4.18+0.03abc



Table 4.7: Mean sum of squares of the effect of processing treatments on protein

content of chickpea and Indian vetch

SOV df Indian vetch

(Lathyrus sativus)

Chickpea

(Cicer arietinum)

Processing treatments 5 1.69* 0.17 NS

Error 12 0.70 0.54

Total 17

* =Significant (P<0.05) NS =Non significant (P>0.05)

Table 4.8: Effect of processing treatments on protein content of chickpea and Indian vetch


(Lathyrus sativus)

Chickpea‡*

(Cicer arietinum)

Raw 29.68+0.68a 21.40+0.42

Soaking 29.17+0.53ab 21.30+0.55

Autoclaving 28.76+0.29ab 21.10+0.40

Controlled fermentation 28.11+0.65ab 21.01+0.51

Natural fermentation 27.75+0.27b 20.71+0.25

Germination 28.02+0.27b 21.11+0.33



Table 4.9: Mean sum of squares of the effect of processing treatments on crude

fiber content of chickpea and Indian vetch

SOV df Indian vetch

(Lathyrus sativus)

Chickpea

(Cicer arietinum)

Processing treatments 5 0.003NS 0.004NS

Error 12 0.002 0.004

Total 17

NS=Non significant (P>0.05)

Table 4.10: Effect of processing treatments on crude fiber content of chickpea and Indian vetch


(Lathyrus sativus)

Chickpea‡*

(Cicer arietinum)

Raw 0.98+0.05 1.09+0.02

Soaking 0.94+0.02 1.08+0.07

Autoclaving 0.97+0.01 1.05+0.01

Controlled fermentation 0.92+0.02 1.01+0.05

Natural fermentation 0.94+0.01 1.03+0.01

Germination 0.89+0.02 1.01+0.03



variation to this level. In Indian vetch the crude fiber content in processed flours

ranged from 0.89 - 0.94 g/100g. This change was analyzed to be non significant

(P>0.05). On the similar lines, processed chickpea contained 1.01 to 1.08 g/100g

crude fiber.

4.1.2.6. Nitrogen free extract (NFE)

Mean sum of squares and means regarding NFE content in raw and processed flours

of Indian vetch and chickpea have been presented in Tables 4.11 and 4.12,

respectively. The NFE values in Indian vetch were calculated to be high in both

controlled and natural fermented and germinated flours (68.7, 69.1 and 68.9 g/100g,

respectively). Unprocessed Indian vetch flour sample possessed low level of NFE

(66.9±0.79 g/100g). Whereas NFE values for soaked and autoclaved Indian vetch

flours were at par to each other. The NFE content changed non-significantly

(P>0.05) in chickpea. The results for NFE in raw and processed chickpea flour

samples ranged from 74.9 to 75.7 g/100g. The minimum level among these was

attained by unprocessed chickpea flour sample.

4.1.3. Discussion

Moisture content of the decorticated Indian vetch and chickpea flours varied slightly

significantly (P>0.05). All the treatments except raw grains were subjected to

drying operation in order to prepare flour for later use in bar making. This drying

resulted in the flours with moisture content even less than the original raw flour.

Achieving dry state of flours is of utmost importance for their shelf stability. At low

moisture levels, the flours possessed low water activity hindering any microbial

growth. Moreover, dry flours are devoid of moisture required for the spore growth

and physiological activity.

It was noted that moisture level below 8% could be called as strongly bound water

and becomes unavailable for even seed respiration. However, the damage due to

imbibing water becomes high at this moisture level (Vertucci and Leopold, 1984).

The moisture levels studied in the Indian vetch and chickpea flours were similar to

that found in Siclian genotypes (6.47-7.53%) (Patane, 2006) and new Indian

varieties of chickpea (5.62-8.17%) (Singhai and Shrivastava, 2006).

Table 4.11: Mean sum of squares of the effect of processing treatments on nitrogen fee extract (NFE) of chickpea and Indian vetch

SOV df Indian vetch

(Lathyrus sativus)

Chickpea

(Cicer arietinum)

Processing treatments 5 8.13* 1.92NS

Error 12 1.77 1.87

Total 17

NS=Non significant (P>0.05)

*= Significant (P<0.05)

Table 4.12: Effect of processing treatments on nitrogen fee extract (NFE) of chickpea and Indian vetch


(Lathyrus sativus)

Chickpea‡*

(Cicer arietinum)

Raw 66.9+0.79b 74.9+0.65

Soaking 67.8+0.98ab 75.4+1.27

Autoclaving 68.0+0.23ab 75.4+0.82

Controlled fermentation 68.7+0.73a 75.4+0.42

Natural fermentation 69.1+1.06a 75.7+0.82

Germination 68.9+0.50a 75.5+0.40



The data shows that ash content of the soaked samples were the lowest among all

treated flours. The leaching of minerals from the seeds during soaking could be the

reason. Ash content in the decorticated raw chickpea flour samples is found lower

than the reported values of ash in chickpea cultivars. It ranged from 2.50-3.06

g/100g in Indian varieties (Singhai and Shrivastava, 2006) and 2.27-2.93 g/100g in

Siclian genotypes (Patane, 2006). Whereas, the ash content in decorticated Indian

vetch flour also followed the pattern of possessing lesser amount of ash content than

reported in literature i.e. 2.9 g/100g (Kuo et al., 1995), 2.7 g/100g (Hanbury et al.,

2000), 3.5 g/100g (Ramachandran et al., 2005) and 3.43 g/100g (Rehman et al.,

2008).

The outer seed coat or testa is known as the site of mineral concentration in grains.

Decrease in levels of ash in the raw and processed legume samples during this study

could be due to the decortication of legumes during their processing to subsequent

use in making food bars. It was observed that the legumes contained a large portion

of water soluble ash which has the tendency to leach out during hydro processing of

seeds. In gram seed (Cicer arietinum), the ratio of water soluble to insoluble ash

was 1.94:1.00 (Singhai and Shrivartava, 2006). Indian vetch and chickpea contain a

notably varied percentage of crude fat content. Indian vetch possesses rather low

level of crude fat. It is also true for most of the legumes e.g. broad bean, common

bean, field pea, garden pea and lentil which contained 2.99, 2.19, 3.19, 3.13 and

3.16 g/100g of ether extract, respectively (Grela and Gunter, 1995). However, crude

fat content ranged between 5.12 to 8.57 g/100g in some chickpea cultivars with the

mean value 6.0±0.9 g/100g (Patane 2006). The crude fat content in chickpea was

somewhat lower than these values but are found in agreement to the fat content

determined in Indian chickpea varieties (4.18-4.92 g/100g) (Singhai and

Shrivartava, 2006). The fat present in chickpea is rich in essential fatty acids i.e.

linolenic and linoleic acids, that exhibits hypocholesterolemic effect on

consumption by human (Singh, 1988). Indian vetch has been reported to contain low

level (0.7%) of fat (Dhiman et al., 1983), 0.8% (Low et al., 1990), 1.2% (Kuo et al.,

1995), 1.31% (Rehman et al., 2007) and 1.35% (Rehman et al., 2006). Crude fat

levels studied in this study were in close proximity to these findings.

Autoclaving and soaking processes have negligible effect on crude fat content of the

legume flours (Gupta et al., 1987). Fat level remained unchanged also during

germination. Similar observations were made by Torres et al. (2007) during

germination of pigeon pea seeds. As far as fermentation effect on crude fat of

legume samples is concerned, natural and controlled fermentation behaved

differently. Crude fat levels of the controlled fermented flours of Indian vetch and

chickpea remained unchanged but a significant reduction (P<0.05) in crude fat

content was noted in natural fermented legume flours. The bacterial growth could be

responsible for this effect because bacterial species contain lipase activity at

variable levels. During natural fermentation of legumes, Staphylococcus species

produce notably high content of extra cellular enzyme lipase (Odunfa and

Komolafe, 1989). It has been noted that lipase activity of Lactobacillus helveticus is

limited (Torriani et al., 1994). When adjunct cultures of different bacterial species

were compared for lipase activity in reduced fat Edam cheese, the Lactobacillus

helveticus culture showed low lipase activity (Tungjaroenchai et al., 2004). The

lypolytic ability of Lactobacillus cultures is considered even lower than natural

cultures. It was substantiated in a study where ether extractable fat contents

determined after Latobacillus plantarum and Lactobacillus fermentum fermentations

were 5.49±0.02 and 4.71±0.01%, respectively which was found to reduce to

3.48±0.001% in natural fermented maize cowpea ogi (Oyarekua et al., 2008).

The legumes under study (Indian vetch and chickpea) contain quite different protein

content. Chickpea contains lowest protein content among legumes (Sotelo et al.,

1987). Other legumes possess higher levels of protein such as garden pea (24.28%),

common bean (26.48%), field pea (27.5%), horse bean (29.34%) and lentil (30.25%)

(Grela and Gunter, 1995). The protein content in Siclian genotypes of chickpea was

studied to range between 18.6 to 20.5 g/100g with an average of 19.5 g/100g

(Patane, 2006). However, the protein content of chickpea cultivars grown in

Pakistan ranges between 22.89% and 24.82% (Iqbal et al., 2006). In Indian

cultivars, protein level was slightly lower than Pakistani cultivars and fluctuated

between 19.68 to 22.75 g/100g (Singhai and Shrviastava, 2006).

Although, Indian vetch is an underutilized crop but contains a high proportion of

protein. Its protein level is one of the best among the members of family

leguminosae. Only lupin and soybean are considered as truly having higher protein

content (35.1 and 37.64%, respectively) than Indian vetch (Hanburry et al., 2000;

Grela and Gunter, 1995). The Indian vetch was analyzed to contain 29.68% protein

in the present study. This value is slightly lower than reported by Kuo et al. (1995)

(35.9% protein), however, higher than reported by some other authors (Dhiman et

al., 1983; Low et al., 1990; Rehman et al., 2006; Rehman et al., 2007). They

reported protein level ranging from 25.6% to 28.32%, on dry weight basis.

Processing has the effect of variable nature on the protein content of legume seeds

or flours. Soaking and autoclaving have no effect on the protein concentration of

chickpea or Indian vetch. It has been supported by the Gupta et al. (1988).

Germination caused a slight decrease in crude protein content of Indian vetch and

chickpea but the effect was non-significant (P>0.05). It was noted that proteolysis

occured during sprouting which could result in an increase in non-protein nitrogen

and free amino acids (Sangronis et al., 2004). However, analytical method

employed in this study relies on determination of total nitrogen for protein

calculation (AOAC, 2000). Thus, the change in the form of nitrogen by breaking

peptide bonds does not affect the crude protein level (Torres et al., 2007).

Controlled and natural fermentation reduced the protein content significantly

(P<0.05). The reduction of protein level after natural fermentation was also noticed

in bean (Granto et al., 2002) and cowpea (Granito et al., 2005), whereas, in cowpea

no change in protein level has been reported (Akinyele and Akinlosotu, 1999).

Reduction in protein content due to fermentation could be attributed to proteolysis

that results in the production of volatile ammonia which is a characteristic of

fermentation of protein rich foods (Diawara et al., 1998). The ammonia production

is common feature of fermentation of some African proteinous foods i.e. davadava,

soumbala and netetu (Omafuvbe et al., 2000; Beaumont, 2002). The loss of nitrogen

into environment in the form of ammonia partially reduces protein content. Other

reason could be the metabolism of amino acids into organic acids, alcohols and

aldehydes (Feron and Wache, 2006).

Legumes are among the richest sources of crude fiber in human diet (Heller and

Hackler, 1978). The crude fiber content is noticeably high in various legumes i.e.

common bean (4.82%), broad bean (8.51%), field pea (4.86%), garden pea (5.09%),

horse bean (8.94%), kidney bean (4.64%) and lentil (4.17%) (Grela and Gunter,

1995). Chickpea cultivars possess high crude fiber level with a wide variability. In a

Sicilan genotypes of chickpea, it was noted as high as 5.05 g/100g (Patane, 2006).

Similarly, crude fiber data has shown inconsistency during different studies. The

level of crude fiber in chickpea ranged between 5.3-5.9% (Dhiman et al., 1983; Low

et al., 1990; Kuo et al., 1995). However, type of chickpea affected level of crude

fiber. In the Kabuli chickpea, it was estimated as 5.9 g/100g, whereas in desi

chickpea having thick coat, its concentration increased to 8 g/100g (Khalil et al.,

2007). Tthe crude fiber content in some Indian chickpea desi cultivars was noted to

vary between 7.8±0.82 and 12.7±0.92%. However, kabuli cultivar possessed 4.6%

crude fiber only (Kaur et al., 2005). It is obvious that crude fiber content depends on

thickness of seed coat, because it is present principally in the outer seed testa (Grela

and Gunter, 1995). For technological application, legumes with thinner seed coat are

preferred (Singh, 1985). Other alterative could be removal of hull through some

processing techniques. However, some new hybrid varieties of Cicer aritinum were

analyzed to contain 0.26-1.43 g/100g crude fiber with an average of 0.90 g/100g

(Singha and Shrivatama, 2006). In the present study, chickpea and Indian vetch

seeds decorticated at 10% level were found to possess 1.09±0.02% and 0.98±0.05%

crude fiber respectively. This reduction in crude fiber content makes these flours

technologically more valuable for use in food products.

Germination was found to affect crude fiber content of neither chickpea nor Indian

vetch. Khalil et al. (2007) observed similar results in the sprouted chickpea seeds.

The natural fermentation process committed a marginal reduction in crude fiber

level of legume flours. These results were found to be in close proximity to the

findings of Ramachandran et al. (2005) who observed reduction in crude fiber in

Indian vetch as a result of fermentation. Moreover, there was no change observed in

crude fiber during controlled fermentation. The variation in the fermentation effect

on crude fiber may be attributed to the ability of involved microorganisms to react

with crude fiber. It is known that crude fiber by definition is resistant to extraction

by mild acid (0.225N HCl). Moreover, microbes employed for fermentation, contain

different plasmids associated with digestion of substrate. There is a direct

relationship between crude fiber present in a substrate and fermentation capacity of

microbe. Lesser is the crude fiber, better performance of fermentation process

(Ponnampalam et al., 2004). In the present study controlled fermentation has

exhibited poor performance to alter crude fiber level in legume flour substrate.

Carbohydrates are considered as substances that remain after protein, ash, crude

fiber and ether extract have been determined and are also called as nitrogen free

extract (NFE). These are rich source of energy for living organisms. NFE content

varies widely among different legumes which depend upon variation in other major

nutrients. Variation in crude protein and fat contents in legumes affects NFE to a

large extent. Particularly, it was true in soybean that contained 37.6% protein and

22.9% fat, having only 18.2% NFE (Grela and Gunter, 1995). Pentaclethra

morcophylla rich in crude protein (33.7%) and crude fat (51.50%) contains only

3.66% NFE. Similarly, Afzelia africana a tropical African legume with 37.15%

crude fat owe only 32.3% NFE (Ajah and Madubuike, 1997).

Among popular legumes, NFE is abundantly found (68.63% and 68.46%,

respectively) in kidney bean and garden pea due to their low protein levels (<22%)

(Grela and Gunter, 1995). Same is the case of chickpea which holds low level of

protein (21.4%). Thus, NFE determined in this study was high for chickpea (68.5%).

NFE content reported in this study are even better than those reported in Siclian

genotypes of chickpea (62.4%) (Patane et al., 2006). On the other hand, NFE value

reported for Indian vetch is slightly low (59.4%) mainly due to the presence of high

content of protein (29.68%). This value is at par to the NFE content in Indian vetch

reported by various researchers (57.55-61.34%) (Low et al., 1990; Kuo et al., 1995;

Rehman et al., 2007a; Rehman et al., 2007b).

Soaking and autoclaving have insignificantly affected NFE value in both legumes.

Whereas, NFE level altered to a modest degree in rest of the processing treatments

applied to Indian vetch. It has been reported that a number of changes may take

place in grains during fermentation or germination process. For instance during

natural fermentation, starch has been hydrolyzed into maltose due to α-amylase

activity of microorganisms for catering their energy needs. Similarly, soluble dietary

fiber is also partly utilized during the process (Granito et al., 2005). It was noted

that disaccharides i.e. sucrose and fructose, decreased as a result of fermentation

process (Granito et al., 2002). Germination process might also carry α-amylase

activity (Torres et al., 2007). Such activity could cause slight variation in available

as well as insoluble or resistant fractions of carbohydrate. In germination process,

soluble sugars mount as a result of oligosaccharide hydrolysis during sprouting

(Dey, 1985). Thus, although form of sugar changes but total carbohydrate level

remains unchanged. Despite the variations in carbohydrate level of legumes

suggested by earlier researchers due to fermentation and germination process, the

results obtained in this study sketch different picture. The reason is quite obvious as

computation of NFE is dependent upon quantity of other constituents in a sample. It

resulted in high level of NFE in fermented and germinated Indian vetch flours even

greater than raw samples.

4.1.4. Conclusion

Under the influence of processing all proximate composition parameters of Indian

vetch except crude fiber content altered significantly (P<0.05). However, in chickpea,

NFE did not vary also. It is important to note that during these processing treatments,

protein, the main focus point of the study remained unchanged.

4.2. EFFECT OF PROCESSING ON ANTINUTRITIONAL FACTORS OF

LEGUMES

4.2.1. Objective

Despite the presence of rich amount of protein, the presence of antinutritional factors

i.e. proteolytic inhibitors, phytates, polyphenols and lathyrogens affect the nutritional

quality of a legume. In this study, different food processing technologies including

fermentation and germination were employed as a strategy to overcome problem of

antinutritional factors in legumes. Major antinutritional factors e.g. trypsin inhibitors,

tannin, total phenols, phytates and β-ODAP in raw and processed chickpea and Indian

vetch samples were analyzed to determine the effect of processing treatments.

4.2.2. Results

4.2.2.1. Tannin content

The mean values for the effect of processing treatments on tannin are presented in

Table 4.14. The analysis of variance of the data (Table 4.13) shows the highly

significant differences (P<0.05) for tannin content of legumes (Indian vetch and

chickpea) as result of effect of processing methods.

In Indian vetch (Lathyrus sativus) best reduction in tannin content was recorded for

controlled fermentation (22%) closely followed by natural fermentation (18%) and

germination (15%). Raw Indian vetch sample possessed the maximum concentration

of tannin (0.822 g/100g). Soaking and autoclaving altered the tannin level to a little

extent. Similar pattern of results was followed by chickpea processing. However, in

chickpeas, natural fermentation decreased the tannin to a matchable extent with

controlled fermentation, both reduced tannin 30% and 28%, respectively.

Germination was the second best treatment, contributing 15% reduction of tannin.

4.2.2.2. Total phenols

The data for processing treatments effect on total phenolic content in Indian vetch

and chickpea is given in Table 4.16. The results were analyzed statistically through

analysis of variance technique (Table 4.15).

Table 4.13: Mean sum of squares of the effect of processing treatments on tannin content of Indian vetch and chickpea

SOV df Indian vetch

(Lathyrus sativus)

Chickpea

(Cicer arietinum)


Error 12 0.0012 0.0007

Total 17


Table 4.14: Effect of processing treatments on tannin content of Indian vetch and chickpea


(Lathyrus sativus)

Chickpea‡*

(Cicer arietinum)

Raw 0.822+0.019a 0.713+0.025a

Soaking 0.719+0.013bc 0.638+0.006bc

Autoclaving 0.769+0.006ab 0.676+0.016ab

Controlled fermentation 0.639+0.017d 0.502+0.008d

Natural fermentation 0.673+0.021cd 0.512+0.018d

Germination 0.694+0.032cd 0.607+0.012c

‡g/100g on dry wt. basis, estimated in dehulled flour + SE (Standard Error)


Table 4.15: Mean sum of squares of the effect of processing treatments on total phenol content of Indian vetch and chickpea

SOV df Indian vetch

(Lathyrus sativus)

Chickpea

(Cicer arietinum)


Error 12 0.005 0.005

Total 17


Table 4.16: Effect of processing treatments on total phenol content of Indian vetch and chickpea


(Lathyrus sativus)

Chickpea‡*

(Cicer arietinum)

Raw 2.12+0.02a 1.97+0.02a

Soaking 1.74+0.01c 1.59+0.06b

Autoclaving 1.88+0.08b 1.68+0.13b

Controlled fermentation 0.95+0.04e 0.76+0.02d

Natural fermentation 1.23+0.03d 0.98+0.09c

Germination 1.17+0.02d 0.91+0.05c

‡g/100g on dry wt. basis, estimated in dehulled flour + SE (Standard Error)


The total phenolic contents of two legumes i.e. Indian vetch and chickpea were

considerably affected by processing treatments. Indian vetch flour contained higher

amount of total phenols (2.12±0.02 g/100g) than chickpea flour (1.97±0.002

g/100g). After processing, total phenol in Indian vetch was reduced at variable level.

Soaking the Indian vetch seeds for 9 hours reduced total phenols (18%) better than

dry autoclaving (11%). The best decline in total phenolic content among all

processing treatments was observed in controlled fermentation (55%). Total phenol

determined in control fermented sample was 0.95±0.004 g/100g. Germination

processing produced next best results i.e. 45% reduction in total phenols closely

followed by natural fermentation showing 42% decline.

Chickpea processing showed similar trend in total phenol reduction (Table 4.16).

However, soaking and autoclaving treatments did not differed significantly (P>0.05)

from each other for total phenol reduction tendency (19 and 15%, respectively). On

the same pattern, natural fermentation and germination processing were close to

each other in their ability to reduce total phenols in chickpea (50 and 54%,

respectively). Controlled fermentation produced better decline (61%) in phenolics in

chickpea. It exhibited even better ability to reduce total phenol in chickpea than

Indian vetch (55%).

4.2.2.3. Trypsin inhibitor activity (TIA)

Trypsin inhibitor contents in raw and treated legume (Indian vetch and chickpea)

seeds are presented in Table 4.18. Results of statistical analysis of the data are given

in Table 4.17.

Indian vetch flour underwent a great reduction in trypsin inhibitor activity (TIA)

under the influence of processing techniques. Raw dehulled flour carried

13.09±0.417 TIU/g on dry weight basis. It was not affected notably by soaking

(P>0.05). However, autoclaving at 121oC and 15 lb pressure for 15 min caused a

significant reduction (P<0.05) in TIA (51%). This TIA reducing effect was further

enhanced to 80% when the autoclaved Indian vetch flour was subjected to

controlled fermentation. Thus, a net 29% further decrease in TIA could be

attributed to controlled fermentation. Natural fermentation decreased TIA in the

tone of 38%. Although, germination substantially eliminated the TIA of Indian

vetch flour,

Table 4.17: Mean sum of squares of the effect of processing treatments on trypsin inhibitor activity of Indian vetch and chickpea

SOV df Indian vetch

(Lathyrus sativus)

Chickpea

(Cicer arietinum)

Processing treatments 5 48.46** 19717**

Error 12 0.493 114

Total 17


Table 4.18: Effect of processing treatments on trypsin inhibitor activity of Indian vetch and chickpea


(Lathyrus sativus)

Chickpea‡*

(Cicer arietinum)

Raw 13.09+0.42a 280.2+3.44a

Soaking 12.68+0.69a 262.5+1.85a

Autoclaving 6.46+0.21d 174.8+3.54c

Controlled fermentation 2.56+0.07e 58.2+1.89d

Natural fermentation 8.11+0.30c 163.8+3.19c

Germination 10.37+0.45b 225.9+4.30b

‡ TIU/g on dry wt. basis, estimated in dehulled flour + SE (Standard Error)


however, its TIA reduction was not that good in comparison to other processing

techniques. Raw chickpea flour contains 21 fold high level of TIA in comparison to

raw Indian vetch samples. Whereas controlled fermentation processing of both

flours widened this gap to 22.5 fold.

Chickpea flour followed the TIA reduction pattern almost similar to that of Indian

vetch but with a little difference as in chickpea, the net decrease of TIA after both

types of fermentation was in close proximity. After controlled and natural

fermentation processing, 41 and 42% reduction was noted respectively.

4.2.2.4. Phytic acid

The results for effect of processing treatments on phytic acid content of Indian vetch

and chickpea are given in Table 4.20. The data was subjected to analysis of variance

(Table 4.19) showing considerable differences among the phytic acid content of raw

and processed legume flours (Indian vetch and chickpea).

In their raw forms, both legumes hold quite variable levels of phytic acid i.e.

734.2+30.95 mg/100g in Indian vetch and 171.3+2.34 in chickpea. High phytate level

in these legumes demands considerable attention thus it may be reduced

significantly.

The controlled fermentation decreased phytic acid down to a significantly low level

(357 mg/100g) as compared to germination and natural fermentation processing in

Indian vetch. The mean values show that the controlled fermentation imparted 51%

reduction, while germination and natural fermentation caused 43 and 38%

reduction, respectively in phytic acid contents of Indian vetch. Soaking and

autoclaving resulted in non significant reduction of phytate (P>0.05).

Similarly, in chickpea, maximum reduction in phytate was observed in controlled

fermentation (59%) followed by germination and natural fermentation (38% and

34%, respectively). There was a slight reduction in phytate content observed in the

autoclaving treatment in which about 9% decrease was noted in phytate content than

the original sample.

4.2.2.5. β-ODAP

The mean values for the effect of different processing treatments on the β-ODAP

Table 4.19: Mean sum of squares of the effect of processing treatments on phytic

acid content of Indian vetch and chickpea

SOV df Indian vetch

(Lathyrus sativus)

Chickpea

(Cicer arietinum)

Processing treatments 5 48.46** 19717**

Error 12 0.493 114

Total 17


Table 4.20: Effect of processing treatments on phytic acid content of Indian vetch and chickpea


(Lathyrus sativus)

Chickpea‡*

(Cicer arietinum)

Raw 734.2+30.95a 171.3+2.338a

Soaking 723.6+5.040a 167.5+4.313ab

Autoclaving 698.1+29.29a 156.3+2.823b

Controlled fermentation 357.6+4.930c 69.5+2.182d

Natural fermentation 456.8+17.40b 112.9+6.357c

Germination 418.5+11.11b 106.6+3.262c

‡ g/100g on dry wt. basis, estimated in dehulled flour + SE (Standard Error)


Table 4.21: Mean sum of squares of the effect of processing treatments on β-ODAP content of Indian vetch

SOV df Indian vetch

(Lathyrus sativus)

Chickpea

(Cicer arietinum)

Processing treatments 5 0.0082** ND

Error 12 0.0002 ND

Total 17


ND= Not detected

Table 4.22: Effect of processing treatments on β-ODAP content of Indian vetch


(Lathyrus sativus)

Chickpea‡*

(Cicer arietinum)

Raw 0.386+0.003a ND

Soaking 0.311+0.006bc ND

Autoclaving 0.335+0.009b ND

Controlled fermentation 0.241+0.012e ND

Natural fermentation 0.269+0.014de ND

Germination 0.283+0.012cd ND

‡ g/100g on dry wt. basis, estimated in dehulled flour + SE (Standard Error)


level of Indian vetch are given in Table 4.22. The statistical analysis is shown in

Table 4.21.

β-ODAP content in Indian vetch samples treated through different processing

techniques varied significantly (P<0.05). Indian vetch was initially carrying a high

level of β-ODAP (0.386±0.003 mg/100g) which reduced significantly by soaking

(0.311±0.006 mg/100g) and autoclaving (0.335±0.009 mg/100g). A higher

reduction in β-ODAP level of Indian vetch (0.145 mg/100g) was observed in

controlled fermentation conditions followed by natural fermentation and

germination processing. These two techniques reduced β-ODAP at 30 and 27%

respectively, at par to each other. As β-ODAP presence is a matter of great concern

in Indian vetch, as it specifically exists in this legume. It was not detected in

chickpea.

4.2.3. Discussion

Tannin is present abundantly in the legume seeds. These are produced by

condensation of simple phenolics and are secondary metabolites having variety of

molecular structures and are generally divided into hydrolysable and condensed

proanthocyanidins (polymers of flavon-3-ols). These are specifically present in the

outer coat of the seeds (Haslam, 1989).

The tannin concentration varies among under utilized food legumes i.e. 1.56-1.70

g/100g in Mucuna purines (valvet bean) seeds accessions (Ezeagu, 2002) and 2.35

g/100g in Bauhinia purpurea (Vijayakumar, 2007). Beachpea contains higher

amount of condensed tannin (11.6 g/100g) than greenpea (72.0 mg/100g), Canadian

grass pea (109 mg/100g) and Indian grasspea (1.54 g/100g) (Chavan et al., 2001). In

cowpea accessions, tannins ranged from 0.12 to 2.38% (Asante et al., 2004).

Process variables and microorganisms play a vital role in the reduction of tannins.

During this study, mixed culture of Lactoabacillus helveticus and Streptococcus

thermophillus decreased the tannin to a maximum extent. These results were in line

to earlier findings of Moreno et al. (2004) who observed that optimum fermentation

time and temperature are quite essential to obtain maximum tannin reduction in

chickpea. They also confirmed the ability of a starter, Rhizopus oligosporus to

decrease the tannin during solid state fermentation of chickpea tempeh flour.

Various studies have reported the reduction in tannin content of legumes during

soaking (Vijayakumari et al., 1996; Vijayakumari et al., 2007). However, in present

study, soaking was taken as the preparatory step of germination process. Hence,

soaking conditions were not as much rigorous to cause high reduction. The tannin

decrease during soaking might be attributed to leaching into soaking medium

(Vijayakumari, 1997).

The formation of insoluble complexes between tannin and proteins makes them

unavailable for extraction during analysis (Uzogara et al., 1990). Autoclaving of wet

substrate usually shows increase in tannin content due to degradation of protein

tannin interactions. However, dry autoclaving of legume flours did not owe such

ability.

Germination of Indian vetch (Lathyrus sativus) and chickpea (Cicer arietenum)

reduced the tannin not better than fermentation processes. Tannin destruction during

germination is usually attributed to enzymatic hydrolysis by polyphenolase (Reddy

et al., 1985). Germination processing of dehulled Indian vetch and chickpea

experienced a significant decrease (P<0.05) in tannin content. This reduction could

be due to the leaching out tannin from surface by soaking and wetting of seeds

during germination steps and enzymatic hydrolysis. After dehulling the cotyledon

contains only a meager amount of tannin.

Indian vetch and chickpea are legumes with dark colored seed coat, inheriting high

amount of total phenols. These are the largest group of secondary metabolites in

plant based foods and represent more than 6000 identified substances. These have

contrast physiological and functional characteristics. Their action as antioxidant is

considered as beneficial in foods, where these act as supplementary food additives

as well as living body where these are oxidized in preference to other food

constituents or cellular components and tissues (Robards et al., 1999; Yoshida et al.,

1999; Miura et al., 2001). However, these pose as anti-nutritional factors (Kroll et

al., 2003) by hindering with enzymatic digestion of food proteins and interfering in

the bioavailability of the minerals (Towo et al., 2006). The high content of total

phenolics also interferes with digestion and absorption of dietary carbohydrates and

other nutrients such as vitamin B12. These can also cause damage to the mucosa of

the digestive tract (Liener, 1994).

In the present investigations, the total phenols in decorticated Indian vetch flour

were higher when compared with a popular legume, the chickpea, processed in

similar manner (2.12±0.2 vs 1.97±0.2 g/100g). Some other underutilized legumes

also contain high content of total phenols i.e. 2.25 g/100g in Bauhinia purporea

(Vijayakumari et al., 2007), 7.7 g/100g in Mucuna pruriens var. pruriens, 6.1

g/100g in Mucuna pruriens var. utilis, 3 g/100g in Sesbania cannalina (Siddhuraju

et al., 2002; Adebowale et al., 2005; Vadivel and Janardhanan, 2005).

Soaking decreased the level of polyphenolic compounds to a significant level

(P<0.05). The loss of phenolics may be attributed to the leaching of these

substances into soaking medium from seed coat (Vijayakumari et al., 1997).

However, in this study, distilled water used as soaking medium was mostly

absorbed by the grains. Moreover, the analyses are based on the decorticated flour

samples. Therefore, in the present study polyphenols reduction percentage was not

as high as reported by other researchers (Vijayakumari et al., 1996; Vijayakumari et

al., 2007).

Autoclaving of the legumes altered the polyphenol values to a small extent. In a

similar kind of study on jackbean (Canavalia ensiformis L. DC), dry heat stood

ineffective in reducing the polyphenols (Babar et al., 1988). Whereas, a significant

reduction (P<0.05) in polyphenols in some legumes i.e. Dolichos lablab var vulgaris

(Vijayakumari et al., 1995), Lathyrus sativus (Srivastav and Khokhar, 1996) and

Vigna radiate (Kataria et al., 1989) was observed when these were subjected to

soaking followed by thermal processing.

When the water soaked seeds were subjected to germination for 36 hours, it reduced

the polyphenols significantly (P<0.05) both in Indian vetch and chickpea. The

reduction percentages of polyphenols in Indian vetch and chickpea were 45% and

54%. This decrease in polyphenols during germination may be attributed to the

polyphenol oxidaze based enzymatic hydrolysis (Jood et al., 1987). During

germination, an increase in polyphenol peroxidase (PPO) activity is noted by Rao

and Deorthale (2006). Highest PPO activity was studied for black gram and lowest

in chickpea. Seed cotyledon contributes towards almost whole of the PPO activity.

The PPO activity may be routed to reduce polyphenol content of germinating

legume seeds in this study. Germination has been found effective in reducing

polyphenol content at variable fashion in different legumes such as Lathyrus sativus

(Srivastava and Khokhar, 1996), fababean, ricebean (Saharan et al., 2002), jackbean

(Babar et al., 1988), chickpea (Muzquiz et al., 2004), peas (Alonso et al., 1998) and

mungbean (Barroga et al., 1985).

The phenolic compounds reduction was found high, in the controlled fermentation

as well as natural fermentation. A number of cultures are used to decrease

polyphenols present in substrate i.e. S. diastaticus with L fermentum (Khetarpaul

and Chauhan, 1990) in sprouted pearl millet substrate, L. casei and L. plantarum in

a food mixture substrate carrying tomato pulp and sprouted green gram paste among

ingredients (Sindhu and Khetarpaul, 2003). They were drastically reduced in

substrate with sequential culture fermentation by S. boulardii followed by L. casei

or L. plantarum. The change in sequential culture fermentation was higher than

single culture fermentation (Sindhu and Khatarpaul, 2003). The reduction in

polyphenols during fermentation has been reported by various researchers including

Antony and Chandra (1998), Bartolome et al. (1997) and Granito et al. (2002).

Contrarily solid state fermentation of black bean by GRAS fungi i.e. Aspergillus

awamori, Aspergillus oryzae, Aspergillus sojae and Rhizopus azygorporous enhance

the polyphenol content of substrate (Lee et al., 2008). However, fermentation of

sprouted pearl millet substrate by Saccharomyces diarataticus with Lactobacillus

brevis (SdLb) enhance whereas S. cerevisiae with L. bravis and S. cerevisiae with L.

fermentum did not alter the total polyphenol level.

The loss of polyphenols during fermentation could be attributed to polyphenol

peroxidase activity of seeds and microflora (Dhankher and Chouhan, 1987). Acidic

environment results in abstraction of hybrid ions and rearrangement of phenolic

structures (Porter et al., 1986; Chen et al., 2001). It has also been suggested that

during fermentation addition of water may facilitate the polyphenols to polymerize

or interact with proteins to render themselves become unavailable for extractability

during analysis (Beta et al., 2000).

Trypsin inhibitors have the ability to inhibit the proteolytic activity of digestive

enzyme trypsin which leads to decrease in amino acid availability (Liener and

Kakade, 1980). These may be of two types namely Bowman-Birk type double

headed trypsin inhibitors or Kunitz type single headed trypsin inhibitors. The

aforementioned trypsin inhibitor has the ability to inhibit trypsin and chymotrypsin

enzymes simultaneously. Variety of the legumes e.g. Phaseolus vulgaris, Pisum

sativum and Vigna mungo possess this kind of trypsin inhibitor. However, studies

showed that Indian vetch contains mostly Kunitz type trypsin inhibitors which are

able to interfere only with trypsin (Deshpande and Danodaran, 1990).

In this study, soaking decreased TIA non significantly. Similar results were noted

for kidney bean (Deshpande and Cheryan, 1983) and faba bean (Sharma and Seghal,

1991). The ability of soaking medium to reduce trypsin inhibitors could be enhanced

by increasing its alkalinity (Siddhuraju and Becker, 2001b). Substantial amount of

trypsin inhibitors have been reported to leach out from legume seeds soaked in a salt

solution (Mulimani and Paramjyothi, 1995). Whereas, neutral and acidic pH have

less tendency to soften the coat of legumes having hard seed coat like Indian vetch

and chickpea.

Trypsin inhibitors are heat labile (Dipietro and Liener, 1989). It was noted that

different forms of heating have almost equal tendency to affect trypsin inhibitors.

Similar reduction in trypsin inhibitors in legumes by autoclaving (83.67%), boiling

(82.27%) and microwave cooking (80.50%) were recorded (El-Adawy, 2002). All of

these processes involved the wet processing of legumes. Whereas, dry autoclaving

of legumes in present study, resulted in lower reduction in TIA than previously

reported by El-Adawy (2002).

The processing of legume seeds by controlled fermentation exhibits greater net

reduction in trypsin inhibitor activity than natural fermentation. The reason was the

essential incorporation of autoclaving of medium in controlled fermentation

processing steps. In many previously reported cases, there is no trypsin inhibitor left

available after autoclaving to act upon by microbes in controlled fermentation

(Chompreeda and Filds, 1984). In African yam bean, Lactobacillus plantarum

reduces the residual TIA from significant level to complete (Azeke et al., 2005).

The TIA in Indian vetch and chickpea underwent a high decrease due to natural

fermentation. Similar findings have been accomplished by Tabera et al. (1995) and

Shimelis and Rakshit (2007). The TIA reduction rate was 38 and 42% in Indian

vetch and chickpea, respectively. This value is little higher than TIA decrease noted

for cowpea i.e. upto 20% (Granito et al., 2005) and 27% (Doblado et al., 2003).

However, this rate is lower than the decrease in TIA attained by Granito et al.

(2002) for lentil (58-71%). This variation could be acknowledged to the individual

characteristic of the beans under fermentation, microflora involved and processing

conditions. Granito et al. (2002) attained higher reduction as the fermentation liquid

was removed during their study. Thus, soluble proteins and TIA was also discarded.

Germination of the Indian vetch and chickpea brought significant reduction

(P<0.05) in the trypsin inhibitor content (21 and 19% respectively). This decrease

may be due to mobilization and breakdown of trypsin inhibitor inside the cotyledon

of seeds. Similar findings were reported in different legumes. A lower reduction in

trypsin inhibitor has been achieved in lentil i.e. (7-18%) during 6 days germination

(Frias et al., 1995). In soybean, only 12% TIA is reduced after 12 days fermentation

(Chandrasiri et al., 1987).

A similar trend of low reduction in TIA by germination was found in moth bean

(Khokhar and Chauhan, 1986) and Phaseolus calcaratus (Chau and Cheung, 1997).

However, still some controversy exists in the literature about the effect of

germination on TIA. Legume type and processing conditions have a great role to

play in this regard (Liener and Kakade, 1980). After 5 days germination, TIA was

reduced by 62.9% in great northern bean (Phaseolus vulgaris L.) (Sathe et al.,

1983). A higher reduction in TIA under the influence of germination was observed

in these legumes by Shimelis and Rakshit (2007). Contrarily, Muzquiz et al. (2004)

reported no change in TIA in Vicia faba and Cicer arietinum during germination

process.

