Effect of processing on Chemical Composition and
Effect of processing on Chemical Composition and biological value of cowpeas By Gaafar Abdelatif Nugdallah B.Sc. (Agric.), Al Azhar University – 1977 M.Sc. (Agric.) University of Khartoum - 1996 Supervisor: Prof. Abdullahi Hamid El Tinay A Thesis Submitted to the University of Khartoum in fulfillment for the requirements of the degree of Doctor of Philosophy (Agric.) Department of Food Science and Technology Faculty of Agriculture, University of Khartoum October – 2003 1 DEDICATION This humble effort is dedicated with all love and gratitude to our prophet Mohamed (Peace and Blessing be upon him) through whom Allah the Al Mighty says in Glorious Quran. In the name of Allah, the Beneficent the Merciful ﺑﺴﻢ اﷲ اﻟﺮﺣﻤﻦ اﻟﺮﺣﻴﻢ 24. Then let man look at his food, (And how We provide it) 25. For that We pour forth Water in Abundance, 26. And We split the earth In fragments, 27. And produce therein grain, 28. And Grapes and The fresh vegetation, 29. And Olives and Dates, 30. And enclosed Gardens, Dense with lofty trees, 31. And fruits and Fodder, 32. A provision For you and 2 (24) ﻓﻠﻴﻨﻈﺮ ﺍﻹﻧﺴﺎﻥ ﺇﱃ ﻃﻌﺎﻣﻪ (25) ﺃﻧﺎ ﺻﺒﺒﻨﺎ ﺍﳌﺎﺀ ﺻﺒﹰﺎ (26) ﰒ ﺷﻘﻘﻨﺎ ﺍﻷﺭﺽ ﺷﻘﺎ (27) ﻓﺄﻧﺒﺘﻨﺎ ﻓﻴﻬﺎ ﺣﺒﺎ (28) ﻭﻋﻨﺒﹰﺎ ﻭﻗﻀﺒﺎ (29) ﻭﺯﻳﺘﻮﻧﹰﺎ ﻭﳔﻼ (30) ﻭﺣﺪﺍﺋﻖ ﻏﻠﺒﺎ (31) ﻭﻓﺎﻛﻬﺔ ﻭﺃﺑﺎ (32) ﻣﺘﺎﻋﹰﺎ ﻟﻜﻢ ﻭﻷﻧﻌﺎﻣﻜﻢ your cattle Sura Abasa, Ayat (24 – 32). ZttytÜ TABLE OF CONTENTS Page Dedication.................................................................................................................................. ii List of Contents …............................................................................................................... iii List of Tables............................................................................................................................ viii List of Structure ..................................................................................................................... ix List of Figures …..................................................................................................................... x Acknowledgement ................................................................................................................. xi Abstract ..................................................................................................................................... xii Arabic Abstract xx .................................................................................................................. CHAPTER ONE: INTRODUCTION ..................................................................... 1 1.1. Importance of cowpea................................................................................................. 2 1.2. Classification of cowpea............................................................................................. 4 1.3. Antinutritional factors................................................................................................. 5 1.4 Processing of cowpeas.................................................................................................. 7 1.5 Utilization of cowpea.................................................................................................... 8 1.6. Production of cowpea................................................................................................ 10 3 Objective of the study ......................................................................................................... 11 CHAPTER TWO: LITERATURE REVIEW........................................ 12 2.1. Nutritional value of cow pea .................................................................................. 13 2.2. Protein fractionation.................................................................................................... 16 2.2.1. Nitrogen solubility (NS) ........................................................................................ 16 2.2.2. Protein fractions classification............................................................................ 17 2.2.2.1 Albumins and globulins..................................................................................... 19 2.2.2.2. Prolamin..................................................................................................................... 20 2.2.2.3 Glutelin....................................................................................................................... 21 2.2.2.3.2. G1 - glutelin......................................................................................................... 22 2.2.2.3.2. G2 - glutelin......................................................................................................... 22 2.2.2.3.3 G3 - glutelin.......................................................................................................... 23 2.2.2.4. Insoluble protein................................................................................................... 24 2.3 Anti-nutritional factors................................................................................................. 25 2.3.1. Chemical nature......................................................................................................... 26 2.3.1.1. Chemical nature of tannin................................................................................. 26 2.3.1.2. Chemical nature of phytic acid....................................................................... 30 2.3.1.3 Chemical nature of the inhibitors.................................................................... 33 4 2.3.2 Anti-nutritional effect..................................................................................... 33 2.3.2.1 Anti-nutritional effect of the Inhibitors........................................................ 33 2.3.2.2 Anti-nutritional effect of the tannin................................................................ 36 2.3.2.3 Anti-nutritional effect of phytic acid............................................................ 38 2.3.3 Anti-nutrients content .............................................................................................. 41 2.3.3.1 Tannin content of cowpea.................................................................................... 41 2.3.3.2 Trypsin inhibitor activity..................................................................................... 44 2.3.3.3 Content of phytic acid......................................................................................... 47 2.4. Processing........................................................................................................................ 48 CHAPTER THREE: MATERIALS AND METHODS………………......… 64 3.1 Material.............................................................................................................................. 64 3.1.1 Food materials.............................................................................................................. 64 3.1.1.1 Chemical and reagents..................................................................................... 64 3.1.2 Apparatus....................................................................................................................... 64 3.2 Methods.............................................................................................................................. 65 3.2.1 Preparation of cowpea samples........................................................................... 65 3.2.1.1 Cleaning...................................................................................................................... 65 3.2.1.2 Autoclaving................................................................................................................ 65 3.2.1.3 Roasting....................................................................................................................... 66 3.2.1.4 Germination of cowpea samples..................................................................... 5 66 3.2.1.5 Cooking of sprouted cowpea samples............................................................ 67 3.3 Analytical methods......................................................................................................... 67 3.3.2 Protein fractionation.................................................................................................. 67 2.3.3. In-vitro protein digestibility IVPD.................................................................... 71 3.3.4 Determination of tannin in raw and treated samples................................ 74 3.3.5 Determination phytic acid..................................................................................... 77 3.3.6 Determination of trypsin inhibitory factor....................................................... 81 3.3.7 Statistical analysis....................................................................................................... 82 CHAPTER FOUR: RESULTS AND DISCUSSION………………..……......… 83 4.1 Proximate composition................................................................................................ 84 4.2 Protein fraction................................................................................................................ 86 4.1.3. Ash content................................................................................................................... 87 4.1.4. Fiber content................................................................................................................ 88 4.1.5. Oil content................................................................................................................... 89 4.2. Protein fraction .............................................................................................................. 89 4.2.1 Globulin fractions........................................................................................................ 89 4.2.2 Albumin fraction........................................................................................................ 90 4.2.3. Prolamin fraction........................................................................................................ 93 4.2.4 G1-glutelin fraction..................................................................................................... 93 4.2.5 G2-glutelin fraction..................................................................................................... 94 6 4.2.6. G3-glutelin fraction.................................................................................................... 95 4.2.7 Residue fraction........................................................................................................... 96 4.3. Anti-nutrients ................................................................................................................... 101 4.3.1. Tannins............................................................................................................................ 101 4.3.2. Trypsin inhibitor activity....................................................................................... 102 4.3.3. Phytic acid..................................................................................................................... 102 4.3.4 In-vitro protein digestibility IVPD...................................................................... 104 CHAPTER FIVE SUMMARY, CONCLUSION AND RECOMMENDATIONS…………………………………………………………….……...... 111 Recommendation........................................................................................................................ 113 REFERENCES……………..…………….………………………….……......…………………...... 114 7 LIST OF TABLES Table Title No. 1. Protein extraction Procedure for sequence Ao and Bo................. 70 2. Proximate composition of some cowpea preparations................... 85 3. Effect of germination on protein fractions of cowpea cultivars 4. Effect of cooking on protein fractions of germinated cowpea cultivars............................................................................................................... 5. 98 99 Phytic acid (mg/100g dry weight), Tannins (g/100g dry weight), Trypsin inhibitor activity (TUI/mg protein) of raw and processed cowpea varieties and IVPD......................................... 6. 100 Effect of varying concentration of treated sample (roasting, autoclaving) + raw cowpea seed flour on IVPD............................... 106 7. Effect of autoclaving and roasting on protein fractions of cowpea cultivars............. ............. ............. ............. ............. ............. .......... 8 110 LIST OF STRUCTURES Struc. Title No. 1. Glalotannin......................................................................................................... 28 2. Gallic acid........................................................................................................... 28 3. Hexahydroxy-diphenic acid (Ellagitannin) ......................................... 28 4. Ellagic acid (Dilact) ...................................................................................... 5. Flavanzol (Catechin) ..................................................................................... 29 6. Flavan 3-4-diol (leucoantho-cyanidin) ................................................ 7. 3-Hydroxy flavylium (Anthocyanidin) ................................................. 29 8. Apossible structure of grain sorghum condensed tannin............... 29 I Myo – inositol..................................................................................................... 32 II Phytate ions.......................................................................................................... 32 III Phytate ions.......................................................................................................... 32 IV Polymeric structutre.......................................................................................... 32 V Phytate ions......................................................................................................... 32 9 28 29 LIST OF FIGURES Fig. 1 2 Page Catchen concentration mg/ml relationship between optical density at 500nm and catechin concentration................................. 76 Phytic acid standard curve ................... ................... ................... .......... 80 10 Acknowledgements I wish to express my deep sense of gratitude and sincere thanks to my supervisor A.H. El Tinay for his continuous interest helpful, guidance, encouragement, criticisms, advice and supervision throughout the progress of this work. My sincere thanks and due to my colleagues and technical staff and workers of the Department of Food Science and Technology for their assistance, in particular to Mr. El Habib and Ms. Asma, Mr. Abass and Madam Ihsan. Best regards are due to Mr. Abdelhamed for his unfailing patience and skill in typing the manuscript. I am grateful to my family members who, with great patience, tolerated all the inconveniences resulting and for being of help in this work that no words can express my feelings towards them. Lastly, my thanks are extended to others who offered help in one way or another. Above all my special praise and unlimited thanks are to Allah, who helped me and gave me health and patience to complete this study. 11 ABSTRACT Two cowpea cultivar (Vigna unguiculata) namely "Ain Elgazal" and "Buff" were obtained from Elobeid Research Station and were used in this study. The seeds were analyzed for their chemical composition, protein soluble fractions, in-vitro protein digestibility (IVPD), phytic acid, trypsin inhibitor activity and tannin content of raw, boiled, roasted, autoclaved, germinated for four days, and germinated cooked samples. The proximate composition showed that moisture content ranged from 4.4% to 5.9% for Ain Elgazal cultivar and ranged from 4.4% to 5.5% for Buff cultivar. Protein content ranged from 25.6% to 31.0% for Ain Elgazal cultivar and ranged from 24.6% to 28.9% for Buff cultivar. Ash content ranged from 3.2% to 4.3% for Ain Elgazal cultivar and ranged from 3.5% to 4.3% for Buff cultivar. Fibre content ranged from 2.5% to 3.2% for Ain Elgazal cultivar and ranged from 2.2% to 3.1% for Buff cultivar. Oil content ranged from 1.5% to 1.6% for Ain Elgazal cultivar and ranged from 1.6% to 1.7% for Buff cultivar. All the processing treatments had little effect on oil content and significantly (P ≤ 0.05) increased protein content, ash content and fibre content for both cultivars. The raw and germinated samples were fractioned, the major protein fraction Globulin showed significant (P ≤ 0.05) decrease, they were 87.5%, 80.6%, 76.4%, 71.4% and 70.2% for Ain Elgazal raw seeds, the first, second, third and fourth days respectively. Similarly for buff raw and germinated seeds, they were 89.8, 81.3, 78.3, 75.6 and 72.7% respectively. The Albumin fraction significantly (P < 0.05) 12 decreased they were 4.0%, 3.8%, 2.3%, 1.9% and 2.5% for Ain Elgazal raw seeds, the first, second, third and fourth days respectively. Similarly for Buff raw and germinated seeds, 3.6%, 1.5%, 2.2%, 2.2% and 1.2% respectively. The prolamin fraction for Ain Elgazal raw and germinated seeds were 4.0%, 3.3%, 4.0%, 1.6% and 4.6% respectively. Similarly for buff raw and germinated seeds were 4.5%, 2.7%, 2.8%, 3.9% and 1.4% respectively. The G1-glutelin fraction for Ain Elgazal raw and germinated seeds showed significant (P ≤ 0.05) decrease, they were 2.4%, 2.1%, 1.3%, 0.8% and 1.7% respectively. Similarly for Buff raw and germinated seeds were 2.3%, 1.2%, 1.3%, 2.1% and 1.3% respectively. The G2-glutelin fraction for Ain Elgazal raw and germinated seeds were 2.4%, 2.1%, 1.9%, 1.4% and 1.7% respectively. For Buff raw and germinated seeds were 2.5%, 6.4%, 5.1%, 3.9% and 4.5% respectively. The G3-glutelin fraction showed significantly (P ≤ 0.05) increase, they were 4.8%, 9.8%, 12.0%, 13.5% and 10.4% for Ain Elgazal raw seeds, the first, second, third and fourth days respectively. Similarly for Buff, they were 4.5%, 10.0%, 12.5%, 11.1% and 11.7% respectively. The insoluble protein fraction showed significant (P ≤ 0.05) increase, they were 1.2%, 2.5%, 2.1%, 9.2% and 6.0% for Ain Elgazal raw seeds, the first, second, third and fourth days respectively. Similarly for Buff raw and germinated seeds were 1.2%, 1.7%, 1.9%, 3.6% and 6.1% respectively. Cooking significantly (P ≤ 0.05) reduced globulin, they were 16.4%, 19.0%, 18.1%, 18.0% and 17.8% for Ain Elgazal raw first, second, third and fourth days respectively. Similarly for Buff raw and germinated seeds were 15.7%, 19.9%, 22.0%, 22.6% and 23.0% 13 respectively. Cooking significantly (P ≤ 0.05) reduced Albumin, they were 1.9%, 2.8%, 3.1%, 1.8% and 1.4% for Ain Elgazal raw, first, second, third and fourth days respectively. Similarly for Buff raw and germinated seeds were 2.2%, 1.6%, 1.6%, 2.1% and 1.3% respectively. Cooking significantly (P ≤ 0.05) increased prolamin fraction, they were 4.2%, 6.3%, 4.8%, 5.1%, and 5.0% for Ain Elgazal raw, first, second, third and fourth days respectively. Similarly for Buff raw and first and then decreased second, third and fourth days they were 4.6%, 5.1%, 2.7%, 2.7% and 1.8% respectively. Cooking significantly (P ≤ 0.05) decreased G1-glutelin fraction they were 2.0%, 1.4%, 1.3%, and 1.3% for Ain Elgazal raw, first, second, and third days respectively, but it increased to 2.6% in the fourth day. Similarly for Buff raw and germinated seeds were 2.7%, 1.2%, 1.1%, 1.6% and 1.0% respectively. Cooking significantly (P ≤ 0.05) increased G2-glutelin fraction, they were 3.4%, 4.4%, 4.4%, 4.0% and 5.7% for Ain Elgazal raw, first, second, third and fourth days respectively. Similarly for Buff raw and germinated seeds were 5.1%, 7.5%, 7.5%, 7.9% and 8.8% respectively. Cooking significantly (P ≤ 0.05) increased G3-glutelin fraction, they were 63.8%, 61.0%, 62.0%, 61.6% and 57.0% for Ain Elgazal raw first, second, third and fourth days respectively. Similarly for Buff raw and germinated seeds were 69.3%, 59.0%, 62.3%, 60.1% and 62.1% respectively. Cooking significantly (P ≤ 0.05) increased the insoluble protein fraction, they were 5.5%, 8.0%, 8.8%, 9.6% and 10.2% for Ain Elgazal raw, first, second, third and fourth days respectively. Similarly for Buff raw and germinated seeds were 5.5%, 6.6%, 6.5%, 6.1% and 6.5% respectively. 