Fortification of Wheat Flour with Decorticated Pigeon Pea Flour and Protein Isolate for Bakery Products

Transcription

1 Fortification of Wheat Flour with Decorticated Pigeon Pea Flour and Protein Isolate for Bakery Products By HAYAT ABD ELRAHMAN HASSAN ABD ELATIEF B.Sc Food Technology University of Tanta, Egypt, (1982) M.Sc. Agric. University of Khartoum (1994) A Thesis Submitted to the University of Khartoum in fulfillment of the Requirements for the Degree of Doctor of Philosophy in Agric. Supervisor: Prof. Abd Elmoneim Ibrahim Mustafa Co-supervisor: Prof. Abd Elhalim Rahma Ahmed University of Khartoum Faculty of Agriculture Department of Food Science and Technology April 2006

2 بسم االله الرحمن الرحيم قال تعالى في محكم تنزيله " ولقد ا تينا داو ود وسليمان علما وقالا الحمد الله الذي فضلنا على كثير من عباده المو منين " صدق االله العظيم سورة النمل الا ية ( 15) و قال رسول االله صلى االله عليه وسلم ) من سلك طريقا يلتمس فيه علما سهل االله له طريقا ا لى الجنة ( ب

3 DEDICATION To: My Late Husband, Azhari Makki; Who is still alive, My parents, sisters and brothers, My son Mohammed Al Makki, And daughters; Fatima Alzhraa and Israa, My dearest sister s son Azhari, To all of them who have been eagerly waiting to see the fruitful end to this effort With love Hayat ج

4 ACKNOWLEDGEMENTS With all due humbleness and gratitude I render ultimate thanks and special praise to Allah (Almighty) who gave me health, power and patience to accomplish and conduct this research. My deepest thanks and appreciations are genuinely expressed to my supervisor Prof. Abd Elmoneim Ibrahim Mustafa for his continuous assistance, valuable advices, patience and encouragement through out the course of this study. Special thanks and sincere appreciations are due to my co-supervisor Prof. Abd Elhalim Rahma Ahmed for his interest, guidance, considerable effort, encouragement and unlimited help and advices. Sincere appreciations and gratitude are extended to Prof. Azhari A. Hamada, Agricultural Research Corporation, Ministry of Science and Technology, for sponsoring I remain indebted to the SSMO with respect to Dr. Abd Elgadir Mohammed Abd Elgadir for providing a chance in their laboratories to conduct and complete this work. Special thanks are due to Prof. Abdel Raheem Al Hussien, and Dr. Denna, Central Lab, Ministry of Science and Technology in Soba for the assistance to conduct the amino acids analysis. I would like to express my sincere appreciation to my colleagues in Food Research Centre (FRC), especially the Grain Technology Department. My deepest thanks to Mr. Babiker Ibrahim and Miss. Samah Awad for their continuous help. Thanks are also extended to the staff of the Dept. of Food Science and Technology, Faculty of Agriculture, University of Khartoum My thanks are also offered to Mr. Salah Mohammed Osman for carrying out the statistical analysis and typing and printing of the manuscript. God bless all those who helped me and placed their valuable time and knowledge during the course of the study. د

5 CONTENTS Page No. Dedication Acknowledgements List of Plates and Flow Charts List of Figures List of Tables Abstract Arabic Abstract iii iv xi xii xvii xx xxiii CHAPTER I: INTRODUCTION 1 CHAPTER II: LITERATURE REVIEW 2.1 Wheat and wheat classification The role of wheat in human nutrition Protein quality and amino acids composition of wheat Quality factors for the farmer Quality factors for the miller Quality factors for the baker Quality factors for the consumer Rheological properties of wheat flour and composite flour doughs Nature, origin and production of pigeon pea The role of pigeon pea in human nutrition Protein quality and amino acids composition of pigeon pea The anti-nutritional factors of pigeon pea Protease inhibitors Amylase inhibitors Phytolectins Polyphenoles Oligosaccharides Pigeon pea processing Effect of pigeon pea processing on its nutritive value 19 ه

6 و Page No Dehulling/decortication of pigeon pea Cooking Germination Fermentation Medicinal uses of pigeon pea Functional properties of pigeon pea seed protein Supplementation value of pigeon pea in cereal-based products Bread Bread ingredients Bread making process Mixing Dividing and rounding Fermentation Baking Cooling Biscuit Biscuit ingredients Classification of biscuit Biscuit making process Mixing Shaping and baking 34 CHAPTER III: MATERIALS AND METHODS 3.1 Materials Food samples Chemicals and reagents Methods Preparation of decorticated pigeon pea flour Preparation of pigeon pea protein isolate Preparation of wheat flour and composite flour blends Analytical methods 41

7 Page No Moisture content Ash content Crude protein Fat content Carbohydrates Minerals content Anti- nutritional factors of pigeon pea Tannins content Phytic acid content Rheological characteristics of dough Falling number test Gluten quality and quantity test Farinograph characteristics Extensograph characteristics Fermentograph characteristics Functional properties of wheat flour and composite flour blends Water retention capacity Bulk density Fat absorption capacity Processing of bread samples Evaluation of bread quality Bread volume Bread weight Bread specific volume Sensory evaluation of loaf bread Processing of biscuit samples Evaluation of biscuit quality 59 ز

8 Page No Biscuit weight Biscuit spread ratio Sensory evaluation of biscuits Protein and energy evaluation methods Amino acid profile Chemical score of essential amino acids Caloric value Statistical analysis of data 62 CHAPTER IV: RESULTS AND DISCUSSION 4.1 Proximate composition and mineral content of the raw materials Proximate composition of wheat flour Proximate composition of pigeon pea flour Mineral matter contents Effect of pigeon pea seeds decortication on the anti-nutritional factors The effect on tannins content The effect on phytic acid Falling number of wheat flours as affected by inclusion of DPPF and PPPI The effect on bread wheat flour The effect on biscuit wheat flour Gluten quality and quantity of wheat flours as affected by inclusion of DPPF and PPPI The effect on bread wheat flour The effect on biscuit wheat flour Farinogram characteristics of doughs 77 ح

9 Page No Farinograms of doughs prepared from bread wheat flour and composite flour blends Farinograms of doughs prepared from biscuit wheat flour and composite flour blends Extensograms characteristics of doughs prepared from Bread and biscuit wheat flours and composite flour blends Fermentograms feature of doughs prepared from Bread and biscuit wheat flours and composite flour blends Functional properties of wheat flours as affect by inclusion of DPPF and PPPI The effect on bread wheat flour The effect on biscuit wheat flour Physical and chemical characteristics of wheat loaf bread containing decorticated pigeon pea flour and pigeon pea protein isolate Loaf bread specific volume Chemical composition and energy value Amino acids composition Amino acids chemical score Mineral matter content Sensory evaluations of wheat loaf bread containing different levels of DPPF and PPPI Physical and chemical characteristics of wheat flour biscuit containing DPPF and PPPI Biscuit spread ratio Chemical composition and energy value Amino acids composition Amino acids chemical score Mineral matter content 157 ط

10 Page No Sensory evaluation of wheat biscuit containing decorticated pigeon pea flour and pigeon pea protein isolate 159 CHAPTER V: CONCLSUIONS AND RECOMMENDATIONS 5.1 Conclusions Recommendations 163 REFERANCES 167 APPENDICES 193 ي

11 LIST OF PLATES AND FLOW CHARTS Plate Page No. 2: Whole grains of pigeon pea 38 4: Decorticated pigeon pea (Dicotyledons or splits) 38 6: Decorticated pigeon pea flour 38 8: Pigeon pea protein isolate 38 10: Loaf bread prepared from wheat flour and decorticated pigeon pea flour blends : Loaf bread prepared from wheat flour and pigeon pea protein isolate blends : Biscuit prepared from wheat flour and decorticated pigeon pea flour blends : Biscuit prepared from wheat flour and pigeon pea protein isolate blends 143 Flow Charts 1: Showing the steps of decortication of pigeon pea seeds 39 2: Preparation of pigeon pea protein isolate 40 3: Procedures of biscuit making 60 ك

12 LIST OF FIGURES Figures Page No. 1: Standard curve of a farinogram and evaluation points 50 2: Standard curve of an extensogram and evaluation points 53 3: Farinogram of dough prepared from 100% bread wheat flour 80 4: Farinogram of dough prepared from a blend of 95% bread wheat flour and 5% DPPF 80 5: Farinogram of dough prepared from a blend of 90% bread wheat flour and 10% DPPF 81 6: Farinogram of dough prepared from a blend of 85% bread Wheat Flour and 15% DPPF 81 7: Farinogram of dough prepared from a blend of 80% bread wheat flour and 20%DPPF 82 8: Farinogram of dough prepared from a blend of 75% bread wheat flour and 25% DPPF 82 9: Farinogram of dough prepared from bread wheat flour and PPPI (protein level 15%) 83 10: Farinogram of dough prepared from bread wheat flour and PPPI (protein level 20%) 83 11: Farinogram of Dough Prepared from Bread Wheat Flour and PPPI (protein level 25%) 84 12: Farinogram of dough prepared from 100% biscuit wheat flour 87 13: Farinogram of dough prepared from a blend of 95% biscuit wheat flour and 5% DPPF 87 ل

13 Figures Page No. 14: Farinogram of dough prepared from a blend of 90% biscuit wheat flour and 10% DPPF 88 15: Farinogram of dough prepared from a blend of 85% biscuit wheat flour and 15% DPPF 88 16: Farinogram of dough prepared from a blend of 80% biscuit wheat flour and 20% DPPF 89 17: Farinogram of dough prepared from a blend of 75% biscuit wheat flour and 25% DPPF 89 18: Farinogram of dough prepared from biscuit wheat flour and PPPI (protein level 15%) 90 19: Farinogram of dough prepared from biscuit wheat flour and PPPI (protein level 20%) 90 20: Farinogram of dough prepared from biscuit wheat flour and PPPI (protein level 25%) 91 21: Extensogram of dough prepared from 100% bread wheat flour 95 22: Extensogram of dough prepared from a blend of 95% bread wheat flour and 5 % DPPF 95 23: Extensogram of dough prepared from a blend of 90% bread wheat flour and 10 % DPPF 96 24: Extensogram of dough prepared from a blend of 85 % bread wheat flour and 15% DPPF 96 25: Extensogram of dough prepared from a blend of 80% bread wheat flour and 20% DPPF 97 26: Extensogram of dough prepared from 100% biscuit wheat flour 97 م

14 Figures Page No. 27: Extensogram of dough prepared from a blend of 95 % biscuit wheat flour and 5% DPPF 98 28: Extensogram of dough prepared from a blend of 90% biscuit wheat flour and 10% DPPF 98 29: Extensogram of dough prepared from a blend of 85 % biscuit wheat flour and 15% DPPF 99 30: Extensogram of dough prepared from a blend of 80% biscuit wheat flour and 20% DPPF 99 31: Fermentogram showing CO 2 production of dough prepared from 100% bread wheat flour : Fermentogram showing CO 2 production of dough prepared from 95% bread wheat flour and 5% DPPF : Fermentogram showing CO 2 production of dough prepared from a blend of 90% bread wheat flour and10% DPPF : Fermentogram showing CO 2 production of dough prepared from a blend of 85% bread wheat flour and 15% DPPF : Fermentogram showing CO 2 production of dough prepared from a blend of 80% bread wheat flour and 20% DPPF : Fermentogram showing CO 2 production of dough prepared from a blend of 75% bread flour and 25% DPPF : Fermentogram showing CO 2 production of dough prepared from bread flour and PPPI (protein level 20%) : Fermentogram showing CO 2 production of dough prepared from 100 % biscuit wheat flour : Fermentogram showing CO 2 production of dough prepared from a blend of 95 % biscuit wheat flour and 5% DPPF 107 ن

15 Figures Page No. 40: Fermentogram showing CO 2 production of dough prepared from a blend of 90% biscuit wheat flour and 10% DPPF : Fermentogram showing CO 2 production of dough prepared from a blend of 85% biscuit wheat flour and 15% DPPF : Fermentogram showing CO 2 production of dough prepared from a blend of 80% biscuit wheat flour and 20% DPPF : Fermentogram showing CO 2 production of dough prepared from a blend of 75% biscuit wheat flour and 25% DPPF : Fermentogram showing CO 2 production of dough prepared from biscuit wheat flour and PPPI (protein level 20%) : Fermentogram showing CO 2 production of dough prepared from biscuit wheat flour and PPPI (protein level 25%) : Amino acid profile of the standard curve : Amino acid profile of 100 % wheat flour bread : Amino acid profile of wheat flour bread containing5% DPPF : Amino acid profile of wheat flour bread containing 10% DPPF : Amino acid profile of wheat flour bread containing 15 %DPPF : Amino acid profile of wheat flour bread containing 20 %DPPF : Amino acid profile of wheat flour bread containing 25 %DPPF : Amino acid profile of wheat flour bread containing PPPI (protein level 15%) : Amino acid profile of wheat flour bread containing PPPI (protein level 20%) 129 س

16 Figures Page No. 55: Amino acid profile of wheat flour bread containing PPPI (protein level 25%) : Amino acid profile of 100 % wheat flour biscuit : Amino acid profile of wheat flour biscuit containing 5 % DPPF : Amino acid profile of wheat flour biscuit containing 10 % DPPF : Amino acid profile of wheat flour biscuit containing 15 % DPPF : Amino acid profile of wheat flour biscuit containing 20% DPPF : Amino acid profile of wheat flour biscuit containing 25% DPPF : Amino acid profile of wheat flour biscuit containing PPPI (protein level 15 %) : Amino acid profile of wheat flour biscuit containing PPPI (protein level 20 %) : Amino acid profile of wheat flour biscuit containing PPPI (protein level 25%) 154 ع

17 LIST OF TABLES Table Page No. 1: The dietary nutrients of pigeon pea 13 2: Proximate composition and minerals content of raw materials (on dry matter basis) 65 3: Effect of pigeon pea decortication on the anti-nutritional factors 68 4: Falling number of wheat flour intended for bread making as affected by inclusion of DPPF and PPPI 70 5: Falling number of wheat flour intended for biscuit making as affected by inclusion of DPPF and PPPI 72 6: Gluten quality and quantity of bread wheat flour as affected by inclusion of DPPF and PPPI 74 7: Gluten quality and quantity of biscuit wheat flour as affected by inclusion of DPPF and PPPI 76 8: Farinograms characteristics of bread wheat flour containing DPPF and PPPI 79 9: Farinograms characteristics of biscuit wheat flour containing DPPF and PPPI 86 10: Extensograms characteristics of bread wheat flour as affected by inclusion of DPPF and PPPI 93 11: Extensograms characteristics of biscuit wheat flour as affected by inclusion of DPPF and PPPI 94 12: Fermentograms feature of doughs prepared from bread wheat flour and composite flour blends : Fermentograms of feature doughs prepared from biscuit wheat flour and composite flour blends 102 ف

18 Table Page No. 14: Functional properties of bread wheat flour as affected by inclusion of DPPF and PPPI : Functional properties of biscuit wheat flour as affected by inclusion of DPPF and PPPI : Loaf bread specific volume of wheat flour as affected by inclusion of DPPF and PPPI : Chemical composition and energy value of wheat flour bread containing different levels of DPPF and PPPI : Amino acid profile of proteins of wheat flour bread containing different levels of DPPF and PPPI (on dry basis) : Effect of pigeon pea supplementation on amino acids chemical score of wheat flour bread : Mineral matter content of wheat flour bread (mg/100 g) containing DPPF and PPPI : Sensory evaluation of wheat flour loaf bread containing DPPF : Sensory evaluation of wheat flour loaf bread containing PPPI : Spread ratio of wheat flour biscuit as effected by inclusion of DPPF : Spread ratio of wheat flour biscuit as affected by inclusion of PPPI : Chemical composition and energy value of wheat flour biscuit containing different levels of DPPF and PPPI : Amino acid profile of proteins of wheat flour biscuit containing ص

19 different levels of DPPF and PPPI (on dry basis) 149 Table Page No. 27: Effect of pigeon pea supplementation on amino acids chemical score of wheat flour biscuit : Mineral matter content of wheat flour biscuit (mg/100 g) containing DPPF and PPPI : Sensory evaluation of wheat flour biscuit containing decorticated pigeon pea flour : Sensory evaluation of wheat flour biscuit containing pigeon pea protein isolate 161 ق

20 ABSTRACT Australian wheat (Triticum aestivum) flour (Extraction rate 72%); for bread (BrdF) and biscuit (BisF) were used as a base mixed with decorticated pigeon pea (Cajanus cajan L.) flour (DPPF) and pigeon pea protein isolate (PPPI) for making fortified bread and biscuit. Ratios of DPPF used in wheat flour for making bread and biscuit were ranging between 5 to 25%, where as ratios of PPPI in wheat flour for making bread and biscuit were adjusted to protein levels of 15, 20 and 25 %. All blends were then examined for rheological and functional properties as well as proximate composition, apparent nutritive value of protein and organoleptic quality of the end products. The wet gluten of wheat flour was significantly (P 0.05) reduced with increase incorporation of DPPF (from to 25.50%); (from to 23.18%) and inclusion of PPPI (from 29.0 to 24.50%) and (from 29.2 to 22.5%) for BrdF and BisF, respectively. Increasing levels of DPPF resulted in a significant (P 0.05) increase in falling number from 614 to 663 sec and from 614 to 697 sec.; while PPPI significantly (P 0.05) decreased the falling number from 523 to 458 and 388 sec; also from 515 to 478 and 443 sec. for (15, 20 and 25% protein levels) for (BrdF) and (BisF), respectively. Water retention capacity (WRC) and bulk density (BD) were significantly (P 0.05) increased by addition of DPPF to wheat flour. Water absorption values range were 65.9 to 65.1% and 63.7 to 61.6 %; dough development time was 5.0 to 4.0 min and 4.4 to 4.0 min; dough stability was 4.0 to 2.4 min and 4.1 to 2.3 min for BrdF and BisF, respectively, compared to their respective control. On the other hand, ر

21 addition of PPPI resulted in an increase in water absorption range of 66.3 to 74.8 and 66.7 to 71.0%; dough development time of 7.5 to 12.8 min and 4.5 to 7.3 min and dough stability of 2.6 to 8.6 min and 1.7 to 5.8 min for BrdF and BisF, respectively. The extensogram showed that the dough strength, extensibility and dough resistance to extension were decreased with increasing levels of DPPF and PPPI for the BrdF and BisF. The Fermentograms showed that the time taken to produce CO 2 through the first and the second hours of fermentation were 45 and 40 min for BrdF dough; and for different blends of DPPF and PPPI ranged from 43 to 58 min and 35 to 55 min. On the other hand, values obtained for BisF dough were 52 and 43 min and for different blends range were 48 to 60 min and 36 to 50 min, respectively. Incorporation of DPPF in BrdF beyond 10% had a negative effect on loaf bread specific volume (3.20cc/g). However, incorporation of PPPI showed significant increase (P 0.05) and higher values of bread specific volume were 4.63, 4.72 and 3.897cc/g for (15, 20 and 25% protein levels), respectively, compared to wheat bread (control) (3.45 cc/g). Higher values of spread ratios of biscuit for 10 and 20% DPPF in the blends were and 6.288, respectively, compared with wheat flour biscuit Incorporation of PPPI in the blends showed no significant differences on the spread ratios of the biscuit. Bread supplemented with DPPF was found significantly (P 0.05) more acceptable in all quality attributes at 10% level of supplement, compared to the control bread. Addition of PPPI at 15% protein level in BrdF was found superior in all its sensory characteristics compared to the control bread. ش

22 Significant differences (P 0.05) in color were observed in biscuit supplemented with PPPI at 25% protein level, and also in odor, after taste and overall quality for biscuit supplemented at 20% protein level. Bread supplemented with DPPF significantly increased (P 0.01) in ash, protein and caloric values with high levels of DPPF. Highest levels of DPPF (25%) resulted in the highest protein and calories (16.97% and kcal/100 g, respectively) compared with 14.62% and kcal/100 g for wheat bread. Inclusion of PPPI significantly increased (P 0.01) the protein content to 16.19, 21.16, and % for protein levels 15, 20 and 25%, respectively and the caloric values reduced from to kcal/100 g for 15 and 20% protein level, respectively. Biscuit supplemented with DPPF and PPPI showed significant increase (P 0.05) in ash, protein with high levels of incorporation; while significant decrease (P 0.05) in carbohydrates and caloric value were found when biscuit wheat was supplemented with high level of PPPI. Levels of essential amino acids in supplemented products increased except for methionine and valine, with increasing levels of DPPF. The protein quality has improved and lysine chemical scores increased gradually from 50% for wheat bread to 71% when supplemented with high levels of DPPF and from 62 to 74 and 98% when supplemented with PPPI ( 15, 20 and 25% protein level), respectively. Incorporation of DPPF in wheat flour improved its protein quality and lysine chemical scores increased gradually from 35 for wheat biscuit to 60% for (25% DPPF) blend and from 58 to 62 and 85 in blends containing PPPI 15, 20 and 25% protein levels, respectively. ت

23 و% بسم االله الرحمن الرحيم ملخص الا طروحة تم خلط دقيق القمح الا سترالي (استخلاص %72) الخاص بالخبز (BrdF) والخاص بالبسكويت( BisF ) كمكون ا ساسي مع دقيق اللوبيا العدسي المقشور (DPPF) ومستخلص (مفرزة) بروتينات اللوبيا العدسي (PPPI) كل على حداه لا نتاج خبز وبسكويت مدعم.تراوحت نسبة ا ضافة DPPF بين %25-5 بينما تم ضبط نسبة البروتين با ضافة PPPI ا لى مستوى % لنوعي الدقيق. تم تقييم الخواص الريولوجية والخواص الوظيفية كما تم تقدير المكونات الكيمياي ية والقيمة الغذاي ية للبروتينات بالا ضافة ا لي التقييم الحسي للخبز والبسكويت المنتج منهما. ظهر النقص المعنوي ( P 0.05 )في كمية الجلوتين الرطب في دقيق القمح المخلوط با ضافة DPPF وتراوحت بين الى %25.50 و الى ولمستخلص البروتين بين 29.0 الى الى %24.5 لدقيق الخبز ودقيق البسكويت على التوالي. زيادة DPPF في الدقيق المخلوط ا دت ا لى زيادة معنوية في رقم السقوط لدقيق الخبز تراوح بين ثانية وللبسكويت بين ثانية بينما ظهر ا نخفاض ا معنوي ا (0.05 P) في رقم السقوط با ضافة PPPI من 523 ا لى 458 و 388 ثانية وكذلك و 443 ثانية لنسب البروتين و %25 في دقيق الخبز والبسكويت على التوالي. ظهرت زيادة معنوية (0.05 P) في المقدرة على الا حتفاظ بالماء WRC) ( وكثافة الكتلة (BD) عند ا ضافة DPPF) )لكل من دقيق الخبز و البسكويت. مقدرة دقيق القمح على ا متصاص الماء با ضافة DPPF) )تراوحت بين 65.9 ا لى % 65.1 و 63.7 ا لى %61.6; وزمن تطور العجينة 5.0 ا لى 4.0 دقيقة و 4.4 ا لى 4.0 دقيقة ;وثبات العجينة 4.0 ا لى 2.4 دقيقة و 4.1 ا لى 2.3 دقيقة لكل من دقيق الخبز والبسكويت على التوالي مقارن ة بالشاهد لكل منهما.من جانب ا خر لوحظ زيادة مقدرة دقيق القمح لا متصاص الماء با ضافة( PPPI ) تراوحت بين 66.3 ا لى %74.8 و 66.7 ا لى %71.0; وزمن تطور العجينة 7.5 ا لى 12.8 دقيقة و 4.5 ا لى 7.3 دقيقة; وثبات العجينة 2.6 ا لى 8.6 دقيقة و 1.7 ا لى 5.8 دقيقة لكل من دقيق الخبز والبسكويت على التوالي. ث