Even complete elimination of trypsin inhibitors from legumes has been reported

(Udedibie and Carlini, 1998). However, dry heating has not elsewhere been reported

as complete destroyer of trypsin inhibitors. Earlier, researchers have mentioned that

trypsin inhibitors in beans, cowpea, black gram (Sathe and Salunkhe, 1984), faba

bean (Vidal Valverde et al., 1997) and Mucuna beans (Siddhuraju and Becker,

2001a) was not completely destroyed by dry heating. Although, dry heating caused

greater reduction in trypsin inhibitor activity during present study but complete

destruction was not achieved.

As the legumes contain high amount of phytate, thus appropriate techniques are

quite essential to implement prior to legume’s consumption. Different food

processing methods i.e. soaking, malting, fermentation, cooking, addition of

exogenous enzymes have been tried to decrease phytic acid level of legumes

(Lonnerdal, 2000; Phillippy and Wyatt, 2001; Reddy and Sathe, 2002). However

traditional cooking methods i.e. soaking, hydrothermal cooking etc. have not been

able to show any significant decrease in the phytate content of grains (Nam, 2001).

Thus some treatments in addition to traditional methods are considered necessary

for considerable reduction of phytates.

Fermentation and germination of legumes have been proven as the best strategy to

reduce the phytate level in Indian vetch as well as chickpea. The microorganisms

used in the legume processing reduced the phytate to a great extent by a virtue of

their ability to produce phytase with simultaneous lowering down the pH of

substrate. A number of microorganism have been documented to exhibit phytase

activity including bacteria i.e. Escherichia coli (Greiner, 1993), Klebsiella terrigena

(Greiner, 1997), Lactobacillus plantarum (Kerovuo and Tynkkyneu, 2000), fungi

i.e. Aspergillus spp. (Dvorakova et al., 1997) and yeast i.e. Saccharomyces

cereviciae (Nakamura et al., 2000), Schwassuimyces castelli (Lambrechts et al.,

1992). Moreover, the endogenous phytase also exists in the grains (Fredrikson et al.,

2001).

The phytate reduction in legumes is the product of best suitable process temperature

and pH combination for optimum phytase activity. The phytate degradation

improves when the pH is decreased in the substrate media (Fredlund et al., 1997).

The pH optimum varies for phytase depending upon its source. It differs for fungi,

cereal (wheat) and legume (pea) with the values of 3.5, 5 and 7.5 pH, respectively

(Boisen, 1982; Fredrikson et al., 2001). In legumes (peas and beans), a high activity

of the phytase has been recorded at 37oC (Scott, 1991). Thus, the conditions set for

controlled fermentation during present study (5.5-6.0 pH and 37oC temperature)

exhibited the great reduction in phytate. Moreover, presence of tannin content in

flours, also inhibit phytase activity (Sandburg and Svanberg, 1991). The flours used

in this study are dehusked and posses small content of tannin. Therefore, phytase

activity was at its bloom during the fermentation processing of legume flours.

Phytic acid is a source of phosphorus and cations for the seeds that begin to sprout. It

is also a source of phosphates and inositol, which can both be generated by the

hydrolysis mediated by the phytases during germination. Thus, germination has been

proven a useful method for considerable decrease in phytate level in legumes.

However, in present study it was not ranked as the best. The main reason for this

could be short germination period (36 h) chosen to avoid the fungal growth on seed

surfaces that occurs hereafter. This growth could make the legume grains inedible

from sensory point of view.

Earlier studies have reported a period of 6-8 days slow reduction for different

legumes, before maximum decrease in phytase activity was noted during

germination (Kyriakidis et al., 1998). As during the initial period of germination

phytase activity was low, thus a nominal decrease in phytate was observed in this

study. Further, decrease in phytate content could be achieved by either modifying

the process conditions or by choosing multiple processes. Elkhalil et al. (2001) used

both malting and natural fermentation process alternatively and achieved up to the

83% reduction in phytate.

Indian vetch contains significant amount of β-ODAP (P<0.05). The presence of this

toxin in the Indian vetch is the cause of outbreak of neurolathyrism epidemics in

different parts of the world such as in Afghanistan in 2001 and Ethopia during 1997

(Getahun et al., 2005). This problem hinders the general acceptance of Indian vetch

by public. However, different processing techniques have the tendency to eliminate

or reduce β-ODAP. During present study, autoclaving marginally reduced the toxin

level. It was noted that when the legume flour was extruded at high temperature,

better reduction of β-ODAP was achieved (Grela et al., 2001). Soaking reduced the

β-ODAP about 19% which is below the reduction levels achieved by earlier studies

(Tekle-Haimanot et al. 1993; Padmajparasad et al., 1997; Rehman et al., 2006). The

difference being the decanting of soaking medium many times during process of

detoxification (Rehman et al., 2006) or boiling of the soaking solution

(Padmajaparasad et al., 1997). Furthermore, different alkaline solutions were also

employed to facilitate the toxin removal (Urga et al., 1994). Even, gentle soaking of

Lathyrus seeds for 3 min in large volume of water and decanting the excess water

reduced the β-ODAP by 30% (Tekle-Haimanot et al., 1993). Whereas cooking of

Lathyrus sativus seeds pretreated by salt solution and wood ash extract showed up

to 50% β-ODAP reduction on drainage of the soaking medium (Urga et al., 1994).

However, during this study, soaking was adopted as intermediate step of

germination process during which major portion of soaking water was absorbed by

the seeds, leaving very little to get chance of seeping out of water soluble β-ODAP.

It is thus, suggested that due to the soluble nature of β-ODAP when multiple

processing steps are incorporated in food processing, Indian vetch becomes

detoxified to a large extent. However, direct consumption of untreated raw Indian

vetch flour could be hazardous.

The β-ODAP was reduced by the fermentation processes either controlled (38%) or

natural (27%). These values were better than earlier findings of Ramachandran et al.

(2005). They achieved 24.2% reduction in β-ODAP in Indian vetch with Bacillus sp.

while, in the present study, Lactobacillus helveticus and Streptococcus

thermophillus were utilized to processit. However, solid state fermentation provided

more reduction in β-ODAP level. Aspergillus oryzae and Rhizopus oligosporus used

in succession for this purpose provided 82-97% reduction in β-ODAP (Yigzaw et

al., 2001).

As the β-ODAP is water soluble thus during germination it reduced to considerable

extent. During 36 hours of germination and due to the repeated watering, β-ODAP

may relocate and after decortications, it reduced to a higher extent (30%).

In addition to direct analysis of β-ODAP in processed foods, another way could be

to analyze its efficacy. The prolonged consumption of β-ODAP certainly results into

neurolathyrism. During a case control study, it was noted that raw, roasted and

boiled seed’s consumption increased the risk of neurolathyrism in the subjects.

Whereas, fermented pancake, unleavened bread and gravy preparation does not.

Thus, certain factors are considered to be associated with lower toxicity of Lathyrus

processing. Fermentation inactivated the toxin in fermented pancake preparation.

The β-ODAP could also leach out during gravy preparatory steps. Cereals addition

to the products diluted the toxicity capacity of the β-ODAP present in product

(Getahun et al., 2005). Thus Indian vetch rich in β-ODAP could safely be utilized

after processing as a candidate in cereal-legume complementation.

4.2.4. Conclusion

In Indian vetch and chickpea flours, maximum decrease in antinutritional factors

was observed for controlled fermentation treatment followed by germination and

natural fermentation. There was a slight drop in antinutritional contents observed in

the autoclaving treatment than the raw sample. This study suggests that best

nutritional quality legume flours with limited antinutritional levels could be

produced by mixed culture fermentation process.

4.3. EFFECT OF PROCESSING ON HCl-EXTRACTABILITY OF

MINERALS IN LEGUME SEEDS

4.3.1. Objective

Minerals have multiple and complex interactions within the food matrix. Processing

usually exerts a positive impact through separation or partitioning of minerals, or

through the destruction of inhibitors. The minerals extractable in 0.03 N HCl, the

concentration of acid found in human stomach, is an index of their in-vitro

bioavailability from foods. Raw and processed legume flours i.e. Indian vetch and

chickpea were analyzed to determine the influence of processing on HCl-

extractability of mineral.

4.3.2. Results

4.3.2.1. Copper

Copper is an integral part of many enzymes, some of which are essentially required

for iron metabolism. There is a direct correlation between the dietary Zn/Cu ratio and

the incidence of cardiovascular disease (Cabrera et al., 2003).

The HCl-extractability of copper (Cu) in raw and processed legume flours i.e.

Indian vetch and chickpea differed significantly (Table 4.23). The higher results for

HCl-extractability of Cu were attained by the controlled fermentation of Indian

vetch while raw Indian vetch got the least extractability (Table 4.24). The Indian

vetch samples when compared with each other for their ability to respond to HCl-

extractability showed that controlled fermentation provided Indian vetch flour with

65% better extractability than autoclaved Indian vetch flour, whereas natural

fermentation provided Indian vetch flour with 62% improved extractability in

comparison to raw Indian vetch. Similarly, a high percentage (56%) was obtained

for germinated Indian vetch flour than soaked one. When comparison was made for

chickpea flour samples, the percentages of improved Cu extractability for these

three comparison groups were 57, 44 and 37%, respectively. In chickpea, again

controlled fermentation produced the flour with high rate of copper extractability

(55.36±1.05%).

Table 4.23: Mean sum of squares for effect of processing treatments on HCl-extractability of copper in Indian vetch and chickpea

SOV df Vetch

(Lathyrus sativus)

Chickpea

(Cicer arietinum)


Error 12 3.68 4.82

Total 17


Table 4.24: Effect of processing treatments on HCl-extractability of copper in Indian vetch and chickpea

Processing treatments Vetch‡*

(Lathyrus sativus)

Chickpea‡*

(Cicer arietinum)

Raw 33.33+1.25d 29.06+0.29d

Soaking 37.04+0.35c 32.61+1.13cd

Autoclaving 37.69+0.48c 33.91+1.51c

Controlled fermentation 62.02+1.50a 55.36+1.05a

Natural fermentation 59.06+1.13a 41.74+1.28b

Germination 51.91+1.38b 44.83+1.81b

‡ Percent of metal present in dehulled flour + SE (Standard Error)


4.3.2.2. Manganese

The principal function of the manganese is the activation of numerous essential

enzymes, such as the enzymes which are known to use the energy of ATP (Salisbury

and Ross, 1969).

Statistical analysis of data showed the significant difference (P<0.05) between

processing treatments results for HCl-extractability of manganese (Mn) (Table 4.25)

which improved under the influence of processing of legume flours. Mn

extractability was observed at low level in untreated Indian vetch flour (Table 4.26).

However, it was substantiality improved by the processing through controlled

fermentation using mixed strains culture Lactobacillus helveticus and Streptococcus

thermophilus. Natural fermentation and germination increased HCl-extractability of

Mn at par to each other. Soaking also produced the similar result. The results for

chickpea regarding HCl-extractability were found on the same pattern to Indian

vetch. However, soaking could not produce as good results as observed in soaked

Indian vetch samples.

4.3.2.3. Magnesium

Magnesium (Mg) also contributes to bone health and bone density and is essential in a

wide range of metabolic reactions (Shils, 1996). It is involved as a co-factor in at least

300 enzymatic steps, many of which are linked to energy metabolism; thus, optimum

magnesium level is believed to be critical for proper maintenance of body weight and

the prevention of syndromes related to cardiovascular disease (Grundy et al., 2006).

HCl-extractability of Mg for both legumes i.e. Indian vetch and chickpea varied

highly significantly (P<0.05) under the effect of various processing treatments as

shown in Table 4.27. Processing of the Indian vetch and chickpea produced clear cut

groups of high and low Mg extractability (Table 4.28). Natural fermentation,

controlled fermentation and germination were the processes, which enhanced the

HCl-extractability to a considerable degree. On comparison, it was noted that

natural fermentation increased this attribute in Indian vetch and chickpea at quite

high levels i.e. 34 and 58%, respectively than raw flour. Germination improved the

HCl-extractability of Indian vetch and chickpea at 20 and 22% in comparison to

soaked seed samples.

Table 4.25: Mean sum of squares for effect of processing treatments on HCl-extractability of manganese in Indian vetch and chickpea

SOV df Vetch

(Lathyrus sativus)

Chickpea

(Cicer arietinum)

Processing treatments 5 73.6* 125.4**

Error 12 18.2 19.4

Total 17


Table 4.26: Effect of processing treatments on HCl-extractability of manganese in Indian vetch and chickpea


(Lathyrus sativus)

Chickpea‡*

(Cicer arietinum)

Raw 78.21+3.48bc 60.80+0.76c

Soaking 81.82+0.70ab 61.08+0.68c

Autoclaving 72.90+2.25c 64.21+2.45bc


Natural fermentation 83.97+2.23ab 70.98+3.65ab

Germination 81.79+3.67ab 70.10+2.25ab



Table 4.27: Mean sum of squares for effect of processing treatments on HCl-extractability of magnesium in Indian vetch and chickpea

SOV df Vetch

(Lathyrus sativus)

Chickpea

(Cicer arietinum)


Error 12 8.62 2.34

Total 17


Table 4.28: Effect of processing treatments on HCl-extractability of magnesium in Indian vetch and chickpea


(Lathyrus sativus)

Chickpea‡*

(Cicer arietinum)

Raw 41.00+0.80b 31.00+0.58e

Soaking 43.00+1.09b 37.00+0.72c

Autoclaving 44.00+1.17b 34.00+0.74d

Controlled fermentation 50.00+2.29a 45.00+1.64b

Natural fermentation 55.00+1.81a 49.00+0.66a

Germination 52.00+2.34a 43.00+0.39b



4.3.2.4. Sodium A primarily Na+-dependent electrochemical gradient is needed for transmembrane

transport and electrical membrane processes. Therefore it is not surprising that

concentrations of Na+ are particularly tightly regulated (Biesalski and Grimm, 2005).

Its balanced intake is highly adovacated by nutritionists.

The effect of processing on HCl-extractability of sodium (Na) in Indian vetch and

chickpea produced significant results (Table 4.29). Controlled fermentation

improved HCl-extractability (71.15±3.12% and 82.01±3.39%, respectively) higher

than all other processing treatments (Table 4.30). Whereas untreated legumes i.e.

Indian vetch and chickpea showed low extractability for Na (56.10±0.8% and

62.02±1.66%, respectively). Soaking and autoclaving changed the HCl-

extractability insignificantly (P>0.05) in both legumes. Although, natural

fermentation and germination were observed, as the treatments which could improve

the HCl-extractability of these legumes in a notably significant manner (P<0.05) but

these were not better than controlled fermentation.

4.3.2.5. Potassium

Potassium (K) is an important intracellular cation in the body that plays a vital role in

the maintenance of energy metabolism, cell membrane potential and membrane co-

transport of other ions (Luft, 1996). Because of its role in these processes, optimum

potassium intake is vital for the contraction of muscle groups such as the heart.

The data regarding HCl-extractability of potassium as affected by processing

treatments in Indian vetch and chickpea showed significant variation (P<0.05) as

given in Table 4.31. In both legumes i.e. Indian vetch and chickpea, a clear cut

segregation of treated flours with high and low HCl-extractability values was

estimated (Table 4.32). In Indian vetch, treated flours group comprised of controlled

fermented, natural fermented and germinated flours with high HCl-extractability

ranging from 65-69%. The raw Indian vetch along soaked and autoclaved samples

falls into low HCl-extractability group, having values 49-56%.

Similar pattern of results was achieved in chickpea, where high HCl-extractability

values ranged from 71 to 76% and low mineral HCl-extractability flours carried the

values in the range of 55-59%. Consequently, in chickpea overall better HCl-

extractability of potassium was achieved.

Table 4.29: Mean sum of squares for effect of processing treatments on HCl-

extractability of sodium in Indian vetch and chickpea

SOV df Vetch

(Lathyrus sativus)

Chickpea

(Cicer arietinum)


Error 12 11.1 23.4

Total 17


Table 4.30: Effect of processing treatments on HCl-extractability of sodium in Indian vetch and chickpea


(Lathyrus sativus)

Chickpea‡*

(Cicer arietinum)

Raw 56.10+0.84d 62.02+1.66d

Soaking 59.37+1.95cd 65.01+3.60cd

Autoclaving 58.00+2.08cd 66.01+1.98cd


Natural fermentation 63.97+1.66bc 78.99+3.48ab

Germination 67.01+0.89ab 73.00+1.90bc



Table 4.31: Mean sum of squares for effect of processing treatments on HCl-extractability of potassium in Indian vetch and chickpea

SOV df Vetch

(Lathyrus sativus)

Chickpea

(Cicer arietinum)


Error 12 17.6 14.6

Total 17


Table 4.32: Effect of processing treatments on HCl-extractability of potassium in Indian vetch and chickpea


(Lathyrus sativus)

Chickpea‡*

(Cicer arietinum)

Raw 49.00+1.39b 55.00+0.55b

Soaking 56.00+2.58b 59.00+0.78b

Autoclaving 50.00+2.38b 58.00+0.80b


Natural fermentation 65.00+2.67a 71.00+2.89a

Germination 68.00+1.77a 76.00+2.92a



4.3.2.6. Zinc

Zinc (Zn) plays a crucial role in energy metabolism, as it is involved in numerous

catalytic, structural or regulatory processes (Cousins, 1996). It prohibits oxidative

damage to human body by acting as a co-factor of the superoxide dismutase enzyme

involved in protection against oxidative processes. The metal is also required for

DNA and RNA synthesis (Shils et al., 1994). It is vital to endothelial cell integrity and

plays an important role in vascular endothelial barrier function (Hennig et al., 1992).

The total amount of this element in foods and its HCl-extractability jointly contribute

towards net delivery of Zn.

The results for the effect of processing treatments on HCl-extractability of zinc in

chickpea and Indian vetch differed highly significantly (P<0.05) when these were

subjected to statistical evaluation (Table 4.33). The least HCl-extractability was

attained by raw Indian vetch samples (52.88±0.68%), obvious from Table 4.34.

Soaking and autoclaving remained at par in respect to their effect on Zn HCl-

extractability (56.99±1.71 and 52.05±1.38%, respectively). Controlled fermentation

achieved higher HCl-extractability of Zn (79.94±3.78%). The results differed from

raw samples by 27.06%. It was followed by HCl-extractability of Zn affected by

natural fermentation (75.90±2.47%) differing by 23.02 percentage points and

germination (71.14±3.29%) showing the difference of 18.26 percentage points. On

the other hand in chickpea, controlled fermentation, natural fermentation and

germination did not differ significantly (P>0.05) for their effect on the Zn HCl-

extractability. The results for these treatments ranged between 77.78 to 81.25%

extractability.

4.3.2.7. Iron

Iron (Fe) is a major constituent of blood, being a component of haemoglobin, and thus

plays a crucial role in oxygen delivery throughout the body (Yip and Dallman, 1996).

Due to its redox potential, iron is also involved in many heme- containing compounds

or iron sulphur enzymes that are essential for electron transportation, respiration and

energy metabolism (Wood et al., 2007). It is an essential element and physiological

losses of it must be compensated regularly. Three factors affect the iron needs: the

total amount in the diet, the type of Fe compound, and the other diet components

Table 4.33: Mean sum of squares for effect of processing treatments on HCl-extractability of zinc in Indian vetch and chickpea

SOV df Vetch

(Lathyrus sativus)

Chickpea

(Cicer arietinum)


Error 12 18.2 17.4

Total 17


Table 4.34: Effect of processing treatments on HCl-extractability of zinc in Indian vetch and chickpea


(Lathyrus sativus)

Chickpea‡*

(Cicer arietinum)

Raw 52.88+0.68c 47.78+0.68c

Soaking 56.99+1.71c 53.01+0.98bc

Autoclaving 52.05+1.38c 56.12+3.14b


Natural fermentation 75.90+2.47ab 78.89+0.60a

Germination 71.14+3.29b 77.78+4.27a



Table 4.35: Mean sum of squares for effect of processing treatments on HCl-extractability of iron in Indian vetch and chickpea

SOV df Vetch

(Lathyrus sativus)

Chickpea

(Cicer arietinum)


Error 12 4.59 7.80

Total 17


Table 4.36: Effect of processing treatments on HCl-extractability of iron in Indian vetch and chickpea


(Lathyrus sativus)

Chickpea‡*

(Cicer arietinum)

Raw 46.00+0.95d 33.93+1.31c

Soaking 54.07+1.37c 38.00+0.54c

Autoclaving 54.99+0.36c 35.04+0.81c


Natural fermentation 66.92+1.62b 60.93+2.06b

Germination 68.91+1.71b 63.01+2.40b



(Hallberg et al., 1998). These factors determine the iron efficacy and HCl-

extractability in the in-vivo systems.

Analysis of variance technique which was applied to HCl-extractability of iron data

showed highly significant results (Table 4.35). A high level of Fe extractability was

noted (73.02±0.86%) in Indian vetch by controlled fermentation (Table 4.36).

However, when the fermentation and germination processing results were compared

to their corresponding preparatory treatment results, natural fermentation in

comparison to raw flour achieved higher extractability (46%). Controlled

fermentation in comparison to autoclaving showed 35% increase in the level of Fe

HCl-extractability in Indian vetch. Germination got 28% better HCl-extractability

than soaking treatments. In chickpea flours, these three comparison groups showed

Fe HCl-extractability at 79, 82 and 80%, respectively. However, as far as individual

treatment is concerned, high level of HCl-extractability was achieved by controlled

fermentation (69.03±0.71%). Natural fermentation and germination produced next

best results (60.93±2.06% and 63.01±2.40%, respectively) not varying significantly

from each other (P>0.05).

4.3.2.8. Phosphorous

Among the macronutrients, phosphorus is a major element in hydroxyapatite, a key

inorganic constituent of bone, and is also a critical part of several cellular compounds

e.g. phospholipids, phosphoproteins, nucleic acids and adenosine triphosphate (ATP)

(Arnaud and Sanchez, 1996). Thus, it plays basic roles in human structure and

metabolism.

The HCl-extractability of phosphorus was improved significantly (P<0.05) by the

processing treatments but to a medium extent (Table 4.37). All of the treatments

produced the flours with variable phosphorus HCl-extractability (Table 4.38). The

high level of HCl-extractability of phosphorus was achieved by controlled

fermentation (59.99±0.91%), germination (54.00±2.11%) and natural fermentation

(52.00±2.62%) with results in close proximity to each other. Indian vetch flour

without any treatment showed minimum HCl-extractability of phosphorus

(29.00±0.14%) closely followed by soaking (33.01±0.51%) and autoclaving

(35.01+0.44%). Similarly in chickpea, two segregated groups of treated flours were

Table 4.37: Mean sum of squares for effect of processing treatments on HCl-extractability of phosphorous in Indian vetch and chickpea

SOV df Vetch

(Lathyrus sativus)

Chickpea

(Cicer arietinum)


Error 12 6.30 7.97

Total 17


Table 4.38: Effect of processing treatments on HCl-extractability of phosphorous in Indian vetch and chickpea


(Lathyrus sativus)

Chickpea‡*

(Cicer arietinum)

Raw 29.00+0.14d 34.99+1.65b

Soaking 33.01+0.51cd 39.00+1.75b

Autoclaving 35.01+0.44c 36.98+1.19b


Natural fermentation 52.00+2.62b 47.99+1.91a

Germination 54.00+2.11ab 47.00+2.06a



attained with respect to their HCl-extractability values for phosphorus. The high

HCl-extractability groups comprised of flours generated from both fermentation

and germination treatments. The results for these were in close proximity to each

other ranging from 52.01 to 47.00%. The second group showed low HCl-

extractability of phosphorus from 39.00 to 34.99%.

4.3.3. Discussion

Minerals may persist in the legumes in free as well as bound forms. There have

many kinds of interaction with food components. Minerals bind to acidic side

chains, phosphoserine groups of peptides or phytate-protein complexes. These also

have tendency to bind to pectin, lignins and hemicelluloses. Divalent cations are

involved in the catecholic complexation or bound by polymerization reactions of

tannins and polyphenols. Other antinutritional factors such as phytate and saponins

have capacity to engage minerals into bonds with themselves (Watzke, 1998). In

different unprocessed legumes, minerals possess HCl-extractability at variable

extent depending upon their interactions.

Processing of legumes seeds or their flours may alter the HCl-extractability of

minerals in conspicuously significant manner (P<0.05). Soaking has been found to

cause changes in minerals and their corresponding HCl extractabilities. They were

leached out into water which was discarded before utilization of legumes. In the

absence of further treatment, the HCl-extractability of minerals was recorded in the

soaked seeds at par with unprocessed seeds. Similarly, dry autoclaving being the dry

heat method applied to legume flours was unable to alter the HCl-extractability of

majority of minerals at remarkable levels.

HCl-extractability of divalent minerals has been attributed to the reduction of

antinutrients like phytates (Duhan et al., 1989; Kaur and Kapoor, 1990) and

polyphenols (Jood et al., 1987; Sharma and Sehgal, 1992) which otherwise form

insoluble complexes (Vohra et al., 1965) with divalent cations such as calcium, iron,

zinc and copper (Khan et al., 1988). A significant negative correlation was noticed

with antinutrients viz. phytic acid, polyphenols, saponins and trypsin inhibitors with

copper and zinc extractability in the processed and cooked pigeon pea seeds (Duhan

et al., 2004). A high prevalence of iron deficiency anemia exists in the populations

of developing countries mainly due to the poor HCl-extractability of iron caused by

the inhibitors such as phytates, tannin and fiber in plant based fruits (Indumadhavi

and Agte, 1992). The phytate forms a catalytically inactive iron chelate, thus proves

to be a powerful inhibitor of iron driven hydroxyl radical formation (Graf et al.,

1984). To improve iron absorption, the phytate levels should not be exceeded above

0.5 μmol/g (Eklund-Jonsson et al., 2006).

Further, there are many factors investigated that affect the HCl-extractability of

minerals. An indigenous food mixture of rice flour, whey, sprouted green gram paste

and tomato pulp (2:1:1:1, w/w) was fermented with two types of fermentation cultures

such as single culture fermentation involving L. casei, L. plantarum and sequential

culture fermentation employing S. boulardii, L. casei, S. boulardii and L. plantarum.

As a results these, HCl-extractability of minerals viz. iron (54-67%), calcium (22-

32%), sodium (25-30%) and potassium (17-24%) has been improved (Sindhu et al.,

2005). In rabadi which is a fermented food prepared from pre-cooked wheat flour and

buttermilk mixture which was fermented at different temperatures and time periods

improved the HCl-extractability of divalent cations. This higher extractability of Ca,

Fe, Zn and Mn can be attributed to the decreased content of phytic acid (Gupta and

Khetarpaul, 1993).

In legumes, the HCl-extractability of minerals was greatly affected owing to

reduction in phytate by fermentation followed by a concomitant improvement in the

HCl extractabilities of minerals. A higher level of HCl-extractability of both major

and minor minerals could be attained with increased incubation time. It could be

basically due to the fact that more phytase will be available to solubilize phytate

present in flour (Idris et al., 2005). Similarly, on germination, phytase activates cause

the degradation of phytic acid (Shrivastava, 1994). It reduces the phytic acid and

polyphenol contents of the grains to a significant degree (Abdelrahaman et al., 2007).

Moreover, a negative correlation was found between HCl extractabilities of zinc,

calcium, copper, manganese and iron from rice-defatted soy flour blend and phytic

acid content (Rakhi and Khetarpaul, 1995). Also, Inositol hexaphosphate and

pentaphosphates are the forms of phytate which exert negative impact on the HCl-

extractability of divalent ions. One phytate molecule possesses capacity to bind upto

six divalent cations and one cation can possibly bridge at least two phytic acid

molecules, depending upon the redox state (Graf and Easton, 1990). Breakdown of

these phytate forms into lower phosphates have been found to reduce capability of

binding divalent metal ions (Lonnerdal, 2000). Thus, phytase production during

fermentation and germination plays its role in the improvement of HCl-

extractability of minerals especially Zn, Cu, Fe and Ca.

As far as phosphorus extractability is concerned, the phytate is the storage form of

these minerals under the influence of processing treatments. During the fermentation

and germination processing of phytate rich flours inositol hexaphosphate

hydrolyses, releasing free inorganic phosphorus and myoinositol phosphates (IP5 to

IP1) or inositol (Hotz and Gibson, 2001). Thus, liberated phosphorous becomes

available for HCl-extractability.

Whereas Mn and Mg are extracted not differently by processing treatments. These

minerals are even not bound by antinutritional factors. Thus antinutriton lowering

effect of fermentation and germination has insignificant effect on HCl-extractability

of these minerals and their results remained in conformance to those of other

processing treatments.

4.3.4. Conclusion

It has been noted that processing steps such as soaking and dry autoclaving are

unable to contribute towards improvement in HCl-extractability of minerals to a

significant extent. However, fermentation (controlled and natural) as well as

germination improved the HCl-extractability of minerals in both legumes to a great

extent.

4.4. BIOLOGICAL EVALUATION OF PROTEIN MEALS FOR MAKING

NUTRIENT DENSE FOOD BAR

4.4.1. Objective

Biological evaluation is the best tool for judging the quality of protein, since

numerous factors decide the ultimate quality of the protein in-vivo. Chickpea has

shown better biological value than some popular legumes through the rat balance

method. The relative nutritional importance of raw and processed Indian vetch against

any popular legume such as chickpea has never been determined. Conversely, studies

on nutritive value of fermented or germinated chickpea and Indian vetch flours

supplemented food meals are also limited. The present study was under taken for

biological evaluation of the protein qualities of food meals comprising raw or

processed Indian vetch flours components in comparison to chickpea.

4.4.2. Results

4.4.2.1. Feed /protein intake

The statistical analysis for feed intake of rats fed on different diet groups has shown

significant variations (Table 4.39). The means pertaining to feed intake are given in

Table 4.40. It was observed that the casein diet was consumed maximally, followed

by the germinated Indian vetch and chickpea flour blended meals, while non-protein

diet was minimally consumed (99.6 g). The mean feed intake during test period was

165.0 g for casein group followed by G-CBM (152.5 g) and G-VBM (141.9 g), on a

dry weight basis. Data revealed that there were no significant differences (P>0.05)

between feed intake of germinated and controlled fermented flour blended meals.

Among test proteins, the feed intake of raw Indian vetch and chickpea flour blended

meal groups of rats was observed minimum and did not differ significantly. Protein

intake of casein and G-CBM diet groups was maximum (16.42 and 15.19 g,

respectively) and did not differ significantly (P>0.05) among them followed by G-

VBM, C-CBM and N-CBM diet groups.

4.4.2.2. Weight gain

The statistical analysis indicated considerable weight gain by rat groups fed on

different protein meal diets (Table 4.39). The average body weight gain in 14 days as

shown in Table 4.40 was found maximum for standard diet (50.16 g). It was followed

by G-CBM and G-VBM (42.04 and 37.91 g, respectively). In contrast average loss of

Table 4.39: Mean sum of squares for feed intake, protein intake and weight gain values in rats

SOV df Feed intake Protein

intake Weight gain Fecal

protein output

Processing treatments

8 666.2** 6.621** 138.86** 1.303**

Error 9 67.0 0.683 1.94 0.003

Total 17


Table 4.40. Feed intake, protein intake and weight gain values in rats

Treatments Feed intake g/ rat/ 14days

Protein intake g/ rat/ 14days

Weight gain g/ rat/ 14days

Fecal protein output

g/ rat/ 14daysCAS 165.0+5.29a 16.42+0.53a 50.16+0.51a 2.25+0.09a

R-VBM 106.7+6.35f 10.63+0.65f 22.76+0.37g 1.32+0.02e

G-VBM 141.9+7.81bc 14.13+0.80bc 37.91+1.12c 1.20+0.07f

C-VBM 128.8+7.08cde 12.82+0.72cde 32.34+0.65e 1.82+0.05c

N-VBM 119.8+6.65def 11.91+0.63def 28.41+0.56f 2.12+0.05b

R-CBM 116.6+7.58ef 11.59+0.79ef 26.78+1.38f 1.60+0.10d

G-CBM 152.5+8.25ab 15.19+0.78ab 42.04+1.18b 1.18+0.03f

C-CBM 139.1+9.96bcd 13.82+0.99bcd 35.72+2.10cd 1.79+0.05c

N-CBM 133.5+12.4bcde 13.28+1.29bcde 33.48+2.72de 0.46+0.01g

CNT 99.6+7.95fg 0.74+0.09g -16.503+1.29h 0.25+0.01h

Means followed by different letters, within a row, are significantly different P < 0.05.

CAS = Casein, R-VBM = Raw flour vetch bar meal, C-VBM = Controlled fermented flour vetch bar meal, N-VBM= Naturally fermented flour vetch bar meal, G-VBM= Germinated flour vetch bar meal, R-CBM = Raw flour chickpea bar meal, C-CBM = Controlled fermented flour chickpea bar meal, N-CBM = Naturally fermented flour chickpea bar meal, G-CBM Germinated flour chickpea bar meal, CNT = No protein

weight was 16.50 g in the non-protein diet. Statistical analysis revealed that weight

gain for the casein diet was significantly higher than all test proteins (P<0.05). The

weight gain was significantly lower for both raw flour groups and N-VBM.

4.4.2.3. Feed efficiency

Efficiency could be defined as the gain in body weight per unit feed intake. Mean sum

of squares and means regarding feed efficiency of rats fed on different diet groups

have shown significant differences among diet groups as presented in Table 4.41 and

4.42, respectively. Among the test diets, R-CBM and N-VBM showed significantly

low feed efficiencies (P<0.05), and minimum feed efficiency (0.213) was observed

for R-VBM diet. Results showed that the feed efficiency for casein was 0.304

followed closely by G-CBM (0.276).

4.4.2.4. Feed utilization (FU)

The ratio of feed intake to gain in body weight is called feed utilization. Statistical

analysis showed significant (P<0.05) variation among rat groups fed on various bar

meals (Table 4.41). Mean values given in Table 4.42 revealed good feed utilization in

casein (3.29), G-CBM (3.63), G-VBM (3.74) and C-CBM (3.89). C-VBM and N-

CBM also possessed reasonable feed utilization values, not differing from each other

(3.98 and 3.99, respectively). Maximum feed utilization was observed for R-VBM,

followed by R-CBM.

4.4.2.5. Protein utilization (PU)

Protein utilization is ratio of protein intake to gain in body weight. Statistical results

pertaining to protein utilization of rats group fed on different diets are presented in

Table 4.40. The statistical difference for PU values in the test protein diets and casein

was observed where minimum protein utilization was observed for R-VBM. The

results (Table 4.41) showed that the PU value for G-CBM was 0.361 which was in

close proximity to casein (0.327) having minimum protein utilization value.

4.4.2.6. Protein efficiency ratio (PER)

Protein efficiency ratio is gain in body weight per unit protein intake. Statistical

results pertaining to protein efficiency ratio (PER) of different diets indicate that PER

was significantly affected by the experimental diets (Table 4.43). The results revealed

Table 4.41: Mean sum of squares for feed efficiency, feed utilization and protein utilization values in rats

SOV df Feed efficiency

Feed utilization

Protein utilization

Processing treatments 8 0.001** 0.341** 0.003**

Error 9 0.00004 0.012 0.0001

Total 17


Table 4.42. Feed efficiency, feed utilization and protein utilization values in rats

Treatments Feed efficiency Feed utilization Protein utilization

CAS 0.304+0.007a 3.29+0.07f 0.327+0.007f

R-VBM 0.213+0.009g 4.69+0.20a 0.467+0.021a

G-VBM 0.267+0.006c 3.74+0.10de 0.373+0.011de

C-VBM 0.251+0.009e 3.98+0.14cd 0.396+0.015cd

N-VBM 0.237+0.008f 4.22+0.15bc 0.419+0.014bc

R-CBM 0.230+0.003f 4.35+0.06b 0.433+0.007b

G-CBM 0.276+0.007b 3.63+0.09e 0.361+0.008e

C-CBM 0.257+0.003cd 3.89+0.05d 0.387+0.005de

N-CBM 0.251+0.003de 3.99+0.05cd 0.397+0.006cd


CAS = Casein, R-VBM = Raw flour vetch bar meal, C-VBM = Controlled fermented flour vetch bar meal, N-VBM= Naturally fermented flour vetch bar meal, G-VBM= Germinated flour vetch bar meal, R-CBM = Raw flour chickpea bar meal, C-CBM = Controlled fermented flour chickpea bar meal, N-CBM = Naturally fermented flour chickpea bar meal, G-CBM Germinated flour chickpea bar meal

Table 4.43: Mean sum of squares for protein efficiency ratio (PER) and net protein retention (NPR) in rats

SOV df PER NPR


Error 9 0.004 0.002

Total 17


Table 4.44. Protein efficiency ratio (PER) and net protein retention (NPR) in rats

Treatments PER NPR

CAS 3.06+0.06a 4.06+0.01a

R-VBM 2.15+0.09f 3.69+0.07d

G-VBM 2.69+0.07bc 3.89+0.05b

C-VBM 2.53+0.09cd 3.82+0.06bc

N-VBM 2.39+0.07de 3.77+0.04bcd

R-CBM 2.31+0.04e 3.74+0.02cd

G-CBM 2.77+0.06b 3.86+0.04b

C-CBM 2.59+0.03c 3.78+0.03bcd

N-CBM 2.52+0.04cd 3.77+0.06bcd



that the PER value for casein was 3.06, followed by G-CBM (2.77), G-VBM (2.69)

and C-CBM (2.58) (Table 4.44).

4.4.2.7. Net protein retention (NPR)

Net protein retention is defined as the ratio of sum of weight gain of test protein group

and weight loss of non-protein group to that of protein intake of test protein group.

Statistical results pertaining to net protein utilization of different diets prepared from

different bar meals are shown in Table 4.43. The data indicated that net protein

retention differed significantly (P<0.05) due to the difference in experimental diets.

Statistical difference for NPR values in the test protein diets and casein was

established from data. However, controlled and natural fermentation of both legumes

bar meal diets showed non significant difference (P>0.05) among them. The results

given in Table 4.44 revealed that the NPR value for both germinated bar meals (3.85)

was in close proximity to casein (4.06) having maximum net protein retention value.

4.4.2.8. Corrected protein efficiency ratio (C-PER)

Corrected protein efficiency ratio is defined as ratio of PER of test protein to that of

standard protein multiplied by standard value of reference proteins. The statistical

results regarding corrected protein efficiency ratio (C-PER) of different diets

presented in Table 4.45 indicate that C-PER differ significantly (P<0.05) among

different experimental diets. The corrected PER for meal diets with germinated

legume flours was in good correlation to standard diet (Table 4.46). The C-PER

values of G-VBM and G-CBM indicated that these bar meals contained high quality

proteins. The meal diet with raw Indian vetch flour ranked at the bottom of the C-PER

ranking order with value of 1.75. Statistical analysis revealed that standard protein

had significant (P<0.05) higher C-PER than test protein diets. The test proteins also

differed highly significantly among themselves.

4.4.2.9. Relative protein efficiency ratio (RPER) and Relative net protein ratio

(RNPR)

The analysis of variance for relative protein efficiency ratio (RPER) and relative net

protein ratio (RNPR) (Table 4.45) revealed that parameters varied significantly

(P<0.05) due to compositional difference in diets prepared by incorporating processed

chickpea/ Indian vetch flours. RPER and RNPR values for meal diets with raw Indian

Table 4.45: Mean sum of squares for corrected protein efficiency ratio (C-PER),

relative protein efficiency ratio (RPER) and relative net protein retention (RNPR) in rats

SOV df C-PER RPER RNPR

Processing treatments 8 0.096** 153.79** 13.51**

Error 9 0.0005 0.73 0.75

Total 17


Table 4.46. Corrected protein efficiency ratio (C-PER), Relative protein efficiency ratio (RPER) and Relative net protein retention (RNPR) in rats

Treatments C-PER RPER RNPR

CAS 2.50+0.00a 100+0.14a 100+0.14a

R-VBM 1.75+0.04h 70+1.70h 91+1.14d

G-VBM 2.20+0.01c 88+0.57c 95+0.89b

C-VBM 2.07+0.04e 83+1.27e 94+1.27bc

N-VBM 1.95+0.02f 78+0.99f 93+0.78bcd

R-CBM 1.89+0.01g 76+0.28g 92+0.28cd

G-CBM 2.27+0.01b 91+0.21b 95+0.57b

C-CBM 2.12+0.02d 85+0.71d 93+0.35bcd

N-CBM 2.06+0.01e 83+0.35e 93+1.13bcd



vetch flour (R-VBM) were 70 and 91, respectively (Table 4.46) with difference of

23%, whereas for meal diets with raw chickpea flour (R-CBM) had less difference

(about 17%). Considering the above criteria, G-CBM ranked as best quality meal with

difference of 4 followed by G-VBM, having difference of 7% between RPER and

RNPR values.