14 Autoclaving at 120ºC under 15 psi for 30 min for Ain Elgazal raw seeds significantly (P ≤ 0.05) reduce globulin fraction from 87.5% to 29.2% and albumin from 4.0% to 1.2% but increased prolamin from 4.3% to 4.5%, reduced G1-glutelin from 2.3% to 1.2% and G2-glutelin from 2.4% to 1.2% and increased G3-glutelin from 4.8% to 59.5% and insoluble protein fraction from 1.2% to 8.0%. Autoclaving at 150ºC under 20 psi for 30 min for Ain Elgazal raw seeds significantly (P ≤ 0.05) reduce globulin fraction from 87.5% to 27.6%, albumin fraction from 4.0% to 1.2% but increased prolamin from 4.3% to 4.8%, decreased G1-glutelin from 2.3% to 2.0%, increased G2-glutelin from 2.4% to 4.0%, increased G3-glutelin from 4.8% to 60.2% and increased insoluble protein fraction from 1.2% to 6.0%. Roasting Ain Elgazal raw seeds at 90ºC for 60 min significantly (P ≤ 0.05) reduce globulin fraction from 87.5% to 85.0%, albumin showed changeless, prolamin fraction reduced from 4.3% to 3.6%, G1-glutelin fraction reduced from 2.3% to 2.0%, G2-glutelin fraction reduced from 2.4% to 1.2%, G3-glutelin fraction increased from 4.8% to 6.0% and insoluble protein fraction increased from 1.2% to 3.0%. Roasting Ain Elgazal raw seeds at 120ºC for 60 min significantly (P ≤ 0.05) reduced globulin fraction from 87.5% to 41.0%, albumin fraction reduced from 4.0% to 1.2%, prolamin fraction reduced from 4.3% to 4.2%, G1-glutelin fraction reduced from 2.3% to 1.2%, G2glutelin fraction increased from 2.4% to 3.2%, G3-glutelin fraction increased from 4.8% to 48.0% and insoluble protein fraction increased from 1.2% to 6.0%. Autoclaving at 120ºC under 15 psi for 30 min for Buff raw seeds significantly (P ≤ 0.05) reduced globulin fraction from 15 89.8% to 32.0%, albumin fraction from 3.6% to 1.0%, prolamin from 4.5% to 3.0%, decreased G1-glutelin from 2.3% to 2.0%, G2-glutelin fraction increased from 2.5% to 3.0%, G3-glutelin increased from 4.5% to 55.0% and insoluble protein fraction increased from 1.2% to 6.0%. Autoclaving at 150ºC under 20 psi for 30 min for Buff raw seeds significantly (P ≤ 0.05) reduce globulin fraction from 89.5% to 36.0%, albumin fraction from 3.6% to 1.0% prolamin fraction from 4.5% to 4.0%, G1-glutelin from 2.3% to 1.2%, G2-glutelin increased from 2.5% to 3.2%, G3-glutelin increased from 4.5% to 50.8% and increased insoluble protein fraction from 1.2% to 7.0%. Roasting Buff raw seeds at 90ºC for 60 min significantly (P ≤ 0.05) decreased globulin fraction from 89.5% to 88.0%, albumin showed changeless, prolamin fraction decreased from 4.5% to 4.0%, G1-glutelin from 2.3% to 2.2%, G2-glutelin fraction increased from 2.5% to 2.8%, G3glutelin fraction showed changeless, insoluble protein fraction increased from 1.2% to 1.4%. Roasting Buff raw seeds at 120ºC for 60 min significantly (P ≤ 0.05) decreased globulin fraction from 89.8% to 45.5%, albumin fraction from 3.6% to 1.0%, prolamin fraction increased from 4.5% to 6.0%, G1-glutelin decreased from 2.3% to 2.0%, G2-glutelin fraction increased from 2.5% to 3.0%, G3-glutelin fraction increased from 4.5% to 42.0% and insoluble protein fraction increased from 1.2% to 6.0%. Samples were analyzed for phytic acid, tannin and trypsin inhibitor activity. Germination reduced phytic acid from 310.3 (mg/100g dry weight) of raw seeds to 286.1 mg/100g, 248.9 mg/100g, 201.7 and 139.8mg/100g for Ain Elgazal germinated seeds in the first, 16 second, third and fourth days respectively. Similarly for Buff raw seeds phytic acid was 376.3 which reduced by germinated to 346.2, 301.0, 225.7 and 180.7mg/100g for the first, second, third and fourth days respectively. Cooking showed significant (P ≤ 0.05) decreased of phytic acid for Ain Elgazal raw and germinated seeds, they were 290.3mg/100g, 268.9mg/100g, 229.0mg/100g, 181.5mg/100g and 128.6mg/100g for raw, first, second, third and fourth days respectively. Similarly for Buff they were 353.7mg/100g, 311.6mg/100g, 270.9mg/100g, 205.4mg/100g and 162.6mg/100g for raw, first, second, third and fourth days respectively. Autoclaving at 150ºC under 20 psi for 30 min reduced phytic acid for Ain Elgazal raw seeds from 310.3 to 300.0mg/100g. Similarly for Buff raw seeds autoclaving reduced phytic acid from 376.3 to 350.0mg/100g. Roasting at 120ºC for 60 min reduced phytic acid for Ain Elgazal raw seeds from 310.3 to 301.0mg/100g. Similarly for Buff roasting reduced phytic acid from 376.3 to 352.0mg/100g. Germination significantly (P ≤ 0.05) decreased tannins from 0.48g/100g of Ain Elgazal raw seeds to 0.36g/100g, 0.30g/100g, 0.24g/100 and 0.20g/100g in the first, second, third and fourth days respectively. Similarly for Buff raw seeds tannins was 0.50g/100g it reduced by germination to 0.42g/100g, 0.36g/100g, 0.30g/100g and 0.22g/100g in the first, second, third and fourth days respectively. Autoclaving at 150ºC under 20 psi for 30 min reduce tannins for Ain Elgazal raw seeds from 0.48g/100g to 0.38g/100g. Similarly for Buff autoclaving reduce tannins from 0.50g/100g to 0.40g/100g. Roasting at 120ºC for 60 min reduced tannins for Ain Elgazal raw 17 seeds from 0.48g/100g to 0.36g/100g. Similarly for Buff roasting reduced tannins from 0.50g/100g to 0.42g/100g. Cooking showed significant (P ≤ 0.05) further reduction of tannins for Ain Elgazal raw and germinated seeds, they were 0.26g/100g, 0.18g/100g, 0.15g/100g, 0.12g/100g and 0.10g/100g for raw, first, second, third and fourth days respectively. Similarly for Buff they were 0.30g/100g, 0.22g/100g, 0.20g/100g, 0.18g/100g and 0.11g/100g for raw, first, second, third and fourth days respectively. Germination significantly (P ≤ 0.05) reduced trypsin inhibitor activity for Ain Elgazal raw seeds from 22.0 TUI/mg protein of raw to 11.8 TUI/mg protein, 10.6 TUI/mg protein, 8.0 TUI/mg and 8.0 TUI/mg protein for first, second, third and fourth days respectively. Similarly for Buff they were 25.0 TUI/mg protein, 12.5 TUI/mg protein, 10.0 TUI/mg protein, 9.6 TUI/mg and 9.0 TUI/mg protein for raw, first, second, third and fourth days respectively. Cooking autoclaving at 150ºC under 20 psi for 30 min and roasting at 120ºC for 60min eliminates trypsin inhibitor activity. Germination improved in-vitro protein digestibility (IVPD), it were 73.4%, 75.3%, 77.9%, 80.4% and 84.4% for Ain Elgazal raw seeds, first, second, third and fourth days, respectively. Similarly for Buff they were 74.2%, 75.7%, 79.3%, 82.4% and 83.6% respectively. Cookin germinating seeds showed significant (P < 0.05) further increase of in-vitro protein digestibility, they were 86.2%, 87.2%, 87.5%, 88.8%, and 88.5% for Ain Elgazal raw, first, second, third and fourth days respectively. Similarly for Buff were 85.4%, 86.3%, 86.6%, 87.9% and 88.3% for raw, first, second, third and fourth days respectively. Autoclaving at 150ºC under 20 psi for 30min increased 18 IVPD for Ain Elgazal raw seeds from 73.4% to 86.0% and increased by roasting at 120ºC for 60min to 84.0% Similarly Buff increased by autoclaving from 74.2% to 87.0% and by roasting to 84.0%. Effect of substrate on in-vitro protein digestibility was carried out using mixed samples. Increasing the proportion of heat treated material in comparison to raw material significantly (P ≤ 0.05) increased in-vitro protein digestibility. For Ain Elgazal the proportion were 0.192g raw materials plus 0.192 heat treated material, 0.096g plus 0.282g, 0.048g plus 0.336g, 0.0 plus 0.384g, 0.288g plus 0.096g, 0.336g plus 0.048g and 0.384g plus 0.0g. The heat treated material were roasted at 90ºC and 120ºC for 30min, 45min and 60min. The material were autoclaved at 115.5ºC under 10psi and at 120ºCunder 15 psi for 15 min, 30 min and 45min. The IVPD ranged from 73.4% to 84.0% and 73.4% to 84.2 for raw plus roasted mixed samples respectively. The IVPD ranged from 73.4% to 82.0%, 73.4% to 83.0%, 73.4% to 84.0%, 73.4% to 85.0%, 73.4 to 85.5% and 73.4% to 86.2 for raw plus autoclaved (mixed samples) for Ain Elgazal respectively. Similarly for Buff IVPD were ranged from 74.2% to 79.7%, 74.2% to 79.9%, 74.2% to 79.8%, 74.2% to 83.8%, 74.2 to 84.0% and 74.2% to 84.6 for raw plus roasted (mixed samples) respectively. The IVPD ranged from 74.2% to 80.2%, 74.2% to 84.8%, 74.2% to 84.8%, 74.2% to 85.0%, 74.2 to 85.0% and 74.2% to 85.4 for raw plus autoclaved (mixed samples) respectively. ﺑﺴﻢ اﷲ اﻟﺮﺣﻤﻦ اﻟﺮﺣﻴﻢ 19 ﺧﻼﺻﺔ ﺍﻷﻃﺮﻭﺣﺔ ﺖ ﰲ ﻫﺬﻩ ﺍﻟﺪﺭﺍﺳﺔ ﺻﻨﻔﲔ ﻣﻦ ﺍﻟﻠﻮﺑﻴﺎ ﺍﻟﻄﻴﺐ ﺃﹸﺳﺘﺠﻠﺒﺖ ﻣﻦ ﻫﻴﺌﺔ ﺍﻟﺒﺤﻮﺙ ﺍﻟﺰﺭﺍﻋﻴﺔ ﳏﻄﺔ ﺃﹸﺳﺘﺨﺪِﻣ ﺍﻷﺑﻴﺾ ﻭﻫﻲ ﻋﲔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻭﺍﻟﻌﻴﻨﺔ ﺑﻒ. ﰎ ﻋﻤﻞ ﺍﻟﺘﺤﻠﻴﻞ ﺍﻟﺘﻘﺮﻳﱯ ﻟﻠﻌﻴﻨﺎﺕ ﻭﻛﺬﻟﻚ ﲡﺰﺋﺔ ﺍﻟﱪﻭﺗﲔ ﺣﺴﺐ ﺧﺎﺻﻴﺔ ﺍﻟﺬﻭﺑﺎﻥ ﻭﰎ ﻫﻀﻢ ﺍﻟﱪﻭﺗﲔ ﻣﻌﻤﻠﻴﹰﺎ ﻭﺗﻘﺪﻳﺮ ﲪﺾ ﺍﻟﻔﺎﻳﺘﻚ ﻭﻧﺸﺎﻁ ﻣﺜﺒﻂ ﺇﻧﺰﱘ ﺍﻟﺘﺮﺑﺴﲔ ﻭﺍﻟﺘﺎﻧﲔ ﻟﻠﻌﻴﻨﺎﺕ ﺍﳋﺎﻡ ﻭﺍﻟﱵ ﰎ ﺗﻮﺯﻳﻌﻬﺎ ﳌﺪﺓ ﺃﺭﺑﻌﺔ ﺃﻳﺎﻡ ﻭﻟﻠﻤﻌﺎﻣﻠﺔ ﻃﺮﺩﻳﹰﺎ ﺑﺎﻟﻄﺒﺦ ﺍﻟﺘﻘﻠﻴﺪﻱ ﻭﺍﶈﻤﺼﺔ ﻭﺍﻟﻄﺒﺦ ﲢﺖ ﺿﻐﻂ ﺑﺎﻟﺒﺨﺎﺭ ﻭﺍﻟﻌﻴﻨﺎﺕ ﺍﻟﱵ ﰎ ﺗﺰﺭﻳﻌﻬﺎ ﰒ ﻃﺒﺨﺖ. ﺃﻭﺿﺤﺖ ﻧﺘﺎﺋﺞ ﺍﻟﺘﺤﻠﻴﻞ ﺍﻟﺘﻘﺮﻳﱯ ﺃﻥ ﻧﺴﺒﺔ ﺍﻟﺮﻃﻮﺑﺔ ﺗﺮﺍﻭﺣﺖ ﺑﲔ %5.9 – 4.4ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻭﺗﺮﺍﻭﺣﺖ ﺑﲔ %5.5-4.4ﻟﻠﻌﻴﻨﺔ ﺑﻒ .ﻭﺗﺮﺍﻭﺣﺖ ﻧﺴﺒﺔ ﺍﻟﱪﻭﺗﲔ ﺑﲔ %30.0-25.6ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻭﺗﺮﺍﻭﺣﺖ ﺑﲔ %28.9-24.6ﻟﻠﻌﻴﻨﺔ ﺑﻒ .ﳏﺘﻮﻯ ﺍﻟﻌﻴﻨﺎﺕ ﻣﻦ ﺍﻟﺮﻣﺎﺩ ﺗﺮﺍﻭﺡ ﺑﲔ %4.3-3.2ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻭﺗﺮﺍﻭﺡ ﺑﲔ %4.3-3.5ﻟﻠﻌﻴﻨﺔ ﺑﻒ .ﻭﺗﺮﺍﻭﺡ ﳏﺘﻮﻯ ﺍﻷﻟﻴﺎﻑ ﺑﲔ %3.2-2.5ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻭ %3.1-2.2ﻟﻠﻌﻴﻨﺔ ﺑﻒ .ﳏﺘﻮﻯ ﺍﻟﺰﻳﺖ ﺗﺮﺍﻭﺡ ﺑﲔ %1.6-1.5ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻭ %1.7-1.6ﻟﻠﻌﻴﻨﺔ ﺑﻒ .ﻛﻞ ﺍﳌﻌﺎﻣﻼﺕ ﻛﺎﻧﺖ ﺫﺍﺕ ﺗﺄﺛﲑ ﻗﻠﻴﻞ ﻋﻠﻰ ﳏﺘﻮﻯ ﺍﻟﺪﻫﻦ ﺑﻴﻨﻤﺎ ﻛﺎﻧﺖ ﻫﻨﺎﻙ ﺯﻳﺎﺩﺓ ﻣﻌﻨﻮﻳﺔ ﰲ ﳏﺘﻮﻯ ﺍﻟﱪﻭﺗﲔ ﻭﺍﻟﺮﻣﺎﺩ ﻭﺍﻷﻟﻴﺎﻑ ﻟﻠﻌﻴﻨﺘﲔ. ﰎ ﲡﺰﺋﺔ ﺑﺮﻭﺗﲔ ﺍﻟﻠﻮﺑﻴﺎ ﺍﳋﺎﻡ ﻭﺍﻟﱵ ﰎ ﺗﺰﺭﻳﻌﻬﺎ ﻭﺃﻇﻬﺮ ﺍﳉﻠﻮﺑﻴﻮﻟﲔ ﺗﻨﺎﻗﺺ ﻣﻌﻨﻮﻱ ﰲ ﺣﺪﻭﺩ ﺛﻘﺔ ) (0.05ﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ %70.2 ، %71.4 ، %76.4 ، %80.6 ، %87.5 :ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﺍﳋﺎﻡ ﻭﺍﳌﺰﺭﻋﺔ ﰲ ﺍﻟﻴﻮﻡ ﺍﻷﻭﻝ ﻭﰲ ﺍﻟﻴﻮﻡ ﺍﻟﺜﺎﱐ ﻭﰲ ﺍﻟﻴﻮﻡ ﺍﻟﺜﺎﻟﺚ ﻭﰲ ﺍﻟﻴﻮﻡ ﺍﻟﺮﺍﺑﻊ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ ﻭﺑﺎﳌﺜﻞ ﻛﺎﻥ ﻫﻨﺎﻙ ﺗﻨﺎﻗﺺ ﻣﻌﻨﻮﻱ ﰲ ﺣﺪﻭﺩ ﺛﻘﺔ ) (0.05ﰲ ﺍﻟﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻵﰐ، %75.6 ، %78.3 ، %81.3 ، %89.8 : %72.6ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ ﻫﺬﺍ ﺑﺎﻟﻨﺴﺒﺔ ﻟﻠﱪﻭﺗﲔ ﺍﻷﻛﱪ ﻭﻫﻮ ﺍﳉﻠﻮﺑﻴﻮﻟﲔ. ﻭﻛﺬﻟﻚ ﻧﺴﺒﺔ ﺍﻷﻟﺒﻴﻮﻣﲔ ﺗﻨﺎﻗﺺ ﻣﻌﻨﻮﻱ ﰲ ﺣﺪﻭﺩ ﺛﻘﺔ ) (0.05ﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ، %4.0 : %2.5 ، %1.9 ، %2.3 ، %3.8ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻭﺑﺎﳌﺜﻞ ﻛﺎﻥ ﻫﻨﺎﻙ ﺗﻨﺎﻗﺺ ﻣﻌﻨﻮﻱ ﰲ ﺣﺪﻭﺩ ﺛﻘﺔ ) (0.05ﰲ ﺍﻟﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻵﰐ %1.2 ، %2.2 ، %2.2 ، %1.5 ، %3.6 :ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ. 20 ﻭﻛﺎﻧﺖ ﻧﺘﺎﺋﺞ ﺍﻟﱪﻭﻻﻣﲔ ﰲ ﺍﻟﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻛﺎﻵﰐ، %1.6 ، %4.0 ، %3.3 ، %4.0 : %4.6ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻧﺖ ﻧﺘﺎﺋﺠﻪ ﻛﺎﻵﰐ %1.4 ، %3.9 ، %2.8 ، %2.7 ، %4.5 :ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ. ﻭﻗﺪ ﺃﻇﻬﺮ ﺍﻟﱪﻭﺗﲔ ﺝ-1ﺟﻠﻮﺗﻠﲔ ﺗﻨﺎﻗﺺ ﻣﻌﻨﻮﻱ ﰲ ﺣﺪﻭﺩ ﺛﻘﺔ ) (0.05ﻭﻛﺎﻧﺖ ﻧﺘﺎﺋﺠﻪ ﰲ ﺍﻟﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻛﺎﻵﰐ %1.7 ، %0.8 ، %1.3 ، %2.1 ، %2.4 :ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ %2.1 ، %1.3 ، %1.2 ، %2.3 :ﻭ %1.3ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ. ﻭﻛﺎﻧﺖ ﻧﺘﺎﺋﺞ ﺍﻟﱪﻭﺗﲔ ﺝ -2ﺟﻠﻮﺗﻠﲔ ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻛﺎﻵﰐ، %1.9 ، %2.1 ، %2.4 : %1.7 ، %1.4ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ، %5.1 ، %6.4 ، %2.5 : %3.9ﻭ %4.5ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ. ﺍﻟﱪﻭﺗﲔ ﺝ-3ﺟﻠﻮﺗﻠﲔ ﺃﻇﻬﺮ ﺯﻳﺎﺩﺓ ﻣﻌﻨﻮﻳﺔ ﰲ ﺣﺪﻭﺩ ﺛﻘﺔ ) (0.05ﻭﻛﺎﻧﺖ ﻧﺘﺎﺋﺠﻪ ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻛﺎﻵﰐ %13.5 ، %12.0 ، %9.8 ، %4.8 :ﻭ %10.4ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ %11.1 ، %12.5 ، %10.0 ، %4.5 :ﻭ %11.7ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ. ﻛﻤﺎ ﺃﻇﻬﺮﺕ ﻧﺘﺎﺋﺞ ﺍﻟﱪﻭﺗﻴﻨﺎﺕ ﻏﲑ ﺍﻟﺬﺍﺋﺒﺔ ﺯﻳﺎﺩﺓ ﻣﻌﻨﻮﻳﺔ )ﰲ ﺣﺪﻭﺩ ﺛﻘﺔ (0.05ﰲ ﺍﻟﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻭﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ %9.2 ، %2.1 ، %2.5 ، %1.2 :ﻭ %6.0ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ %3.6 ، %1.9 ، %1.7 ، %1.2 :ﻭ %6.1ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ. ﻛﺎﻥ ﻫﻨﺎﻙ ﺗﻨﺎﻗﺺ ﻣﻌﻨﻮﻱ )ﰲ ﺣﺪﻭﺩ ﺛﻘﺔ (0.05ﻟﻠﺠﻠﻮﺑﻴﻮﻟﲔ ﻧﺘﻴﺠﺔ ﻟﻄﺒﺦ ﺍﻟﻌﻴﻨﺎﺕ ﻏﲑ ﺍﳌﺰﺭﻋﺔ ﻭﺍﳌﺰﺭﻋﺔ ﰲ ﺍﻟﻴﻮﻡ ﺍﻷﻭﻝ ،ﺍﻟﺜﺎﱐ ،ﺍﻟﺜﺎﻟﺚ ﻭﺍﻟﺮﺍﺑﻊ ﻛﺎﻵﰐ %18.0 ، %18.1 ، %19.0 ، %16.4 :ﻭ %16.8ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ .ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ، %19.9 ، %15.7 : %22.6 ، %22.5ﻭ %23.0ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ .ﻛﺬﻟﻚ ﻛﺎﻥ ﻫﻨﺎﻟﻚ ﺗﻨﺎﻗﺺ ﻣﻌﻨﻮﻱ )ﰲ ﺣﺪﻭﺩ ﺛﻘﺔ (0.05 ﻟﻸﻟﺒﻴﻮﻣﲔ ﻭﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ %1.8 ، %3.1 ، %2.8 ، %1.9 :ﻭ %1.4ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻏﲑ ﺍﳌﺰﺭﻋﺔ ﻭﺍﳌﺰﺭﻋﺔ ﰲ ﺍﻟﻴﻮﻡ ﺍﻷﻭﻝ ،ﺍﻟﺜﺎﱐ ،ﺍﻟﺜﺎﻟﺚ ﻭﺍﻟﺮﺍﺑﻊ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ .ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ: 21 %2.1 ، %1.6 ، %1.6 ، %2.2ﻭ %1.3ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ .ﻛﻤﺎ ﺃﻇﻬﺮ ﺍﻟﱪﻭﻻﻣﲔ ﺯﻳﺎﺩﺓ ﻣﻌﻨﻮﻳﺔ ﻧﺘﻴﺠﺔ ﻟﻠﻄﺒﺦ ﻟﻠﻌﻴﻨﺎﺕ ﻏﲑ ﺍﳌﺰﺭﻋﺔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻭﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ %5.1 ، %6.3 ، %4.2 ﻭ %5.0ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ .ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻧﺖ ﻫﻨﺎﻟﻚ ﺯﻳﺎﺩﺓ ﰲ ﺍﻟﻌﻴﻨﺔ ﺍﻟﻐﲑ ﻣﺰﺭﻋﺔ ﻭﺍﳌﺰﺭﻋﺔ ﰲ ﺍﻟﻴﻮﻡ ﺍﻷﻭﻝ ﻛﺎﻵﰐ %5.1 ، %4.6ﰒ ﺗﻨﺎﻗﺺ ﰲ ﺍﻟﺜﺎﱐ ﻭﺍﻟﺜﺎﻟﺚ ﻭﺍﻟﺮﺍﺑﻊ ﻭﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ، %2.7 ، %2.7 : %1.8ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ .ﺝ-1ﺟﻠﻮﺗﻴﻠﲔ ﺃﻇﻬﺮ ﺗﻨﺎﻗﺾ ﻣﻌﻨﻮﻱ ﰲ ﺍﻟﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﺍﻟﻐﲑ ﻣﺰﺭﻋﺔ ﻭﺍﳌﺰﺭﻋﺔ ﰲ ﺍﻟﻴﻮﻡ ﺍﻷﻭﻝ ،ﺍﻟﺜﺎﱐ ،ﻭﺍﻟﺜﺎﻟﺚ ﻭﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ %1.3 ، %1.6 ، %1.4 ، %2.0 :ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ ﰒ ﺃﻇﻬﺮ ﺯﻳﺎﺩﺓ ﰲ ﺍﻟﻴﻮﻡ ﺍﻟﺮﺍﺑﻊ ﺇﱃ .%2.6ﻭﻛﺬﻟﻚ ﰲ ﺍﻟﻌﻴﻨﺔ ﺑﻒ ﺃﻇﻬﺮ ﺝ-1ﺟﻠﻮﺗﻴﻠﲔ ﺗﻨﺎﻗﺺ ﻣﻌﻨﻮﻱ ﻭﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ %1.6 ، %1.1 ، %1.2 ، %2.7 :ﻭ %1.0ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ. ﺃﻇﻬﺮ ﺝ-2ﺟﻠﻮﺗﻴﻠﲔ ﺯﻳﺎﺩﺓ ﻣﻌﻨﻮﻳﺔ ﻋﻨﺪ ﻃﺒﺦ ﺍﻟﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﺍﻟﻐﲑ ﻣﺰﺭﻋﺔ ﻭﺍﳌﺰﺭﻋﺔ ﰲ ﺍﻟﻴﻮﻡ ﺍﻷﻭﻝ ، ﺍﻟﺜﺎﱐ ،ﺍﻟﺜﺎﻟﺚ ﻭﺍﻟﺮﺍﺑﻊ ﻭﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ %4.0 ، %4.4 ، %4.4 ، %3.4 :ﻭ %5.7ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ. ﻭﺑﺎﳌﺜﻞ ﰲ ﺍﻟﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ %7.9 ، %7.5 ، %7.5 ، %5.1 :ﻭ %8.8ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ. ﺃﻇﻬﺮ ﺝ-3ﺟﻠﻮﺗﻴﻠﲔ ﺯﻳﺎﺩﺓ ﻣﻌﻨﻮﻳﺔ ﻋﻨﺪ ﻃﺒﺦ ﺍﻟﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻭﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ، %63.8 : %61.6 ، %62.0 ، %61.0ﻭ %57.0ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ .ﻭﺑﺎﳌﺜﻞ ﰲ ﺍﻟﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ: %61.0 ، %62.4 ، %59.5 ، %69.3ﻭ %62.1ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ .ﻭﻛﺬﻟﻚ ﺃﻇﻬﺮﺕ ﺍﻟﱪﻭﺗﻴﻨﺎﺕ ﻏﲑ ﺍﻟﺬﺍﺋﺒﺔ ﺯﻳﺎﺩﺓ ﻣﻌﻨﻮﻳﺔ ﻋﻨﺪ ﻃﺒﺦ ﺍﻟﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻭﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ، %8.8 ، %8.0 ، %5.5 : %9.6ﻭ %10.2ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ ﻭﺑﺎﳌﺜﻞ ﻛﺎﻧﺖ ﻧﺘﺎﺋﺞ ﺍﻟﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻵﰐ، %6.5 ، %6.6 ، %5.5 : %6.1ﻭ %6.5ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ. ﻋﻨﺪ ﻃﺒﺦ ﺍﻟﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻏﲑ ﺍﳌﺰﺭﻋﺔ ﻃﺒﺦ ﺭﻃﺐ ﲢﺖ ﺿﻐﻂ 15ﺭﻃﻞ/ﺑﻮﺻﺔ 2ﰲ ﺩﺭﺟﺔ ﺣﺮﺍﺭﺓ ﻡ ﳌﺪﺓ 30ﺩﻗﻴﻘﺔ ﺃﻇﻬﺮ ﺍﳉﻠﻮﺑﻴﻮﻟﲔ ﺗﻨﺎﻗﺼﹰﺎ ﻣﻌﻨﻮﻳﹰﺎ )ﰲ ﺣﺪﻭﺩ ﺛﻘﺔ (0.05ﻣﻦ %87.5ﺇﱃ 120º %29.2 ﻭﺍﻷﻟﺒﻴﻮﻣﲔ ﻣﻦ %4.0ﺇﱃ %1.2ﺑﻴﻨﻤﺎ ﺯﺍﺩ ﺍﻟﱪﻭﻻﻣﲔ ﻣﻦ %4.3ﺇﱃ %4.5ﻭﺗﻨﺎﻗﺺ ﺝ-1ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %2.3ﺇﱃ %1.2ﻭﺝ-2ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %2.4ﺇﱃ %1.2ﻭﺯﺍﺩ ﺝ-3ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %4.8ﺇﱃ %59.5 22 ﻭﺍﻟﱪﻭﺗﻴﻨﺎﺕ ﻏﲑ ﺍﻟﺬﺍﺋﺒﺔ ﻣﻦ %1.2ﺇﱃ %8.0ﻭﻋﻨﺪ ﻃﺒﺦ ﻧﻔﺲ ﺍﻟﻌﻴﻨﺔ ﻃﺒﺦ ﺭﻃﺐ ﲢﺖ ﺿﻐﻂ 20ﺭﻃﻞ ﻡ ﳌﺪﺓ 30ﺩﻗﻴﻘﺔ ﺇﳔﻔﺾ ﺍﳉﻠﻮﺑﻴﻮﻟﲔ ﻣﻦ %87.5ﺇﱃ º %27.5ﻟﻠﺒﻮﺻﺔ ﺍﳌﺮﺑﻌﺔ ﰲ ﺩﺭﺍﺟﺔ ﺣﺮﺍﺭﺓ 150 ﻭﺍﻷﻟﺒﻴﻮﻣﲔ ﻣﻦ %4.0ﺇﱃ %1.2ﻭﺯﺍﺩ ﺍﻟﱪﻭﻻﻣﲔ ﻣﻦ %4.3ﺇﱃ %4.8ﻭﺗﻨﺎﻗﺺ ﺝ-1ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %2.3ﺇﱃ %2.0ﻭﺗﺰﺍﻳﺪ ﺝ-2ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %2.4ﺇﱃ %4.0ﻭﺝ-3ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %4.8ﺇﱃ %60.2 ﻡ ﳌﺪﺓ ºﻭﺍﻟﱪﻭﺗﻴﻨﺎﺕ ﻏﲑ ﺍﻟﺬﺍﺋﺒﺔ ﻣﻦ %1.2ﺇﱃ %6.0ﻭﻋﻨﺪ ﲢﻤﻴﺺ ﻧﻔﺲ ﺍﻟﻌﻴﻨﺔ )ﺍﻟﻄﺒﺦ ﺍﳉﺎﻑ( ﻋﻨﺪ 90 60ﺩﻗﻴﻘﺔ ﺗﻨﺎﻗﺺ ﺍﳉﻠﻮﺑﻴﻮﻟﲔ ﺗﻨﺎﻗﺼﹰﺎ ﻣﻌﻨﻮﻳﹰﺎ )ﰲ ﺣﺪﻭﺩ ﺛﻘﺔ (0.05ﻣﻦ %87.5ﺇﱃ %85.0ﻭﺃﻇﻬﺮ ﺍﻷﻟﺒﻴﻮﻣﲔ ﺛﺒﺎﺗﹰﺎ ﻭﺗﻨﺎﻗﺺ ﺍﻟﱪﻭﻻﻣﲔ ﻣﻦ %4.3ﺇﱃ %3.6ﻭﺝ-1ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %2.3ﺇﱃ %2.0ﻭﺝ-2 ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %2.4ﺇﱃ .%1.2ﻭﺗﺰﺍﻳﺪ ﺝ-3ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %4.8ﺇﱃ %6.0ﻭﺍﻟﱪﻭﺗﻴﻨﺎﺕ ﻏﲑ ﺍﻟﺬﺍﺋﺒﺔ ﻣﻦ ﻡ ﳌﺪﺓ 60ﺩﻗﻴﻘﺔ ﺃﻇﻬﺮ ﺍﳉﻠﻮﺑﻴﻮﻟﲔ %1.2ºﺇﱃ .%3.0ﻭﻋﻨﺪ ﲢﻤﻴﺺ ﻧﻔﺲ ﺍﻟﻌﻴﻨﺔ ﰲ ﺩﺭﺟﺔ ﺣﺮﺍﺭﺓ 120 ﺗﻨﺎﻗﺼﹰﺎ ﻣﻌﻨﻮﻳﹰﺎ )ﰲ ﺣﺪﻭﺩ ﺛﻘﺔ (0.05ﻣﻦ %87.5ﺇﱃ %41.0ﻭﺍﻷﻟﺒﻴﻮﻣﲔ ﻣﻦ %4.0ﺇﱃ %1.2 ﻭﺍﻟﱪﻭﻻﻣﲔ ﻣﻦ %4.3ﺇﱃ %4.2ﻭﺝ-1ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %2.3ﺇﱃ %1.2ﻭﺗﺰﺍﻳﺪ ﺝ-2ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %2.4ﺇﱃ %3.2ﻭﺝ-3ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %4.8ﺇﱃ %48.0ﻭﺍﻟﱪﻭﺗﻴﻨﺎﺕ ﻏﲑ ﺍﻟﺬﺍﺋﺒﺔ ﻣﻦ %1.2ﺇﱃ .%6.0 ﻡ ﳌﺪﺓ 30ﺩﻗﻴﻘﺔ ﻟﻠﻌﻴﻨﺔ ºﻭﻋﻨﺪ ﺍﻟﻄﺒﺦ ﺍﻟﺮﻃﺐ ﲢﺖ ﺿﻐﻂ 15ﺭﻃﻞ/ﺑﻮﺻﺔ 2ﰲ ﺩﺭﺟﺔ ﺣﺮﺍﺭﺓ 120 ﺑﻒ ﺃﻇﻬﺮ ﺍﳉﻠﻮﺑﻴﻮﻟﲔ ﺗﻨﺎﻗﺼﹰﺎ ﻣﻌﻨﻮﻳﹰﺎ )ﰲ ﺣﺪﻭﺩ ﺛﻘﺔ (0.05ﻣﻦ %89.8ﺇﱃ %32.0ﻭﺍﻷﻟﺒﻴﻮﻣﲔ ﻣﻦ %3.6ﺇﱃ %1.0ﻭﺍﻟﱪﻭﻻﻣﲔ ﻣﻦ %4.5ﺇﱃ %3.0ﻭﺝ-1ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %2.3ﺇﱃ %2.0ﻭﺗﺰﺍﻳﺪ ﺝ-2ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %2.5ﺇﱃ %3.0ﻭﺝ-3ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %4.5ﺇﱃ %55.0ﻭﺍﻟﱪﻭﺗﻴﻨﺎﺕ ﻏﲑ ﺍﻟﺬﺍﺋﺒﺔ ﻣﻦ %1.2ﺇﱃ %6.0ﻭﻋﻨﺪ ﻃﺒﺦ ﻧﻔﺲ ﺍﻟﻌﻴﻨﺔ ﻃﺒﺦ ﺭﻃﺐ ﲢﺖ ﺿﻐﻂ 20ﺭﻃﻞ ﻟﻠﺒﻮﺻﺔ ﺍﳌﺮﺑﻌﺔ ﰲ ﺩﺭﺍﺟﺔ ﺣﺮﺍﺭﺓ ﻡ ﳌﺪﺓ 30ﺩﻗﻴﻘﺔ ﺗﻨﺎﻗﺺ ﺍﳉﻠﻮﺑﻴﻮﻟﲔ ﺗﻨﺎﻗﺼﹰﺎ ﻣﻌﻨﻮﻳﹰﺎ )ﰲ ﺣﺪﻭﺩ ﺛﻘﺔ (0.05ﻣﻦ %89.8ﺇﱃ 150º %36.0 ﻭﺍﻷﻟﺒﻴﻮﻣﲔ ﻣﻦ %3.6ﺇﱃ %1.0ﻭﺍﻟﱪﻭﻻﻣﲔ ﻣﻦ %4.5ﺇﱃ %4.0ﻭﺝ-1ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %2.3ﺇﱃ %1.2ﻭﺗﺰﺍﻳﺪ ﺝ-2ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %2.5ﺇﱃ %3.2ﻭﺝ-3ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %4.5ﺇﱃ %50.8ﻭﺍﻟﱪﻭﺗﻴﻨﺎﺕ 23 ﻡ ﳌﺪﺓ 60ﺩﻗﻴﻘﺔ ﺃﻇﻬﺮ ºﻏﲑ ﺍﻟﺬﺍﺋﺒﺔ ﻣﻦ %1.2ﺇﱃ .%7.0ﻭﻋﻨﺪ ﲢﻤﻴﺺ ﻧﻔﺲ ﺍﻟﻌﻴﻨﺔ ﰲ ﺩﺭﺟﺔ ﺣﺮﺍﺭﺓ 90 ﺍﳉﻠﻮﺑﻴﻮﻟﲔ ﺗﻨﺎﻗﺼﹰﺎ ﻣﻌﻨﻮﻳﹰﺎ )ﰲ ﺣﺪﻭﺩ ﺛﻘﺔ (0.05ﻣﻦ %89.8ﺇﱃ %88.0ﻭﺃﻇﻬﺮ ﺍﻷﻟﺒﻴﻮﻣﲔ ﺛﺒﺎﺗﹰﺎ ﻭﺗﻨﺎﻗﺺ ﺍﻟﱪﻭﻻﻣﲔ ﻣﻦ %4.0ﺇﱃ %4.5ﻭﺝ-1ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %2.3ﺇﱃ %2.2ﻭﺗﺰﺍﻳﺪ ﺝ-2ﺟﻠﻮﺗﻴﻠﲔ ﻣﻦ %2.5 ﺇﱃ .%2.8ﻭﺝ-3ﺟﻠﻮﺗﻴﻠﲔ ﺃﻇﻬﺮ ﺛﺒﺎﺗﹰﺎ ﻭﺯﺍﺩﺕ ﺍﻟﱪﻭﺗﻴﻨﺎﺕ ﺍﻟﺬﺍﺋﺒﺔ ﻣﻦ %1.2ﺇﱃ .%1.4ﻋﻨﺪ ﲢﻤﻴﺺ ﺍﻟﻌﻴﻨﺔ ﻡ ﳌﺪﺓ 60ﺩﻗﻴﻘﺔ ﺃﻇﻬﺮﺕ ﺗﻨﺎﻗﺺ ﻣﻌﻨﻮﻱ ﰲ ﺍﳉﻠﻮﺑﻴﻮﻟﲔ ﻣﻦ ºﺑﻒ ﻏﲑ ﺍﳌﻌﺎﻣﻠﺔ ﰲ ﺩﺭﺟﺔ ﺣﺮﺍﺭﺓ 120 %89.8ﺇﱃ %45.5ﻭﺍﻷﻟﺒﻴﻮﻣﲔ ﻣﻦ %3.6ﺇﱃ %1.0ﻭﺍﻟﱪﻭﻻﻣﲔ ﺯﺍﺩ ﻣﻦ %4.5ﺇﱃ %6.0ﻭﺝ-1 ﺟﻠﻮﺗﻴﻠﲔ ﻧﻘﺺ ﻣﻦ %2.3ﺇﱃ %2.0ﻭﺝ-2ﺟﻠﻮﺗﻴﻠﲔ ﺯﺍﺩ ﻣﻦ %2.5ﺇﱃ %3.0ﻭﺝ-3ﺟﻠﻮﺗﻴﻠﲔ ﺯﺍﺩ ﻣﻦ %4.5ﺇﱃ %42.0ﻭﺍﻟﱪﻭﺗﻴﻨﺎﺕ ﻏﲑ ﺍﻟﺬﺍﺋﺒﺔ ﺯﺍﺩﺕ ﻣﻦ %1.2ﺇﱃ .%6.0 ﰎ ﺇﺧﺘﺒﺎﺭ ﺍﻟﻌﻴﻨﺎﺕ ﺍﳌﻌﺎﻣﻠﺔ ﻣﻘﺎﺑﻞ ﺍﻷﺧﺮﻯ ﻏﲑ ﺍﳌﻌﺎﻣﻠﺔ ﳊﻤﺾ ﺍﻟﻔﺎﻳﺘﻚ ﻭﺍﻟﺘﺎﻧﲔ ﻭﻣﺜﺒﻂ ﺇﻧﺰﱘ ﺍﻟﺘﺮﺑﺴﲔ. ﻭﻗﺪ ﻭﺟﺪ ﺇﳔﻔﺎﺽ ﻣﻌﻨﻮﻱ )ﰲ ﺣﺪﻭﺩ ﺛﻘﺔ (0.05ﰲ ﲪﺾ ﺍﻟﻔﺎﺗﻴﻚ ﻭﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ: 310.3ﻣﻠﺠﻢ100/ﺟﻢ ﻋﻠﻰ ﺃﺳﺎﺱ ﺍﻟﻮﺯﻥ ﺍﳉﺎﻑ386.1 ،ﻣﻠﺠﻢ100/ﺟﻢ 248.9 ،ﻣﻠﺠﻢ100/ﺟﻢ ، 201.7ﻣﻠﺠﻢ100/ﺟﻢ ﻭ 139.8ﻣﻠﺠﻢ100/ﺟﻢ ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻏﲑ ﺍﳌﻌﺎﻣﻠﺔ ،ﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻡ ﻭﺍﺣﺪ ، ﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻣﲔ ،ﺍﳌﺰﺭﻋﺔ ﻟﺜﻼﺛﺔ ﺃﻳﺎﻡ ﻭﺍﳌﺰﺭﻋﺔ ﻷﺭﺑﻌﺔ ﺃﻳﺎﻡ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ .ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ: 376.3ﻣﻠﺠﻢ100/ﺟﻢ 346.2 ،ﻣﻠﺠﻢ100/ﺟﻢ 301.0 ،ﻣﻠﺠﻢ100/ﺟﻢ 225.7 ،ﻣﻠﺠﻢ100/ﺟﻢ ﻭ 180.7ﻣﻠﺠﻢ100/ﺟﻢ ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻏﲑ ﺍﳌﻌﺎﻣﻠﺔ ،ﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻡ ﻭﺍﺣﺪ ،ﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻣﲔ ،ﺍﳌﺰﺭﻋﺔ ﻟﺜﻼﺛﺔ ﺃﻳﺎﻡ ﻭﺍﳌﺰﺭﻋﺔ ﻷﺭﺑﻌﺔ ﺃﻳﺎﻡ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ. ﻭﻋﻨﺪ ﻃﺒﺦ ﺍﻟﻌﻴﻨﺎﺕ ﺍﳌﺰﺭﻋﺔ ﻭﻏﲑ ﺍﳌﺰﺭﻋﺔ ﺃﻇﻬﺮ ﲪﺾ ﺍﻟﻔﺎﻳﺘﻚ ﺗﻨﺎﻗﺺ ﻣﻌﻨﻮﻱ ﰲ ﺍﻟﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻭﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ290.3 :ﻣﻠﺠﻢ100/ﺟﻢ 268.9 ،ﻣﻠﺠﻢ100/ﺟﻢ 229.0 ،ﻣﻠﺠﻢ100/ﺟﻢ ، 181.5ﻣﻠﺠﻢ100/ﺟﻢ ﻭ 128.6ﻣﻠﺠﻢ100/ﺟﻢ ﻟﻠﻌﻴﻨﺔ ﻏﲑ ﺍﳌﺰﺭﻋﺔ ،ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻡ ﻭﺍﺣﺪ ،ﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻣﲔ ،ﺍﳌﺰﺭﻋﺔ ﻟﺜﻼﺛﺔ ﺃﻳﺎﻡ ﻭﺍﳌﺰﺭﻋﺔ ﻷﺭﺑﻌﺔ ﺃﻳﺎﻡ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ .ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ: 353.7ﻣﻠﺠﻢ100/ﺟﻢ 311.6 ،ﻣﻠﺠﻢ100/ﺟﻢ 270.9 ،ﻣﻠﺠﻢ100/ﺟﻢ 205.4 ،ﻣﻠﺠﻢ100/ﺟﻢ ﻭ 24 162.6ﻣﻠﺠﻢ100/ﺟﻢ ﻟﻠﻌﻴﻨﺔ ﻏﲑ ﺍﳌﻌﺎﻣﻠﺔ ،ﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻡ ﻭﺍﺣﺪ ،ﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻣﲔ ،ﺍﳌﺰﺭﻋﺔ ﻟﺜﻼﺛﺔ ﺃﻳﺎﻡ ﻭﺍﳌﺰﺭﻋﺔ ﻷﺭﺑﻌﺔ ﺃﻳﺎﻡ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ. ﻭﻋﻨﺪ ﻃﺒﺦ ﺍﻟﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻏﲑ ﺍﳌﺰﺭﻋﺔ ﻃﺒﺦ ﺭﻃﺐ ﲢﺖ ﺿﻐﻂ 20ﺭﻃﻞ ﻟﻠﺒﻮﺻﺔ ﺍﳌﺮﺑﻌﺔ ﰲ ﺩﺭﺍﺟﺔ ﻡ ﳌﺪﺓ 30ﺩﻗﻴﻘﺔ ﺇﳔﻔﺾ ﲪﺾ ﺍﻟﻔﺎﻳﺘﻚ ﻣﻦ 310.3ﻣﻠﺠﻢ100/ﺟﻢ ﺇﱃ º 300.0ﺣﺮﺍﺭﺓ 150 ﻣﻠﺠﻢ100/ﺟﻢ ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﻏﲑ ﺍﳌﺰﺭﻋﺔ ﺇﳔﻔﺾ ﲪﺾ ﺍﻟﻔﺎﻳﺘﻚ ﻣﻦ 376.3ﻣﻠﺠﻢ100/ﺟﻢ ﺇﱃ ﻡ ﳌﺪﺓ 60ﺩﻗﻴﻘﺔ ﺍﳔﻔﺾ 300.0ºﻣﻠﺠﻢ100/ﺟﻢ .ﻭﻋﻨﺪ ﲢﻤﻴﺺ ﺍﻟﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﰲ ﺩﺭﺟﺔ ﺣﺮﺍﺭﺓ 120 ﲪﺾ ﺍﻟﻔﺎﻳﺘﻚ ﻣﻦ 310.3ﻣﻠﺠﻢ100/ﺟﻢ ﺇﱃ 301.0ﻣﻠﺠﻢ100/ﺟﻢ ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﺇﳔﻔﺾ ﲪﺾ ﺍﻟﻔﺎﻳﺘﻚ ﻣﻦ 376.3ﻣﻠﺠﻢ100/ﺟﻢ ﺇﱃ 352.0ﻣﻠﺠﻢ100/ﺟﻢ .ﻭﺃﻇﻬﺮﺕ ﺍﻟﺘﺎﻧﻴﻨﺎﺕ ﺍﳔﻔﺎﺿﹰﺎ ﻣﻌﻨﻮﻳﹰﺎ ﻋﻨﺪ ﺗﺰﺭﻳﻊ ﺍﻟﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻭﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ 0.48 :ﺟﻢ100/ﺟﻢ 0.36 ،ﺟﻢ100/ﺟﻢ 0.30 ، ﺟﻢ100/ﺟﻢ 0.24 ،ﺟﻢ100/ﺟﻢ 0.20 ،ﺟﻢ100/ﺟﻢ ﻟﻠﻌﻴﻨﺔ ﻏﲑ ﺍﳌﺰﺭﻋﺔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻡ ﻭﺍﺣﺪ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻣﲔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﺜﻼﺛﺔ ﺍﻳﺎﻡ ﻭﺍﳌﺰﺭﻋﺔ ﻷﺭﺑﻌﺔ ﺃﻳﺎﻡ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ .ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ ﻛﺎﻵﰐ 0.50 :ﺟﻢ100/ﺟﻢ 0.42 ،ﺟﻢ100/ﺟﻢ 0.36 ،ﺟﻢ100/ﺟﻢ 0.30 ،ﺟﻢ100/ﺟﻢ ، 0.22ﺟﻢ100/ﺟﻢ ﻟﻠﻌﻴﻨﺔ ﻏﲑ ﺍﳌﺰﺭﻋﺔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻡ ﻭﺍﺣﺪ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻣﲔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﺜﻼﺛﺔ ﺃﻳﺎﻡ ﻭﺍﳌﺰﺭﻋﺔ ﻷﺭﺑﻌﺔ ﺃﻳﺎﻡ .ﻭﻋﻨﺪ ﻃﺒﺦ ﺍﻟﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻏﲑ ﺍﳌﺰﺭﻋﺔ ﻃﺒﺦ ﺭﻃﺐ ﲢﺖ ﺿﻐﻂ 20ﺭﻃﻞ ﻟﻠﺒﻮﺻﺔ ﺍﳌﺮﺑﻌﺔ ﰲ ﻡ ﳌﺪﺓ 30ﺩﻗﻴﻘﺔ ﺇﳔﻔﻀﺖ ﺍﻟﺘﺎﻧﻴﻨﺎﺕ ﻣﻦ 0.48ﺟﻢ100/ﺟﻢ ﺇﱃ 0.38ﺟﻢ100/ﺟﻢ ºﺩﺭﺍﺟﺔ ﺣﺮﺍﺭﺓ 150 ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﺇﳔﻔﻀﺖ ﺍﻟﺘﺎﻧﻴﻨﺎﺕ ﻣﻦ 0.50ﺟﻢ100/ﺟﻢ ﺇﱃ 0.40ﺟﻢ100/ﺟﻢ .ﻭﻋﻨﺪ ﲢﻤﻴﺺ ﻡ ﳌﺪﺓ 60ﺩﻗﻴﻘﺔ ﺍﳔﻔﻀﺖ ﺍﻟﺘﺎﻧﻴﻨﺎﺕ ﻣﻦ 0.48ﺟﻢ100/ﺟﻢ ﺇﱃ ºﺍﻟﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﰲ ﺩﺭﺟﺔ ﺣﺮﺍﺭﺓ 120 0.36ﺟﻢ100/ﺟﻢ ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﺇﳔﻔﻀﺖ ﺍﻟﺘﺎﻧﻴﻨﺎﺕ ﻣﻦ 0.50ﺟﻢ100/ﺟﻢ ﺇﱃ 0.42 ﺟﻢ100/ﺟﻢ .ﻭﺃﻇﻬﺮﺕ ﺍﻟﺘﺎﻧﻴﻨﺎﺕ ﺗﻨﺎﻗﺺ ﻣﺘﺰﺍﻳﺪ ﻋﻨﺪ ﻃﺒﺦ ﺍﻟﻌﻴﻨﺔ ﺑﻒ ﻏﲑ ﺍﳌﺰﺭﻋﺔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻡ ﻭﺍﺣﺪ ،ﻳﻮﻣﲔ ،ﺛﻼﺛﺔ ﺃﻳﺎﻡ ﻭﺃﺭﺑﻌﺔ ﺃﻳﺎﻡ ﻭﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ 0.26 :ﺟﻢ100/ﺟﻢ 0.18 ،ﺟﻢ100/ﺟﻢ 0.15 ، ﺟﻢ100/ﺟﻢ 0.12 ،ﺟﻢ100/ﺟﻢ 0.10 ،ﺟﻢ100/ﺟﻢ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ .ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ 25 ﻛﺎﻵﰐ 0.30 :ﺟﻢ100/ﺟﻢ 0.22 ،ﺟﻢ100/ﺟﻢ 0.20 ،ﺟﻢ100/ﺟﻢ 0.18 ،ﺟﻢ100/ﺟﻢ ، 0.11ﺟﻢ100/ﺟﻢ ﻟﻠﻌﻴﻨﺔ ﻏﲑ ﺍﳌﺰﺭﻋﺔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻡ ﻭﺍﺣﺪ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻣﲔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﺜﻼﺛﺔ ﺃﻳﺎﻡ ﻭﺍﳌﺰﺭﻋﺔ ﻷﺭﺑﻌﺔ ﺃﻳﺎﻡ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ. ﻛﻤﺎ ﺃﻇﻬﺮ ﻧﺸﺎﻁ ﺇﻧﺰﱘ ﺍﻟﺘﺮﺑﺴﲔ ﺇﳔﻔﺎﺽ ﻣﻌﻨﻮﻱ ﺑﺎﻟﺘﺰﺭﻳﻊ ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻭﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ: 22.0ﻭﺣﺪﺓ/ﻣﻠﺠﻢ 11.8 ،ﻭﺣﺪﺓ/ﻣﻠﺠﻢ 10.6 ،ﻭﺣﺪﺓ/ﻣﻠﺠﻢ 8.0 ،ﻭﺣﺪﺓ/ﻣﻠﺠﻢ 8.0 ،ﻭﺣﺪﺓ/ﻣﻠﺠﻢ ﻟﻠﻌﻴﻨﺔ ﻏﲑ ﺍﳌﺰﺭﻋﺔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻡ ﻭﺍﺣﺪ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻣﲔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﺜﻼﺛﺔ ﺃﻳﺎﻡ ﻭﺍﳌﺰﺭﻋﺔ ﻷﺭﺑﻌﺔ ﺃﻳﺎﻡ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ. ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ 25.0ﻭﺣﺪﺓ/ﻣﻠﺠﻢ 12.5 ،ﻭﺣﺪﺓ/ﻣﻠﺠﻢ 10.0 ،ﻭﺣﺪﺓ/ﻣﻠﺠﻢ 9.6 ، ﻭﺣﺪﺓ/ﻣﻠﺠﻢ 9.0 ،ﻭﺣﺪﺓ/ﻣﻠﺠﻢ ﻟﻠﻌﻴﻨﺔ ﻏﲑ ﺍﳌﺰﺭﻋﺔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻡ ﻭﺍﺣﺪ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻣﲔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﺜﻼﺛﺔ ﺃﻳﺎﻡ ﻼ ﺑﺎﳌﻌﺎﻣﻼﺕ ﺍﳊﺮﺍﺭﻳﺔ ﺍﳌﺨﺘﻠﻔﺔ. ﻭﺍﳌﺰﺭﻋﺔ ﻷﺭﺑﻌﺔ ﺃﻳﺎﻡ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ .ﻭﰎ ﺗﻜﺴﲑ ﻣﺜﺒﻂ ﺇﻧﺰﱘ ﺍﻟﺘﺮﺑﺴﲔ ﺗﻜﺴﲑﹰﺍ ﻛﺎﻣ ﹰ ﻭﻗﺪ ﰎ ﺗﻘﺪﻳﺮ ﺩﺭﺟﺔ ﻫﻀﻢ ﺍﻟﱪﻭﺗﲔ ﻣﻌﻤﻠﻴﹰﺎ ﻟﻠﻌﻴﻨﺎﺕ ﺍﳌﺰﺭﻋﺔ ﻭﻏﲑ ﺍﳌﺰﺭﻋﺔ ﻭﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ: %80.4 ، %77.9 ، %75.3 ، %73.4ﻭ %84.4ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻏﲑ ﺍﳌﺰﺭﻋﺔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻡ ﻭﺍﺣﺪ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻣﲔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﺜﻼﺛﺔ ﺃﻳﺎﻡ ﻭﺍﳌﺰﺭﻋﺔ ﻷﺭﺑﻌﺔ ﺃﻳﺎﻡ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ .ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ: %82.4 ، %79.3 ، %75.7 ، %74.2ﻭ %83.6ﻟﻠﻌﻴﻨﺔ ﻏﲑ ﺍﳌﺰﺭﻋﺔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻡ ﻭﺍﺣﺪ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻣﲔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﺜﻼﺛﺔ ﺃﻳﺎﻡ ﻭﺍﳌﺰﺭﻋﺔ ﻷﺭﺑﻌﺔ ﺃﻳﺎﻡ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ .ﻭﻋﻨﺪ ﻃﺒﺦ ﺍﻟﻌﻴﻨﺎﺕ ﺍﳌﺰﺭﻋﺔ ﺃﻇﻬﺮﺕ ﻗﻴﻤﺔ ﻫﻀﻢ ﺍﻟﱪﻭﺗﲔ ﺯﻳﺎﺩﺓ ﻣﻌﻨﻮﻳﺔ ﻭﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ %88.8 ، %87.5 ، %87.2 ، %86.2 :ﻭ%88.5 ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻏﲑ ﺍﳌﺰﺭﻋﺔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻡ ﻭﺍﺣﺪ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻣﲔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﺜﻼﺛﺔ ﺃﻳﺎﻡ ﻭﺍﳌﺰﺭﻋﺔ ﻷﺭﺑﻌﺔ ﺃﻳﺎﻡ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ .ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﻛﺎﻧﺖ ﺍﻟﻨﺘﺎﺋﺞ ﻛﺎﻵﰐ %87.9 ، %86.6 ، %86.3 ، %85.4 :ﻭ%88.3 ﻟﻠﻌﻴﻨﺔ ﻏﲑ ﺍﳌﺰﺭﻋﺔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻡ ﻭﺍﺣﺪ ﻭﺍﳌﺰﺭﻋﺔ ﻟﻴﻮﻣﲔ ﻭﺍﳌﺰﺭﻋﺔ ﻟﺜﻼﺛﺔ ﺃﻳﺎﻡ ﻭﺍﳌﺰﺭﻋﺔ ﻷﺭﺑﻌﺔ ﺃﻳﺎﻡ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ. ﻡ ﳌﺪﺓ 30ﺩﻗﻴﻘﺔ ºﻋﻨﺪ ﺍﻟﻄﺒﺦ ﺍﻟﺮﻃﺐ ﲢﺖ ﺿﻐﻂ 20ﺭﻃﻞ ﻟﻠﺒﻮﺻﺔ ﺍﳌﺮﺑﻌﺔ ﰲ ﺩﺭﺍﺟﺔ ﺣﺮﺍﺭﺓ 150 ﺯﺍﺩﺕ ﻗﻴﻤﺔ ﻫﻀﻢ ﺍﻟﱪﻭﺗﲔ ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻏﲑ ﺍﳌﺰﺭﻋﺔ ﻣﻦ %73.4ﺇﱃ %86.0ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﺯﺍﺩﺕ ﻡ ﳌﺪﺓ 60ﺩﻗﻴﻘﺔ ﺯﺍﺩﺕ ºﻣﻦ %74.0ﺇﱃ .%87.0ﻭﻋﻨﺪ ﲢﻤﻴﺺ ﺍﻟﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﰲ ﺩﺭﺟﺔ ﺣﺮﺍﺭﺓ 120 26 ﻗﻴﻤﺔ ﻫﻀﻢ ﺍﻟﱪﻭﺗﲔ ﻣﻦ %73.4ﺇﱃ %84.0ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﺯﺍﺩﺕ ﻗﻴﻤﺔ ﻫﻀﻢ ﺍﻟﱪﻭﺗﲔ ﻣﻦ %74.2ﺇﱃ .%84.0 ﻭﻗﺪ ﲤﺖ ﺩﺭﺍﺳﺔ ﺗﺄﺛﲑ ﺍﻟﺴﺒﺴﺘﺮﺍﺕ ﻋﻠﻰ ﻗﻴﻤﺔ ﻫﻀﻢ ﺍﻟﱪﻭﺗﲔ ﺑﺎﺳﺘﺨﺪﺍﻡ ﻋﻴﻨﺎﺕ ﳐﻠﻮﻃﺔ ﻣﻦ ﺍﻟﻌﻴﻨﺔ ﺍﳌﻌﺎﻣﻠﺔ ﺣﺮﺍﺭﻳﹰﺎ ﻣﻊ ﺍﻟﻌﻴﻨﺔ ﻏﲑ ﺍﳌﻌﺎﻣﻠﺔ ﻭﻛﺎﻧﺖ ﺍﳋﻠﻄﺔ ﰲ ﺍﻟﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻋﻠﻰ ﺍﻟﻨﺤﻮ ﺍﻷﰐ0.192 :ﺟﻢ 0.096 ، ﺟﻢ 0.048 ،ﺟﻢ 0.00 ،ﺟﻢ 0.288 ،ﺟﻢ 0.336ﺟﻢ ﻭ 0.384ﺟﻢ ﻣﻦ ﺍﻟﻌﻴﻨﺔ ﺍﳋﺎﻡ ﻣﻊ 0.192 ﺟﻢ 0.288 ،ﺟﻢ 0.336 ،ﺟﻢ 0.384 ،ﺟﻢ 0.096 ،ﺟﻢ 0.048 ،ﺟﻢ ﻭ 0.00ﺟﻢ ﻣﻦ ﻡ ºﻡ ﻭ º120ﺍﻟﻌﻴﻨﺎﺕ ﺍﳌﻌﺎﻣﻠﺔ ﺣﺮﺍﺭﻳﹰﺎ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ .ﻭﻗﺪ ﻛﺎﻧﺖ ﺍﳌﻌﺎﻣﻠﺔ ﺍﳊﺮﺍﺭﻳﺔ ﻟﻠﻌﻴﻨﺎﺕ ﲢﻤﻴﺼﹰﺎ ﰲ ﺩﺭﺟﺔ 90 ﻡ ﲢﺖ ﺿﻐﻂ º 10ﳌﺪﺓ 45 ، 30ﻭ 60ﺩﻗﻴﻘﺔ ﺑﺎﻹﺿﺎﻓﺔ ﻋﻠﻰ ﻃﺒﺦ ﺭﻃﺐ ﰲ ﺩﺭﺟﺔ ﺣﺮﺍﺭﺓ 115.5 ﻡ ﲢﺖ ﺿﻐﻂ 15ﺭﻃﻞ/ﺑﻮﺻﺔ 2ﳌﺪﺓ 45 ، 30ﻭ 60ﺩﻗﻴﻘﺔ ﻭﻗﺪ ﺗﺮﺍﻭﺣﺖ ﻧﺘﺎﺋﺞ ﻫﻀﻢ ºﺭﻃﻞ/ﺑﻮﺻﺔ 2ﻭ 120 ﺍﻟﱪﻭﺗﲔ ﺑﲔ %84.2-73.4 ، %84.0-%73.4 :ﺑﺎﻟﻨﺴﺒﺔ ﻟﻠﻌﻴﻨﺎﺕ ﺍﶈﻤﺼﺔ ﻛﻤﺎ ﺗﺮﺍﻭﺣﺖ ﰲ ﺍﻟﻄﺒﺦ ﺍﻟﺮﻃﺐ ﺑﲔ %82.0-73.4ﻭ -73.4 ، %85.0-73.4 ، %84.0-73.4 ، %83.0-73.4 %85.5ﻭ %86.2-73.4ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ .ﻭﺑﺎﳌﺜﻞ ﻟﻠﻌﻴﻨﺔ ﺑﻒ ﺗﺮﺍﻭﺣﺖ ﻗﻴﻤﺔ ﻫﻀﻢ ﺍﻟﱪﻭﺗﲔ ﻣﻦ %84.0-74.2 ، %83.8-74.2 ، %79.8-74.2 ، %84.6-74.2 ، %79.7-74.2ﻭ %84.6-74.2ﺑﺎﻟﻨﺴﺒﺔ ﻟﻠﻌﻴﻨﺎﺕ ﺍﶈﻤﺼﺔ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ ،ﻭﺗﺮﺍﻭﺣﺖ ﰲ ﺍﻟﻄﺒﺦ ﺍﻟﺮﻃﺐ ﻣﺎ ﺑﲔ -74.2 -74.2 ، %85.0-74.2 ، %85.0-74.2 ، %84.8-74.2 ، %84.8-74.2 ، %80.2 %85.4ﻟﻠﻌﻴﻨﺔ ﻋﲔ ﺍﻟﻐﺰﺍﻝ ﻋﻠﻰ ﺍﻟﺘﻮﺍﱄ. 27 CHAPTER ONE INTRODUCTON 28 1. INTRODUCTON 1.1. Importance of cowpea: Cowpeas (Vigna unguiculata and Vigna Senensis) are important grain legumes in East and West Africa as well other developing countries (Dovlo et al. 1976). The beans are also known by other names such as blackeye beans, southern peas, colossus peas and crowther peas. The pulse is indigenous to Africa (Okigbo, 1986), though it is now grown in other continents, such as Central America (Bressani et al. 1961) as well as North and South America and Asia. Higher meat prices during recent years and the need for protein- rich foods have led people in the most developing countries to shift their consumption to cowpeas and other grain legumes. Cowpea is grown mainly for the seed and sometimes pods in West Africa, India and South America, but it is grown for both seeds, pods and leaves in East Africa. It is also utilized as fodder and as quick growing cover-crop. It improves soil fertility because of its ability to fix nitrogen efficiently (up to 240 kg N per hectare) and can leave a fixed- N deposit in the soil of up to 60 – 70 kg/ha for the succecding crop (Rechie, 1985; Kachare, et al. 1988). Like other grain legumes, cowpeas are a good source of energy, proteins (amino acids), vitamins, minerals and deitary fiber. Legumes are sometimes refserred to as “poor man’s meat” or the “rich man’s vegetable” ( Walker, 1981). Cowpea is most widely consumed legume in Nigeria as it represents a cheap source of dietary protein. Of the different species of bean consume in the country, cowpeas in particular have attracted attention as possible home grown sources of protein and successive selections have made it possible to introduce several high yielding varieties with desirable packages of nutrients and with resistance to pest 29 and microbial infections (Ologhobo et al, 1983). Cowpea are major sources of protein in developing countries where consumption of certain animal foods is taboo because of religious or customary beliefs. This trend has been obsorved in parts of northern Nigeria where pork and pork products, horse meat, donkey meat and meat of camels and asses are avoided by muslims. Cowpeas are a major source of thiamin and niacin and also contain reasonable amount of other watersoluble vitamins, riboflavin, pyridoxine and folacin. In addition, they supply the essential minerals, calcium, magnesium, potassium, iron, zinc and phosphorus (Aykroyed et al. 1982). In Sudan, people relied mostly on other available leguminous crops (Ahmed and Nour, 1990). Cowpea proteins provide the three esential amino acids lysine threonine and methionine, (Dhankher et al 1990. Sosulski, 1987; Fashakin, 1986; Philips, 1987; Saeed, 1977; El Hardallou, 1980). Intense efforts to find alternative sources of proteins from plants adapted to adverse conditions are being conducted around the world (Siddhuraju et al. 1995; Bravo et al., 1994; Bhattacharya et al. 1994). Despite the importance of cowpea (Vigna unguculata L. Walp.) as afood crop in tropical and subtropical regions, especially in West Africa (Rachie, 1973; R.D phillips 1982 a), little work has been carried out on the characterization of its major seed proteins in comparison to other legume species such as Glycine max or Phaseolus vulgaris. A major globulin protein was identified by Joubert (1957). and (Carasco et al. 1978) reports further details of protein subunits present in both mature and developing cowpeas. Sefa–Dedeh and Stany, 1979a,b, reports further details of the solubility parameters of cowpea proteins. 1.2. Classification of Cowpea: 30 Cowpea belongs to the family leguminosae . It is known as black eye beans or southern peas, colossus peas and crowpeas . The pulse is indingenous to Africa though it is now grown in other non African countries (Uzogara, 1992a, b). 31 1.3. Antinutritional factors: Legumes are generally known to contain various natural constitunets which affect their nutritional quality. Some of these components are proteins which inhibit specific enzyme activities for example the inhibitors of protease and amylases. Orhers are haemagglutinins, saponins, tannins and antivitamins (Witaker and Feeney, 1973; Liener, 1969). Cowpeas contain antinutritients such as polyphenols, tripsin inhibitors, lectins and phytates which may decrease protein digestibility and reduce protein quality (Bressani and Elias, 1978). The availability of nutrients in plant foods may be affected by natural complexing agents. This is particularly true in the case of seeds containing phytic acid, such as legumes, cereals, and oil seeds. In fact, because of its highly reactive structure, at different pH levels phytic acid can complex proteins as well as mono- and divalent cations . Phytic acid and its chemical and nutritional effects have been extensively reviewed (Cheryan 1980; Reddy et al. 1982). From a nutritional point of view, many studies have concentrated on the metal ion chelating property of phytic acid (Evdman 1981), its binding of zinc and formation of less soluble complexes that reduce zinc availability (Prasad, 1979; Morris and Ellis, 1980). The interaction of phytic acid with proteins has been studied mainly in soy beans. Such studies describe the formation of phytate protein complexes, their effects on protein solubility and related properties, and methods for phytic acid removal (Okubo et al., 1975, 1976; O’ Dell and DeBoland, 1976; De Rham and Jost 1979; Omosaiye and Cheryan 1979; Honing et al. 1984). However, the nature of the phytate- protein interaction is not completely understood, and its nutritional effects on protein availability still 32 needs clarification. Phytate–protein binding is affected by several factors, such as the characteristics of the protein matrix (Carnoval E., et al .1987). However, the acceptability and utilization of legumes as food has been limited due to the presence of relatively high concentrations of certain antinutritional factors such as lectins, protease inhibitors, α-amylase inhibitors, allergens, polyphenols, and phytic acid (Liener, 1994). Phytic acid, the hexaphosphate ester of myoinositol, is a major phosphorus storage constituent of most cereals, legumes and oilseeds, The amount of phytic acid is these products varies from 0.5 to 6% and accounts for between 50 and 90% of the total phosphorus. The ability of phytate to complex with proteins and with minerals and the consequences of these interactions have attracted considerable interest from both chemical and nutritional view points (Kevin et al. 1987). Tannins are high molecular weight polyphenolic compounds that have the ability to bind with proteins, through hydrogen bonding with peptides linkages, tannins precipitate proteins from aqueous solutions rendering plant proteins relatively indigestible and reducing enzyme activity (Van sumere et al. 1975). Although heat treatment will effectively eliminate most of these undesirable substances; careful control of processing conditions is essential to prevent both functional as well as nutritional damage to the protein. On the other hand, the breeding of varieties or strains of cowpea with low levels of one or more of the antinutritional factors offers a much more satisfactory long- term solution to this problem (Ologhobo et al. 1983). 1.4 Processing of cowpeas: Processing of cowpeas and legumes in general is essential to make them nutritious, nontoxic, palatable and acceptable. The constraints to maximum 33 utilization of cowpeas can be overcome by appropriate processing technology. Processing can be classified into domestic and industrial techniques. The domestic processing techniques that are practiced in villages in developing countries include dehulling, grinding, soaking, germination, fermintation, addition of salts, wet and dry heat treatments, cooking and roasting. Theses processes can also be achieved in the food industry. The industrial processing techniques that are not common in the villages include canning, roasting, extrusion cooking, formation of protein concentrates and isolates and texturized vegtable proteins. Processing can further be divided into primary and secondary processes. Primary processes yield storable products to be used as and when required and include smoaking, dehulling, grinding and milling. Secondary processes are involved in the preparation of final consumer products from cowpeas and include various forms of heat treatment, such as boiling, steaming, cooking, alkaline treatment, roasting and deep fat frying (Uzogara et al. 1992). 1.5 Utilization of cowpea: The potential of cowpeas to contribute more to African or tropical diets has been investigated (IDRC, 1973) with an emphasis on reducing postharvest losses (Beucchat, 1983), on developing appropriate technologies to alleviate the heavy labor imputs required in many traditional preparations (Reichert et al. 1979; Hudda, 1983). Cowpeas were chosen as the source of nutrients because of their relatively high protein content and their amino acid profile, which is both superior to and complementary to that of cereal grains (Dovlo et al. 1976; Rechie, 1985). Protein- energy malnutrition of infants is one of the major nutritional problems in the world. It is due to several causes including lack of 34 weaning foods, the preparation of weaning foods with inadequate protein content, and to the use of foods too low in energy density to satisfy the needs of the growing infants (Oyus et al., 1985). A weaning food commonly used in Nigeria is composed largely of sorghum with limiled amount of dried–milk powder, usually in the ratio 5:1. Such mixtures have been shown to be poor in protein content and quality (Akinrele and Bassir, 1967; Oyeleke, 1977) Amore suitable weaning food has been prepared based on soybean and maize flour enriched with vitamins (Akinrele et al., 1970). An attempt was made to improve the quality of the popular mixture by replacing part of the sorghum with cowpeas, Vigna unguiculatea, (Oyus et al., 1985). Cowpea is being successfully used in child-feeding programme (Ologhobo, 1983). In tropical Africa cowpeas are primarily used in the form of dry seed cooked as a pulse in large variety of dishes. Preference is for brown, white or cream seeds with a small eye and wrinkled or rough seed coat. In many areas of both West and East Africa the tender green leaves are cooked like spinach or as relish. Green beans or cut green pods are used as avegetable of secondary importance. Cowpeas are also grown for fodder, ground cover or green manure but to a much lesser extent than for pulse. Cowpea is practically never grown as a sole crop in Africa. In Asia the pulse uses of cowpeas are important primarily in the drier regions such as India where increasing amounts of crop are used in dhal. In South America cowpeas are grown mainly as pluse. In the USA dry seed is grown mainly in California and Western Texas, whereas green peas for canning and freezing (Rachie, 1985). 1.6. Production of Cowpea: 35 Worldwide cowpea production in 1981 was estimated at 2.27 million tonnes from 7.7 million hectares. Cowpeas are grown extensively in 16 African countries, with this continent producing two–thirds of the total. Two countries– Nigeria and Niger- prduce 850,000 t annually or 49.3 per cent of the world crop. The second–highest producing country is Brazil where 600,000 t of dry seeds or 26.4 percent of the worldwide total was produced in 1981. Other major producers in Africa include Burkina Faso (95,000t), Ghana (57,000t), Kenya (48,000 t). Uganda (42,000t ), and Malawi (42,000 t). Tanzania, Senegal and Togo each produces annually from 20,000 ton to 20.000 ton. Current estimates of production vary widely according to source, but the statistics are probably conservative for example, Asian production, including that of long beans as a vegetable, may be under estimated by a factor of 10 or at a level of about 1 millon hectares, concentrated in India, Sirlanka, Burma, Bangladesh, Philippines, Indonesia, Thailand, Pakistan, Nepal, China and Malaysia. India alone is estimated to cultivate more than half or 500,000 ha for dry seed fodder, green pods and green manure. Similarly, production estimates may be low for Africa and the Western Hemisphere where cowpeas are traditionally included as associated crops in peasant – farming system. Thus, realistic production levels may approach or exceed 2.5 million tonnes of dry seeds on about 9 million hectares. The only developed country producing large amounts of cowpeas is USA. (60.000 ton). Low yields are significant attribute of production estimates, particularly in Africa and Asia where 240-300 kg/ha are typical (K.O Rachie et al., 1985). In Sudan the yield is 1070 – 1190 kg /hectare. Objectives: The obectives of this study were to assess: 36 The effect of antinutritional factors (tannin, phytic acid , and trypsin inhibitors) on cowpea protein digestibility. The effect of dry cooking (roasting), wet cooking (autclaving) on antinutritional factors as well as protein digestitibity. The effect of germination on antinutritional factors and protein digestibility. The effect of heat tretment on protein fractions and protein digestibility. 37 CHAPTER TWO LITERATURE REVIEW 38 2. LITERATURE REVIEW 2.1. Nutritional value: Cowpea is eaten in the form of dry seeds, green pods and tender green leaves (Rachie 1985). Like other grain legumes, cowpeas are a good source of energy (Carbohydrate range was 54.4–63.6 g/100g, fat ether extract range was 1.2–1.4 g /100g). Protein range was 23.1–31.3 g/100g, Vitamins, thiamin range was 0.77–0.8 mg/100g, riboflavin range was 2.5 –2.0 mg/100g. Niacin range was 3.48–2.8 mg/100g, minerals (ash) range was 3.5-3.0g/100g and deitary fiber (crude fiber) range was 6.30–0.64 g/100g, amino acids range were lysine 6.6–8.1, Histidine 2.9–4.47, Arginine 5.4–8.0, Threonine 3.6-45, valine 4.9–57, Isoleucine 4.2–4.8, leucine 7.6–8.5, Tyrosine 2.2–3.6, Phenylalanine 5.5 – 6.2, Methionine 1.5 – 2.3, crude protein 23.1–31.3 (Walker, 1981). They are useful sources of good quality protein during hungry season. Cowpea contain about 20-30 % protein. But protein digestibility is low in the beans (Onigbinde et al. 1990). The food value of cowpeas is highly rated by the nutritionists, as they can provide supplementary proteins to traditional diets based on cereals, starchy roots and tubers (Aykroyd et al., 1982; Matthews 1989). Cowpeas are rich in lysine and other essential amino acids but low in sulphur amino acids (Bressani 1985; Kochhar et al., 1988; Armu 1990). The high lysine content of cowpea makes it an excellent improver of the protein quality of foods low in lysine, such as cereals, which are low in lysine but rich in sulphur amino acids. The quality of a food legume is highest when such food contains high level of sulphur amino acids (Bressani et al. 1980). 39 Maximum nutritional benefits are therefore achieved by complementing cereals with cowpeas in the right amounts so that cereal-cowpea mixes yield amino acid scores closer to the FAO/WHO/UNU standard found in meats, fish and egg. Addition of methionine to cowpea protein (Bressani 1985; Sherwood et al., 1954) significantly increased protein quality estimated by biological value (BV) and net protein utilization (NPU). Cowpeameal (CM) BV % was 58.17 ± 2.31 and NPU% was 50.6 ± 1.83, CM + cystine BV% was 80.25 ± 1.87 and NPU% was 72.74 ± 0.94, CM + cystine + methionine BV % was 94.61 ± 1.26 and NPU % was 82.12 ± 1.07, CM + methionine BV% was 95.84 ± 1.45 and NPU % was 81.46 ± 0.87, Albumin BV % was 101.72 ± 2.54 and NPU% was 99.52 ± 1.85. Cowpea – cereal mixtures provide the highest quality protein at a weight ratio of 45 parts cereal to 15 parts cowpea (Bressani 1985). Cowpeas are a major source of protein, water soluble vitamin and essential minerals in developing countries (Aykroyd et al., 1982).This is important, since in most developing countries milk is hardly an important part of the diet; the need for calcium can be met by consuming cowpeas and other vegetable foods. The low sodium content of cowpeas makes it a good food for individuals on low sodium diets, while their high potassium content should be of special interest to those individuals who take diurectics to control hypertension and who need increased intake of K+ to replace that excreted. Cowpeas are also low in fat and contain no cholestrol. Immature cowpea seeds are good source of vitamin A, beta carotene and vitamin C (Eheart et al. 1948). Cowpeas also have a great 40 potential in up grading traditional weaning foods based on cereal paps (Odum et al. 1981; Oyeleke et al. 1985). The beans are sometimes fed as the main food to infants in developing countries unless such infants show intolerance to the cowpea diet. A food product for weaning small children made of 75 % cereal grain and 25% cowpea would be about 13% good quality protein (Bressani, 1985). Cowpea are relatively cheap compared to meat foods and, as they have a high (50 – 65%) carbohydrate content, act as high energy foods for peasants and nomadic farmers (Longe 1980). Cowpeas also add variety to monotonous high carbohydrate staples common in the tropics. Starch contributes about 30 –50% of cowpea carbohydrate and as in other food legumes over 50% of the starch is in the form of amylose. Srinavasa– Rao (1976) showed that high amylose content caused slow digestibility. However, carbohydrate digestibility of cawpea was increased in vitro by baking, roasting and germination and these processes might also facilitate in vivo carbohydrate digestibility (Srinavasa–Rao 1976; Geervani et al., 1981; Reddy et al. 1984; Carbezas et al . 1982). In addition, cowpeas also contain some indigestible sugars known as oligosaccharides. 41 These oligosaccharides, stachyose, raffinose and verbaseose cause gas or flatus in some individuals who consume cowpeas (Uzogara et al . 1992a). 2.2. Protein fractionation 2.2.1. Nitrogen solubility (NS): Cowpea protein extractability at the isoelectric point 40% of the extractable protein; unlike most legumes which showed a protein extractability of about 10% or less at the isoelectric point. This high extraction at the isoelectric pH has been attributed to presence of neutral salts in the extraction buffers or the composition of the proteins since some albumins and globulins may not precipitate at the isoelectric pH (4.4) which affected by ionic strength and pH (Sefa – Dedeh et al. 1979). Solubility is an important property governing the functional behavior of proteins and their potential application to food processing . Dennaturation implicates damages to functionality and is usually measured as a loss of solubility. Generally soluble proteins posses superior functional attributes for moat applications in food processing. Protein functionality is dependant on hydrophobic, electrostatic, and steric parameters of the proteins, which are essential for defining the protein structure (Nakai, 1983). 2.2.2. Protein fractions classification:Protein from cowpea seeds can be recovered in sex solubility fractions. Fraction I contained salt soluble protein globulins, the value for fraction I ranges from 65.7 to 79.7 %. Fraction II contains water soluble protein albumins which ranged from 4.0 to 12.3 % Fraction III contained alcohol soluble protein. Prolamin from 1.4 to 4.0 % . Fraction IV contained G1-glutelins range from (0.9 to 3.0% . Fraction V. contains G2–glutelins range from 1.4 to 2.9% . Fraction VI contains G3–glutelin range from 9.0 to 14.0% and insoluble protein rangs from 0.5 to 3.0% (Nugdallah and El Tinay, 1997). 