24 ا ظهرت نتاي ج الا كستنسوجراف والمتمثلة في قوة العجينة والتمدد ومقاومة العجينة للتمدد ا نخفاض ا ملحوظ ا مع زيادة نسبة ا ضافة كل من DPPF وPPPI لكل من دقيق الخبز ودقيق البسكويت. ا ما بالنسبة لنتاي ج الفيرمنتوجرام ظهرت ا ختلافات واضحة في زمن ا نتاج غاز ثاني ا كسيد الكربون في الساعة الا ولى والثانية من زمن التخمير وقد كان زمن تخمير العجينة بالنسبة لدقيق الخبز 45 و 40 دقيقة ولمخلوط الدقيق تراوح بين 43 ا لى 58 دقيقة و 35 ا لى 55 دقيقة ا ما بالنسبة لدقيق البسكويت بين 48 و 43 دقيقة ولمخلوط الدقيق 48 ا لى 60 دقيقة و 36 ا لى 53 للخبز ا ضافة ا كثر من %10 3 ( 3.20 سم /جم).بينما ا ضافةPPPI للخبز و الخبز المنتج من الدقيق الشاهد البسكويت المدعم با ضافة DPPF ا ظهر تا ثيرا سلبيا معنويا ا ظهرت زيادة معنوية (P 0.05) (P 0.05) على التوالي. على الحجم النوعي في الحجم النوعي سم /جم لنسب البروتين % على التوالي مقارن ة مع 3 (3.45 سم /جم). DPPF ا ظهرت النتاي ج زيادة معنوية (P 0.05) في درجة الا نتشار وا علاها كانت (6.262) عند نسبة ا ضافة % 10 و % با ضافة % 20 مقارن ة مع درجة الا نتشار للدقيق الشاهد(( ). الخبز المدعم با ضافة DPPF %10 والقبول العام مقارن ة مع دقيق القمح الشاهد. كان الا فضل جودة معنوي ا ا ضافة (P 0.05) PPPI عند مستوى البروتين الخبز كانت الا كثر جودة في كل من المظهر العام النكهة الطعم والقوام مقارن ة بالشاهد. بروتين( ظهر نقص معنوي (%15) (P 0.05) (% 25 بروتين (%20). بدقيق في البسكويت المدعم با ضافة وكذلك في الراي حة ومتبقيات الطعم والتفضيل العام في في الخواص في دقيق PPPI في اللون عند مستوى البسكويت المدعم ا لى نسبة ظهرت زيادة معنوية في نسبة الرماد والبروتين والسعرات الحرارية في الخبز بزيادة التدعيم DPPF حيث كانت ا على نسبة للبروتين %16.97 وللسعرات الحرارية كيلوكالوري/ 100 جرام)عند ا على مستوى ا ضافة من DPPF مقارن ة بالبروتين والسعرات الحرارية للشاهد %14.62 و كيلوكالوري/ 100 جرام على التوالي. عند ا ضافة البروتين معنويا في المنتج النهاي ي ا لي % % على التوالي)وكذلك انخفضت السعرات الحرارية من عند PPPI زادت نسبة الي كيلوكالوري/ 100 جرام عند (مستوى بروتين %على التوالي). (مستوى بروتين و خ

25 البسكويت المدعم با ضافة DPPF و PPPI ظهرت فيه زيادة معنوية في كل من الرماد والبروتين بارتفاع مستوى التدعيم لكل منهما بينما ظهر ا نخفاض معنوي في نسبة الكاربوهيدرات والسعرات الحرارية بزيادة مستوى التدعيم بالمستخلص البروتيني. تحسن مستوى كل الا حماض الا مينية الا ساسية في الخبز المدعم عدا الميثيونين والفالين با ضافة DPPF وتحسنت جودة البروتين وزادت الدرجات الكيمياي ية للايسين من % 50 للخبز الشاهد ا لى % 71 عند ا على مستوى تدعيم وكذلك ا لى مستويات % عند التدعيم با ضافة (15 PPPI 20 و %25 بروتين) على التوالي هنالك ا يض ا تحسن في جودة البروتين في البسكويت المدعم حيث ا رتفعت الدرجات الكيمياي ية للايسين من %35 لبسكويت الشاهد ا لى %60 عند ا على مستوى من ا ضافة DPPF وا لى مستويات و 85 %عند ا ضافة PPPI بمستويات البروتين و %25 على التوالي. ذ

26 CHAPTER ONE INTRODUCTION Food legumes, particularly dried pulses play an important role in both crop production system and human nutrition. They form an important component of the diets of people in many developing countries of Asia and Africa. Grain legumes are traditionally consumed as human foods along with cereals in various forms. Among food crops, legumes contain the highest amount of protein, generally twice the level formed in cereals. Pulses, which belong to the group of protein-rich vegetable foods, are particularly rich in the amino acid lysine, yet are usually deficient in the sulfur-containing amino acids like methionine. Pulses are cheaper source of proteins when compared to animal proteins. Clinically, pulses were reported to reduce levels of cholesterol and glucose in blood Soni et al. (1982). Among food legumes, pigeon pea (Cajanus cajan L.) is commonly grown and consumed in tropical and subtropical regions in the world. Pigeon pea is a valuable source of low-cost vegetable protein, minerals and vitamins and occupies a very important place in human nutrition. On the other hand, cereal grains i.e. wheat, sorghum, rice, millet, etc. are staple foods consumed by the population. Cereal grains are nutritionally characterized by respectively low levels of proteins, and are deficient in some essential amino acids, among which lysine is the most important, but have adequate amounts of sulfur-containing amino acids. Hence, essential amino acid(s) deficient in cereals are complemented with addition of legumes to cereals. 1

27 Supplementation i.e. fortification or enrichment of cereals with legumes has been advocated as a way of combating protein-energy malnutrition (PEM) problems in developing countries. Legumes as a supplement bring to cereal-based diets a variety of taste and texture. Availability of certain technologies with respect to pigeon pea processing (e.g. decortication, protein concentration and isolation etc) encourages the use of these legumes in fortification of cereal-based products. Such a move is expected to extend the use of pigeon pea towards preparation of protein rich cereal-based foods, which can meet both nutritional and culinary purposes. The objectives of this study are: - The use of decorticated pigeon pea flour and decorticated pigeon pea protein isolate in development of protein rich - baked products namely bread and biscuits, suitable for general and specific nutritional purposes. - To study the effect of incorporation of pigeon pea flour and its protein isolate on the sensory evaluation and quality of bread and biscuits produced. 2

28 CHAPTER TWO LITERATURE REVIEW 2.1 Wheat and wheat classification Wheat is one of the most widely cultivated cereal crops in the world. Cereals, such as wheat, sorghum, millet etc. are belonging to the class Monocotyledons (Kent, 1983; Walton, 1988). Wheat is a member of the family Gramineae, and has the botanical name of Triticum. Flour millers are the major wheat processors; other processors include breakfast food manufactures, bulgur producer, food manufactures, and industrial users. Wheat has a predominant role in the grain trade and is utilized as food (67%), feed (20%), seed (7%) and industrial products (6%) as stated by Oleson (1994). Wheat is considered as one of the main food crop in Sudan. It ranks after sorghum as a staple diet especially in urban centers. FAO (1999) reported that wheat could be grown in a relatively wide range of climatic conditions especially in temperate climate and susceptible to disease in warm, humid regions. In 1999 and 2000 Sudan production of wheat was 172,000 and 214,000 metric tons, respectively (FAO, 2000). The wheats are divided into three groups:- [1] Triticum aestivum; include the varieties milled mainly to produce flour that is used mainly for breads, sweet yeast goods, cakes, pastry products, blended mixes, cookies and crackers. [2] Triticum durum, it is used extensively for the manufacture of macaroni. Its principal use is in production of semolina, coarsely ground and highly purified. Durum wheat is used for pasta products (macaroni, spaghetti, vermicelli, noodles, etc). 3

29 [3] Triticum compactum, or club wheat; flours milled from this class are too soft for bread making. Wheats are classified according to the season in which the seed is sown into winter and spring wheat. Wheat may also be classified by the color of their seed coat, conventionally known as red, yellow or white (Hoseney et al., 1988). Wheat grain may further be considered as being strong/hard, medium or soft. Wheat kernel texture differentiates cultivars of hard and soft wheat classes. Such classification is useful since it predicts milling times and energy requirements as reported by Miller et al. (1984); Pomeranze et al. (1985) and possibly by loaf volumes (Symes, 1969). Pomeranze et al. (1985) stated that many ways can be used to determine hardness and softness of bulk wheat. Location of growth also affects texture and individual kernels from a cultivar grown in one location also vary in hardness. A hard wheat kernel requires greater force to cause it to disintegrate than does soft wheat kernel. The flour obtained from hard wheat has a coarser particle size than soft wheat. Also hard wheat grinds much faster than soft wheats (Miller et al., 1991). Structurally, wheat can be divided into three main parts; the bran coatings, the germ, or embryo, and the endosperm. The bran coatings constitute approximately 14.5% of the wheat grain as reported by Anon (1987). The germ is approximately 2.5% of the grain and is the wheat plant in embryo. The endosperm, which is approximately 83% of the grain, from which the white flour is milled. 2.2 The role of wheat in human nutrition Wheat and wheat food are a major source of nutrients for people in many regions of the world. Wheat is a source of carbohydrates, proteins, 4

30 vitamins and minerals when consumed as a major component of the diet. Betchart (1982) reported that the endosperm consists mainly of starch and significant proportion of many minerals and vitamins. Nutrients are generally found in the highest concentrations in the germ and in the aleurone cells surrounding the starchy endosperm. Significant quantities of minerals and vitamins are lost when whole wheat is milled to produce white endosperm flour because the outer layers of bran are removed along with aleurone cells and germ. Extraction flour of 72-75% contains from as little as 20% to about 60% of the B vitamins originally present in whole-wheat flour (FAO, 1970). 2.3 Protein quality and amino acids composition of wheat The protein quality and quantity are both considered factors that govern the potential use of wheat or flour (Pomeranz and Mac Masters, 1970).The protein quality needed for bread making differs considerably from that required for pastries or pasta products as stated by Orth and Bushuk (1972); Schmidt (1973) and Hoseney et al. (1969). The protein content of wheat is highly influenced by the environmental conditions, grain yield and available nitrogen as well as the variety group as mentioned by George (1973). The protein content of wheat fractions is greatest in germ as reported by Kordylas (1991) followed by middling, bran, whole-wheat flour, and white flour in decreasing order (Miladi et al., 1972). There is generally a gradient increasing protein and decreasing starch per cell from the inner to the outer region of the endosperm (FAO, 1999). Within types of wheat, the hard wheats are generally higher in protein than the soft wheat, as reported by Kulp et al. (1980). The protein content of hard red spring was %, % for hard red winter and 5

31 8-11% for soft red winter. Schruben (1979) and Kent-Jones and Amose (1967) reported that when wheat is milled, the protein content decreased as extraction rate decreases; which reflects the removal of germ and aleurone-containing bran which are relatively rich in protein (Pederson and Eggum, 1983). Blackman and Payen (1987) reported that strong and weak flours produce dough, which have different mixing properties, this difference is due mainly to the quality and quantity of protein. Although the proteins in the wheat endosperm are nutritionally inferior to the non-endosperm proteins, they possess the unique and distinctive property of forming gluten when wetted and mixed with water. The protein quantity (crude protein) has been measured by the classic Kjeldahl method. Protein quality criteria are related to the gluten protein of the flour protein. Branlard and Dardevet (1985) reported the diversity of grain proteins and bread wheat quality. Most quality evaluations of wheat flour are based on the flours unique property of contributing visco-elastic characteristics to dough. Mac Ritchie (1984) and Pyler (1983) reported that the gluten proteins are essential for bread production because elasticity and extensibility are considered most important in bread making. Glutenins appear to contribute to mixing time, strength and elasticity, whereas gliadins contribute to extensibility and stickiness. Gliadine make up roughly 70% of the total protein in wheat flour (Huebner and Wall, 1976). Osborne fraction, divided the proteins of wheat kernel into four solubility classes: albumins, soluble in water; globulins, soluble in salt solutions, but insoluble in water; gliadins, soluble in 70-90% alcohol; and glutenins, insoluble in neutral aqueous solutions, saline or alcohol. Cereal proteins are nutritionally poor because they are deficient in essential amino acids such as lysine, threonine and tryptophan (Chung 6

32 and Pomeranze, 1985). Lysine is the first limiting amino acid in all wheat flours and wheat fractions, except germ. Amino acid composition indicates protein quality when compared with amino acid profile. Patterns such as the FAO provisional amino acid scoring pattern and suggested pattern of amino acid requirements for infants, children and adults FAO/WHO/UNU (1985) have been used to evaluate wheat protein quality (Betchart, 1978; Bodwell, 1985 and Young and Pellett, 1985). The growing conditions were mentioned since they affect the amino acid composition, protein contents and the nutritional values of wheat grain. As reported by Eppenorfer et al. (1985), lysine as a percentage of dry matter was found to increase with increasing levels of nitrogen. Bodwell (1981) compared various scores based on amino acid composition and estimates of protein nutritional value for a large number of protein sources, as assessed in human. Wolzak et al. (1981a) stated that the traditional biological assays were compared with the chemical methods that are normally used for estimating the protein quality in food. The protein efficiency ratio (PER) was mentioned to correlate better with the chemical parameters than the net protein ratio (NPR). Among the chemical methods, the amino acid score was regarded as the best of the chemical parameters. It is generally agreed that there are proteins in the wheat endosperm that serve storage function and they are rapidly broken down to amino acids and peptides upon germination of the seeds to provide a source of nitrogen for the proteins being synthesized for use by the developing embryo (Varner, 1965). The storage proteins in wheat are characterized by extremely high contents of glutamine (one-third or more of the total amino acids residues) and of proline (one-seventh or more of total residue). Woychick et al. (1961) reported that storage proteins make 7

33 up the greater part of gluten, prepared by washing starch from dough; but gluten also contains albumins, membrane proteins as reported by Simmonds (1972) and presumably; proteins associated with cell walls. These non-storage proteins usually have amino acids composition that differs substantially from those of storage proteins. Gluten contains about 5-10% lipids and 10-15% carbohydrates in addition to protein as stated by Kasarda et al. (1971). Betchart (1978) reported that protein quality of white bread improves when the bread is supplemented with amino acids such as lysine and therionine. Wheat quality represents several measurable characteristics that are significant in terms of end use. Mailhot and Patton (1988) reported that flour quality can be defined as the ability of the flour to produce a uniformly good end product under conditions agreed to by supplier and the customer. Flour quality factors can be divided into two basic groups; those are inherent in the wheat as the result of the genetic; and changes that brought about by growing conditions, including fertilization, weather (drought, heat, forest, rainfall, etc.) and disease; and those might be affected during the process of converting the wheat into flour Quality factors for the farmer - High yielding varieties. - Resistant varieties to diseases. - Well-adapted varieties (to climate and soil conditions). - Adequate moisture content Quality factors for the miller - Low content of screenings (Dockage, Besatz). - Sound, well-developed and undamaged kernels. 8

34 - High extraction rate. - Adequate kernel hardness. - Adequate moisture content Quality factors for the baker - Adequate protein content and protein quality (gluten and gluten index). - Equal and constant flour types (granulation, enzyme pattern (α-amylase activity). - Adequate level of damaged starch. - Well-balanced proportion between gas production. - Gas retention and high rate of water absorption Quality factors for the consumer - Wholesome appearance. - Well-balanced in nutrients content. - Free of toxic or harmful compounds or impurities. - Naturalness of end products. 2.4 Rheological properties of wheat flour and composite flour doughs Rheological properties of dough are important to baker for two reasons; first, they determine the behavior of dough pieces during mechanical handling, such as dividing, rounding and molding. Second, they affect the quality of finished loaf of bread. The term composite flour is used to describe flour made by blending non-wheat flour with wheat flour and used for production of 9

35 baked goods traditionally made from wheat only. The non-wheat component can be flour form other cereals, namely barley, maize, millet, oats or sorghum; starches from roots or tubers; or high-protein flours, concentrates isolates from oilseeds and legumes (Civetta, 1974). In general, the bread making potential decreases linearly with the proportion of wheat flour. The proportion of non-wheat flour that the wheat flour can effectively carry depends on the inherent strength of the wheat flour (Youssef and Buskuk, 1986).The method of preparation can increase or diminish the functionality of protein isolates in bread doughs. Youssef and Bushuk (1986) reported that, two high protein products prepared from faba beans by different procedures had widely different functionality in bread making. However, Bloksma and Bushuk (1988), stated that the characteristics of the dough are depending on the type of flour, quality and quantity, ingredients used and mixing conditions. Hoseney and Rogers (1993) reported that the wheat flour dough is able to retain gas, which is essential for production of baked products with a light texture. Determination of the rheological properties of dough is part of the quality assessment of the flour. Instruments designed for this purpose have been reviewed by Shuey (1975) and by Bloksma and Bushuk (1988). Extensibility of dough must be maintained long enough under baking conditions to permit sufficient oven rise as reported by Bloksma (1990a) and Bloksma and Bushuk (1988). Extensibility is enhanced by the presence of many un-branched, long-chain glutenin molecules as reported by Bloksma (1990a). Kieffer (2003) mentioned that the resistance is positively related to baked volume. The full bread making potential of the dough is attained only at the optimum point of dough development as 10

36 stated by Faubion and Hoseney (1990). Beyond the point of optimum mixing, resistance to extension no longer increased and the dough starts to breakdown as reported by Spies (1990). Bloksma (1990b) mentioned that a long dough development time of flour is considered an indication of good baking performance. 2.5 Nature, origin and production of pigeon pea Pigeon pea, Cajanus cajan (L. Mill sp.) also called red gram or tuar is a member of the sub-tribe Cajaninae, Tribe Phaseoleae and family Leguminasea. Van der Maesen (1980) concluded that evidence point to an Indian origin of the pigeon pea and Africa is a secondary centre of origin. Pigeon pea is inherently a perennial, erect, bushy plant. It is also cultivated as annual crop, with a range of maturity days. It can grow over 3 m tall, has woody stems, and a long tap root. The dry seeds vary greatly with respect to their size (100 seed mass ranged 2-24 g), shape (round, oval, or flattened), or color (white, brown, red, purple or black). The seed coat is smooth, and the cotyledons are light yellow. Pigeon pea is the fifth most important pulse crop in the world after bean, pea, chickpea and broad bean. Pigeon pea is commonly grown and consumed in developing countries, particularly those located in tropical and subtropical regions of the world, between Latitudes 30 S to 30 N at elevations from sea level to 2000 m. India accounts for more than 90% of the world supply of pigeon pea (ICRISAT, 1986). Other major producing countries are Myanmar, Nepal, Kenya, Malawi, Tanzania and countries of Caribbean regions (Singh, 1992). In India, pigeon pea is cultivated on 3.82 million hectares with an annual production of 2.88 million metric tons (Anon, 1997). In Sudan, pigeon pea (known locally as Lubia Addassy ) is traditionally grown in North and Central Sudan as a very minor crop. It is 11

37 usually grown a long the irrigation canals in the Gezira or to demarcate the borders of small holdings in Northern Sudan along the Nile. Sometimes it is used as a living fence in dairy farms, or as intercrop, hedges and windbreakers. There is no information about area cultivated, either their magnitude or total annual production as reported by Elawad and Osman (1993). The popular variety of pigeon pea in Sudan is the brown colored seeds of long duration. The main season of production is winter, although some is grown in rainy season. Pigeon pea seeds are usually offered boiled with some salt added or sugar and it is a casual dish during the Muslims fasting month; Ramadan. The remainder of the plant is used as feed for livestock and other different uses. 2.6 The role of pigeon pea in human nutrition Pigeon pea cotyledons contribute about 85%, the embryo about 1%, and the seed coat 14% to the total seed mass. The distribution of the dietary nutrients in different parts of mature pigeon pea seeds is presented in Table 1. The chemical constituents of pigeon pea seed govern its nutritive value. There is a wide variability in the reported chemical constituents of pigeon pea (Tripathi et al., 1975; Sharma, et al., 1977; Narasimha and Desikachar, 1978; Manimek et al., 1979; Singh et al., 1984c and Mohammed, 2002); most of these variability can be attributed to differences in analytical methods, and to the origin of the samples. Variations in sample origin include differences in sampling time after harvest, the cultivar used, and the environment where the crop was grown. 12

38 Table 1.The dietary nutrients of pigeon pea Constituents Green Seed Mature Seed Dhal Protein (%) Protein digestibility (%) Trypsin inhibitor (units mg -1 ) Starch (%) Starch digestibility (%) Amylase inhibitor (units mg -1 ) Soluble sugars (%) Flatulence factors (g/100 g -1 soluble sugar) Crude fiber (%) Fat (%) Minerals and trace elements (mg g dry matter) Calcium Magnesium Copper Iron Zinc Vitamins (mg g fresh weight of edible portion ) Carotene (Vit. A g ) Thiamin (Vit. B1 0.3 Riboflavin (Vit. B2) 0.3 Niacin 3.0 Ascorbic acid (Vit. C) 25.0 Source: Faris et al

39 The starch and protein are the principal constituents of pigeon pea seed. Singh et al. (1984c) showed that the starch content of the cotyledons of several cultivars belonging to different maturity groups ranged between 51.4 and 58.8% with a mean of 54.7%. Mohammed (2002) reported values of starch content of whole and decorticated pigeon pea seeds as and 67.28%, respectively Protein quality and amino acids composition of pigeon pea The protein quality is of prime importance in the pigeon pea products used for human food as stated by Salunkhe et al. (1986). Srivastava et al. (1999) reported that the grain forms an important source of protein with 18% protein content. The protein quality of pigeon pea seeds is a function of the amount of its protein, the essential amino acids in that protein, and the protein digestibility (Singh and Eggum, 1984). Hulse (1977) found that the protein content of pigeon pea seed samples ranged between 18.5 and 26.3% with a mean value of 21.5%. Singh and Jambunathan (1981a) found the protein content of 43 varieties of pigeon pea ranged between 17.9 and 24.3% for whole seeds, and between 21.1 and 28.1% for dhal samples. Mohammed (2002) found the protein content of Sudanese variety, season 2000/01, to be and 21.62% for whole and decorticated pigeon pea, respectively. Protein fractions play an important role in determining the overall amino acid composition of the seed proteins. Storage proteins; i.e. globulins constitute about 65% of the total seed protein of pigeon pea cotyledons (Singh and Jambunathan, 1982). These globulins have fewer sulphur amino acids than other seed proteins, and thus limit the nutritive value of pigeon pea protein. Albumin fractions, although representing a small proportion of the total proteins, are a very rich source of methionine 14

40 and cystine. The glutelin fraction is a better source of sulphur amino acid than the globulin fraction. Eggum and Beames (1983) reported lower values of amino acids, i.e. methionine, cystine and tryptophan in pigeon pea than in other legumes. A negative relationship is usually found in legumes between protein percentage and methionine content per unit of protein (Bliss and Hall, 1977). The negative relationship could be improved by protein breeding for protein quality as stated by Singh and Eggum (1984). The true protein digestibility (TD) of pigeon pea significantly increased with cooking as reported by Singh et al. (1990). The biological value (BV) of cooked samples decreases in both whole seed and dhal, whereas the net protein utilization (NPU) of cooked samples increases and that may be due to increase in protein digestibility. The TD of raw whole seed (nearly 60%) and dhal sample (over 70%) indicated a large increase in TD when pigeon pea is made into dhal (Singh et al., (1990) The anti-nutritional factors of pigeon pea The majority of food legumes plants including pigeon pea have the capacity to synthesize certain biologically active substances commonly considered to be anti-nutritional factors since they have been shown to affect animal and human nutrition (Liener, 1979). Pigeon pea is consumed by million of people in developing countries without any harmful effect. This indicates that their deleterious and anti-nutritional effects were partly or wholly removed by processing. The commonly observed anti-nutritional factors of pigeon pea are protease inhibitors, amylase inhibitors, oligosaccharides, phytolectins and polyphenoles. 15