4.4.3. Discussion

There are certain factors that affect the feed intake of rats such as amino acid balance

of diet (Peter and Harper, 1985), fat contents (Le Magnen, 1983), sucrose contents

and antinutritional factors (Nestares et al., 1996). Moreover, the taste and smell of

food also affects the diet consumption by the subjects (Bos et al., 2000). The low

weight gain could be attributed to the antinutritional factors and toxic constituents

present in the legumes (Mortuza et al., 2000). The other factors responsible for low

weight gain for diets with raw legume flours are the low sulfur amino acid content

and low digestibility of the protein (Huyghebaert et al., 1979) and carbohydrate

(Mercier, 1979). The fortification of wheat based foods with protein rich non-wheat

plant material results in the gain in body weight of rats. The consumption of diets

carrying processed legumes helps to increase the weight of rats. Cooked whole pea

seeds can also contribute an increase in body weight of rats (Shah, 1991). The gain in

weight of the rats fed on gram-wheat flour samples is attributed to improve the

proximate and mineral composition of the blends (Anwar, 1980). Moreover,

substantial reduction in antinutritional factors during processing may be the reason to

gain in body weight (Nestares et al., 1996).

The standard PER of casein is suggested as 2.5 (Chapman and Mitchell, 1959). The

PER values for casein usually range between 2.15 and 3.31 with a mean of 2.79 (Bos

et al., 2000). The casein diet value for this study was found to fall between the earlier

recorded values for casein diets. Plant proteins are categorized into three groups for

PER, high PER (> 1.6), medium PER (< 1.6 to >1.0) and low PER (< 1.0) (Hsu et al.,

1978). In the present study, all the test diets and standard protein diet had high PER.

The protein efficiency ratio depends on essential amino acids composition and their

digestibility in human/animals (Jansen, 1978). The PER values of test diets, close to

the casein diet indicate adequate combination of essential amino acids in the test diets.

Germinated chickpea flour based diets closely followed by diets carrying germinated

Indian vetch flour were found high in PER values during this study. The germinated

flours may be compared well with casein, might be due to increase in the level of

amino acids (Fernandez and Barry, 1988). It is observed that processing improves the

PER of legumes. Cooking significantly enhances PER value of peas (Nagra and

Bhatty, 2007). Biological studies on rats show lower PER value (1.46) for diets

containing grams. Another diet on equi-proteneous basis got PER 1.56 when 20%

gram flour was incorporated in wheat flour (Shehta and Fryer, 1970). It is therefore

suggested that combining different sources of proteins improves PER value due to

complementary effect of proteins (Abid et al., 1991). A composite diet carrying wheat

flour, groundnut and Bengal gram got high PER value (2.18). This high value has

been attributed due to the contribution of protein of Bangal gram in the diet (Danial et

al., 1964).

Net protein retention (NPR) method for determining biological evaluation of any food

has been considered as an improvement over PER method. The reason being the NPR

method credits protein used for growth and maintenance instead of PER which

encounters the protein involved in growth only (Sarwar, 1997). Relative net protein

retention (RNPR) method is a modified method (based on weight gain) and generates

results similar to biological value (BV) and net protein utilization (NPU) method

(Young and Pellet, 1980). These methods do not always rank the proteins in the same

order as the PER method. The RNPR values for poor quality proteins are found much

higher than the RPER values (Mitchell et al., 1989). The casein has been considered

as the protein source with high RPER and RNPR (Sarwar, 1997).

In present study, high quality protein sources i.e. processed flour blends, either

carrying germinated or fermented legume flours agreed closely with previous studies.

It has been noted that meal diets carrying raw legume flours hold a wide gap between

RPER and RNPR ratios. Moreover, the RPER values for these meals were especially

low.

Thus it could be suggested that antinutritional factors present in the raw legumes

flours have significantly affected the protein performance. As these factors could

adversely affect the nutrients utilization and exert growth depression in the animals

(Sarwar, 1997). The enzymatic (proteases) breakdown of legume proteins during

germination leads to the formation of polypeptides, oligopeptides and free amino

acids. Moreover it has also been suggested that germination lowers the level of

antinutritional factors such as trypsin inhibitors, tannins and phytates (Gupta and

Sehgal, 1991). It may contribute towards improvement of nutritional indices of diets

carrying processed legumes (Nielsen, 1991).

4.4.4. Conclusion

This study was undertaken for evaluation of biological value of the protein of diets

containing raw and processed chickpea and Indian vetch flours which were used for

the preparation of nutritionally rich bars. The indigenous food processing

technologies such as controlled and natural fermentation and germination were used

to improve the protein quality of legumes. The diets containing germinated legume

flours (G-VBM and G-CBM) were ranked as the best quality meals on the basis of

their nutritional indices. The values for RPER and RNPR are in close proximity to

each other pertaining to processed legumes which indicate good quality of protein of

their blends. It could thus be concluded that high nutritional value food bars would be

produced by using meals having processed Indian vetch/chickpea flours.

4.5. PROTEIN DIGESTIBILITY-CORRECTED AMINO ACID SCORE

(PDCAAS) METHOD FOR PROTEIN QUALITY EVALUATION OF

MEALS

4.5.1. Objective

The Protein Digestibility-Corrected Amino Acid Score (PDCAAS) method is an

approved assay to evaluate protein quality of plant based foods (Young and Pellett,

1991). As PDCAAS method is based on human amino acid requirements, thus it is

recommended by agencies for evaluating the protein quality of foods intended for

human consumption (FDA, 1993). The true digestibility of protein scores corrected

for amino acid take into account three critical parameters of protein quality

evaluation: the essential amino acid profile of food protein, its digestibility, and its

ability to supply the essential amino acids in the amounts required by humans (Henley

and Kester, 1994). Thus, the protein meals were evaluated for their complementation

potential in this study.

4.5.2. Results

Statistical analysis of data regarding essential amino acid contents in food bar meals

containing processed Indian vetch (Lathyrus sativus) and chickpea (Cicer arietinum)

flours indictaed that there was a non-significant difference (P>0.05) between

treatments for essential amino acids except isoleucine and sulfur containing amino

acid group (methionine + cysteine) (Table 4.47). Mean values for essential amino acid

contents in food bar meals are presented in Table 4.48. The amino acids in food bar

meals i.e. histidine, leucine, lysine, threonine, valine and phenylalanine + tyrosine

differed non-significantly (P>0.05) and the values were in the range of 20.21+0.46 to

20.76+0.62, 89.16+2.10 to 91.99+6.02, 72.42+1.66 to 75.01+1.10, 36.75+0.38 to

37.66+1.10, 49.92+1.37 to 50.84+0.62 and 87.47+5.16 to 91.66+0.98 mg/g protein,

respectively. However, a momentous effect of incorporated legume type on isoleucine

and methionine + cysteine values of food bar meals was observed. The maximum

isoleucine content (55.62+0.91 mg/g) was observed in raw flour chickpea bar meal

that was statistically at par with controlled fermented chickpea flour bar meal

(55.18+1.79 mg/g) and germinated flour chickpea flour bar meal (55.01±1.96 mg/g).

The food bar meals incorporated with Indian vetch behaved alike and isoleucine

centents varied from 51.62+0.59 to 52.51+0.97 mg/g of protein. Overall, the

treatments containing chickpea flour contained higher isoleucine contents as compare

to Indian vetch flour incorporated food bar meals.

As far as methionine + cysteine are concerned, food bars meals having chickpea flour

contained higher contents as compared to bar meals having vetch. The highest

methionine + cysteine contents were recorded in raw flour chickpea bar meals

(57.53+1.93 mg/g) that decreased non-significantly (P>0.05) in fermented and

germinated chickpea flour bar meals. However, methionine + cysteine were recorded

in the range of 51.44+4.36 to 52.22+1.63 mg/g in food bar meals containing raw and

processed vetch flours.

The analysis of variance (Table 4.49) for true protein digestibility (TPD) and protein

digestibility-corrected amino acid score (PDCAAS) in rats fed on food bar meals

containing processed Indian vetch and chickpea flours showed that these values

differed highly significantly (P<0.05). Mean TPD values are presented in Table 4.50.

The highest value for total protein digestibility (93.3) was observed in C-CBM, while

the lowest (81.2) was noted with raw flour vetch bar (R-VBM). Total protein

digestibility studied in germinated flour vetch bar meal, naturally fermented flour

vetch bar meal, raw flour chickpea bar meal, germinated flour chickpea bar meal and

naturally fermented flour chickpea bar meal gained 91.4, 92.6, 86.8, 83.9, 91.1 and

88.4 values, respectively.

Mean values observed in rats for the parameter of PDCAAS when fed Indian vetch

and chickpea flours enriched bar exhibited in Table 4.50. The highest value for

PDCAAS (1.00) was recorded with naturally fermented flour vetch bar meal and C-

CBM, followed by germinated vetch bar meal (0.99), germinated flour chickpea bar

meal (0.97), whereas the lowest value was observed with raw flour vetch bar meal

(0.89).

4.5.3. Discussion

4.5.3.1. Role of amino acids

Although, legumes are rich source of protein, but they are also deficient in sulfur

containing amino acids such as cysteine and methionine (Messina, 1999). This dearth

of particular amino acids has been a consistent feature of almost all legumes. On the

Table 4.47: Mean sum of squares for essential amino acid contents in Indian vetch (Lathyrus sativus) and chickpea (Cicer arietinum) flours

incorporated food bar meals

SOV df Histidine Isoleucine Leucine Lysine Threonine Valine Methionine + cysteine

Phenylalanine + tyrosine

Processing treatments 7 0.098 7.860** 3.537 3.112 0.278 0.265 24.695** 9.148

Error 16 0.348 1.901 14.479 4.329 1.692 2.462 5.503 12.440

Total 23

Table 4.48: Essential amino acid contents (mg/g) for Indian vetch (Lathyrus sativus) and chickpea (Cicer arietinum) flours incorporated food

bar meals

Amino acids R-VBM G-VBM C-VBM N-VBM R-CBM G-CBM C-CBM N-CBM

Histidine 20.76+0.62 20.55+0.55 20.70+0.67 20.45+0.31 20.52+0.69 20.34+0.69 20.42+0.62 20.21+0.46

Isoleucine 52.51+0.97bc 52.15+1.77bc 52.41+1.49bc 51.62+0.59c 55.62+0.91a 55.01+1.96a 55.18+1.79a 54.68+0.83ab

Leucine 90.07+1.26 89.16+2.10 89.61+5.36 89.52+1.61 91.99+6.02 91.25+4.59 91.44+3.93 91.44+2.40

Lysine 73.15+2.31 72.86+3.64 72.79+1.61 72.42+1.66 75.01+1.10 74.71+2.34 74.56+1.28 74.26+1.54

Threonine 37.23+1.06 36.86+1.31 37.01+1.83 36.75+0.38 37.66+1.10 37.44+0.42 37.36+1.27 37.17+2.05

Valine 50.84+0.62 50.49+1.52 50.73+2.31 50.33+1.52 50.60+1.68 50.19+2.02 50.45+0.76 49.92+1.37 Methionine + cysteine 52.22+1.63b 51.70+1.86b 51.74+2.08b 51.44+4.36b 57.53+1.93a 57.10+1.53a 57.19+1.24a 56.61+2.64a

Phenylalanine + tyrosine 88.64+5.31 87.89+3.68 88.34+3.24 87.47+5.16 91.66+0.98 91.06+2.90 91.41+2.59 90.93+2.14

Means followed by different letters, within a row, are significantly different (P < 0.05).

R-VBM = Raw flour vetch bar meal, C-VBM = Controlled fermented flour vetch bar meal, N-VBM= Naturally fermented flour vetch bar meal, G-VBM= Germinated flour vetch bar meal, R-CBM = Raw flour chickpea bar meal, C-CBM = Controlled fermented flour chickpea bar meal, N-CBM = Naturally fermented flour chickpea bar meal, G-CBM Germinated flour chickpea bar meal

Table 4.49: Mean sum of squares for true protein digestibility (TPD) and Protein

Digestibility-Corrected Amino Acid Score (PDCAAS) for Indian vetch (Lathyrus sativus) and chickpea (Cicer arietinum) flours incorporated food bar meals

SOV df TPD PDCAAS


Error 16 17.901 0.001

Total 23


Table 4.50: True protein digestibility (%) and Protein Digestibility-Corrected

Amino Acid Score (PDCAAS) in rats for given meals for Indian vetch (Lathyrus sativus) and chickpea (Cicer arietinum) flours incorporated food bar meals

Treatments TPD (%) PDCAAS

R-VBM 81.2+2.3c 0.89+0.03c

G-VBM 91.4+4.6ab 0.99+0.05a

C-VBM 92.6+2.2a 1.00+0.05a

N-VBM 86.8+4.5abc 0.94+0.05abc

R-CBM 83.9+5.7bc 0.91+0.04bc

G-CBM 91.1+5.7ab 0.97+0.02ab

C-CBM 93.3+3.5a 1.00+0.02a

N-CBM 88.4+3.8abc 0.95+0.03abc

Means followed by different letters, within a column, are significantly different (P < 0.05).

R-VBM = Raw flour vetch bar meal, C-VBM = Controlled fermented flour vetch bar meal, N-VBM= Naturally fermented flour vetch bar meal, G-VBM= Germinated flour vetch bar meal, R-CBM = Raw flour chickpea bar meal, C-CBM = Controlled fermented flour chickpea bar meal, N-CBM = Naturally fermented flour chickpea bar meal, G-CBM Germinated flour chickpea bar meal

other hand, wheat and wheat products have short supply of lysine (Horn and

Schwartz, 1961). This amino acid is vital for normal body function. Methionine acts

as cysteine precursor, an important muscle constituent and fuel source of body.

Cysteine catabolism yields glutathione and taurine both of which act as antioxidants.

Taurine has central role in calcium transport and neuronal cell development. It also

regulates the membrane potential (de Poll et al., 2005). Lysine catabolism on the other

hand, generates carnitine and glutamine as intermediate products. It plays vital role in

mitochondrial oxidation of long chain fatty acids and reduces chronic stress induced

anxiety. Thus, during the course of development of a diet with good quality protein,

the balance of these amino acids should be considered to be maintained and their

deficiencies should be roofed up.

The deficiency of these amino acids is reported at variable degree. In various Indian

vetch samples, methionine is quite low and reported to vary between 0.24 g/100g to

0.82 g/100g protein (Lain et al. 1963; Cai et al., 1984; Low et al., 1990; Kuo et al.

1995). However, this legume is rich in lysine and carries as high as 9.65 g lysine/

100g protein (Lain et al., 1963). Vetch has been reported to carry other essential

amino acids in balanced quantities. Their quantities are reported in Indian vetch as

histidine 2.61%, isoleucine 4.41%, leucine 6.90%, phenylalanine 4.49%, threonine

4.08%, tyrosine 2.45% and valine 4.90% (Low et al., 1990). Whereas in another study

these amino acids were observed as histidine 3.47%, isoleucine 4.82%, leucine

8.60%, phenylalanine 3.89%, threonine 5.15%, tyrosine 2.92% and valine 5.08%

(Kuo et al., 1995). The amino acid levels determined in other legumes were also

found low level in sulfur conyaining amino acids (Roy and Spencer, 1989; Hanbury et

al., 2000). Similar patern of low sulfur amino acid contents was noted in chickpea.

Methionine was reported to range between 0.8 g/100g to 1.1 g/100g protein in

different chickpea cultivars of Pakistan. Howevr, these cultivars were quite rich in

lysine i.e. 6.7-7.0 g/100g protein. Essential amino acids reported for Pakistani

chickpea cultivars are as histidine 2.9-3.0%, isoleucine 4.5-4.8%, leucine 8.1-8.5%,

phenylalanine 5.0-5.3%, threonine 2.7-3.0% and valine 4.1-4.6% (Haq et al., 2007).

As far as amino acid profile of wheat is concerned, winter soft wheat is rich in sulfur

containing amino acids i.e. methionine (1.9%) and cysteine (2.1%) but deficient in

lysine (3.5%). It possesses different amino acids at adequate levels i.e. histidine 2.5%,

isoleucine 3.5%, leucine 7.3%, phenylalanine 4.8%, threonine 3.2% and valine 4.8%.

(Ejeta et al., 1987). Whey protein isolate, a byproduct of whey recovered from the

cheese manufacturing process carrys quite rich amino acid profile: histidine 2.2%,

isoleucine 5.8%, leucine 10.8%, phenylalanine 3.3% threonine 7.2% and valine 5.8%.

It posses sulfur containing amino acids to a considerable level i.e. methionine and

cysteine, 1.9 and 2.3%, respectively. It has also been gifted with good supply of lysine

(9.6%).

Essential amino acids histidine and threonine were observed as limiting amino acids

for protein bar meals carrying vetch and chickpea flours, respectively and was the

focal point for PDCAAS calculations. The incorporation of chickpea or vetch

seeds/flours has an effect on amino acid profile of the resultant food bar, whereas

individual amino acid levels, were least affected by processing. The efficiency of

increased soluble proteins in substrate depicts in the rat assays for true digestibility.

Germination was found to have a little effect on amino acids profile of cowpea

(Nnanna and Philips, 1989). This kind of results has been attained because of mild

germination treatment (24 hrs at 25oC or 30oC). However an increase in lysine content

was noted after 6 days germination (Fageer et al., 2004). Thus germination could be

attributed to show an increase in essential amino acids (Taylor, 1983) especially the

deficient one, the lysine (Dalby and Tsai, 1976) after prolonged germination. In the

present study short germination period resulted in no change in amino acid profile of

legumes.

It is also reported that decortication reduces the total protein content of grains;

however, it has insignificant effect on essential amino acid profile of the sample

(Adebooye and Singh, 2007). The baking process has the ability to degrade amino

acids. This degradation has been attributed to strecker reaction during high

temperature heating process (Khalique et al., 2004). The thermal break down of

amino acids produces methylbutanol, methional and acetyl pyroline. This can

contribute upto 10-20% reduction in amino acid content during baking of sourdough

breads (Gobbetti et al., 1995). However, the baking procedure adopted for bar

preparation is less intense i.e. baking at 150oC for 20 minutes in the presence of little

water. It may contribute nothing in amino acid breakdown.

The Lactobacillus helveticus have different enzymes and able to break down the

proteins into amino acids further into volatile compounds. These enzymes include a

cell wall bound proteinase of the serine type (Zevaco and Gripon, 1988) and other

peptidases (Christensen et al., 1995). The microbial metabolism could convert amino

acids into certain alcohols i.e. methylbutanol, 3-(methylthio)-propan-1-ol or

phenyletahnol (Gobbetti et al., 1995). However, in the present study the mixed culture

of Lactobacillus helveticus and Streptococcus thermophillus reduced a modest content

of amino acids. Similar findings were achieved for Lactobacillus sanfranciscensis

during souring of dough. It was noted that L. sanfranciscensis needs peptides not the

amino acids for its growth. The peptides could be metabolized in the cytosol of

bacteria. Therefore, the total amino acids contents of the substrate have been found

least affected (Thiele, 2002).

In the present study, the complementation of wheat based ingredients i.e. roasted

flour, farina and rusk crumb with processed chickpea or Indian vetch at 4:1 ratio

helped to over come the deficiency of these amino acids at a great extent. The

addition of whey protein isolate at 7% to the blend of wheat and legume meals further

enhanced the total protein concentration of the bars as well as increased the level of

amino acids considered generally as limiting ones in plant based diets.

4.5.3.2. In-vivo assay for true protein digestibility

The other factor of importance for PDCAAS calculation is true protein digestibility

(TPD) value. In the present study, TPD values varied for meals carrying different

processed legume flours. The TPD value is usually dependent upon the protein

source. Generaly, animal based food sources such as egg, milk and meat hold >95%

TPD value. The plant based diets scoring TPD values close to the animal based diets

include wheat gluten and soy protein isolates. It was noted that when the cell wall

constituents were removed, the TPD value was marginally increased as the TPD for

whole grain cereals, soybean, peas and chickpea has been reported to be low e.g. 80-

90%. Whereas, foods with tough plant cell walls (millet and sorghum flours),

breakfast cereals and mixed diet rich in ground whole cereals grains, legumes

carrying anti-nutritional factors and heat treated breakfast cereals possess TPD value

50-80% only (Millward, 1999). In beans (Phaseolus vulgaris), the presence of

antinutritional factors has resulted in poor digestibility value (30%). This low TPD

value could be improved to considerably high value by processing techniques i.e.

fermentation.

In the present study, fermentation by mixed culture of microbes yielded high TPD

values. An increase in the TPD value for fermented rice or flour blend products was

noted. This increase can be attributed to the protease activity (Goyal, 1991). Similarly,

an improvement in TPD value for fermented soybean was observed (Boralkar and

Reddy, 1985) because fermentation has the ability to decrease tannin content of the

globulin fraction of sorghum thus resulting in increase in its TPD value (Khalifa and

Zinay, 1994). Germination of legumes has been found to affect TPD value to a

significant extent. A high TPD value was noted for germinated beans (87%) (Kannan

et al., 2001). This improvement in TPD value could be attributed to the modification

of storage proteins. Moreover, germination process, with the help of proteases causes

protein mobilization which leads to the formation of polypeptides, oligopeptides and

free amino acids that facilitate digestibility of protein (Nielsen, 1991). It has also been

reported that germination reduces the level of antinutritional factors i.e. trypsin

inhibitors, tannin, phytates (Gupta and Sehgal, 1991) and lectins (Smets et al., 1985).

This reduction in antinutritional factors could also lead to improve TPD in legume

based diets.

The thermal processing of protein sources has variable effects on protein digestibility

of diets. It was noted that autoclaving of skim milk (121oC at 15 lb pressure for 20

minutes) reduced the TPD value, whereas similar treatment to soybean meal and

black beans resulted in the significant increase of TPD. Thus, heat processing having

a pronounced reduction effect on antinutritional factors may increase TPD (Sarwar,

1997). In the present work, it was noted the bar meals containing certain quantity of

raw legume flour may lead to low level of TPD and the meal carrying processed one,

results in improved TPD.

4.5.3.3. Protein digestibility-corrected amino acid score method (PDCAAS)

The principal merit of PDCAAS is its design based on human amino acid

requirements, which makes it more appropriate for human than a method based on the

amino acid requirements of animals. True protein digestibility and first limiting amino

acid content of diet are the factors used for calculation of PDCAAS (FDA, 1993,

Henley and Kester, 1994 and Dewey, et al, 1996). Thus, it has been reported that

PDCAAS suffers badly due to shortage of a particular amino acid among amino acid

profile of that diet. The PDCAAS of cereals is quite low (0.69) due to low quality

proteins. The PDCAAS of wheat is particularly low (0.42) attributing to lysine

deficiency (Schaafsma, 2000). Same is true for cereal products which owe low

PDCAAS values e.g. nixtamalized maize flour based tortilla (0.54), extruded maize

flour based tortilla (0.55) (Dorado et al., 2008). The legumes usually deficient in

sulphur amino acids and carrying antinutritional factors hold better PDCAAS 0.89,

while soybean having even higher value (1.0). The animal based products e.g. milk,

meat and egg contain good PDCAAS (1.0) (Kizlansky and Lopez, 2006).

However, it was observed that complementing cereal meal with legume could cause

significant improvement (P<0.05) in biological value of resulting diet and generates

quite good PDCAAS value. A food mixture produced from combination of maize (45-

50%) and cowpeas (35-40%), further fortified by either peanut or soybean scored high

PDCAAS i.e. 0.72-0.82 (Wilmot et al., 2001). Protein quality evaluation of tempeh

prepared from chickpea flour got the PDCAAS 0.92 (Bejarano et al., 2008). The

PDCAAS for chickpea dip with taminah has been observed 0.70 (Faris and Takruri,

2002). The combination of wheat flour, chickpea and milk powder scored PDCAAS

0.73 (FAO/WHO, 1989). The PDCAAS value recommended by FAO/WHO (1991) is

> 0.6 to fulfill the amino acid needs of pre-school age children (2-5 years). However,

for the development of a food commodity intended to be used in special

circumstances such as emergency relief should carry PDCAAS value > 0.8

(FAO/WHO, 1989).

In this study, food bars prepared from processed chickpea/Indian vetch flour in

combination with cereal meal, supplemented by whey protein isolate got variable

PDCAAS values. In both raw chickpea and Indian vetch flour bars, the PDCAAS

values were low. All other treatments got good PDCAAS values (more than > 0.94).

The low PDCAAS for bars containing raw flours could be attributed to their low

TPD. The presence of antinutritional factors may also contribute in this regard.

4.5.4. Conclusion

The essential amino acid contents of all food bar meals were determined using HPLC

technique. The food bar meals showed significant variation among samples for

isoleucine and sulfur containing amino acids i.e. methionine and cysteine. High

contents of these amino acids were studied in meals carrying chickpea flours. When

this amino acid profile was compared with amino acid requirement criteria for 2-5

years old children, it was observed that for both kinds of meal groups this criterion

was comfortably matched. High true protein digestibility (TPD) was achieved by food

bars carrying controlled fermented Indian vetch and chickpea flour meals, followed

closely by germinated flour possessing meals. The computed protein digestibility

corrected amino acid score (PDCAAS) was >0.9 for aforementioned meals.

4.6. SELECTION OF A PROTOTYPE NUTRIENT DENSE FOOD BAR

4.6.1. Objectives

Bars were produced by incorporating same constituents except one variable i.e.

differently processed Indian vetch or chickpea flours. The parameters such as

proximate composition, calorific values, In-vitro protein digestibility (IVPD), In-vitro

starch digestibility (IVSD) and sensory characteristics of bars were employed to

provide technical bases for selection of a prototype bar for further studies.

4.6.2. Results

4.6.2.1. Proximate analysis

Proximate composition includes estimation of moisture, crude fat, crude protein,

crude fiber and total ash contents of the samples. The results for proximate analysis of

food bars showed that various processing treatments have non-significantly

influenced (P>0.05) the moisture, crude fat, crude fiber and total ash contents (Table

4.51). The moisture contents of food bars were recorded in the range of 3.62±0.09 to

3.90±0.13%, total ash content 0.80±0.01 to 0.84±0.03%, crude fat 16.55±0.80 to

17.16±0.56%, crude fiber 2.29±0.05 to 2.38±0.07% and NFE 67.56±1.92 to

68.15±3.76 % (Table 4.52). However, protein contents were highly significantly

(P<0.05) affected by incorporation of processed Indian vetch and chickpea flours.

The lowest protein content (11.69±0.50%) was found in naturally fermented chickpea

flour bars and the highest (12.63±0.18%) in raw Indian vetch flour bars.

4.6.2.2. Calorific values

The results revealed that various processing treatments showed non-significant effect

(P>0.05) on calorific values of food bars (Table 4.51). The lowest energy value

(1909.8 KJ/100g) was recorded in Indian vetch bar containing naturally fermented

flour which had non-significant (P>0.05) difference from the highest value (1922.0

KJ/100g) that was found in chickpea bar having naturally fermented flour (Table

4.52).

4.6.2.3. In-vitro protein digestibility (IVPD)

The processing of the flours non-significantly influenced (P>0.05) the IVPD of food

bars (Table 4.53). The mean values given in Table 4.54 showed that lowest IVPD

Table 4.51: Mean sum of squares for proximate composition of Indian vetch (Lathyrus sativus) and chickpea (Cicer arietinum) flours

incorporated food bars

SOV df Moisture Total ash Crude fat Crude protein

Crude fiber NFE Energy value

(KJ) Processing treatments 7 0.028 0.001 0.153 0.579* 0.003 0.156 49.198

Error 16 0.028 0.001 0.575 0.176 0.005 7.429 9119.21

Total 23 Table 4.52: Mean proximate composition and energy values for Indian vetch (Lathyrus sativus) and chickpea (Cicer arietinum) flours

incorporated food bars

Treatments Moisture Ash‡ Fat‡ Protein‡* Crude fiber‡ NFE‡ Energy value (KJ)

R-VBM 3.90+0.13 0.83+0.02 16.67+1.14 12.63+0.18a 2.31+0.07 67.56+1.92 1912.5+119.0 C-VBM 3.64+0.17 0.81+0.02 16.61+0.83 12.56+0.30a 2.33+0.03 67.75+2.42 1912.1+42.1 N-VBM 3.69+0.19 0.80+0.03 16.55+0.80 12.49+0.75ab 2.34+0.05 67.82+3.35 1909.8+87.0 G-VBM 3.77+0.19 0.81+0.01 16.78+1.20 12.52+0.23ab 2.29+0.05 67.60+0.69 1915.3+57.9 R-CBM 3.79+0.18 0.84+0.03 16.91+0.28 11.77+0.18bc 2.35+0.15 68.13+1.98 1915.8+63.6 C-CBM 3.62+0.09 0.82+0.03 17.10+0.27 11.73+0.59bc 2.38+0.07 67.97+0.77 1919.6+77.1 N-CBM 3.67+0.18 0.82+0.02 17.16+0.56 11.69+0.50c 2.31+0.04 68.02+4.42 1922.0+182.6 G-CBM 3.65+0.18 0.80+0.01 16.95+0.26 11.74+0.19bc 2.36+0.06 68.15+3.76 1917.1+52.6

‡ g/100g on dry wt basis + SE (Standard Error) *Any two means not sharing a letter in a column differ significantly at P<0.05 (LSD) (n=3) R-VB Raw Indian vetch flour bar, C-VB Controlled fermented Indian vetch flour bar, N-VB Naturally fermented Indian vetch flour bar, G-VB Germinated Indian vetch flour bar, R-CB Raw chickpea flour bar, C-CB Controlled fermented chickpea flour bar, N-CB Naturally fermented chickpea flour bar, G-CB Germinated chickpea flour bar

(74.2±4.7%) was of raw flour Indian vetch bar, whereas the highest (78.5±5.9%)

IVPD was of germinated flour chickpea bars.

4.6.2.4. In-vitro starch digestibility (IVSD)

The mean sum of squares given in Table 4.53 showed that the processing of flours

affected IVSD of food bars highly significantly (P<0.05). The highest (369±23.0 mg

maltose/g) IVSD was found in controlled fermented flour chickpea bars followed by

363±14.0 mg maltose/g in naturally fermented flour chickpea bar, 358±8.0 mg

maltose/g in naturally fermented flour Indian vetch bar and 356±16.0 mg maltose/g in

germinated flour chickpea bar (Table 4.54). The raw flour Indian vetch bars had the

lowest IVSD (312±6.0 mg maltose/g).

4.6.2.5. Sensory characteristics

Sensory study of processed Indian vetch and chickpea flour incorporated food bars

revealed that processing treatments influenced the sensory perception for these bars

significantly (P<0.05) (Table 4.55). The mean sensory scores awarded to various

parameters are presented in Table 4.56. The highest scores (5.4±0.34) for texture were

awarded to germinated flour Indian vetch bars and the lowest (5.1±0.22) to raw flour

chickpea bars. The appearance of germinated flour Indian vetch bars was also the best

with mean scores of 5.4±0.27 while the flavor of controlled fermented flour chickpea

bars was liked the most with mean scores of 5.9±0.30 followed by controlled

fermented flour Indian vetch bars, germinated flour chickpea bars and germinated

flour Indian vetch bars with mean score values of 5.8±0.22, 5.8±0.33 and 5.7±0.13,

respectively. The taste of controlled fermented flour chickpea bars was awarded the

highest scores (5.9±0.23) followed by controlled fermented flour Indian vetch bars

with mean values of 5.7±0.23. Regarding the overall acceptability, controlled

fermented flour chickpea bars and controlled fermented flour Indian vetch bars were

liked the most with mean scores of 5.6±0.12 and 5.6±0.18, respectively. The naturally

fermented flour chickpea and Indian vetch bars were liked the least for almost all the

sensory parameters.

Table 4.53: Mean sum of squares for In-vitro digestibilities of Indian vetch

(Lathyrus sativus) and chickpea (Cicer arietinum) flours incorporated food bars

SOV df In-vitro protein digestibility (%)

In-vitro starch digestibility

(mg maltose/g)

Processing treatments 7 7.761 1252.661**

Error 16 17.465 273.50

Total 23


Table 4.54: In-vitrodigestibilities of Indian vetch (Lathyrus sativus) and chickpea

(Cicer arietinum) flours incorporated food bars

In-vitro protein digestibility (%)

In-vitro starch digestibility (mg maltose/g)*

R-VB 74.2+4.7 312+6.0c

C-VB 75.7+4.7 346+17.0ab

N-VB 74.9+5.3 358+8.0a

G-VB 75.1+1.2 321+17.0bc

R-CB 77.1+2.8 340+23.0abc

C-CB 77.9+4.4 369+23.0a

N-CB 77.7+2.0 363+14.0a

G-CB 78.5+5.9 356+16.0a


R-VB Raw Indian vetch flour bar C-VB Controlled fermented Indian vetch flour bar N-VB Naturally fermented Indian vetch flour bar G-VB Germinated Indian vetch flour bar R-CB Raw chickpea flour bar C-CB Controlled fermented chickpea flour bar N-CB Naturally fermented chickpea flour bar G-CB Germinated chickpea flour bar

Table 4.55: Mean sum of squares for sensory studies of Indian vetch (Lathyrus

sativus) and chickpea (Cicer arietinum) flours incorporated food bars

SOV df Texture* Appearance* Flavor* Taste* Overall acceptability*


7 0.138** 0.177** 2.914** 3.033** 1.139**

Error 88 0.037 0.051 0.077 0.075 0.084

Total 95


Table 4.56: Mean values for sensory scores of Indian vetch (Lathyrus sativus) and

chickpea (Cicer arietinum) flours incorporated food bars

Treatments Texture* Appearance

* Flavor* Taste* Overall

acceptability*

R-VB 5.3+0.19abc 5.1+0.27c 5.0+0.17bc 4.7+0.17e 5.0+0.10cd

C-VB 5.4+0.13ab 5.3+0.28ab 5.8+0.22a 5.7+0.23ab 5.6+0.18a

N-VB 5.2+0.18bcd 5.2+0.13bc 4.8+0.16cd 4.5+0.18e 4.9+0.30d

G-VB 5.4+0.34a 5.4+0.27a 5.7+0.13a 5.3+0.18cd 5.4+0.19ab

R-CB 5.1+0.22d 5.3+0.27ab 5.2+0.17b 5.2+0.47d 5.2+0.33bc

C-CB 5.2+0.07cd 5.3+0.22ab 5.9+0.30a 5.9+0.23a 5.6+0.12a

N-CB 5.2+0.09cd 5.1+0.09c 4.7+0.43d 4.7+0.18e 4.9+0.52d

G-CB 5.3+0.18abcd 5.3+0.19ab 5.8+0.33a 5.5+0.38b

c 5.5+0.34a


R-VB Raw Indian vetch flour bar C-VB Controlled fermented Indian vetch flour bar N-VB Naturally fermented Indian vetch flour bar G-VB Germinated Indian vetch flour bar R-CB Raw chickpea flour bar C-CB Controlled fermented chickpea flour bar N-CB Naturally fermented chickpea flour bar G-CB Germinated chickpea flour bar

4.6.3. Discussion

In order to improve nutritional properties of cereal products, these could be fortified

with supplements from different high protein sources, such as fish protein

concentrate, soy flour, soy protein isolates, milk and milk products, cotton seed

meal, egg albumin, whey protein, yeast protein concentrate (Nielsen et al., 1980),

oil seed flour, oil seed meal and legume flours (Awan et al., 1996). The

complementation of cereal meal with whey protein isolate and a legume meal was

investigated in this study for its capability to provide a balanced diet. Indian vetch

has been found as a suitable candidate for wheat supplementation in the

development of a food bar. Indian vetch contributed higher protein level to the bars

than chickpea. The incorporation of cowpea flour to traditional wheat based

Nigerian snack foods (cake, chin-chin and puff-puff) and supplementation of green

pea, yellow pea, chickpea and lentil to wheat for spaghetti production increased the

protein level of the products (Akubor, 2004; Zhao et al., 2005). The granola bar

developed by the addition of common black and red bean (Phaseolus vulgaris) also

resulted in improved protein and fiber contents (Maurer et al., 2005). The inclusion

of peanut flour, mesquite cotyledon and soy resulted in increased protein content of

food bars (de Penna et al., 1993; Escobar et al., 1998; Estevez et al., 2000).

Fat is an integral part of food bar because it is a rich source of energy. Moreover, it

plays a crucial role along sweeteners in sticking together the ingredients of a food

bar (Escobar et al., 1994). This agglutination process gives the bars a firm and

compact texture; a must characteristic of emergency relief rations (IOM, 2002).

Moreover, fat plays its role in increasing the palatability of a food bar. However,

high level of unsaturated fatty acids could lead to auto-oxidation resulting in

rancidity (Nawar, 1996). Therefore, complete precautions should be taken to avert

rancidity.

The balanced level of carbohydrates in a diet is quite necessary. The optimization of

food bars for protein and fat levels, results in the product with optimum level of

carbohydrates (Bower and Whitten, 2002). A soy based candy bar contained 58.7%

carbohydrates despite the presence of 12.4% protein and 9% fat (de Penna et al.,

1993). The presence of variety of ingredients could share the carbohydrate fraction

of a food bar e.g. cane sugar, liquid glucose, wheat meals, legume meals, dairy

ingredients etc. In the present study, wheat flour, farina, rusk crumb, Indian

vetch/chickpea flour, liquid glucose, cane sugar contributed towards carbohydrate

fraction of bar. However, whey protein isolate is considered to be carrying a little of

it.

Food bars due to their carbohydrate, fat and protein contents are considered as the

foods with high caloric value. These can thus be served for a number of purposes

e.g. sports, emergency situation, snack food, nutritional serving etc. Each situation

requires variable protein, fat and carbohydrate ratios as well as caloric density. For

instance, sportsmen need a food bar with high caloric density with mineral

supplementation to fulfill the depleted energy as well as electrolyte needs in most of

the sports. The bars used in relief purpose should carry nutrient dense characteristic

to over come nutrient deficiencies. However, bars serving the snack food purpose

are usually carrying high level of carbohydrates due to presence of some chocolate

and sweetener ingredients.

An energy bar developed for sportsmen supplied 520 kcal/100g with calorific

distribution percentages 8:40:52 for protein, fat and carbohydrate, respectively. The

ingest of two bars of hundred grams each was considered to provide recommended

intake for the most frequently practiced sports (de Penna et al., 1992). Whereas, a

soy-based candy bar contributed 375.2 kcal/100g shared by protein (14%), fat (22%)

and carbohydrate (65%) (de Penna et al., 1993). In order to fulfill the nutritional

needs of a person, recommended baseline content per 1000 kcal diet for protein, fat

and carbohydrates are 34 g, 39-50 g and 100-125 g, respectively (NRC, 1989). In

the present study, a high energy value (1922 KJ/100g) for controlled fermented

Indian vetch flour food bar is being shared by protein (10.3%), carbohydrate

(56.7%) and fat (33.0%). It is obvious that the ratio of fat and carbohydrates is quite

important to determine the energy density level of food bars. These food bars

provide complete nutritional requirement of an individual in a single package. Due

to balanced distribution of nutrients, regular consumption offer great liberty to

consumer from nutritional deficiencies.