42 Cowpea proteins classification depends on selective solubility of proteins in defferent extraction solvents According to Fruton et al. (1959) proteins are divided into five major groups, albumins which are water–soluble, globulins which are salt-soluble, prolamins which are 70–80% ethanol–soluble, glutelins which are sodium hydroxide– soluble, and sclero–proteins which are insoluble in aqueous solvents. Osborne (1924) reported that globulins are the major storage proteins of legumes and they require appreciable salt concentration for solubilization and they account for 50–70% of the total seed proteins. Other studies on legume proteins have reached the same conclusion (Romero et al., 1975; Cjakrborty et al., 1979). Chan-CW. et al (1994), reported that the abundance of cowpea CV. California Blackeye No.5 seed protein fractions was in the following order: globulins > albumins > glutelins > prolamins. The globulins contained 4 major polypeptides with molecular mases of 65, 60, 59 and 50 KDa, 3 of which were covalently bound with carbohydrate. The albumins cotained 4 major polypeptides with molecular masses of 99, 91 32 and 30 KDa. The alkali- soluble glutelin fraction was mainly composed of polypetides in the molecular mass range 44-62 KDa. Polypeptides of molecular mass 105, 62,59 and 54 KDa, were found in the prolamin fraction. The glutelin and prolamin fractions were high in essential amino acids compared with the other 2 fractions. 2.2.2.1 Albumins and globulins: 43 Albumins and globulins are very heterogeneous and differed in polypeptide composition. Extraction with water and then with NaCl to solubilize what is termed albumins and globulins respectively. These descriptions are inaccurate since water also extracts the lower molecular weight nitrogen (LMWN) as well as albumin. In addition, because of endogenous salt in the grain some globulins are also extracted (Wilson et al., 1981). The globulins constituted 82.6% of the total seed proteins and the albumins 8.6%. Albumins were richest (3.76 g/100 g protein) in methionine, (Dhankher et al., 1990). The cross contamination of the water–and salt soluble proteins of legumes attributed mainly to the native ionic strength of been flours and the low flour–to–solvant ratio. Thus a sharp distinction between albumins and globulins on the basis of the solubility can not made (Bhatty, 1982). Water – and salt- soluble proteins together accounted for 63 – 83 % of the total nitrogen of chickpeas, cowpeas and dry beans (Deshpande et al., 1987) . Six chickpeas were analyzed for their protein fractions, albumin content ranged from 8.4 to 12.3% and globulin content ranged from 53.4 to 60.3 % (Dhawan et al., 1992).Several genetic lines of field bean seeds were anayzed for electrophoretic patterns of the globulin and albumin fractions. Globulin constituted the major fraction of seed protein (Pasqualini et al., 1992). The general deficiency of lysine in most cereals e.g sorghum and corn is essentially the consequence of their low content of albumins and globulins (FAO, 1981). Globulins account for 50 – 75% of the total seed protein (Osborne, 1924). Nugdallah and El Tinay (1997) reported that Albumins and globulins ranged form 4.0 – 12% and 66 – 80% respectively. 2.2.2.2. Prolamin: 44 Prolamin can be defined as a portion material extracted at room temperature by aqueous alcohol; free of reductant and salt, from a corn meal deprived of lipids and salt soluble protein. Prolamin proteins are synthesized in the developing endosperm where they form protein bodies–within the rough endoplasmic reticulum because they account for more than half of the total seed protein (Landry and Moureaux, 1981). Prolamin content ranged from 3.1 to 6.9% of total seed protein in chickpea seeds (Dhawan et al., 1991). Prolamin content was 2.6% in cowpea seeds (Dhankher et al. 1990). The ethanol soluble protein fraction was 0.97% in cowpea seeds (Deshpande and Nielsen, 1987). Nugdallah and El Tinay (1997) reported that prolamins ranged from 1.4 – 4.0% for cowpea. 2.2.2.3 Glutelin: Glutelines are defined as including these proteins that are either soluble in dilute aqueous alkali or insoluble in neutral aqueous solutions, saline solutions or alcohol (Osborne, 1924). After albumin, globulin and prolamin proteins were removed from corn, it was found that the addition of 2 – ME to 70% ethanol and 0.5% sodium acetate removed protein from corn (Paulis et al., 1969 ). This protein was thought be zein like in solubility but later it was termed alcohol – soluble reduced glutelin based on the definition that all proteins remaining after removel of salt and alcohol soluble proteins were glutelins (Paulis and Wall, 1971). Glutelins consists of several different polypeptide chains linked by disulfide bonds to form an isoluble three–dimensional matrix (Nielsen et al., 1970 ). Glutelins are associated with lower molecular weight proteins through noncovalent bonding: they consist mainly of two categories of polypeptide linked 45 by disulfide bonds. Alcohol soluble and alcohol insoluble glutelins are two types of polypeplide deposited in different subcellular structures. The alcohol soluble polypeptides resemble prolamins but have significant structural differences (Paulis, 1982). Corn grain contains three glutelin subgroups called G1, G2 , and G3 – glutelin (Landry and Moureaux, 1970). Cowpea cultivars contain three glutelin fractions called G1, G2, G3 - glutelin (Nugdallah and El Tinay, 1997). 2.2.2.3.1. G1-glutelin: G1–glutelin (Zein–Like) has an amino acid composition somewhat similar to zein, but with higher levals of glycine, methionine, histidine and proline and lower levels of a spartic acid leucine and isoleucine, G1 - glutelin was previously recoverd as a part of glutelin fraction, zein apears to be cross – linked to glutelin through disulfide bonds (Paulis et al., 1969). Nugdallah and El Tinay (1997) reporte that G1 – gutelins ranged from 0.9 – 3.0% for cowpea. 2.2.2.3.2. G2 - glutelin: G2 – glutelin (glutelin–like ) isolated at pH 10, can be fractionated on the basis of their extractability at pH 3 (Misra et al. 1972). Acid soluble G2- glutelin exhibits some general characteristics of cereal prolamin. They are rich in proline, glutamic acid or glutamine and typified by high histidine and poor lysine and aspartic acid or asparagine. Moreover, they may be extracted both by acidic and alcoholic media . Indeed, amino acid composition of acid soluble G2 – glutelins and water soluble, alcohol soluble–glutelins are nearly identical (Landary and 46 Moureaux, 1981).Consequently, the acid insoluble G2–glutelins are not removed by alcoholic extraction. Because of their extractability and amino acid composition, especially their relatively high lysine cotent, they may be regarded as being similar to G3 – glutelins. Thus fraction IV contains both acid – soluble and acid – insoluble. G2 – glutelins, rich in histidine, which might be called prolamine–like and glutelin– like, respectively. Amount of G2–glutelins isolated at step (5) depends on conditions used earlier to extract salt – soluble and alcohol – soluble proteins ( L andry and Moureaux, 1981). Nugdallah and El Tinay (1997) reported that G2 – glutelins ranged from 1.4 – 2.9% for cowpea. 2.2.2.3.3 G3 - glutelin There are polypeptide which did not separate clearly on SDS – polyacrylamide gel (Misra et al., 1972). Moreover, G3–glutelins having an amino acid composition similar to that of salt soluble proteins (though richer than them in hydrophobic residues and with lower cysteine content than other glutelin subgroups). It appears to be non extractable by 2-ME in a saline or alcoholic medium or by their combination. The in ability of extract G3–glutelins with such media may be related to noncovalent interpolypeptide bonds (Landry and momreaux, 1981). G3–glutelins exist at the earliest stages of grain development before zein accumulation so they may consist of membrane protein from cell organelles such as mitochondria or ribosomes (Landry and Moureaux, 1976). This hypothesis lends support to the existence of noncovalent bonds in glutelins since hydrophobic 47 interactions stabilize these membranes and their multimeric enzymes. This is also a close Parallel between the decrease in the amount per grain of salt soluble proteins and the increase of G3–glutelins observed during grain maturation. Therefore, G3–glutelins include membrane–bond proteins, some naturally associated salt-soluble proteins and some proteins altered during extraction. As G3 – glutelins could contain membrane proteins, all the more as detergent is necessary to it dissolution (Landary and moureaux, 1981). Alkali soluble protein fraction was 11% in legumes (Des hpande and nielsen, 1987). The glutelin fraction in cowpea seeds was 6.4% of the total seed proteins (Dhankher et al. 1990) . In six chickpeas cultivars analyzed for their protein fractions glutelins content ranged from 19.38% to 24.4% (Dhankher et al., 1990). Nugdallah and El Tinay (1997) reported that G3 – glutelins ranged from 9.0% to 14.0% for cowpea. 2.2.2.4. Insoluble protein: Insoluble protein (residue) was not extracted because it was linked to the cell wall (Wall and Paulis, 1978). Residue may be unextracted glutelin plus variable amounts of globulins and albumins associated with starch and cell debris (Wilson, 1971). Moreover, a small amount of nitrogen remains insoluble after all these extraction procedures. This residue consists mainly of proteins form previously defined groups becoming insoluble due to interaction with lipids, carbohydrates, or polyphenols via oxidation process (Landry and Moureaux, 1981). Nugdalla and El Tinay (1997) reported that insoluble protein ranged from 0.8 – 3.0% for cowpea. 2.3 Anti-nutrional factors: 48 Anti-nutrients are common in many legumes, including cowpeas. Liener (1980), has defined these toxic components in legumes as “those causing physiological response in man or animals when consumed”. These Leguminous anti-nutrients include protase inhibitors, amylase inhibitors, hemagglutinins, allergens, aflatoxins, cyanogenic glycoside, favism factors, lathyrogens, metal binding factors (phytates, oxalates, saponins), anti-vitamins, estrogens, pressoramins, flatulence factors and polyphenols. Cowpea contain anti-ntrients such as polypenols, trypsin inhibitors, lectins and phytate (Bressani and Elias, 1978). 49 2.3.1. Chemical nature: 2.3.1.1. Chemical nature of tannins:Tannins are polymeric phenols of higher molecular weight (M.W. 500– 5000 ) containing sufficient phenolic hydroxyl groups to premit the formation of stable cross–linke with proteins (Swain, 1965). The main distinctions between the two groups arise from their action towards hydrolytic agents, particularly acids. The hydrolysable tannins which have a polyester structure are readily hydrolysed by acids or enzymes into sugars or related polyhydric alcohols and a phenol carboxylic acid and dependent on the nature of the later, a subdivision into gallotannins and elogitannins is also usually made. Thus on hydrolysis, the gallotannins give gallic acid and the ellagitannins hexahdroxydiphenic acid isolated normally as its stable dilactone ellagic acid or acids which can be considered to be derived by simple chemical transformation of ellagitannins such as oxidation, reduction and ring fission. The condensed tannins in contrast do not readily break down with acid, instead they undergo progressive polymerization under the action of acids to yield , the amorphous phlabaphens or tannin reds (Haslam, 1966). The condensed tannins also referred to as procyanidins (Weinges et al. 1969) . and formally as leucoantho-cyanidin (Rossenheim, 1920) because many forms cyanidin upon acid hydrolysis, they are mostly flavans or polymers of flavan 3-ols (Catechin) and/or flavan 3-4 diols (leucoanthocyanidins). Both catechin and leucoanthocyanidins are readily converted by dehydrogenating enzymes or even by very dilute mineral acids at 50 room temperature into flavonoid tannins (Weinges et al. 1968). Heating in acid solution convert leucoantho cyanidins to the corresponding anthocyanidins and brown phlobatannins (Swain, 1959). Tannins are insoluble in nonpolar solvents like ether, chloroform and benzene and are sparingly soluble in ethyl acetate. Because of the presence of a large number of polar groups, they readily dissolve in water and alcohol to form colloidal solutions. Extraction of tannin by aqueous mixture of polar organic solvents depends upon random transfer of individual bonds from the substrate to the competing sites on the solvent molecules (Mc-Leod, 1974). Generally most of tannins appear to be found in few families of the dicotyledons such as the leguminosae (White, 1957). But the condensed tannins (Structure 1 – 8) are more widely distributed in higher plants (Schanderl, 1970). HO H2O – OO C HO HO COO O O C O O C HO HO HO HO HO COO HO HO COO O HO HO HO COO HO Structure 1 Glalotannin HO COOH HO OH HO HO HO HO HO COOH HO HO 51 OH OH OH OH HCOO Structure 3 Hexahydroxy-diphenic Structure 2 Gallic acid 52 O HO Structure 5 Flavanzol (Catechin) O HO Structure 6 Flavan 3-4-diol (leucoantho-cyanidin) HO + O HO Structure 7 3-Hydroxy flavylium (Anthocyanidin) HO O HO HO HO HO HO O HO HO HO HO HO Structure 8 Apossible structure of grain sorghum condensed tannin (Haslam, 1977) HO O HO HO 53 HO 2.3.1.2. Chemical nature of phytic acid: Beans, in common with other seeds, contain metal salts of myoinositolhexaphosphate or phytic acid . Di–and trivalent metal salts of phytic acid are relatively insoluble in water. Ferric phytate is nearly insoluble in acid solution as well as in water. This property has been utilized for the isolation of ferric salts of phytic acid from acid extracts of natural products (Heubner et al. 1914; Young, 1936; McCANCE el al. 1935). Quantitative determination of phytic acid may be based on the analysis of phosphorus or iron in the isolated ferric phytate (Mc CANCE et al. 1935; Crean et al. 1963; Schormuller et al. 1956). Alternatively or indirectly, on the determination of the residual iron in solution after precipitation of ferric phytate from a known concenteration of ferric salt in acid solution (Crean el al., 1963). Determination of phytic acid based on phosphorus analysis of ferric phytate has been reviewed by Schormuller et al. (1956). Wide variations in values for phytic acid were obtained by several investigators who have used different analytical methods (Schormuller et al., 1956; Marrese et al., 1961). Phytic acid, the hexaphosphate ester of myo – inositol (I) , is a major phosphorus storage constituent of most cereals, legumes and oilseeds (Reddy et al. 1982). Phytic acid has twelve ionisabe hydrogen atoms, for which dissociation constants have been measured at 28oC by 31P n.m.r spectroscopy (Costello et al, 1976). The variation of chemical shifts with pH shows that six of the acidic groups (one from each phosphate) have pka values in the range 1.1 – 2.1, three others are weakly acidic with pka values between 5.7 and 7.6 and the remaining three lie in the very weak acid range 10.0 – 12.0. Under physiological pH conditions, therefore, phytic acid is extensively ionised 54 and is capable of interacting strongly with proteins and with metal ions (Reddy et al. 1982). The phytate – protein interactions are thought to be ionic at low pH and mediated by cations, through the formation of phytate – cation- protein complexes, at high pH (Reddy et al. 1982). These interactions lead to reduced protein solubility and a consequent alteration of their solubility–dependent properties such a hydrodynamic behaviour, foaming and emulsifying capabilites, dispersibility in water, etc. Phytate is also a potentially strong ligand and can form stable complexes. Hence the phosphate groups may chelate as in structure (II) to give 4–membered ring complexes (Anderson et al., 1977), alternatively two or more phosphate groups from the same or from different phytate ions may complex to one metal cation giving structure (III) and the polymeric structure (IV), respectively, or a phosphate group may serve to bridge two metal ions as in (V) (Jones et al., 1977). To date there exists little structural information on these complexes and there is clearly a need for X – ray crystallographic investigations to fill this void (Kevin et al. 1987). OPO3H2 OPO3H2 OPO3H2 H2O3PO OPO3H2 OPO3H2 Structure I O M O O || || (PO3H)5 – inositol – O – P O –OM – O P HO O O P OH Insitol – (PO3H)4 Structure (III) O 55 Structure (II) 2.3.1.3 Chemical nature of the inhibitors:Most of the elicited protease inhibitors are proteins. Trypsin inhibitors appear to be ubiquitous in all tissues, and they have been most intensively studied in the legumes and cereals. Soybean seeds contain two types of trypsin inhibitors, the kunitz inhibitor of 21,000 MW that is specific for trypsin only (1:1 complex) and the Bowman – Brik inhibitor of 8300 MW that binds independently and simultaneously to trypsin and chymotrypsin (1:1:1 complex) .Most legumes contain the Bowman – Brik type inhibitors, with considerable homology among them, while most legumes do not produce the Kunitz-type inhibitor. The Kunitz inhibitor, with two disulfide bonds. About 1h of cooking is required to completely inactivate the Bowman – Brik inhibitor, unless a reducing compound, such as cysteine, is added. Several isoinhibitors of typsin are often found in higher plants. The legume inhibitors are known to be significant, nutritionally, at least in some animals, (Owen et al., 1996). 2.3.2 Anti-nutritional effect: 2.3.2.1 Anti-nutritional effect of the Inhibitors: Enzymes are responsible for the myriad reactions associated with reproduction, growth, and maturation of all organisms. In most cases, these are desired activities. In some cases, too much enzyme 56 activity, such as polyphenol oxidase– caused browning, can lead to major losses in fresh fruits and vegetables. In many humans, absence of or too little of an enzyme is responsible for many genetically related diseases. Microbially caused diseases present another problem . The best way to treat these types of diseases is through inhibition of one or more key enzymes of microorganisms, resulting in their death. The inhibitors might complete reversibly with substrates or inhibitor might form acovalent bond with active site groups (affinity labling inhibitor), or the compound might be treated as substrate and be catalyzed to product that, while still in the active site. The last type is the most specific and desirable in medicine and food because in hibitor can be targeted specially for enzyme. Enzymes continue to catalyze reactions in raw food materials after they reach maturity. These reactions can lead to loss of color, texture, flavor and aroma, and nutritional quality. Therefore, there is need for control of these enzymes to stabilize the product as food. Enzyme inhibitors are aslo important in the control of insects and microorgansims that attack raw food. They also are used as herbicides in the control of unwanted weeds, grasses and shrubs. Enzyme inhibitors are an important means of controlling enzyme activity . An enzyme inhibitor is any compound that decrease 57 intial velocity (Vo) when added to the enzyme – substrate reaction. There are many enzyme inhibitors, both naturally occurring and synthetic. Some inhibitors bind reversibly to enzymes and others form irreversibly, covalent bonds with the enzymes. Some inhibitors are large proteins or carbohydrates, and others are as small as HCN. Products of enzyme – catalyzed reactions can be inhibitory. Change in pH can alter activity by making conditions less optimum for enzyme activity. Elevated temperatures can decrease enzyme activity by denaturing some of the enzyme, but at the same time increasing the velocity of conversion of substrate to product by the active enzyme. Most enzymologists do not consider either of these variable to be enzyme inhibitors. Denaturation of the enzyme eliminates its activity, and this can be accomplished by shear forces, very high pressures, irradiation, or miscible organic solvents. Enzyme activity can also be decreased by chemical modification of essential active site groups of the enzyme. Enzymes are aslo inactive when their substrate(s) are removed. All of these inhibitroy approaches are valid ways of controlling enzyme activities in foods (Owen et al. 1996). Protease inhibitors in food are subtances that have the ability to inhibit proteolytic activity 58 of certain enzymes (Liener et al., 1980). They are present in cowpeas and other legume seeds (Kocchar et al., 1988; Dellagata et al., 1989). Their importance lies in their possible adverse effect on nutritive value of plant proteins. Plant breeders in their effort to produce insect resistant varieties of cowpea have sometimes increased levels of trypsin inhibitors (Gatehous et al., 1979). These toxicants lower protein quality by decreasing PER. Protease inhibitors, can be reduced by soaking and dehulling the seeds followed by heating (Ogun et al., 1989) 2.3.2.2 Antinutritional effect of the tannin: These toxicants lower digestibility (Liener 1976), protein efficiency ratio (PER) and overall nutnitive value of uncooked or improperly cooked seeds and can cause diarrhea and vomitting (Anon 1976). Cowpeas contain Anti-nutrients such as polyphenols which may decrease protein digestibility and reduce protein quality (Bressani et al. 1978). Elias et al. (1979), obtained lower PER for beans combined with cooking water than for drained beans alone. They suggested that tannins and/or other pigments interfered with protein utilization. Elias et al. (1979) also found that tannin concentration was high in colored seed coats but low in white–coated seeds. However, Radke et al. (1981) observed that PER values were 59 identical for the white variety of blackeye beans with and without cook water. Tannin also lowers protein digestibility in cowpeas (Laurena et al. 1984; 1986). The unfavorable influences of tanins on nutritional properties of cowpea have bean discussed by Price et al. (1980). The adverse effects of tannins may be related to the fact that tannins interfere with protein digestion, affecting digestive action of trypsin and alpaha amylase either by binding the enzmes themselves or by binding dietary protein