41 Protease inhibitors The protease (trypsin and chemotrypsin) inhibitors are widely distributed in plants and it has been recognized for many years that the nutritive value and protein digestibility of many plant proteins, particularly those derived from legumes, are very poor unless they are cooked or subjected to some other form of heat treatment as stated by Liener (1969). In comparison with soybeans, peas and common beans, chickpea and pigeon pea offer fewer problems as far as these factors are concerned (Gallardo et al., 1974; Pak, 1974; Sumathi and Patabiraman, 1976; Hettiarochchy and Kantha, 1982). Hettiarochchy and Kantha (1982) showed that 33.4 mg trypsin inhibitor/g of soybean sample, 22.1 mg/g of pigeon pea and 1.9 mg/g of chickpea. Singh and Jambunathan (1981b) reported that the low protein digestibility of some wild pigeon pea is attributed to their high levels of protease inhibitors. The inhibitory activities of pigeon pea are completely destroyed only when subjected to heat under acidic condition as reported by Sumathi and Patabiraman (1976) Amylase inhibitors Jaffe et al., (1973) reported that pigeon pea seed extracts showed remarkably higher amylase inhibitor activity (22-45 units/g) in comparison with chickpea (4-6 units/g). The heat-labile natures of amylase inhibitors were studied by Grannum (1979). Some inhibition of starch digestion by amylase inhibitors may be expected when unheated seeds of pigeon pea are eaten Phytolectins Phytolectins are toxic factors that interact with glycoprotein on the surface of red blood cells and causing them to agglutinate. Grant et al. 16

42 (1983) reported that the hemagglutinating properties of several legume seeds have indicated that chickpea and pigeon pea have low lectin activity, i.e. below the toxicity level. Hettiarochchy and Kantha (1982) reported that phytolectin levels were 100 unit/g of sample for green gram and black gram, 400 units/g of sample for chickpea and pigeon pea, and 800 units/g of sample for cowpea and soybean. A complete destruction of hemagglutinanting activity was achieved in pigeon pea by autoclaving at 121 C for 30 minutes as reported by Ochetin and Bogere (1983), and moist heat treatment was essential to indicative the hemagglutinating activity Polyphenoles Polyphenoles refer to a complex family of phenolic compounds, which are widely distributed in plants, and their level varies greatly even between the cultivars of the same species. Genetic factors, as well as environmental conditions largely influence their presence in plant foods. Several functions have been attributed to polyphenoles; like antipathogenic, anti-herbivore and allelopathic properties (Brice and Morrison, 1982 and Ray and Hastings, 1992). Bravo (1998) reported that phenolic compounds are partially responsible for the sensory and antinutritional quality of plant foods. Polyphenoles of dry beans decreased protein digestibility by making protein partially unavailable or by inhibiting digestive enzymes as mentioned by Bressani and Elias (1979). Pigeon pea contains considerable amounts of polyphenolic compounds that are genetically variable as reported by Singh (1984). The pholyphenolic compounds that inhibit the activity of digestive enzymes; trypsin, chemotrypsin and amylase, are higher in chickpea and pigeon pea cultivars with dark seed- 17

43 coat color (Singh, 1984). On the other hand, Price et al. (1980) reported that tannins are present at low concentration (0-0.2%) in pigeon pea. Such processing practices as decortications, soaking, germination and cooking influences the levels of polyphenolic compounds of pigeon pea. Decortication of pigeon pea has been reported to reduce polyphenolic compounds by 90% (Rao and Deothale, 1982). Soaking (water discard) followed by cooking before consumption is suggested as a means of removing harmful effects of pholyphenolic compounds in the regions where these pulses are consumed as whole seeds Oligosaccharides The oligosaccharides; starchyose, raffinose and verbascose contribute to flatulence in man and animals (Rackis, 1975). Flatulence is characterized by the production of high amounts of carbon dioxide, hydrogen, and small amounts of methane gas. Savitri and Desikachar (1985) found that there were noticeable differences in the contents of these sugars among the cultivars of pigeon pea. The raffinose content of 12 pigeon pea cultivars ranged between 0.3 and 1.8% (Rao and Belavady, 1978). That study also reported a large variation in the combined stachyose and verbascose content of these cultivars. Singh et al. (1984) reported that these three sugars together constitute about 53% of the total soluble sugars in pigeon pea. The levels of flatulence-causing sugars increased as the seed matured in pigeon pea (Singh et al., 1984). Germination followed by cooking brought about 70% reduction in the levels of total oligosaccharides in pigeon pea as reported by Iyengar and Kulkarni (1977). 18

44 2.7 Pigeon pea processing The dry whole seed of pigeon pea possesses a fibrous seed coat or testa (husk or skin). The seed coat is indigestible and sometimes causes a bitter taste (Singh and Singh, 1992). Pigeon pea is traditionally processed into consumable forms by different methods, which can be divided into two categories; primary processing or dehulling, which converts whole seed into dhal, i.e. decorticated dry split cotyledons; and secondary processing that involves three major treatments; cooking (include soaking, boiling, frying, roasting), germination and fermentation. The dehulling operation is usually performed in two steps; the first involves loosening the husk from the cotyledons, and second removing the husk from the cotyledons and splitting them using roller machine or stone shakki (quren), (Araullo, 1974; Singh and Jambunathan, 1981a) Effect of pigeon pea processing on its nutritive value Dehulling/decortication of pigeon pea Proper dehulling of legumes for human nutrition essentially related to efficient separation of the seed coat from the cotyledons as mentioned by Singh and Jambunathan (1981). Rao and Deothale (1982) and Singh (1984; 1993) reported that polyphenoles, which are mostly present in the seed coat, are significantly reduced by dehulling. Removal of hull facilitates a reduction of fiber and tannin contents and improvement in the appearance, texture, cooking quality, palatability and digestibility of the grain legumes. Dehulling also improves the protein quality (Kon et al., 1973 and Desphande et al., 1982). Dehulling usually removes the germ along with the husk, thus important dietary nutrients such as protein, calcium, iron and zinc are lost (Singh et al., 1989a). Losses depend on the 19

45 method of dehulling, and the grain characteristics of the pigeon pea cultivars Cooking Cooking makes legumes edible by making them tender and also aids in flavor development (Salunkhe and Kadam, 1989 and Ruiz et al., 1996). Cooking improves the bioavailability of nutrients and also partially or wholly destroys some of the anti-nutritional factors (Salunkhe, 1982). Starch digestibility is improved by moist heat treatment as reported by Geervani and Theophilus (1981a). Although cooking improves the nutritional quality, prolonged cooking results in a decrease in protein quality and a loss in vitamins and minerals. A major beneficial effect of cooking on pigeon pea is the destruction of the protease inhibitors that interfere in protein digestibility. These inhibitors are completely destroyed when heated under acidic conditions as reported by Sumathi and Pattabiraman (1976). Preliminary soaking followed by dry heat treatment only, partially inactivates the trypsin inhibitors as stated by Contreras and Tagle (1974). The essential amino acids of pigeon pea do not change noticeably during cooking, except for a possible slight decrease in lysine (Singh et al., 1989a) Germination The nutritional quality of grain legumes is affected by germination (Lee and Karunanithy, 1990; Hsu et al., 1980 and El-Mahady and El- Sebaidy, 1982).Soaking followed by germination considerably reduces the activity of the trypsin inhibitors in pigeon pea. Phytic acid forms insoluble compounds with essential minerals such as calcium, iron, 20

46 magnesium and zinc. Salunkhe (1982) reported that germination can reduce or eliminate appreciable amounts of phytic acid in pigeon pea and improves bioavailability of its minerals. Osman (2001) studied the effect of germination on pigeon pea seeds (baladi cultivars) and found that the crude protein increased from 20.2 to 22.7%. The protein digestibility was significantly improved from 61.1 to 83.1% as a result of germination. Germination also cause significant changes in protein fractions of pigeon pea and altered the level of insoluble proteins. On the other hand, Onimawo and Asugo (2004) found that germination decreased the crude protein content from to 21.19%, fat from 6.75 to 5.25% and crude fiber from 7.37 to 7.0% Fermentation Fermented foods are essential components of diets in many parts of the world because of their higher nutritive value and organoleptic characteristics as reported by Stainkraus and Van Veen (1971). Fermentation causes a general improvement in the nutritional value of legumes, brings about desirable changes in taste and texture and in the breakdown of some of the anti-nutritional endogenous compounds (Zamora and Fields, 1979; and Akpapunam and Achinewhu, 1985). Tempeh is a traditional Indonesian food that is prepared by fermenting, soaked, dehulled and cooked legumes seed with a Rhizopus mould (Buckle and Iskandar, 1989). During soaking, bacterial fermentation takes place a long with mould fermentation, synthesizes enzymes that decompose proteins, carbohydrates and lipids, thus improving the digestibility, nutritional value and palatability of the legume seed (Buckle and Iskandar, 1989). Salunkhe et al. (1985) reported that pigeon pea in combination with cereals; fermented and cooked, could become more popular as health 21

47 foods. Pigeon pea can be used with or without soybean to prepare Tempeh (Vaidehi and Rathanamani, 1988). Fermentation increases the levels of soluble nitrogen and soluble sugars in pigeon pea (Faris and Singh, 1990). Trypsin and chemotrypsin inhibitors activity of pigeon pea were decreased significantly as a result of fermentation (Rajalakshmi and Vanaja, 1976); and Buckle and Iskandar (1989) observed no significant differences in the amino acid composition after fermentation of pigeon pea seeds. 2.8 Medicinal uses of pigeon pea Pigeon pea has several uses as medicine. It is used in ayurveda as volerant; a medicine that heals wounds and sores; as an astringent; a medicine that stops bleeding by constricting the tissues, and as a medicine that cures diseases of the lungs and chest. It also works as antihelminthic to destroy internal worms (Faris and Singh, 1990). Prema and Kurup (1973) reported that pigeon pea protein intake in rats on a high-fatcholesterol diet had a marked decrease in total and free cholesterol, phospholipid, and triglyceride contents in their blood serum. Pigeon pea causes reversion of sickled cells in patients suffering from sickle-cell anaemia (Ekeke and Shode, 1985). 2.9 Functional properties of pigeon pea seed protein Functionality is any property of a substance, other than its nutritional value, that affects its utilization (Pour-El, 1979). Proteins have molecular properties that contribute to functionality of food ingredients (Huebner et al., 1977; Ryam, 1977 and Finney, 1978). Genetic and agronomic factors, as well as processing condition, alter protein properties of seeds and, in turn, change the functional behavior of their products (Cherry et al., 1975 and Huebner et al., 1977). The proteins glutenin and gliadin are responsible for most of the functional, or bread 22

48 making, properties of wheat flour. Various oxidizing and reducing agents are used to alter sulfhydryle and disulfide bonds in these wheat proteins, producing varying bread quality. Onimawo and Asugo (2004) studied the effect of germination on the functional properties of pigeon pea flour and reported that the water absorption capacity (WAC) was significantly improved (P<0.05) by %, while oil absorption capacity was significantly decreased by 47.75%. The emulsion activity (2.3%) and foaming capacity (38%) were also reported to be decreased by germination. The least gelatin concentration for the germinated and ungerminated pigeon pea flours was 8 and 10%, respectively. Mizubuti et al. (2000) studied the functional properties of pigeon pea flour and pigeon pea protein concentrate. The solubility of both samples was superior to 70% at ph above 6.7 and below 3.5. The water and oil absorption were 1.2 and 1.07 ml/g of sample and 0.87 and 1.73 ml/g of flour and protein concentrate, respectively. The maximum concentrations of flour and protein concentrate needed for gelation was 20 and 12%, respectively Supplementation value of pigeon pea in cereal-based products The supplementary value of legumes is generally estimated by comparing the amino acid composition of mixed diets with standard references proteins (FAO/WHO, 1973). Legumes as a supplement bring to cereal-based diets a variety of taste and texture. They supplement cereals for minerals and vitamins of the B-complex (Aykroyd and Doughty, 1982), but most important is that they complement the essential amino acids in cereals (Hulse, 1989). The ratio of cereal protein to legume protein is 70:30 in Latin America, 75:25 in Africa and the Near East, and 90:10 in Southeast Asia as stated by (Hulse, 1989). The mutual 23

49 compensation is closest to ideal when the ratio by weight of cereal to legume is roughly 70:30, in which proportion each provides above equal parts by weight of protein (Hulse, 1989). Pigeon pea protein is a rich source of lysine, but is usually deficient in sulphur-containing amino acids, methionine and cystine; it thus supplements the essential amino acids in cereals as reported by Gopalan et al. (1971). Pigeon pea improves the amino acid scores for lysine in rice and wheat-based diets; and for threonine, leucine, and isoleucine if the proportion of pigeon pea in the diet is increased to 70:30 cereals: pigeon pea (Gopalan et al., 1971). Daniel et al. (1970) found that incorporation of 8.5% pigeon pea dhal in a rice diet, and 16.7% in a finger millet diet improved the diets nutritive value. Kurien et al. (1971) reported that the nutritive value of the Kaffir (maize) and wheat-based diets was considerably improved when supplemented with pigeon pea. Mustafa (1977) noticed that, for high protein biscuit using peanut flour as protein supplement, the biscuit protein was raised to 28% and when fed to school children their growth rate was recorded very high. Mustafa et al. (1986) improved the nutritive value of cookies and bread by adding cowpea protein isolate to raise wheat flour protein to 15 and 20%. The cookies made from the blends had spreading ratio of 7.12 and 6.27, receptively, compared with the control-spread ratio of Pigeon pea has rarely used in baked foods and confectionary products. Legumes are used in bread and cookies for protein enrichment. Nath et al. (1960) prepared biscuit with protein hydrolyzates of Cajanus indicu having protein. Gayle et al. (1986) reported that physical, sensory and nutritional characteristics of breads supplemented with pigeon pea flour up to 25% showed no significant differences (P<0.05) 24

50 compared with unsupplement bread. Malted pigeon pea flour can constitute up to 40% (by weight) of flour to make protein-rich cookies of food baking quality as reported by Vaidehi et al. (1985). Pigeon pea flour was added to rice flour to produce proteinenriched rice cookies (Prasetyo, 1988). He reported that for every 10% addition of pigeon pea flour the protein content of cookies increased by about 12% of total protein compared to that of the control (100% rice). Azman (1988) reported that supplementing milled rice flour with pigeon pea flour up to 30% increased protein content of the extruded product from 9.1% to 13.2%. The products were still acceptable to consumer and panelists. Mueses et al. (1993) fortified biscuit with pigeon pea flour more than 75 % level and found it unacceptable but those with 75% wheat flour and 25% pigeon pea flour had high acceptability, appearance and nutritive value. Pigeon pea flour was substituted at levels of 0, 5, 10, 15, 20 and 25% to wheat flour and whole wheat meal flour for bread and chapatti making, respectively, as reported by Harinder et al. (1999). Blends were prepared up to 50% for cookies making. Increasing levels of pigeon pea in blends significantly increased the protein and mineral of baked products. The bread from 10% pigeon pea flour blend with 2-3% vital gluten and 0.5% SSL had high loaf volume and loaf quality. Blends containing 15% pigeon pea flour were acceptable for chapatti, and pigeon pea flour with 0.25 % sodium stearoyl lactylate (SSL) were acceptable for cookies making. Eh (1999) reported that biscuits produced from 65% millet flour and 35% pigeon pea flour resulted in high nutritional value, highest scores for flavor, texture and general acceptability. Mohammed (2002) reported that the amino acids analysis confirmed the great beneficial effects of pigeon pea supplementation on 25

51 faterita sorghum nutritional value. Incorporation of pigeon pea flour into faterita sorghum flour at a ratio of 35:65 overcome lysine deficiency in feterita sorghum protein and increased the protein score from 37% in feterita flour to 91% feterita-pigeon pea composite flour Bread All over the world, bread means food and life. It requires no further preparation once purchased. Bread, a nutritionally dense food, is high in complex carbohydrates, which give the body a sustained energy (Bennion, 1967). Herbst (1995) reported that bread is a staple since prehistoric times. It has originated in Egypt about 3500 BC as reported by Joswellman (2003). The migration from countries site inward the urban has imposed the spread out of the habit of eating bread and thus increase the consumption (Dendy, 1992). The large consumption of bread led to the development of a well-organized baking industry. Bread acts as a vehicle for protein and vitamin-rich materials such as meat and cheese. In addition to that, the solid state of bread facilitates its transportation Bread ingredients Bread is made from four essential ingredients; namely flour, yeast, water and salt. Sugar and fat are optional ingredients. Flour, is the most important ingredient as it is the structure builder for bread. American hard red winter and red spring wheat and Australian prime hard wheat with at least 12.5% protein content are suitable for bread making (NCFM, 2003). Fance (1976) reported that all flours differ in their capacities for absorbing water in bread making. Water in the dough comes next to flour in order of importance. The functions of water in bread making are formation of the elastic gluten and 26

52 dissolving of soluble ingredients such as salt and ascorbic acid, etc. In the presence of water, starch in the dough gets wet and swells and subsequently rendered digestible when it gets gelatinized (cooked) during baking. Also the enzymes activity occurs in the presence of water (UN/ECA, 1998). Yeast (Saccharomyces cervisiae) is a unicellular microorganism of the fungus type, which depends on the presence of water, air and food sources for its growth and development. The growth process involves the breakdown of sugars by the enzymes in the yeast into carbon dioxide and alcohol i.e. fermentation. The carbon dioxide produces a leavening effect, forms small bubbles in the dough, and causes the dough to rise and extend in volume (Matz, 1968). Most of the alcohol is driven off during baking, while the remainder contributes to flavor the bread. Common salt or table salt used for food preparation is the type used for bread making. Salt enhances the bread flavor, strengthens the dough and development of crust color (UN/ECA, 1998) Bread making process Mixing The mixing of bread dough has three functions. First, the ingredients are transformed into a mass that is homogeneous. Second, flour proteins swell and organize in such away that they impart to the dough the desired gas retention; dough development.third, air is occluded, forming gas cells that grow in size (Baker and Mize, 1941). Upon continued mixing, the dough becomes extremely extensible and sticky and consequently difficult to handle; then it is over mixed (Pomeranz, 1988). 27

53 Dividing and rounding The dough is divided (scaled into individual portions of predetermined weight. Then rounding followed the dividing steps. When the dough piece leaves the divider, it is irregular in shape with sticky cut surface. Rounding forms a continuous new surface skin and thereby prevents gas from escaping (UN/ECA, 1998) Fermentation The objectives of fermentation (gas production) are to bring the dough to an optimum condition for baking. During fermentation, dough development is continued; that is, gas retention is improved, if this has not been achieved by mixing (Bloksma and Bushuk, 1988). Punching and/or molding increases the number of gas cells. At the end of the final proof, the dough must contain a large volume of gas and must yet have sufficient gas retention left for oven rise. The fermentation of the dough is divided into two steps; first fermentation or bulk fermentation, which occurs during the time from the end of mixing to the molding of the first loaf. Second fermentation i.e. proofing or final fermentation, which occurs between the molding of the first loaf and placing it in the oven(un/eca,1998).the bulk fermentation has great effect on the rheological characteristics of the dough and conditioning of the gluten by making it extensible; for the bread to have a good volume. On the other hand, the second fermentation has less influence on the rheological characteristics of the dough and aims at allowing the dough to expand to its optimum by fermentation before baking it in the oven (UN/ECA, 1998). There are many factors influencing the fermentation which include; quantity and quality of the yeast, the level of salt content, temperature of 28

54 the dough, consistency of the dough and the fermentation power of the flour, which is related to the enzymes activity (amylase) that produce sugars from flour starch (UN/ECA, 1998) Baking The purpose of baking is to transform the well-fermented dough into bread. Bloksma (1986) reported that temperature increase, in the interior up to 100 C and in the crust above 100 C. During baking, three important changes in dough properties take place; first the dough expands further; the volume of gas increases (oven rise). Second, the predominantly fluid dough is transformed into a predominantly solid bread crumb or crust. Third, the foam structure of the dough with separate gas cells is transformed into a sponge structure with interconnected gas cells (Baker, 1939). Stokes (1971) reported that in the first stage of baking, the yeast continues to produce carbon dioxide even at an increase rate, until it is inactivated by heat at a temperature of about 50 C. As a result of the temperature rise, dough viscosity first decreases, and no transformations take place. In the center of the dough piece, viscosity is further decreased by the transport of water baking from the outer to the inner parts (Sluimer and Krist-Spit, 1987). Above 60 C, dough viscosity increases rapidly as a result of the swelling of the starch granules and the exudation of amylase from them. The enormous increase in viscosity is mainly caused by starch gelatinization and the effect is increased by the polymerization of glutenins (Le Grys et al., 1981 and Schofield et al., 1986). At the surface of the dough piece, starch gelatinization and evaporation of water cause the formation of a semi rigid crust. Browning occurs as a result of the high temperature and may be caused by chemical 29

55 reactions other than those of the Maillard type (Ziderman and Friedman, 1985). Flavor compounds are also formed. Baking temperature and time should be chosen according to the size and weight of the bread. Small-size bread needs higher temperature than large-size bread. At the normal baking temperature of 250 C the baking time is 20 to 25 min. for a loaf of 280 g. For small loaves of 80 g to 100 g the baking time is about 12 to 15 min. at 260 C Cooling Sluimer and Krist-Spit (1987) reported that during cooling, water vapor diffuses from the center of the loaf outward and is replaced by air flowing inward. The latter flow is possible because of the transformation into sponge structure. Joswellman (2003) reported that the loaf is full of saturated steam, which must be given time to evaporate. The whole loaf is cooled to about 35 C before slicing and wrapping can occur without damaging the loaf Biscuit Biscuit is a popular item in the diet of weaned infant and young children. The word biscuit comes from the old French bescoit, which means twice cooked (Griswold, 1962 and Herbst, 1995). In the United States of America it is called cookies and crackers as reported by Wade (1988). Kulp (1994) reported that cookies' baking is one of the oldest of human arts, whose origin is lost in the live light of prehistoric times, which for today s sophisticated automated industry, turning out cookies products in virtually endless flow and packaging. The biscuit industry in Sudan started in the early sixties with three pioneer factories namely Satty, Karam and Kambal. The essential biscuit 30

56 production in Sudan was 12,500 tons in the year 1996, increased gradually up to 39,400 tons in 2000 (El-Shiekh, 2004) Biscuit ingredients Soft wheat flour with about 9-9.5% protein is normally used in biscuit making (NCFM, 2003). If strong flours are being used, more shortening and sugar must be added to obtain an acceptable texture. Water is recognized as a toughener. Stiffer dough has a less welldeveloped system for retaining gases plus greater resistance to expansion. It is impossible to specify in a recipe an amount of liquid that will be appropriate for all flours (Matz, 1968). The best way is to find an amount that gives soft dough with the flour to be used (Griswold, 1962). Sweeteners are very important to the cookie formula. Lorenz (1994) reported that the types of dry sugars; sucrose, dextrose, lactose and brown sugar, and liquid sugar, such as corn syrup high fructose, invert syrup, molasses and honey were used in cookies manufacturing. Wade (1988) reported that the addition of sugar to the biscuit dough has the effect of reducing the amount of water required in the dough. As the sucrose content increases, it acts as a hardening agent, making cookies more crisp and firm. However, when in a solution it tends to act as a softening agent when used at moderate levels, helping to hold water in the finished products. Fats, such as butter, shortening or oils are essential ingredients in baking (Phillips, 2003). Lorenz (1994) reported that shortening has four primary functions in cookies: lubrication, aeration, eating quality and spreading. The principal classes of fats and oils used in cookie production are butter, shortening and margarine. Wade (1988) recommended that a few biscuit dough contain or does not contain sugar, but all the recipes 31