A number of enzymatic assays are reported for estimation of IVPD. These include

pepsin digest residue index (Sheffuer et al., 1956), the pepsin pancreatin index

method (Akenson and Stachmann, 1964), the Ford and Stalter’s gel filtration

method (1966), the pepsin pancreatin digest dialysat index (Mauron, 1973), the

multi digestion method (Satterlee et al., 1979) and the pepsin pancreatin digestion

(Gauthier et al., 1982). During this study, pepsin pancreatin digestion method was

used to estimate IVPD.

It was observed that food bars carrying the fermented legume flours resulted in

improved IVSD and IVPD. It could be attributed to degradation of protein and

starch fractions under the influence of microbes. It was observed in Bacillus subtilis

fermented soybean that on the expense of protein fractions (5200-7000 Da),

peptides (200-1,100 Da) were produced. Similarly, fermentation process degraded

polysaccharides into oligosaccharides of <5 kDa. Increased fermentation time

resulted in an increased level of intermediate compounds from about 500-10,000 Da

(Kiers et al., 2000). Whereas, processing of soybean with Bacillus subtilis resulted

in a 60-fold increase in free amino acids which constituted 26% of total amino acids

content (Sarkar et al., 1997). Natural fermentation resulted improvement in IVPD

which could be attributed mainly due to the reduction in anti-nutritional factors.

Khamir (local bread) fermentation resulted in increased IVPD in sorghum cultivars

(Osman, 2004).

Further, processing of legumes improves the IVPD by destroying protease

inhibitors, thermal denaturation of protein to open the protein structure and

destroying or digesting globulins which are highly resistant to protease in natural

form (Siddhuraju and Becker, 2001a; Fagbemi et al., 2005). The digestibility of

food proteins can thus be affected by food processing treatments e.g. heating,

chemical application, exposure to high pressure, fermentation etc. (Takagi et al.,

2003).

The earlier concept regarding starch was that it can completely be digested and

absorbed. However, different kind of fractions of starch are unable to digest e.g.

retrograded amylase, ungelatized starch granules of the B-type found in green

banana and raw potato, physically enclosed starch such as present in beans and

chemically modified or dry heated food starches (Asp, 1995). The digestibility of

starch could also be affected by the cell wall structural features (Tovar et al., 1991;

Kataria et al., 1992) and anti nutrients such as amylase inhibitors (Lajolo et al.,

1991), phytates and tannins (Yadav and Kheterpaul, 1994). An increase in

digestibility after thermal treatment of legume flour could be attributed to disruption

of cell, thus freeing the encapsulated starch, starch gelatinization, physical

disintegration of seeds (Tovar et al., 1991) as well as reduction in anti-nutrient

factors (Bishnoi and Khetarpaul, 1994). Moreover, process of fermentation and

germination are considered able to reduce anti-nutritional factors of legumes e.g.

phytates, amylase inhibitors and tannins (Khalil and Mansour, 1995) which results

in the improvement of IVSD.

The decortication of grains improves the IVSD in resulting products whereas

presence of fibrous material declined the IVSD in a bran enriched toast in

comparison to white bread and French toast (Fontvieille et al., 1988). Legumes are

the food that contain significant amount of incompletely digested starch (Englyst et

al., 1992; Muir and O’Dea, 1992).

There exists a correlation between degree of starch gelatinization and IVSD in

wheat products (Ross et al., 1987). However, a wheat product with low water: flour

ratio and moderate baking temperature treatment results in less starch gelatinization

and consequently results in low IVSD (Wooton and Chaudhry, 1980). Further, the

presence of high levels of sucrose and fat are also considered to decrease starch

gelatinization (Hoseney et al., 1977; Wootton and Bamunarachchi, 1980). A

positive correlation is also present between glycemic index and percentage starch

In-vitrodigestibility (Ross et al., 1987).

The sensory quality is an important dimension of the total product quality and is

evaluated by the human senses of sight, smell, taste, hearing and touch (Meilgaard

et al., 1991). The sensory quality in this study was determined by hedonic

evaluation. The hedonic pleasure is one of the strongest motivations behind repeated

food choice (Arvola et al., 1999).

Among sensory quality attributes, taste is reported to be the most important criterion

(Wandel and Bugge, 1997; Schifferstein and Oude Ophuis, 1998; Torjusen et al.,

2001). In the present study, a similar pattern of preference was attained. The food

bars with natural fermented flours were ranked the lowest for taste which

deteriorated overall acceptability of bars as well. The superior importance of taste

for product acceptance is in line with priority of sensory attributes for product

choice or purchase on the part of consumers (Lappalainen et al., 1998; Grankivist

and Biel, 2001; Magnusson et al., 2001).

The term flavour covers the impressions perceived via the chemical senses form a

product in the mouth (Meilgaard et al., 1991). Thus, flavor includes the olfactory

perceptions caused by volatile substances perceived in the nasal cavity, the

gustatory perception caused by soluble substances as basic taste in the mouth, and

chemical feeling factors (astringency, spice heat, cooling, bite) stimulating the nerve

ends. Whereas term taste deals with, sensation produced by stimulation of the taste

buds on the tongue (Heinio, 2003).

The sensory evaluation of a food bar accounts appearance, texture, taste, flavor and

overall acceptability parameters. It was noted that sensory aroma and flavors have

high influence on consumer liking, followed by textural features and appearance for

cereal bars. The preference was linked to filling flavor, sweetness, chew and

crunchy texture (Bower and Whitten, 2000). As far as color features are concerned,

the darker products are less preferred due to their unattractive color. It was observed

that bars containing black bean scored low for color attribute in comparison to red

bean bars (Maurer et al., 2005). Moreover the preference for certain ingredients

always stands high, these include chocolate (Boustani and Mitchell, 1990) and nuts

(Estevez et al., 1995).

To minimize the effect of certain flavor and taste masking ingredients on the real

ingredients during sensory study, no flavor was added. It helped to investigate the

actual preference for processed legumes, being the only variable during the study.

4.6.4. Conclusion

As the part of selection criteria, certain parameters were analyzed for nutrient dense

food bars. It is envisaged that in-vitro protein digestibility (IVPAD) and in-vitro

starch digestibility (IVSD) for bars were affected by incorporation of processed

Indian vetch and chickpea flours. High IVSD was gained by processed flours in both

legumes rather than raw flours whereas high IVPD value was achieved by bars

carrying germinated chickpea flour. It was noted that all sensory attributes especially

flavor, taste and overall acceptability deteriorated on incorporation of natural

fermented flour in bars. Whereas controlled fermented flour containing bars were

preferred most. The proximate composition data shows that Indian vetch bars

contributed 12.52-12.63% protein and 16.55-16.78% fat. However chickpea bars

contributed marginally less protein (11.69-11.77%) than bars with Indian vetch flour.

The investigation showed that bars provided high energy value as well i.e. 1909-1922

KJ/100 g. On the basis of these selection criteria along biological evaluation of meals,

formulation containing fermented Indian vetch flour was choosen to prepare bars for

storage studies.

4.7. SCREENING OF NATURAL ANTIOXIDANT EXTRACTS FROM

FOOD PROCESSING WASTE MATERIALS

4.7.1. Objectives

The use of synthetic antioxidants is a norm in food industry to improve shelf

stability of fat rich food products. However, certain fears concerning damage to

body parts and carcinogenesis have been found associated with the use of certain

synthetic antioxidants e.g. butylated hydroxyanisole (BHA) and butylated

hydroxytoluene (BHT). It has fostered researchers to explore alternative natural

antioxidants to replace synthetic ones. Present work was undertaken to identify

effective natural antioxidant from cheap food industry wastes. The waste materials

selected for the study were: peanut skin, almond skin, pomegranate peel and onion

scales. Antioxidant compounds were extracted using different solvents and their

efficiency was explored through determination of free radial (DPPH) scavenging

activity and β-carotene bleaching assay over extended storage intervals.

4.7.2. Results


The extraction rate of antioxidants from various food processing wastes was highly

significantly influenced (P<0.05) by different extraction treatments (Table 4.57). The

highest extraction rates were observed with 80% ethanol solution in case of almond

skin (8.5±0.20), onion scales (9.8±0.40), peanut skin (18.1±1.10) and pomegranate

peels (16.9±0.80) followed by ethanol and methanol (Table 4.58). The lowest

extraction was performed with water. As a whole, the extraction rate of antioxidants

was found the highest in case of peanut skin followed by pomegranate peels, onion

scales and almond skin.

4.7.2.2. Total phenols

The total phenol contents in different food processing wastes were found highly

significantly affected (P<0.05) by various extraction treatments (Table 4.59). The

highest total phenol contents in almond skin (42.0±2.20) and onion scales

(76.0±2.50) were extracted by using ethanol followed by 80% ethanol solution. In

case of peanut skin (111.3±5.6) and pomegranate peel (132.7±3.1), the highest

Table 4.57: Mean sum of squares for extraction rate of food processing waste

materials under influence of different solvents

SOV df Almond skin

Onion scales

Peanut skin

Pomegranate peel


4 18.099** 12.936** 27.780** 80.796**

Error 10 0.076 0.236 1.024 0.390

Total 14


Table 4.58: Extraction rate (%) of food processing waste materials under influence

of different solvents

Solvents Almond skin Onion scales Peanut skin Pomegranate peel

Water 2.6+0.20d 4.6+0.10d 10.3+0.70d 3.5+03.0d

Ethanol 80% 8.5+0.20a 9.8+0.40a 18.1+1.10a 16.9+0.80bc

Ethanol 8.3+0.50ab 9.0+0.80b 15.7+1.10b 14.5+0.30a

Methanol 7.9+0.20b 9.2+0.10ab 16.3+1.40b 14.3+0.70c

Acetone 6.4+0.10c 8.5+0.60c 13.1+050c 11.7+0.80ab

Means followed by different letters, within a row, are significantly different (P < 0.05).

Table 4.59: Mean sum of squares for polyphenols in various solvent extracts from

food processing waste materials

SOV df Almond skin

Onion scales

Peanut skin

Pomegranate peel


4 231.0** 366.9** 330.5** 4364.0**

Error 10 2.988 6.566 30.68 35.162

Total 14


Table 4.60: Total polyphenols content (mg/g) in various solvent extracts from food

processing waste materials

Solvents Almond skin Onion scales Peanut skin Pomegranate peel

Water 20.0+0.60d 48.0+1.40d 85.1+5.8c 36.6+0.50c

Ethanol 80% 38.0+1.80bc 65.0+1.70b 111.3+5.6a 132.7+3.1a

Ethanol 42.0+2.20a 76.0+2.50a 102.3+5.0ab 125.8+7.5a

Methanol 35.0+1.70c 56.0+4.30c 109.7+7.8a 98.5+7.7b

Acetone 40.0+1.90ab 53.0+1.80c 98.7+1.6b 107.9+7.1b


phenol contents were recorded by extracting with 80% ethanol (Table 4.60).

The peanut skin and pomegranate peels contained the higher mean phenol contents

than almond skin and onion scales.

4.7.2.3. Antioxidant activity

The mean sum of squares presented in Table 4.61 show that the application levels and

treatments influenced the antioxidant activity of different food processing waste

extracts significantly (P<0.05). The antioxidant activity was significantly higher

(P<0.05) in case of high application level (50 µg/ml). Among the food wastes, the

pomegranate peels extract showed the highest mean activity as an antioxidant

followed by onion scales, peanut skin and almond skin, respectively (Table 4.62). The

similar trend is depicted by the graphical presentation (Fig 4.1) of results. The

processing of raw materials with 80% ethanol solution produced the extract with the

highest antioxidant activity followed by ethanol and acetone.

4.7.2.4. Radical scavenging activity

The radical scavenging activity of food processing waste extracts was highly

significantly influenced (P<0.05) by the processing treatments as well as application

levels (Table 4.63). The higher application level (50 µg/ml) resulted in considerably

higher activity of radical scavenging than 25 µg/ml application level. The

pomegranate peel extract showed the highest mean activity among various food

sources followed by peanut skin, onion scales and almond skin, respectively. The

extracts obtained by ethanol 80% solution were at the highest level of radical

scavenging activity whereas those extracted by water showed the lowest values (Table

4.64). Similar trend is depicted in Fig 4.2 which shows that the pomegranate peels

extract and extraction with 80% ethanol were at the top in terms of radical scavenging

activity.

4.7.3. Discussion


Plant food stuff is a treasure house of antioxidant compounds. These compounds

include more than 5000 polyphenols such as phenolic acids, flavonoids,

bioflavonoids, isoflavones and tannins. Other important group of about 500

naturally occurring plant based antioxidant is carotenoids including lycopene,

Table 4.61: Mean sum of squares for antioxidant activity of various solvent

extracts from food processing waste materials

SOV df Almond skin

Onion scales

Peanut skin

Pomegranate peel

Application level (A) 1 662.7** 1968.3** 1306.8** 2594.7**

Processing treatments (P) 4 202.8** 175.95** 64.05** 316.2**

AxP 4 8.70 11.55 8.55 17.7

Error 20 12.0 6.60 15.20 18.9

Total 29


Table 4.62: Mean values for antioxidant activity (%) of various solvent extracts from food processing waste materials

Food processing waste materials Application level

Antioxidant activity

Water Ethanol 80% Ethanol Methanol Acetone Means

Almond skin

50 µ/ml 58 73 66 64 67 60.9C

25 µ /ml 46 63 60 56 56

Means 52C 68A 63B 60B 61.5B

Onion scales

50 µ/ml 64 76 73 80 69 64.3B

25 µ /ml 52 61 54 62 52

Means 58C 68.5A 63.5B 71A 60.5BC

Peanut skin

50µ/ml 64 75 70 68 73 63.4B

25 µ/ml 54 60 58 55 57

Means 59C 67.5A 64AB 61.5BC 65AB

Pomegranate peel

50 µ/ml 75 89 87 86 78 73.7A

25 µ /ml 53 71 67 73 58

Means 64B 80A 77A 79.5A 68B

Fig 4.1: Antioxidant activity of various solvent extracts from food processing

waste materials

0102030405060708090

100

Water Ethanol80%

Ethanol Methanol Acetone

Solvents

Ant

ioxi

dant

act

ivity

(%)

Almond skin Onion scales Peanut skin Pomegranate peel

Table 4.63: Mean sum of squares for DPPH quenching activity of various solvent extracts from food processing waste materials

SOV df Almond skin

Onion scales

Peanut skin

Pomegranate peel

Application level (A) 1 907.5** 2650.8** 1732.8** 3564.3**

Processing treatments (P) 4 272.25** 277.05** 99.45** 439.05**

AxP 4 35.25 14.55 8.55 13.05

Error 20 15.40 14.40 16.50 22.90

Total 29


Table 4.64: Mean values for DPPH quenching activity (%) of various solvent extracts from food processing waste materials

Food processing waste materials Application level

DPPH

Water Ethanol 80% Ethanol Methanol Acetone Means

Almond skin

50 µ/ml 65 78 73 70 74 66.5D

25 µ /ml 46 69 66 62 62

Means C55.5 A73.5 AB69.5 B66 B68

Onion scales

50 µ/ml 72 83 79 86 74 69.4C

25 µ /ml 50 69 58 67 56

Means C61 A76 B68.5 A76.5 BC65

Peanut skin

50µ/ml 73 86 80 77 83 72.2B

25 µ/ml 61 69 66 62 65

Means C67 A77.5 AB73 BC69.5 AB74

Pomegranate peel

50 µ/ml 78 94 91 94 83 77.1A

25 µ /ml 52 73 70 76 60

Means C65 A83.5 A80.5 A85 B71.5

Fig 4.2: DPPH scavenging capacity of various solvent extracts from food

processing waste materials

0

20

40

60

80

100

Water Ethanol80%

Ethanol Methanol Acetone

Solvents

DPP

H s

cave

ngin

g ca

paci

ty(%

)

Almond skin Onion scales Peanut skin Pomegranate peel

carotene and xanthophylls (oxygen-containing carotenoids) (Larson, 1997; Cadenas and

Packer, 2002). Almost all plant origin foods contain antioxidants but the rich sources

are fruits, spices, tea, vegetables etc. (Kelawala and Ananthanarayan, 2004). A number

of by-products are generated during the processing of agriculture produces. It has been

noted that among them grape pomace (Lee et al., 2003), rice hull (Murthy et al., 2002)

almond skin (Harrison and Were, 2007), pomegranate peels (Negi and Jayaprakasha,

2003), onion scales (Nuntila et al., 2002), peanut skin (Yu et al., 2006) are the good

sources of phenolic compounds. These extracts carry a bunch of compounds exhibiting

antioxidant activity. It has been noted that these compounds in combination with some

other antioxidant source perform better than their individual performance to inhibit

lipid peroxidation (Jaswir et al., 2005).

A variety of plant based antioxidants have been evaluated in the food stuff for their

effectiveness. A combination of extracts from drumstick leaves (Moringa oleifera),

raisin (Vitis vinifera) and amla (Emblica officanalis) has been evaluated in biscuits for

the synergistic effect of these extracts (Reddy et al., 2005). Fenugreek seeds and ginger

rhizomes have been applied to beef patties for controlling lipid oxidation during cold

storage (Mansour and Khalil, 2000). Similarly, antioxidant compounds of plant origin

e.g. benzoin, catechin, chlorogenic acid, ferulic acid and quercetin have been explored

for their potential to extend shelf life of extruded oat cereals (Viscidi et al., 2004).

The main features for selection of a material (solvent) to use for extraction of

antioxidants include ease in extraction, extraction rate, antioxidant activity of the

extract, availability of material and effect on sensory attributes of food. Food

processing waste materials are abundantly produced during processing operations and

have huge potential for extraction of the antioxidants. To extract the antioxidants from

plant materials, a variety of extraction procedures are available. These procedures range

from simple aqueous extraction to CO2 supercritical fluid extraction (Suhaj, 2006). It is

necessary to explore suitable extraction procedure for antioxidants before their

commercial application. The antioxidant extraction rate from plant materias depends on

the nature and quantity of solvents employed and agitation rate during extraction (Hsu

et al., 2006). The yield of the antioxidants depends largely on polarity of the solvents

which could eventually be attributed to polar nature of the antioxidants. Moreover, the

extraction of antioxidants depends on type of phenolics as well. A number of

hydrolysable tannins present in extract possess higher extraction rate as several

hydrophilic phenolic hydroxyl groups with strong polarity may ease the process. In the

present study, aqueous extraction was compared with extraction through different

solvents such as methanol, ethanol, acetone and 80% ethanol. Among these solvents

used in this study, water holds the highest polarity followed by 80% ethanol, ethanol,

methanol and acetone. However, after preliminary trials, hexane was omitted from this

study because hexane being of non-polar nature owes least solubility of antioxidants

which results in low extraction capacity. Thus, it was used for prior removal of fat from

the samples.

Acetone has the ability to open the bonds between tannin molecules and proteins in

plant tissues and tannin becomes instantly dissolved in acetone (Zhang et al., 2007).

Resultantly acetone exhibits good ability to extract antioxidants among different

solvents for certain materials.

A comprehensive review on extraction of antioxidants from spices advocates the use of

alcoholic solvents (Suhaj, 2006). Majority of antioxidants have good solubility in

methanol and ethanol. Moreover, due to their polar nature these two solvents have wide

applicability in antioxidant extraction procedures (Siddhuraju and Becker, 2003; Zhou

and Yu, 2004). Further, methanol alcoholyses the ester bonds of polyphenols (Sun,

1992). For commercial use ethanol should probably be preferred over methanol as

eventual residues of solvents would be of low toxicity (Suhaj, 2006; Sultana et al.,

2007). In the present study, 80% ethanol solution provided the yield at par to that of

methanol. However, from food safety point of view, 80% ethanol solution was selected

as a choice for further study.

4.7.3.2. Screening of natural antioxidant extract

In this study, pomegranate peels extract showed better results for antioxidant activity,

total polyphenols and free radical scavenging activity. The pomegranate peels have

been recognized as one of the richest sources of antioxidants (Ben et al., 1996). When

peel, pulp and seed fractions of 28 kinds of fruits commonly consumed in China were

compared, the pomegranate peel was found to contain the highest antioxidant activity

as determined by ferric reducing antioxidant power (FRAP) assay (Guo et al., 2003).

The pomegranate peels extract contains several antioxidants contributing towards its

antioxidant activity including phenolics, flavonoids, proanthocyanidins and ascorbic

acid. It has been reported that among phenolics pomegranate peel contains ellagic acid

and gallic acid (Nasr et al., 1996; Chidamabaramurthy et al., 2002). It was noted that

when methanolic extract of pomegranate peels was compared with standard of butylate

hydroxyanisole (BHA) for DPPH scavenging activity, the aforementioned performed

better (Negi and Jayaprakasha, 2003). Given the higher value of phenolics present in

peel extract, it is not surprising that pomegranate peel exhibits high free radical

scavenging activity. Various tannins are also present in pomegranate peels extract e.g.

ellagitannin, gallotannin, pumicalagin and punicalin (Singh et al., 2002; Nagi et al.,

2003; Seeram et al., 2005). The role of flavonoids and proanthocyanidins could not be

ruled out towards contribution to the antioxidant activity presented by pomegranate

peels extract (Li et al., 2006).

A positive correlation exists between antioxidant activity by β-carotene linoleate model

system of pomegranate peels extract and phenolic contents (Singh et al., 2002). The

antioxidant activity of peel extract could also be compared with punicalagin, a chief

pomegranate polyphenol. It was found that peel extract carries a higher antioxidant

activity than that of punicalagin alone. It shows the presence of a synergetic effect

between different phenolic compounds present in peel extract (Kulkarni et al., 2004).

Onion scales are rich source of flavonols especially querectin (Suh et al., 1999). The

quercetins are present both as aglycon and aglycosides. Outer scales are devoid of

kaempferol which is abundantly present in inner core of onion (Bilyk et al., 1984). The

antioxidant activity of almond skin largely depends upon the flavones along many other

phenolics. Isorhamnetin rutinoside, isorhamnetin glycoside, kaempferol rutinoside,

kaempferol glucoside are the four flavone glycosides reported in almond seed coats

(Frison-Norrie and Sporns, 2002; Wijeratne et al., 2006). Other polyphenols found in

almond skin include quercetin in glycoside form, naringenin, catechin, protocatchuic

acid, vanillic acid and a benzoic acid derivative (Sang et al., 2002; Chen et al., 2005).

Peanut kernel processing to produce roasted peanut snack yields a plenty of peanut

skin. This by-product holds a good antioxidant potential. The galaxy of antioxidants

present in peanut skin comprises of three main classes of polyphenols such as phenolic

acids including chlorogenic acid, caffeic acid, coumaric acid and ferulic acid,

flavonoids including epigallocatechin, epicatechin, catechin gallate, epicatechin gallate

and stilbene resveratrol (Yu et al., 2005).

Lou et al. (1999) studied six A-type procyanidins in peanut skin. These dimmers have

probably more antioxidant capacity than phenolic monomers. Whereas resveratrol due

to its high metal chelating capability as well as free radical scavenging capacity

contributes a lot towards antioxidant activity of peanut skin extract. The potential

synergistic effect of flavonoids and resveratrol may contribute to the high antioxidant

activity of peanut skin (Yu et al., 2005).

Scavenging of the stable radical 2,2-diphyenyl-1-picrylhydrozyl (DPPH) has been

considered as a valid method to determine scavenging activity of an antioxidant extract

(Suhaj, 2006). DPPH carries deep violet color which exhibits absorption maxima 515-

528 nm. It losses its chromophore and becomes yellow when it receives a proton from

any hydrogen donor, mainly from phenolics. Therefore, there exists a direct relationship

between the quantity of antioxidants present in a sample and change in color of DPPH

(Sanchez-Moreno et al., 1999). This method has been applied to characterize antioxidant

activities of quercetin from onion skin (Suh et al., 1999), corn cob (Sultana et al., 2007),

wheat bran (Zhou and Yu, 2004), rice bran (Iqbal et al., 2005) and grape seed (Murthy et

al., 2002).

β-carotene bleaching is a free radical mediated phenomenon resulting from the

hydropheroxides formed from linoleate. In the absence of an antioxidant β-carotene

rapidly decolorizes (Singh et al., 2002). The extraction of cinnamon fruit extract by water

at 100 ppm level compared well with antioxidant activity of BHA (200 ppm) through β-

caroten linoleate model system (Jayaprakasha et al., 2007). This method was also

effectively employed to determine the antioxidant activity of Teucrium sp. (Panovska et

al., 2005), pomegranate peels and seeds (Singh et al., 2002) and cinnamon (Jayaprakasha

et al., 2007).

4.7.4. Conclusion

Food processing waste materials were extracted using different solvents to get the

antioxidant extracts. It could be concluded that 80% aqueous ethanol (v/v) solution is

effective in extracting food processing waste materials. Total polyphenol content of

extracts varied from each other at high pace. Mean value for pomegranate peal was

higher than other materials. Similarly, the extraction through ethanol and 80% ethanol

solution yielded best polyphenol level. Whereas, higher values of DPPH scavenging were

achieved by pomegranate peel among all solvent extracts. The β-carotene bleaching

capacity expressed in antioxidant activity was also much better in pomegranate peels

extract than extracts from other materials.

4.8. OPTIMIZATION OF ANTIOXIDANT EXTRACT LEVELS FOR A SHELF STABLE BAR USING RESPONSE SURFACE METHODOLOGY (RSM)

4.8.1. Objective

Usually synthetic antioxidants are used to decelerate oxidation processes in fat rich foods.

The health hazards associated with these synthetic antioxidants has prompted the

researchers to explore their natural alternatives. Food processing waste materials i.e.

almond skin, pomegranate peel, onion scales and peanut skin carry appreciable amount of

natural antioxidants and have the potential to be recognized as cheap source of these

compounds. Amla (Emblica officinalis) fruit extract is another good source of high

antioxidant activity. In order to optimize the levels of these variables, experiment was

designed by using Response Surface Methodology (RSM). The antioxidant combinations

are hypothesized to possess a sovereign or cumulative effect on the desired responses.

4.8.2. Results

The food bars were prepared taking into account the best formulation, incorporating

Emblica officinalis extract, Punica granatum peel extract and citric acid at variable

levels. Response Surface Methodology (RSM) was used to relate storage responses to

independent variables i.e. Emblica officinalis extract, a best performing extract from food

processing wastes and citric acid. Fifteen bar treatments were generated using a Box-

Behnken design with 3 variables and 3 levels for each variable. 13 different formulations

were produced in total and the central point was evaluated in triplicate. A second-order

polynomial models were fitted for independent variables i.e. Emblica officinalis extract

(X1), Punica granatum peel extract (X2) and citric acid (X3). The regression equations

and coefficients were determined from multiple regression analysis of storage data

regarding different parameters at 0, 30, 60, 90 ands 120 days storage intervals.

Effect of antioxidant levels on dependent responses during storage

4.8.2.1. Free fatty acids content

The responses for free fatty acids content from Box-Behnken design were fitted with

second order polynomial equations (Table 4.65). The statistical significance of the

model equation was evaluated by the F-test for analysis of variance (ANOVA), which

showed that the regression is statistically significant at 99% (P<0.05) confidence

level (Table 4.66). The calculated F-values for free fatty

Table 4.65: Coefficients of variables in models for free fatty acids in bars during

storage and their respective R2

Predictors Regression Coefficients

0 Days 30 Days 60 Days 90 Days 120 Days

Constant 0.800** 1.633** 2.633** 3.510** 4.860**

X1 -0.000 -0.371* -0.624* -0.890** -1.041**

X2 0.000 -0.418* -0.768* -1.260** -1.579**

X3 -0.000 0.004 -0.014 -0.103 -0.038

X12 0.000 0.341* 0.447* 0.449* 0.653**

X22 -0.000 0.208 0.364 0.419* 0.633**

X32 -0.000 -0.114 -0.028 -0.021 -0.075

X1X2 0.000 -0.293* 0.530* 0.463* 0.085*

X1X3 0.000 0.020 0.008 0.013 0.083*

X2X3 0.000 -0.073 -0.015 -0.043 0.028

R2 (%) 0.000 95.8% 92.4% 99.3% 99.9%

X1= Emblica officinalis extract, X2= Punica granatum peel extract, X3= Citric acid Table 4.66: Analysis of variance for the full regression of model

SOV df Mean squares

Regression 9 0.000 0.389* 1.125* 2.367** 3.513**

Residual Error 5 0.000 0.030 0.166 0.030 0.004

Total 14

** Highly significant at 95% confidence level (P<0.01)

* Significant at 95% confidence level (P<0.05)

Fig 4.3: Effect of independent variables (X1, X2) on free fatty acids in nutrient dense food bars during storage

a) 60 days Z= 2.62 - 0.624 X1 - 0.767 X2 + 0.449 X1*X1 + 0.367 X2*X2 + 0.530 X1*X2

EMBLICAPUNICA

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

2.5

3.5

4.5

5.5

6.5

2.577 2.941 3.306 3.671 4.035 4.4 4.765 5.13 5.494 5.859 above

b) 120 days Z= 4.81 - 1.04 X1 - 1.58 X2 + 0.658 X1*X1 + 0.638 X2*X2 + 0.0850 X1*X2

EMBLICAPUNICA

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

4

5

6

7

8

9

10

4.09 4.675 5.261 5.847 6.433 7.018 7.604 8.19 8.776 9.362 above

X2 X1

X2 X1


a) 60 days Z= 2.86 - 0.624 X1 - 0.014 X3 + 0.419 X1*X1 - 0.056 X3*X3 + 0.007 X1*X3

EMBLICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

2.5

3.5

4.5

5.5

6.5

2.688 2.841 2.993 3.145 3.298 3.45 3.602 3.755 3.907 4.06 above

b) 120 days Z= 5.25 - 1.04 X1 - 0.038 X3 + 0.604 X1*X1 - 0.124 X3*X3 + 0.083 X1*X3

EMBLICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

4

5

6

7

8

9

10

4.839 5.096 5.352 5.609 5.866 6.122 6.379 6.636 6.892 7.149 above

X3 X1

X3 X1


a) 60 days Z= 2.91 - 0.768 X2 - 0.014 X3 + 0.330 X2*X2 - 0.062 X3*X3 - 0.015 X2*X3

PUNICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

2.5

3.5

4.5

5.5

6.5

2.514 2.693 2.872 3.051 3.23 3.41 3.589 3.768 3.947 4.126 above

b) 120 days Z= 5.26 - 1.58 X2 - 0.038 X3 + 0.582 X2*X2 - 0.125 X3*X3 + 0.028 X2*X3

PUNICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

4

5

6

7

8

9

10

4.382 4.744 5.106 5.469 5.831 6.193 6.555 6.917 7.28 7.642 above

X3 X2

X3 X2

acids level in bars at different storage periods imply that models are statistically

significant (P<0.05). The coefficient of determination (R2) were calculated to be

more than 92.4% indicating that the models could explain this much variability in data.

The data showed that Emblica officinalis extract (X1) and Punica granatum peel extract

(X2) contributed towards reduced level of free fatty acids in bars at 60 and 120 days

storage intervals. Third variable i.e. citric aid was studied to have no effect on free fatty

acids level of nutrient dense food bars. The effect of linear terms of X1 and X2 were

statistically significant (P<0.05) for free fatty acids content of bars at all storage

intervals whereas, the X2 quadratic term was found significant at 90 and 120 days

storage intervals. The interaction of first two variables (X1X2) showed significant effect

on free fatty acids level at various storage intervals. Another interaction term i.e. X1X3

also exhibited significant variation at 120 days storage period. Due to the application of

ingredients with similar free fatty acids level in all experimental treatments showed that

no model could be generated for free fatty acids at 0 days storage.

Three dimensional response surface plots were generated on the basis of the model

equations, to investigate the linear and interactive effects of variables. The effect of

Emblica officinalis extract (X1) and Punica granatum peel extract (X2) concentrations in

nutrient dense food bars for free fatty acids at 60 and 120 days storage intervals is

shown in Fig. 4.3, which demonstrates that both variables contributed towards reduced

level of free fatty acids i.e. 1.5 and 3.9 mg/g in bars at 60 and 120 days storage intervals

respectively. On the other hand, Fig. 4.4 and 4.5 exhibited almost similar surfaces and

contours. These graphs indicated a clear effect of three independent variables.

4.8.2.2. Peroxide value

The coefficients of variables for peroxide value of bars during storage in models and their

respective coefficients of determination (R2) are given in Table 4.67. The analysis of

variance for full regression of models is shown in Table 4.68. The ANOVA table

depicted significant effect of variables on peroxide values of bars during storage

intervals. The R2 showed the measure of fitting the data were very high (>97.6%) for

peroxide value of bars at 30, 60, 90 and 120 days storage intervals, revealing adequacy or

strength of the models. Similar results for peroxide values of

Table 4.67: Coefficients of variables in models for peroxide value of bars during




Constant 0.300 1.967** 3.767** 6.000** 8.200**

X1 -0.000 -0.600** -1.350** -2.050** -2.775**

X2 0.000 -0.663** -1.475** -2.613** -3.123**

X3 -0.000 -0.113 -0.050 -0.038 -0.038

X12 0.000 0.154 0.592* 0.838* 1.263*

X22 -0.000 0.129 0.092 0.463 0.738

X32 -0.000 -0.321 -0.208 -0.438 -0.763

X1X2 0.000 -0.050 -0.300 -0.650 -0.900

X1X3 -0.000 0.000 -0.250 0.000 0.000

X2X3 0.000 -0.125 -0.150 -0.075 0.075

R2 (%) 0.000 98.1% 98.2% 97.6% 97.6%


SOV df Mean squares

Regression 9 0.000 0.793* 3.806* 10.464* 18.723*

Residual Error 5 0.000 0.028 0.126 0.468 0.187

Total 14



Fig 4.6: Effect of independent variables (X1, X2) on peroxide value of nutrient dense food bars during storage

a) 60 days Z= 3.64 - 1.35 X1 - 1.47 X2 + 0.608 X1*X1 + 0.108 X2*X2 - 0.300 X1*X2

EMBLICAPUNICA

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

2

3

4

5

6

7

8

1.602 2.205 2.807 3.41 4.012 4.615 5.217 5.819 6.422 7.024 above

b) 120 days Z= 7.73 - 2.78 X1 - 3.41 X2 + 1.32 X1*X1 + 0.796 X2*X2 - 0.900 X1*X2

EMBLICAPUNICA

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

4

6

8

10

12

14

16

3.326 4.593 5.861 7.128 8.396 9.663 10.93 12.198 13.465 14.733 above

X2 X1

X2 X1

Fig 4.7: Effect of independent variables (X1, X3) on peroxide value of nutrient

dense food bars during storage

a) 60 days Z= 3.82 - 1.35 X1 - 0.050 X3 + 0.585 X1*X1 - 0.215 X3*X3 - 0.250 X1*X3

EMBLICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

2

3

4

5

6

7

8

2.682 3.05 3.417 3.784 4.152 4.519 4.887 5.254 5.622 5.989 above

b) 120 days Z= 8.65 - 2.78 X1 - 0.04 X3 + 1.21 X1*X1 - 0.82 X3*X3 + 0.00 X1*X3

EMBLICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

4

6

8

10

12

14

16

6.552 7.269 7.986 8.703 9.419 10.136 10.853 11.57 12.287 13.003 above

X3 X1

X3 X1

Fig 4.8: Effect of independent variables (X2, X3) on peroxide value of nutrient dense food bars during storage

a) 60 days Z= 4.13 - 1.48 X2 - 0.050 X3 + 0.046 X2*X2 - 0.254 X3*X3 - 0.150 X2*X3

PUNICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

2

3

4

5

6

7

8

2.167 2.549 2.93 3.312 3.693 4.075 4.456 4.838 5.219 5.601 above

b) 120 days Z= 8.98 - 3.41 X2 - 0.04 X3 + 0.64 X2*X2 - 0.86 X3*X3 + 0.08 X2*X3

PUNICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

4

6

8

10

12

14

16

5.366 6.229 7.092 7.954 8.817 9.68 10.543 11.406 12.268 13.131 above

X3 X2

X3 X2

all bar treatments due to use of same kind of raw materials, resulted no regression model

at 0 day interval. The significant results (P<0.05) were achieved for linear terms of first

two variables (Emblica officinalis and Punica granatum extracts) as well as quadratic

term of Emblica officinalis extract. Third variable (citric acid) caused non-significant

change (P>0.05). The changes in levels of two variables i.e. Emblica officinalis and

Punica granatum extracts exhibited a sharp decline in peroxide value, whereas citric acid

present in combination with these extracts contributed almost nothing.

The effect of changes in the levels of two variables i.e. Emblica officinalis and Punica

granatum extracts on the peroxide value of food bars at 60 and 120 days storage

intervals is depicted in Fig. 4.6. The surface and contour plots exhibited a sharp decline

in peroxide value under the influence of Emblica officinalis and Punica granatum

extracts, whereas citric acid present in combination with these extracts contributed a

modest part towards change in peroxide level of bars during storage (Fig 4.7, 4.8).

4.8.2.3. Appearance

The regression coefficients are shown in Table 4.69 as well as correlation coefficients

obtained for all five models related to appearance of nutrient dense food bars under the

influence of independent variables (Emblica officinalis and Punica granatum extracts

and citric acid) at five storage intervals (0, 30, 60, 90 and 120 days). The regression

coefficient for these models (R2 = 67.7%, 61.7%, 98.05, 57.0% and 72.9% respectively)

are high enough for a well fitted response surface models. Analysis of variance (Table

4.70) showed a non-significant variation for full regression. Therefore, all linear,

quadratic and correlation terms for appearance of food bars during storage intervals

were non-significantly affected by the independent variables.