into an indigestible form (Bressani et al. 1982) .Tannins can also be complexed with vitamin B12 causing a decrease in absorption of the vitamin in rats. Cooking drecreased tannin and increased in vitro protein digestibility in cowpeas (Laurena et al. 1984; Uzogara et al. 1990a). Various workers (Akinyele 1989; Ogum et al. 1989), observed increased losses in tannin when cowpeas soaked and cooked. Tannin loss may be due to heat degradation of the tannin molecules or formation of water soluble complexes between tannin and other tissue molecules of the bean. Such water–soluble complexes could leach out into the cook liquor. Uzogara et al. (1990a) observed increased removal of tannins in beans cooked in alkaline solutions especially under pressure cooking. Tannins form complexes with proteins, carbohydrates and other polymers in food as well as with certain metals such as iron under suitable conditions of 60 oncentration and pH (Goldstein et al. 1965). Lease et al. (1969), reported a marked decreased in blood haemoglobin in rats fed 5% tannins. They proposed that this phenomenon was due to the formation of tannin–iron complex which reduce the availability of iron. 2.3.2.3 Antinutritional effect of phytic acid: Phytic acid is common in cowpea and other legumes and is the principal storage form of phosphorus in many dry beans. Phytic acid occurs as acomplex (phytin with divalent cations or monovalent cations in discrete regions of the beans and accounts for up to 80 % of the total phosphorus content (Reddy et al. 1982). Most of the phytates in dry beans are located in the cotyledons and not in the seed coat. Anti-nutritonal concern about the presence of phytates in dry bean arises from the fact that phytate decreases the bioavailabilty of essential minerals (Ca, Mg, Mn Zn, Fe, Cu) and may posssibly interfere in the utilization of proteins due to phytate–protein and phytate–mineral– protein complexes (Oberleas et al., 1981). Under physiological conditions these complexes may be insoluble thereby making proteins unavailable for proteolysis in humans and animals. It has been shown that phytate can inhibit enzymes such as alpha amylase, pepsin and trypsin under in vitro, conditions (Reddy et al, 61 1982), which may further, reduce substrate utilization. In foods high in phytate, zinc may not be readily available for absorption since a phytate: zinc molar ratio of above 20 is reported to be associated with chemical zinc deficiency (Oberleas et al., 1981). The hard-to–cook (HTC) defect is a condition whereby bean cotyledons absorb water but fail to soften during boiling (Stanley et al 1985; Ramcharran et al., 1985; Paredes–Lopez et al., 1989; Hentges et al., 1991). Development of HTC defect is not well understood and various factors may cause the defect and leads to increased cooking time in beans. HTC defect lead to increased consumption of cooking fuel that may be scarce and expensive in developing countries. This may limit utilizing cowpeas and other dry grain legumes for prevention of protein energy malnutrition in these countries. It also places people who rely on dry beans for dietary protein at a nutritional disadvantage. The most frequently advanced hypotheses for explanation of the HTC defect are (a) the middle lamella–pectin– cation–phytate mechanism of Mattson (1949); (b) the dual enzyme (phytase + pectin methyl estrase) mechanism of Jones et al., (1983); (c) the cross- linking of phenolics and / or protein in middle lamella theory as proposed by Hincks et al. (1987) and (d) the cross – linking of phenolics and /or protein in middle lamella theory as proposed by 62 Hincks et al. (1987). and Vindiola et al. (1986).; (e) the decreased solubility of starch and protein theory (Akinyele et al., 1986; Hentges et al., 1991). Basically, phytic acid located in the protein bodies of bean cotyledons chelates divalent cations (Ca, Mg). At high temperature and high relative humidity conditions in legumes with high moisture content, there is increased metabolic activity, phytase activation and membrane degardation. Phytase hydrolyzes phytin in cotyledon cells to release bound Ca and Mg, which migrate from the cotyledon cells to the middle lamella. At the same time, pectin methyl esterase (PME) in the middle lamella hydrolyzes pectin to pectic acid and pectinic acid and methanol. The divalent cations that migrated to the middle lamella now react with the released pectinic acid, forming insoluble Ca or Mg- pectinates that firms the middle lamella and cements cells together . Decreased pectin solubility and low phytate content have been correlated to poor cookability in HTC bean (Vindiola et al., 1986). 63 2.3.3 Anti-nutrients Content: 2.3.3.1 Tannin content of cowpea: Most plant tissues contain a wide range of secondary products such as polypenols. The significance of these secondary plant products is a matter of wids especulation and it has been suggested that some may serve to protect the plant from pests, diseases and natural predators (Hulse et al., 1980). Tannic acid contents. Expressed as percentages of bean dry weight, in raw and processed cowpea varieties. The ranges are: for raw whole beans, 0.42–0.78%; for autoclaved beans. 0.33–0.67 % ; for cooked beans, 0.23–0.42% for soaked beans, 0.37 – 0.69% and for germinated beans,0.29–0.56%. Cooking and germination decreased tannins contents by 31.0– 47.3 and 23.8 37.0%, respectively. Autoclaving was not as effective as cooking and germination and losses obtained ranged between 13.8 in ‘kano 1696’ and 38.3% in Nigeria B7 (Anthony et al., 1984). Two varieties had tannins 2.7 to 25 mg/g raw, of which 7.5 to 48% was destroyed by boiling, 7.4 to 34% (8 varieties) by pressurecooking and 57, 62 or 64% (3 varieties) by soaking for 48 h befor boiling, which was done for red kidney bean, black cowpea and rice bean. The varieties with the most tannin white cowpea seemed to be 64 the best of those legumes for feed efficiency, destruction of TIA on cooking and low tannin content. Tannin content of cowpea cultivars was 1.24–1.42 mg/g. There was no significant correlation between IVPD and tannin content (Ene– obong, 1995). Tannin content in Red cowpea (RCP) flours ranged from 3.0 to 4.5 mg/g sample. These quality parameters indicated for (RCP), dry processing generally produced product superior to those obtained from wet processing (Ningsanond et al., 1989). It has been hypothesized that part of hard – to – cook (HTC) defect in cowpeas is due to decreases in solubility and thermal stability of intraccllular proteins during storage since coagulated proteins would limit water to starch and pervent full swelling during cooking (Keshum et al., 1993). Porridges prepared from extruded millet and press- dried cowpea had high nutritional quality with acceptable properties for weaning foods (e.g an intermediate consistency, smooth texure and pleasant color and flavor). Treatment with sorghum malt allowed the prparation of more fluid products (Almeida, Dominguez et al. 1993). Somples of 15 Nigerian cowpea cultivars were analysed. Hotsoaking and cooking significantly reduced trypsin inhibitor. Phytic 65 acid levels were reduced by cold–soaking and hot soaking (Ogun, 1989). No clear variations in protein quality were observed between boild and pressure cooked samples. For the biological parameters Protein Efficiency Ratio (PER), Net Protein utilization NPU & Invitotro protein Digestibility (IVPD) studied raw and cooked chickpea samples gave the highest values followed by cowpea and peas (Hashimy et al., 1985). Studies were conducted on chickpeas (Cicer arietinum), horse gram (Dolichos biflorus) and cowpea (Vigna sinensis), processed by 6 methods; boiling, pressure cooking, puffing, frying, germination and germination+cooking. For cowpea most of these treatments improved IVPD (El Faki et al., 1984). Study was carried out on 15 local and improved cowpea genotypes grown in 12 environments, comprising 3 Nigerian locations over 3 seasons per location. Genotype effects were strongest in controlling trypsin inhibitor activity while environment was the major source of variation for tannins, haemagglutinin and phytic acid contents. Results, indicated that a cowpea genotype grown and consumed safely in one environment can be poisonous when grown and consumed in another. Genotype x environment effects were 66 significant for tannins, haemagglutinins and trypsin inhibitor concentrations (Oluwatosin, 1999). The cream and speckled African yam bean contains more TI (30.9 and 25.3 mg/g) than pigeon pea (7.5–14.1 mg/g ) and cowpea (9.8–20.5 mg/g). Apart from the white cowpea cultivar, they contained more tannin (1.2–1.4 mg/g) than pigeon pea (0.14-0.97 mg/g) and African yam bean (0.7–1.2 mg/g). Phytate was lowest in the African yam bean (6.3–7.5 mg/g) than pigeon pea (8.3–11.3 mg/g) and cowwpea (8.4–9.9 mg/g). Phytic acid contributed 67.0-74.0% of the phosphorous in the African yam bean, 66 – 75% in pigeon pea and 54.0–59.0% in cowpea. The IVPD of African yam bean (73.3 ±0.7%) was lower (P ≤ 0.05) than those of pigeon pea (76.34 ± 0.2%) and cowpea (77.8 ± 0.4%) There was a negative correlation between trypsin inhibitor and IVPD (r = - o.63, p ≤ 0.05). There was no significant correlation between IVPD and phytate and tannin contents. There was a positive correlation between protein content and IVPD(r+ 0.69) for the legumes under study (Ene-Obong, 1995). 2.3.3.2 Trypsin inhibitor activity TI activities in the raw, autoclaved, cooked, soaked and germinated cowpea varieties ranged between 19.6 and 28.2 TUI/mg 67 protein (TUI, trypsin units inhibited). Autoclaving and cooking resulted in a complete loss of activity, while soaking decreases inhibitor activity by 22.8–42.4%. In the germinated samples, percentage losses were highest in Igbira and west breed where TI activites were reduced to 8.4 and 11.0 TUI/mg protein, corresponding to 59.1 and 57.2% losses, respectively. (Anthony et al., 1984). The combined effects of soaking, germination and temperature on cowpea on trypsin inhibitor activity (TIA) was 4.28 mg/g. (Wang et al., 1997). The inhibitation of human and bovine pancreatic trypsin, Chymotrypsin and total proteolytic activity by extracts from 5 samples of red cowpea (Vigna unguiculata) were studied. The thermal liability of the inhibitors was also assessed . The raw cowpea samples had a trypsin inhibitor activity (TIA) level of 14.3 mg trypsin inhibited/g samlpe. Inhibitation of proteolytic activity was influenced by the type and source of pancreatic enzymes. At all levels of raw cowpea extract concentration, bovine trypsin was inhibited to a signifcantly greater extent than was human trypsin. With regards to processing effects, almost complete inactivation of inhibitors was achieved by cooking whole cowpea seeds after soaking and dehulling, while only partial inactivation occurred when raw cowpea was milled into flour before cooking. It is concluded that effective control of the inhibitory 68 activities in cowpea for maximum nutritional benefits can therefore be achieved by soaking, dehulling and cooking whole cowpea seeds (Nti et al., 1996). Trypsin inhibitor (TI) of cultivars of cowpea (Vigna unguiculata) were 9.8–20.5 mg/g . There was a negative correlation between TI and IVPD. These legumes may pose no serious problems to populations consuming them especially when heat treatment is applied before consumption (Ene-Obong, 1996). The availability of protein in Italian cultivars of beans chickpeas, peas lentils, cowpeas an soyabeans was estimated by quantifying anti-nutritional factors: trypsin inhibitors, tannins, phytic acid and dietary fibre. Beans and soyabeans had the largest amounts of trypsin inhibitors, 64.5 and 13.5 UTI/mg, and contained over 1% phytic acid; lentils had the lowest, 2.4 UTI/mg, and 0.44% phytic acid. Beans had high tannin contents and had the lowest protein digestibility value ranging from 71.3 to 78.5%, whereas the other legumes had an average protein digestibility of 83%. Cooking increased the digestibility of beans. Protein digestibility was found to be correlated with tannin and phytic acid contents (Carnovale et al., 1992). The mature seeds of legumes bought in the philippines were of 11 varieties (species of Psophocarpus, Phaseolus and probably cajanus and vigna). Protein in dry matter ranged from 20 to 30 % raw and 18 69 to 40% after being boiled until soft, which took 30 min to 2h according to variety. TIA units were 2 to 41/mg raw, of which 68 to 97% was destroyed by boiling by 76 to 97% for pressure – cooking at 10 lb/in2 for soft, 15 min to 2h. (Beltran et al., 1983). 2.3.3.3 Content of phytic acid: Germination of cowpea seeds greatly lowered the phytic acid content. The decrease amounted to 51.6% in Prima and 43.4% in Adzuki .Soaking decreased phytic acid content of seeds by 19.4 to 280%, and cooking by 7.7–11.7%. Autoclaving had very slight and maximum loss did not exceed 7.2% in Nigeria B7 (Anthony et al., 1984; Beltran et al., 1983). Juice was obtained from 2 cultivars of cowpeas by hot water extraction, cold water exraction or boiling for 10 min befor cold water extraction. Trypsin inhibiting units/ml ranged from 4.5 to 6%. Phytic acid content ranged from 61.8 – 80.5 mg/100g and IVPD ranged from 60.5 – 81.8% for 2 cowpea varieties extraction obtained by hot water and cold water eraction (Akinyele, 1991). Raw cowpea seeds and cooked cowpea seeds were analysed for protein, Sulphur amino acids and trypsin inhibitor (TI). Trypsin inhibitor ranged from 26.7 to 66.2 TI units/mg proteins in raw seeds and from 3.8 to 8.1 TI units /mg protein in cooked seeds, with the % loss on cooking ranging from 77.7 70 to 92.5%. Protein contents ranged from 20.8 to 26.4% (by wt.). The study of the 2 varieties indicated that the cotyledon contains most of the TI and that TI levels in the cotyledon are generally unaffected by soaking. No correlation was found between TI levels and bruchid (a storage pest) resistance (Catta et al., 1989). Phytate content of cowpea cultivars were 8.4–9.92 mg/g. Phytic acid contributed 54–59% of the phosphorous in cowpea. There was no significat correlation between IVPD and phytate content (Ene-Obong, 1995). 2.4. Processing: The appearent insignificant changes in the proximate composition (DM) with the exception of ether exctract and moisture content show that the processed were efficient in terms of nutrient retention (Akiny et al., 1989) Batches of cowpeas were (i) cooked at 100 degree ºC for approx 1.5h, (ii) germinated in moist cotton wool at room temperature for 72 h; or (iii) fermented for 72 h. Cooking caused slight decrease in phyic acid and crude protein germination caused considerable decrease in phytic acid (Akpapuman et al., 1985). The preparation of cowpeas, by soaking for 1h, dehulling, cooking for 45 min and sun drying to 15% 71 moisture, destroyed trypsin inhibitor activity almost entirely (Abbey et al., 1988) Cowpeas seeds were germinated at 25- 30oC for 24h, in-vitro protein digestibility was determined, it did not improve significantly by germination or decortication but was improved by cooking (Nnanna and Phillips, 1989). Two cowpea (Vigna unguiculata) variety 3 legume spp. (Centrosema pubescens, Psophocarpus tetragonolobus, Cajanus cajan), 3 sorghum (Sorghum bicolor) and 2 millet (Digitara spp). were autoclaved for 20 min (30 min for winged bean) at 21 1b/inc2. Cooked products were analysed for tannin contents. Assayable polyphenol levels were generally higher in cereals than legumes (1.76–8.21 vs. 0.3–4.4 % as tannins). The dark colored sorghum seed coats contained the highest levels. In comparison to raw foods cooked products contained substantially less tannin (up to 71% loss on cooking). due to the heat destruction and complexation of protein/tannin. For nutritional purposes light colored sorghum seeds and moist cooking are recommended (Ekpenyong, 1985). Cowpea is grown in newly reclaimed lands which are slightly to moderately saline. Both dry mature seeds and snap pods are popular as food . 72 Effect of growth regulators and reflecting antitranspirant on chemical composition and nutritional quality of raw and cooked cowpea seeds after soaking in different media was studied. Foliar application with gibberellic acid (GA3) and white wash (reflecting antitranspirant, CaCO3, 6% suspension) especially under saline conditions, improved the nutritive value of raw cowpea seeds by increasing total protein, carbohydrate, total free amino acids and in vitro protein digestibility as well as reducing anti-nutritional factors, i.e trypsin inhinitor activity, phytic acid and tannins. Cooking in water containing NaCl (2%) after soaking in hot water for 12 h showed the lowest content of protein, carbohydrate and antinutritional factors and the highest content of fibre (Bakr et al., (1991). The albumin + globulin fraction increased (P ≤ 0.05) for corn germinated seeds, accompanied by a decrease in the prolamin zein fraction while the G1-glutelim fraction decreased (P ≤ 0.05) as reported by (El Khalifia et al., 1996). The protein content decreased during germination; the albumin and glutelin, increased by 61.5 and 57.0% respectively, while a 54.6% decrease was noted in the prolamin fraction. The globulin fraction increased at the beginning of germination but decreased at the end. Germination significantly (P ≤ 0.05) increased the crude protein, 73 nitrogen solubility and in-virtro protein digestibility but decreased the fat, phytic acid and polyphenol contents of the pumpkin (Telfairia occidentalis) seeds (Giami et al., 1999; El Khalifa and El Tnay, 1999) reported that albumin, globulin and residue increased significantly (P ≤ 0.05) for two germinated sorghum cultivars for 72h, but the glutelin decreased significantly (P ≤ 0.05). For chickpea (Cicer arietinum) germinated seed for 6 days little variation was observed on total nitrogen content, however, the non protein nitrogen was doubled. A decrease of 19-1% and 20.6% was obsorved in total globulin and albumin fractions, respectivaly. The trypsin inhibitor activity showed a little drop after 6 days of germination, indicating a possible increase on digestibility of the proteins (Neves et al., 2001). Suda et al (2000) reported that protein fractions in wild poinsettia (Euphorbia heterophylla), exhibit different degradation patterns during germination and initial seedling development; gobulins being continuously degraded after the start of imbibition whereas saltsoluble fractions were degraded between 36 and 72 hours, and albumins between 60 and 84 hours. Globulin depletion is accompanied by increase in free amino acids in the endosperm whereas intense albumin depletion did not occur. This result suggests that during albumin depletion there was a rapid transfer of amino acids 74 to the growing embryo. Fiel et al. (2003) reported that the globulin fractions of cooked faba beans ranged from 36.6 to 55.0% compared to 69.5-78.1% for the uncooked seeds. The Albumin fractions of cooked faba beans ranged from 0.6 to 1.0% compared to 1.4- 3.4% for the uncooked. The prolamin fractions of cooked faba beans ranged from 2.6 to 5.9 % compared to 2.1–4.1% for the uncooked seeds. The G1- glutelin fractions of cooked faba beans ranged from 1.5 to 2.3% compared to 0.9–2.2 for uncooked seeds. The G2-glutelin fractions of cooked faba beans ranged from 4.1–7.8% compared to 1.9 – 6.2 % for uncooked seeds. The G3–glutelin fractions of cooked faba beans ranged from 27.1 to 45.3% compared to 8.9-14.4% for the uncooked seeds. Residues of cooked seeds ranged from 2.4 to 3.6% compared to 1.83.4 for the uncooked seeds. Use of germenated cowpeas in food is fast gaining popularity in Nigeria an other west African countries (Papunam et al. 1985; Ologhobo et al. 1986; Obizoba 1989). Germination or sprouting improves proteins, carbohydrates, vitamins, minerals and overall nutritional values of legumes and leads to reduction in some toxicants (Vanderstop 1981; Boralkar et al . 1985). Germination leads to an increase in enzymes (Nnanna and Phillips, 1989) as well as free amino acids. Vitamins such as ascorbic acid 75 riboflavin, niacin, choline and biotin are increased by germination (Nnanna and Phillips, 1988). However, thiamin and pantothenic acid did not change while folic acid diminished by germination. Germination leads to a slight decrease in trypsin inhibitor activity, and ample decrease in starch and flatulence-causing oligosaccharides in cowpeas (Ologhobo et al., 1986; Nnanna and Phillips, 1988). During germination, reducing sugar content increases while polyphenol and phytate levels are reduced (Chen et al. 1977). There is general increase in nutritive value of germinated cooked cowpeas (Obizoba 1989). Germination imparts a characteristic agreeable flavor to cowpeas probably because of amides released during germination(Kurien 1987). The germinated cowpeas can be dried and cooked later into sweet savory dishes with good nutritional qualities (Uzogara et al. 1992). Cowpea cultivars with the greatest peak viscosities showed low stabilities to extended cooking. Roasting depressed paste viscosity properties of all the cowpea cultivars studied. Tannin concentrations were 0.3 – 6.9 and 7.2 – 116 mg CE/g flour from whole cowpea seeds and seed coats respectively, increasing with intensity of seed color. Although dehulling removed 98% of the tannin content of raw cowpeas, improvement in protein quality as a result of dehulling was 76 observed for only the highly–pigmented in Maroon–red variety. Roasting significantly improved digestibility. Roasted cowpeas possess adequate nutritional and functional qualities as protein supplements in cereal – based weaning foods (Plahar, 1997). Six commonly consumed legumes in India were subjected to the following cooking processes: roasting (160oC), germination (24) h, pressure cooking (15-lb for 15 min), germination and roasting. Reduction of phytic acid was apparent when roasting (43%) or germination (36%) were used; pressure cooking did not achieve any significant differences. Each legume produced different results. Lentils had the least phytic acid degradation along with the cowpea, comparatively the moth bean and the bengal gram exhibited the most. Changes in the solubility of the minerals were larger in the seeds that had been germinated and then rosted but were not significant (P ≤ 0.01). It is concluded that roasting and germination are effective in reducing levels of phytic acid and enhancing mineral solubility (Vaishale et al., 1998). Roasting and pressure cooking increased the net protein ratio values of the cawpea, canavalia and lupin flours although the values were lower than that for casein. Both treatments decreased trypsin inhibitor activities. Tannins decreased slightly in cowpea and by 38 and 68% in canavalia and lupin flours, respectively. 