57 examined contain an appreciable amount of added fat. The addition of fat has the effect of reducing the amount of water required to make a dough with a workable consistency and making the product more tender. A use of hydrogenated vegetable oil cream is satisfactoy (Matz, 1968 and Phillips, 2003). In baked goods, milk and milk derivatives are used for color improvement, water absorption and spread control properties, and flavor. The dried product is preferred because of the convenience of use and their storing stability. Wade (1988) noticed that fresh whole milk is replaced successively by evaporated or condensed milk, whole milk powder, and skimmed milk powder and currently by dematerialized whey powder. The presence of amino acids promote the browning reaction during baking, which is responsible for the development of the color and flavor of biscuits. In chemical leavened foods, the source of carbon dioxide is either sodium bicarbonate or ammonium bicarbonate. Addition of ammonium bicarbonate enhances the spread as well as opens the structure to provide some lift, and the addition of sodium bicarbonate makes a rapid increase in width and also affects the thickness. Whiteley (1971) reported that to improve texture, bite and appearance, it is necessary to achieve some forms of aeration e.g. mechanical, biological and chemical. Stauffer (1994) noticed that leavening of cookies produces two results: an increase in total volume of the cookie and an alteration of the spread ratio. Cookie is leavened by steam, CO 2 from the decomposition of added soda and ammonium bicarbonate. Soda is added to cookies doughs both to raise the ph and to give some leavening effect through release of CO 2 in the oven. Also ammonium bicarbonate is frequently used in cookies doughs, 32

58 particularly the moist types such as rotary cookies, to increase the volume Classification of biscuit types Biscuits are broadly classified as hard dough or soft dough origin. The soft dough group includes all sweet biscuits and the hard dough biscuits fall naturally into three sections fermented doughs, puffed doughs, and the semi-sweet doughs (Whiteley, 1971). Hoseney (1986) and Moreth (1994) classified cookies by the way the dough is placed on the baking band, to four general types: firstly rotary mold cookies, for this type, the dough is forced into molds on a rotating roll. As the roll completed a half turn, the dough is extracted from the cavity and placed on the band for baking. During baking, the cookies should neither rise nor spread and formulations are characterized by fairly high sugar and shortening levels and very low amount of water. Secondly, cutting machine cookies, the dough is made into a continuous sheet and the product cut out from it. The formula contains much more water and relatively low sugar and the gluten is developed in this system. Thirdly, wire-cut cookies in which the dough is extruded through an orifice and cut to size usually by a wire. Wire-cut cookies rise and spread as they are baked. Fourthly waffer cookies, the flour used is usually of low extraction rate. Baking system contains closed-plate that resembles waffle irons. After baking the product is cut into blocks and the filling is placed between the various layers. 33

59 Biscuit making process Mixing There are two basic methods of mixing soft dough as reported by Whiteley (1971), the first method is known as creaming and the second method is known as all-in method or continuous method. Moreth (1994) implied that even when all other factors (i.e. raw materials, machining, baking, etc.) remain constant, changes in the mixing operation results in changes in the finished products. Wade (1988) reported that the majority of batch mixers used for preparation of biscuit dough fall into one of two main categories, vertical spindle or horizontal drum mixers. Lehman et al. (1994) reported that the factors affecting variation in cookies spread are summarized as follows: firstly, causes of increased cookie spread, flour with low protein content and low protein quality, uses of fluid shortening, small particle size of sugar, high percentage of sugar syrup in formula, high level of leaving, single stage vs. multistage mixing, high percentage of moisture added to formula, low initial or slowly rising heat during baking and low fat-high sugar ratio. Second possible causes of decreased cookie spread were; flour with high protein, chlorinated flour, use of plasticized shortening, large particle size sugar, high fat-low sugar ratio, use of multistage mixingcreaming method, low percentage of water added, high initial oven heat during baking and high amount of water absorbing ingredients Shaping and baking There are two ways to shape the dough, roll and cut, or drop. The rolling and kneading results with flakiness biscuit; not sticky, and has sufficiently developed gluten. After rolling, the biscuit dough is cut into 34

60 shape, 2-3 inches in diameter and the pieces are placed in a greased baking sheet. The dropping method, drops the dough in an irregularly shape into grease baking sheet by highly floured fingertips as the dough is more sticky. The shaped dough then put in a well-preheated oven 250 C (Phillip, 2003). Whiteley (1971) noticed that to make biscuit palatable, baking is essential, and is achieved by transferring heat from a heat source to the biscuit. Wade (1988) recommended those two properties of the product which are; its color and its moisture generally judge the completion of baking process content. 35

61 CHAPTER THREE MATERIALS AND METHODS 3.1Materials Food samples Australian wheat flour Triticum sp. (extraction rate 72 %) of two types; bread flour made from hard wheat; production date October, 2003 and biscuit wheat flour; production date September, The wheat flours {small packs (1 kg)} were purchased from the agent of Sayga Flour Mills in Khartoum North. Pigeon pea (Cajanus cajan L.) brown colored seeds (Plate 2) were purchased from local market during the harvest season of 2002/2003. The added materials used in processing i.e. yeast, salt, sugar, shortening, ascorbic acid and skim milk were purchased from the local market Chemicals and reagents All chemicals and reagents used were of analytical grade obtained from Food Research Centre (FRC) and Faculty of Agriculture, University of Khartoum. 3.2 Methods Preparation of decorticated pigeon pea flour Pigeon pea was decorticated into its dicotyledons (dhal/splits), according to the methods of Hassan and Bureng (1996). Pigeon pea was soaked in water for 3 hrs. The excess water was discarded and the soaked seeds were tempered (conditioned) by covering the soaked seeds with permeable cloth, for 18 hrs; overnight. The conditioned seeds were sun 36

62 dried for 2-3 days, then the dried seeds were decorticated using stone mill (Appendix/Plate 3). The seed coat was removed by aspiration machine (Schule GMBH Humburg Johr). Undecorticated seeds and green cotyledons were sorted manually and removed out. The clean decorticated seeds (Plate 4) were ground into flour using an Efficient Universal Pulverizer, capacity ( kg); flour fineness ( mesh) (Appendix / Plate 5). Decorticated pigeon pea flour (DPPF) (Plate 6), was tightly packed in polyethylene bags and stored in a freezer (-20ºC) until needed for investigation. Flow chart (1) showing the steps of decortication of pigeon pea seeds Preparation of pigeon pea protein isolate Pigeon pea protein isolate (PPPI) was prepared according to the method of Mustafa et al. (1986). Pigeon pea flour was blended with phosphate buffer (ph 6.8). The ratio of buffer/flour was 10:1 using 250 g of flour each time. The flour was mixed with the buffer in a 4L Erlenmeyer flask and left for 20 hrs in a refrigerator at about 2ºC. The mixture was then blended in a Warning blender at medium speed for 15 min. The blended material was filtered through a nylon sieve (150 µm); the residue was re-blended for another 2 min. and filtered. The combined filtrate was then centrifuged, (Type , serial No ); (Appendix /Plate 7), at 1000 rpm for 1 min. to precipitate the starch. The filtrate containing the solubilized protein was transferred to a 4L beaker to which 5% hydrochloric acid was added, drop by drop, during stirring with a magnetic stirrer until the ph dropped to 4.5, at which point a turbid precipitate had been reached. The beaker and its contents were transferred to a refrigerator (about 2 C) and left for 60 min. The precipitate curd was washed with distilled water twice using centrifugation to separate the curd. The protein curd was transferred and dried at room temperature 37

63 Plate (2): whole pigeon pea Grain Plate (4): Decorticated Pigeon pea (dicotyledons / splits) Grain Plate (6): Decorticated Pigeon Flour (DPPF) Plate (8): Pigeon pea Protein Isolate (PPPI) 38

64 Pigeon pea seeds (Brown colored) Cleaning and Grading Shrunken seeds, mud and stones Excess water Discard Soaking in water (3 hours) Tempering of soaked seeds (18 hours) Sun-drying (2-3 days) Decortication (Using stone mill) Bran (husk/coat) Splits (dhal) Broken seeds and powder Milled into flour (DPPF) 1: Flow chart showing steps of decortication of pigeon pea seeds 39

65 Phosphate buffer Decorticated pigeon pea flour (DPPF) Milling Decorticated pigeon pea seeds Mixing (Buffer / DPPF) 10:1 Keeping in refrigerator (20 hours at 2 C) Blending for 15 min. Re- blending (2 min.) Filtration through 150µm Centrifugation 1000 rpm/min Addition of HCl (5%) Keeping in refrigerator (60 min.) Precipitate curd Discard Water by centrifugation Washing twice with dist. water Drying (25-30ºC) Mixing and Grinding Pigeon Pea Protein Isolate (PPPI) 2: Flow chart showing preparations of protein isolate (PPPI) 40

66 (25-30 C). The dry protein isolate was then mixed, ground (Plate 8) and then stored in a tight polyethylene bag in the freezer until used. The protein content of the protein isolate was determined by the micro- Kjeldahl method. Flow chart (2) showing the procedure for preparing pigeon pea protein isolate Preparation of wheat flour and composite flour blends The wheat flour types were removed from the packs, each type was homogenized separately in a homogenizer standard (No. YY ), (Appendix/Plate 1) then packed and tied separately in polyethylene bags and stored in a freezer (-20 C) until needed. Decorticated pigeon pea flour (DPPF) and pigeon pea protein isolate (PPPI) were added to wheat flour for bread and wheat flour for biscuit separately. The percentages of DPPF added to the two types of wheat flours were 5, 10, 15, 20 and 25%. On the other hand, PPPI was blended with bread and biscuit flour to increase the protein levels to 15, 20 and 25%, Pearson Square was used to reach these levels of protein. These levels of protein corresponded by weight to 1.2, 6.2, 11.2 and 1.7, 6.7 and 11.7 grams of protein isolate for bread and biscuit flour, respectively. All the blends prepared were used for bread and biscuit processing Analytical methods The analytical methods were carried out for the two types of flour samples, pigeon pea samples (Decorticated & Un-decorticated) and all blends, besides bread and biscuit samples processed Moisture content The moisture content was determined according to the method of AACC (1983) using Buhler Rapid Tester (Type MLI-1000). Ten grams 41

67 of sample were placed on a pan, when the oven temperature reached 130 C. After 20 minutes, the reading of moisture percentage was directly obtained from scale reading Ash content The ash content of the sample was measured according to the method of the AOAC (1990) using the muffle furnace (Model Tipoforno ZA No Gel Ran 1001). An amount of 2-3 grams of samples was weighed into a clean pre-dried and weighed porcelain crucible and placed in a temperature-controlled furnace at C for complete ashing (until white grey color). The crucible with its ash was transferred directly to a desiccator, cooled at room temperature, weighed and calculated as percent of original weight of sample. Ash content (%) = Weight of ash x 100 x 100 Weight of sample (100-%moisture) Crude protein The crude protein content was determined in all samples by micro- Kjeldahl method according to the AOAC (1990). The sample was accurately weighed (0.2 g) and transferred to the Kjeldahl digestion flask. Copper sulphate-sodium sulphate catalyst was added with 3.5 mls concentrated sulphuric acid. Then the flask was placed into a Kjeldahl digestion unit until a colorless digest was obtained. The flask was left to cool at room temperature, and then the contents were placed into the distillation apparatus. Twenty mls of 40% NaOH were added, the ammonia evolved was received in 10 mls of 2% boric acid solution. The trapped ammonia was titrated against HCl (0.1N) in the presence of 2-3 drops of indicator (Bromo Cresol green and methyl red) until a brown reddish color was observed. Crude protein was calculated as follows: 42

68 Where: Crude protein (%) = (S-B) x 0.1 x x factor x ml HCl (0.1N) = 1.4 mg nitrogen S = sample titer B = blank titer Weight of sample (100-%moisture) Factor = protein conversion factor 5.7 for wheat flour, and 6.25 for other grains Fat content Crude fat was determined according to the standard method of AOAC (1990). A sample of 2-3 g was weighed into an extraction thimbles and covered with cotton; that was previously cleaned with petroleum ether, then the sample and a pre-dried and weighed Erlenmeyer flask containing about 100 ml petroleum ether were attached to the extraction unit. The extraction was carried for 8 hrs at C. At the end of the distillation period, the flask was disconnected from the unit and then was redistilled. Later, the flask with the remaining crude ether extract was put in an oven at 105 C for about an hour. Cooled in a desiccator, reweighed and the dried extract was recorded as crude fat (%DM) according to the following formula:- Crude fat (%) = Dry extract weight (g) x 100 x 100 Weight sample (100 %moisture) Carbohydrates The carbohydrates were calculated by difference. The sum of moisture, fat, protein and ash contents was subtracted from 100 as described by West et al. (1988). 43

69 Minerals content According to the AOAC (1990), minerals content i.e. calcium, iron, copper and magnesium were determined using atomic absorption spectroscopy (model Perkin Elmer). One gram of a dried, ground sample was weighed in a porcelain crucible, placed in a muffle furnace and ashed at 550 C overnight. The ash was then cooled and dissolved in 5 ml of 20% HCl. The solution was then warmed, filtered into 50 ml volumetric flask and the solution was diluted to volume with deionized water and mixed well. Standard conditions were used to determine the concentration of the elements of interest. Standards were prepared by suitable dilution of the stock standard solutions. The samples were diluted, if necessary, to bring the concentration of the element of interest into a suitable range for analysis. To overcome potential anionic interferences when determining calcium and magnesium, the final sample dilution and all standards and blanks should contain 1% (w/v) lanthanum Anti-nutritional factors of pigeon pea Tannins content The rapid visual technique (Price et al., 1978) was used to quickly distinguish between zero, low, intermediate and high tannin sample by development of shades of yellow, green and blue color. Quantitative estimation of tannins was carried out using modified vanillin-hcl in methanol and 1% vanillin/methanol, the reagent was prepared daily by mixing equal volume of 1% vanillin/methanol fresh and 8% conc. HCl/methanol, it was discarded if a trace of color appeared. D(+) catechin was used to prepare the standard curve; this was done by adding 100 mg of D(+) catechin to 50 ml of 1% HCl/methanol, from this 44

70 stock solution, various dilutions were prepared, 5 ml of vanillin/hcl reagent (0.5%) were added to 1 ml of each dilution. The absorbance was recorded using spectrophotometer (Jenway 6305UV/Vis. Spectrophotometer). At 500 nm after 20 min. at 30 C from addition of reagent, the absorbance was blotted against catechin concentration. A 0.2 g of the sample was placed in a test tube. Then 10 ml of 1% vanillin/hcl/ methanol were added. The test tube was stoppered and continuously shaken for 20 min. in a mechanical shaker (Gerhardt Schuttel-machine R010, type R010, Gerhardt Bonn App. NO ) and then centrifuged at 2500 rpm for 5 min. (Bench Centrifuge BTL made in England). One ml of the supernatant after centrifugation was pipetted into each of the tubes and then proceeding as was described in standard above. For zero setting, before absorbance was read, 1 ml blank solution was mixed with 5 ml 4% conc. HCl and 5 ml vanillin reagent in a test tube and incubated for 20 min. at 30 C (blank) (incubator RO-8 Memmerr Germany). Absorbance at 500 nm was read on spectrophotometer and concentration of condensed tannin was determined from the standard curve. Tannin concentration was expressed as catechin equivalent (CE) as follows:- Where: C.E% = C x 10 x C = concentration corresponding to optical density 10 = volume of extract (ml) 200 = sample weight (mg) 45

71 Phytic acid content The phytic acid was determined according to the method described by Wheeler and Ferrel (1971). Phytic acid was extracted in low acid media, then precipitate as ferric phytate by addition of FeCl 3, which was converted to Fe (OH) 3 by the addition of NaOH. The ferric hydroxide was dissolved as Fe (NO 3 ) 3 through addition of HNO 3. Fe (NO 3 ) 3 was measured optically. The phytate phosphorous was calculated assuming a 4:6 iron: phosphorous ratio. Two grams of milled dried sample were weighed in 125 ml conical flask. Fifty mls of 3% trichloro-acetic acid (TCA) were added to the flask, and then put into a mechanical shaker for 3 hrs. The suspension was centrifuged for 5 min. at 2500 rpm. Ten mls from the clear supernatant were transferred into a test tube (40 mls) with 4 mls of FeCl 3 solution (2 mg Fe ++ ions/ml TCA). The tube was heated in a boiling water bath for 45 min., then cooled and centrifuged for min. at 2500 rpm. After decantation, the precipitate was washed, transferred into a test tube containing 25 mls TCA (3%) and dispersed well in water -bath for 15 min. at 100 C. Then, the tube cooled, centrifuged, and the remaining precipitate was washed once again and dispersed well in distilled water with 3 mls of NaOH (1.5N). The total volume was made up to 30 mls and the sample was kept in a water-bath for 30 min. at 100 C and immediately filtered through a filter paper Whatman No. 2. The remaining precipitate was washed again with hot distilled water and quantitatively transferred into 100 mls volumetric flask with 40 mls hot NHO 3 (3.2N) and the final volume was made up to 100 ml. Then, 0.5 mls aliquot was taken into a 10 ml volumetric flask and 20 mls of potassium thiocyanate (1.5N) was added and the volume was made up to the mark with distilled water and immediately read in a 46

72 spectrophotometer (Jenway 6305 UV/ vis. Spectrophotometer) within 1 min. at a wavelength of 480 nm. Calculations: A standard curve of different Fe (NO 3 ) 3 concentrations under the same conditions was plotted to calculate the Fe +3 concentrations. The phytate phosphorous was calculated from the Fe +3 concentration assuming 4:6 iron: phosphorus molar ratio Mls standard Conc. Ppm Fe A Conc./A=K Mean K = Phytic acid = 6 x A x mean K x 20 x 10 x 50 x 100 mg P/100 g sample x 2 Where: A = optical density Rheological characteristics of dough Falling number test Falling number (FN) is defined as the time in seconds required stirring and allowing stirrer to fall a measured distance through a hot aqueous flour gel undergoing liquefaction. A falling number apparatus (FN 1400, type 1402, No. 2317) was used to measure the activity of α- amylase in wheat flour and composite flour according to the method No B of AACC (1999). The corrected weight of sample (14% moisture 47

73 basis) was placed into a dried falling number tube. Twenty five mls of water were added at 22±2ºC with pipette. The tube was stoppered and shaked up and down until mixed. Viscometer-stirrer was used to scrape down slurry coating upper part of tube, and all slurry from stopper was scraped. The tube and with viscometer-stirrer was inserted into the boiling water bath and locked into position. The test automatically starts, the sample was stirred for 60 seconds, and the viscometer stirrer was stopped in an up position, released and sinked under its own weight through the uniform gelatinized suspension. The time in seconds for the stirrer to fall through the suspension was recorded as the falling number (seconds). The required flour sample weight (RW) is obtained from the correction tables of sample weight to 14% moisture basis corresponding to 7 g. No change is made in the quantity of the water used (25 mls). Where: RW (g) = 7 x (100 14) (100-m) RW = the required flour sample weight used for determination. m = actual moisture percentage of the flour sample Gluten quality and quantity Wet gluten, dry gluten and gluten index were determined on 10 grams flour samples by the glutomatic system (Perten Instruments). The system comprises three components: Glutomatic 2200; for the wet gluten, centrifuge 2015; for the gluten index and glutork 2020 drier; for determination of dry gluten content. The system is approved for gluten index standard method of the AACC No. 158, (1999). The wet gluten percentage was calculated from the following equation:- Wet gluten (%) = gluten ball weight x 100 Weight of sample 48

74 Gluten index determination was carried by placing the wet gluten in a sieve cassette inside a centrifuge (2015), after running for 30 seconds, the centrifuge was automatically stopped and the passed gluten through the sieve was weighed to obtain the gluten index from the following equation:- Gluten index % = Total wet gluten wt. passed gluten wt. x 100 Total wet gluten wt. The dry gluten was determined by using Glutork (2020) instrument by placing the wet gluten in the centre bottom plate of the Glutork and closed, after 4 minutes the Glutork was automatically stopped and the dry gluten was removed and weighed, the dry gluten percentage was obtained from the following equation:- Dry gluten ( %) = weight of dry gluten x 100 Weight of sample Farinograms characteristics The rheological properties of wheat flour and composite flour were determined using the Brabender Farinograph E according to the standard method of the AACC No (1999). The sample of flour (300 g, on 14% moisture basis) was placed into a clean mixer. The Farinograph was switched on at 63 rpm for one minute, then the distilled water was added from a special burette (at deviation from the 500 units consistency, the correct water absorption can be calculated from the deviation; 20 units deviation correspond to 0.5% water, if the consistency is higher than 500 FU, more water is needed and vice-versa. When the appropriate amount of water is added from a burette; a curve with maximum consistency, defined as 500 FU is obtained. Individual values derived from the Farinograph Fig. 1 are as follows:- 49

75 Fig. 1: Standard curve of a farinogram and its evaluation points 50

76 - Water absorption (%) The percent of water that results in a dough consistency of 500 Farinograph units (FU).It prescribes the quantity of water, which has to be added to the flour during the production. - Dough development time (or peak time) The dough development time is defined as a time in minutes until a diagram has reached its maximum (dough is fully developed). - Dough stability Dough stability is defined as a time difference (to close 0.5 min.) between the points were the top of the curve first intersects the 500 FU line (arrival time) and the point where the curve leaves 500 FU line (departure time). - Softening of the dough The softening (weakening) of the dough is obtained in Farinogram units after 12 minutes of the development time. - Farinograph quality number The Farinograph quality number measures the length of the curve in (mm) from the beginning to the point at which the curve has decreased by 30 Farinograph units from the 500 FU Extensograms characteristics The dough extensibility was determined by using the Brabender Extensograph according to the standard method of the AACC No (1999). The Extensograph records a force-time curve for a test piece of dough stretched until it breaks. Characteristics of force-time curves or Extensograms are used to assess the general quality of flour and its response to improving agents. 51

77 Dough was prepared as described before in the Farinograph. The amount of water (at 30ºC) was filled from the burette into an Erlenmeyer flask and 6 gm of salt (sodium chloride of recognized analytical quality) was dissolved. The salt solution was added quickly after1 min. premixing; within 25 sec., as slow water addition influences the dough development time. The dough was mixed for 1 min., and then the Farinograph stopped for 5 min. After the completion of mixing, two pieces of 150±0.1 g dough was scaled off and revolved in the Extensograph rounder with automatic shutoff after 20 revolutions. The dough was centered on shaping unit and rolled into cylindrical test piece; clamped in lightly greased dough holders, the test pieces on dough holders were stored in humidified chamber. After rest period of 45 min. from end of shaping; the cradle holding test sample was placed on the balance arm of the Extensograph. A hook was pulled through the dough piece at a constant speed, which was thus stretched until the dough breaks. By means of the balance system, the load acting onto the dough during this procedure was measured and recorded. The resulting diagram; extensogram showed the force, which the dough opposed to the stretching force as a function of the stretching time i.e. the stretching length. The dough of the first test was removed from the holder and reshaped. As before, allowed next period of 45 min. before stretching. The dough pieces were tested again after 90 and 135 min. The four most common measurements made on force- time charts, or extensograms Fig.1 are as follows:- - Resistance to extension The resistance to extension is the height of the Extensogram at a constant deformation of the dough. The value is determined at the point 52

78 Fig. 2: Standard Curve of an Extensogram and its evaluation points 53

79 where the curve start rising. The results are given in EU with a precision of 5 EU. - Extensibility The extensibility of the dough is a distance in mm from the beginning of the stretching until the breaking of the test piece. - Energy The energy measures the area under the curve in cm 2. The value (energy) describes the work applied for stretching the dough and is a measure for the flour quality. - Ratio The ratio is the quotient of resistance and extensibility. Ratio = Resistance to extension (EU) Extensibility (mm) Fermentograms characteristics The Fermentograph is an instrument for measuring the gas production, carbon dioxide (CO 2 ) of fermenting dough. The main purpose of such tests is to check the quality of yeasts, besides, the influence of the recipe and of baking improvers. The operating principle of the Brabender Fermentograph according to its manual is based on measuring the lift of a piece of dough during yeast fermentation due to the production of carbon dioxide (CO 2 ). The dough sample was placed in a rubber balloon, which was then placed into the temperature-controlled water bath. The change in volume due to CO 2 production caused certain buoyancy, which was measured as a loss in weight by means of measuring spring and recorded on a mechanical line recorder. The dough was prepared in the Brabender Farinograph (E). The amount of water is sufficient to obtain a consistency of 500 FU (Farinograph units) at a dough temperature of 30 C. About 54