4.8.2.4. Taste

Taste of the food bars was not affected by the influence of independent variables over

four months storage (Table 4.72). The linear term X1 has significant effect on taste

(P<0.05). It contributed negatively at 0 and 15 days intervals, whereas by its role towards

rancidity avoidance in food bars, treatments carrying high level of X1 scored better for

taste (Table 4.71). The quadratic term of X1 has negative effect on taste of the bars. The

coefficients of determination (R2) showed that the models covered more

Table 4.69: Coefficients of variables in models for appearance of bars during storage

and their respective R2



Constant 5.500** 5.600** 5.600** 5.466** 5.533**

X1 -0.050 0.013 -0.013 -0.000 -0.013

X2 -0.013 0.075 0.000 0.013 -0.050

X3 -0.013 -0.013 0.063 0.063 0.063

X12 0.038 0.025 -0.050 -0.058 0.033

X22 -0.038 -0.100 0.025 0.067 0.008

X32 0.063 0.025 -0.000 0.067 0.033

X1X2 0.050 0.025 -0.025 -0.025 0.025

X1 X3 -0.050 -0.000 -0.050 -0.025 0.000

X2 X3 0.125 -0.075 0.025 0.000 0.025

R2 (%) 67.7% 61.7% 58.0% 57.0% 72.9% X1= Emblica officinalis extract, X2= Punica granatum peel extract, X3= Citric acid Table 4.70: Analysis of variance for the full regression of model

SOV df Mean squares

Regression 9 0.145 0.013 0.007 0.009 0.007

Residual Error 5 0.013 0.015 0.020 0.012 0.005

Total 14


Table 4.71: Coefficients of variables in models for taste of bars during storage and

their respective R2



Constant 5.666** 5.566** 5.433** 5.433** 5.366**

X1 -0.188** -0.038 0.113* 0.137* 0.150*

X2 0.013 0.075* 0.087 0.088 0.075

X3 -0.025 -0.038 0.000 0.000 -0.025

X12 -0.108* -0.096 -0.142 -0.142 -0.146

X22 -0.058 -0.071 -0.092 -0.092 -0.096

X32 0.0167 0.004 -0.017 0.033 0.104

X1 X2 0.050 0.000 -0.050 -0.100 -0.125

X1 X3 -0.025 -0.025 -0.075 -0.025 0.025

X2 X3 0.075 0.050 0.025 0.075 0.075

R2 (%) 95.2% 79.2% 77.9% 85.7% 83.9%


SOV df Mean squares

Regression 9 0.378 0.130 0.296 0.385 0.477

Residual Error 5 0.019 0.034 0.084 0.064 0.092

Total 14



Fig 4.9: Effect of independent variables (X1, X2) on taste of nutrient dense food bars during storage

a) 60 days Z= 5.51 + 0.0875 X1 + 0.125 X2 - 0.0385 X1*X1 - 0.0635 X2*X2 + 0.100 X1*X2

EMBLICAPUNICA

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

5.05

5.15

5.25

5.35

5.45

5.55

5.002 5.055 5.107 5.159 5.212 5.264 5.317 5.369 5.421 5.474 above

b) 120 days Z= 5.22 + 0.238 X1 + 0.225 X2 - 0.0769 X1*X1 - 0.0519 X2*X2 + 0.0750 X1*X2

EMBLICAPUNICA

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

4.84.95.05.15.25.35.45.55.6

4.767 4.833 4.9 4.967 5.033 5.1 5.167 5.233 5.3 5.367 above

X2 X1

X2 X1


a) 60 days Z= 5.40 + 0.0875 X1 - 0.0375 X3 - 0.0250 X1*X1 + 0.125 X3*X3 - 0.0750 X1*X3

EMBLICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

5.05

5.15

5.25

5.35

5.45

5.55

5.066 5.107 5.148 5.188 5.229 5.27 5.311 5.351 5.392 5.433 above

b) 120 days Z= 5.17 + 0.238 X1 + 0.0125 X3 - 0.071 X1*X1 + 0.029 X3*X3 - 0.100 X1*X3

EMBLICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

4.84.95.05.15.25.35.45.55.6

4.939 4.987 5.035 5.083 5.131 5.179 5.227 5.275 5.323 5.371 above

X3 X1

X3 X1


a) 60 days Z= 5.42 + 0.125 X2 - 0.0375 X3 - 0.0519 X2*X2 + 0.123 X3*X3 + 0.0500 X2*X3

PUNICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

5.05

5.15

5.25

5.35

5.45

5.55

5.186 5.22 5.254 5.288 5.322 5.356 5.39 5.425 5.459 5.493 above

b) 120 days Z= 5.15 + 0.225 X2 + 0.0125 X3 - 0.044 X2*X2 + 0.031 X3*X3 - 0.025 X2*X3

PUNICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

4.84.95.05.15.25.35.45.55.6

5.007 5.054 5.101 5.148 5.195 5.242 5.289 5.336 5.383 5.431 above

X3 X2

X3 X2

than 77.9% variability in data. It is evident from data that at low level of Emblica

officinalis and Punica granatum extracts while keeping third variable at coded value on

medium range (0), the taste of bars got high scores. However, during the course of

study due to oxidation of fat, the taste of food bars was deteriorated. The X1 in

combination with X3 showed that low levels of Emblica officinalis extract got better

scores, whereas due to the onset of rancidity, taste score pattern tilted towards variable

combinations containing high level of Emblica officinalis and Punica granatum extracts

which scored better for taste attribute.

The surface plots for taste attribute of nutrient dense food bars draw a better sketch of

data. It is evident from Fig 4.9 that at low level of Emblica officinalis and Punica

granatum extracts, the taste of bars got high scores, while keeping third variable at

coded value on medium range (0). However, during the course of study due to

oxidation of fat, the taste was deteriorated. Thus, the surface plots for taste tilted

reversely at 60 and 120 days intervals of storage (Fig.4.10). The X1 in combination with

X3 showed that low levels of Emblica officinalis extract got better scores (Fig. 4.11).

However, due to the onset of rancidity, taste score pattern tilted towards variable

combinations containing high level of Emblica officinalis and Punica granatum

extracts. These bars scored better for taste.

4.8.2.5. Flavor

Regression coefficients of variables in different models for flavor of bars during storage

are given in Table 4.73. The analysis of variance applied to flavor’s data is presented in

Table 4.74. Coefficient of determination (R2) showed that >83.0% variability was

covered by the models. The overall data for flavor of bars remained non-significantly

varying (P>0.05). The linear terms for X1 and X2 significantly affected the flavor over

30, 60, 90 and 120 days storage intervals. For these storage periods the interactive term

X1X2 also contributed a significant role in flavor. Positive sign for linear terms

indicates that with an increase in X1 and X2 levels, flavor score increased but portion of

each extract could vary. Interactive term of these two variables also has positive effect

on flavor perception of judges.

The effect of changes in levels of two variables i.e. Emblica officinalis and Punica

granatum extracts on the flavor attribute of food bars over 60 and 120 days storage

intervals is depicted in Fig. 4.6. The surface and contour plots exhibited an increase in

flavor score under the influence of Emblica officinalis and Punica granatum extracts

whereas citric acid contributed a little (Fig 4.7, 4.8).

Table 4.73: Coefficients of variables in models for flavor of bars during storage and

their respective R2



Constant 5.700** 5.500** 5.300** 5.200** 5.100**

X1 0.013 0.138** 0.125** 0.238** 0.275**

X2 0.000 0.100* 0.150** 0.263** 0.275**

X3 -0.013 -0.013 -0.000 -0.000 0.025

X12 0.025 -0.025 -0.025 -0.038 -0.038

X22 0.050 0.000 0.025 0.013 -0.038

X32 0.075 0.075 0.075 0.138 -0.038

X1X2 0.025 0.075* 0.100* 0.125* 0.125*

X1X3 -0.000 -0.000 0.000 0.000 0.025

X2X3 -0.025 -0.025 0.000 -0.050 0.025

R2 (%) 83.0% 85.6% 93.7% 96.4% 98.5%


SOV df Mean squares

Regression 9 0.004 0.031 0.041 0.128 0.144

Residual Error 5 0.002 0.010 0.005 0.009 0.004

Total 14

** Highly significant at 95% confidence level (P<0.01) * Significant at 95% confidence level (P<0.05)

Fig 4.12: Effect of independent variables (X1, X2) on flavor of nutrient dense food bars during storage

a) 60 days Z= 5.35 + 0.125 X1 + 0.150 X2 - 0.0308 X1*X1 + 0.0192 X2*X2 + 0.100 X1*X2

EMBLICAPUNICA

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

5.25

5.35

5.45

5.55

5.65

5.195 5.241 5.286 5.332 5.377 5.423 5.468 5.514 5.559 5.605 above

b) 120 days Z= 5.08 + 0.275 X1 + 0.275 X2 - 0.0346 X1*X1 - 0.0346 X2*X2 + 0.125 X1*X2

EMBLICAPUNICA

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

4.6

4.8

5.0

5.2

5.4

5.6

5.8

4.616 4.734 4.853 4.971 5.089 5.208 5.326 5.445 5.563 5.682 above

X2 X1

X2 X1


a) 60 days Z= 5.32 + 0.125 X1 - 0.0000 X3 - 0.0269 X1*X1 + 0.0731 X3*X3 + 0.0000 X1*X3

EMBLICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

5.25

5.35

5.45

5.55

5.65

5.185 5.219 5.254 5.289 5.324 5.358 5.393 5.428 5.462 5.497 above

b) 120 days Z= 5.08 + 0.275 X1 + 0.0250 X3 - 0.035 X1*X1 - 0.035 X3*X3 + 0.025 X1*X3

EMBLICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

4.6

4.8

5.0

5.2

5.4

5.6

5.8

4.708 4.775 4.842 4.909 4.976 5.044 5.111 5.178 5.245 5.312 above

X3 X1

X3 X1


a) 60 days Z= 5.28 + 0.150 X2 - 0.0000 X3 + 0.0269 X2*X2 + 0.0769 X3*X3 + 0.0000 X2*X3

PUNICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

5.25

5.35

5.45

5.55

5.65

5.192 5.234 5.277 5.319 5.361 5.403 5.445 5.488 5.53 5.572 above

c) 120 days Z= 5.08 + 0.275 X2 + 0.0250 X3 - 0.035 X2*X2 - 0.035 X3*X3 + 0.025 X2*X3

PUNICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

4.6

4.8

5.0

5.2

5.4

5.6

5.8

4.708 4.775 4.842 4.909 4.976 5.044 5.111 5.178 5.245 5.312 above

X3 X2

X3 X2

4.8.2.6. Texture

The models were developed for texture of bars as affected by independent variables

during 120 days storage. The regression coefficients of variables in models are shown

in Table 4.75. The statistical analysis by applying analysis of variance technique to the

full regression of model (Table 4.76) showed non- significant effect (P>0.05) of

variables. However linear terms of first variable (X1) were observed to negatively

change the texture of bars at all storage intervals, whereas quadratic term of citric acid

(X32) has a positive effect. When interaction of these two terms (X1X3) was studied, it

was found negative, whereas the interactive term for second and third variable (X2X3)

positively affected the texture of nutrient dense food bars over all storage intervals. The

coefficient of determination was studied as above 57%, therefore it could be assured

that models are adequately fitted.

4.8.2.7. Overall acceptability

The coefficients of regression and correlation for models generated for effect of

independent variables (Emblica officinalis and Punica granatum extracts and citric

acid) on overall acceptability of bars during storage are revealed in Table 4.77.

Analysis of variance for full regression models showed significant results (Table 4.78)

for 60, 90 and 120 days intervals. It is noted that first two variables have negative effect

on overall acceptability of bars at 0 days, whereas a positive effect was noted for these

variables during 60, 90 and 120 days storage. The quadratic terms of X1 also showed

significant but negative effect. The R2 value exhibited a low variability in data

(<15.5%). When citric acid was kept constant at 0 coded values, both Emblica

officinalis and Punica granatum extracts contributed towards improved overall

acceptability during 60 and 120 days storage. On the other hand, it was noted that citric

acid contributed negligibly in food bars for overall acceptability.

A parabolic surface plot (Figs. 4.15, 4.16, 4.17) for overall acceptability of food bars

suggests that optimum levels for two variables (X1 and X2) exist at higher concentration

side.

4.8.2.8. Moisture content

The coefficients of variables in models for moisture of bars at different storage intervals

have been given in Table 4.79. It shows that any linear, quadratic and interactive term

was not influenced by concentration of independent variables at

Table 4.75: Coefficients of variables in models for texture of bars during storage and

their respective R2



Constant 5.666** 5.700** 5.700** 5.567** 5.633**

X1 -0.050 -0.000 -0.012 -0.000 -0.025

X2 -0.012 0.075 0.000 -0.013 -0.050

X3 -0.012 -0.000 0.062 0.063 0.050

X12 0.004 0.013 -0.050 -0.058 0.020

X22 -0.070 -0.088 0.025 0.067 0.020

X32 0.029 0.013 0.000 0.067 0.020

X1X2 0.050 0.025 -0.025 -0.025 0.025

X1X3 -0.050 -0.025 -0.050 -0.025 -0.025

X2X3 0.125 0.075 0.025 0.000 0.025

R2 (%) 72.3% 77.5% 58.0% 57.0% 77.3%


SOV df Mean squares

Regression 9 0.014 0.011 0.006 0.009 0.006

Residual Error 5 0.010 0.066 0.020 0.013 0.003

Total 14


Table 4.77: Coefficients of variables in models for overall acceptability of bars during




Constant 5.666** 5.567** 5.367** 5.367** 5.300**

X1 -0.113* 0.038 0.125** 0.175** 0.175**

X2 -0.000 0.075* 0.113** 0.125** 0.125**

X3 -0.0125 -0.038 0.013 -0.000 -0.000

X12 -0.058 -0.083 -0.096* -0.108* -0.113*

X22 -0.033 -0.058 -0.021 -0.058 -0.063

X32 0.0417 0.0167 0.029 0.042 0.038

X1X2 0.025 0.025 0.000 0.000 -0.025

X1X3 0.000 -0.050 -0.050 -0.000 0.025

X2X3 0.025 0.025 0.025 0.050 0.075

R2 (%) 84.5% 86.3% 92.1% 96.4% 94.9%


SOV df Mean squares

Regression 9 0.132 0.121 0.279* 0.443** 0.464**

Residual Error 5 0.024 0.019 0.024 0.017 0.025

Total 14



Fig 4.15: Effect of independent variables (X1, X2) on overall acceptability of nutrient dense food bars during storage

a) 60 days Z= 5.54 + 0.0500 X1 + 0.0750 X2 - 0.0423 X1*X1 + 0.0077 X2*X2 + 0.0250 X1*X2

EMBLICAPUNICA

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

4.8

5.0

5.2

5.4

5.6

4.688 4.766 4.844 4.922 4.999 5.077 5.155 5.233 5.311 5.389 above

b) 120 days Z= 5.40 + 0.112 X1 + 0.0875 X2 - 0.0250 X1*X1 - 0.0250 X2*X2 + 0.0500 X1*X2

EMBLICAPUNICA

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

4.95.05.15.25.35.45.55.65.7

4.86 4.92 4.98 5.04 5.099 5.159 5.219 5.279 5.339 5.399 above

X2 X1

X2 X1


a) 60 days Z= 5.51 + 0.0500 X1 + 0.0250 X3 - 0.0385 X1*X1 + 0.0615 X3*X3 - 0.0250 X1*X3

EMBLICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

4.95.05.15.25.35.45.55.65.7

4.973 5.019 5.066 5.113 5.159 5.206 5.252 5.299 5.345 5.392 above

b) 120 days Z= 5.38 + 0.112 X1 + 0.0250 X3 - 0.0231 X1*X1 + 0.0019 X3*X3 - 0.0250 X1*X3

EMBLICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

4.8

5.0

5.2

5.4

5.6

4.977 5.031 5.086 5.141 5.195 5.25 5.305 5.359 5.414 5.469 above

X3 X1

X3 X1


a) 60 days Z= 5.48 + 0.0750 X2 + 0.0250 X3 + 0.0154 X2*X2 + 0.0654 X3*X3 + 0.0250 X2*X3

PUNICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

4.95.05.15.25.35.45.55.65.7

5.108 5.135 5.162 5.19 5.217 5.244 5.271 5.299 5.326 5.353 above

b) 120 days Z= 5.38 + 0.0875 X2 + 0.0250 X3 - 0.0231 X2*X2 + 0.0019 X3*X3 + 0.0250 X2*X3

PUNICACIT_ACID

Z

-1.2-0.8

-0.40.0

0.40.8

1.2

-1.2-0.8

-0.40.0

0.40.8

1.2

4.8

5.0

5.2

5.4

5.6

5.078 5.12 5.161 5.203 5.244 5.286 5.327 5.369 5.41 5.452 above

X3 X2

X3 X2

Table 4.79: Coefficients of variables in models for moisture of bars during storage and

their respective R2



Constant 4.486** 4.647** 4.557** 4.543** 4.453**

X1 -0.044 -0.039 -0.111 -0.056 -0.014

X2 0.002 0.021 -0.030 0.004 0.018

X3 -0.064 0.030 0.009 -0.030 -0.124

X12 -0.030 -0.156 -0.092 0.028 0.063

X22 0.083 -0.096 0.001 0.018 0.110

X32 -0.015 -0.084 0.053 -0.039 -0.067

X1X2 0.035 0.055 0.025 -0.085 0.003

X1X3 -0.003 -0.038 0.062 -0.538 0.015

X2X3 0.010 0.073 0.025 0.043 0.068

R2 (%) 27.4% 35.4% 51.6% 23.2% 64.7%


SOV df Mean squares

Regression 9 0.009 0.012 0.019 0.009 0.025

Residual Error 5 0.045 0.040 0.032 0.056 0.024

Total 14


different storage intervals. The coefficient of determination (R2) fitted poorly for models

corresponding to moisture content at 0, 30 and 90 days storage. R2 for 60 and 120 days

storage were found to be adequately fitted (<50%). The analysis of variance of full

regression of models as given in Table 4.80 showed non-significant variation (P>0.05)

in data. The values for moisture content of bars during storage varied between 4.3-

4.8%, whereas not even a single treatment reported the value above 4.8%.

4.8.2.9. Chroma Value

The coefficients of variables in models for chroma values of nutrient dense food bars

during storage are given in Table 4.81. The analysis of variance for full regression of

models has been presented in Table 4.82 which remained non-significantly (P>0.05)

affected during storage intervals under the influence of independent variables. It was

noted that all of the linear, quadratic and interaction terms were non-significantly affected

by the independent variables. However, the positive sign of first two variables (X1 and

X2) and their interactions exhibited a little increase in chroma value under the influence

of these two variables.

The coefficients of determination (R2) were noted > 66.3% for chroma value of bars at

different storage intervals, indicating that model could explain 66.3% or above

variability.

4.8.2.10. Hue Angle

Regression coefficients in models for hue angle values of bars at different storage

intervals have been presented in Table 4.83. An analysis of variance for hue angle

response is presented in Table 4.84. Regression analysis for different models depicted

that the fitted quadratic models accounted for over 71% of the variation in experimental

data, which shows the strength of the models. Multiple regression equations showed

that neither linear nor quadratic or interaction terms were significantly (P<0.05)

affected by variables at different storage intervals. The magnitude and sign of these

terms showed that similarly to chroma value, the hue angle value increased under the

influence of first two variables, however the magnitude was quite low. Similarly, all the

quadratic terms and interactions terms of Emblica officinalis and Punica granatum

extracts also contributed towards an increase in hue angle value. A slight increase in

hue angle has been visible under the interactive effect of Emblica officinalis and Punica

granatum extracts.

Table 4.81: Coefficients of variables in models for chroma value of bars during




Constant 2667.67 2628.00 2625.33 2606.00 2666.00

X1 05.37 5.75 25.12 10.750 9.38

X2 48.63 26.63 18.63 37.00 48.75

X3 -29.50 -49.88 -11.25 -42.75 -51.88

X12 -12.46 -11.75 8.12 8.88 4.25

X22 51.54 82.50 62.71 23.88 -3.00

X32 74.79 70.50 111.46 78.37 71.75

X1X2 40.25 31.75 45.75 47.75 29.25

X1X3 -24.00 -17.25 -6.00 -0.250 -54.00

X2X3 58.00 39.00 38.50 -25.75 11.25

R2 (%) 66.3% 69.1% 78.7% 97.00% 71.5%


SOV df Mean squares

Regression 9 8724 88.31 8900 6904.7 8457

Residual Error 5 7994 711.8 4325 384.1 6054

Total 14

Table 4.83: Coefficients of variables in models for hue angle value of bars during




Constant 76.60 77.10 76.77 76.50 77.67

X1 0.188 0.075 0.263 0.050 0.125

X2 0.325 0.463 0.150 0.250 0.475

X3 -0.388 -0.213 -0.188 -0.375 -0.425

X12 0.113 0.715 0.067 0.200 0.029

X22 0.638 0.350 0.442 0.250 0.070

X32 0.663 0.400 0.767 0.800 0.579

X1X2 0.250 0.275 0.325 0.150 0.225

X1X3 -0.025 -0.225 -0.200 -0.100 -0.225

X2X3 0.200 0.250 0.075 -0.200 0.075

R2 (%) 87.3% 76.9% 93.4% 96.9% 71.9%


SOV df Mean squares

Regression 9 0.627 0.449 0.482 0.499 0.566

Residual Error 5 0.164 0.243 0.060 0.029 0.398

Total 14

4.8.3. Discussion

Optimization of variables for a shelf stable nutrient dense food bar

The optimization process for antioxidants levels was based on results obtained for sensory

characters like flavors, taste, and overall acceptability and rancidity analysis for free fatty

acids and peroxide values for nutrients dense food bars. The computation of the optimal

levels of antioxidants was performed using a multiple response procedure called

desirability (Derringer and Such, 1980). This optimization procedure takes into account

desires and priorities for each variable. This combination resulted in maximum taste, flavor

and overall acceptability scores, whereas minimum free fatty acid content and peroxide

value were targeted for optimization process. Desirability scores could range from 0.0 for

undesirable to 1.0 for very desirable.

The second order polynomial models were fitted for independent variables:

Y= β0 + β1X1 + β2X2 + β3X3 + β11X22 + β22X2

2 + β33X32+ β12X12 + β13X13 + β23X23

In this equation:

Y = Dependent variable to be measured

β0 = Regression coefficient for treatment effect

β1 = Regression coefficient for X1



X1 = Coded level of Emblica officinalis extract

X2 = Coded level of Punica granatum extract

X3 = Coded level of citric acid content

A number of techniques are available to find out the best levels of input variables, which

in turn optimize their responses (Box and Draper, 1987). The most straight forward way

to undertake it is to draw the surface or contour plots of the fitted models. In this study,

during data analysis, surfaces and counters were drawn with the assistance of computer

software, Statistica. Three levels, each of Emblica officinalis extract, Punica granatum

extract and citric acid at different rates were used. The levels were coded as -1, 0 and +1.

The relationship between coded (X) and experimental levels (x) of Emblica officinalis

extract, Punica granatum extract and citric acid are as below.

2

X1 = x1 - 0.75 0.75 X2 = x2 - 1.0 1.0 X3 = x3 - 0.03 0.03 Whereas X1, X2 and X3 are the coded scales for Emblica officinalis extract, Punica

granatum extract and citric acid. For estimating a second degree polynomial, different

second order designs are proposed e.g. central composite design, Box-Behnken designs

(Box and Darper, 1987). The Box-Behnken fractional factorial design is often preferred

for product optimization, while evaluating sensory attributes, since interaction parameter

estimates are not completely confounded and the design is considerably smaller than that

of other fractional factorial design (Dean and Voss, 1999). These designs are rotateable

or nearly rotateable (Montgomery, 1991). The centre points for all model parameters must

be repeated (Dean and Voss, 1999), the model required 15 treatments tested by each

panellist instead of a larger pool of 27 treatments for 33 factorial design.

The nutrient dense food bars contain high content of fat (>16%) and low level of

moisture (<4%). The limited moisture content and proper packaging makes them safe

from microbial deterioration, however rancidity could occur due to presence of high level

of fat. Lipid peroxidation is the main cause of lipids and lipid-containing foodstuff’s

deterioration. The degree of lipid oxidation can be measured by using chemical and/or

physical methods. Under normal storage conditions, hydroperoxides and free fatty acids

are the key products of this process, detected in traditional tests. The development of

these compounds during storage thus contributes towards deteriorated consumer’s

acceptance for the rancid foodstuffs. Increase in total amount of free fatty acid during

storage might be attributed to activities of lipases as well as lipolytic acylhydrolases present

in flour (Morrison, 1978).

It was noted that control of free fatty acid content is associated with processing treatments

but increases with presence of high moisture level in the products and extended storage

period. Development of hexanal due to oxidation degradation of unsaturated fatty acids is

responsible for rancidity problem (Molteberg et al., 1995). The rancidity in food stuff could

be controlled through the use of antioxidants. The antioxidant sources Emblica officinalis

and Punica granatum extracts as well as citric acid were selected in order to estimate

their efficacy to keep the nutrient dense food bars stable during storage. It is known that

pomegranate peel extract is a source of useful antioxidants. The selection criteria of

Punica granatum extract was its better antioxidant activity and free radical scavenging

capacity among food processing waste extracts, trailed during study. The pomegranate

peel contains substantial amount of certain polyphenols such as ellagic tannins, ellagic

acid and gallic acid (Gil et al., 2000).

Amla (Emblica officinalis) fruit extract has been well known for its antioxidant activity. It

contains a number of antioxidant compounds e.g. geraniin, quercetin 3-β-D

glycopyranoside, isocorilagin, quercetin and kaempferol. Amongst, geraniin showed the

highest antioxidant activity (4.5 IC50 value for DPPH assay) (Liu et al., 2008). The tannins

present in the amla include low molecular weight hydrolyzable tannins e.g. emblicanin A

and emblicanin B, pedunculagin and punigluconin (Chandhuri, 2004). Moreover, gallic

acid and tannic acid are identified as the major antioxidant component in its polyphenolic

fraction (Kumaran and Karunakran, 2006). This fruit is also known as one of the richest

sources of ascorbic acid and contains about 0.40% (w/w) ascorbic acid. The ascorbic acid

contributes 45-70% of total antioxidant activity which is exhibited by the fruit extract

(Scartezzini et al., 2006). The rest of antioxidant activity has been attributed to the

phenolics. Also this fruit possess more free phenolics (126 mg/g) than the bound ones (30

mg/g) contributing towards high antioxidant activity.

Citric acid is found almost in all plant and animal species. It can chelate metal ions by

forming bonds between the metal and the carboxyl or hydroxyl groups of the citric acid

molecule (Anon., 1985). This acid can effectively retard the oxidative deterioration of

lipids in foods and is commonly added to vegetable oils after deodorization (Gordon,

1990). It shows some synergistic effect when used in combination with other natural

antioxidant extracts. A rosemary extract showed 2.61% synergistic effect with citric acid

while retarding rancidity in sunflower oil (Hras et al., 2000).

The antioxidant extracts of Emblica officinalis and Punica granatum are considered to

avoid oxidation in a food system. The synergistic effect of these extracts has been noted in

the present study. The descriptor of this effect has been the interaction (X1X2) of first two

variables Emblica officinalis and Punica granatum. Their interaction showed significant

effect on free fatty acids level at various storage intervals thus contributed towards

extended shelf life of food bars. The interactive term X1X2 also contributed a significant

role in improved sensory properties e.g. flavor, of food bar. The mechanisms by which

natural antioxidants are involved in the control of autoxidation processes are different. The

Emblica officinalis extract acts as radical scavenger, Punica granatum extract acts as

radical scavenger as well as ascorbic acid present in it acts as oxygen scavenger, whereas

citric acid act as a chelating agent (Kochhar and Rossell, 1990). Response surface

methodology adopted for the study exhibited this effect quite precisely.

To optimize the antioxidant levels for shelf stable food bar, Response Optimizer function

of statistical program Minitab (ver. 14) was used. For the optimization process maximum

taste, flavor and overall acceptability scores, whereas minimum free fatty acid contents

and peroxide values were targeted.

Free fatty acid level in a fat rich food during storage is a good predictor of shelf life of

that food. Response and contour plots (Fig. 4.3-4.5) showed that minimum free fatty acid

level achieved by the bars was 3.64 mg/g. The optimization process envisaged that the

shelf stable food bar with minimum free fatty acid could be achieved by incorporating

Emblica officinalis extract 0.95%, Punica granatum extract 2.5% and citric acid 0.06% at

desirability level of 1.

The fat rancidity as expressed by peroxide value has been a feature of prime importance

in optimization process of levels of independent variables. The surface and contours (Fig.

4.6-4.8) depicted that the minimum peroxide value (3.9 meq/kg) was achieved by adding

1.5% Emblica officinalis extract, 2.0% Punica granatum extract and 0.03% citric acid

during trail and was set as target for optimization. The upper limit for peroxide value was

set at 10 meq/kg and lower at 1 meq/kg. At the desirability level 1, it was predicted that

Emblica officinalis extract 0.95%, Punica granatum extract 2% and citric acid 0.06% can

achieve the targeted value.

As far as sensory characteristics of food bars are concerned, over the period of four months

storage, the taste, flavor and overall acceptability were the sensory parameters which

showed a declining trend in the absence or presence of low levels of independent variables.

Taste and flavor of food bars are directly related to development of free fatty acids and

peroxide value in the products (Rao et al., 1995; Elahi, 1997). The low scores for taste and

flavor simultaneously contributed towards a decrease in overall liking for the food bars.

Moisture gain is a factor which can contribute towards decline in overall acceptability of

cereal products, but it is not the case experienced here. The moisture gain is non-

significantly affected among various treatments over whole period of study which exhibited

integrated packaging conditions for all treatments. Therefore, parameters linked with

rancidity stood accountable for decrease in overall liking of food bars. The fat rancidity in

food bars has inverse relationship with presence of antioxidants and their dosage. There

exists a direct relationship between peroxide value and taste of food products. It has been

reported that at peroxide value below 10 meq/kg, no noticeable rancidity odor and taste

could be recognized by tasters (Gladovic et al., 1997).

The surface and counter plots for taste at 60 and 120 days (Fig. 4.9, 4.10, 4.11) show the

optimum levels of three independent variables i.e. Emblica officinalis extract, Punica

granatum extract and citric acid were used for achieving acceptable quality attributes for

shelf stable bars. Fig 4.9 depicted 5.5 as maximum score for the taste of nutrient dense

bars at 120 days storage. This value was selected as target value for taste attribute. The

optimized levels for three variables were generated as: Emblica officinalis extract 1.26%,

Punica granatum extract 1.64% and citric acid 0.06% at the desirability level 1. When

these values were put in regression model for taste at 120 days, the computed value

attained was 5.5 too.

The Figs 4.12 - 4.14 presented the surfaces and contours for the flavor attribute of

nutrient dense food bars at the mid (60 days) and end (120 days) of storage study. The

flavor attribute for food bars got 5.6 score at independent variables levels 0.75, 2.0 and

0.06% as well as 1.50, 2.0 and 0.03% for Emblica officinalis extract, Punica granatum

extract and citric acid, respectively. Taking 5.6 as target value, the optimized level of

independent variables has been calculated as Emblica officinalis extract 1.33% and

Punica granatum extract 1.80%, whereas the optimization process eliminated citric acid

from combination at high desirability level (1.0).

The overall acceptability is an important attribute for the selection of best combination of

independent variables for nutrient dense food bars with acceptable sensory perception.

The surfaces and contour plots for this attribute (Fig. 4.15 - 4.17) depicted that judges

allotted 5.5 score to the bar carrying Emblica officinalis extract 0.75%, Punica granatum

extract 2.0% and citric acid 0.06%. This value was set as the target value and optimized

response for overall acceptability of bars was generated, while considering 5.0 as lower

score and 6.0 as upper value in the calculation. The optimized levels of independent

variables were studied as Emblica officinalis extract 0.87%, Punica granatum extract

2.0% and citric acid 0.06% at desirability level of 1.

It is evident from the data that each attribute suggests different optimization level for

each independent variable. The response optimization function of Minitab program was

commissioned to reach a cumulative result. For the target values of taste, flavor, overall

acceptability, free fatty acid and peroxide level, the optimized variable levels at high

desirability (0.99) were as follows:

Emblica officinalis extract = 1.05%

Punica granatum extract = 1.85%

Citric acid = 0.059%

As far as other quality attributes are concerned the color of the nutrient dense food bars

was noted using a CIF-Lab uniform color space. L* indicated lightness a* showed

chromaticity on a green (-) to red (+) axis, and b* chromaticity on a blue (-) to yellow (+)

axis (Rocha and Morais, 2003). The numerical presentation of these values into hue angle

and chroma value is a norm (Francis, 1980). In this study positive values for a* and b*

were observed. These depicted the presence of red and yellow color in the bars. In

roasted ingredients of food bar a typical lightly reddish yellow shade was visible which

contributed towards similar instrumental readings. The storage intervals as well as

independent variable were unable to cause any change in food bar color. The change in

sensory color score during storage has been reported by some workers for cereal products.

Elahi (1997) reported a gradual decrease in color of biscuits prepared from wheat-chickpea

composite flour. Butt (2006) reported variation in sensory color score for cake rusk during

storage. However, in present study subjective attribute, the appearance, describing the color

of bars resulted in neither change during storage, nor objective analysis pertaining to color

i.e. hue angle and chroma value, showed a significant variation in data.

The incorporation of ingredients in a food product with antioxidant activity delays or

inhibits the rancidity onslaught. Red palm oil was found to keep cake rusks shelf stable

upto 28 days due to its high contents of tocopherols, trienols and carotenoids (Butt et al.,

2006). Turmeric, betel leaves, clove, lemon grass and Garcinia atriviridis extended the

shelf life of butter cake to 4 weeks in comparison to BHA contributing three weeks shelf

stability (Lean and Mohamad, 1999).

4.8.4. Conclusion

It has been observed that the rancidity problem in nutrient dense food bars could effectively

be addressed through incorporation of Emblica officinalis 1.05%, Punica granatum extract

1.86% and citric acid 0.059% in food bar formulation. Antioxidative effect of Emblica

officinalis and Punica granatum extracts is pronounced. Response surface methodology

was noted as best tool to distinguish the interactive effects of independent variables.

Chapter-5

Summary

Increas ing demand for nutri t ious meals and snacks , have prompted

the food researchers to develop new products that combine

convenience and nutr i t ion. Food bars have shown to be snack foods

of good sensory character is t ics due to their contents of prote ins ,

l ip ids and carbohydrates . To improve the prote in qual i ty of food

products d if ferent s trategies have been proposed . One of such

s trategy i s cereals and legumes complementat ion . This project

included th is concept to develop food bars wi th augmented prote in

qual i ty . The use of indigenous food process ing technologies such as

natural fermentat ion , control led fermentat ion and germinat ion

could enhance the ef fect iveness of this s trategy.

A cheap rather less invest igated source of h igh prote in port ion, the

Indian vetch (Lathyrus sa t ivus L. ) and a popular legume, the

ch ickpea (Cicer ar i tenum ) were se lected for augmentat ion . Various

process ing techniques i .e . control led fermentat ion, natural

fermentat ion and germinat ion were used to reduce the

ant inutri t ional factors i . e . tryps in inhibi tors , α -amylase inhib i tors ,

tannins , and phytates in these legumes and β -ODAP in Indian vetch

only . The prepared bars were character ized for the ir energy value ,

sensory at tr ibutes , in-v i t ro protein and starch digest ib i l i t ies .

Whereas , prote in ef f icacy s tudies including Prote in Digest ib i l i ty-

Corrected Amino Acid Score (PDCAAS) method was used to

evaluate the prote in qual i ty of the bars . This project was a lso

des igned to explore the potent ia l o f ant iox idant extracts from food

process ing waste materia ls on shel f s tabi l i ty of food bars . Dif ferent

ant iox idant extracts were f irs t ly screened for their act iv i ty and

then Response Surface Methodology (RSM) was used to establ ish

the re lat ionships between the ant iox idant extract levels and the

responses i .e . phys ico-chemical and sensory attr ibutes , re levant to

the s torage qual i ty during four months of s torage .

Statistical analysis for the effect of processing treatments on moisture and total ash

contents of Indian vetch and chickpea shows highly significant results. The moisture

level in the processed flours was less than the raw ones, whereas soaking showed the

minimum ash value under the influence of some leaching phenomenon. Similarly crude

fat level differed significantly. The raw flours of both legumes contained high fat level.

NFE varied notably in Indian vetch but not in chickpea. The processing of legumes

affected crude protein level to some extent. The minimum value for protein was noted for

natural fermentation, which could be attributed to nitrogen loss of the substrate during

processing. However crude fiber was at low level because of decortications of legumes

prior or afterward processing and was least affected.

The anti-nutritional factors were analyzed to investigate influence of processing treatment

on their level. Tannin and polyphenolics, both were affected by processing treatment and

least values for these were attained by the controlled fermentation process except for

phenolics level in Indian vetch. Here natural fermentation and germination showed low

values for tannin and phenolics. The polyphenol peroxidase is considered to play its role

in this regard. Similarly trypsin inhibitor activity and phytic acid results varied

considerably under the influence of processing of Indian vetch and chickpea. The

controlled fermentation showed best control of these two nutrients. The phytase could be

responsible for phytic acid reduction in this case. β-ODAP in Indian vetch was greatly

reduced by fermentation treatments than other processing treatments.

The mineral HCl-extractability (an index of bioavailability) of processed and raw Indian

vetch and chickpea flours was determined. It varied considerably in all mineral cases. For

copper, HCl-extractability values 62 and 55% were noted in controlled fermented

samples of both legumes, respectively, whereas for manganese, the HCl-extractability

noted for Mn was 84 and 71% in controlled fermented flours. Mg extractability of natural

fermented samples was increased from 41% in raw to 55% and 31% to 49% in Indian

vetch and chickpea respectively. Thus chickpea experienced higher gain in Mg HCl-

extractability. Sodium was changed marginally by processing and high HCl-extractability

for Na was attained by controlled fermentation followed by natural fermentation and

germination. In K, Zn, Fe and P, higher HCl-extractability values were achieved by

fermented and germinated samples at par to each other and lower ones by raw flours.

However a few exceptions also occurred, for instance, iron was affected less by natural

fermentation and germination than controlled fermentation processes in both legumes.

As the part of selection criteria, certain parameters were analyzed including in-vitro

protein and starch digestibilities, sensory characteristics, proximate composition and

calorific value of bars. In-vitro protein digestibility (IVPAD) and in-vitro starch

digestibility (IVSD) for nutrient dense bars were affected by the incorporation of

processed Indian vetch and chickpea flours. High IVPD values were achieved by food

bars carrying germinated chickpea flour whereas high IVSD was gained by processed

flours in both legumes rather than raw flours. It was noted that all sensory attributes

especially flavor, taste and overall acceptability deteriorated on the incorporation of

natural fermented flour in food bars. Whereas controlled fermented flour containing bars

were preferred most. The proximate composition of all the food bars differed non-

significantly except for crude protein content. High protein level was noted in the bars

carrying Indian vetch flours. The proximate composition data showed that Indian vetch

bars contributed protein 12.52-12.63% and fat 16.55-16.78%. However chickpea bars

contributed protein only 11.69-11.77%, marginally less than food bars with Indian vetch

flours. All the bars contained moisture level below 4%. This investigation showed that

these bars provided energy value 1909-1922 KJ/100 g which reflects that these food bars

are calorific dense as well.

Statistical analysis showed significant variation among food bar meals for all nutritional

indices. Feed intake, protein intake, weight gain and fecal protein output were the

parameters on the basis of which the nutritional indices were computed. All these differed

variably from each other. The rat groups fed on raw and natural fermented flour

containing bar meals performed poorly, however casein diet was at the top. For net

protein retention (NPR), both germinated and controlled fermented flour possessing bar

meals ranked high and were at par to each other. Similar trend was observed for PER. For

corrected and relative PER and relative NPR values, germinated food bar meals were

ranked at top. Relative PER and relative NPR values in close proximity to each other as

evident in the controlled fermented flours containing food bars are an indicator of good

quality protein.

The essential amino acid contents of all food bar meals were determined using HPLC

equipped with florescence detector. The food bar meals showed significant variation

among samples for isoleucine and sulfur containing amino acids i.e. methionine and

cysteine. High contents of these amino acids were studied in meals carrying chickpea

flours. When this amino acid profile was compared with amino acid requirement criteria

for 2-5 years old children, it was observed that for both kinds of meal groups this

criterion was comfortably matched. Statistical analysis of data exhibited significant

difference between results for true protein digestibility (TPD) and protein digestibility

corrected amino acid score (PDCAAS). High TPD was achieved by food bars carrying

controlled fermented Indian vetch and chickpea flour meals, followed closely by

germinated flour possessing meals. The computed PDCAAS value was 1 for

aforementioned meals whereas this value was observed around 0.98 for meals containing

germinated flour.

Food processing waste materials were extracted using different solvents to get the

antioxidant extracts. The statistical analysis showed variation among solvents towards

ease to extract antioxidants. Ethanol 80% solution in water was found effective in

extracting all materials from yield point of view. Total polyphenol content of extracts

varied from each other at high pace. Mean value for pomegranate peal extract was higher

than other material’s extracts. Similarly the extraction through ethanol and 80% ethanol

solution yielded best polyphenol level. Statistical analysis of data showed considerable

variation among treatments for DPPH scavenging activity as well as β-carotene bleaching

assay. Higher values of DPPH scavenging were achieved by pomegranate peel among all

solvent extracts, whereas β-carotene bleaching capacity was also much better in

pomegranate peel extracts than extracts from other materials.