77 Apparent digestibility of lupin protein was similar to that for casein at roasting temperatures up to 200oC and after autoclaving. Values were slightly lower for the other flours. True digestibility values were similar. Available lysine was not significantly affected by treatment. Jibaja et al. (1988). Wet–heat treatments such simple boiling (atmospheric preasure, 100oC) and pressurized boiling (higher than 100oC) reduced the polyphenol content of mature dark red seeds of cowpea (Vigna unguiculata) c.v UPL CP3 by 61 to 80% and increased protein digestibility in vitro (IVPD) by 6 to 25%. Pressurized steaming (higher than 100oC) removed 48 to 83% of the polyphenols but increased IVPD by only 1.1 to 4.2%. Dry heat as exemplified by roasting and microwave treatment inactivated 58 to 71% of the tannins but increased IVPD by only 1%. All the heat treatments were effective in removing or inactivating polyphenol although different IVPD values resulted (Laurena et al., 1987). Six types of fingermillet (Eleusine coracana)–based tempe were developed by incorporating either common beans, groundnuts, cowpeas, mung beans, chickpeas, sesame and/or their mixtures and fermented by Rhizopus oligosporus. The proximate and mineral composition was not changed significantly by fermentation. Tempe processing reduced the tannin and hydrogen cyanide contents by 55.2–75-7, and 71.0- 86.2% respectively. The in- 78 vitro protein digestibility was improved (Mugula et al. 1999). Soaking cowpea seeds (25oC, 24h) reduced trypsin inlibitor activity by 20%, whereas boiling of soaked seeds decreased TIA by 85%. A slight increase in TIA occurred in soaked cooked and fermentation for 18h cowpeas (Prinyawiwat kul et al., 1996). Though soaking significantly reduced the content of tannins alone in Vigna aconitifolia, total free phenolics and tannins were markedly reduced in Vigna sinensis. Greater loss of total free phenolics as well as tannins occurred under autoclaving compared to soaking and cooking in both legumes investigated. In Vigna aconitifolia, soaking in distilled water for 6h and cooking for 30 min reduced the phytic acid content by up to 43%. Maximum reduction in the level of phytic acid (36%) was observed in distilled water soaking compared to cooking and autoclaving in Vigna sinensis. Limited loss in content of phytic acid was noticed under autoclaving compared to soaking and cooking in four pulses studied. However, IVPD of Vigna aconifolia and Vigna sinensis was enhanced to 12.5 and 14.8%, respectively, under autoclaving. Of all the processing methods, autoclaving seemed to be the most efficient for reduction in content of the anti-nutrients, except phytic acid. (Vijayakumari et al., 1998). A decreasing trend was noted in phytic 79 acid and trypsin inhibitor of cowpea flour when processed into milk, followed by a further reduction at the end of fermentation (Sanni et al., 1999). All cowpea varieties contained typsin inhibitors, lectins, hydrogen cyanide, tannins and phytic acid. Autoclaving destroyed all the trypsin inhibitors and lectins and significantly reduced HCN but tannic and phytic acids were heat stable. In raw samples, values obtained for these compounds, in sequence, were 10.9 to 33.7 trypsin inhibitor/units mg/protein, 28.6 to 76.6 haemagglutinating units/mg protein, 1.6 to 3.9mg/100g, 0.2 to 0.4mg/g and 422.3 to 543.4mg/100g. respectively. Autoclaved samples contained HCN 0.73 to 1.32 mg/100g, tannins 0.17 to 0.36 mg/g., and phytic acid 314.97 to 420.54 mg/100g. The amount of total phosphorus that remained bound to phytic acid and was thus unavailable ranged from 29.60 to 33.15%. Antinutritional factor concentrations differed between varieties (P ≤ 0.001)(Oke et al., 1996). Autoclaving completely eleminated trypsin in hibitor, haemagglutinin and hydrogen cyanide (HCN), whereas it reduced (P ≤ 0.01) the levels of phytic and tannins in the cowpeas. In raw and autoclaved forms, the insect susceptible cowpeas were better (P ≤ 0.05) utitized by rats than their resistant counterparts, a condition which was attributable to lower levels of anti-nutrients in the susseptible varrieties. In the raw cowpeas, trypsin 80 inhibitor, haemagglutinin and HCN were significant variables affecting cowpea protein utilization, while in autoclaved samples, tannic and phytic acids were important though nonsignificant factors. It is concluded that autoclaving at 105oC under 15 psi for 30 min improved the protein quality of the insect susceptible more than the insect resistant cowpea varieties (Umoren et al., 1997). Dry heating and autoclaving reduced the antinutritional components significantly. The in vitro protein digestibilities of raw, dry–heated and autoclaved legume seeds (Acacia nilotica) were 61.2, 77.4 and 80.2%, respectively (Siddhuraju et al., 1996). Germination decreased the phytic acid content of chickpea (Cicer arietinum L.) and pigeonpea (Cajanus cajan L.) seeds by > 60% and that of mung bean (Vignaradiata), urd bean (Vigna mungo) and soybean (Glycine max) by about 40 % . Fermentation decreased phytic acid content by 26– 39% in all these legumes with exception of pigeonpea in which it was decreased by 50%. Autoclaving and roasting were more effective in decreasing phytic acid in chickpea and pigeonpea than in urd bean, mug bean and soyabean. Germination greatly increased the in vitro protein digestibility (IVPD). IVPD was only slightly increased by roasting and autoclaving of all legumes. Germination and fermentation also decreased the total dietary fibre (TDF) in all legumes. 81 Autoclaving and roasting caused slight increase in TDF values. All the processing treatments had little effect on calcium, magnesium and iron contents (Chitra et al., 1996). Dry heating as well as autoclaving significantly reduced anti-nutritional factors ofthe little–known legume, velvet bean (Mucuna pruriens L. DC.) .Protein efficiency ratio, true protein digestibility, biological value, net protein utilization and utilizable protein were significantly improved by autoclaving compared with dry heating, the values of true protein digestibility and net protein utilization of dry–heated samples were significantly higher than the raw samples (Perumal– Siddhuraju et al., 1996). Soaking for 12h was most effective in reducing cooking time, tannin and phytate concentrations; it also improved IVPD. Prolonged soaking (24h) decreased calcium and iron values by 19 and 35%, respectively. Dehulling showed that Ca, Fe, Mg and Zn were concentrated in seed coat of African yambean. Dehulling reduced tannin content but had no significant effect on phytate content or in vitro protein digestibility except for seeds soaked for 12h before Dehulling for 24h increased crude protein content by 16 % (P ≤ 0.05) . Blanching and roasting increased IVPD by 8–11% . Fermentation had no effect on crude protein, Ca Fe. Mg or Zn but significantly reduced phytate content. Fermentation had no advantage over heat treatment 82 with respect to improving IVPD (Ene- Obong et al., 1996). NaHCO3 solution and autoclaving significantly reduced the contents of total free phenolics (85-88%) compared to raw seeds. Autoclaving (45 min) reduced tannins by up to 72%. Soaking seemed to have limited effect in eliminating phytohaemgglutinating activity whereas autoclaving (45 min) seemed to eliminate the haemagglutinating activity completely. The reduction in phytic acid was found to be some what greater in distilled water soaking (28%) compared to NaHCO3 solution (22%). Only a limited loss in phytic acid was observed under cooking as well as autoclaving. Loss of HCN was greater under autoclaving (87%) compared to the other processes studied. Of the 3 sugers analysed, soaking reduced the level of verbascose more than that of stachyose and raffinose. Autoclaving reduced the content of oligosacccharides more efficiently (67–86%) than ordinary cooking (53–76%) . Autoclaving improved the in vitro protein digestibility (IVPD) significantly (13%). Of all the different water and hydrothermal treatments studied autoclaving seemed to be the most eficeient method in improving IVPD and eliminating the anti-nutrients investigated except phytic acid (Vijayakumari et al., 1995). Fermentation of various rice and bengal gram dhal blends prepared by mixing them in different proportion at 35oC for 18h brought about a 83 significant decline in phytic acid content to extent of 23 to 36% over the control. Incorporation as well as fermentation improved the starch and protein digestibility (in-vitro) of all the rice – bengal gram dhal mixures. Improvement in starch and protein digestibility is related to the reduction in phytic acid content, as this anti-nutrient is known to inhibit amylolysis and proteolysis, A significant negetive correlation was found between phytic acid and digestibility of stach and protein (Anshu – Sharma et al., 1995). Anti-nutnitional factors such as lectins where higher in raw and boiled nunas (Phaseolus vulagaris) samples than in roasted nunas, while tannin levels did not change from raw to roasted treatment. Overall, in vitro digetibility was slightly lower for toasted nunas than boiled dry bean (Beem et al., 1992) . The application of dry heat to the Red gram (pigeonpea) seeds and meal was not effective in inactivating the trypsin inhibitory activity (TIA) and chymotrypsin inhibitory activity (CIA). Soaking for 24h followed by cooking for 20 min was effective in distroying the TIA and CIA (Mulimani et al., 1994). Indigenous fermentation of coarsely ground dehulled black- gram dhal slurry at 25,30 and 35oC for 12 and 18 h reduced the concentrations of phytic acid and polyphenols (P ≤ 0.05) The unfermented legume batter had high amounts of phytic acid (1000 mg/100g) and poly 84 phenols (998 mg/100g), and these were reduced to almost half in the protuct fermented at 35oC for 18h. In vitro digestibility of starch and protein was improved (P ≤ 0.05) with increase in the temperature and period of fermentation. A significant and negative correlation was found between the in vitro digestibility and anti-nutrients contents further strengthens these findings (Yadav et al., 1994). Four Kabuli type varieties were evaluated, the trypsin inhibitor activity (TIA) of the varieties was reduced by germination or by cooking the seeds. Reduction in TIA on sprouting in the variety ILC 116 was significantly lower than in the other varieties, but this characteristic was not seen when it was cooked. True digestibility of the protein correlated negatively with TIA and positively with protein content (Savage et al., 1993). Processing, especially presoaking followed by boiling in water or in alkaline solutions at atmospheric pressure, reduces the phytic acid content of cowpeas (Ogun et al. 1989; Uzogara et al. 1990a) while pressure cooking or autoclaving caused less loss of phytic acid (Uzogara et al. 1990a; Ologhobo et al. 1984). 85 CHAPTER THREE MATERIALS AND METHODS 86 3. MATERIALS AND METHODS 3.1 Material: 3.1.1 Cowpea seeds: The dry cowpea seeds (Vigna unguiculate L. Walp) of the two cultivar were obtained from El Obeid Research Station. 3.1.1.1 Chemicals and Reagents: Potassium sulphate, cupric sulphate, sulphuric acid, sodium hydroxide, boric acid, hydrochloric acid, methyl red, ethanol (EOH), petroleum ethen (b.p. 60–80ºC) soddium chloride, isopropyl alcolhol (PrOH), sudium borate, sodium dodecyl sulphate (SDS), 2-mercaptoethanol (2-ME), vanillin, methanol, catechin, potassium hydroxide sodium carbonate, trichloroacetic anid, casein, sodium dihydrogen phosphate, sodium hydrogen phosphate, potassium thiocyanate, ferrichitrate, hexane, formaldehyde, sodium azide, bromocersolgreen. All chemicals used in the study were of analytical grade. 3.1.2 Apparatus: Centrifuge, (PTL limted, London), Gerhardt lieraeus shaker, spectrophotometer, (Unicam Instruments), Cambridge water bath, Heraeus Hanau mettler sensitive balance, Soxhelt extracting apparatus, magnetic stirrer, steamed out apparatus, oven,mill, autoclave, heater, incubator, keldahl apparatus, thermometer, burette, pipette, volumetric flasks, beakers, conical flasks, measuring cylinders, test tubes, Bunsen burner, aluminum foil, stand , funnel, filter paper (Whatman No.1), pH meter, deep freezer were utilized. 3.2 Methods: 87 3.2.1 Preparation of cowpea samples:3.2.1.1 Cleaning About 5 kg of mature and dry seeds of cowpea (Vigna unguiculata L. Walp) were obtained from El Obeid Research station and were cleaned thoroughly; freed from foreign matter, broken seeds, and immature seeds. The seeds were stored in plastic containers at room temperature. 3.2.1.2 Autoclaving: About 2 kg of seeds were autoclaved at 15 1b, or 20 1b for 15, 30, 45 minOne hundred seeds (100) were placed in 500 ml conical flask, 150 ml. distilled water were added to the flask. Then flasks were placed in the autoclave. After autoclaving, liquied was separated, and the autoclaved seeds were dehydrated in a hot air oven at 50 oC to constant weight and were powdered, defatted and kept for further use. 88 3.2.1.3 Roasting: About 2 kg of seeds were roasted at 90oC or 120oC for 30 , 45 and 60 min., the roasted seeds were powdered by micro hammer mill with 0.5 mm mesh size. The flour was placed in a conical flask and n-hexane was added, the solvent to flour ratio was 10:1. The mixture was stirred for 16 h and was then filtered and washed again with n- hexane and was air dried overnight at room temperature and kept in clean bottles for further use. 3.2.1.4 Germination of Cowpea samples: Germination was carried out according to the method of Bhise et al. (1988) .Broken grains were removed by hand. The seeds were soaked with about 3 volumes of distilled water overnight at room temperature, with two changes of water during the day to remove dirt and extra husks. The wet grains were then soaked in 1-2 volumes of 0.2% formaldehyde solution for 40 min to retard mold growth during germination. The soaked grains were then washed with distilled water several times and soaked in water for 20 min to remove residual formaldehyde. These seeds were spread evenly (about 1 cm thick) in plastic tray, with plenty of air space, covered with cheese cloth and germinated in an air circulating incubater at 30 ± 2oC for 1, 2, 3, and 4 days. Water was sprinkled on the grains every 12h to avoid drying; non germinated and moldy seeds were discarded. Sprouted cowpea samples were dried at 50 oC to constant weight and finely ground. Samples were taken to fractionate the protein on the basis of solubility, and to determine in-vitro protein digestibility (IVPD), phytate and trypsin inhibitor acctivity (TIA) 89 3.2.1.5 Cooking of sprouted cowpea samples: Samples were cooked according to the mothod of ICAITI (1978). One hundred seeds were placed in 150 ml boiling flask and boiled in distilled water for 45 min. Cooked seeds were dried at 50oC to constant weight and finely ground. Samples were taken to fractionate the protein on the basis of solubility to determined IVPD, phytate and trypsin inhibitor activity (TIA) 3.3 Analytical methods: 3.3.1 Proximate analysis: Proximate analysis was determined using the official methods (AOAC, 1990) . The nitrogen contents of the flours were determined using micro Kjeldahl method (Pearson, 1970). 3.3.2 Protein fractionation:Cowpea proteins can be separated into five fractions by selective extraction method (Landry and Moureaux, 1970, 1976, 1981, 1982). 3.5g. defatted samples were kept in suspension with 35 ml extractant by magnetic stirring in 50ml centrifugal tubes. The duration and number of extractions with solvent and identification of protein fractions were carried out as follows: Step I: The first fraction (globulin) The powdered sample (3.5g) was mixed with 35ml of 0.5M NaCl for 3 extraction times (60,30, 30 min) at 4oC. The total supernatants collected were 105 ml Step II: The second fraction (albumin): 90 The residue was mixed with 35 ml of distilled water for 2 extraction times (15, 15 min) at 4oC. The total volume collected was 70 ml. Step III :- The third fraction (prolamins): The resdiual material was stirred with 60% ethanol twice for 30 min at 20oC and then at 60oC for 30 min, followed by extraction with 55% isopropanol at 20oC three times (60, 30, and 15 min. with stirring). The total volume collected was 210 ml. Step IV: The fourth fraction (G1–glutelins): The residue was extracted with 60% ethanol plus 0.6 % 2mercaptoethanol (2-ME) and stirred twice for 30 min (20oC), then extracted with 55% PrOH containg 2-ME (0.6%) at 20oC twice for 30 min. The total volume collected was 140 ml. Step. V:- The fifth fraction (G2- glutelins): Borate buffer, pH 10 (0.0125 M N2aB4O7 – 10H2O and 0.02 M NaOH). 2g of sodium borate were dissolved in 250 ml distilled water and 0.4g of sodium hydroxide in 250 ml distilled water were added and minxed together, and was adjusted to pH 10 using either NaOH or HCl) with 0.6% 2.ME and 0.5 M NaCl were used with stirring for 60,30 and 30 min (20oC ). The total volume collected was 105 ml. Step VI:- The sixth fraction (G3- glutelins), Borate buffer, pH 10 with 0.6% (2-ME) 2- mercaptoethanol and 0.5%sodium dodecyl sulphae (SDS) were used with stirring for 60, 30, and 15 min (20oC). The total volume collected was 105 ml. 91 Fractions I and II contained the albumins and globulins, the free amino acids and small peptide fragments. Fraction III contained the prolamin. Fraction IV contained G1- glutelins. Fraction V contained G2 –glutelins. Fractian V contained G3 – glutelins. The solid material was isolated from extractants by centrifugation at 30000 g for 15 min. For each solvent, supernatants were combined to give the total extract. The nitrogen content of each of these six fractions was determined by mircro–Kjeldahl method. The residue left after extraction was also analysed for nitrogen content (Table 1). 92 Table 1 . Protein Extraction Procedure for sequence Ao and Bo Step Total Volume (extract) Extractant 1 105ml Nacl,O,5Mwater (4oC) 2 3 4 70 ml 105 ml 105 ml 70 ml 70 ml 5 105 ml 6 105ml 7 o Water (4 C) EtOH,60%(20oC) Time of Extraction No. of Protein (min) Fraction groups 60,30,30, I Globulins 15,15 II Albumins III Prolamins IV G1-glutelins 60, 30, 30 V G2-glutelins 60,30,15 VI G3-glutelins ------ ----- 30.30.30 And then at 60oC 60,30,30 2-Pr.OH 55%(20oC) EtOH,60%±2ME O.6%(v/v)(20oC)2PrOH,55%±2MEO.6% (v/v)(20oC) NaCl,0.5M,PH10±2ME. 0.6% (v/v)(20oC) NaDodSO4,0.5%(W/v) PH10±2ME 0.6%(v/v)(20oC) ------- 93 30.30 30,30 Insoluble Proteins Calcultions: Soluble protein% = Protein fraction% = T.F × N × TV × 14 × 6.25 × 100 a × b × 1000 So lub le protein × 100 total protein of sample Where:T.F: Titre N: Normality of HCl. TV: Total volume of the aliquot extracted. 14: Each ml of HCl is equivalent to 14 mg nitrogen. A:Number of ml of aliquot taken for digestion: (10) B: Number of g sample extracted : (3.5 g). 1000: To convert from g to mg. 2.3.1.3. In-vitro protein digetibility (IVPD): IVPD was carried out according to Saunder et al , 1970). A 0.2g of the sample was placed in a 50ml centrifuge tube, 15 ml of 0.1 N HCl containing 1.5mg pepsin, were added and the tube was incubated at 37oC for 3h. The suspension was then neutralized with 3.3ml , 0.5 N NaOH, then treated with 4 mg of pancreatin in 7.5 ml of 0.2 M. phosphate buffer (pH= 8.0), containing 0.005 M sodium azide; the mixitre was then gently shaken and incubated at 37oC for 24h. After incubation the sample was treated with 10 ml, 10% trichloroacetic acid (TCA), and centrifuged at 50.000g, for 20 min at room temperature. Nitrogen in 94 the supernatant was estimated using the micro Kjeldahl method. Digestibility was calculated using the formula: Protein Digestibility % = Nitrogen in sup ernatnt × 100 Nitrogen in sample The mixed samples were prepared as follows: Assuming 25.6% protein in (A) samples and 24.6% protein in (B) samples taking 0.39g and 0.41g sample containing 0.1g protein for digestion. Theefor the mixed samples of raw material with treated samples would be carried out as the below sequences: 1. Ain Elgazal (A) Cultivar Sample weight = 0.1 × 100 = 0.39g 25.6 (Sample weight) (Protein content) Auto/Roasted Raw Total protein 0.384g + 0.00g 0.1g A1 0.336g + 0.048g 0.1g A2 0.288g + 0.096g 0.1g 0.00g + 0.384g 0.1g A4 0.048g + 0.336g 0.1g A5 0.096g + 0.288g 0.1g A6 0.192g + 0.192g 0.1g A0 A3 Control Control 95 2- BUFF (B) Cultivar, Sample weight = 0.1 × 100 = 0.41g 24.6 (Sample weight) (Protein content) Auto/Roasted Raw Total protein 0.4g + 0.00g 0.1g B1 0.3g + 0.1g 0.1g B2 0.35g + 0.05g 0.1g 0.00g + 0.4g 0.1g B4 0.05g + 0.358g 0.1g B5 0.1g + 0.3g 0.1g B6 0.2g + 0.2g 0.1g B0 B3 Control Control 96 3.3.4 Determination of tannin in raw and treated samples: Quantitative estimation of tannin for each sample was carried out using the modified vanillin-HCl in methanol method as described by Price et al. (1978). The specific reagent for the determination encompasses equal volumes of 1% vanillin in methanol (w/v) and 8% conc. HCl in methanol (v/v). These are to be mixed just prior to use and were rejected when ever a trace of colour appears. The manipulation is in the sequence of: 0.2g of the ground sample placed into a 100 ml conical flask. Ten ml of 1% HCl in methanol (v/v) were added, shaken for 20 min using mechanical shaker and centrifuged at 2500 rpm. One ml of the supernatant was pippetted into a test tube and 5 ml of vanillin HCl reagent were added. The optical density was read using a colorimeter (Lab System Analyzergfilters, J. Mitra and Bros. Pvt. Ltd.) at 500 nm after 20 minutes incubation at 30oC. For zero setting of the colorimeter 1 ml of ablank (1% HCl in methanol). solution was mixed with 5 ml 4% HCl in methanol (v/v) and 5ml vanillin – HCl reagent in a test tube. The bank mixture was incubated at 30oC for 20 min. Calculations: C E% = C × 10 × 100 =C×5 200 9 Where: C = Concentration corresponding to optical density. 