80 20% of the total amount of water was titrated from the burette into a glass container in which 5 g of sodium chloride was dissolved. Another 20% of water was poured from the same burette into a glass container in which 8 g of yeast was dissolved. The sample was fitted in the Farinograph mixer. The dissolved salt solution was poured first over the flour and then added the yeast solution. The addition of salt and yeast solution must not take more than 1 min. The mixer was stopped, covered and the dough reposed for 5 min. The dough was mixed again for 2 min. About 400 g of the dough was filled in a rubber balloon with controlled valve. The rubber balloon was pressed until all air inside has escaped, and the valve was closed. The rubber balloon containing the dough was inserted into the temperature-controlled water bath (30 C) and the zero point was adjusted. After a fermentation period of 1 hr., the dough in the rubber balloon was kneeled very strongly, in order to remove the carbon dioxide that was produced, through the valve. Then close the valve again, and the procedure was repeated in order to remove the very last bit of CO 2.Again the pointer of the recorder must match the zero line. The test time for flour generally depends on the quality of the yeast, on the local baking schedules and on the gluten strength of the respective flour. The test time in the Fermentograph for wheat flour and composite flour were extended for 2 hr. The Fermentograph directly records the carbon dioxide in cm 3.The steeper the curve, the more intensive the development of carbon dioxide Functional properties of wheat flour and composite flour blends Water retention capacity The water retention capacity (WRC) was measured for the flours and composite flour (blends) by the method of Lin et al. (1974).With modification described by Quinn and Beuchat (1975). A 10% H 2 O 55

81 suspension (3 g/30 ml) was stirred in a 50 ml centrifuge tube using a glass rod for 2 min. After 30 min. equilibrium, the tube was centrifuged for 20 min. at 4400 rpm. The freed water was carefully decanted into a graduated cylinder, and the volume was recorded. The WRC was expressed as milliliters of water retained by 100 g sample Bulk density The bulk density was determined by the method of Wang and Kinsella (1976). A 10 g sample of material was placed in a graduated cylinder (25 ml) and packed by gently tapping the cylinder on the bench top (10 times) from a reasonable height (~5-8 cm). The final volume of the sample was recorded and the bulk density is expressed as gram sample per milliliters volume occupied Fat absorption capacity The fat absorption capacity (FAC) of the samples was measured by a modified method of Lin et al. (1974). A 4 g material was treated with 20 ml of refined edible oil (specific gravity 0.9) in a 50 ml centrifuge tube. The suspension was stirred with a glass rod for 5 min. and the contents were allowed to equilibrate for further 25 min. The suspension was then centrifuged at 4400 rpm for 20 min. and the volume of the fat was measured. The FAC was expressed as milliliters of fat-absorbed by 100 g sample Processing of bread samples The bread-baking test for assessing the quality of wheat flour and composite flour blends were carried out according to the method of Badi et al. (1978). Bread flour sample (100% wheat),decorticated pigeon pea flour DPPF (5, 10, 15, 20 and 25% w/w) in wheat flour, and pigeon pea 56

82 protein isolate (PPPI) (15, 20 and 25% protein levels) were prepared into bread. The ingredients used in bread making were as follows: Item Quantity (g) Flour 250 Yeast 2.5 Salt 1.5 Sugar 2.5 The amount of water used for the wheat flour and composite flour was according to the water absorption of the Farinograph. All the ingredients were weighed and mixed to form a dough in Mono-Universal Laboratory dough mixer for 5 min. at medium speed. The dough was allowed to rest for 10 min. at room temperature (30 C), then it was scaled to three portions of 120 g each. The three dough portions were made into round balls and allowed to rest for another 15 min. Then molded up, put into pans, placed in the fermentation cabinet for final proof between min. The fermented dough samples were baked in Simon Rotary Test Oven at C with saturation of steam for min. The loaves were then left to cool The loaves were sliced with an electric knife, part of the slices were kept closed in polyethylene bags at room temperature for sensory evaluation, in the same day. Some of the slices were dried at room temperature (38±2 C) for 24 hr. and kept for analytical work Evaluation of bread quality The bread made from wheat bread flour and different blends were cooled at room temperature (38±2 C) for an hour after baking and quality measures were made on triplicate loaves as follows: 57

83 Bread volume The loaf volume expressed in cubic centimeters was determined by seed displacement method according to Pyler (1973). The loaf was placed in a container of known volume into which small seeds (millet seeds) were run until the container is full. The volume of seeds displaced by the loaf is equal to the loaf volume Bread weight The loaf weight of bread was taken in grams Bread specific volume The specific volume of the loaf was calculated according to the AACC standard method (1999) by dividing loaf volume (cc) by its weight (g) Sensory evaluation of loaf bread Loaf bread samples were assessed organoleptically by the ranking test according to the procedure described by Ihekoronye and Ngoddy (1985). Fifteen semi-trained assessors were provided coded samples (Appendix) and asked to evaluate the general appearance, flavor, taste, texture and overall quality of the loaf bread. Sum of ranks were then statistically interpreted according to the same ranking test described by the same authors Processing of biscuit samples Biscuits were prepared according to Vatsala and Harids Rao (1991) method. Control sample of wheat flour (biscuit flour) and blends with decorticated pigeon pea flour (DPPF) and pigeon pea protein isolate (PPPI) were prepared by substituting DPPF in ratios of 0, 5, 10, 15, 20, 58

84 and 25% (w/w) and by increasing protein levels to 15, 20 and 25% by using PPPI. The formula used in biscuit processing were as follows: Ingredient Quantity(g) Biscuit flour 100 Sugar powder 30 Shortening 30 Skim milk powder 2 Sodium chloride 1 Sodium bicarbonate 0.4 Ammonium bicarbonate 1.5 Glucose 2 Water 15 ml The ingredients were weighed for 200 g of flour. Sugar powder, shortening, skim milk powder and glucose were creamed in Hobart N-50 mixer (Appendix /Plate 9) with a flat beater for 3 min. at 61 rpm. Salt, ammonium bicarbonate and sodium bicarbonate were dissolved separately in part of required water and added to the cream. Mixing was continued for 8 min. at 125 rpm to obtain a homogenous cream. Finally, flour was added and mixed for 3 min. at 61 rpm, and then the dough was sheeted to a thickness of 3.5 mm with the help of an aluminum plate form and a frame. The piece dough was transferred to an aluminum tray. The biscuits were baked in an electric oven maintained at 205 C for 8.5 min., the baked units were cooled, packed in polyethylene bags and stored for further analysis. Flow chart (3) showing the procedures of biscuit making Evaluation of biscuit quality Biscuit weight Biscuits were weighed (10 biscuits) and the weights were recorded. 59

85 Weighing Sugar (powder) Shortening Skim milk Creaming (3 min 61 rpm) Mixing for 8 min at 125 rpm Salt Ammonium bicarbonate and Sodium bicarbonate Adding of flour (Mix 3 min at 61 rpm) Sheeting of dough Cutting of Dough Baking at 205 C for 8.5 Cooling Packing Storing Fig. 3: Flow chart showing the procedures of biscuit making 60

86 Biscuit spread ratio Biscuits were evaluated for the spread ratio according to the following equation: Spread ratio = Width of the biscuit Thickness of the biscuit Sensory evaluation of biscuits Evaluation of biscuits made from wheat and composite flours; with DPPF and PPPI, were carried out. Fifteen semi-trained assessors were provided coded samples (Appendix) and asked to evaluate the general appearance, color, after taste, texture and overall quality of the biscuits according to the scoring (Hedonic) scale of 5 points., described by Ihekoronye and Ngoddy (1985).A key table was given to the panelists guided them to score according to it Protein and energy evaluation methods Amino acid profile The amino acids composition of all samples was determined according to the official methods by using High Performance Liquid Chromatography (HPLC) Sykam system (Model S7130). The system is equipped with a programmable auto injector. The samples were prepared by placing 200 mg of each sample in hydrolysis tubes. Five milliliters of 6N-hydrochloric acid were added to each and tightly closed. The tubes were incubated at 100 C for 24 hours. The hydrolysate of each sample was then filtered using 125 mm filter paper. A 200 µl of the filtrates were evaporated at 140 C for about an hour. A diluted buffer (PH 3.5) was added to the dried samples and then the samples were ready for analysis. 61

87 The HPLC system was calibrated with a standard amino acid kit solution and then the sample hydrolysate was injected into the HPLC analyzer system with an auto injector Chemical scores of essential amino acids The essential amino acids of the raw materials and cereal-based baked products (biscuit and bread) were compared as percent of the amount recommended by FAO/WHO/UNU (1985) Caloric value The energy values of the bread and biscuit were calculated based on Atwater factors for protein, fat and carbohydrates as indicated by Leung (1968): Fat factor = 8.37 (kcal/g) Protein factor = CHO factor = 3.87 (kcal/g) 4.12 (kcal/g) 1 kcal = (Kj) Statistical analysis of data Data generated was subjected to the Statistical Package for Social Sciences (SPSS). Means (±SD) were tested using One-factor Analysis of Variance, and then separated using Duncan s Multiple Range Test (Mead and Gurnow, 1983). 62

88 CHAPTER FOUR RESULTS AND DISCUSSION 4.1 Proximate composition and mineral content of the raw Materials Proximate composition of wheat flour The proximate composition of the two types of Australian wheat flours (bread and biscuit) is shown in Table 2. Bread and biscuit flours were found to contain and 88.47% dry matter, 0.65 and 0.63% ash, 13.8 and 13.2% protein, 1.41 and 1.5% fat, and 73.05% carbohydrates on dry matter basis, respectively. These results appear to be in a good agreement with those reported previously by Pyler (1973); Kent-Jones and Amose (1967); Schruben (1979) and Eltoum(2004). The protein content of wheat is very highly influenced by environmental conditions, grain yield and available nitrogen as well as the variety genotype as reported by George (1973).Generally, the protein content for bread flour ranges between 12-14% and for biscuit flour between % as reported by Zeleny (1971) Proximate composition of pigeon pea flour From the results in Table 2, the whole seeds of pigeon pea and decorticated pigeon pea flour were found to have and 91.47% dry matter; and 23.73% protein; 3.96 and 3.73% ash; 1.50 and 1.38 fat and and 62.94% carbohydrates on dry matter basis, respectively. Hulse (1977) found that the protein content of pigeon pea seed samples ranged between 18.5 and 26.3% with a mean value of 21.5%. Singh and Jambunatham (1981a) found that the protein content of 43 commonly cultivated varieties ranged between 17.9 and 24.3% for whole seeds, and between 21.1 and 28.1% for decorticated (Dhal) samples. 63

89 Dahiya et al. (1977) reported a high environmental influence on protein content, and a negative correlation between yield and percentage-seed protein. Mohammed (2002) reported values of 3.63 and 3.37% ash, and 21.62% protein and 2.41 and 2.07% fat for whole and decorticated pigeon pea flour, respectively. Most variability of chemical constituents of pigeon pea can be attributed to differences in analytical methods, and to the origin of samples. Variation in sample origin includes differences in sampling, time after harvest, the cultivar used, and the environment where crop was grown. Decortication of pigeon pea seeds resulted in an increase of protein and carbohydrates and reduction of fat and ash. These results were in agreement with the results obtained by Mohammed (2002) Mineral matter content Table 2 shows the mineral matter content of bread wheat flour (BrdF), biscuit wheat flour (BisF), pigeon pea whole, decorticated pigeon pea flour (DPPF) and pigeon pea protein isolate (PPPI). Bread and biscuit flours were found to contain 55.8 and mg/100 g calcium; 4.33 and 4.40 mg/100 g iron; 3.55 and 3.50 mg/100g cupper and and mg/100 g magnesium on dry matter basis, respectively. Values obtained in this study were higher than the values obtained by Lorenz et al. (1986) for hard and soft wheat. Values of bread (hard) and biscuit (soft) flour were close in mineral contents, and biscuit flour gave higher values of Ca, Fe and Mg than bread flour. Betchart (1988) found that many factors influence mineral concentrations, which include; type and variety of wheat, field location, milling methods and analytical methods. 64

90 Table 2: Proximate composition and mineral matter of raw materials (on dry matter basis) Types of flour Moisture (%) Ash (%) Protein* (%) Fat (%) CHO** Mineral (mg/100 g) (%) Ca Fe Cu Mg Bread flour (BrdF) Biscuit flour (BisF) Whole pigeon pea flour(wppf) Decorticated pigeon pea flour(dppf) Pigeon pea protein isolate(pppi) * Factor of wheat flour N x 5.7; and for pigeon pea N x 6.25 ** CHO calculated by different 65

91 Whole pigeon pea seed mineral content obtained were; , 18.33, 3.65and mg/100 g for Ca, Fe, Cu and Mg on dry matter basis, respectively. Singh et al. (1968) reported higher values of calcium (296 mg/100 g) and lower value of iron (6.7 mg/100 g D.M) compared with the results obtained in this study. Also Faris et al. (1987) reported close values of Ca ( mg/100 g) and lower values of iron (3.9 mg/100 g), Mg (122.0 mg/100 g) and Cu (1.3 mg/100 g). Decortication of pigeon pea seeds resulted in decrease of Ca, Fe and Mg contents. Mineral values of decorticated pigeon pea flour obtained were 77.60, 16.94, 3.85 and mg/100 g for Ca, Fe, Cu and Mg on dry matter basis, respectively. The values obtained were higher than the values obtained by Faris et al. (1987) for split seeds. Singh and Jambunathan (1990) stated that considerable amounts of calcium (about 20%) and iron (about 30%) were removed due to the decortication of pigeon pea seeds, and it does not affect protein quality and amino acids. Pigeon pea protein isolate (PPPI) mineral values obtained were 13.8, 26.06, and mg/100 g for Ca, Fe, Cu and Mg on dry matter basis, respectively. Values of Ca and Mg were lower than the values of whole and decorticated pigeon pea flour; while iron and cupper values were higher in PPPI. It was concluded that from the results obtained, decorticated pigeon pea and pigeon pea protein isolate will be a good source of protein and minerals and consequently a good supplement to wheat flour. 4.2 Effect of pigeon pea seeds decortication on the anti-nutritional factors The effect on tannins content Table 3 shows the anti-nutritional factors i.e. tannins of decorticated and un-decorticated (whole) pigeon pea seeds. The tannins 66

92 content of un-decorticated pigeon pea was 0.13 mg /100 gm, while decorticated pigeon pea gave a value of 0.03 mg /100 gm material. Similar values were reported by Singh (1988) for ten cultivars, and also Mohammed (2002) obtained similar results. Babiker et al., (1993) reported tannin contents of some cultivars of faba bean to be in the range of %. Tannins commonly known as polyphenols; inhibit the activity of digestive enzymes, are higher in chickpea and pigeon pea cultivars with dark seed-coat color as reported by Singh (1984). On the other hand, Price et al. (1980) reported that tannins have not been detected in chickpea but are present at very low concentration ( %) in pigeon pea, although not detected by earlier worker (Habib et al., 1976). Decortication has been reported to influence the polyphenolic compounds and eliminate their levels. Rao and Deosthale (1982) reported that decortication of chickpea and pigeon pea has been reported to reduce polyphenolic compounds by 90%. Soaking (water discard) followed by cooking before consumption is suggested as a mean of removing harmful effects of polyphenolic compounds in the regions where these pulses are consumed as whole seeds, as stated by Rao and Deosthale (1982) The effect on phytic acid content Phytic acid results obtained in this study for whole and decorticated pigeon pea flour were and mg/100 g (DM), respectively (Table 3).These values obtained were lower than the values obtained by Mohammed (2002) who reported for whole and mg/100 g for decorticated pigeon pea flour. 67

93 Table 3: Tannins and phaytic acid content in pigeon pea as affected by decortication Pigeon Pea Flour Tannins (mg/100 gm material) Phytic acid (mg/100 gm) Un-decorticated pigeon pea flour Decorticated pigeon pea flour

94 Phytic acid was mentioned to be stored mainly in the aleurone layer and to a lesser extent in the germ. Thus, milling and/or decortication and germination were greatly found to reduce the amount of phytates (Lasztity and Lastztity, 1990). Dendy (1995) mentioned that, the level of phytate -polyphenols in sorghum was not higher than those reported for wheat, barley and corn but significantly lower than those reported for soybeans and other oilseeds. 4.3 Falling number of wheat flours as affected by inclusion of DPPF and PPPI 4.3.1The effect on bread wheat flour The effect of decorticated pigeon pea flour (DPPF) and pigeon pea protein isolate (PPPI) on falling number (FN) of bread flour (BrdF) is shown in Table 4. Falling number values i.e. alpha-amylase activity of bread flour (control) was found to be 554 seconds. Substitution of DPPF in BrdF increased the values of falling number and values ranged from 614 to 663 sec. Statistical analysis showed no significant differences (P 0.05) between the ratios of 5,10 and 25% of DPPF in BrdF. Highly significant differences (P 0.05) were observed between 15 and 20% of DPPF in bread wheat flour. On the other hand, substitution of pigeon pea protein isolate decreased the falling number gradually and caused reduction to 523, 458 and 388 sec for pigeon pea protein isolate substitution for (15, 20 and 25% protein levels), respectively. 69

95 Table 4: Falling number of wheat flour intended for bread making as affected by inclusion of DPPF and PPPI Flour Blends* Falling Number(Sec.) 100% BrdF(Control) 554.0±10.44 c 95% BrdF + 5% DPPF 627.0±08.54 b 90% BrdF + 10% DPPF 614.7±03.06 b 85% BrdF + 15% DPPF 663.0±02.65 a 80% BrdF+ 20% DPPF 654.7±02.89 a 75% BrdF + 25% DPPF 621.7±04.93 b BrdF + PPPI(Blend 15% protein) BrdF + PPPI(Blend 20% protein) BrdF + PPPI(Blend 25% protein) 523.0±01.00 d 458.0±05.00 e 388.3±13.50 f Calculated F-ratio * C.V% 1.23% Lsd 5% # Mean values (±SD) having different superscript letters in the column differ significantly (P 0.05). Where: BrdF: DPPF: PPPI: Bread flour Decorticated pigeon pea flour Pigeon pea protein isolate 70

96 4.3.2 The effect on biscuit wheat flour The falling number results (alpha-amylase activity) of decorticated pigeon pea flour and pigeon pea protein isolate are shown in Table 5. No significant differences (P 0.05) were observed between the levels of 5 and 15% of DPPF; highly significant differences (P 0.05) were observed in the levels of 10, 20 and 25% DPPF. Substitution of 20 and 25% DPPF decreased the values of the falling number. Incorporation of PPPI significantly (P 0.05) decreased the falling number gradually and values were 515, 478 and 443 for (15, 20 and 25% protein levels) in the blends, respectively. Kaldy and Rubenthaler (1987) found that the falling number of soft white winter and spring wheat ranged between 380 to 451 sec and 111 to 479 sec, respectively. From the results obtained above, it could be observed that; the values of falling number for BrdF and different blends with DPPF were relatively high (low alpha-amylase) and most of the blends were higher than the falling number of wheat flour; that may be attributed to the increase of DPPF in the blends. In contrast, addition of PPPI improved the alpha-amylase activity by decreasing the level of falling number in the blends. Alpha-amylase may be added to wheat flour to achieve any desired level of enzyme activity. The optimum level of enzyme activity is ultimately governed by the end use of the flour and the type of processing involved in the end use, as mentioned by Mailhot and Patton (1988). 71

97 Table 5: Falling number of wheat flour intended for biscuit making as affected by inclusion of DPPF and PPPI Flour Blends* Falling Number(Sec.) 100% BisF(Control) ±7.57 b 95% BisF + 5% DPPF ±16.5 b 90% BisF + 10% DPPF ±6.00 a 85% BisF + 15% DPPF ±7.00 b 80% BisF + 20% DPPF ±6.00 c 75% BisF + 25% DPPF ±7.00 c BisF+ PPPI(Blend 15% protein) BisF. + PPPI(Blend 20% protein) BisF. + PPPI(Blend 25% protein) ±7.51 d ±12.0 e ±1.00 f Calculated F-ratio C.V% 1.45% Lsd 5% # Mean values (±SD) having different superscript letters in the column differ significantly (P 0.05). Where: BisF: Biscuit flour DPPF: Decorticated pigeon pea flour PPPI: Pigeon pea protein isolate 72

98 4.4 Gluten quality and quantity of wheat flours as affected by inclusion of DPPF and PPPI The effect on bread wheat flour Gluten quantity (wet and dry) and gluten quality (gluten index) percentages of doughs prepared from decorticated pigeon pea flour (DPPF) and pigeon pea protein isolate (PPPI) with Australian bread wheat flour are presented in Table 6. The wet gluten percentage of bread wheat flour (control) was 32.25%. Incorporation of DPPF in wheat bread gave values of wet gluten ranged from 25.5 to 32.45%. No significant differences (P 0.05) were observed by addition of 5% DPPF to wheat flour. Increasing levels of DPPF resulted in the significant decrease (P 0.05) in wet gluten; and no significant differences were observed in the levels of 15 and 20% DPPF in bread wheat flour. On the other hand, incorporation of PPPI gave values of wet gluten ranged from 24.5 (for 25 % protein) to 29.8% (for 15 % protein). These values were significantly lower (P 0.05) than the wet gluten of bread wheat flour (32.25 %) The wet gluten content of the whole flour of Sudanese cultivars was found in the range of 26.2 to 31.9% as reported by Mohammed (2000). Ahmed (2005) found the wet gluten content of the flour of Sudanese cultivars to be in the range of to 29.81%. The dry gluten percentage of bread wheat flour was found to be 10.65%. Increasing levels of DPPF and PPPI gave values ranged from for 5% to 8.1% for 25% DPPF; and from 10.0 to 8.4% dry gluten for the incorporation of PPPI. No significant differences (P 0.05) were observed due to incorporation of 5% DPPF in wheat flour. Significant decrease (P 0.05) has occurred in dry gluten percentage of bread flour 73

99 Table 6: Gluten quality and quantity of bread wheat flour as affected by inclusion of DPPF and PPPI Blends* 100% BrdF(control) 95% BrdF + 5% DPPF 90% BrdF + 10% DPPF Wet gluten (%) Dry gluten (%) Gluten index (%) ±0.25 a ±0.15 a ±0.48 a ±0.05 a ±0.05 ab ±1.95 bc ±0.30 b 9.950±0.05 bc ±5.87 bc 85% BrdF+ 15% DPPF ±0.05 c 9.700±0.10 cd ±2.07 c 80% BrdF+ 20% DPPF ±0.35 c 9.250±0.15 de ±5.96 bc 75% BrdF+ 25% DPPF ±0.30 e 8.100±0.10 f ±1.06 c BrdF + PPPI (Blend 15%protein) BrdF + PPPI (Blend 20% protein) ±0.80 c ±0.50 bc ±0.50 b ±0.65 d 9.000±0.50 e ±0.50 bc BrdF + PPPI (Blend 25% protein) ±0.50 f 8.400±0.40f ±0.50 b Calculated F-ratio * C.V% 1.48% 3.00% 4.55% Lsd 5% # Mean values (±SD) having different superscript letters in columns differ significantly (P 0.05). Where: BrdF: Bread flour DPPF: Decorticated pigeon pea flour PPPI: Pigeon pea protein isolate 74