The food bars were prepared taking into account the best formulation, incorporating

fermented Indian vetch flour along cereal meal. Emblica officinalis extract (X1), Punica

granatum peel extract (X2) and citric acid (X3) at different levels were incorporated for

investigating their impact on shelf stability of food bars. This combination resulted in

maximum taste, flavor and overall acceptability scores, whereas minimum free fatty acid

content and peroxide value were targeted for optimization process. The results carrying

such kind of results were possessing upto 1.0 desirability scores whereas undesirable

results scored even down to 0.0. Second-order polynomial models were fitted for

independent variables on storage data at various intervals regarding dependent

parameters. High coefficient of determination (R2) values for models (>50%) showed the

adequacy of these models.

Free fatty acids and peroxide values of nutrient dense food bars decreased with an

increase in Emblica officinalis (X1) and Punica granatum (X2) levels. Response and

contour plots depicted that minimum free fatty acid level achieved by the bars was 3.64

mg/g. The optimization process envisaged that the shelf stable food bar with low free

fatty acid content could be produced by incorporating Emblica officinalis extract

0.95%, Punica granatum extract 2.5% and citric acid 0.06%.

The effect of changes in levels of independent variables on peroxide value of food bars

over 0, 60 and 120 days storage intervals exhibited a sharp decline in peroxide value

under the influence of Emblica officinalis, Punica granatum extracts and citric acid. The

surface and contours depicted that the minimum peroxide value (3.9 meq/kg) was

achieved by adding 1.5% Emblica officinalis extract, 2.0% Punica granatum extract and

0.03% citric acid during trail and was set as target for optimization. At the desirability

level 1, it was predicted that Emblica officinalis extract 0.95%, Punica granatum extract

2% and citric acid 0.06% can achieve the targeted value.

The sensory data for appearance, texture, taste and flavor of food bars remained non-

significantly varying. However, linear terms for X1 and X2 and the interactive term of

these two variables (X1X2) significantly affected the flavor over 30, 60, 90 and 120

days storage intervals. The flavor attribute for food bars got 5.6 score at independent

variables levels 0.75, 2.0 and 0.06% as well as 1.50, 2.0 and 0.03% for Emblica

officinalis extract, Punica granatum extract and citric acid, respectively. Taking 5.6 as

target value, the optimized level of independent variables has been calculated as

Emblica officinalis extract 1.33% and Punica granatum extract 1.80%, whereas the

optimization process eliminated citric acid from combination at high desirability level

(1.0).

The linear term X1 has significant effect on taste by its role towards rancidity avoidance

in food bars and scored better for this attribute at later storage intervals (60, 90 and 120

days). The surface and counter plots for taste depicted 5.5 as maximum score for the

taste of nutrient dense bars at 120 days storage. This value was selected as target value

for taste attribute. The optimized levels for three variables were generated as: Emblica

officinalis extract 1.26%, Punica granatum extract 1.64% and citric acid 0.06% at the

desirability level 1.

Analysis of variance for full regression models for overall acceptability of bars showed

significant results for 60, 90 and 120 days intervals. It is noted that first two variables

have a positive effect during these storage intervals. The quadratic terms of X1 also

produced significant but negative effect. The surfaces and contour plots for this attribute

depicted that judges allotted 5.5 score to the bar carrying Emblica officinalis extract

0.75%, Punica granatum extract 2.0% and citric acid 0.06%. This value was set as the

target value and optimized response for overall acceptability of bars was generated. The

optimized levels of independent variables were studied as Emblica officinalis extract

0.87%, Punica granatum extract 2.0% and citric acid 0.06% at desirability level of 1.

It is evident from the data that each attribute suggests different optimization level for

each independent variable. It has been observed that the rancidity problem in nutrient

dense food bars could effectively be addressed through incorporation of Emblica

officinalis 1.05%, Punica granatum extract 1.86% and citric acid 0.059% in food bar

formulation.

Conclusions

The processing of legumes through controlled fermentation and germination

improved nutritional and sensory properties of legume flours.

β-ODAP a big concern factor in vetch was effectively reduced through

fermentation and germination.

The mineral HCl-extractability (an index of mineral bioavailability) of legume

flours increased through fermentation and germination.

The food bars with incorporation of fermented vetch flour exhibited good

biological values as well as sensory perception.

The PDCAAS value of 1.0 was achieved for food bars with controlled fermented

and germinated vetch and chickpea flour meals.

The pomegranate peel extract exhibited better antioxidant properties among

extracts from food processing waste materials.

The food bars were produced with extended shelf life through addition of Punica

granatum, Emblica officinalis extracts and citric acid.

The synergistic effect of Punica granatum and Emblica officinalis extracts was

evident from study.

Recommendations

The under utilized legume crops should be explored for their extensive food use.

Strategies should be devised to reduce the anti-nutritional factors in underutilized

legume crops.

The natural antioxidant extracts should be investigated for their technological and

functional benefits especially for their use in fat rich food products.

A range of food bars should be developed using indigenous resources with

enhanced consumer’s acceptability to nourish the masses.

References AACC. 2000. Approved Methods of American Association of Cereal Chemists. The

American Association of Cereal Chemists, Inc. St. Paul. Minnesota.

Abdelrahaman, S.M., H.B. Elmaki, W.H. Idris, A.B. Hassan, E.E. Babiker and A.H. El-Tinay. 2007. Antinutritional factor content and hydrochloric acid extractability of minerals in pearl millet cultivars as affected by germination. Int. J. Food Sci. Nutr. 58:16-17.

Abegaz, B.M., G. Alemayehu and Y. Yigzaw. 1997. Lathyrus and Lathyrism, a Decade of Progress, Addis Ababa, Ethiopia; University of Ghent, Ghent, Belgium. p. 75-77.

Abid, A.A., M. Anwar, S. Rasool and M.E. Babar. 1991. Effect of different levels of gram flour supplementation on the nutritive improvement of wheat flour. Sarhad J. Agric. 7(2):27-31.

Acosta, O., F. Viquez, E. Cubero and I. Morales. 2006. Ingredient levels optimization and nutritional evaluation of a low-calorie blackberry (Rubus irasuensis Liebm.) jelly. J. Food Sci. 71(5):S390-S394.

Adams, L.S., N.P. Seeram, B.B. Aggarwal, Y. Takada, D. Sand and D. Heber. 2006. Pomegranate juice, total pomegranate ellagitannins and punicalagin suppress inflammatory cell signaling in colon cancer cells. J. Agric. Food Chem. 54:980-985.

Adebooye, O.C. and V. Singh. 2007. Effect of cooking on the profile of phenolics, tannins, phytate, amino acid, fatty acid and mineral nutrients of whole-grain and decorticated vegetable cowpea (Vigna unguiculata L. Walp). J. Food Qual. 30(6):1101-1120.

Adebowale, Y.A., A. Adeyemi and A.A. Oshodi. 2005. Variability in the physicochemical and antinutritional attributes of six Mucuna species. Food Chem. 89:37-48.

Ajah, P.O. and F.N. Madubuike. 1997. The proximate composition of some tropical legume seeds grown in two states in Nigeria. Food Chem. 59(3):361-365.

Akalu, G., G. Johansson and B.M. Nair. 1998. Effect of processing on the content of β-N-oxalyl-α,β-diaminopropionic acid (β-ODAP) in grasspea (Lathyrus sativus) seeds and flour as determined by flow injection analysis. Food Chem. 62 (2):233-237.

Akenson, W. R. and M.A. Stahmann. 1964. A pepsin pancreatin digest of protein quality evaluation. J. Nutr. 83:257-264.

Akinyele, I.O. and A. Akinlosotu. 1999. Effect of soaking, dehulling and fermentation on the oligosaccharides and nutrient content of frijol (Vigna unguiculata). Food Chem. 41:43-53.

Akobundu, C.N., C.N. Ubbaonu and C.E. Ndupuh. 1988. Studies on the baking potencial of non-wheat composite flours. J. Food Sc. Tech. 25(4):211-214.

Akubor, P.I. 2004. Protein contents, physical and sensory properties of Nigerian snack foods (cake, chin-chin and puff-puff) prepared from cowpea-wheat flour blends. Int. J. Food Sci. Tech. 39(4):419-424.

Alam, M.I. and A. Gomes. 2003. Snake venom neutralization by Indian medicinal plants (Vitex negundo and Emblica officinalis) root extract. J. Ethnophar. 86:75-80.

Al-Hooti S., J.S. Sidhu, J. Alotaibi, H. Alameeri and H. Qabazard. 1997. Date bars fortified with almonds, sesame seeds, oat flakes and skim milk powder. Plant Foods Hum. Nutr. 51: 125–135.

Alizadeh M., M. Hamedi and A. Khosroshahi. 2005. Optimizing sensorial quality of Iranian white brine cheese using response surface methodology. J. Food Sci. 70(4):S299-303.

Alonso, R., E. Orue and F. Marzo. 1998. Effects of extrusion and conventional processing methods on protein and antinutritional factor contents in pea seeds. Food Chem. 63(4):502-512.

Al-Rehaily, A.J., T.A. Al-Howiriny, M.O. Al-Sohaibani and S. Rafatullah. 2002. Gastroprotective effects of “amla” Emblica officinalis on in vivo test models in rats. Phytomed. 9:515-522.

Al-Saikhan, M.S., L.R. Howard and J.C. Miller Jr. 1995. Antioxidant activity and total phenolics in different genotypes of potato (Solanum tuberosum L.). J. Food Sci. 60:341-343, 347.

Ammawath, W., Y.B.C. Man, S. Yusof and R.A. Rahman. 2002. Effects of type of packaging material on physicochemical and sensory characteristics of deep-fat-fried banana chips. J. Sci. Food Agric. 82(14):1621-1627.

Anderson J.C., A.O. Idowu, U. Singh and B. Singh. 1994. Physicochemical characteristics of flours of faba bean as influenced by processing methods. Plant Foods Hum. Nutr. 45(4):371-379.

Anderson, J.C. and B.D. Jones. 1999. Principal factor analysis of extruded sorghum and peanut bar changes during accelerated shelf-life studies. J. Food Sci. 64(6):1059-1063.

Anon. 1985. Ullmann's encyclopedia of industrial chemistry (5th ed.). VCH, Weinheim. p. 103-108.

Antony, U. and T. S. Chandra. 1998. Antinutrient reduction and enhancement in protein, starch and mineral availability in fermented flour of finger millet (Eleusine coracana). J. Agric. Food Chem. 46 (7):2578-2582.

Anwar, M. 1980. Studies on the determination of optimum level of gram flour supplementation on the nutritive value of wheat protein. M.Sc. Thesis. Dept. Food Technol., Univ. Agri., Faisalabad, Pakistan.

AOAC. 2000. Official methods of analysis. 17th ed., Assn. of Official Analytical Chemists. Arlington, VA, USA.

Arnaud, C.D. and S.D. Sanchez. 1996. Calcium and phosphorus. p. 245-255. In J. E. Zigler and L. J. Filer (eds.) Present Knowledge in Nutrition. 7th ed. ILSI Press, Washington, D. C.

Arts, I.C.W. and P.C.H. Hollman. 2005. Polyphenols and disease risk in epidemiologic studies. Am. J. Clin. Nutr. 81(Suppl):317S-325S.

Arvola, A., L. Lahteenmaki and H. Tuorila. 1999. Predicting the intent to purchase familiar and unfamiliar cheeses: the effect of attitudes, expected liking and food neophobia. Appetite. 32(1):113-126.

Asante, I.K., H. Adu-Dapaah and P. Addison. 2004. Seed weight and protein and tannin contents of 32 cowpea accessions in Ghana. Trop. Sci. 44(2):77-79.

Asp, N.G. 1995. Classification and methodology of food carbohydrates as related to nutritional effects. Am. J. Clin. Nutr. 61(suppl):930S-937S.

Attia, R.S., A.M.E.T. Shehata, M.E. Aman and M.A. Hamza. 1994. Effect of cooking and decortication on the physical properties, the chemical composition and the nutritive value of chickpea (Cicer arietinum L.). Food Chem. 50:125-131.

Aviram, M., L. Dorafeld, M. Rosenblat, N. Volkova, M. Kaplan, R. Coleman, T. Hayek, D. Presser and B. Fuhrman. 2000. Pomegranate juice consumption reduces oxidative stress, atherogenic modifications to LDL and platelet aggregation: studies in humans and in atherosclerotic apolipoprotein E-deficient mice. Am. J. Clin. Nutr. 71:1062-1076.

Aw, T.L. and B.G. Swanson. 1985. Influence of tannin on Phaseolus vulgaris protein digestibility and quality. J. Food Sci. 50:67-71.

Awan, J.A., M.I. Siddique and A.H. Gilani. 1996. Fortification of wheat flour. Pak. J. Food Sci. 6:39-50.

Azeke, M.A., B. Frtzdorff, H. Buening-Pfaue, W. Holzapfel and T. Betsche. Nutritional value of African yam bean (Sphenostylis stenocarpa L.): Improvement by lactic acid fermentation. J. Sci. Food Agric. 85(6):963-970.

Babar, V.S., J.K. Chavan and S.S. Kadam. 1988. Effects of heat treatments and germination on trypsin inhibitor activity and polyphenols in jack bean. Plant Foods Hum. Nutr. 38:319-324.

Barroga, F.B., Laurena, A.C. and E.M.T. Mendoza. 1985. Polyphenols in mungbean; determination and removal. J. Agric. Food Chem. 33:1006-1009.

Barry J.L., J.M. Chourot, F. Kozlowski and A. David. 1989. In vitro fermentation of neutral monosaccharides by ruminal and human fecal microflora. Acta. Vet. Scand. S86:93-95.

Bartolome, B., I. Estrella and T. Hernaandez. 1997. Changes in phenolic compounds in lentils (Lens culinaris) during germination and fermentation. Z. Leben. Unters. Forsch. A. 205:290-294

Batra, V.I.P., R. Vasishta and K.S. Dhindsa. 1986. Effect of heat and germination on trypsin inhibitor activity in lentil and pigeon pea. J. Food Sci. Technol. 23:260-263.

Beaumont, M. 2002. Flavouring composition prepared by fermentation with Bacillus spp. Int. J. Food Microbiol. 75:189-196.

Bejarano, P.I.A., N.M.V. Montoya, E.O.C. Rodríguez, J.M. Carrillo, R.M. Escobedo, J.A.L. Valenzuela, J.A.G. Tiznado and C.R. Moreno. 2008. Tempeh flour from chickpea (Cicer arietinum L.) nutritional and physicochemical properties. Food Chem. 106(1):106-112.

Bellido, L.L. 1994. Grain legumes for animal feed. p. 273-288. In J.E.H. Bermejo and J. León (eds.) Neglected crops:1492 from a different perspective. FAO Plant Production and Protection Series No. 26. FAO, Rome.

Ben, N.C., N. Ayed and M. Metche. 1996. Quantitative determination of the polyphenolic content of pomegranate peel. Z. Lebens. Unter. Fors. A. 203:374-378.

Bender, A.E. 1978. Food processing and nutrition. Academic Press, London. p. 219-254.

Beta, T., L.W. Rooney, L.T. Marovatsanga and J.R.N. Taylor. 2000. Effect of chemical treatments on polyphenols and malt quality in sorghum. J. Cereal Sci. 31:295-302.

Bhagya, B., K.R. Sridhar and S. Seena. 2006. Biochemical and protein quality evaluation of tender pods of wild legume Canavalia cathartica of coastal sand dunes. Live. Res. Rural Develop. 18:1-20.

Bhatia, A. and N. Khetarpaul. 2002. Effect of fermentation on phytic acid and in vitro availability of calcium and iron of `doli ki roti'—an indigenously fermented indian bread. Ecol. Food Nutr. 41(3):243-253.

Bhattacharya, A., A. Kumar, S. Ghosal and S.K. Bhattacharya. 2000. Effect of bioactive tannoid principles of Emblica officinalis on iron-induced hepatic toxicity in rats. Phytomedicine. 7(2):173-175.

Bhattacharya, S.K., A. Bhattacharya, K. Sairam and S. Ghosal. 2002. Effect of bioactive tannoid principles of Emblica officinalis on ischemia-reperfusion-induced oxidative stress in rat heart. Phytomedicine. 9(2):171-174.

Bhatty, N., A.H. Gilani and S.A. Nagra. 2000. Effect of cooking and supplementation on nutritional value of gram (Cicer atietinum). Nutr. Res. 20(2):297-307.

Biesalski, H.K. and P. Grimm. 2005. Pocket atlas of nutrition. Georg Thieme Verlag, Stuttgart, Germany.

Bilyk, A., P.L. Cooper and G.M. Sapers. 1984. Varietal differences in distribution of quercetin and kaempferol in onion (Allium cepa L.) tissue. J. Agric. Food Chem. 32:274-276.

Bishnoi, S. and N. Khetarpaul. 1994. Protein digestibility of vegetable and field peas (Pisum sativum): varietal differences and effect of domestic processing and cooking. Plant Foods Hum. Nutr. 46:71-76.

Block, G., B. Patterson and A. Subar. 1992. Fruit, vegetables and cancer prevention:a review of the epidemiological evidence. Nutr. Cancer. 18:1-29.

Boisen, S. and R. Djurtoft. 1982. Protease inhibitor from barley embryo inhibiting trypsin and trypsin-like microbial proteases. Purification and characterisation of two isoforms. J. Sci. Food Agric. 33(5):431-440.

Boralkar, M. and N.S. Reddy. 1985. Effect of roasting, germination and fermentation on the digestiblity of starch and protein present in soy bean. Nutr. Rep. Int. (USA). 31(4):833-836.

Bos, C., C. Gaudichon and D. Tomé. 2000. Nutritional and physiological criteria in the assessment of milk protein quality for humans. J. Am. Coll. Nutr. 19:191S-205S.

Boustani P. and V.W. Mitchell. 1990. Cereal bars: a perceptual, chemical and sensory analysis. Br. Food J. 92(5):17-22.

Bower, J.A. and R. Whitten. 2000. Sensory characteristics and consumer liking for cereal bar snack foods. J. Sens. Stud. 15(3):327-345.

Box, G.E.P. and N.R. Draper.1986. Empirical model-building and response surface, John Wiley and Sons, Inc., New York.

Box, G.E.P., W.G. Hunter and J.S. Hunter. 1978. Statistics for experimenters. An introduction to design, data analysis and model building. John Wiley and Sons. New York. p. 672.

Bressani, R. 1993. Grain quality of common beans. Foods Rev. Int. 9:237-297.

Bressani, R. and J.L. Sosa. 1990. Effect of processing on the nutritive value of Canavalia Jackbeans (Canavalia ensiformis L.). Plant Foods Hum. Nutr. 40:207-214.

Bressani, R. and L.G. Elias. 1980. The nutritional role of polyphenols in beans. p. 61. In J.H. Hulse (ed.) Polyphenols in cereals and legumes. International Development Research Center, Ottawa, Canada.

Bressani, R., B.R. Gomez, A. Garcia and L.G. Elias. 1987. Chemical composition, amino acid content and protein quality of Canavalia seeds. J. Sci. Food Agric. 40:17-23.

Brisske L. K., S.Y. Lee, B.P. Klein and K.R. Cadwallader. 2004. Development of a prototype high-energy, nutrient-dense food product for emergency relief. J. Food Sci. 69(9):S361-S367.

Butt M.S., J. Rasool and K. Sharif. 2006. Preparation and characterisation of cake rusks by using red palm oil fortified shortening. Food Sci. Tech. Int. 12(1):85–90.

Cabrera C., F. Lloris, R. Gimenez, M. Olalla and M.C. Lopez. 2003. Mineral content in legumes and nuts: contribution to the Spanish dietary intake. Sci. Total Environ. 308:1-14

Cadenas, E. and L. Packer. 2002. Handbook of antioxidants. Marcel Dekker Inc., NY.

Cai, W.T., Z.X. Li and Y.Z. Chen. 1984. Studies on Lathyrus sativus. J. Lanzhou Univ. (Nat. Sci.) 20:79-85.

Campbell, C.J. and C.J. Briggs, 1987. Registration of low neurotoxin content Lathyrus germplasm LS8246. Crop Sci. 27:821.

Cao, G., H.U. Muccitelli, C. Sanchez-Moreno and R.L. Prior. 2001. Anthocyanins are absorbed in glycated forms in elderly women:a pharmacokinetic study. Am. J. Clin. Nutr. 73:920-926.

Chandrasiri, V., H.M. Bau, C. Villaume, F. Giannangeli, D. Lorient and L. Mejean. 1987. Effet de la germination de la graine de soja sur la composition et la valeur nutritionnelle de sa farine. Sci. Aliments. 7:139-150.

Chapman, G. and H.H. Mitchell. 1959. Evaluation of protein in foods: a method for the determination of protein efficiency ratio. Canad. J. Biochem. Phys. 37:679.

Chau, C.F. and P.C.K. Cheung. 1997. Effect of various processing methods on antinutrients and in vitro digestibility of protein and starch of two Chinese indigenous legume seeds. J. Agric. Food Chem. 45:4773-4776.

Chaudhuri, R.K. 2004. Standardised extract of Phyllanthus emblica: a skin lightener with anti-aging benefits. Proc. PCIA Conference, Guangzhou, China 9-11 March 2004.

Chavan, U.D., F. Shahidia and M. Naczkb. 2001. Extraction of condensed tannins from beach pea (Lathyrus maritimus L.) as affected by different solvents. Food Chem. 75:509-512

Cheftel, J.C., J.L. Cuq and D. Lorient. 1996. Amino acids, peptides and proteins. In O.R. Fennema (ed.) Food chemistry. Marcel Dekker Inc., NY.

Chen, C.Y., P.E. Milbury, K. Lapsley and J.B. Blumberg. 2005. Flavonoids from almond skins are bioavailable and act synergistically with vitamins C and E to enhance hamster and human LDL resistance to oxidation. J. Nutr. 135(6):1366-1373.

Chen, Y.Z., Z.X. Li, F.H. Lu, X.G. Bao, S.Z. Liu, X.C. Liu, G.W. Zhang and Y.R. Li. 1992. Studies on the screening of low toxic species of Lathyrus, analysis of toxins and toxicology. J. Lanzhou Univ. (Nat. Sci.) 28:93-98.

Chen, Z.U., Q.Y. Zhu, D. Tsang and Y. Huang. 2001. Degradation of green tea catechins in tea drinks. J. Agric. Food Chem. 49:477-482.

Chidamabaramurthy, K.N., G.K. Jayaprakasha and R.P. Singh. 2002. Antioxidant activity of pomegranate peel extracts in vivo models. J. Agric. Food Chem. 50:4791-5.

Chitra U., U. Singh and V.P. Rao 1996. Phytic acid, in vitro protein digestibility, dietary fiber, and minerals of pulses as influenced by processing methods. Biomed. Life Sci. 49(4):307-316.

Chompreeda, P.T. and M. Fields. 1984. Effect of heat and natural fermentation on amino acids, flatus producing compounds, lipid oxidation and trypsin inhibitor in blends of soybean and cornmeal. J. Food Sci. 49:563-565.

Christensen, J.E., D. Lin, A. Palva and J.L. Steele. 1995 Sequence analysis, distribution and expression of an aminopeptidase N-encoding gene from Lactobacillus helveticus CNRZ32. Gene. 155:89-93.

Cohn, D.F. and M. Streifler. 1983. Intoxication by chickling pea (Lathyrus sativus): nervous system and skeletal findings. Arch. Toxic. 53(6):190-193.

Cornell, J.A. 1990. Experiments with mixtures:design, models and the analysis of mixture data. 2nd ed. Wiley, New York, USA.

Cousins, R.J. 1996. Zinc. p. 293-306. In E.E. Ziegler and L.J. Filer Jr. (eds.). Present knowledge in nutrition. 7th ed. ILSI Press. Washington, DC.

Cuadrado C., G. Ayet, L.M. Robredo, J. Tabera, R. Villa, M.M. Pedrosa, C. Burbano and M. Muzquiz. 1996. Effect of natural fermentation on the content of inositol phosphates in lentils. Z. Lebensm. Unters. Forsch. A. 203(3):268-271.

Dalby, A. and C.Y. Tsai. 1976. Lysine and tryptophan increases during germination of cereal grains. Cereal Chem. 53:222-226.

Danial, V.A., R. Leela, S.V. Rao, K. Hariharem, K. Indiramma, M. Swaminathan and H.A.B. Parpia. 1964. Mutual and amino acid supplementation of proteins. Nutritive value of the proteins of blends of wheat, groundnut, soybean, gram, sesame and skim milk powder fortified with limiting essential amino acid. J. Nutr. Diet. 1:293-298.

de Leon J.M., M.L.C. Nosthas and C.P. Salomon. 2000. Obtaining a fermented chickpea extract (Cicer arietinum L.) and its use as a milk extensor. Arch. Latin. Nutr. 50(2):157-63.

de Paula H., R.C. Santos, M.E. Silva, E.C.S. Glória, M.L. Pedrosa, N.A.V. Almeida, A.S.V. Costa and L.C.C. Malaquias. 2004. Biological evaluation of a nutritional supplement prepared with QPM maize cultivar BR 473 and other traditional food items. Braz. Arch. Biol. Technol. 47(2):247-251.

de Penna E.W., A. Bunger, M. Sansur, L. Lopez and R. Santana. 1993. Development of soy-based protein candy bars for athletes. Arch. Latin. Nutr. 43(3):241-7.

de Penna E.W., A. Infante, A. Suarez, L. Lopez, R. Santana and H. Torti. 1992. Development of an energy food for athletes. Arch. Latin. Nutr. 42(3):331-44.

de Poll, M.C.G.V., Y.C. Luiking, C.H.C. Dejong and P.B. Soeters. 2005. Encyclopedia of Human Nutrition. p. 92-100. B. Caballero, L. Allen and A. Prentice (eds.) Amino acids/specific functions. Elsevier Ltd., Oxford, UK.

Dean, A.M. and D.T. Voss. 1999. Design and analysis of experiments. Springer-Verlag, New York. p 740.

Demirekler, P., G. Sumnu and S. Sahin. 2004. Optimization of bread baking in a halogen lamp-microwave combination oven by response surface methodology. Eur. Food Res. Technol. 219:341-347.

Derringer, G. and R. Suich. 1980. Simultaneous optimization of several response variables. J. Qual. Tech. 12(4):214-19.

Deshpande, S.S. and M. Cheryan. 1983. Changes in the phytic acid, tannin and trypsin inhibitor activity on soaking of dry beans. Nutr. Rep. Int. 27:371-378.

Deshpande, S.S. and S. Damodaran. 1990. Food legumes: chemistry and technology. Adv. Cereal Sci. Technol. 10:147.

Deshpande, S.S. and S.S. Nielsen. 1987. In vitro digestibility of dry bean (Phaseolus vulgaris L.) proteins: the role of heat-stable protease inhibitors. J. Food Sci. 52:1330-1334.

Desphande, S. and M. Cheryan. 1983. Changes in the phytic acid, tannins and trypsin inhibitor activity on soaking of dry beans (Phaseolus vulgaris L.). Nutr. Rep. Int. 27:371-377.

Dewey, K.G., G. Beaton, C. Fjeld, B. Lonnerdal and P. Reeds. 1996. Protein requirements of infants and children. Eur. J. Clin. Nutr. 50:S119-S150.

Dey, P.M., 1985. D-Galactose containing oligosaccharides. p. 53-129. In P.M. Dey (ed.) Biochemistry of storage carbohydrates in green plants. Academic Press, London, UK.

Dhankher, N. and B.M. Chauhan. 1987. Effect of temperature and fermentation time on phytic acid and polyphenol content of Rabadi- a fermented pearl millet food. J. Food Sci. 52:828-829.

Dhiman, T.R., V.K. Sharma and M.P. Narang. 1983. Evaluation of ‘Khesari dhal’ (Lathyrus sativus) in calf starter. Agric. Wastes. 8:1-8.

Dhurandhar, N.V. and K.C. Chang. 1990. Effect of cooking on firmness, trypsin inhibitors, lectins and cystinekysteine content of navy and red kidney beans (Phaseolus vulgaris). J. Food Sci. 55:470-474.

Diawara, B., L. Sawadogo, W.K.A. Amoa-Awua and M. Jakobsen. 1998. Quality system for the production of soumbala. The HACCP System. Waitro, Taastrup.

Dinelli, G., A. Bonetti, M. Minelli, I. Marotti, P. Catizone and A. Mazzanti. 2006. Content of flavonols in Italian bean (Phaseolus vulgaris L.) ecotypes. Food Chem. 90:105-114.

Dipietro, C.M. and I.E. Liener. 1989. Heat inactivation of the kunitz and Bowman Birk soybean protease inhibitors. J. Agric. Food Chem. 37:39-44.

Doblado, R., J. Frias, R. Munoz and C. Vidal-Valverde. 2003. Fermentation of Vigna sinensis var. carilla flours by natural microflora and Lactobacillus species. J. Food Prot. 66(12):2313-2320.

Donangelo, C.M., L.C. Trugo, M.F. Trugo and B.O. Eggum. 1995. Effect of germination of legume seeds on chemical composition and on protein and energy utilization in rats. Food Chem. 53:23-27.

Dorado, R.G., A.E.A. Rodríguez, J.M. Carrillo, J.L. Cervantes, J.A.G. Tiznado, J.A.L. Valenzuela, O.P. López and C.R. Moreno. 2008. Technological and nutritional properties of flours and tortillas from nixtamalized and extruded quality protein maize (Zea mays L.). 85(6):808-816.

Duhan A., N. Khetarpaul and S. Bishnoi. 2002. Changes in phytates and HCl-extractability of calcium, phosphorus and iron of soaked, dehulled, cooked and sprouted pigeon pea cultivar (UPAS-120). Plant Foods Hum. Nutr. 57:275-284.

Duhan A., N. Khetarpaul and S. Bishnoi. 2002. Content of phytic acid and HCl-extractability of calcium, phosphorus and iron as affected by various domestic processing and cooking methods. Food Chem. 78:9-14.

Duhan A., N. Khetarpaul and S. Bishnoi. 2004. HCl-extractability of zinc and copper as affected by soaking, dehulling, cooking and germination of high yielding pigeon pea cultivars. J. Food Comp. Anal. 17(5):597-604.

Dvorakova, J., O. Volfova and J. Kopecky. 1997. Characterization of phytase produced by Aspergillus niger. Folia. Microbiol. 42:349-352.

Ejeta, G., M.M. Hassen and E.T. Mertz. 1987. In vitro digestibility and amino acid composition of pearl millet (Pennisetum typhoides) and other cereals. Proc. Natl. Acad. Sci. USA. 84:6016-6019.

Ekanayake, S., E.R. Jansz and B.M. Nair. 2000. Nutritional evaluation of protein and starch of mature Canavalia gladiata seeds. Int. J. Food Sci. Nutr. 51:289-294.

Eklund-Jonsson C., A.S. Sandberg and M.L. Alminger. 2006. Reduction of phytate content while preserving minerals during whole grain cereal tempe fermentation. J. Cereal Sci. 44(2):154-160.

El Faki H.A., L.V. Venkatarama and H.S.R. Desikachar 1984. Effect of processing on the in vitro digestibility of proteins and carbohydrates in some Indian legumes. Plant Foods Hum. Nutr. 34(2):127-133.

El-Adawy, T.A. 2002. Nutritional composition and anti-nutritional factors of chickpeas (Cicer arietinum L.) undergoing different cooking methods and germination. Plant Food Hum. Nutr. 57:83-97.

Elahi, H.H. 1997. Use of emulsifier for the production of biscuits from composite flour. MSc Thesis, Department of Food Technology, University of Agriculture, Faisalabad, Pakistan.

Elkhalil, E.A.I., A.H. El-Tinay, B.E. Mohamed and E.A.E. Elsheikh. 2000. Effect of malt pretreatment on phytic acid and in vitro protein digestibility of sorghum flour Department of Food Science and Technology, Faculty of Agriculture, University of Khartoum, Shambat, Sudan.

El-Mahdy, A.R., E.K. Moustafa and M.S. Mohamed. 1981. Trypsin inhibitor activity in Vicia faba beans. Food Chem. 7:63-71.

ElMaki, H.B., S.M. AbdelRahaman, W.H. Idris, A.B. Hassan, E.E. Babiker and A.H. El Tinay. 2007. Content of antinutritional factors and HCl-extractability of minerals from white bean (Phaseolus vulgaris) cultivars: influence of soaking and/or cooking. Food Chem. 100(1):362-368.

Englyst, H.N., S.M. Kingman and J.H. Cummings. 1992. Classification and measurement of nutritionally important starch fractions. Eur. J. Clin. Nutr. 46:S33-S50.

Escobar, B., A.M. Estevez and M.A. Guinez. 2000. Storage of cereal bars with mezquite cotyledon (Prosopis chilensis (Mol) Stuntz). Arch. Latin. Nutr. 50(2):152-6.

Escobar, B., A.M. Estevez, A. Tepper and M. Aguayo. 1998. Nutritional characteristics of cereal and peanut bars. Arch. Latin. Nutr. 48(2):156-159.

Escobar, B., A.M. Estevez, M. Vasquez, E. Castillo and E. Yanez. 1994. Cereal bars with peanut and puffed amaranthus: chemical composition and stability in accelerated storage. Arch. Latin. Nutr. 44(1):36-40.

Estevez, A.M., B. Escobar and V. Ugarte. 2000. Use of mezquite cotyledon (Prosopis chilensis (Mol) Shuntz) in the manufacturing of cereal bars. Arch. Latin. Nutr. 50(2):148-51.

Estévez, A.M., B. Escobar, M. Vásquez, E. Castillo, E. Araya and I. Zacarías. 1995. Cereal and nut bars, nutritional quality and storage stability. Plant Foods Hum. Nutr. 47(4):309-317

Ezeagu, I.E., B. Maziya-Dixon and G. Tarawali. 2002. Seed characteristics and nutrient and antinutrient composition of 12 Mucuna accessions from Nigeria. Trop. Subtrop. Agroeco. 1(2/3):129-139.

Fagbemi, T.N., A.A. Oshodi and K.O. Ipinmoroti. 2005. Processing effects on some antinutritional factors and in vitro multienzyme protein digestibility (IVPD) of three tropical seeds: breadnut (Artocarpus altilis), cashewnut (Anacadium occidentale) and fluted pumpkin (Telfairia occidentalis). Pak. J. Nutr. 4:250-256.

Fageer, A.S.M., E.E. Babikr and A.E. El Tinay. 2004. Effec of malt pretreatment and/or cooking on phytate and essential amino acids contents and in vitro protein digestibility of corn flour. Food Chem. 88:261-265.

FAO/WHO. Food and Agriculture Organization-World Health Organization. 1991. Protein Quality Evaluation. Report of a Joint FAONHO expert consultation, Food and Nutrition Paper 51, FAO, Rome.

FAO/WHO/UNU. Food and Agriculture Organization-World Health Organization-United Nations University. 1985. Energy and protein requirements, report of a joint FAO/WHO/UNU expert consultation. WHO Tech. Rep. Ser. 724, WHO, Geneva, Switzerland.

Faris, M.A.E. and H.R. Takruri. 2002. Study of the effect of using different level of taninah (sesame butter) on the protein digestibility-corrected amino acid score (PDCAAS) of chickpea dip. J. Sci. Food Agric. 83:7-12.

FDA. 1993. Food labeling, general provisions, nutrition labeling, label format, nutrient content claims, health claims, ingredient labeling, state and local requirements and exemptions, final rules. Fed. Regist. 5893:2101-2106.

Fennema O.R. 2000. Food chemistry. Marcel Dekker Inc., NY.

Fernandez, M.L. and J.W. Berry. 1988. Nutritional evaluation of chickpea and germinated chickpea flours. Plant Food Hum. Nutr. 38(2):127-134.

Fernandez, M.M., P. Aranda, M. Lopez-Jurado, G. Urbano, E. Estrella, C. Sotomayor, C. Diaz, M. Prodanov, J. Frias and C. Vidal-Valverde. 1993. Effect of processing on some antinutritive factors of faba beans: influence on protein digestibility and food intake in rats. p. 467-471. In A.F.B. van der Poel, J.H. Huisman and S. Saini (eds.) Recent advances of research in antinutritional factors in legume seeds. Wageningen Perss, Wageningen, The Netherlands.

Feron, G. and Y. Wache. 2006. Microbial biotechnology of food flavour production. p. 400-429. In K. Shetty, G. Paliyath, A. Pometto and R.E. Levin (eds.) Food biotechnology. CRC Press, Boca Raton, FL. USA.

Fontvieille, A.M., F. Bornet, S.W. Rizkalla, P. Le Francois, P. Pichard, N. Desplanque, A. Chevalier, M. Letanoux, A. Verel and G. Tchobroutsky. 1988. In vitro and in vivo digestibility and metabolic effects of 3 wheat-flour products (white bread, french toast (rusk) and french toast bran-enriched) in normal subjects. Diab. Metab. 14(2):92-96.

Ford, D. E. and D.N. Salter. 1966. Analysis of enzymatically digested food protein by sephadex gel filtration. Br. J. Nutr. 20:843-849.

Ford, J.R., G.C. Mustakas and R.D. Schmutz. 1978. Phytic acid removal from soybeans by lipid protein concentrate process. J. Am. Oil Chem. Soc. 55:371-374.

Francis, FJ. 1980. Colour quality evaluation of horticultural crops. Hort. Sci. 15:58-59.

Fredlund, K., M. Larrson, I. Marklinder and A. Sandberg. 1997. Phytate reduction in whole grains of wheat, rye, barley and oats after hydrothermal treatment. J. Cereal Sci. 25:83-91.

Fredrikson M., M.L. Alminger, N.G. Carlsson and A.S. Sandberg. 2001. Phytate content and phytate degradation by endogenous phytase in pea (Pisum sativum). J. Sci. Food Agric. 81(12):1139-1144.

Frias, J., C. Diaz-Pollan, C.L. Hedley and C. Vidal-Valverde. 1995. Evolution of trypsin inhibitor activity during germination of lentils. J. Agric. Food Chem. 43:2231-2234.

Frias, J., C. Vidal-Valverde, C. Sotomayor, C. Diaz-Pollan and G. Urbano. 2000. Influence of processing on available carbohydrate content and antinutritional factors of chickpeas. Eur. Food Res. Tech. 210(5):340-345.

Frison-Norrie, S. and P. Sporns. 2002. Identification and quantification of flavonol glycosides in almond seedcoats using MALDI-TOF MS. J. Agric. Food Chem. 50:2782-2787.

Gauthier, S.F., C. Vachon, J.D. Jones and L. Savoie. 1982. Assessment of protein digestibility by in vitro enzymatic hydrolysis with simultaneous dialysis. J. Nutr. 112:1718-1725.

Getahun H., F.W. Lambe and P. van der Stuyft. 2002. ABO blood groups, grass pea preparation and neurolathyrism in Ethiopia. Trans. Royal Soc. Trop. Med. Hyg. 96:700-703.

Getahun, H., A. Mekonnen, R. Teklehaimanot and F. Lambein. 1999. Epidemic of neurolathyrism in Ethiopia. Lancet. 354:306-307.

Getahun, H., F. Lambein, M. Vanhoorne and P. Van der Stuyft. 2005. Neurolathyrism risk depends on type of grass pea preparation and on mixing with cereals and antioxidants. Trop. Med. Int. Health. 10(2):169-178.

Ghosal, S., V.K. Tripathi and S. Chauhan. 1996. Active constituent of Emblica officinalis: part 1, the chemistry and antioxidant effects of two new hydrolysable tannins, emblicanin A and B. Indian J. Chem. 35B:941-948.

Gil, M. I., F.A. Tomas-Barberan, B. Hess-Pierce, D.M. Holcroft and A.A. Kader. 2000. Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. J. Agric. Food Chem. 48:4581-4589.