10 = Volume of extract in ml 200= Sample weight in mg. The standard curve of tannin: 97 The standard curve of tannim was traced via a procedure in which 100 mg D(+) – catechin has been added to 50 ml 1% HCl in methanol in 50 ml volumetric flask, giving a concentration of 2 mg catechin per 1 ml. A series of 0.0, 0.02, 0.04, 0.06, 0.08, and 0.1mg catechin / ml solutions were prepared according to the plan. Aliquots of 0-ml , 0.5ml, 1.0 ml, 1.5 ml, 2.0 ml,and 2.5 ml were pippetted from the stock solution into a series of 50ml volumetnic flasks. Then 1% HCl in methanol were added up to mark. 1 ml portions from each volumetric flask of the different concentrations were taken into test tubes, and five ml vanillin – HCl reagent were added. The optical density was read in a colorimeter at 500 nm after 20 minutes incubation at 30oC from addition of the reagents. For zero setting 1ml blank solution was mixed with 5ml 4% HCl in methanol and 5 ml vanillin-HCl reagent in test tube, and was incubated at 30oC for 20 minutes. A linear relationship between catchen concentration and optical density obtained as shown in Fig. 1. 3 Optical density at 500nm 2.5 2 1.5 1 98 3.3.5 Determination of phytic acid: The determination of phytic acid was carried out according to the method of Wheeler and Ferrel (1971) . One gram of finely ground sample was weighed into a 125 ml conical flask, extracted with 50 ml 3% TCA (w/v) for 30 min with mechanical shaking. Then the suspension was centriguged at 3000 rpm. Ten ml aliquots of the supernatant were transferred into 50 ml boiling tubes. Then 4ml of FeCl3 were added (2mg ferric iron per ml 3% TCA), centrifuged at 3000 rpm for 15 minutes and the clear supernatant was decanted carefully. The precipitate was then washed twice by despersing well into 25 ml 3% TCA heated in boiling water bath for 5- 10 minutes and Then centrifuged. Washing was repeated once with water. The precipitate was cautiously dispersed in few ml distilled water enriched with 3ml 1.5 N NaOH with mixing. The volume was made approximately to 30 ml with distilled water and heated in boiling water bath for 30 minutes. The content of the tube were filtered hot (quantitatively) through filter paper (Whatman No.1) and the filtrate was discarded. The precipitate was dissolved with 40 ml hot 3.2 N HNO3 into a 100 ml volumetric flask . The filter paper was washed with several portions of distilled water, collecting the washings in the same flask. The contents in the flask were cooled to room temperature and diluted to volume with distilled water. Five ml aliquots were transferred to another 100 ml volumetric flask and diluted to approximately 70 ml with distilled water. Then, 20ml of 1.5M (potassium thiocyanate) KSCN were added, completing the volume up to the mark. The intensity of color was immediately assessed (within one minute) in a colorimeter 99 (Lab System Analyzer. 9 giters, J Metra and Bros. Pvt Ltd.) at 480 nm .A blank probe was run with each set of samples. Calculation: The iron content was calculated from a prepared Fe (NO3)3 standard curve. Then the phytate phosphorous (P) was calculated from the iron (Fe)(of a 4:6 Fe: P molecular ratio). Standard curve of phosphate was plotted from a procedure including: 0.4321g of ferric nitrate (Fe(NO3)3) dissolved in distilled water in 1 liter volumetric flask up to the mark. Five mllileters of this solution were taken into 50 ml volumetric flask and the volume made up to the mark with distilled water, giving concentration of 10 ppm of ferrous (Fe). Concentrations of 0, 1, 2, 3, 4 , and 5 ppm were preparded by taking 0, 5, 10, 15, 20, and 25 ml from 10 ppm ferous solution into a series of 50 ml volumetric flasks. Then distilled water was added up to the mark. Five mllileters aliquots from the standards were pippetted into a 100 ml volumetric flasks, and diluted up to 70 ml with distilled water. Then 20 ml of 1.5 M KSCN were added , completing the volume with distilled water. The density of color was read at 480 nm immediately (within one min) in a colorimeter (Lab System Analyzer 9 filters, J. Mitra and Bros. Pvt. Ltd.). A standard curve was obtained by plotting concentrations against the corresponding readings of colorimeter giving a straight line. The phytic phosphorus was calculsted from the ferric ion concentration assuring 4 : 6 iron : phosphorus molar ratio. MI standard ml Fe+3 conc. ppm A Conc/A = K 5 1 0.09 11 10 2 0.18 11 100 15 3 0.20 15 20 4 0.28 14.25 25 5 0.30 12.25 K = 13.6 Phytic % = 6 A × mean k × 20 × 10 × 50 × 100 × 4 1000 × 2 Where: A = optical density K= Con A 30 Absorbance at 480 nm 25 20 15 10 101 5 = 9648.0 × A 3.3.6. Determination of trypsin inhibitory activity: The extract of the samples was obtained by shaking 4g of the sample with 40ml of 0.1 M phosphate buffer pH 7.5 (16ml of 0.2 M NaH2PO4 and 84 ml Na2HPO4 (0.2M) diluted to 200ml) for 3 hours using a mechanical shaker (Shutted machine R010) at room temperature. The extracts were then centrifuged at ambient temperature at 3000 rpm. for 20 minutes. The supernatants were diluted two times and then used for analysis. For the assay of enzymatic activity, the casein substrate was used for determining trypsin inhibitor activity in the crude preparations (phosphate buffer extracts). A 2% casein solution in phosphate buffer (0.1M pH 7.5) was used as substrate, while the enzyme used as trypsin (Beef pancreas), 5 mg/ml. The incubation mixture consisted 0.5 ml trypsin. 2ml 2% casein, 0.9 ml phosphate buffer (0.1M pH 7.5) 0.4 ml HCl solution (0.001 M) and 0.2 phosphate buffer extract. In all cases the total volume of incubation mixture were kept at 4 ml. Incubation was carried out at 37ºC for 20 minutes, after which 6.0 ml of TCA solution was added to stop the reaction and corresponding blanks were run concurrently. In this method one trypsin unit (TU) is arbitararity defined as an increase of 0.01 absorbance unit at 280 nm in 20 minutes for 10 ml of reaction mixture under there conditions and the trypsin inhibitor activity as the number of trypsin units inhibited (Ray and Ras, 1971). 3.3.7 Statistical analysis: 102 Each sample was analyzed in triplicate and the figures were then averaged. Data were assessed by analysis of variance (ANOVA), (Snendecor and Cochran, 1987) and by Duncan’s multiple Range Test (DMRT) with a probability p ≤ 0.05 (Dumcan, 1955). 103 CHAPTER FOUR RESULRS AND DISCUSION 104 4. RESULRS AND DISCUSION 4.1 Proximate composition of cowpea seeds: 4.1.1. Moisture content: The chemical composition of cowpea seeds is shown in Table 2. The results are expressed as on dry matter basis (DMB). Data obtained showed that, moisture content for Ain Elgazal cultivar raw uncooked seeds, uncooked 24h germinated seeds, uncooked 48h germinated seeds, uncooked 72h germinated seeds, uncooked 96h germinated seeds, raw cooked seeds, roasted seeds, autocalved seeds, cooked 24h germinated seeds, cooked 48h germinated seeds, cooked 72h germinated seeds, and cooked 96h germinated seeds were 5.9%, 4.9%, 4.5%, 4.4% 4.4%, 4.8%, ,4.7%, 4.6%, 4.6%, 4.5%, 4.5%, and 4.4% respectively. Similarly for Buff cultivar raw uncooked seeds, uncooked 24h germinated seeds, uncooked 48h germinated seeds, uncooked 72h germinated seeds, uncooked 96h germinated seeds, raw cooked seeds, roasted seeds, autocalved seeds, cooked 24h germinated seeds, cooked 48h germinated seeds, cooked 72h germinated seeds, and cooked 96h germinated seeds were 5.5%, 4.8%, 4.6%, 4.5% 4.5%, 5.2%, 4.8%, 4.6%, 4.5%, 4.6%, 4.5% and 4.4% respectively. Data obtained showed that germination and heat treatment significantly (P ≤ 0.05) decreased, moisture content. This disagreed with that reported by Akinyele (1989). 105 Table 2. Proximate composition of some cowpea preparations Cultivar Treatment Ain Elgazal cultivar Moisture % Protein % Ash % Fiber % Oil % Moisture % Protein % Ash % Fiber % Oil % Raw uncooked seeds Germinated 24 h. (uncooked) Germinated 48 h. (uncooked) Germinated 72 h. (uncooked) Germinated 96 h. (uncooked) Raw cooked Roasted seed Autoclaved seeds Germinated 24 h. (cooked) Germinated 48 h. (cooked) Germinated 72 h. (cooked) Germinated 96 h. (cooked) 5.9±0.1a 25.6±0.01e 3.2 2.5 1.5 5.5±0.01a 24.6±0.01e 3.5 2.2 1.6 4.9±0.2b 26.2±0.02c 3.4 2.6 1.5 4.8±0.02b 25.0±0.0d 1.5 c 4.5±0.01 c 4.4±0.01 c 4.4±0.00 c 28.6±0.1 b 30.7±0.2 a 3.6 3.8 4.0 2.7 2.9 3.0 1.6 1.6 4.6±0.02 d 4.5±0.01 d 4.5±0.01 2.3 1.6 27.4±0.01 3.7 2.4 1.6 28.4±0.01 b 3.8 2.5 1.6 30.6±0.01 a 3.9 2.6 1.7 de 4.0 2.8 1.7 4.0 3.1 1.6 5.2±0.01 23.6±0.02 4.7±0.1b 25.9±0.01c 4.0 3.1 1.6 4.8±0.1a 24.0±0.02d 4.0 3.0 1.7 c 26.0±0.02 c c 4.0 3.0 1.7 26.2±0.02 c 4.6±0.0 25.9±0.01 4.0 4.6±0.03 c 4.5±0.01 d 27.5±0.02b 4.5±0.01 d b 4.2 31.0±0.01a 4.3 4.4±0.01e 28.0±0.01 4.1 c 4.2 3.1 3.0 3.2 1.6 1.6 a 3.6 c c 4.8±0.2 a 27.5±0.0 c Buff cultivar c 4.6±0.01 d 4.5±0.00 c 25.0±0.01 25.0±0.0 c 4.2 3.1 1.7 27.4±0.03 b 4.2 3.1 1.7 ab 4.3 3.1 1.7 4.3 3.1 1.7 1.6 4.6±0.03 3.2 1.6 d 4.5±0.1 28.4±0.01 3.2 1.6 4.4±0.01e 28.9±0.00a Values are means (±SD). Means not sharing a common superscript letter in a column for the same cultivar are significantly different at (P < 0.05) as assessed by Duncan's Multiple Range Test. 106 4.1.2 Protein Content: Data obtained showed that protein content for Ain Elgazal cultivar raw uncooked seeds, uncooked 24h germinated seeds, uncooked 48h germinated seeds, uncooked 72h germinated seeds, uncooked 96h germinated seeds, raw cooked seeds, roasted seeds, autocalved seeds, cooked 24h germinated seeds, cooked 48h germinated seeds, cooked 72h germinated seeds, and cooked 96h germinated seeds were 25.6%, 26.2%, 27.5%, 28.6%, 30.7%, 25.9%, 25.9%, 26.0%, 26.2%, 27.5%, 28.0% and 31.0% respectively. For Buff cultivar raw uncooked seeds, uncooked 24h germinated seeds, uncooked 48h germinated seeds, uncooked 72h germinated seeds, uncooked 96h germinated seeds, raw cooked seeds, roasted seeds, autocalved seeds, cooked 24h germinated seeds, cooked 48h germinated seeds, cooked 72h germinated seeds, and cooked 96h germinated seeds were 24.6%, 25.0%, 27.4%, 28.4%, 30.6%, 23.6%, 24.0%, 25.0%, 25.0%, 27.4%, 28.4% and 28.9% respectively. Data obtained showed that germination and heat treatment significantly (P ≤ 0.05) increased, protein content. These results reported by Akpapuman et al., (1985). And Naves et al. (2001), bur were similar to those reported by Giami et al. (1999); Obong, (1995); Vanderstop 107 (1981; Boralk et al. (1985); Alonso et al. (1999), Akinyele (1989); Perumal-Siddhuraju et al. (1996). 4.1.3. Ash content: Data obtained showed that ash content for Ain Elgazal cultivar raw uncooked seeds, uncooked 24h germinated seeds, uncooked 48h germinated seeds, uncooked 72h germinated seeds, uncooked 96h germinated seeds, raw cooked seeds, roasted seeds, autocalved seeds, cooked 24h germinated seeds, cooked 48h germinated seeds, cooked 72h germinated seeds, and cooked 96h germinated seeds were 3.2%, 3.4%, 3.6%, 3.8%, 4.0%, 4.0%, 4.0%, 4.0%, 4.1%, 4.2%, 4.2% and 4.3% respectively. For Buff cultivar raw uncooked seeds, uncooked 24h germinated seeds, uncooked 48h germinated seeds, uncooked 72h germinated seeds, uncooked 96h germinated seeds, raw cooked seeds, roasted seeds, autocalved seeds, cooked 24h germinated seeds, cooked 48h germinated seeds, cooked 72h germinated seeds, and cooked 96h germinated seeds were 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.0%, 4.0%, 4.2%, 4.2%, 4.3% and 4.3% respectively. Data obtained showed that processing significantly (P ≤ 0.05) increased, ash content, a results which are similar that reported by Giami et al. (1999), Obong (1995) Vanderstop (1981), Boralk et al. (1985); Alonso et al. (1999), Akinyele (1989). 108 4.1.4. Fibre content: Data obtained showed that fibre content for Ain Elgazal cultivar raw uncooked seeds, uncooked 24h germinated seeds, uncooked 48h germinated seeds, uncooked 72h germinated seeds, uncooked 96h germinated seeds, raw cooked seeds, roasted seeds, autocalved seeds, cooked 24h germinated seeds, cooked 48h germinated seeds, cooked 72h germinated seeds, and cooked 96h germinated seeds were 2.5%, 2.6%, 2.7%, 2.9%, 3.0%, 3.1%, 3.1%, 3.1%, 3.0%, 3.2%, 3.2% and 3.2% respectively. For Buff cultivar raw uncooked seeds, uncooked 24h germinated seeds, uncooked 48h germinated seeds, uncooked 72h germinated seeds, uncooked 96h germinated seeds, raw cooked seeds, roasted seeds, autocalved seeds, cooked 24h germinated seeds, cooked 48h germinated seeds, cooked 72h germinated seeds, and cooked 96h germinated seeds were 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.8%, 3.0%, 3.0%, 3.1%, 3.1%, 3.1% and 3.1% respectively. Data obtained showed that processing significantly (P ≤ 0.05) increased, fibre content, these results were different from those reported by Akinyele (1989) but similar to that reported by Perumal-Siddhuraju et al. (1996). 109 4.2.5 Oil content: Data obtained showed that oil content for Ain Elgazal cultivar ranged from 1.5% to 1.6% and for Buff cultivar ranged from 1.6% to 1.7% that showed insignificant (P ≤ 0.05) increased in oil conttent for both cultivar. These results were different from those reported by Akinyele (1989). 4.2. Protein fractions: 4.2.1. Globulin fractions: As shown in Tables 3, 4 and 5, raw germinated, raw germinated cooked, autoclaved and roasted seeds were fractionated. The major protein fraction globulin showed significant (P ≤ 0.05) increase they were 87.5%, 80.6%, 76.4%, 71.4%, and 70.2% for Ain Elgazal raw, the first, second, third, and fourth day respectively. Similarly for Buff they were 89.8%, 81.3%, 78.3%, 75.6% and 72.7% respectively, a result which are similar to that reported by Suda et al. (2000) and Neves et al. (2001). Cooking significantly (P ≤ 0.05) reduced globulin they were 16.4%, 19.0%, 18.1%, 18.0%, 17.8% and 17.8% for Ain Elgazal raw the first, second, third and fourth days respectively. Similarly for Buff they were 15.7%, 19.9%, 22.0%, 22.6% and 23.0% respectively, a result which are simlar to that reported by Nugdallah and El Tinay (1997); Fiel et al. (2003). 110 Autoclaving at 120ºC under 15 psi for 30 minutes and at 150ºC under 20 psi for 30 minutes for Ain Elgazal raw seeds significantly (P ≤ 0.05) reduced globulin fraction from 87.5% - 29.2% and 27.6% respectively. Similarly for Buff raw seeds autoclaving at 120ºC under 15 psi for 30 minutes and at 150ºC under 20 psi for 30 minutes significantly (P ≤ 0.05) reduced globulin fraction from 89.8% - 32.0% and 36.0% respectively. Dry heating as well as cooking and autoclaving significantly (P ≤ 0.5) reduced globulin fraction. Roasting Ain Elgazal raw seeds at 90ºC/60 min and 120ºC/60 min significantly (P ≤ 0.05) decreased globulin fraction from 87.5% to 85.0% and 41.0% respectively. Similarly for Buff raw seeds roasing at 90ºC/60 min and 120ºC/60 min significantly (P ≤ 0.05) decreased globulin fraction from 89.8% to 88.0% and 45.5% respectively, a result which are similar to that reported by Nugdallah and El Tinay (1997); Fiel (2003). 4.2.2. Albumin fraction: As shown in Tables 3, 4 and 5, raw germinated, raw germinated cooked, autoclaved and roasted seeds were fractionated. The albumin fraction significantly (P ≤ 0.05) decreased, they were 4.0%, 3.8%, 2.3%, 1.9%, and 2.5% for Ain Elgazal raw, the first, second, third, and fourth day respectively. Similarly for Buff they were 3.6%, 1.5%, 111 2.2%, 2.2% and 1.2% respectively, a result which are similar to that reported by Nugallah and El Tinay (1997), Sud et al. (2000); Neves et al. (2001), but were different from those reported by Giami et al. (1999); and El Khalifa and El Tinay (1999). Cooking significantly (P ≤ 0.05) reduced albumin, they were 1.9%, 2.8%, 3.1%, 1.8%, and 1.4% for Ain Elgazal raw the first, second, third and fourth days respectively. Similarly for Buff they were 2.2%, 1.6%, 1.6%, 2.1% and 1.3% respectively. Autoclaving at 120ºC under 20 psi for 30 minutes for Ain Elgazal raw seeds significantly (P ≤ 0.05) reduced albumin fraction from 4.0%-1.2% and 1.2% respectively. Similarly for Buff raw seeds autoclaving at 120ºC under 15 psi for 30 minutes and at 150ºC under 20 psi for 30 minutes significantly (P ≤ 0.05) reduced globulin fraction from 3.6% - 1.0% and 0.9% respectively. Roasting as well as cooking and autoclaving significantly (P ≤ 0.5) reduced albumin fraction. Roasting Ain Elgazal raw seeds at 90ºC/30 min albumin in fraction showed changeless, but roasting at 120ºC/60 min significantly (P ≤ 0.05) reduced albumin fraction from 4.0% to 1.2%. Similarly for Buff raw seeds roasing at 90ºC/30 min albumin fraction showing changeless, but roasting at 120ºC/60 min significantly (P ≤ 0.05) reduced albumin fraction from 3.6% to 1.0%, a result which are 112 similar to that reported by Nugdallah and El Tinay (1997); Fiel (2003). 4.2.3. Prolamin fraction: As shown in Tables 3, 4 and 5, raw germinated, raw germinated cooked, autoclaved and roasted seeds were fractionated. The prolamin fraction significantly (P ≤ 0.05) decreased, they were 4.0%, 3.3%, 4.0%, 1.6%, respectively. Similarly for Buff raw and germinated seeds they were 4.5%, 2.7%, 2.8%, 3.9% and 1.4% respectively, a result which are similar to that reported by Giami et al. (1999); Khalifa and El Tinay (1999). Cooking significantly (P ≤ 0.05) increased prolamin fraction, they were 4.3%, 6.3%, 4.8%, 5.1%, and 5.0% for Ain Elgazal raw the first, second, third and fourth days respectively. Similarly for Buff they were 4.6%, 5.1%, 2.7%, 2.7% and 1.8% respectively. It showed significant (P ≤ 0.05) increase during the first day of germination but started to decrease significantly (P ≤ 0.05), reaching a minimum value after 96h of germination, a result which are similar to that reported by Nugdallah and El Tinay (1997). Autoclaving at 120ºC under 15 psi for 30 minutes and at 150ºC under 20 psi for 30 minutes for Ain Elgazal raw seeds significantly (P ≤ 0.05) increased prolamin fraction from 4.3% - 4.5% and 4.8% respectively. For Buff raw seeds autoclaving at 120ºC under 15 psi for 30 minutes and at 150ºC under 20 psi for 30 minutes, significantly (P ≤ 0.05) decreased prolamin fraction from 4.5% - 3.0% and 4.0% respectively. Roasting as well as cooking and autoclaving significantly (P ≤ 0.5) decreased prolamin. Roasting Ain Elgazal raw seeds at 90ºC/30 min and at 120ºC/60 min significantly (P ≤ 0.05) reduced prolamin fraction from 4.3% to 3.6%, and 4.2%. For Buff raw seeds significantly (P ≤ 0.05) reduced 113 prolamin from 4.5% to 4.0% but roasting at 120ºC/60min significantly (P ≤ 0.05) increased prolamin fraction from 4.5% to 6.0% respectively. A result which are similar to that reported by Nugdallah and El Tinay (1997). 4.2.4. G1-glutelin: As shown in Tables 3, 4 and 5, the G1- glutelin fraction for Ain Elgazal raw and germinated seeds significantly (P ≤ 0.05) decreased, they were 2.3%, 1.5%, 1.3%, 0.8%, and 1.7%, respectively. For Buff raw and germinated seeds they were 2.3%, 1.2%, 1.3%, 2.1% and 1.3% respectively, a result which are similar to that reported by El Khalifa et al., (1996). Cooking significantly (P ≤ 0.05) decreased G1glutelin fraction, they were 2.0%, 1.4%, 1.3%, 1.3%, and 2.6% for Ain Elgazal raw the first, second, third and fourth days respectively, but started to increase 96h of germinnat. For Buff raw and germinated seeds, it showed significant (P ≤ 0.05) decreased they were 2.7%, 1.2%, 1.1%, 1.6% and 1.0% respectively, a result which are similar to that reported by Nugdallah and El Tinay (1997). Autoclaving at 120ºC under 15 psi for 30 minutes and at 150ºC under 20 psi for 30 minutes for Ain Elgazal raw seeds significantly (P ≤ 0.05) decreased G1-glutelin fraction from 2.3% - 1.2% and 2.0% respectively. For Buff raw seeds autoclaving significantly (P ≤ 0.05) decreased G1-glutelin from 2.3% to 2.0% and 1.2% respectively. 114 Roasting as well as cooking and autoclaving significantly (P ≤ 0.5) decreased G1-glutelinfor Ain Elgazal raw seeds from 2.3% to 2.0% and 1.2% respectively. For Buff raw seeds G1-glutelin significantly (P ≤ 0.05) decreased from 2.3% to 2.2% and 2.0% respectively, a result which are similar to that reported by Nugdallah and El Tinay (1997). 4.2.5. G2-glutelin: As shown in Tables 3, 4 and 5, the G2- glutelin fraction for Ain Elgazal raw and germinated seeds significantly (P ≤ 0.05) decreased, they were 2.4%, 2.1%, 1.9%, 1.4% and 1.7%, respectively, a result which are similar to that reported by El Khalifa et al. (1996). For Buff raw and germinated seeds G2-glutelin showed a significantly (P ≤ 0.05) increased, they were 2.5%, 6.4%, 5.1%, 3.9% and 4.5% respectively, a result which are similar to that reported by El Khalifa et al., (1996). Cooking significantly (P ≤ 0.05) decreased G1-glutelin fraction, they were 3.4%, 4.4%, 4.4%, 4.0%, and 5.7% for Ain Elgazal raw the first, second, third and fourth days respectively. Similarly for Buff raw and germinated seeds, they were 5.1%, 7.5%, 7.5%, 7.9% and 8.8% respectively, a result which are similar to that reported by Nugdallah and El Tinay (1997); Fiel et al. (2003). Autoclaving at 120ºC under 15 psi for 30 minutes significantly (P ≤ 0.05) decrease G2-glutelin from 2.4% to 1.2% and 4.0% 115 autoclaving at 150ºC under 20 psi for 30 minutes significantly (P ≤ 0.05) increased from 2.5% to 3.0% and 3.2% respectively. Roasting Ain Elgazal raw seeds at 90ºC for 60 minutes significantly (P ≤ 0.05) decreased G2-glutelin fraction from 2.4% - 1.2% and roasting the seeds at 120ºC for 60 minutes significantly (P ≤ 0.05) increased G2glutelin from 2.4% to 3.2%. Similarly for Buff raw seeds roasting at 90ºC for 60 minutes and 120ºC for 60 minutes significantly (P ≤ 0.05) increased G2-glutelin from 2.5% to 2.8% and 3.0% respectively, a result which are similar to that reported by Nugdallah and El Tinay (1997); Fiel et al. (2003). 