100 with the increase of DPPF and PPPI. Levels of 5, 10% DPPF and PPPI (15% protein) showed no significant differences in dry gluten in the blends. No significant differences were also observed in the levels of 10, 15% DPPF and PPPI (15% protein). Kulkarni et al. (1987) reported that the percentage of dry gluten ranged from 9.4 to 15.1% for hard red winter wheat. Ahmed (2005) reported values from to 9.5 for Sudanese wheat cultivars. The decreasing levels of wet and dry gluten were attributed to the dilution effect of DPPF and PPPI of gluten in bread wheat flour. The gluten index values of bread wheat were found to be 80.73%. Substitution of DPPF and PPPI in bread wheat resulted in a significant decrease (P 0.05) in gluten index. The values ranged from 60.19% for 25% DPPF to 67.5% for PPPI (both levels 15 and 20% protein). No significant differences (P 0.05) were observed with the substitution of PPPI (15, 20 and 25% protein levels) and (5, 10 and 20%) DPPF, in bread wheat flour. Incorporation of DPPF levels (5, 10, and 20) and PPPI (15, 20 and 25% protein) showed no significant differences (P 0.05) in gluten index values. It was observed that higher values of gluten index were obtained from the blends of PPPI when compared with DPPF incorporation in the bread wheat flour The effect on biscuit wheat flour The effect of decorticated pigeon pea flour (DPPF) and pigeon pea protein isolate (PPPI) on wet, dry and gluten index of biscuit wheat flour (BisF) is shown in Table 7. Wet and dry gluten of BisF (control) was found to be and 10.25%, respectively. Incorporation of DPPF and PPPI resulted in a significant decrease in wet and dry gluten. No significant differences (P 0.05) were observed between 5 and 10% DPPF in wet gluten in the blends; and also between 20% DPPF and PPPI (20% 75

101 Table 7: Gluten quality and quantity of biscuit wheat flour as affected by inclusion of DPPF and PPPI Blends* 100% Biscuit flour (BisF) (control) Wet gluten (%) Dry gluten (%) Gluten index (%) ±0.20 a ±0.05 a 80.29±0.02 a 95% BisF + 5% DPPF ±0.05 b ±0.10 a 67.11±0.29 d 90% BisF+ 10% DPPF ±0.28 b 9.750±0.05 b 62.73±0.11 e 85% BisF+ 15% DPPF ±0.25 c 9.250±0.05 c 76.69±1.63 b 80% BisF+ 20% DPPF ±0.25 d 8.640±0.12 d 74.25±0.71 c 75% BisF+ 25% DPPF ±0.73 e 7.337±0.04 e 74.57±2.51 c BisF + PPPI (Blend 15% protein) BisF + PPPI (Blend 20% protein) ±0.20 c 9.500±0.50 bc 73.57±0.93 c ±0.15 d 9.217±0.20 c 76.50±0.50 b BisF + PPPI (Blend 25% protein) ±0.50 f 7.600±0.20 e 66.6±0.50 c Calculated F-ratio * * C.V% 2.08% 0.94% 2.50% Lsd 5% # Mean values (±SD) having different superscript letters in columns differ significantly (P 0.05). Where: BisF: Biscuit flour DPPF: Decorticated pigeon pea flour PPPI: Pigeon pea protein isolate 76

102 protein level). The values of the dry gluten were significantly decreased and the result obtained were 10.1, 9.75, 9.25, 8.64 and dry gluten at 5, 10, 15, 20 and 25% substitutions of DPPF in biscuit wheat flour. No significant differences were noticed in the dry gluten of the blends contained 15% DPPF and PPPI 15 and 20% protein level. Lower values of dry gluten 7.34 and 7.6% were obtained from 25% DPPF and PPPI (25% protein level) and no significant differences were observed between them. The value of the gluten index of biscuit wheat flour was 80.29%. Increasing levels of DPPF and PPPI resulted in decrease of gluten index, and the values ranged from for 10%DPPF to and for both 15%DPPF and 20%protein level, respectively. Incorporation of DPPF resulted in a significant decrease (P 0.05) of the gluten index in all blends; and 10% DPPF obtained the lower values (62.73%) and both 20% DPPF and PPPI (20% protein) gave the higher values of gluten index. No significant differences (P 0.05) were observed in 20 and 25% DPPF and PPPI (15% protein level). Mohammed (2000) reported that the gluten index values of whole flour of Sudanese wheat ranged from to 82.96%. Ahmed (2005) reported values of gluten index of Sudanese whole wheat cultivars; ranged from for El-Nielain to 92.21% for Debeira. 4.5 Farinogram characteristics of doughs Farinograms of doughs prepared from bread wheat flour and composite flour blends The Farinograph behavior of doughs made from bread wheat flour and the various composite flour blends is presented in Table 8 and shown in figures 3 to 11. Water absorption value for bread wheat flour (control) was 65.3%; this value increased to 65.9% for substitution of 5% 77

103 decorticated pigeon pea flour (DPPF) and to 65.7% for both substitution of 10 and 15% of DPPF. The water absorption then decreased till it reached the lowest value (65.1%) for 75% BrdF/25% DPPF. Higher values of water absorption of supplemented samples could be attributed to the higher water absorption capacity and increasing levels of protein content caused by pigeon pea flour. These findings agreed with the results obtained by Sulieman (2005) who had supplemented wheat flour with chickpea flour and similar trends of water absorption increase were observed. On the other hand, the water absorption of bread wheat flour increased with increasing levels of pigeon pea protein isolate. The highest values of 66.3, 71.6 and 74.8% were observed for the levels of protein (15, 20 and 25%) in the blends, respectively. Incorporation of PPPI resulted in higher values of water absorption than DPPF. Mustafa et al. (1986) and Sulieman (2005) reported similar results. Dough development time for BrdF was 7.0 min. The composite flour gave values ranged from 5.0 min for 5% DPPF and 4.0 min for 20 and 25% DPPF in the blends. However, dough development time for PPPI increased steadily to 7.7min for 15% protein level, 12.8 min for 20% protein and 7.5 min for 25% protein compared to the control (7.0 min). This followed the general trends reported by Anaka and Tipples (1979) who reported that dough development time increased in flours with high protein content. The dough stability time of BrdF value (8.5 min) tended to decrease with the substitution of DPPF although PPPI in blend (15% protein) gave the highest dough stability (8.6 min). 78

104 Table 8: Farinograms characteristics of bread wheat flour containing DPPF and PPPI Flour Blends* Water Absorption (%) Dough Development Time (min) Dough Stability Degree of Softening (FU) Farinograph Quality No.(min) 100% Bread Flour (BrdF) 95% BrdF + 5% DPPF % BrdF+ 10% PPF % BrdF+ 15% PPF % BrdF+ 20% PPF % BrdF+ 25% PPF BrdF + PPPI (Blends15% protein) BrdF + PPPI (Blends 20% protein) BrdF + PPPI (Blends 25% protein) BrdF: Bread wheat flour, DPPF: Decorticated pigeon pea flour, PPPI: Pigeon pea protein isolate 79

105 Fig 3: Farinogram of dough prepared from 100 % bread wheat flour Fig 4: Farinogram of dough prepared from a blend of 95% bread wheat flour and 5% DPPF 80

106 Fig 5: Farinogram of dough prepared from a blend of 90% bread wheat flour and 10% DPPF Fig 6: Farinogram of dough prepared from a blend of 85% bread wheat flour and 15% DPPF 81

107 Fig 7: Farinogram of dough prepared from a blend of 80% bread wheat flour and 20% DPPF Fig 8: Farinogram of dough prepared from a blend of 75% bread wheat flour and 25% DPPF 82

108 Fig 9: Farinogram of dough prepared from a blend of bread wheat flour and PPPI (protein level 15%) Fig 10: Farinogram of dough prepared from a blend of bread wheat flour and PPPI (protein level 20%) 83

109 Fig 11: Farinogram of dough prepared from a blend of bread wheat flour and PPPI (protein level 25%) 84

110 The degree of softening for doughs increased sharply from 28 FU for BrdF to 115 FU for both substitutions of 20 and 25% of DPPF. Addition of PPPI to BrdF also resulted in an increase in the degree of softening from 14 FU to 38 FU for 15 and 25% protein level, respectively (Table 8). The Farinograph quality number values decreased gradually from101 min for BrdF (control) to 53 for the substitution of 75% BrdF/25% DPPF. Substitution of PPPI in BrdF resulted in a decrease to 151, 147 and 96 min for protein levels (15, 20 and 25%), respectively Farinograms of doughs prepared from biscuit wheat flour and composite flour blends The Farinogram behavior of doughs made from biscuit wheat flour (BisF) and the various composite flour blends is presented in Table 9 and shown in figures (12 to 20). Water absorption value for BisF (control) was found to be 63.7%. Replacing BisF with DPPF in the ratios of 5 and 10% gave the same value of water absorption (63.7%). The lowest value of water absorption (61.6%) was noticed at the highest substitution of biscuit flour (75%BisF/25% DPPF). On the contrary the water absorption of biscuit flour, when blended with varying amount of PPPI, increased gradually and gave highest values of 66.7, 69.5 and 71.0% for 15, 20 and 25% protein levels of the blends, respectively; compared with BisF (control) value 63.7%. Dough development time of BisF was 6.7 min. A negative increase was observed in dough development time of BisF with the increase of DPPF; the values decreased gradually from 4.4 min for 95 BisF/5 DPPF to 4.0 min for75bisf/25 DPPF. On the other hand, incorporation of PPPI in BisF affected the dough development time and gave the highest value of 7.3 min for 85

111 Table 9: Farinograms characteristics of biscuit wheat flour containing DPPF and PPPI Flour Blends* Water Absorption (%) Dough Development Time (min) Dough Stability Degree of Softening (FU) Farinograph Quality No.(min) 100% Biscuit Flour (BisF) 95% BisF + 5% DPPF % BisF+ 10% PPF % BisF+ 15%DPPF % BisF + 20% PPF % BisF+ 25%DPPF BisF + PPPI (Blend 15% protein) BisF + PPPI (Blend 20% protein) BisF + PPPI (Blend 25% protein) Where: BisF: Biscuit wheat flour, DPPF: Decorticated pigeon pea flour, PPPI: Pigeon pea protein isolate 86

112 Fig 12: Farinogram of dough prepared from 100% biscuit wheat flour Fig 13: Farinogram of dough prepared from a blend of 95% biscuit wheat flour and 5% DPPF 87

113 Fig 14: Farinogram of dough prepared from a blend of 90% biscuit wheat flour and 10% DPPF Fig 15: Farinogram of dough prepared from a blend of 85% biscuit wheat flour and 15% DPPF 88

114 Fig 16: Farinogram of dough prepared from a blend of 80% biscuit wheat flour and 20% DPPF Fig 17: Farinogram of dough prepared from a blend of 75% biscuit wheat flour and 25% DPPF 89

115 Fig 18: Farinogram of dough prepared from a blend of biscuit wheat flour and PPPI (protein level 15%) Fig 19: Farinogram of dough prepared from a blend of biscuit wheat flour and PPPI (protein level 20%) 90

116 Fig 20: Farinogram of dough prepared from a blend of biscuit wheat flour and PPPI (protein level 25%) 91

117 protein level of 20% and the lowest value of 4.5 min obtained for 15% protein level. The dough stability of 10.1 min for BisF tended to decrease with the increasing of DPPF and PPPI substitution. The dough stability value for DPPF substitution ranged from 4.1 to 2.3 min, and for PPPI ranged from 5.8 to 1.7 min. The degree of softening increased sharply from 21 FU for BisF to 173 FU for 75 BisF/25 DPPF. Lower values were obtained when PPPI was incorporated in the blends; 47, 58 and 69 FU for protein levels of 20, 15 and 25%, respectively. Higher values of Farinograph quality number 118 min for BisF was obtained. DPPF incorporation resulted in decrease, and lower values of 55 min was obtained for 75% BisF/25% DPPF and for PPPI 76, 80 and 90 min for 15, 25 and 20% protein levels, respectively. 4.6 Extensograms characteristics of doughs prepared from bread and biscuit wheat flours and composite flour blends The Extensogram characteristics of doughs prepared from bread wheat flour (BrdF) and biscuit flour (BisF) in different blends; with decorticated pigeon pea flour (DPPF) is shown in Tables 10 and 11, and Figures 21 to 30. The Extensogram measures the extensibility (E), the energy (cm 2 ), the resistance (EU) and the resistance to extension (R/E) i.e. ratio, of the doughs from wheat and different composite flours. The stretching properties of the dough, in particular the resistance to stretching and extensibility characterize the flour quality and, consequently, the baking and the processing properties of corresponding dough. 92

118 Table 10: Extensograms characteristics of bread wheat flour as affected by inclusion of DPPF and PPPI Flour Blends Energy (m 2 ) Resistance (EU) Extensibility (mm) R/E % Bread Flour % BrdF+5%DPPF % BrdF+10% DPPF % Brd.F+15%DPPF % Brd.F+20%DPPF Where: BrdF: DPPF: PPPI: Bread flour Decorticated pigeon pea flour Pigeon pea protein isolate 93

119 Table 11: Extensograms characteristics of biscuit wheat flour as affected by inclusion of DPPF and PPPI Flour Blends Energy (m 2 ) Resistance (EU) Extensibility (mm) R/E % Biscuit Flour % BisF+5%DPPF % BisF+10% DPPF % BisF+15% DPPF % BisF+20% DPPF Where: BisF: Biscuit flour DPPF: Decorticated pigeon pea flour PPPI: Pigeon pea protein isolate 94

120 Fig 21: Extensogram of dough prepared from 100% bread wheat flour Fig 22: Extensogram of dough prepared from a blend of 95% bread wheat flour and 5% DPPF 95

121 Fig 23: Extensogram of dough prepared from a blend of 90% bread wheat flour and 10 % DPPF Fig 24: Extensogram of dough prepared from a blend of 85% bread wheat flour and 15% DPPF 96

122 Fig. 25: Extensogram of dough prepared from a blend of 80% bread wheat flour and 20% DPPF Fig26: Extensogram of dough prepared from 100% biscuit wheat flour 97

123 Fig27: Extensogram of dough prepared from a blend of 95 % Biscuit Wheat Flour and 5% DPPF Fig28: Extensogram of dough prepared from a blend of 90% biscuit wheat flour and 10% DPPF 98

124 Fig29: Extensogram of dough prepared from a blend of 85% biscuit wheat flour and 15% DPPF Fig30: Extensogram of dough prepared from a blend of 80% biscuit wheat flour and 20% DPPF 99

125 From the results obtained; the energy of the dough (dough strength), the dough extensibility and dough resistance to extension were decreased with increasing replacement of the two types of wheat flour (bread and biscuit). This in general agreed with the findings of Jone (1991). It was clear that the strength of bread flour was superior to biscuit flour on all different measures of Extensograph. 4.7 Fermentograms feature of doughs prepared from bread and biscuit wheat flours and composite flour blends The production of carbon dioxide of fermented doughs prepared from bread and biscuit flours and composite flours, over the time (two hours) are presented in Tables 12 and 13 and Figures 31 to 45. The objective of fermentation is to bring the dough to an optimum condition for baking. During fermentation, the yeast converts fermentable sugars into carbon dioxide and ethanol. Not enough fermentable sugars are present in the flour to maintain gas production until baking. The action of amylases on available starch is to supplement and form fermentable sugars during fermentation. The activities of the yeast, beside the influence of the ingredients were determined by the time taken by the yeast to evolve carbon dioxide (using the Fermentograph.) The effect of decorticated pigeon pea flour (DPPF) and pigeon pea protein isolate (PPPI) on wheat flour doughs, to produce carbon dioxide over the time, by yeast, shows that; the two types of wheat showed noticeable differences in the time taken to produce CO 2 through the first and second hours. Although the CO 2 production of the two types of wheat flours (bread and biscuit) was close; the time taken for wheat bread (45 min) and the different blends range was (43 to 58 min) were lower than the time taken for biscuit flour (52 min) with its blends range (48 to 60). 100

126 Table 12: Fermentograms feature of doughs prepared from bread wheat flour and composite flour blends Flour Blends* 100% Bread Flour (BrdF) 95% BrdF + 5% DPPF 90% BrdF+ 10% DPPF 85% BrdF+ 15% DPPF 80% BrdF+ 20% DPPF 75% BrdF+ 25% DPPF BrdF + PPPI (Blend 15% protein) BrdF + PPPI (Blend 20% protein) BrdF + PPPI (Blend 25% protein) First hour fermentation Second hour fermentation Vol. Of Time taken Vol. Of Time taken CO 2 (min) CO 2 (min) ND - ND ND - ND - Where: BrdF: Bread flour, DPPF: Decorticated pigeon pea flour, PPP I: Pigeon pea protein isolate ND: Not Determined 101

127 Table 13: Fermentograms feature of doughs prepared from biscuit wheat flour and composite flour blends Flour Blends* 100% Biscuit Flour (BisF) 95% BisF + 5% DPPF 90% BisF+ 10% DPPF 85% BisF+ 15% DPPF 80% BisF+ 20% DPPF 75% BisF+ 25% DPPF BisF + PPPI (Blend 15% protein) BisF + PPPI (Blend 20% protein) BisF. F. + PPPI (Blend 25% protein) First hour fermentation Second hour fermentation Vol. Of Time taken Vol. Of Time taken CO 2 (min) CO 2 (min) ND - ND ND - Where: BisF: Biscuit flour DPP: Decorticated pigeon pea flour PPPI: Pigeon pea protein isolate 102

128 Fig 31: Fermentogram showing CO 2 production in dough prepared from 100% bread wheat flour Fig 32: Fermentogram showing CO 2 production in dough prepared from a blend of 95% bread wheat flour and5% DPPF 103

129 Fig 33: Fermentogram showing CO 2 production in dough prepared from a blend of 90% bread wheat flour and 10% DPPF Fig 34: Fermentogram showing CO 2 production in dough prepared from a blend of 85% bread wheat flour and 15% DPPF 104

130 Fig 35: Fermentogram showing CO 2 production in dough prepared from a blend of 80% bread wheat flour and 20% DPPF Fig 36: Fermentogram showing CO 2 production in dough prepared from a blend of 75% bread wheat flour and 25% DPPF 105

131 Fig 37: Fermentogram showing CO 2 production in dough prepared from a blend of bread wheat flour and PPPI (protein level 20%) Fig 38: Fermentogram showing CO 2 production in dough prepared from 100 % biscuit wheat flour 106

132 Fig 39: Fermentogram showing CO 2 production in dough prepared from a blend of 95 % biscuit wheat flour and 5% DPPF Fig 40: Fermentogram showing CO2 production in dough prepared from a blend of 90%biscuit wheat flour and 10% DPPF 107

133 Fig 41: Fermentogram showing CO2 production in dough prepared from a blend of 85%biscuit wheat flour and 15% DPPF Fig 42: Fermentogram showing CO2 production in dough prepared from a blend of 80%bicuits wheat flour and 20% DPPF 108

134 Fig 43: Fermentogram showing CO2 production in dough prepared from a blend of 75%biscuit flour and 25% DPPF Fig 44: Fermentogram showing CO2 production in dough prepared from a blend of biscuit wheat flour and PPPI (protein level 20%) 109

135 Fig 45: Fermentogram showing CO2 production in dough prepared from a blend of biscuit wheat flour and PPPI (protein level 25%) 110

136 The second hour of fermentation showed reduction in the time taken to produce CO 2 than the first hour Tables 13 and 14. The activity results of the yeast obtained in this study are in agreement with Bronn (1982) who grouped the yeast according to their gas generation. Yeast need less than 60 minutes for dough rising are of good quality. Yeast that need ( minutes), are moderate and yeast that need over 100 minutes are of poor quality. Makoto et al. (1992) reported that the time needed to produce a balloon full (1000 cc) of the Fermentograph is recorded as indicator of the activity of the yeast strain, shorter time (minutes) indicates high activity while increase in time (minutes giving indication of yeast activity slacken). 4.8 Functional properties of wheat flours as affected by inclusion of DPPF and PPPI The effect on bread wheat flour Table 14 shows the effect of decorticated pigeon pea flour (DPPF) and pigeon pea protein isolate (PPPI) on functional properties of wheat flour for bread (BrdF). The bulk density (BD) of the BrdF, DPPF and PPPI was found to be 0.556, and g/ml, respectively. Incorporation of DPPF and PPPI in BrdF was found significantly (P 0.05) increased the bulk density, the values ranged from g/ml for both 80% BrdF/20% DPPF and 75% BrdF/25% DPPF, to g/ml for BrdF/PPPI (protein level 25%), compared with for BrdF. Although the replacement of both DPPF and PPPI increased the bulk density of the different blends; DPPF substitution decreased the value of bulk density, while PPPI replacement increased the values of bulk density by the increasing levels of substitution. Low bulk density is a desirable characteristic when powdered food materials of high nutrients content are to be packed in a limited space or area. 111

137 Table 14: Functional properties of bread wheat flour as affected by inclusion of DPPF and PPPI Flour Blends* Bulk Density (BD) Water Retention Capacity (WRC)g/100g Fat Absorption Capacity (FAC)g/100g 100% BrdF 0.556±0.00 g 130±10.0 e 105±15.0 b (Control) DPPF 0.713±0.00 b 190±10.0 a 55±5.00 c PPPI 0.667±0.00 c 180±0.00 a 100±20.0 b 95% BrdF + 5% DPPF 0.667±0.00 c 140±0.00 d 90±10.0 b 90% BrdF + 10% DPPF 0.646±0.02 d 150±10.0 c 85±5.00 b 85% BrdF + 15% DPPF 0.625±0.00 e 160±0.00 b 90±10.0 b 80% BrdF + 20% DPPF 0.607±0.02 f 160±0.00 b 90±10.0 b 75% BrdF + 25% DPPF 0.667±0.00 c 160±0.00 b 90±10.0 b BrdF +PPPI 0.625±0.00 e 130±10.0 d 105±15.0 b (Blend 15% protein) BrdF +PPPI 0.667±0.00 c 140±0.00 d 125±5.00 a (Blend 20% protein) BrdF +PPPI 0.714±0.00 a 130±10.0 d 90±0.00 b (Blend 25% protein) Calculated F-ratio * 32.76* C.V% 1.30% 4.55% 11.78% Lsd 5% # Mean values (±SD) having different superscript letters in each columns differ significantly (P 0.05). Where: BrdF= Bread flour, DPPF = Decorticated pigeon pea flour, PPPI = Pigeon pea protein isolate 112

138 The water retention capacity (WRC) of the BrdF, DPPF and PPPI was found to be 130, 190 and 180 ml/100 g, respectively. Increasing levels of DPPF in BrdF resulted in the increase of water retention capacity and the values obtained ranged from 140 ml/100 g for 5% DPPF to 160 ml/100 g for 25% DPPF. The water retention capacity increase attributed to the increase in protein content, which resulted from pigeon pea substitution. Significant differences (P 0.05) were observed in the ratios of 5, 10 and 15% of DPPF; while no significant differences were observed between the ratios of 15, 20 and 25% DPPF and the values of water retention capacity (160 ml/100 g) remained constant. Mizubuti et al. (2000) reported values of water retention capacity of pigeon pea flour and pigeon pea protein concentrates about 120 and 107 ml/100 g, these values are considered lower than the values obtained in this study. The fat absorption capacity (FAC) of BrdF, DPPF and PPPI was found to be 105, 55 and 100 ml/100 g, respectively. No significant differences (P 0.05) were observed in all the blends obtained from the replacement of DPPF and PPPI; except of BrdF/PPPI (20% protein level), which showed significant difference (P 0.05) from the other blends and gave the highest value of fat absorption capacity (125 ml/100 g). Sumner et al. (1981) reported that the low fat absorption capacity of pea-protein preparation suggested the presence of a large protein of hydrophilic than hydrophobic groups on the surface of the protein molecules. Fat absorption capacity was also correlated with protein content. Mizubuti et al. (2000) reported values of fat absorption capacity of pigeon pea flour and pigeon pea protein concentrate as 87 and 173 ml/100 g, respectively The effect on biscuit wheat flour Table 15 shows the effect of decorticated pigeon pea flour (DPPF) and pigeon pea protein isolate (PPPI) on functional properties of biscuit 113