Giovanni M. 1983. Response surface methodology and product optimization. Food Tech. 37(11):41-45, 83.

Gladovic, N., L. Zupancic-Kralj and J. Plavec. 1997. Determination of primary oxidation products of linoleic acid and triacylglycerols. J. Chrom. A. 767(1-2):63-68.

Gobbetti, M., A. Corsetti and J. Rossi. 1995. Interactions between lactic acid bacteria and yeasts in sour dough using a rheofermentometer. World J. Microbial. Biotechnol. 11:225-630.

Gordon, M.H. 1990. The mechanism of antioxidant action in vitro. p. 1-18. In B.J.F. Hudson (ed.) Food antioxidants. Elsevier Applied Science, Essex, UK.

Goyal, R. 1991. Nutritional improvement and utilization of the cereal-legume blends through fermentation. M.Sc. thesis, Haryana Agricultural University, Hissar, India.

Graf, E. and J.W. Easton. 1990. Antioxidant function of phytic acid. Free Rad. Biol. Med. 8:61-69.

Graf, E., J.R. Mahoney, R.G. Bryant and J.W. Easton. 1984. Iron catalyzed hydroxyl radical formation stringent requirement for free iron coordinate site. J. Bio. Chem. 259:3620-3624.

Granito M., A. Torres, J. Frias, M. Guerra and C. Vidal-Valverde. 2005. Influence of fermentation on the nutritional value of two varieties of Vigna sinensis. Eur. Food Res. Technol. 220:176-181.

Granito, M., J. Frias, R. Doblado, M. Guerra, M. Champ and C.Vidal-Valverde. 2002. Nutritional improvement of beans (Phaseolus vulgaris) by natural fermentation. Eur. Food Res. Tech. 214(3):226-231.

Grankivist, G. and A. Biel. 2001. The importance of beliefs and purchase criteria in the choice of eco-labeled food products. J. Environ. Psych. 21(4):405-410.

Greiner, R., E. Haller, U. Konietzny and K.D. Jany. 1997. Purification and characterization of a phytase from Klebsiella terrigena. Arch. Biochem. Biophys. 341:201-206.

Greiner, R., U. Konietzny and K.D. Jany. 1993. Purification and characterization of two phytases from Escherichia coli. Arch. Biochem. Biophys. 303:107-113.

Grela, E.R. and K.D. Gunter. 1995. Fatty acid composition and tocopherol content of some legume seeds. Anim. Feed Sci. Technol. 52:325-331.

Grela, E.R., T. Studziñski and J. Matras. 2001. Antinutritional factors in seeds of Lathyrus sativus cultivated in Poland. Lath. Lath. News. 2:101-104

Grice, H.C. 1986. Safety evaluation of butylated hydroxytoluene (BHT) in the liver, lung and gastrointestinal tract. Food Chem. Toxicol. 24:1127-1130.

Grusak, M.A. and D. Dellapenna. 1999. Improving the nutrient composition of plants to enhance human nutrition and health. Ann. Rev. Plant Phys. Plant Mol. Biol. 50:133-161.

Guo, C.J., J.J. Yang, J.Y. Wei, Y.F. Li, J. Xu and Y.G. Jiang. 2003. Antioxidant activities of peel, pulp and seed fractions of common fruits as determined by FRAP assay. Nutr. Res. 23:1719-1726.

Gupta, C. and S. Sehgal. 1991. Development, acceptability and nutritional value of weaning mixtures. Plant Foods Hum. Nutr. 41:107-116.

Gupta, M. and N. Khetarpaul. 1993. HCl-extractability of minerals from rabadi-a wheat flour fermented food. J. Agric. Food Chem. 41:125-127.

Gupta, Y.P. 1987. Anti-nutritional and toxic factors in food legumes: a review. Plant Foods Hum. Nutr. 37(3):201-228.

Habiba, R.A. 2002. Changes in anti-nutrients, protein solubility, digestibility and HCl-extractability of ash and phosphorus in vegetable peas as affected by cooking methods. Food Chem. 77(2):187-192.

Hallberg, L., L. Hulthen and L. Garby. 1998. Iron stores in man in relation to diet and iron requirements. Eur. J. Clin. Nutr. 52:623-631.

Hanbury, C.D., C.L. White, B.P. Mullan and K.H.M. Siddique. 2000. A review of the potential of Lathyrus sativus L. and L. cicera L. grain for use as animal feed. Anim. Feed Sci. Tech. 87:1-27.

Haq, M.Z.U., S. Iqbal, S. Ahmad, M. Imran, A. Niaz and M.I. Bhanger. 2007. Nutritional and compositional study of Desi chickpea (Cicer arietinum L.) cultivars grown in Punjab, Pakistan. Food Chem. 105(4):1357-1363.

Haque, A., M. Hossain, G. Wouters and F. Lambein. 1996. Epidemiological study of lathyrism in North western districts of Bangladesh. Neuroepidemiology. 15:83-91.

Harrison, K. and L.M. Were. 2007. Effect of gamma irradiation on total phenolic content yield and antioxidant capacity of Almond skin extracts. Food Chem. 102:932-937

Haslam, E. 1989. Plant polyphenols-vegetable tannins revisited. Cambridge University Press, Cambridge, UK.

Haug, W. and H.J. Lantzsch. 1983. Sensitive method for the rapid determination of phytate in cereal and cereal products. J. Sci. Food Agric. 34:1423-1426.

Haytowitz, D.B. and R.H. Matthews. 1983. Effect of cooking on nutritive retention of legumes. Cereal Food World. 28:326-364.

Heiniö, R.L. 2003. Influence of processing on the flavour formation of oat and rye. Academic Dissertation. VTT Technical Research Centre of Finland, Vuorimiehentie, Finland.

Heller, S.N. and L.R. Hackler. 1978. Changes in the crude fiber content of the American diet. Am. J. Clin. Nutr. 31:1510-1514.

Hemalatha S., K. Platel and K. Srinivasan. 2007. Influence of germination and fermentation on bioaccessibility of zinc and iron from food grains. Euro. J. Clin. Nutr. 61:342-348.

Henley, E.C. and J.M. Kester. 1994. Protein quality evaluation by protein digestibility corrected amino acid scoring. Food Tech. 48(4):74-77.

Hennig, B., Y. Wang, S. Ramasamy and C.J. McClain. 1992. Zinc deficiency alters barrier function of cultured porcine endothelial cells. J. Nutr. 122(6):1242-1247.

Hernandez, M., I. Montalvo, V. Sousa and A. Sotelo. 1995. The protein efficiency ratios of 30:70 mixtures of animal: vegetable protein are similar or higher than those of the animal foods alone. J. Nutr. 126(9):574-581.

Horn, P.J. and H.M. Schwartz. 1961. Kaffir corn malting and brewing studies: amino composition of kaffir corn grain and malt. J. Food Sci. 40:65.

Hoseney, R.C., W.A. Atwell and D.R. Lineback. l977. Scanning electron microscopy of starch isolated from baked products. Cereal Foods World. 22:56-60.

Hotz, C. and R.S. Gibson. 2001. Assessment of home-based processing methods to reduce the phytate content and phytate/zinc molar ratio of white maize (Zea mays). J. Agric. Food Chem. 49:692-698.

Hras, A.R., M. Hadolin, Z. Knez and D. Bauman. 2000. Comparison of antioxidative and synergistic effects of rosemary extract with α-tocopherol, ascorbyl palmitate and citric acid in sunflower oil. Food Chem. 71(2):229-233.

Hsu, B., I.M. Coupar and K. Ng. 2006. Antioxidant activity of hot water extract from the fruit of the Doum palm, Hyphaene thebaica. Food Chem. 98:317-328.

Hsu, H.W., N.E. Sutton, M.O. Banjo, L.D. Satterlee and J.G. Kendrick. 1978. The C-PER and T-PER assay of protein quality. Food Tech. 32:69-73.

Huxley, R.R. and H. Neil. 2003. The relationship between dietary flavonol intake and coronary heart disease mortality:a meta analysis of prospective cohort studies. Eur. J. Clin. Nutr. 57:904-908.

Huyghebaert, G., G. Fontaine and G. de Groote. 1979. Broad bean (Vicia faba) as an alternative protein source in feed for broiler chicks. 1. The effect of different thermomechanical treatment. Rev. Agric. 32:1243-1255.

Ibrahim, S.S., R.A. Habiba, A.A. Shatta and H.E. Embaby. 2002. Effect of soaking, germination, cooking and fermentation on antinutritional factors in cowpeas. Nahrung/Food. 46(2):92-95.

Idris, W.H., A.B. Hassan, E.E. Babiker and A.H. El Tinay. 2005. Effect of malt pretreatment on antinutritional factors and HCl extractability of minerals of sorghum cultivars. Pak. J. Nutr. 4 (6):396-401.

Indrani, D. and G.V. Rao. 2001. Optimization of the quality of South Indian parotta by modelling the ingredient composition using the response surface methodology. Int. J. Food Sci. Tech. 36(2):189-197.

Indumadhavi, M. and V. Agte. 1992. Effect of fermentation on ionizable iron in cereal-pulse combinations. Int. J. Food Sci. Tech. 27(2):221-228.

IOM. Inst. of Medicine. 2002. High-energy, nutrient-dense emergency relief food product. National Academy Press, Washington, D.C. p 117.

Iqbal, A., N. Ateeq, I.A. Khalil, S. Perveen and S. Saleemullah. 2006. Physiochemical characteristics and amino acid profile of chickpea cultivars grown in Pakistan. Foodserv. Res. Int. 17(2):94-101.

Iqbal, S., M.I. Bhangar and F. Anwar. 2005. Antioxidant properties and components of some commercially available varieties of rice bran in Pakistan. Food Chem. 93:265-272.

Izzo, M. and K. Niness. 2001. Formulating nutrition bars with inulin and oligofructose. Cereal Foods World. 46:102-106.

James, K.A. and E.L. Hove. 1980. The ineffectiveness of cystine in legume based rat diets. J. Nutr. 110(9):1736-1744.

Jansen, G.R. 1978. Biological evaluation of protein quality. Food Technol. 32:52-56.

Jaswir, I., D.D. Kitts, Y.B.C. Man and T.H. Hassan. 2005. Physico-chemical stability of flaxseed oil with natural antioxidant measures during heating. J. Oleo Sci. 54(2); 71-79.

Jayaprakasha, G.K., P.S. Negi, B.S. Jena and L.J.M. Rao. 2007. Antioxidant and antimutagenic activities of Cinnamomum zeylanicum fruit extracts. J. Food Comp. Anal. 20:330-336

Jirapa, P., H. Normah, M.M. Zamaliah, R. Asmah and K. Mohamad. 2001. Nutritional quality of germinated cowpea flour (Vigna unguiculata) and its application in home prepared powdered weaning foods. Plant Foods Hum. Nutr. 56:203-216.

Johnsen, S., K. Overvad, C. Stripp, A. Tjonneland, S.E. Husted and H.T. Sorensen. 2003. Intake of fruit and vegetables and the risk of ischemic stroke in a cohort of Danish men and women. Am. J. Clin. Nutr. 78:57-64.

Jood S. and A.C. Kapoor. 1997. Improvement in bioavailability of minerals of chickpea and blackgram cultivars through processing and cooking methods. Int. J. Food Sci. Nutr. 48:307-312.

Jood, S., B.M. Chauhan and A.C. Kapoor. 1987. Polyphenols of chickpea and blackgram as affected by domestic processing and cooking methods. J. Sci. Food Agric. 39:145-149.

Joseph, E. and B.G. Swanson. 1994. Protein quality of “Idli” fermented steam cakes prepared from beans (Phaseolus vulgaris) and rice. Nutr. Res. 14(4):553-568.

Kadam, S.S. and R.R. Smithard. 1987. Effects of heat treatments on trypsin inhibitor and hemagglutinating activities in winged bean. Plant Foods Hum. Nutr. 37:151-159.

Kannan S., S.S. Nielsen and C. Mason. 2001. Protein digestibility-corrected amino acid scores for bean and bean-rice infant weaning food products. J. Agric. Food Chem. 49:5070-5074.

Kanner, J. and I. Rosenthal. 1992. An assessment of lipid oxidation in foods. Pure Appl. Chem. 64(12):1959-1964

Kataria, A. and B.M. Chauhan. 1988. Content and digestibility of carbohydrates of mung beans (Vigna radiata L.) as affected by domestic processing and cooking. Plant Foods Hum. Nutr. 38:51-59.

Kataria, A., B.M. Chauhan and D. Punia. 1989. Antinutrients and protein digestibility (in vitro) of mung bean as affected by domestic processing and cooking. Food Chem. 32(1):9-19.

Kataria, A., B.M. Chauhan and D. Punia. 1992. Digestibility of protein and starch (in vitro) of amphidiploids (black gram x mung bean) as affected by domestic processing and cooking. Plant Foods Hum. Nutr. 42:117-125.

Kaur, D. and A.C. Kapoor. 1990. Starch and protein digestibility of rice bean (Vigna umbellata): effect of domestic processing and cooking methods. Food Chem. 38:263-272.

Kaur, M., N. Singh and N.S. Sodhi. 2005. Physicochemical, cooking, textural and roasting characteristics of chickpea (Cicer arietinum L.) cultivars. J. Food Eng. 69(4):511-517.

Keen, C.L., R.R. Holt, P.I. Oteiza, C.G. Fraga and H.H. Schmitz. 2005. Cocoa antioxidants and cardiovascular health. Am. J. Clin. Nutr. 81(Suppl):298S-303S.

Kelawala, N.S. and L. Ananthanarayan. 2004. Antioxidant activity of selected foodstuffs. Int. J. Food Sci. Nutr. 55:6, 511-516.

Kerovuo, J. and S. Tynkkynen. 2000. Expression of Bacillus subtilis phytase in Lactobacillus plantarum 755. Lett. Appl. Microbiol. 30:325-329.

Khalifa, A.O. and A.H. Zinay. 1994. Effect of fermentation on protein fractions and tannin content of low-and hightannin cultivars of sorghum. Food Chem. 49:265-269.

Khalil, A.H. and E.H. Mansour. 1995. The effect of cooking, autoclaving and germination on the nutritional quality of faba beans. Food Chem. 54:177-182.

Khalil, A.W., A. Zeb, F. Mahmood, S. Tariq, A.B. Khattak and H. Shah. 2007. Comparison of sprout quality characteristics of desi and kabuli type chickpea cultivars (Cicer arietinum L.). LWT. 40:937-945.

Khalil, E.A.M. 2004. Antidiabetic effect of an aqueous extract of pomegranate (Punica granatum L.) peels in normal and alloxan diabetic rats. Egyptian J. Hosp. Med. 16:92-99.

Khalique, A., K.P. Lone, T.N. Pasha and A.D. Khan. 2004. Amino acids digestibility of chemically treated acid extruded cooked deflated rice polishing. Mal. J. Nat. 10(2):195-206.

Khan, M.N., K. Almas, A.R. Abid and M. Yaqoob. 1976. The effect of gram flour on the quality of wheat protein. Pak. J. Agric. Sci. 13(2):167-172.

Khan, N., R. Zaman and M. Elahi. 1988. Effect of processing on the phytic acid content of bengal gram (Cicer arietinum) products. J. Agric. Food Chem. 36:1274-1276.

Khatoon, N. and J. Prakash. 2004. Nutritional quality of microwave-cooked and pressure-cooked legumes. Int. J. Food Sci. Nutr. 55:441-448.

Khetarpaul, N. and B.M. Chauhan. 1990. Effect of germination and fermentation on in vitro starch and protein digestibility of pearl millet. J. Food Sci. 55 (3):883-884.

Khokhar, S. and B.M. Chauhan. 1986. Anti-nutritional factors in moth bean (Vigna aconitifolia): varietal differences and effects of methods of domestic processing and cooking. J. Food Sci. 51:591-594.

Khopde, S.M., K.I. Priyadarsini, H. Mohan, V.B. Gawandi, J.G. Satav, J.V. Yakhmi, M.M. Banavaliker, M.K. Biyani and J.P. Mittal. 2001. Characterizing the

antioxidant activity of amla (Phyllanthus emblica) extract. Current Sci. 81(2):185-190.

Kiers, J.L., A.E.A. van Laeken, F.M. Rombouts and M.J.R. Nout. 2000. In vitro digestibility of Bacillus fermented soya bean. Int. J. Food Microbiol. 60(2-3):163-169.

Kim, M. and M.B. Zemel. 1986. In vitro estimation of the potential bioavailability of calcium from sea mustard, milk and spinach under stimulated normal and reduced gastric acid conditions. J. Food Sci. 51:957-959.

Kirilenko O.A., O.A. Linkevich, E.I. Suryaninova and T.A. Lysogor. 1978. Antibacterial properties of juice of various types of pomegranate. Konser. Ovosh. Promy. 12:12-3.

Kirk, J.R. 1984. Biological availabilty of nutrients in processed foods. J. Chem. Educ. 61(4):364.

Kitson R.E. and M.G. Mellon. 1944. Colorimetric determination of phosphorus as molybdivanadophosphoric acid. Ind. Engg. Chem. Anal. Ed. 16(6):379-383.

Kizlansky, A. and L.B. Lopez. 2006. Assessment of protein quality in foods by calculating the amino acids score corrected by digestibility. Nutr. Hosp. 21(1):47-51.

Kmura, M. and Y. Itokawa. 1990. Cooking losses of minerals in foods and its nutritional significance. J. Nutr Sci. Vitaminol. 36(1):S26-32.

Knekt, J. Kumpulainen, R. Jarvinen, H. Rissanen, M. Heliovaara, A. Reunanen, T. Hakulinen and A. Aromaa. 2002. Flavonoid intake and the risk of chronic diseases. Am. J. Clin. Nutr. 76:560-568.

Knuckles, B.E., D.D. Kuzmicky, M.R. Gumbmann and A.A. Betschart. 1989. Effect of myo-inositol phosphate esters on in vitro and in vivo digestion of proteins. J. Food Sci. 54:1348-1350.

Kochhar, S. P., and J. B. Rossell. 1990. Detection, estimation and evaluation of antioxidants in food systems. p. 19-64. In B.J.F. Hudson (ed.) Food antioxidants. Elsevier, New York.

Koleva, I.I., T.A. van Beek, J.P.H. Linssen, A. de Groot and L.N. Evstatieva. 2002. Screening of plant extracts for antioxidant activity: a comparative study on three testing methods. Phyto. Anal. 13(1):8-17.

Kozlowska, H., J. Borowska, J. Fornal, C. Scheneider and H. Schamndke. 1990. Preparation of faba bean (Vicia faba L. minor) products. IV. Effect of hydrothermal treatment of faba bean on the quality of flour. Acta Aliment. Pol. 15:161-165.

Kroll, J., M.R. Harshadrai and R. Sascha. 2003. Reactions of plant phenolics with food proteins and enzymes under special consideration of covalent bonds. Food Sci. Technol. Res. 9(3):205-218.

Kulkarni, A.P., S.M. Aradhya and S. Divakar. 2004. Isolation and identification of a radical scavenging antioxidant - punicalagin from pith and carpellary membrane of pomegranate fruit. Food Chem. 87:551-557.

Kumaran, A. and R.J. Karunakran. 2006. Nitric oxide radical scavenging active components from Phyllanthus emblica L. Plant Foods Hum. Nutr. 61:1-5.

Kuo, Y.H., F. Ikegami and F. Lambein. 1998. Metabolic routes of β-(isoxazolin-5-on-2-yl)-L-alanine (BIA), the precursor of the neurotoxin ODAP (β-N-oxalyl-L-α,β-diaminopropionic acid), in different legume seedlings. Phytochemistry. 49:43-48.

Kuo, Y.H., H.M. Bau, B. Quemener, J.K. Khan and F. Lambein. 1995. Solid-state fermentation of Lathyrus sativus seeds using Aspergillus oryzae and Rhizopus oligosporus sp. T-3 to eliminate the neurotoxin ODAP without loss of nutritional value. J. Sci. Food Agric. 69:81-89.

Kyriakidis, N.B., M. Galiotou-Panayotou, A. Stavropoulou and P. Athanasopoulos. 1998. Increase in phytase activity and decrease on phytate during germination of 4 common legumes. Biotechnol. Lett. 20(5):475-478.

Lajolo, F.M., F.F. Filho and E.W. Menezes. 1991. Amylase inhibitors in Phaseolus vulgaris beans. Food Technol. 9:119-121.

Lambrechts, C., H. Boze, G. Moulin and P. Galzy. 1992. Utilization of phytate by some yeasts. Biotech. Lett. 14:61-66.

Lampe, J. W. 1999. Health effects of vegetables and fruits: assessing mechanism of action in human experimental studies. Am. J. Clin. Nutr. 70:475S-490S.

Land, D.G. and R. Shepherd. 1988. Scaling and ranking methods. p. 155-185. In J.R. Piggott (ed.) Sensory analysis of foods. Elsevier Applied Science, New York, USA.

Lappalainen, R., J. Kearney and M. Gibney. 1998. A pan EU survey of consumer attitude of food nutrition and health: an overview. Food Qual. Pref. 9(6):467-478.

Larson, R.A. 1997. Naturally occurring antioxidants. Lewis Pub., New York, USA.

Le Magnen, G. 1983. Body energy balance and food intake: a neuroendocrine regulatory mechanism. Physiol. Rev. 63:314-386.

Lean L.P. and S. Mohamed. 1999. Antioxidative and antimycotic effects of turmeric, lemon-grass, betel leaves, clove, black pepper leaves and Garcinia atriviridis on butter cakes. J. Sci. Food Agric. 79(13):1817-1822.

Lee, I.H., Y.H. Hung and C.C. Chou. 2008. Solid-state fermentation with fungi to enhance the antioxidative activity, total phenolic and anthocyanin contents of black bean. Int. J. Food Microb. 121(2):150-156

Lee, S.C., J.H. Kim, S.M. Jeong, D.R. Kim, J.U. Ha, K.C. Nam and D.U. Ahn. 2003. Effect of far-infrared radiation on the antioxidant activity of rice hulls. J. Agric. Food Chem. 51(15):4400-4403.

Lestienne, I., C.C. Icard-Vernie, C. Mouquet, C. Picq and S. Treche. 2005. Effects of soaking whole cereal and legume seeds on iron, zinc and phytate contents. Food Chem. 89:421-425.

Li, Y., C. Guo, J. Yang, J. Wei, J. Xu and S. Cheng. 2006. Evaluation of antioxidant properties of pomegranate peel extract in comparison with pomegranate pulp extract. Food Chem. 96:254-260

Liener, I. E. 1994. Implications of antinutritional components in soybean foods. Crit. Rev. Food Sci. Nutr. 34:31-61.

Liener, I.E. and M. Kakade. 1980. Protease Inhibitors. p. 7-71. In I.E. Liener (ed.) Toxic constituents of plant foodstuff. Academic Press, New York, USA.

Lin J.K. and Y.C. Liang. 2000. Cancer chemoprevention by tea polyphenols. Proc. Natl. Sci. Counc. ROC(B):24(1)1-13.

Lindley, M.G. 1998. The impact of food processing o antioxidants in vegetable oils, fruits and vegetables. Trends Food Sci. Tech. 9:336-340.

Liu, K. and P. Markakis. 1987. Effect of maturity and processing on the trypsin inhibitor and oligosaccharides of soybeans. J. Food Sci. 52:222-225.

Liu, X., C. Cui, M. Zhao, J. Wang, W. Luo, B. Yang and Y. Jiang. 2008. Identification of phenolics in the fruit of emblica (Phyllanthus emblica L.) and their antioxidant activities. Food Chem. 109:909-915.

Liu, X.C., G.W. Zhang, Y.R. Li, J.X. Wang and Z.N. Liang. 1989. Toxicological study on grass pea vine (Lathyrus sativus L.) and its toxic-component BOAA. Sci. Agric. Sin. 22:86–93.

Livesey, G. 1990. Energy values of unavailable carbohydrate and diets: an inquiry and analysis. Am. J. Clin. Nutr. 51:6l7-37.

Lodhi, S.A., S. Rehman and N. Huma. 2003. Effect of supplementation of detoxified matri flour on the quality of pan bread. Pak J. Sci. Ind. Res. 46:207-210.

Longe, O.G. 1983. Varietal differences in chemical characteristics related to cooking quality of cowpea. J. Food Proc. Pres. 7:143-150.

Longstaff, M.A. and J.M. McNab. 1991. The effect of concentration of tannin-rich bean hulls (Vicia faba L.) on activities of lipase and a-amylase in digesta and pancreas and on the digestion of lipid and starch by young chicks. Br. J. Nutr. 66:139-147.

Lonnerdal, B. 2000. Dietary factors influencing zinc absorption. J. Nutr. 130:1378S-1383S.

Lou, H.X., Y. Yamazaki, T. Sasaki, M. Uchida, H. Tanaka and S. Oka. 1999. A-type procyanidins from peanut skin. Phytochemistry. 51:297-308.

Low, R.K.C., R.G. Rotter, R.R. Marquardt and C.G. Campbell. 1990. Use of Lathyrus sativus L. (var. Seminisalbi) as a foodstuff for poultry. Brit. Poult. Sci. 31:615-625.

Lowry, O.H., N.J. Rosebrough, A. L. Farr and R. J. Randall. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193 (1):265-275.

Luft, F.C. 1996. Starvation in a sea of plenty: can a “slime loosener” help the critically ill? J. Mol. Med. 74(7):345-346.

Magnusson M.K., A. Arvola, U.K. Koivisto Hursti, L. Aberg and P.O. Sjoden. 2001. Attitudes towards organic foods among Swedish consumers. Br. Food J. 103(3):209-226.

Malick, C.P. and M.B. Singh. 1980. Plant enzymology and Histo enzymology. Kalyani Publications, New Delhi. p. 286.

Manach, C., G. Williamson, C. Morand, A. Scalbert and C. Rémésy. 2005. Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am. J. Clin. Nutr. 81(suppl):230S-242S.

Mansour E.H. 1996. Biological and chemical evaluation of chick pea seed proteins as affected by germination, extraction and α-amylase treatment. Plant Food Hum. Nutr. 49(4):271-282.

Mansour, E.H. and A.H. Khalil. 2000. Evaluation of antioxidant activity of some plant extracts and their application to ground beef parties. Food Chem. 69:135-141.

Marletta, L., M. Carbonaro and E. Carnovale. 1992. In vivo protein and sulphur amino acid availability as a measure of bean protein quality. J. Sci. Food Agric. 59:497-504.

Martin-Cabrejas M.A., B. Sanfiz, A.Vidal, E. Molla, R. Esteban and F.J. Lopez-Andreu. 2004. Effect of fermentation and autoclaving on dietary fiber fractions and antinutritional factors of beans (Phaseolus vulgaris L.). J. Agric. Food Chem. 52:261-266.

Martínez, B., F. Rincón, M.V. Ibáñez and P. Abellán. 2004. Improving the nutritive value of homogenized infant foods using response surface methodology. J. Food Sci. 69(1):SNQ38-43.

Martinez-Tome, M., A. Jimenez, S. Ruggieri, N. Frega, R. Strabbioli and M. Murcia. 2001. Antioxidant properties of mediterranean spices compared with common food additives. J. Food Prot. 64:1412-1419.

Matthaus, B. 2002. Antioxidant activity of extracts obtained from residues of different oilseeds. J. Agric. Food Chem. 50(12):3444-3452.

Maurer, G., G. Fukuda and S. Nielsen. 2005. Development of bean-based granola bars and cereal. Cereal Foods World. 50(1):27-32.

Mauron, J. 1973. The digest dialysat index of food proteins, amino acid composition and nutritive value. In J.W.G. Porter and B.A. Rolls (eds.) Protein in human nutrition. Academic Press, London.

Mbithi S., J.V. Camp, R. Rodriguez and A. Huyghebaert. 2001. Effects of sprouting on nutrient and antinutrient composition of kidney beans (Phaseolus vulgaris var. Rose coco). Eur. Food Res. Tech. 212(2):188-191.

McDonough, F.E., F.H. Steinke, G. Sarwar, B.O. Eggum, R. Bressani, P.J. Huth, W. Barbeau, W.E. Mitchell, G.V. Mitchell and J.G. Phillips. 1990. In vivo rat assay for true protein digestibility: collaborative study. J. Assoc. Off. Anal. Chem. 73(5):801-805.

Meilgaard, M., G.V. Civielle and B.Y. Carr. 1991. Sensory attributes and the way we perceive them. p. 8-22. In Sensory evaluation techniques. CRC Press Inc., Boca Raton, Florida.

Mera, M., A. Montenegro, N. Espinoza and N. Gaete. 2000. Research backs grass pea exports by small Chilean farmers. Lath. Lath. News. 1:31.

Mercier, C. 1979. The α-galactosides of legume seeds. p. 79-90. In Matieres premieres et alimentation des volailles. Institut National de la Recherche Agronomique, Versailles, France.

Mertz, E.T., M.M. Hassen, C. Cairns-Whittern, A.W. Kirleis, L. Tu and J.D. Axtell. 1984. Protein digestibility of proteins in sorghum and other major cereals. Proc. Natl. Acad. Sci. USA. 81:1-2.

Messina, M.J. 1999. Legumes and soybeans: overview of their nutritional profiles and health effects. Am. J. Clin. Nutr. 70(suppl):439S-50S.

Milczak, M., M. Pedzinski, H. Mnichowska, K.S. Urbas and W. Rybinski. 2001. Creative breeding of grasspea (Lathyrus sativus L.) in Poland. Lath. Lath. News. 2(2):85-88.

Miller D.S. and A. E. Bender. 1955. The determination of the net utilization of proteins by a shortened method. Br. J Nutr. 9:382-388.

Millward, D.J. 1999. The nutritional value of plant-based diets in relation to human amino acid and protein requirements. Proc. Nutr. Soc. 58:249-260.

Mitchell, G.V., M.Y. Jenkins and E. Grundel. 1989. Protein efficiency ratios and net protein ratios of selected protein foods. Plant Foods Hum. Nutr. 39(1):53-58.

Miura, Y., T. Chiba, I. Tomita, H. Koizumi, S. Miura, K. Umegaki, Y. Hara, M. Ikeda and T. Tomita. 2001. Tea catechins prevent the development of atherosclerosis in Apoprotein E-deficient mice. J. Nutr. 131:27-32.

Mohan, V.S., V. Nagarajan and C. Gopalan. 1966. Simple practical procedures for the removal of toxic factors in Lathyrus sativus (Khesari dal). Indian J. Med. Res. 54:410-419.

Mole, S. 1989. Polyphenolics and the nutritional ecology of herbivores. p. 191-223. In P.R. Cheeke (ed.) Toxicants of plant origin, phenolic. Vol. IV. CRC Press Inc., Boca Raton, FL, USA.

Molteberg, E.L., G. Vogt, A. Nilsson and W. Frolich. 1995. Effects of storage and heat processing on the content and composition of free fatty acids in oats. Cereal Chem. 72(l):88-93.

Mongeau, X., G. Sanvar, R.W. Peace and R. Brassard. 1989. Relationship between dietary fiber levels and protein digestibility in selected foods as determined in rats. Plant Foods Hum. Nutr. 39:45-51.

Montgomery, D.C. 1991. Design and analysis of experiments. 3rd edn. JohnWiley and Sons., New York. p 649.

Morales, E.R.L., J.F.G. Otero and J. Coretes. 1963. Amino acids in seeds of legumes cultivated in Spain. Rev. Nutr. Anim. 1:24-32.

Moreno, C.R., E.O.C. Rodríguez, J.M. Carrillo, O.G.C. Valenzuela and J.B. Hoyos. 2004. Solid state fermentation process for producing chickpea (Cicer arietinum L) tempeh flour. Physicochemical and nutritional characteristics of the product. J. Sci. Food Agric. 84(3):271-278.

Morrison, W.R. 1978. Cereal lipids. p. 297-304. In Y. Pomeranz (ed.) Advances in cereal science and technology. Vol. II. Am. Assoc. Cereal Chem. St. Paul, MN, USA.

Mortuza, M.G., N. Newaz, M.A. Hossain and M.H. Rahman. 2000. Nutritional response of rats to faba beans (Vicia faba L. minor black 'Kalimattar') and its fractions. Int. J. Food Sci. Nutr. 51(1):5-9.

Moslehuddin, A.B.M., Y.D. Hang and G.S. Stoewsand. 1987. Evaluation of the toxicity of processed Lathyrus sativus seeds in chicks. Nutr. Rep. Intern. 36:851-855.

Mueller, R. and J.K.P. Weder. 1990. Isolation and characterization of two trypsin-chymotripsin inhibitors from lentil seeds (Lens culinaris Medik). J. Food Biochem. 13:39-63.

Muir, J.G. and K. O'Dea. 1992. Measurement of resistant starch: factors affecting the amount of starch escaping digestion in vitro. Am. J. Clin. Nutr. 56:123-127.

Mulimani, V.H. and S. Paramjyothi. 1993. Effect of heat and UV on trypsin and chymotrypsin inhibitor activities in redgram (Cajanus cajan L.). J. Food Sci. Technol. 30:62-63.

Mulimani, V.H. and S. Paramjyothi. 1995. Changes in trypsin and chymotprysin inhibotry activity on soaking of redgram (Cajanus cajan L.) Plant Food Hum. Nutr. 47:185-190.

Murthy, K.N.C., R.P. Singh and G.K. Jayaprakasha. 2002. Antioxidant activities of grape (Vitis vinifera) pomace extracts. J. Agric. Food Chem. 50(21):5909-5914.

Muzquiz, M., T. Welham, P. Altars, C. Goyoaga, C. Cuadrado, C. Romero, E. Guillamon and C. Domoney. 2004. The effect of germination on seed trypsin inhibitors in Vicia faba and Cicer arietinum. J. Sci. Food Agric. 84(6):556-560.

Nagra S.A. and N. Bhatti. 2007. In vivo (rat assay) assessment of nutritional improvement of peas (Pisum sativum L.). East. Med. Health J. 13(3):646-653.

Naik, G.H., K.I. Priyadarsini, R.G. Bhagirathi, B. Mishra, K.P. Mishra, M.M. Banavalikar and H. Mohan. 2005. In vitro antioxidant studies and free radical reactions of triphala, an ayurvedic formulation and its constituents. Phyto. Res. 19(7):582-586

Nakamura, Y., H. Fukuhara and K. Sano. 2000. Secreted phytase activities of yeasts. Biosci. Biotech. Biochem. 64:841-844.

Nam, G.Y. 2001. Analysis of phytate content in Korean food items and effect of cooking on phytate content and zinc bioavailability in Korean diet. Master’s thesis, Seoul National University, Department of Food and Nutrition, Seoul, Korea.

Nanda, V., S. Singh, C.S. Raina, N. Jindal, K. Singh and D.C. Saxena. 2004. Optimization of the process variables for the preparation of processed paneer using response surface methodology. Eur. Food Res. Technol. 218:529-534.

Nasr, C.B., N. Ayed and M. Metche. 1996. Quantitative determination of the polyphenolic content of pomegranate peel. Z. Lebensm. Unter. Forsch. A. 203:374-8.

Nawar, W.W. 1996. Lipids. In O.R. Fennema (ed.) Food chemistry. Marcel Dekker Inc., NY.

Nawwar, M.A.M., S.A.M. Hussein and I. Merfort. 1994. Leaf phenolics of Punica granatum. Phytochemistry. 37:1175-1177.

Negi, P.S. and G.K. Jayaprakasha. 2003. Antioxidant and antibacterial activities of Punica granatum peel extracts. J. Food Sci. 68(4):1473-1477.

Nestares, T., M. Barrionuevo, M. Lopez-Frias, G. Urbano, C. Diaz, M. Prodanov, J. Frias, I. Estrella and C. Vidal-Valverde. 1993. Effect of processing on some antinutritive factors of chickpea: influence on protein digestibility and food intake in rats. p. 487-491. In A.F.B. van der Poel, J.H. Huisman and S. Saini (eds.) Recent advances of research in antinutritional factors in legume seeds. Wageningen Press, Wageningen, The Netherlands.

Nestares, T., M.L. Frias, M. Barrionuevo and G. Urbano. 1996. Nutritional assessment of raw and processed chickpea (Cicer arietinum L.) protein in growing rats. J. Agric. Food Chem. 44:2760-2765.

Nieblas, J.M., A. Sanchez, L.G. Cumplido and I. Higuera-Ciapara. 1991. Effect of packing material and temperature of storage on the quality of maize tortilla. Arch. Latin. Nutr. 41(4):584-94.

Nielsen, M.A., A.K. Summer and L.L. Whalley. 1980. Fortification of pasta with pea flours and air classified pea protein concentrates. Cereal Chem. 57:203-206.

Nielsen, S. S. 1991. Digestibility of legume proteins. Food Technol. 112-118.

Nielsen, S.S. 1998. Color analysis. p. 601-612. In Food analysis. 2nd edn. Aspen Publishers, Inc., Gaithersburg, Md., U.S.A.

Nnanna I.A. and R.D. Phillips. 1989. Amino acid composition protein quality and water-soluble vitamin content of germinated cowpeas (Vigna unguiculata). Plant Foods Hum. Nutr. 39 (2):187-200

Nnanna I.A. and R.D. Phillips. 1989. Amino acid composition protein quality and water-soluble vitamin content of germinated cowpeas (Vigna unguiculata). Plant Foods Hum. Nutr. 39 (2):187-200

Nolan, K.B. and P.A. Duffin. 1987. Effect of phytate on mineral bioavailability. In vivo studies of Mg, Ca2+, Fe3+, Cu2+ and Zn2+ solubilities in the presence of phytate. J. Sci. Food. 40:70-83.

Nozolillo, C. and G.M. de Bezeda. 1984. Browning of lentil seeds, concomitant loss of viability and the possible role of soluble tannins in both phenomena. Can. J. Plant Sci. 64:815-824.

NRC. 1989. Recommended dietary allowances. 10th edn. National Academy Press, Washington, DC.

Nuutila, A.M., K. Kammiovirta and K.M. Oksman-Caldentey. 2002. Comparison of methods for the hydrolysis of flavonoids and phenolic acids from onion and spinach for HPLC analysis. Food Chem. 76:519-525.

Odunfa, S.A. and O.B. Komolafe. 1989. Nutritional characteristics of staphylococcus species from fermenting African locust bean (Parkia biglobosa). Nahrung/Food. 33:607-615.

Omafuvbe, B.O., O.O. Shonukan and S.H. Abiose. 2000. Microbiological and biochemical changes in the traditional fermentation of soybean for ‘soy-daddawa’ - Nigerian food condiment. Food Microbiol. 17(5):469-474.

Osman, M.A. 2004. Changes in sorghum enzyme inhibitors, phytic acid, tannins and in vitro protein digestibility occurring during Khamir (local bread) fermentation. Food Chem. 88:129-134.

Oyarekua, M.A., I.O. Akinyele, S. Treché and A.F. Eleyinmi. 2008. Amylolytic lactic acid bacteria fermentation of maize-cowpea ogi. J. Food Proc. Pres. 32(2):286-305.

Oyeleke, O.A., I.D. Morton and A.E. Bender. 1985. The use of cowpeas (Vigna unguiculata) in improving a popular Nigerian weaning food. Br. J. Nutr. 54:343-347.

Padmajaprasad V., M. Kaladhar and R.V. Bhat. 1997. Thermal isomerisation of β-N-oxalyl-L-α,β-diaminopropionic acid, the neurotoxin in Lathyrus sativus, during cooking. Food Chem. 59(1):77-80.

Palazzolo, G. 2003. Cereal bars: they're not just for breakfast anymore. Cereal Foods World. 48(2):70-72.

Panovska, T.K., S. Kulevanova, I. Gjorgoski, M. Bogdanova and G. Petrushevska. 2007. Hepatoprotective effect of the ethyl acetate extract of Teucrium polium L. against carbontetrachloride-induced hepatic injury in rats. Acta Pharma. 57(2): 241-248.