4.2.6. G3-glutelin: As shown in Tables 3, 4 and 5, the G3- glutelin fraction significantly (P ≤ 0.05) increased, they were 4.8%, 9.8%, 12.0%, 13.5% and 10.4%, for Ain Elgazal raw seeds, the first, second, third and fourth days respectively, a result which are similar to that reported by El Khalifa et al. (1996). For Buff raw and germinated seeds G2-glutelin showed a significantly (P ≤ 0.05) increased, they were 4.5%, 10.0%, 12.5%, 11.1% and 11.7% respectively, a result which are similar to that reported by El Khalifa et al. (1996). Cooking significantly (P ≤ 0.05) decreased G3-glutelin fraction, they were 63.8%, 61.0%, 62.0%, 116 61.6%, and 57.0% for Ain Elgazal raw the first, second, third and fourth days respectively. Similarly for Buff raw and germinated seeds, they were 69.3%, 59.0%, 62.3%, 60.1% and 62.1% respectively, a result which are similar to that reported by Nugdallah and El Tinay (1997); Fiel et al. (2003). Autoclaving at 120ºC under 15 psi for 30 minutes and at 150ºC under 20 psi for 30 minutes significantly (P ≤ 0.05) increased G3-glutelin for Ain Elgazal raw seeds from 4.8% to 59.5% and 60.2% respectively. For Buff seeds G3-glutelin significantly (P ≤ 0.05) increased from 4.5% to 55.0% and 50.8% respectively. Roasting Ain Elgazal raw seeds at 90ºC for 60 minutes and at 120ºC for 60 minutes significantly (P ≤ 0.05) increased G3-glutelin from 4.8% - 6.0% and 48.0% respectively, for Buff raw seeds G3glutelin showed changeless at 90ºC for 60 minutes but at 120ºC for 60 minutes showed significantly (P ≤ 0.05) increased they were 4.5% and 42.0% respectively, a result which are similar to that reported by Nugdallah and El Tinay (1997); Fiel et al. (2003). 4.2.7. Residue fraction: As shown in Tables 3, 4 and 5, residue fraction showed significant (P ≤ 0.05) increased, they were 1.2%, 2.5%, 2.1%, 9.2% and 6.0%, for Ain Elgazal raw seeds, the first, second, third and fourth 117 days respectively, similarly for Buff raw and germinated seeds, they were 1.2%, 1.7%, 1.9%, 3.6% and 6.1% respectively, a result which are similar to that reported by El Khalifa and El Tinay (1999). Cooking significantly (P ≤ 0.05) decreased G3-glutelin fraction, they were 5.5%, 8.0%, 8.8%, 9.6%, and 10.2% for Ain Elgazal raw the first, second, third and fourth days respectively. Similarly for Buff raw and germinated seeds, they were 5.5%, 6.6%, 6.5%, 6.1% and 6.5% respectively, a result which are similar to that reported by Nugdallah and El Tinay (1997); Fiel et al. (2003). Autoclaving at 120ºC under 15 psi for 30 minutes and at 150ºC under 20 psi for 30 minutes significantly (P ≤ 0.05) increased fractions for Ain Elgazal raw seeds from 1.2% to 8.0% and 6.0% respectively. For Buff seeds residue fractions significantly (P ≤ 0.05) increased from 1.2% to 6.0% and 7.0% respectively. Roasting Ain Elgazal raw seeds at 90ºC for 60 minutes and at 120ºC for 60 minutes significantly (P ≤ 0.05) increased residue fraction from 1.2% - 3.0% and 6.0% respectively. For Buff raw seeds residue fraction showed significant (P ≤ 0.05) increased at 90ºC for 60 minutes but at 120ºC for 60 minutes from 1.2% to 1.4% and 6.0% respectively, a result which are similar to that reported by Nugdallah and El Tinay (1997); Fiel et al. (2003). 118 Table 3. Effect of germination on protein fractions of cowpea cultivars Cultivar Ain-Elgazal Buff G3-glutelin Residue % % 2.4±0.00a 4.8±0.07e 1.2±0.00d 106.5 1.5±0.03b 2.1±0.01b 9.8±0.01d 2.5±0.00c 103.6 4.0±0.00b 1.3±0.01c 1.9±0.01c 12.0±0.02b 2.1±0.01c 100.0 1.9±0.03c 1.6±0.00d 0.8±0.00d 1.4±0.02e 13.5±0.1a 9.2±0.01a 99.8 70.2±0.61e 2.5±0.01b 4.6±0.1a 1.7±0.00b 1.7±0.00d 10.4±0.04c 6.0±0.00b 97.1 Raw seeds 89.8±0.07a 3.6±0.01b 4.5±0.12d 2.3±0.03a 2.5±0.00e 4.5±0.04e 1.2±0.00d 108.4 24 81.3±0.13b 1.5±0.00c 2.7±0.21d 1.2±0.00d 6.4±0.04a 10.0±0.03d 1.7±0.00c 104.8 48 78.3±0.04c 2.2±0.00c 2.8±0.00c 1.3±0.07c 5.1±0.03b 12.5±0.04a 1.9±0.01c 104.1 72 75.6±0.02d 2.2±0.00b 3.9±0.00b 2.1±0.00b 3.9±0.04d 11.1±0.12c 3.6±0.01b 102.4 96 72.7±0.03e 1.2±0.01d 1.4±0.00e 1.3±0.13c 4.5±0.00c 11.7±0.00b 6.1±0.01a 98.9 Germination Globulin Albumin Prolamin time, h % % % Raw seeds 87.5±0.02a 4.0±0.15a 24 80.6±0.91b 48 G1-glutelin % G2-glutelin % 4.3±0.25a 2.3±0.09a 3.8±0.06a 3.3±0.03c 76.4±0.03c 2.3±0.03b 72 71.4±0.04d 96 Total proteins Values are means (±SD), Means not sharing a common superscript letter in a column for the same cultivar are significantly different at (P < 0.05) as assessed by Duncan's Multiple Range Test 119 Table 4. Effect of cooking on protein fractions of germinated cowpea cultivars Cultivar Ain-Elgazal Buff G3-glutelin Residue Total proteins % % % 3.4±0.04d 63.8±0.04a 5.5±0.01e 97.2 1.4±0.00c 4.4±0.04b 61.0±0.04b 8.0±0.00d 102.9 4.8±0.16c 1.3±0.00c 4.4±0.04b 62.0±0.07b 8.8±0.04c 102.5 1.8±0.00c 5.1±0.00bc 1.3±0.09c 4.0±0.03c 61.6±0.04b 9.6±0.00b 101.4 17.8±0.04d 1.4±0.01d 5.0±0.16c 2.6±0.00a 5.7±0.41a 57.0±0.00c 10.2±0.01a 99.7 0 15.7±0.02e 2.2±0.00a 4.5±0.00b 2.7±0.02a 5.1±0.07d 69.3±0.04a 5.5±0.01d 105.1 24 19.9±0.04d 1.6±0.02c 5.1±0.02ab 1.2±0.00c 7.5±0.04c 59.0±0.04cd 6.6±0.01a 100.9 48 22.0±0.06c 1.6±0.01c 2.7±0.03c 1.1±0.09c 7.5±0.00c 62.3±0.04b 6.5±0.01b 103.7 72 22.6±0.04b 2.1±0.01ab 2.7±0.00c 1.6±0.00b 7.9±0.02bc 60.1±0.04c 6.1±0.01c 103.1 96 23.0±0.00a 1.3±0.01d 1.8±0.07d 1.0±0.00d 8.8±0.04 62.1±0.03b 6.5±0.01b 104.7 Germination Globulin Albumin Prolamin time, h % % % 0 16.4±0.00e 1.9±0.03c 24 19.0±0.04a 48 G1-glutelin % G2-glutelin % 4.3±0.04d 2.0±0.00b 2.8±0.04b 6.3±0.00a 18.1±0.04b 3.1±0.01a 72 18.0±0.04bc 96 Values are means (±SD). Means not sharing a common superscript letter in a column for the same cultivar are significantly different at (P < 0.05) as assessed by Duncan's Multiple Range Test. 120 Table 5. Effect of autoclaving and roasting on protein fractions of cowpea cultivars Treatment Globulin % Albumin % Prolamin % G1-glutelin % G2-glutelin % G3-glutelin % Residue % Total P. recovered % 87.5±0.02a 29.2±0.4d 27.6±0.2e 4.0±0.015a 1.2±0.1b 1.2±0.1b 4.3±0.25c 4.5±0.1b 4.8±0.1a 2.3±0.09a 1.2±0.0c 2.0±0.0b 2.4±0.0c 1.2±0.4d 4.0±0.0a 4.8±0.07cd 59.5±0.1a 60.2±0.1a 1.2±0.00d 8.0±0.3a 6.0±0.1b 106.5 104.8 105.8 85.0±0.3b 41.0±0.4c 4.0±0.2a 1.2±0.3b 3.6±0.2e 4.2±0.4d 2.0±0.1b 1.2±0.6c 1.2±0.0d 3.2±1.3b 6.0±0.2c 48.0±0.3b 3.0±0.0c 6.0±0.4b 104.8 104.8 89.8±0.07a 32±0.1e 36±0.2d 3.6±0.01a 1.0±0.01b 0.9±0.02c 4.5±0.12b 3.0±0.01d 4.0±0.01c 2.3±0.03a 2.0±0.1c 1.2±0.09d 2.5±0.00d 3.0±0.03b 3.2±0.02a 4.5±0.04d 55.0±0.02a 50.8±0.1b 1.2±0.00d 6.0±0.01b 7.0±0.1a 108.4 102.0 103.1 88.0±0.3b 45.5±1.3c 3.6±0.2a 1.0±0.01b 4.0±0.02c 6.0±0.1a 2.2±0.01b 2.0±0.1c 2.8±0.02c 3.0±0.00b 4.5±0.01d 42.0±0.06c 1.4±0.2c 6.0±0.00b 106.5 105.5 Ain Elgazal Raw seeds Autoclave 15 lb/30 min 20 lb/30 min Roasting 90ºC/60 min. 120ºC/60 min. Buff Raw seeds Autoclave 15 lb/30 min 20 lb/30 min Roasting 90ºC/60 min. 120ºC/60 min. Values are means (±SD) Means not sharing a common superscript letter in a column for the same cultivar are significantly different at (P < 0.05) as assessed by Duncan's Multiple Range Test. 121 4.3. anti-nutrient: 4.3.1. Tannins: As shown in Tables 6 germination significantly (P ≤ 0.05) reduced Tannins from 0.48 g/100g of raw seeds to 0.36g/100g, 0.24g/100g and 0.20g/100g for Ain Elgazal germinated seeds, the first, second, third and fourth days respectively. Similarly for Buff raw seeds, Tannins was 0.50g/100g which significantly (P ≤ 0.05) reduced by germination to 0.42g/100g, 0.36g/100g, 0.30g/100g and 0.22g/100g for the first, second, third and fourth days respectively. Cooking significantly (P ≤ 0.05) reduced Tannins for Ain Elgazal raw and germinated seeds, they were 0.26g/100g, 0.18g/100g, 0.15g/100g 0.12g/100g and 0.10g/100g respectively. Similarly for Buff, cooking significantly (P ≤ 0.05) reduced tannins, they were 0.30g/100g, 0.22g/100g, 0.20g/100g, 0.18g/100g and 0.11g/100g respectively. Autoclaving significantly (P ≤ 0.05) decreased Tannins for Ain Elgazal raw seeds 0.48g/100g, 0.38g/100g, Similarly for Buff raw seedsautoclaving at 150ºC under 20 psi for 30 minutes significantly (P ≤ 0.05) decreased Tannins from 0.50g/100g to 0.40g/100g. Roasting Ain Elgazal raw seeds significantly (P ≤ 0.05) reduced Tannins from 0.48g/100g to 0.36g/100g. Similarly for Buff raw seeds roasting significantly (P ≤ 0.05) decreased Tannins from 0.50g/100g to 122 0.42g/100g, a result which are similar to that reported by Anthony et al. (1984); Ene-Obong (1995); Ningsanond et al. (1989); Ekpenyong (1985); Bakr et al. (1991); Chen et al. (1977); Plahar (1997); Vaishale et al. (1998); Jibaja et al., (1988); Laurena et al. (1987); Mugula et al., (1999); Vijayakumari et al. (1998). Results obtained were different from those reported by Oke et al., (1996) and Beem Van et al., (1992). 4.3.2. Trypsin inhibitor activity (TIA): As shown in Tables 6 germination significantly (P ≤ 0.05) reduced Trypsin inhibitor activity for Ain Elgazal raw and germinated seeds, they were 22.0 TUI/mg protein, 11.8 TUI/mg protein, 10.6 TUI/mg protein, 8.0 TUI/mg protein and 8.0 TUI/mg protein, respectively. Similarly for Buff raw and germinated seeds TIA were 25.0 TUI/mg protein, 12.5 TUI/mg protein, 10.0 TUI/mg protein, 9.6 TUI/mg protein and 9.0 TUI/mg protein, respectively. Cooking autoclaving and raosting eliminates TIA, a result which are similar to that reported by Anthony et al. (1984); Carnovale et al., (1992) Beltran et al., (1983); Iss et al., (1996); Mulimani et al., (1994). 4.3.3. Phytic acid: As shown in Tables 6 germination significantly (P ≤ 0.05) reduced Phytic acid from 310.3 (mg/100g dry weight) of raw seeds to 123 286.1 mg/100g, 248.9 mg/100g, 201.7 mg/100g, and 139.8 mg/100g, for Ain Elgazal germinated seeds, the first, second, third and fourth days respectively. Similarly for Buff raw seeds, phytic acid were 376.3 mg/100mg which significantly (P ≤ 0.05) reduced by germination to 346.2 mg/100g,, 301.0 mg/100g, 225.7 mg/100g, and 180.7 mg/100g, for the first, second, third and fourth days respectively. Cooking significantly (P ≤ 0.05) they decreased Phytic acid for Ain Elgazal raw and germinated seeds, they were 290.3 mg/100g, 268.9 mg/100g, 229.0 mg/100g, 181.5 mg/100g, and 128.0 mg/100g, respectively. Similarly for Buff cooking significantly (P ≤ 0.05) decreased Phytic acid they were, 353.0 mg/100g, 311.6 mg/100g, 270.9 mg/100g, 205.4 mg/100g, and 162.6 mg/100g, respectively. Autoclaving significantly (P ≤ 0.05) decreased Phytic acid for Ain Elgazal raw seeds from 310.3 mg/100g, to 300.0 mg/100g,. similrly for Buff raw seeds autoclaving at 150ºC under 20 psi for 30 minutes significantly (P ≤ 0.05) decreased Phytic acid from 376.3 mg/100g, to 350.0 mg/100g. Roasting at 120ºC for 60 min significantly (P ≤ 0.05) decreased Phytic acid for Ain Elgazal raw seeds from 310.3 mg/100g, to 300.1 mg/100g. Similarly for Buff raw seeds roasting significantly (P ≤ 0.05) decreased Phytic acid from 376.3 mg/100g, to 352.0 mg/100g, a result which are similar to that 124 reported by Anthony et al. (1984); Alonso et al., (1998); Chitra (1996); Anshu et al., (1995); Sanni et al., (1999); Perumal-Siddhuraju et al., (1996); Ene – obong et al., (1996); Yadav et al., (1994); Ogum et al., (1989); Uzogara et al., (1997) Ologhobo et al., (1984); Chem et al., (1997); Vanderstop (1981); Baralker et al., (1985); Alxiny et al., (1991); Ene – Obong (1995); Vijayakumari et al., (1998). Bakr et al., (1991), results obtained were different from those reportedb by Oke et al., (1996). 4.4. In-vitro protein digestibilty (IVPD): 4.4.1. Effect of processing on IVPD: Improvement of protein digestibility after processing could be attributable to the reduction or elimination of different antinutrients. Phytic acid, as well as condenced tannins and polyphenols which are known to interact with protein to form complexes. This interaction increases the degree of cross-linking, decreasing the solubility of proteins and making protein complexes less succeptable to proteolyic attack than the same protein alone, Alonso et al., (2000). As shown in table 6 germination significantly (P ≤ 0.05) improved in-vitro protein digestibility (IVPD), they were 73.4%, 75.3%, 77.9%, 80.4%, and 84.4% for Ain Elgazal raw seeds, the first, second, third, and fourth days respectively. Similarly for Buff, they were 74.2%, 75.7%, 79.3%, 125 82.4% and 83.6% respectively. Cooking germinated seeds showed significantly (P ≤ 0.05) further increase of in-vitro protein digestibility, they were 86.2%, 87.2%, 87.5%, 88.8%, and 88.5% for Ain Elgazl raw seeds, the first, second, third, and fourth days respectively. Similarly for Buff, they were 85.4%, 86.3%, 86.6%, 87.9% and 88.3% for raw seeds, the first, second, third, and fourth days respectively. Autoclaving at 150ºC under 20 psi for 30 min significantly (P ≤ 0.05) increased in vitro protein digestibilty for Ain Elgazal raw seeds from 73.4%, to 86.0% and significantly (P ≤ 0.05) increased by roasting at 120ºC for 60 min from 73.4% to 84.0%. Similrly for Buff raw seeds IVPD significantly (P ≤ 0.05) increased by autoclaving from 74.2% to 87.0% and by roasting from 74.2% to 84.0% respectively, a result which are similar to that reported by Anthony et al. (1988); Nnanno and Phillips (1989); Jibaja et al., (1988). Laurena et al., (1987); Mugula et al., (1996); Chitra (1996); PerumalSiddhuraju et al., (1996); Ene-obong et al., (1996); Vijayakumari et al., (1998); Anshu-Sharma et al., (1995) Yadav et al., (1994); Vanderstop (1981); Baralker et al., (1985). 126 Table 6. Phytic acid (mg/100g dry weight), Tannins (g/100g dry weight), trypsin inhibitor activity (TUI/mg protein) and IVPD of raw and processed cowpea cultivar Treatment Ain Elgazal Raw seeds Germinated 24 h. Germinated 48 h. Germinated 72 h. Germinated 96 h. Autoclaved 20 lb/30 min. Roasting 120ºC/60 min. Buff Raw seeds Germinated 24 h. Germinated 48 h. Germinated 72 h. Germinated 96 h. Autoclaved 20 lb/30 min. Roasting 120ºC/60 min. Tannic acid Uncooked Cooked TIA Uncooked Cooked Phytic acid Uncooked Cooked IVPD Uncooked Cooked 0.48±0.2 0.36±0.02 0.30±0.2 0.24±0.1 0.20±0.02 - 0.26±0.01 0.18±0.5 0.15±0.3 0.12±0.3 0.10±0.01 0.38±0.03 0.36±0.1 22.0±0.5 11.8±0.01 10.6±0.01 8.0±0.1 8.0±0.1 - Nil - 310.3±1.3a 286.1±0.6b 248.9±1.4c 201.7±1.3d 139.8±1.4e - 290.3±1.4a 268.9±1.3b 229.0±1.9c 181.5±1.8d 128.6±0.4e 300.0±0.1 301.0±0.3 73.4±1.33e 75.3±1.44d 77.9±0.4c 80.4±0.4b 84.4±0.3a - 86.2±0.6bc 87.2±0.5b 87.5±0.5b 88.8±0.4a 88.5±0.2a 86.0±c 84.0±d 0.50±0.01 0.42±0.01 0.36±0.01 0.30±0.02 0.22±0.01 - 0.30±0.02 0.22±0.01 0.20±0.02 0.18±0.01 0.11±0.01 40.0±0.1 42.0±0.2 25.0±0.1 12.5±0.1 10.0±0.01 9.6±0.01 9.0±0.2 - Nil - 376.3±0.4a 346.2±01b 301.0±0.2c 125.7±0.1d 180.7±0.3e - 353.7±0.6a 311.6±0.1b 270.9±02c 205.4±0.1d 162.6±0.5e 350±0.1 352±0.1 742±0.5e 75.7±0.6d 79.3±1.86c 82.4±0.4b 83.6±0.3a - 85.4±0.6cd 86.3±0.5a 86.6±0.4ab 87.9±0.4 88.3±0.4c 87.0±0.1b 84.0±0.1e Values are means (±SD). Means not sharing a common superscript letter in a column for the same cultivar are significantly different at (P < 0.05) as assessed by Duncan's Multiple Range Test. 127 4.4.2. Effect of substract on in-vitro protein digestibilty: Effect of substrate on in-vitro protein digestibilty was carried out using mixed proteins of raw with treated sample in various proteins. Increasing the protein of treated in the comparison to raw material as shown in Table 7 significantly (P ≤ 0.05) increased in-vitro protein digestibilty for both cultivars. The rate of increase for the proportional for amount of treated samples. This could attributed to heat inactivation to anti-nutritional factors which are expected to be of higher concentration in raw material. Ain Elgazal raw proportions were 0.384g, 0.336g, 0.288g, 0.00g, 0.048g, 0.096g and 0.192g which were mixed with roasted samples, the proportions of roasted samples were 0.00g, 0.048g, 0.096g, 0.384g, 0.0336g, 0.0282g, and 0.912g the sequences were A0, A1, A2, A3, A4, A5 and A6 respectively. Raw plus autoclaved samples sequences were A7, A8, A9, A10, A11, A12 and A13 respectively. Roasting was at 90ºC and at 120ºC for 30, 45 and 60 min, autoclaving was at 115.5ºC under 10 psi and at 120ºC under 15 psi for 15, 30, and 45 min. In-vitro protein digestibilty for the above sequence were 73.4%, for A0, they were 73.4%, 73.4%, 73.5%, 73.5%, 73.5% 73.6% and 73.8% respectively for A1, they were 73.5%, 73.6%, 73.6%, 73.9%, 73.8% and 74.0% respectively for A2, they 128 were 80.0%, 80.1%, 80.2%, 83.9%, 84.0%, and 84.2% respectively for A3, they were 79.5%, 79.6%, 79.8%, 83.0%, 83.2%, and 83.1% respectively for A4, they were 75.0%, 75.3%, 75.2%, 75.4%, 75.5%, and 76.0% respectively for A5, they were 73.8%, 73.8%, 74.0%, 74.8%, 75.1%, and 75.0% respectively for A6, they were 73.4% for A7, they were 73.8%, 74.0%, 74.2%, 74.1%, 74.0%, and 74.6% respectively for A8, they were 74.9%, 74.8%, 74.8%, 74.2%, 75.0%, and 75.8% respectively for A9, they were 82.0%, 83.0%, 84.0%, 85.0%, 85.5% and 86.2% respectively for A10, they were 81.0%, 81.8%, 82.8%, 83.5%, 83.3%, and 84.0% respectively for A11, they were 75.5%, 75.6%, 75.7%, 75.7%, 76.2%, and 76.3% respectively for A12, they were 74.8%, 74.8%, 74.9%, 74.8%, 75.2%, and 75.0% respectively for A13, Similarly for Buff cultivar the raw proportion were 0.40g, 0.35g, 0.30g, 0.00g, 0.05g, 0.10 and 0.20g which were mixed with roasted samples, the proportion of roasted sample were 0.00g, 0.05g, 0.10g, 0.40g, 0.35g, 0.30g and 0.20g in sequences which were B0, B1, B2, B3, B4, B5 and B6 respectively. Raw plus autoclaved samples sequences were B7, B8, B9, B10, B11, B12 and B13 respectively. In-vitro protein digestibilty for the above sequence were 74.2%, for B0, they were 74.5%, 74.6%, 74.6%, 75.5%, 75.6% and 75.0% respectively for 129 B1, they were 75.0%, 75.1%, 75.0%, 76.0%, 76.0 and 75.3% respectively for B2, they were 79.7%, 79.9%, 79.8%, 83.8%, 84.0%, and 84.8% respectively for B3, they were 78.7%, 78.8%, 78.8%, 82.3%, 82.9%, and 83.0% respectively for B4, they were 76.0%, 76.0%, 76.0%, 76.8%, 76.8%, and 77.2% respectively for B5, they were 74.6%, 74.5%, 74.5%, 75.1%, 75.2%, and 75.0% respectively for B6, they were 74.2% for B7, they were 74.5%, 74.8%, 74.9%, 75.1%, 75.0%, and 75.0% respectively for B8, they were 75.7%, 75.6%, 75.4%, 75.8%, 76.0% and 76.4% respectively for B9, they were 80.2%, 84.8%, 84.8%, 85.0%, 85.0%, and 85.4% respectively for B10, they were 79.0%, 82.0%, 83.0%, 83.0%, 84.0%, and 84.0% respectively for B11, they were 76.0%, 76.5%, 76.8%, 77.0%, 77.1%, and 77.3% respectively for B12, they were 75.0%, 75.0%, 75.0%, 75.5%, 75.6%, and 76.0% respectively for B13, a result which are similar to that reported by Plahar (1997); Vaishale et al., (1998); Jibaja et al., (1988); Laurena et al., (1987); Yadav et al., (1994); Savage et al., (1993); Mugula et al., (1999). 130 Table 7. Effect of Substrate on IVPD: In ivtro protein digestibility of mixed sample weight Roasted sample Autoclaved sample 30min 45min 60min 30min 45min 60min Sequences Buff Ain Elgazal Sequences Sample 15min 30min 45min 15min 30min 45min A0 0.384±0.0 73.4±1.3cd 73.4±1.3cd 73.4±1.3cd 73.4±1.3de 73.4±1.3de 73.4±1.3d A7 73.4±1.3d 73.4±1.3de 73.4±1.3de 73.4±1.3de 73.4±1.3de 73.4±1.3de A1 0.336±0.048 73.4±0.3cd 73.4±0.6cd 73.5±0.4cd 73.5±0.4de 73.6±0.4cde 73.8±0.4cd A8 73.8±0.6d 74.0±0.6d 74.2±0.9de 74.1±0.3d 74.0±0.4d 74.6±0.06de A2 0.288±0.096 73.5±0.5c 73.6±0.5c 73.6±0.4c 73.9±0.3de 73.8±0.6cd 74.0±0.5cd A9 74.9±1.3cd 74.8±0.5cd 74.8±0.4cd 74.2±0.6d 75.0±0.3cd 75.8±0.6cd A3 0.0±0.384 80.0±0.3a 80.1±0.5a 80.2±0.6a 83.9±0.6a 84.0±0.6a 84.2±0.4a A10 82.0±0.6a 83.0±0.5a 84.0±0.6a 85.0±0.4a 85.5±0.6a 86.2±0.4a A4 0.048±0.336 79.5±0.4a 79.6±0.4a 79.8±0.5a 83.0±1.3ab 83.2±1.3ab 83.1±0.3ab A11 81.0±0.5b 81.8±0.5ab 82.8±0.4ab 83.5±0.4b 83.3±0.5b 84.0±0.5b A5 0.096±0.282 75.0±1.3b 75.3±0.5b 75.2±0.4b 75.4±0.6c 75.5±0.5c 76.0±0.4c A12 75.5±0.5c 75.6±0.5c 75.7±0.6c 75.7±0.5c 76.2±0.4c 76.3±0.4c A6 0.192±0.192 73.8±0.4 73.8±0.3c 74.0±1.3bc 74.8±0.4d 75.1±0.6cd 75.0±1.3cd A13 74.8±0.4cd 74.8±0.4cd 74.9±0.6cd 74.8±0.4d 75.2±0.5cd 75.0±0.4cd B0 0.4±0.0 74.2±0.4d 74.2±0.4de 74.2±0.4e 74.2±0.4e 74.2±0.4ed 74.2±0.4cd B7 74.2±0.4de 74.2±0.4de 74.2±0.4e 74.2±0.4e 74.2±0.4e 74.2±0.2cd B1 0.3±0.1 74.5±0.4d 74.6±0.4d 74.6±0.6d 75.5±0.5d 75.6±0.4cd 75.0±0.4d B8 74.5±0.4d 74.8±0.3de 74.9±0.3de 75.1±0.3de 75.0±0.5de 75.0±0.4bc B2 0.35±0.05 75.0±0.4d 75.1±0.4d 75.0±0.4cd 76.0±0.4cd 76.0±0.3d 75.3±0.3d B9 75.7±0.4c 75.6±0.3d 75.4±0.9d 75.8±1.3d 76.0±0.0d 76.4±0.4b B3 0.0±0.4 79.7±0.3a 79.9±0.4a 79.8±0.5a 83.8±0.6a 84.0±0.5a 84.8±0.3a B10 80.2±0.5a 84.8±0.4a 84.8±0.6a 85.0±0.4a 85.0±0.4a 85.4±0.5a B4 0.05±0.35 78.7±0.4b 78.8±0.3b 78.8±0.3b 82.3±0.5b 82.9±1.3b 83.0±0.3b B11 79.0±0.6b 82.0±0.4b 83.0±0.6b 83.0±0.4b 84.0±0.5ab 84.0±0.4a B5 0.10±0.3 76.0±0.4c 76.0±0.4c 76.0±0.4c 76.8±0.4c 76.8±0.6c 77.2±0.6c B12 76.0±0.5c 76.5±0.3c 76.8±0.5c 77.0±0.4c 77.1±0.6c 77.3±1.3b B6 0.2±0.2 74.6±1.3d 74.5±1.3de 74.5±1.3de 75.1±0.4de 75.2±0.6cd 75.0±0.5d B13 75.0±0.4cd 75.0±0.6d 75.0±0.4d 75.5±0.3d 75.6±1.3d 76.0±0.5b Raw (ing) Treated (ing) 90ºC 120ºC 115..5ºC 10psi 120.5ºC 15spi Values are means (±SD). Means not sharing a common superscript letter in a column for the same cultivar are significantly different at (P < 0.05) as assessed by Duncan's Multiple – Range Test. 131 CHAPTER FIVE SUMMARY, CONCLUSION AND RECOMMENDATION 132 CHAPTER FIVE SUMMARY AND CONCLUSIONS Two cowpea cultivars were analyzed for proximate composition. Treatment included cooking, roasting, autoclaving, germinating and cooking plus germinated seeds. The proteins of control and treated sample were fractionated according to solubility behaviour. The results indicated that the albumin plus globulin fractions decreased significantly (P ≤ 0.05) for all treated sample, however, there was high retention of globulin fraction in autoclaved sample compared to ordinary cooked ones. For all treated samples decrease in the albumin and globlin fraction was a companied by significant (P ≤ 0.05) increase in G3-glutelin fraction, the IVPD on processed cowpeas was significantly (P ≤ 0.05) improved. This was more pronounced for cooked germinated and autoclaved samples. This was associated with significant (P ≤ 0.05) reduction of the anti-nutritional factor for all treatment. 133 RECOMMENDATION: The acute shortage of meat and animal proteins in developing countries has made it necessary for consumer in these countries to rely heavily on proteins from legumes spically cowpeas. These beans, which are rich in protiens, B-vitamins and dietary fiber, are important in the diet of people in developing countries where malnutrition is a perennial problem. Attention should be directed to other areas of cowpea processing and utilization. More research should be conducted into ways of reducing anti-nutritional factors, improving IVPD and improving cowpea protiens. Germinated, roasted and autocalved cowpeas possess adequate nutritional qualities as protein supplements in cereal-based weaning foods. 134 REFERENCES 135 REFERENCES A.O.A.C.(1990). 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