139 Table 15: Functional properties of biscuit wheat flour as affected by inclusion of DPPF and PPPI Flour Blends* Bulk Density (BD) Water Retention Capacity (WRC) g/100 g Fat Absorption Capacity (FAC) g/100 g 100% BisF 0.517±0.02 i 140±0.00 e 80±0.00 d (Control) DPPF 0.713±0.00 a 190±10.0 a 55±5.00 e PPPI 0.667±0.00 b 180±0.00 b 100±20.0 b 95% BisF + 5% DPPF 0.667±0.02 g 150±0.00 d 90±0.00 d 90% BisF + 10% DPPF 0.607±0.02 e 155±5.00 c 90±0.00 d 85% BisF + 15% DPPF 0.625±0.00 d 150±10.0 d 90±0.00 d 80% BisF + 20% DPPF 0.646±0.02 c 150±10.0 d 100±0.00 b 75% BisF + 25% DPPF 0.625±0.00 d 118±2.52 g 110±10.0 a BisF +PPPI 0.568±0.02 f 140±0.00 e 95±5.00 c (Blend 15% protein) BisF +PPPI 0.558±0.00 h 140±0.00 e 90±10.0 d (Blend 20% protein) BisF +PPPI 0.625±0.00 d 135±5.0 f 100±0.00 b (Blend 25% protein) Calculated F-ratio * * C.V% 2.00% 3.78% 8.45% Lsd 5% # Mean values (±SD) having different superscript letters in each columns differ significantly (P 0.05). Where: BisF: Biscuit flour, DPPF: Decorticated pigeon pea flour, PPPI: Pigeon pea protein isolate) 114

140 wheat flour (BisF). The bulk density (BD) of the BisF, DPPF and PPPI were found 0.517, and g/ml, respectively. Incorporation of DPPF and PPPI in BisF resulted in a significant increase (P 0.05) in BD of the blends. Values obtained were g/ml for 5% DPPF and g/ml for 25% DPPF in BisF blends. While PPPI incorporation gave values of BD 0.568, and g/ml for protein levels 15, 20 and 25%, respectively. The water retention capacity (WRC) of the BisF, DPPF and PPPI was found to be140, 190 and 180 ml/100g, respectively. Increasing levels of DPPF in BisF resulted in a significant increase (P 0.05) in WRC for all different blends up to 20% DPPF level. Values of WRC ranged from ml/100 g compared with BisF (control ) (140 ml/100 g). No significant differences were observed between 5, 15 and 20% DPPF in the blends. On the other hand, incorporation of PPPI showed no significant differences (P 0.05) and gave the same values of WRC of BisF (control) (140 ml/100 g). Lower values of WRC (118.2 and g/100g) were observed in both 25% DPPF and PPPI (protein level 25%); the highest levels of protein, respectively. The fat absorption capacity (FAC) of BisF, DPPF and PPPI was found to be 80, 55 and 100 ml/100 g, respectively. Incorporation of DPPF showed no significant (P 0.05) differences on the FAC in the ratios of 5, 10 and 15% DPPF. Increasing levels of DPPF to 20 and 25% resulted in the increase of FAC and values obtained were 100 and 110 ml/100 g, respectively. These increases may be attributed to the increase of hydrophobic groups on the surface of the protein molecules. Incorporation of PPPI in BisF resulted in a significant increase in FAC and values obtained were 95, 90 and 100 ml/100 g for 15, 20 and 25% protein levels, respectively. 115

141 It was obvious that, increasing protein levels in the blends, to some extent, resulted in a decrease in WRC and increase in FAC and vise versa. The variations were attributed to hydrophilic and hydrophobic groups in the surface of protein molecules. 4.9 Physical and chemical characteristics of wheat loaf bread containing decorticated pigeon pea flour and pigeon pea protein isolate Loaf bread specific volume The effect of decorticated pigeon pea flour (DPPF) and pigeon pea protein isolate (PPPI) on loaf bread specific volume of wheat bread (control) is shown in Table 16 and plates 10 and 11. Loaf volume of bread baked with up to 10% DPPF was significantly (P 0.05) increased compared with wheat bread (control). The loaf volume then decreased linearly with the proportion of wheat flour with DPPF; from 350 cc for 15% DPPF to 310 cc for 25% DPPF. No significant differences (P 0.05) were observed in bread specific volume for 5 and 10% DPPF replacement. The bread specific volume then decreased gradually till it reached its lowest value (2.86cc/g) for 25% DPPF. These findings agreed with the results obtained by Mustafa et al. (1986) who found that beyond 10% replacement of wheat flour by cowpea flour, the specific volume of the bread decreased. Mustafa (1976) also obtained similar results with 5% soy flour in bread. Dilution of gluten with addition of non-wheat flours to wheat flour has been reported to be associated with loaf volume depression effect of composite flours (DeRuiter 1978; Chavan and Kadam, 1993). On the other hand, incorporation of PPPI resulted in an increase in loaf bread specific volume and gave higher values of 4.63, 4.72 and cc/g for 15, 20 and 25% protein level in the blends, respectively. These 116

142 Table 16: Loaf bread specific volume of wheat flour as affected by inclusion of DPPF and PPPI Flour Blends* 100% BrdF (control) Bread Volume (cc) 3 Bread Weight (g) Bread Specific Volume (cc/g) 370.0±10.00 e 108.6±1.05 b 3.45±0.02 c 95% BrdF + 5% DPPF 391.7±02.89 d 109.1±0.75 b 3.59±0.05 c 90% BrdF+ 10% DPPF 380.0±08.66 f 109.7±0.95 b 3.47±0.11 c 85% BrdF + 15% DPPF 350.0±00.00 g 109.4±0.49 b 3.20±0.02 d 80% BrdF + 20% DPPF 320.0±05.00 h 110.4±1.89 a 2.90±0.06 e 75% BrdF+ 25% DPPF 310.0±08.66 i 108.6±0.06 a 2.86±0.08 e BrdF + PPPI (Blend 15% protein) BrdF + PPPI (Blend 20% protein) 475.0±13.23 b 102.5±0.75 e 4.63±0.15 a 493.3±10.41 a 104.6±0.90 d 4.72±0.14 a BrdF+ PPPI (Blend 25% protein) 420.0±05.00 c 107.8±1.21 c 3.897±0.09 b Calculated F-ratio * * C.V% 2.08% 0.94% 2.50% Lsd 5% # Mean values (±SD) having different superscript letters in each columns differ significantly (P 0.05). Where: BrdF: Bread flour DPPF: Decorticated pigeon pea flour PPPI: Pigeon pea protein isolate 117

143 Plate (10): Loaf Bread and Slices Prepared From Bread Wheat Flour and Decorticated Pigeon pea Flour Where flour blends: A: 100% Bread wheat flour B: 95 % BrdF F/5%DPPF C: 90%Brd F /10%DPPF D: 85%Brd F/ 15%DPPF E: 80%Brd F/20%DPPF F: 75%Brd F/25%DPPf 118

144 Plate (11): Loaf Bread and Slices Prepared From Bread Wheat Flour And pigeon pea protein isolate Where : A: 100% Wheat flour bread B: BrdF F/PPPI (Blends 15% protein) C: BrdF F /PPPI (Blends20% protein) D: BrdF F/ PPPI (Blends25% protein) 119

145 results were in agreement with Bean (1990) who mentioned that dough containing low protein gave lower loaf volume. Perten (1980) reported that the volume of loaf bread decrease was due to the reduction of protein and fiber in the flour. Hestangen and Frolish (1983) and Lukour (1990) reported values of bread volume 355, 376cc for Canadian wheat flour bread. Bread specific volume increased positively (P 0.05) with bread volume. There were highly significant differences (P 0.05) in loaf bread specific volume in the ratios of 15, 20 and 25% DPPF. However, no significant differences were observed in 15, 20% protein level with PPPI replacement; while 25% protein level gave the lowest value (3.897cc/g) of PPPI substitution for bread specific volume. The decrease in bread specific volume attributed to the dilution of gluten and hence resulted in lower gas retention and lower bread volume. The major factor accounting for variation in loaf volume within a variety was protein content; the relation between loaf volume and protein content was essentially linear between 8-18% proteins. From the results obtained, it was concluded that uses of DPPF beyond 10% has a negative effect on loaf bread specific volume. However, incorporation of PPPI shows significant increase and higher values of loaf bread specific volume. In general, the bread making potential decreases linearly with the proportion of wheat flour. Youssef and Bushuk (1986) mentioned that the proportion of non-wheat flour depends on the inherent strength of the wheat flour. The method of preparation can increase or diminish the functionality of protein isolate in bread doughs. Flours from stronger wheat cultivars can carry a higher percentage of the non-wheat products and still meet the specifications of the baked products. 120

146 4.9.2 Chemical composition and energy value Table 17 shows the effect of decorticated pigeon pea flour (DPPF) and pigeon pea protein isolate (PPPI) on chemical composition and energy value of wheat flour bread. Increasing levels of DPPF and PPPI in wheat flour for bread resulted in a significant increase (P 0.01) in ash, protein and fat contents. Values of ash ranged from for 5% to % for 25 % replacement of DPPF, compared with wheat bread (Control) 0.973%. Incorporation of PPPI increased the ash content and values obtained were 0.952, and 1.429% for 15, 20 and 25% protein levels, respectively, compared with control bread (0.973%). Higher incorporation levels for both DPPF and PPPI gave close values of ash, and no significant differences were observed between them. The protein content increased significantly (P 0.01) with increasing levels of DPPF. The protein content of wheat bread was found to be 14.62%. This value increased to 15.18, 15.51, 16.10, and for replacements of 5, 10, 15, 20 and 25% DPPF, respectively. No significant differences were observed between 5 and 10% and between 20 and 25% replacements. Incorporation of PPPI significantly increased (P 0.01) the protein of supplemented bread to 16.19, and 25.86% for protein levels 15, 20 and 25%, respectively. Close values of protein were obtained; and % for 15% DPPF replacement and 15 % protein level for PPPI, respectively. These results of ash and protein obtained were similar to those reported by suleiman (2005), who had supplemented wheat flour with chick pea flour to produce bread. The control wheat bread had the highest content of carbohydrates (77.17%).This value was significantly decreased (P 0.01) with increasing levels of supplementation till it reached 73.05% for the high level (25% DPPF) of replacement. On the other hand, incorporation of PPPI resulted in a significant decrease in carbohydrates, from 77.17% for wheat flour 121

147 Table 17: Chemical composition and energy value of wheat flour bread containing different levels of DPPF and PPPI Flour Blends Dry matter (%) Ash (%) Protein (%) Fat (%) Carbohydrates (%) Caloric value (kcal/100 g) DM 100% Bread wheat (BrdF) d g f 1.38 d a fg 95% BrdF + 5% DPPF ab f e 1.54 cd b de 90% BrdF+ 10% DPPF e e e 1.83 bc c fg 85% BrdF+ 15% DPPF e c d 2.63 a e d 80% BrdF+ 20% DPPF cd b c 2.52 a f c 75% BrdF+ 25% DPPF bc a c 2.70 a g b BrdF + PPPI ab g d 2.42 a d a (Blend 15% protein) BrdF + PPPI a d b 1.75 c h ef (Blend 20% protein) BrdF + PPPI b a a 2.10 b i g (Blend 25% protein) SE± CV (%) , # Mean values having different superscript letters in each column differ significantly (P 0.05). Where: BrdF: Bread flour DPPF: Decorticated pigeon pea flour PPPI: Pigeon pea protein isolate

148 bread to 74.93, and 65.00% for 15, 20 and 25% protein levels, respectively. Significant increase (P 0.01) in caloric values were observed due to incorporation of DPPF; values increased from for 5% DPPF to Kcal/100g for 25% replacement, compared with control wheat bread Kcal/100g. In contrast, incorporation of PPPI resulted in a significant decrease (P 0.01) of caloric value with increasing levels of protein. Values decreased from for 15% protein level to and Kcal/100g for 20 and 25% protein levels, respectively. It is concluded that, increasing levels of DPPF resulted in an increase of caloric value and vise versa for incorporation with PPPI. These finding maybe attributed to the increase of protein coupled with an increase in fat content for the replacement with DPPF; while increasing level of protein and decreasing level of fat for the replacement of PPPI Amino acids composition Amino acid profile of proteins of wheat bread containing different levels of decorticated pigeon pea flour (DPPF) and pigeon pea protein isolate (PPPI) is shows in Table 18 and Figures 46 to 55. Tryptophan is an essential amino acid, and nutritionally important. This amino acid was not determined in the present study as it is not stable to acid hydrolysis. Amino acid composition data provides useful bases for predicting the nutritional values of protein source for humans. As reported above, the protein content of DPPF was 23.73% and PPPI was 81.12%. The amino acid composition (g/100 g protein) of wheat bread (control) and bread containing different levels of DPPF and PPPI revealed some noticeable differences. Levels of lysine, methionine, threonine and tyrosine were relatively low in wheat bread; values obtained were 2.74, 1.62, 2.49 and 2.12 g/100 g protein, respectively. 123

149 Table 18: Amino acid profile of proteins of wheat flour bread containing different levels of DPPF and PPPI* (on dry basis) Flour Blends % levels of DPPF PPPI (%Protein level) /Amino Acid (g/100 g protein) Essential amino acids Histidine (for children) Isoleucine Leucine Lysine Methionine Cystine NP NP NP NP NP NP NP NP NP Phenylalanine Tyrosine Thrionine Tryptophan ND ND ND ND ND ND ND ND ND Valine Non-essential amino acids (NEAA) Alanine Arginine Aspartic acid Glutamic acid Glycine NP Proline Serine * Results of a single determination; ND = Not determined; NP = No peak found 124

150 Fig 46: Amino acid profile of the standard curve Fig 47: Amino acid profile of 100 % wheat flour bread 125

151 Fig 48: Amino acid profile of wheat flour bread containing 5 % DPPF Fig 49: Amino acid profile of wheat flour bread containing 10 % DPPF 126

152 Fig 50: Amino acid profile of wheat flour bread containing 15 % DPPF Fig 51: Amino acid profile of wheat flour bread containing 20 % DPPF 127

153 Fig 52: Amino acid profile of wheat flour bread containing 25 % DPPF Fig 53: Amino acid profile of wheat flour bread containing PPPI (protein level of the blend 15%) 128

154 Fig 54: Amino acid profile of wheat flour bread containing PPPI (protein level of the blend 20%) Fig 55: Amino acid profile of wheat flour bread containing PPPI (protein level of the blend 25%) 129

155 Chung and Pomeranze (1985) reported values closed to those obtained in this study. Lysine-deficiency is considered as a major nutritional problem in wheat protein. According to the literature, this problem could be solved either through direct addition of lysine or supplementation by legumes (Jansen, 1970 and Dendy, 1995). Pigeon pea is found to be an excellent and rich source of amino acid lysine as reported by Singh et al. (1981); Singh and Eggum (1984); Singh et al. (1990); Ahmed and Nour (1990) and Mohammed (2002). Bressani (1972) reported that food legumes are rich source of protein and the nutritional deficiencies of protein arise, in general, from low sulphur amino acids content. Among the non-essential amino acids (NEAAs), wheat bread protein had higher values of glutamic acid (27.474) and proline (14.794) compared to pigeon pea protein. These findings agreed with the results obtained by Chung and Pomeranz (1985) who reported that the common characteristics of protein in cereal grains are that; glutamic acid is a major amino acid (AA) and tryptophan is a minor, and the second major AA is proline. Pigeon pea showed higher values of aspartic acid (11.24%), arginine (5.50), glycine (3.84) and serine (5.27) as reported by Mohammed (2002). Hulse (1976) reported that both lysine and methionine are higher in the outer than the inner fractions in pigeon pea. Also he reported that analysis quoted by different authors showed wide variations in AA content of pigeon pea. Supplementation with DPPF and PPPI resulted in an increase of some essential amino acids of wheat proteins and different blends. Lysine increased gradually from 2.74 for wheat bread to 2.97, 3.19, 3.42, 3.65 and 3.83 g/100g protein when wheat flour was supplemented with 5,10,15,20, and 25%DPPFAlso incorporation of PPPI resulted in increase in lysine content and values 130

156 obtained were 3.37, 4.04 and 5.33g/100g protein for protein level 15, 20 and 25 %, respectively. Methionine, valine, glutamic acid and serine, showed reduction with increasing levels of DPPF. Increasing levels of PPPI resulted in a decrease of proline from to and for 15, 20 and 25% protein levels, respectively; although it was still higher than the value of wheat bread control (14.80 g/100g) Amino acids chemical scores Table 19 shows the effect of decorticated pigeon pea flour (DPPF) and pigeon pea protein isolate (PPPI) supplementation on amino acids chemical scores (CS) of wheat bread. From the results obtained, the essential amino acids chemical scores increased gradually with increasing different substitution levels; for both DPPF and PPPI. Lysine is found to be the first limiting amino acid in wheat bread and all different blends when incorporated with DPPF and PPPI. The protein chemical scores for lysine were increased gradually from 50 for wheat bread to 55, 59, 67, and 71% due to the incorporation of 5, 10, 15, 20 and 25% DPPF respectively. Incorporation of PPPI in wheat bread also resulted in increase in chemical scores for lysine from 62 to74 and 98%for 15, 20 and 25% protein levels, respectively. Higher scores were observed when wheat bread was supplemented with higher levels of PPPI. Threonine was found to be the second limiting amino acid of wheat bread and all other different blends; and its chemical scores increased with increasing levels of both DPPF and PPPI. The chemical scores for thrionine increased from 64 to 87% for incorporation with DPPF; and from 65 to 107% for PPPI compared with wheat bread 62%. Wolzak et al. (1985) mentioned that the most common method for evaluation of protein quality is usually based primarily on its content of a certain essential amino acids that in greatest deficit when it is compared to its amount in a reference protein 131

157 Table 19: Effect of pigeon pea supplementation on amino acids chemical scores of wheat flour bread Flour Blends Amino acid % Levels of decorticated pigeon pea flour (DPPF) % Protein levels with PPPI Recommended AA levels* Essential amino acids Chemical scores of amino acids (%) g/100 g protein Histidine (for children) Isolucine Leucine Lysine Met. + Cys** Phe + Tyr*** Threonine Valine *Dendy ** Phenylalanine and tyrosine ***Methionine and cystine 132

158 In this study, the protein pattern recommended by FAO/WHO/UNU (1985) and Dendy (1995) is used as a reference protein for infants. The results of CS obtained in this study was close to those obtained by Faris and Singh (1990) who reported values of essential amino acids scores of wheat supplemented with pigeon pea flour at ratios of 10, 20 and 30%. They reported that methionine and cystine followed by tryptophan and threonine are the limiting essential amino acids in pigeon pea, whereas lysine is the first limiting amino acid of wheat. Chung and Pomeranze (1985) reported that gluten and its protein fractions are nutritionally inferior to whole wheat protein or to soluble proteins, primarily because of extremely low lysine scores. They also stated that, both lysine and threonine scores are substantially lower in gliadin and its fractions than in glutenin. From the results obtained, it is apparent that pigeon pea improves the amino acids chemical scores for lysine, threonine, leucine and isolucine in wheat based diets if the proportion of DPPF and PPPI increased in wheat pigeon pea backed products Mineral matter content Table 20 shows the effect of decorticated pigeon pea flour (DPPF) and pigeon pea protein isolate (PPPI) on mineral matter of wheat bread. Calcium (Ca) content was found to be mg/100 g in wheat bread (control) on dry basis. Incorporation of decorticated pigeon pea flour resulted in an increase from mg/100 g for 5% DPPF to mg/100 g for 20% DPPF. On the other hand, PPPI obtained values of Ca , and mg/100 g for 15, 20 and 25% protein levels, respectively. It is observed that inclusion of DPPF and PPPI in wheat bread improved the Ca content and hence improves its nutritional value 133

159 Increasing levels of both DPPF and PPPI resulted in an increase in iron (Fe) content. Values obtained ranged from more than 9.72 to mg/100 g (DB) due to incorporation of DPPF, and from to mg/100 g for incorporation of PPPI compared with wheat bread (control) 8.64 mg/100 g. Cupper (Cu) and magnesium (Mg) contents increased gradually with increasing levels of DPPF and PPPI. The values of Cu and Mg of wheat bread (control) were found as 1.54 and mg/100 g, respectively. Highest values of Cu obtained were found beyond 9.14 mg/100 g for 25% DPPF level and 6.27 mg/100 g for PPPI (25% protein). On the other hand, highest values of Mg were found to be and mg/100 g for DPPF (25% level) and PPPI (25% protein), respectively Table Sensory evaluations of wheat loaf bread containing different levels of DPPF and PPPI Sensory evaluations of bread containing decorticated pigeon pea flour (DPPF) and pigeon pea protein isolate (PPPI) are presented in Tables 21 and 22.General appearance, flavor, taste, texture and overall quality of breads made with up to 10% DPPF level of replacement was found significantly better than the control bread Table 21.No significant differences (P 0.05) were observed between 5, 10 and 15% DPPF in all sensory characteristics; except of flavor and taste for 15% DPPF level, which showed significant differences from the other levels. Increasing levels of the replacement of DPPF resulted in a decrease of the organoleptic quality of the bread. However, 25% DPPF level of replacement was significantly lower in appearance, flavor and overall quality. It seems that addition of DPPF affected the flavor and taste, 134

160 Table 20: Mineral matter content of wheat flour bread (mg/100 g) containing DPPF and PPPI* 100% Bread Flour (control) Blends Ca Fe Cu Mg % BrdF+5%DPPF % BrdF+10%DPPF % BrdF+15%DPPF % BrdF+20%DPPF % BrdF+25%DPPF ND ND BrdF+ PPPI (Blend 15% Protein) BrdF+ PPPI (Blend 20% Protein) BrdF+ PPPI (Blend 25% Protein) *On dry matter basis Where: BrdF = Bread flour DPPF = Decorticated pigeon pea flour PPPI = Pigeon pea protein isolate 135

161 which was observed in the significant differences in the various increased levels. This sharp decrease is probably due to the domination of pigeon pea flavor at the increasing levels. These results were in agreement with the findings obtained by Suleiman (2005) who found that at 10% level, chickpea odor was dominant in the loaf. On the other hand, incorporation of pigeon pea protein isolate (PPPI) resulted in significant differences from control bread Table 22. The protein level of 15% in the blend was found significantly better than the control bread and other different blends. Significant decrease (P 0.05) in sensory characteristics was observed when BrdF was supplemented with 20% and 25% protein level in the blends. The high level of protein (25%) in the blends gave the lowest quality attributes of sensory evaluations and overall quality. It is concluded that, there was a significant (P 0.05) effect of replacement of wheat flour with decorticated pigeon pea flour and pigeon pea protein isolate on sensory evaluations. Decorticated pigeon pea flour can be used to produce highly acceptable bread supplemented with up to 10% DPPF. While pigeon pea protein isolate can be used to produce highly acceptable bread with (15%). protein level in the blend. 136

162 Table 21: Sensory evaluations of wheat loaf bread containing decorticated pigeon pea flour Flour Blends* General appearance Sum of ranks* Flavor Taste Texture Overall quality 100% Bread Flour (BrdF) 89 a 73 a 80 a 92 a 94 a 95% BrdF+ 5% DPPF 50 b 49 b 50 b 51 b 56 b 90% BrdF + 10% DPPF 50 b 50 b 51 b 46 b 53 b 85% BrdF + 15% DPPF 48 b 61 a 60 c 50 b 54 b 80% BrdF + 20% DPPF 67 c 80 c 69 c 72 c 75 c 75% BrdF + 25% DPPF 95 a 87 c 87 a 86 a 88 c # Mean values (±SD) having different superscript letters in each columns differ significantly (P 0.05). Where: *BrdF: Bread flour, DPPF: Decorticated pigeon pea protein flour 137