Paredes-Lopez, O. and G.I. Harry. 1988. Food biotechnology review: traditional solid-state fermentations of plant raw materials--application, nutritional significance, and future prospects. Crit. Rev. Food Sci. Nutr. 27(3):159-87.

Pataki, R., I. Bak, P. Kovacs, D. Bagchi, D.K. Das and A. Tosaki. 2002. Grape seed proanthocyanidins improved cardiac recovery during reperfusion after ischemia in isolated rat hearts. Am. J. Clin. Nutr. 75:894-899.

Patane, C. 2006. Variation and relationship among some nutritional traits in Sicilian genotypes of chickpea (Cicer arietinum L.). J. Food Qual. 29:282-293.

Pedó I., V.C. Sgarbieriand and L.C. Gutkoski. 1999. Protein evaluation of four oat (Avena sativa L.) cultivars adapted for cultivation in the south of Brazil. Plant Foods Hum. Nutr. 53(4):297-304.

Peters, J.C. and A.E. Harper. 1985. Adaptation of rats to diets containing different levels of protein: effects on food intake, plasma and brain amino acid concentrations and brain neurotransmitter metabolism. J. Nutr. 115:382-398.

Phillippy, B.Q. and C.J. Wyatt. 2001. Degradation of phytate in foods by phytase in fruit and vegetable extracts. J. Food Sci. 66(4):535-539.

Ponnampalam E., D.B. Steele, D. Burgdorf and D. McCalla. 2004 Effect of germ and fiber removal on production of ethanol from corn. App. Biochem. Biotech. 115(1-3):837-842.

Porter, L.J., L.N. Hrstich and B.G. Chan. 1986. The conversion of roacyanidins and prodelphinidins to cyanidin and delphinidin. Phytochemistry. 25(1):223-230.

Praveen, S., R.P. Joharl and S.L. Mehta. 1994. Cloning and expression of OX-DAPRO degrading genes from soil microbe. J. Plant Biochem. Biotechnol. 3:25-29.

Preet K. and D. Punia. 2000. Proximate composition, phytic acid, polyphenols and digestibility (in vitro) of four brown cowpea varieties. Int. J. Food Sci. Nutr. 51(3):189-193

Rakhi, G. and N. Khetarpaul. 1995. Effect of fermentation on HCl-extractability of minerals from rice-defatted soy flour blend. Food Chem. 50:419-422.

Ramachandran, S., A. Bairagi and A.K. Ray. 2005. Improvement of nutritive value of grass pea (Lathyrus sativus) seed meal in the formulated diet for rohu, Labeo rohita (Hamilton) fingerlings after fermentation with a fish gut bacterium. Biores. Tech. 96:1465-1472.

Rao, P.U. and Y.G. Deosthale. 2006. Polyphenoloxidase activity in germinated legume seeds. J. Food Sci. 52(6):1549-1551.

Rao, T.S.S., M.N. Ramanuja, N. Ashok and H.S. Vibhaker. 1995. Storage properties of whole egg powder incorporated biscuits. J. Food Sci. Tech. 32(6): 470–476.

Reddy, N.R. and M.D. Pierson. 1994. Reduction in antinutritional and toxic components in plant foods by fermentation. Food Res. Int. 27:281-290.

Reddy, N.R. and S.K. Sathe. 2002. Food phytates. CRC Press, Boca Raton, USA.

Reddy, N.R., C.V. Balakrishnan and D.K. Salunkhe. 1978. Phytate phosphorus and mineral changes during germination and cooking of black gram (Phaseolus mungo) seeds. J. Food Sci. 43:540-543.

Reddy, N.R., M.D. Pierson, S.K. Sathe and D.K. Salunkhe. 1985. Dry bean tannins: A review of nutritional implications. J. Am. Oil Chem. Soc. 62(3):541-549.

Reddy, N.R., M.D. Pierson, S.K. Sathe and D.K. Salunkhe. 1989. Occurrence, distribution, content and dietary intake of phytate. p. 39-56. In N.R. Reddy, M.D. Pierson, S.K. Sathe and D.K. Salunkhe (eds.) Phytates in cereals and legumes. CRC Press, Boca Raton, FL, USA.

Reddy, V., A. Urooj and A. Kumar. 2005. Evaluation of antioxidant activity of some plant extracts and their application in biscuits. Food Chem. 90:317-321

Rehman S.U., J.R. Piggott, M.M. Ahmad, S. Hussain, N. Ahmad and P.O. Darko. 2008. Preparation and evaluation of pizza cheese made from blend of vetch-bovine milk. Int. J. Food Sci. Tech. 43(5):770-778.

Rehman, S., A. Paterson, S. Hussain, I.A. Bahtti and M.A.R. Shahid. 2006a. Influence of detoxified Indian vetch (Lathyrus sativus L.) on sensory and protein quality characteristics of composite, flour chapatti. J. Sci. Food Agric. 86:1172-1180.

Rehman, S., A. Paterson, S. Hussain, M.A. Murtaza and S. Mehmood. 2007. Influence of partial substitution of wheat flour with vetch (Lathyrus sativus) flour on quality characteristics of doughnuts. LWT-Food Sci. Tech. 40:73-82.

Rehman, S., B.H. Shah, S.Z.H. Kazmi and S.T. Muntaha. 2006b. Effect of incorporation of matri skim milk powder on dough rheology and bread sensory quality. Indus. J. Plant Sci. 3:805-809.

Rehman, S., M.I. Siddique, J.A. Awan and H.E. Ullha. 1997. Nutritional impact of matri (Lathyrus sativus) on biscuits. J. Agric. Res. 35:453-459.

Rehman, S., S. Hussain, A. Randhawa and M.S. Din. 2004. Physico-chemical characteristics of matri-skim milk blend ice cream. Food Sci. 25:83-86.

Rissanen, T.H., S. Voutilainen, J.K. Virtanen, B. Venho, M. Vanharanta and J. Mursu 2003. Low intake of fruits, berries and vegetables is associated with excess mortality in men: the Kuopio ischaemic heart disease risk factor (KIHD) study. J. Nutr. 133:199-204.

Robards, K., P.D. Prenzler, G. Tucker, P. Swatsitang and W. Glover. 1999. Phenolic compounds and their role in oxidative processes in fruits. Food Chem. 66:401-436.

Rocha, A.M.C.N. and A.M.M.B. Morais. 2003. Shelf life of minimally processed apple (cv. Jonagored) determined by color changes. Food Control. 14(1):13-20.

Roldan, S.G., A.C. Ludolph, J. Hugon, M. Hens, D. Mateo, G.E. Kisby and P.S. Spencer. 1994. Lathyrism in Spain: progressive central nervous system deficits more than 45 years after onset. p. 10-25. In B.M. Abegaz, R.T. Haimanot, V.S. Palmer and P.S. Spencer (eds.) The grass pea and lathyrism. Proc. Sec. Int. Lath./Lath. Conf. Ethiopia, Third World Medical Research Foundation, New York.

Ross, S.W., J.C. Brand, A.W. Thorburn and A.S. Truswell. 1987. Glycemic index of processed wheat products. Am. J. Clin. Nuir. 46:631-5.

Roy, D.N. and P.S. Spencer. 1989. Lathyrogens. p. 170-201. In P.R. Cheeke (ed.) Toxicants of plant origin. Vol. III. Proteins and amino acids. CRC Press, Boca Raton, FL, USA.

Roy, D.N. and S.P. Rao. 1971. Evidence, isolation, purification and some properties of a trypsin inhibitor in Lathyrus sativus. J. Agric. Food Chem. 19(2):257-259.

Rozan, P., R. Lamghari, M. Linder, C. Villaume, J. Fanni, M. Parmentier and L. Mejean. 1997. In vivo and in vitro digestibility of soybean, lupine and rapeseed meal proteins after various technological processes. J. Agric. Food Chem. 45:1762-1769.

Rubio L.A., M. Muzquiz, C. Burbano, C. Cuadrado and M.M. Pedrosa. 2002. High apparent ileal digestibility of amino acids in raw and germinated faba bean (Vicia faba) and chickpea (Cicer arietinum) based diets for rats. J. Sci. Food Agric. 82(14):1710-1717.

Rudra M.P.P., M.R. Singh, M.A. Junaid, P. Jyothi and S.L.N. Rao. 2004. Metabolism of dietary ODAP in humans may be responsible for the low incidence of neurolathyrism. Clin. Biochem. 37(4):318-322.

Sabu M.C. and R. Kuttan. 2002. Anti-diabetic activity of medicinal plants and its relationship with their antioxidant property. J. Ethnopharma. 81(2):155-160.

Saharan K., N. Khetarpaul and S. Bishnoi. 2001. HCl-extractability of minerals from ricebean and fababean: influence of domestic processing methods. Inn. Food Sci. Emerg. Tech. 2(4):323-325.

Saharan, K., N. Khetarpaul and S. Bishnoi. 2002. Anti-nutrients and protein digestibility of fababean and ricebean as affected by soaking, dehulling and germination. J. Food Sci. Technol. 39(4):418-422.

Sai Ram, M., D. Neetu, P. Deepti, M. Vandana, G. Ilavazhagan, D. Kumar and W. Selvamurthy. 2003. Cytoprotective activity of amla (Emblica officinalis)

against chromium (VI) induced oxidative injury in murine macrophages. Phyto. Res. 17:430-433.

Saikia, P., C.R. Sarkar and I. Borua. 1999. Chemical composition, antinutritional factors and effect of cooking on nutritional quality of rice bean [Vigna umbellata (Thunb; Ohwi and Ohashi)]. Food Chem. 67(4):347-352.

Salisbury F.B. and C. Ross. 1969. Mineral nutrition in plants. In Plant physiology. Wadsworth Publishing Company, Inc., Belmont, CA, USA.

Sanchez-Moreno, C., J.A. Larrauri and F. Saura-Calixto. 1999. Free radical scavenging capacity and inhibition of lipid oxidation of wines, grape juices and related poly-phenolic constituents. Food Res. Int. 32:407-412.

Sandberg A.S. and U. Svanberg. 1991. Phytate hydrolysis by phytase in cereals; effects on in vitro estimation of iron availability. J. Food Sci. 56(5):1330-1333.

Sandberg, A.S. 2002. Bioavailability of minerals in legumes. Br. J. Nutr. 88(3):S281-S285

Sang, S., K. Lapsley, W.S. Jeong, P.A. Lachance, C.T. Ho and R.T. Rosen. 2002. Antioxidative phenolic compounds isolated from almond skins (Prunus amygdalus Batsch). J. Agric. Food Chem. 50(8):2459-2463.

Sangronis, E., M. Rodríguez, R. Cava and A. Torres. 2006. . Eur. Food Res. Tech. 222(1-2):144-148.

Sarkar, P.K., L.J. Jones, G.S. Craven, S.M. Somerset and C. Palmer. 1997. Amino acid profiles of kinema, a soybean fermented food. Food Chem. 59:69-75.

Sarwar G. 1997. The protein digestibility-corrected amino acid score method overestimates quality of proteins containing antinutritional factors and of poorly digestible proteins supplemented with limiting amino acids in rats. J. Nutr. 127:758-764.

Sarwar G., R.W. Peace, H.G. Botting and D. Brulé. 1989. Digestibility of protein and amino acids in selected foods as determined by a rat balance method. Plant Foods Hum. Nutr. 39(1):23-32.

Sarwar, G. 1987. Digestibility of protein and bioavailability of amino acids in foods: effects on protein quality assessment. World Rev. Nutr. Diet. 54:26-70.

Sarwar, G. and F.E. McDonough. 1990. Evaluation of protein digestibility corrected amino acid score method for assessing protein quality of foods. J. Assoc. Off. Anal. Chem. 73 (3):347-356.

Sarwar, G. and R.W. Peace. 1986. Comparisons between true digestibility of total nitrogen and limiting amino acids in vegetable proteins. J. Nutr. 116:1172-1184.

Sarwar, G., F.W. Sosulski and J.M. Bell. 1975. Protein nutritive value of legume-cereal blends. Can. Inst. Food Sci. Tech. J. 8:109-112.

Sathe, S.K. and D.K. Salunkhe. 1984. Technology of removal of unwanted components of dry beans. CRC Critc. Rev. Food Sci. Nutr. 21:263-287.

Satterlee, L.D., H.F. Marshall and J.M. Tennyson. 1979. Measuring protein quality. J. Am. Oil Chem. Soc. 56:103-109.

Savage, G.P. and D.R. Thompson. 1993. Effect of processing on the trypsin inhibitor content and nutritive value of chickpeas (Cicer arietinum). p. 435-440. In A.F.B. van der Poel, J.H. Huisman and S. Saini (eds.) Recent advances of research in antinutritional factors in legume seeds. Wageningen Press, Wageningen, The Netherlands.

Scalbert, A. and G. Williamson. 2000. Dietary intake and bioavailability of polyphenols. J. Nutr. 130:2073S-2085S.

Scartezzini, P. and E. Speroni. 2000. Review on some plants of Indian traditional medicine with antioxidant activity. J. Ethnophar. 71:23-43.

Scartezzini, P., F. Antognoni, M.A. Raggi, F. Poli and C. Sabbioni. 2006. Vitamin C content and antioxidant activity of the fruit and of the Ayurvedic preparation of Emblica officinalis Gaertn. J. Ethnopharma. 104(1-2):113-118.

Schaafsma G. 2005. The protein digestibility-corrected amino acid score (PDCAAS) -a concept for describing protein quality in foods and food ingredients: a critical review. J. Assoc. Off. Anal. Chem. 88(3):988-994

Schanderi, S.H. 1970. Methods in food analysis. Academic Press, New York. p. 709.

Schifferstein, H.N.J. and P.A.M. Oude Ophuis. 1998. Health related determinants of organic food consumption in Netherland. Food Qual. Pref. 9(3):119-133.

Schuster, R. 1988. Determination of amino acids in biological, pharmaceutical, plant and food samples by automated precolumn derivatization and high-performance liquid chromatography. J. Chrom. 431:271-284.

Scott, J.J. 1991. Alkaline phytase activity in nonionic detergent extracts of legume seeds. Plant Physiol. 95:1298-1301.

Seena, S., K.R. Sridhar and B. Bhagya. 2005. Biochemical and biological evaluation of an unconventional legume, Canavalia maritima of coastal sand dunes of India. Trop. Subtrop. Agro. 5:1-14.

Seeram, N.P., M. Aviram, Y. Zhang, S.M. Henning, L. Feng, M. Dreher and D. Heber. 2008. Comparison of antioxidant potency of commonly consumed polyphenol-rich beverages in the United States. J. Agric. Food Chem. 56:1415-1422.

Seeram, N.P., R. Lee, M.L. Hardy and D. Heber. 2005. Rapid large scale purification of ellagitannins from pomegranate husk, a byproduct of the commercial juice industry. Sep. Purif. Technol. 41:49-55.

Shah B.A. 1991. Nutritional evaluation of pigeon pea and its cooking characteristics. M.Sc. Thesis, Punjab University, Lahore.

Sharma A. and A.C. Kapoor. 1996. Levels of antinutritional factors in pearl millet as affected by processing treatments and various types of fermentation. Plant Foods Hum. Nutr. 49(3):241-252.

Sharma A. and N. Khetarpaul. 1994. Effect of whey fermentation on the HCI-extractability of calcium, iron and phosphorus in various rice-bengalgram dhal blends. Nahrung/Food. 40(3):147-150.

Sharma, A. and S. Sehgal. 1991. Priximate composition and protein fractions of fababean (Vicia faba). Bull. Grain Technol. 29(2):104-107.

Sharma, A. and S. Sehgal. 1992. Effect of domestic processing cooking and germination of trypsin inhibitor activity and polyphenol content of faba bean (Vicia faba). Plant Foods Hum. Nut. 42:127-129.

Sharma, S., K.S. Sekhon and H.P.S. Nagi. 1999. Suitability of durum wheat for flat bread production. J. Food Sci. Tech. 36:61-62.

Sheffner, A. L., G.A. Eckffeldt and H. Spector. 1956. The pepsin-digestresidu (PDR) amino acids index of net protein utilization. J. Nutr. 60:105-110.

Shehta, N.A. and B.A. Fryer. 1970. Effect of protein quality of supplementing wheat flour with chickpea flour. Cereal Chem. 47:663-669.

Shils M.E., J.A. Olson, M. Shike and A.C. Ross. 1994. Modern nutrition in health and disease. 8th edn. Lea and Febiger, Malvern, PA, USA.

Shils, M.E. and R.K. Rude. 1996. Deliberations and evaluations of the approaches, endpoints and paradigms for magnesium dietary recommendations..J. Nutr. 126(9):2398S-2403S.

Shimelis, E.A. and S.K. Rakshit. 2007. Influence of natural and controlled fermentation on α-galactosides, antinutrients and protein digestibility of beans (Phaseolus vulgaris L.). Int. J. Food Sci. Tech.

Shrivastava S. 1994. Study on the effects of processing on nutrients and non-nutrient composition of Lathyrus sativus. MSc Thesis, CCS Haryana Agricultural University, Hisar, India.

Siddhuraju, P. and K. Becker. 2001a. Effect of various domestic processing methods on antinutrients and in vitro protein and starch digestibility of two indigenous varieties of Indian tribal pulse, Mucuna pruriens var. utilis. J. Agric. Food Chem. 49:3058-3067.

Siddhuraju, P. and K. Becker. 2001b. Rapid reversed phase high performance liquid chromatographic method for the quantification of L-DOPA (1-3, 4 dihydroxyphenylalanine) non-methylated and methylated tetrahydroisoquinoline compounds from Mucuna beans. Food Chem. 72:389-394.

Siddhuraju, P. and K. Becker. 2003. Antioxidant properties of various extracts of total phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa oleifera) leaves. J. Agric. Food Chem. 51:2144-2155.

Siddhuraju, P., O. Osoniyi, H.P.S. Makker and K. Becker. 2002. Effect of soaking and ionizing radiation on various antinutritional factors of seeds from different species of an unconventional legume, Sesbania and a common legume, green gram (Vigna radiata). Food Chem. 79:273-281.

Sindhu, S.C. and N. Khetarpaul. 2003. Fermentation with one step single and sequential cultures of yeast and lactobacilli: Effect on antinutrients and digestibilities (in vitro) of starch and protein in an indigenously developed food mixture. Plant Foods Hum. Nutr. 58(3):1-10.

Sindhu, S.C., N. Khetarpaul and A. Sindhu. 2005. Effect of probiotic fermentation on carbohydrate and mineral profile of an indigenously developed food blend. Acta Alim. 34(1):41-47.

Singh, R.P., K.N.C. Murthy and G.K. Jayaprakasha. 2002. Studies on the antioxidant activity of pomegranate (Punica granatum) peel and seed extracts using in vitro models. J. Agric. Food Chem. 50:81-86.

Singh, S., C.S. Raina, A.S. Bawa and D.C. Saxena. 2004. Sweet potato-based pasta product: optimization of ingredient levels using response surface methodology. Int. J. Food Sci. Tech. 39:191-200.

Singh, U. 1985. Nutritional quality of chickpea (Cier arietinum L.): current status and future research needs. Plant Foods Hum. Nutr. 35:339-351.

Singh, U. 1988. Anti-nutritional factors of chickpea and pigeonpea and their removal by processing. Plant Foods Hum. Nutr. 38(3):254-261.

Singh, U., M.S. Kherdekar and R. Jambunathan. 1982. Studies on desi and kabuli chickpea (Cicer arietinum L.) cultivars, the levels of amylase inhibitors, levels of oligosaccharides and in vitro starch digestibility. J. Food Sci. 47:510-512.

Singhai, B. and S.K. Shrivastava. 2006. Nutritive value of new chickpea (Cicer arietinum) varieties. J. Food Agric. Environ. 4(1):48-53.

Smartt, J. 1990. Pulses of the classical world. p. 190-200. In R.J. Summerfield and E.H. Ellis (eds.) Grain legumes: evaluation and genetic resources. Cambridge Univ. Press, Cambridge, UK.

Smets, G., E. van Driessche and S. Beckman. 1985. Ultrastructural localization of pea lectin in the embryo and cotyledon during development by the colloidal-gold method. p. 453. In Lectins. Vol. IV. de Gruyter, Berlin, Germany.

Smith Jr., S.C., J. Allen, S.N. Blair, R.O. Bonow, L.M. Brass, G.C. Fonarow, S.M. Grundy, L. Hiratzka, D. Jones, H.M. Krumholz, L. Mosca, R.C. Pasternak, T. Pearson, M.A. Pfeffer and K.A. Taubert . 2006. AHA/ACC Guidelines for secondary prevention for patients with coronary and other atherosclerotic vascular disease: update. Am Coll Cardiol. 47:2130-2139.

Sogi, D.S., R. Bhatia, S.K. Garg and A.S. Bawa. 2005. Biological evaluation of tomato waste seed meals and protein concentrate. Food Chem. 89:53-56.

Sotelo, A., F. Flores and M. Hernandez. 1987. Chemical composition and nutritional value of Mexican varieties of chickpea (Cicer arietinum L.). Plant Foods Hum. Nutr. 37:299-306.

Spencer, P.S. 1989. The grass pea: threat and promise. Third World Medical Research Foundation, New York. p. 244.

Spencer, P.S., A.C. Ludolph and G.E. Kisby. 1993. Neurologic diseases associated with use of plant components with toxic potential. Environ. Res. 62:106-113.

Spencer, P.S., D.N. Roy, A. Ludolph, M.P. Dwivedi, D.N. Roy, J. Hugon and H.H. Schaumburg. 1986. Lathyrism: evidence for the role of the neuroexcitatory amino acid BOAA. Lancet. 2:1066-1067.

Sripriya, A.U. and T.S. Chandra. 1997. Changes in carbohydrate, free amino acids, phytate and HCl extractability of minerals during germination and fermentation of finger millet (Eleusine coracana). Food Chem. 58(4):345-350.

Srivastava, S. and S. Khokhar. 1996. Effects of processing on the reduction of β-ODAP (β-N-Oxalyl-L-2,3-diaminopropionic acid) and anti-nutrients of khesari dhal, Lathyrus sativus. J. Sci. Food Agric. 71(1):50-58.

Steel, R.G.D., J.H. Torrie and D.A. Dickey. 1997. Principles and procedures of statistics. a biometrical approach. 3rd edn. McGraw Hill Book Co. Inc., New York.

Steinkraus, K.H. 1996. Indonesian tempe and related fermentations. p. 7. In K.H. Steinkraus (ed.) Handbook of indigenous fermented foods. 2nd edn. Marcel Dekker Inc., NY.

Su, J.F., C.J. Guo, J.Y. Wei, J.J. Yang, Y.G. Jiang and Y.F. Li. 2003. Protection against hepatic ischemia-reperfusion injury in rats by oral pretreatment with quercetin. Biomed. Environ. Sci. 16:1-8.

Suh, H.J., J.M. Lee, J.S. Cho, Y.S. Kim and S.H. Chung. 1999. Radical scavenging compounds in onion skin. Food Res. Int. 32:659-664.

Suhaj, M. 2006. Spice antioxidants isolation and their antiradical activity: a review. J. Food Comp. Anal. 19:531-537.

Sultana, B., F. Anwar and R. Przybylski. 2007. Antioxidant potential of corncob extracts for stabilization of corn oil subjected to microwave heating. Food Chem. 104:997-1005.

Sun, D.W. 1992. Plant tannin chemistry. China Forestry Press, Beijing, China. p. 13.

Suschetet, M. 1975. Influence of tannic acid on the hepatic content of vitamin A in rats fed a vitamin A-containing diet or vitamin A-deficient diet. C. R. Seances SOC. Biol. Ses Fil. 169:970-978.

Tabera J., J. Frias, I. Estrella, R. Villa and C. Vidal-Valverde. 1995. Natural fermentation of lentils: Influence of time, concentration and temperature on protein content, trypsin inhibitor activity and phenolic compound content. Z. Labensm. Unters. Forsch. 201(6):587-591.

Takagi, K., R. Teshima, H. Okunuki and J.I. Sawada. 2003. Comparative study of in vitro digestibility of food proteins and effect of preheating on the digestion. Biol. Pharm. Bull. 26(7):969-973

Takeoka, G. R. and L.T. Dao. 2003. Antioxidant constituents of almond [Prunus dulcis (Mill.) D.A. Webb] hulls. J. Agric. Food Chem. 51(2):496-501.

Tanaka, T., G.I. Nonaka, I. Nishioka and I. Nishioka. 1986. Tannins and related compounds XLI. Isolation and characterization of novel ellagitannins, punicacarteins A, B, C and D and punigluconin from the bark of pomegranate. Chem. Pharm. Bull. 34:656-663.

Taylor, J.R.N. 1983. Effect of malting on the protein and free amino nitrogen composition of sorghum. J. Sci. Food Agric. 34:885-892.

Tekele-Haimanot, R., B.M. Abegaz, E. Wuhib, A. Kassina, Y. Kidane, N. Kebede, T. Alemu and P.S. Spencer. 1993. Pattern of Lathyrus sativus (grass pea) consumption and β-N-Oxalyl-L-2,3-diaminopropionic acid (β-ODAP) content of food samples in the lathyrism endemic region of northwest Ethiopia. Nutr. Res. 13:1113-1126.

Temple, N.J. and K.K. Gladwin. 2003. Fruit, vegetable and the prevention of cancer: research challenges. Nutrition. 19:467-470.

Thiele, C., M.G. Ganzle and R.F. Vogel. 2002. Contribution of sourdough lactobacilli, yeast and cereal enzymes to the generation of amino acids in dough relevant for bread flavor. Cereal Chem. 79:45-51.

Thompson, L.U., A.V. Tenebaum and H. Hiu. 1986. Effects of lectins and mixing of proteins on rate of protein digestibility. J. Food Sci. 51:150-152.

Torjusen, H., G. Lieblein, M. Wandel and C.A. Francis. 2001. Food system orientation and quality perception among consumers and producers of organic food in Hadmark County, Norway. Food Qual. Pref. 9(3):207-216.

Torres, A., J. Frias, M. Granito and C. Vidal-Valverde. 2007. Germinated Cajanus cajan seeds as ingredient in pasta products: chemical, biological and sensory evaluation. Food Chem. 101:202-211.

Torriani, S., M. Vescovo and G. Scolari. 1994. An overview on Lactobacillus helveticus. Annal. Microbiol. Enz. 44(1):163-191.

Tovar, J., A. de Francisco, I. Bjorek and N.G. Asp. 1991. Relationship between microstructure and in vitro digestibility of starch in precooked leguminous seed flours. Food Struct. 10(1):19-26.

Towo, E., E. Matuschek and U. Svanberg. 2006. Fermentation and enzyme treatment of tannin sorghum gruels: effects on phenolic compounds, phytate and in vitro accessible iron. Food Chem. 94:369-376.

Trugo, L.C., A. Farah and N.M.F. Trugo. 1993. Germination and debittering lupin seeds reduce R-galactosides and intestinal carbohydrate fermentation in humans. J. Food Sci. 58:627-630.

Trugo, L.C., L.A. Ramos, N.M.F. Trugo and M.C.P. Souza. 1990. Oligosaccharide composition and trypsin inhibitor activity of Phaseolus vulgaris and the effect of germination on the alpha-galactoside composition and fermentation in the human colon. Food Chem. 36:53-61.

Tungjaroenchai W., M. A. Drake and C. H. White. 2004. Influence of adjunct cultures on ripening of reduced fat Edam cheeses. J. Dairy Sci. 84(10):2117-2124.

Udedibie, A.B.I. and C.R. Carlini. 1998. Braizlian Mucuna pruriens seeds (velvet bean) lack hemagglutinating activity. J. Agric. Food Chem. 46:1450-1452.

Urbano, G., M. Lopez-Jurado, J. Hernandez, M. Fernandez, M.C. Moreu, J. Frias, C. Diaz-Pollan, M. Prodanov and C. Vidal-Valverde. 1995. Nutritional assessment of raw, heated and germinated lentils. J. Agric. Food Chem. 43:1871-1877.

Urga, K., F. Alemu and M.G. Tsadik. 1994. Influence of processing methods on cooking time and nutritional quality of grass pea. p. 105-118. In B.M. Abegaz, R.T. Haimanot, V.S. Palmer and P.S. Spencer. (eds.) The grass pea and lathyrism. Proceedings of the Second International Lathyrus/Lathyrism Conference in Ethiopia, Third World Medical Research Foundation, New York. p. 105-118.

Uzogara, S.G., I.D. Morton and J.W. Daniel. 1990. Changes in some antinutrients of cowpeas (Vigna unguiculata) processed with ‘kanwa’ alkaline salt. Plant Foods Hum. Nutr. 40:249-258.

Vadivel, V. and K. Janardhanan. 2005. Nutritional and antinutritional characteristics of seven south Indian wild legumes. Plant Foods Hum. Nutr. 60:69-75.

Vadivel, V. and M. Pugalenthi. 2007. Biological value and protein quality of raw and processed seeds of Mucuna pruriens var. utilis. Live. Res. Rural Devel. 19(7):Article 97. http://www.cipav.org.co/lrrd/lrrd19/7/vadi19097.htm (accessed on March 27, 2008).

Vatsala, C.N., D.C. Saxena and P.H. Rao. 2001. Optimization of ingredients and process conditions for the preparation of puri using response surface methodology. Int. J. Food Sci. Technol. 36:407-414.

Velasco J., S. Marmesat, G.M. Ruiz and M.C. Dobarganes. 2004. Formation of short-chain glycerol-bound oxidation products and oxidised monomeric triacylglycerols during deep-frying and occurrence in used frying fats. Eur. J. Lipid Sci. Tech. 106(11):728-735.

Vertucci, C.W. and A.C. Leopold. 1984. Bound water in soybean seed and its relation to respiration and imbibitional damage. Plant Phys. 75:114-117.

Vidal-Valverde, C., J. Frias, C. Diaz-Pollan, C. Fernandez, M. Lopezz-Jurado, G. Urbano. 1997. Influence of processing on trypsin inhibitor activity of faba beans and its physiological effects. J. Agric. Food Chem. 45:3559-3564.

Vidal-Valverde, C., J. Frias, I. Estrella, M.J. Gorospe, R. Ruiz and J. Bacon. 1994. Effect of processing on some antinutritional factors of lentils. J. Agric. Food Chem. 42:2291-2295.

Vidal-Valverde, C., J. Frias, M. Prodanov, J. Tabera, R. Ruiz and J. Bacon. 1993. Effect of natural fermentation on carbohydrates, riboflavin and trypsin inhibitor activity of lentils. Z. Leben. Unters. Forsch. A. 197(5):449-452.

Vijayakumari, K., M. Pugalenthi and V. Vadivel. 2007. Effect of soaking and hydrothermal processing methods on the levels of antinutrients and in vitro protein digestibility of Bauhinia purpurea L. seeds. Food Chem. 103:968-975.

Vijayakumari, K., P. Siddhuraju and K. Janardhanan. 1995. Effects of various water or hydrothermal treatments on certain antinutritional compounds in the seeds of the tribal pulse, Dolichos lablab var. vulgaris L. Plant Foods Hum. Nutr. 48:17-29.

Vijayakumari, K., P. Siddhuraju and K. Janardhanan. 1996. Effect of different post-harvest treatments on antinutritional factors in seeds of the tribal pulse, Mucuna pruriens (L.). Int. J. Food Sci. Nutr. 47:263-272.

Vijayakumari, K., P. Siddhuraju and K. Janardhanan. 1997. Chemical composition, amino acid content and protein quality of the little known legume Bauhinia purpurea L. J. Sci. Food Agric. 73:279-286.

Viscidi, K.A., M.P. Dougherty, J. Briggs and M.E. Camire. Complex phenolic compounds reduce lipid oxidation in extruded oat cereals. Lebensm. Wiss. Technol. 37:789-796.

Vohra, P., G.A. Gray and F.H. Kratzer. 1965. Phytic acid metal complexes. Proc. Soc. Exp. Biol. Med. 120:447-452.

Wandel, M. and A. Bugge. 1997. Environmental concern in consumer evaluation of food quality. Food Qual. Pref. 8(1):19-26.

Watzke, H.J. 1998. Impact of processing on bioavailability examples of minerals in foods. Trends Food Sci. Tech. 9:320-327.

Weder, J.K.P. and I. Link. 1993. Effect of treatments on legume inhibitor activity against human proteinases. p. 481-485. In A.F.B. van der Poel, J.H. Huisman and S. Saini (eds.) Recent advances of research in antinutritional factors in legume seeds. Wageningen Perss, Wageningen, The Netherlands.

Wichi, H.P. 1988. Enhanced tumour development by butylated hydroxyanisole (BHA) from the prospective of effect on forestomach and oesophageal squamous epithelium. Food Chem. Toxicol. 26:717-723.

Wijeratne, S.S.K., M.M. Abou-Zaid and F. Shahidi. 2006. Antioxidant polyphenols in almond and its coproducts. J. Agric. Food Chem. 54(2):312-318.

Wilmot Y.M., R.D. Phillips and J.L. Hargrove. 2001. Protein quality evaluation of cowpea-based extrusion cooked cereal/legume weaning mixtures. Nutr. Res. 21(6):849-857.

Wondimu, A. and N.G. Malleshi. 1996. Development of weaning foods based on malted, popped, and roller-dried barley and chickpea. Food Nutr. Bull. 17:169-174.

Wood, J.A. and M.A. Grusak. 2007. Nutritional value of chickpea. p. 101-142. In R. Redden, W. Chen, B. Sharma and Y. Yadav (eds.) Chickpea breeding and management. Oxford University Press, New York, USA.

Wootton, M. and A. Bamunarachchi. 1980. Application ofdifferential scanning calorimetry to starch gelatinisation. III Effect of sucrose and sodium chloride. Starch. 32:126-9.

Wootton, M. and M.A. Chaudhry. 1980. Gelatinization and in vitro digestibility of starch in baked products. J. Food Sci. 45:1783-4.

Wu, W.U., W.P. Williams, M.E. Kunkel, J.C. Acton, Y. Huang, F.B. Wardlaw and L.W. Grimes. 1995. True protein digestibility and digestibility-corrected amino acid score of red kidney beans (Phaseolus vulgaris L.). J. Agric. Food Chem. 43:1295-1298.

Yadav, S. and N. Khetarpaul. 1994. Indigenous legume fermentation: effect on some antinutrients and in vitro digestibility of starch and protein. Food Chem. 50:403-406.

Yigzaw, Y., L. Gorton, T. Solomon and G. Akalu. 2004. Fermentation of seeds of teff (Eragrostis teff), grass-pea (Lathyrus sativus) and their mixtures: aspects of nutrition and food safety. J. Agric. Food Chem. 52:1163-1169.

Yigzaw, Y., N. Larsson, L. Gorton, T. Ruzgas and T. Solomon. 2001. Liquid chromatographic determination of total and beta-N-oxalyl-L-alpha,beta-diaminopropionic acid in Lathyrus sativus seeds using both refractive index and bioelectrochemical detection. J. Chrom. A. 929(1-2):13-21.

Yip, R. and P.R. Dallman. 1996. Iron. p. 277-292. In E.E. Ziegler, L.J. Filer Jr. (eds.) Present knowledge in nutrition. 7th ed. ILSI Press, Washington, DC.

Yoshida, H., T. Ishikawa, H. Hosoai, M. Suzukawa, M. Ayaori, T. Hisada, S. Sawada, A. Yonemura, K. Higashi, T. Ito, K. Nakajima, T. Yamashita, K. Tomiyasu, M. Nishiwaki, F. Ohsuzu and H. Nakamura. 1999. Inhibitory effect of tea flavonoids on the ability of cells to oxidize low density lipoprotein. Biochem. Pharmacol. 58:1695-1703.

Young, V. and P.L. Pellett. 1991. Protein evaluation, amino acid scoring and the Food and Drug Administration’s Proposed Food Labeling Regulations. J. Nutr. 121:145-155.

Yu, F., P.J. Moughant, T.N. Barry and W.C. McNabb. 1996. The effect of condensed tannins from heated and unheated cottonseed on the ileal digestibility of amino acids for the growing rat and pig. Br. J. Nutr. 76:359-371.

Yu, J., M. Ahmedna and I. Goktepe. 2005. Effects of processing methods and extraction solvents on concentration and antioxidant activity of peanut skin phenolics. Food Chem. 90:199-206.

Zamora, A.F. and M.L. Fields. 1979. Nutritive quality of fermented cowpeas (Vigna sinensis) and chickpeas (Cicer arietinum). J. Food Sci. 44 (1):234-236.

Zevaco C. and J.C. Gripon. 1988. Properties and specificity of a cell-wall proteinase from Lactobacillus helveticus. Lait 68:393-408.

Zhang, Q., D. Jia and K. Yao. 2007. Antiliperoxidant activity of pomegranate peel extracts on lard. Nat. Prod. Res. 21(3):211-216

Zhao, Y.H., F.A. Manthey, S.K.C. Chang, H.J. Hou and S.H. Yuan. 2005. Quality characteristics of spaghetti as affected by green and yellow pea, lentil and chickpea flours. J. Food Sci. 70(6):S371-S376.

Zhou, K. and L. Yu. 2004. Effect of extraction solvent on the wheat bran antioxidant activity estimation. LWT-Food Sci. Tech. 37:717-721.

APPENDIX-I

DIET COMPOSITION

Overall (%) Protein 10 Corn oil sufficient for 9 Cellulose 5 Salt mixture 5 Vitamin mixture 2 Corn starch sufficient for 100 Minerals (g) CaHPO4 430 KCl 100 NaCl 100 MgO 10.5 MgSO4 50 Fe2O3 3 FeSO4.7H2O 5 Trace elements 10 Corn starch sufficient for 1000 Vitamins (mg) Vitamin A 2000 UI Vitamin D3 250 UI Vitamin B1 2.0 Vitamin B2 1.5 Vitamin B3 7.0 Vitamin B6 1.0 Vitamin B7 15.0 Vitamin B12 0.005 Vitamin C 80.0 Vitamin E 17.0 Vitamin K3 4.0 Vitamin PP 10.0 Choline 136.0 Folic acid 0.5 Acid PABacid 5.0 Biotin 0.03 Corn starch sufficient for 1000

APPENDIX-II

HEDONIS SCALE RANKING METHOD Score sheet

N a me o f t h e P r o d u c t

N a me o f t h e J u d g e

D a t e :

Sample Co lo r F lavor Tas t e Tex tu re T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15

S ig n a tu r e o f j u d g e

I n s t r u c t io n f o r J u d g e s 1 . Ex a mi n e t h e s a mp l e , w i th r e sp ec t t o t h e s t a t ed q u a l i t y q u e s t i o n u s in g t h e

f o l l o w in g s c a l e :

E x t r e me l y P o o r 1 V e r y P o o r 2 P o o r 3 B e lo w f a i r a b o v e p o o r 4 F a i r 5 B e lo w g o o d a b o v e f a i r 6 G o o d 7 V e r y G o o d 8 E x c e l l en t 9

2 . E v a lu a t e f o r co lo r b y v i s u a l o b s e r v a t i o n . Ch e w th e s a m p l e i n mo u th an d s co r e f o r f l a v o r a n d t a s t e a n d t e x tu r e .

3 . B e f o r e p r o c e ed in g t o t h e n ex t s amp l e , r i n s e mo u th w i th w a t e r . D o n o t d i s t u r b t h e o r d e r o f t h e s a mp le s , a n d d o n o t c o n s u l t w i th o th e r j u d g e s .

Date post:	09-May-2020
Category:	Documents
Upload:	others
View:	3 times
Download:	0 times

Omer Mukhtar Tararprr.hec.gov.pk/jspui/bitstream/123456789/34/1/145S.pdf · To my paranymphs Taj...

Documents