163 Table 22: Sensory evaluations of wheat loaf bread containing pigeon pea protein isolate Flour Blends* 100% Bread Flour (control) BrdF + PPPI (Blend 15% protein) BrdF + PPPI (Blend 20% protein) BrdF + PPPI (Blend 25% protein) General appearance Sum of ranks* Flavor Taste Texture Overall quality 59 a 34 a 42 a 42 a 39 a 31 a 31 a 31 b 35 b 30 b 35 b 51 b 45 a 47 a 45 a 65 a 74 c 72 c 66 c 66 c # Mean values (±SD) having different superscript letters in each columns differ significantly (P 0.05). Where: *BrdF: Wheat flour, PPPI: Pigeon pea protein isolate 138

164 4.11 Physical and chemical characteristics of wheat biscuit containing DPPF and PPPI Biscuit spread ratio The effect of decorticated pigeon pea flour on the physical characteristics i.e. width (cm), thickness (cm) and spread ratio of wheat biscuit are shown in Table 23 and (plate 12). The width of the biscuit (control) was cm. This value increased gradually with the incorporation of DPPF till it reached the highest value of and cm for both levels of 80% BisF/20% DPPF and 75% BisF/25% DPPF, respectively. Highest value of thickness cm was also obtained for 75% BisF/25% DPPF blends. The biscuit made from the blends had spread ratios ranged from 6.08 for 95% BisF/5% DPPF to and for replacement of 20 and 25 % DPPF, respectively. The increase in protein content due to incorporation of DPPF did not restrict the spread ratio of the biscuits; on the contrary, it increased the spread ratio noticeably. As reported by Lehman et al. (1994) causes of increased cookie spread ratio are low protein content with poor protein quality and low initial or slowly rising heat during baking. No significant differences (P 0.05) were observed in the spread ratio values; 6.014, 6.087, and for 0, 5, 15 and 25 DPPF in the blends, respectively. Higher values of spread ratio and obtained from 10 and 20% of DPPF in the blends, respectively; and no significant differences (P 0.05) were shown for spread ratio of these levels 139

165 Plate (12): Biscuit Prepared From Biscuit Wheat Flour and Decorticated Pigeon pea Flour Where flour blends: A: 100% Biscuit Wheat flour B: 95 % BisF/5%DPPF C: 90%BisF /10%DPPF D: 85%Bis F/ 15%DPPF E: 80%Bis F/20%DPPF F: 75%Bis F/25%DPPf 140

166 Table 23: Spread ratio of wheat flour biscuit as affected by inclusion of DPPF 100% BisF (control) Flour Blends* Width (cm) Thickness (cm) Spread ratio (width/thick) 5.238±0.11 d 0.871±0.07 d 5.849±0.25 c 95% BisF + 5% DPPF 5.449±0.08 c 0.949±0.06 a 6.608±0.35 d 90% BisF+ 10% DPPF 5.467±0.18 c 0.883±0.02 c 6.262±0.20 a 85% BisF+ 15% DPPF 5.600±0.10 b 0.926±0.03 b 6.084±0.24 b 80% BisF+ 20% DPPF 5.782±0.18 a 0.920±0.02 b 6.288±0.27 a 75% BisF + 25% DPPF 5.798±0.14 a 0.960±0.04 a 6.059±0.22 b Calculated F-ratio * C.V% 2.47% 4.88% 4.28% Lsd 5% # Mean values (±SD) having different superscript letters in each columns differ significantly (P 0.05). Where: * BisF: Biscuit flour DPPF: Decorticated pigeon pea flour 141

167 El Shiekh (2004) reported similar value of spread ratio (6.16), while Eltoum (2004) reported higher values of spread ratio (8.08) for biscuit made from Australian wheat flour. Kulp (1994) reported that fats with higher levels of solids tend to have the least effect on spread ratio; while fats with very low solids gave a greater effect. The effect of pigeon pea protein isolate on physical characteristics of wheat biscuit (control) and blends were shown on Table 24 and plate 13. Incorporation of PPPI resulted in an increase in biscuit width from to 5.317, and cm for 15, 20 and 25% protein level in the blends, respectively. Statistical analysis showed no significant differences in the spread ratios of biscuit from wheat flour and composite flour blends. The spread ratio of biscuits decreased, as the pigeon pea protein isolate (protein levels) increased compared with cm for wheat biscuit (control).the values ranged from cm for BisF/PPPI (protein level 15%) to cm for BisF/PPPI (protein level 25%). Generally, the increase in protein content restricted the spread ratio of biscuits. This agreed with Tsen (1976) who reported that fortifying with protein rich food additives can drastically reduce cookie spread and increase thickness Chemical composition and energy evaluation Table 25 shows the effect of inclusion of decorticated pigeon pea flour (DPPF) and pigeon pea protein isolate (PPPI) on chemical composition and energy values of wheat flour biscuit. The ash content was significantly increased (P 0.01) with the increasing levels of replacement of DPPF. The ash content of wheat flour biscuit (control) was found to be 0.917%, this value increased to 0.952, 0.994, 1.042, and 1.292% for 5, 10, 15, 20 and 25% DPPF level in the blends, respectively. No significant differences were observed between 5%, 10% 142

168 Plate (13): Biscuit Prepared from Biscuit Wheat Flour and Pigeon pea protein isolate Where: A: 100% Wheat Biscuit B: Bis F/PPPI (Blendes 15% protein) C: Bis F /PPPI (Blends20% protein) D: Bis F/ PPPI (Blends25% protein) 143

169 Table 24: Spread ratio of wheat flour biscuit as affected by inclusion of PPPI Flour Blends BisF (control) BisF +PPPI (Blend 15% protein) BisF. +PPPI (Blend 20% protein) width (cm) Thickness (cm) Spread ratio (width/thick) 5.238±0.11 d 0.871±0.07 b 5.849±0.25 a 5.317±0.18 c 0.924±0.04 c 5.758±0.23 a 5.411±0.11 b 0.969±0.07 a 5.606±0.38 a BisF +PPPI (Blend 25% protein) 5.466±0.14 a 0.931±0.05 c 5.809±0.24 a Calculated F-ratio n.s C.V% 2.59% 6.53% 4.86% Lsd 5% # Mean values (±SD) having different superscript letters in each columns differ significantly (P 0.05). Where: BisF: Biscuit flour PPPI: Pigeon pea protein isolate 144

170 Table 25: Chemical composition and energy value of wheat flour biscuit containing different levels of DPPF and PPPI Flour Blends Dry matter (%) Ash (%) Protein (%) Fat (%) Carbohydrates (%) Caloric value (kcal/100 g) DM 100% Biscuit Flour(BisF) cd e g d a bc 95% BisF + 5% DPPF c e g d a bc 90% BisF+ 10% DPPF de de f cd b bc 85% BisF+ 15% DPPF cd cd e bc c bc 80% BisF+ 20% DPPF a b d b d ab 75% BisF+ 25% DPPF a a c b e c BisF + PPPI b cd de a d a (Blend 15% protein) BisF. + PPPI e c b b f d (Blend 20% protein) BisF + PPPI f b a bc g e (Blend 25% protein) SE± CV (%) # Mean values having different superscript letters in each column differ significantly (P 0.05). Where: BisF: Biscuit flour DPPF: Decorticated pigeon pea flour PPPI: Pigeon pea protein isolate

171 levels of replacement and the control. On the other hand, incorporation of PPPI showed increase in ash content when compared with wheat flour biscuit. Values obtained were 1.049, and1.179% for protein levels 15, 20 and 25%, respectively. No significant differences were noticed between 15 and 20% protein levels. Similar values were obtained for ash content for 15% DPPF replacement and 15% protein level for PPPI; and no significant differences were found between them. Increasing levels of DPPF resulted in a significant increase (P 0.01) in protein content. Values obtained were found to be 13.91, 14.78, 15.31, and for 5, 10, 15, 20 and 25% DPPF, compared with wheat flour biscuit protein (13.62%). On the other hand, increasing levels of PPPI resulted in a significant increase in protein content and values obtained were found to be 15.62, and % for 15, 20 and 25 protein levels, respectively. No significant differences were observed between the control and 5% replacement; also between 15% DPPF (15.31%) and 15 % Protein level (15.62%). Values of fat obtained were between for 5% DPPF and 14.52% for 25% replacement; and for high level of protein (25% protein); by inclusion of PPPI, compared with wheat biscuit control (14.20% protein). Incorporation of DPPF resulted in a significant decrease (P 0.01) of carbohydrates from 67.00% for 5% DPPF to the value of 63.91% for 25% replacement compared with wheat flour biscuit (67.31%).While incorporation of PPPI resulted in a significant decrease of carbohydrates and values obtained were 64.83, and for 15, 20 and 25% protein levels, respectively. 146

172 No significant differences were observed in the caloric values when DPPF was incorporated in wheat flour up to the level of 15% replacement, compared with wheat flour biscuit. Values ranged from to kcal/100g. Incorporation of PPPI in biscuit wheat flour resulted in a significant decrease (P 0.01) of caloric value of supplemented biscuit. Values obtained were 450.6, and kcal/100g for 15, 20 and 25% protein levels, respectively. The results obtained above were in agreement with those reported by Sulieman et al (2003) who reported that supplemented cookies with cowpea increased ash, protein and fat with increasing levels of replacement Amino acids composition General profile of amino acids (AAs) of wheat biscuit containing different levels DPPF and PPPI is shown in Table 26 (Figures 56 to 64). The levels of essential amino acids (g/100 g protein) of wheat biscuit (control) was found lower in lysine (1.92), methionine (1.91), cystine (0.75) and tyrosine (2.40). These values obtained from wheat biscuit were close to the values of amino acids obtained from wheat bread used in this study. Incorporation of DPPF and PPPI in wheat biscuit resulted in an increase of essential amino acids for all the blends; except methionine and valine, which showed reduction with increasing levels of DPPF. Values obtained for methionine decreased gradually from 1.91for wheat biscuit to 1.87, 1.83, 1.78, 1.74 and 1.70 g/100 g protein; for incorporation of 5,10,15,20 and25%, respectively. Valine also decreased from 4.91 for wheat biscuit to 3.56 g/100 g protein for replacement of 25% DPPF and to 2.70, 3.51 and 3.86 for PPPI; protein levels 15, 20 and 25%, respectively.among the non-essential amino acids, aspartic acid (2.96), glutamic acid (11.365) and proline (12.96) were also found lower than the values obtained from wheat bread. This decrease may be 147

173 attributed to the lower values of gluten (glutenin and gliadin) in wheat biscuit. The latter two amino acids are also considered higher when compared with those of pigeon pea flour. From the results obtained it is also observed that lysine, which is considered deficit in wheat flour was increased gradually from 1.92 in wheat biscuit to 2.19,2.45,2.72,2.99 and3.26 g/100 g protein for 5,10,15,20 and 25 %DPPF replacement. Also incorporation of PPPI increased lysine to 3.17, 3.37 and 4.62 for protein levels (15, 20 and 25%), respectively. It is concluded that pigeon pea flour and protein isolate will be a good supplement for production of fortified biscuit. 148

174 Table 26: Amino acid profile of proteins of wheat flour biscuit containing different levels of DPPF and PPPI* (On dry basis) Flour Blends % levels of DPPF PPPI (%Protein level) Amino Acid (g/100 g protein) Essential amino acids Histidine (for children) NP Isoleucine Leucine 6.82 NP Lysine Methionine Cystine NP 0.77 Phenylalanine Tyrosine Thrionine Tryptophan ND ND ND ND ND ND ND ND ND Valine 4.91 NP Non essential Amino Acids (NEAA) Alanine 4.33 NP Arginine NP NP NP NP NP NP NP NP NP Aspartic acid NP 3.69 Glutamic acid Glycine NP 0.36 NP 1.11 NP NP Prolin Serine NP NP NP NP NP NP - * Results obtained from one determination; ND = Not determined; NP = No peak found. 149

175 Fig 56: Amino acid profile of 100 % wheat flour biscuit Fig 57: Amino acid profile of wheat flour biscuit containing 5 % DPPF 150

176 Fig 58: Amino acid profile of wheat flour biscuit containing 10 % DPPF Fig 59: Amino acid profile of wheat flour biscuit containing 15 % DPPF 151

177 Fig 60: Amino acid profile of wheat flour biscuit containing 20% DPPF Fig 61: Amino acid profile of wheat flour biscuit containing 25% DPPF 152

178 Fig 62: Amino acid profile of wheat flour biscuit containing PPPI (protein level of the blend 15 %) Fig 63: Amino acid profile of wheat flour biscuit containing PPPI (protein level of the blend 20%) 153

179 Fig 64: Amino acid profile of wheat flour biscuit containing PPPI (protein level of the blend 25%) 154

180 Amino acids chemical score The essential amino acids chemical scores (EAACs) of wheat biscuit and different blends containing DPPF and PPPI is shown in Table 27. The chemical scores were calculated based on a comparison with the reference pattern recommended by FAO/WHO/UNU (1985) and Dendy (1995). From the results obtained, the EAACs increased gradually with supplementation with DPPF and PPPI, except methionine plus cystine which showed reduction in CS from 76% in wheat biscuit which decreased gradually to 70% with incorporation of 25% DPPF, as pigeon pea is usually deficient in sulphur-containing amino acids; methionine and cystine. Singh et al. (1981) reported that methionine and cystine are partly destroyed during hydrolysis. Boulter and Thompson (1976) reported that from the nutrition point of view, the levels of both methionine and cystine are important and should be considered together. Lysine is found to be the first limiting amino acid in wheat biscuit and the different blends with DPPF and PPPI. The lysine chemical scores of wheat biscuit (35%) increased gradually and values obtained were 40,45,50,55 and 60% for 5,10,15,20 and 25%, respectively. Inclusion of PPPI resulted in increase in lysine chemical scores, and values were found to be 58, 62 and 85% for protein levels15, 20 and 25%, respectively. Thrionine was also found to be the second limiting amino acid in wheat biscuit and the biscuit of other blends and its chemical scores increased from 60 for wheat biscuit to 70% for the incorporation of 155

181 Table (27): Effect of pigeon pea supplementation on amino acids chemical scores of wheat flour biscuit Flour Blends Amino acids % Levels of decorticated pigeon pea flour % Protein levels with PPPI Recommended levels* Essential amino acids Chemical scores of amino acids (%) g/100 g protein His Isoleu Leu Lys Met. + Cys** Phe. + Tyr.*** Thr Val * Dendy

182 25% DPPF. Higher scores of lysine were observed when wheat biscuit was supplemented with PPPI. It is concluded that lysine and sulphur containing amino acids were considered as two of the important protein components in cereals and legumes based diet, as they tend to complement each other Mineral matter content Table 28 shows the effect of DPPF and PPPI on mineral matter of wheat biscuit. Calcium (Ca) content was found to be mg/100 g in wheat biscuit (control) on dry matter basis. Incorporation of DPPF resulted in an increase of Ca values from to and mg/100 g (DM) for replacement of (5, 10 and 15%) DPPF. Inclusion PPPI increased the calcium content from to and 98.9 mg/100 g (DM) for (15, 20 and 25% protein levels), respectively. Iron (Fe) and magnesium (Mg) contents were increased with increasing levels of DPPF and PPPI. Values of Fe and Mg of wheat biscuit obtained were 3.07 and mg/100 g (DM), respectively. Highest values of Fe and Mg obtained from replacement of 25% DPPF were 9.14 and mg/100 g (DM), respectively and highest values of Mg obtained from inclusion of PPPI (25% protein) was found to be mg/100 g (D.M). Cupper decreased gradually with increasing levels of DPPF. Value obtained for wheat biscuit (control) was found to be 5.34 mg/100 g (DM). Lowest value obtained was (2.03 mg/100 g) for 25% DPPF. On the other hand, incorporation of PPPI resulted in an increase of Cu and values obtained were and mg/100 g for 20 and 25% protein content, respectively. 157

183 Table 28: Mineral matter content of wheat flour biscuit (mg/100 g)* containing DPPF and PPPI Blends Ca Fe Cu Mg 100% Biscuit Flour (control) % BisF+5%DPPF % BisF+10%DPPF % BisF+15%DPPF % BisF+20%DPPF % BisF+25%DPPF BisF+ PPPI (Blend 15% Protein) BisF+ PPPI (Blend 20% Protein) BisF+ PPPI (Blend 25% Protein) ND *On dry matter basis Where: - BisF = Biscuit flour - DPPF = Decorticated pigeon pea flour - PPPI = Pigeon pea protein isolate 158

184 4.12 Sensory evaluation of biscuit containing decorticated pigeon pea flour and pigeon pea protein isolate Sensory evaluation of biscuit containing DPPF and PPPI are presented in Tables 29 and 30. No significant differences were observed in color, after taste and texture in all blends prepared with DPPF compared with biscuit (control). Increasing levels of DPPF resulted in increase of the scores of the color. The values obtained were ranged from 3.9 for 95% BisF/5% DPPF to 3.1 for 75% BisF/25% DPPF compared with biscuit control (3.2). No significant differences (P 0.05) were observed in all different blends in color. These high scores in color may be attributed to the increased levels of protein of pigeon pea flour, which reacts with carbohydrates (Millard s reaction) and also the creamy color of pigeon pea cotyledons affects the color, which may be preferred by the panelists. On the other hand, after taste and texture decreased gradually with increasing the levels of supplementation with DPPF. Odor scores of biscuit significantly (P 0.05) decreased with increasing levels of DPPF from 4.0 for biscuit (control) to 3.9, 3.7, 3.6 and 3.3 for 5, 10, 15 and 25% DPPF in the blends, respectively. There was a significant decline in the overall quality and acceptability of biscuit made from DPPF at different levels supplementation; probably due to the domination of pigeon pea flavor. From these results, it was concluded that biscuit containing DPPF could be introduced with satisfactory performance when compared with 159

185 Table 29: Sensory evaluation of wheat biscuit containing decorticated pigeon pea flour Flour Blends* Color Odor After taste Texture Overall quality 100% BisF (control) 3.2±1.61 a 4.0±1.56 a 3.9±1.46 a 4.0±1.07 a 4.0±1.41 a 95% BisF + 5% DPPF 3.9±0.92 a 2.9±0.96 c 3.5±1.06 a 3.7±1.10 a 3.3±1.05 b 90% BisF + 10% DPPF 4.0±1.07 a 3.7±1.18 b 3.5±1.06 a 3.7±0.82 a 3.4±0.83 b 85% BisF + 15% DPPF 3.9±0.99 a 3.6±1.35 b 3.7±1.44 a 3.8±1.01 a 3.3±1.33 b 80% BisF + 20% DPPF 3.7±1.29 a 3.3±1.10 b 3.3±1.05 a 3.5±0.83 a 3.1±1.13 b 75% BisF + 25% DPPF 3.1±1.51 a 4.0±0.65 a 3.5±1.13 a 3.2±1.21 a 2.9±1.22 c Calculated F-ratio 1.292n.s n.s 1.069n.s C.V% 34.73% 32.68% 33.95% 27.81% 35.22% Lsd 5% # Mean values (±SD) having different superscript letters in each columns differ significantly (P 0.05). Where: (BisF: Biscuit flour, DPPF: Decorticated pigeon pea protein flour) Table 2.Key of description Color Odor After taste Texture Golden brown (5) Desirable (5) Pleasant (5) Crispy (5) Uniformity (4) Normal (3-4) Normal (3-4) Hard (3-4) Brownish (3-3) Off Flavor (1-2) Off taste (1-2) Brittle(1-2) 160

186 Table 30: Sensory evaluation of wheat biscuit containing pigeon pea protein isolate Flour Blends* Color Odor After taste Texture Overall quality BisF (control) 3.9±0.80 a 3.9±1.16 a 4.2±1.01 a 4.3±0.96 a 4.1±1.13 a BisF + PPPI (Blend 15%protein) 3.5±0.83 a 3.8±0.94 a 3.7±1.03 a 3.7±1.10 b 3.6±0.74 b BisF + PPPI (Blend 20%protein) 3.8±1.21 a 2.7±1.18 b 2.7±1.44 b 3.4±1.06 c 2.8±1.26 c BisF + PPPI (Blend 25%protein) 2.3±1.67 b 3.9±1.03 a 4.4±0.74 a 4.5±0.74 a 3.5±1.13 b Calculated F-ratio C.V% 35.06% 30.20% 28.79% 24.57% 30.75% Lsd 5% # Mean values (±SD) having different superscript letters in each columns differ significantly (P 0.05). Where: BisF: Biscuit flour, PPPI: Pigeon pea protein isolate) Table 2.Key of description Color Odor After taste Texture Golden brown (5) Desirable (5) Pleasant (5) Crispy (5) Uniformity (4) Normal (3-4) Normal (3-4) Hard (3-4) Brownish (3-3) Off Flavor (1-2) Off taste (1-2) Brittle(1-2) 161

187 parameters obtained in the reference key, and hence could be used in all quality attributed (Appendix). On the other hand, incorporation of PPPI was not significantly (P 0.05) affected the color, up to BisF/PPPI blend (protein level 20%) Table 30. Significant increase was observed in BisF/PPPI blend (25% protein level) and lower scores in color were obtained (2.3). Odor and after taste were not significantly affected by the supplementation of PPPI in 15 and 25% protein level in the blends. Low scores and significantly differences at (P 0.05) were observed in odor, after taste and overall quality of BisF/PPPI (protein level 20%) blends. High scores were observed in blends of 25% protein level in after taste and texture and these may be attributed to the reaction with carbohydrate and high protein in the blends (caramilization and Miallard s reaction effect). From the results obtained above; it was observed that cookies made from the flour blend with 20% protein were rated inferior to the control, cookies made from flour blend with 15% and 25% protein level were still within the acceptable range. It was concluded from all the results obtained that inclusion of decorticated pigeon pea flour and pigeon pea protein isolate in cookies at specific level can be carried out successfully without any noticeable changes in desirable organoleptic properties of the end product, but better process methods may be needed to maintain quality. Such high-protein cookies could be given to children in drought areas to combat malnutrition. The children of these areas are already acquainted with the pigeon pea flour taste; however, proper formulation should resolve undesirable changes. 162

188 CHAPTER FIVE CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions Rheological characteristics of bread and biscuit wheat doughs have been improved with incorporation of DPPF and PPPI. Incorporation of DPPF and PPPI in wheat flour has improved the nutritional value of bread and biscuit produced i.e. increase in ash (mineral), protein, fat, and caloric values with increasing levels of replacement. Deficiencies of essential amino acids, lysine and sulphur containing amino acids, in wheat and pigeon pea have been supplemented and their chemical scores have been improved in bread and biscuits produced. Bread supplemented with DPPF (10 %) and PPPI (protein level 15 %) were found to be more acceptable in all quality attributes than wheat flour bread and wheat flour biscuit. 5.2 Recommendations Inclusion of DPPF and PPPI in biscuits formulae at certain level can be carried out successfully without noticeable changes in desirable characteristics of the end product, but better process methods may be needed to improve quality and promote good flavor. The developed high protein bread and biscuit could be given to needy children or to vulnerable groups suffering from 163

189 malnutrition or to any other needy groups, particularly those suffering from food shortage stress (famines, crises...etc). More research is needed to cover the following areas:- The economical feasibility of production for the products on a pilot scale should be explored. In-vivo testing of the fortified products to insure the nutritional values among malnourished children in Sudan. Microbiological studies of the products should be carried out. The shelf - life of these products need to be studied. 164

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216 Plate (1): Homogenizer For Flour Mixing (No. YY 219) Plate (3): Stone mill Used for Pigeon pea Decortication 192

217 Plate (5): Efficient Universal Pulverizer (capacity kg) used for milling of decorticated pigeon pea Plate (7): Centrifuge (type ) Used for Separation of Pigeon pea Protein isolate 193

218 Plate (8): Hobert Mixer N