\\\\\\\\\\\\\ \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\i “1 3 1293 10529 6994 ’ ”J'— '1 J c I 1 m. I lLlfiseAEEERIr Micit‘na skate { University ~. A This is to certify that the dissertation entitled Genetic, Physico-Chemicai and Structural Parameters Affecting Texture of Dry Edibie Beans presented by Agbo, N' 21 Georges has been accepted towards fulfiHrnent of the requirements for the Ph.D. degree“, Food Science and Human Nutr. //c/M:Z fl /1c $4 1’? ”/6 Dr. Mark A. Uebersax Major professor Date 11-11-82 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 4___* MSU LIBRARIES RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES wiii SW be charged if book is returned after the date stagged below. 741 0’5") gily iffiflgll: .. . urn-"W , r J”; 5 1993. V0 .' a i 7 “figgws GENETIC, PHYSICO-CHEMICAL AND STRUCTURAL PARAMETERS AFFECTING TEXTURE OF DRY EDIBLE BEANS By Agbo N'Zi Georges A DISSERTATION Submitted to Michigan State University in partiaI fuifiiiment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1982 ABSTRACT GENETIC, PHYSICO—CHEMICAL AND STRUCTURAL PARAMETERS AFFECTING TEXTURE OF DRY EDIBLE BEANS By Agbo N'Zi Georges The purpose of the present study was to develop funda- mental information concerning physical and chemical characteristics of dry bean (Phaseolus vulgaris L.) seeds and relate them to cooking quality. Two dry bean isolines San-Fernando (a black seeded cultivar of tropical origin) and Nap-2 (a white seeded mutant from San-Fernando) presuma- bly differing only in seed coat color were compared for proximate analysis, soaking and cooking, and flour charac- teristics. Sanilac, a navy bean cultivar used widely in dry bean studies was employed as a control. Proximate composition of Nep-Z and San-Fernando showed no significant differences for moisture, protein, fat, starch and ash contents. Cumulative proximate composition over the three cr0p years (l978, 1979 and l980) showed significant differences in fat contents only and Nep-Z had higher fat values than San-Fernando. In the cotyledon, a-oligosaccharide contents were similar in all three genotypes with stachyose being in the greatest quantity. The oligosaccharides were not detected in the seed coat of any of the three cultivars used. Agbo N'Zi Georges The rate of water absorption was lower in San-Fernando than Nep-Z and Sanilac. After beans were thermally pro- cessed, sample of Sanilac and Nep-Z had higher washed drained weights and lower texture than for San-Fernando. No significant differences were detected among the strains for moisture content after soaking and processing. The visco- sity of bean flour indicated high initial pasting tempera- tures with continuously rising pasting curves for all strains. The removal of the bean seed coat increased the viscosity potential of Sanilac and San-Fernando flours. However, Nep-Z flour showed a decreased curve pattern. Variability among genotypes was observed under Scanning Electron Microscope for seed structural characteristics. Nep-2 had partially occluded micropyle, slightly elevated hypocotyl area, and a "plastic-like" seed coat with holes while San—Fernando had closed micropyle, elevated hypocotyl area, rigid seed coat without holes, firmly packed parenchyma cells organization in the dry seeds, and partially liberated starch granules in the soaked and processed seeds. To my Mother For her patience and moral support ii ACKNOWLEDGEMENTS I wish to express my gratitude to my major professor, Dr. M. Uebersax, for his guidance and patience during this study and preparation of this dissertation. I address my sincere thanks to Dr. G. Hosfield of the Department of Crops and Soil Science, and his wife Marie, for their particular concern and moral support in the accomplishment of this work. I extend my appreciation to Dr. P. Markakis, Dr. L. Dawson, and Dr. I. Gray of the Department of Food Science" and Human Nutrition, and Dr. R. Herner of the Department of Horticulture, for serving on my graduate committee with their suggestions and reviewing of this manuscript. I express my deep sense of appreciation and love to my dear mother and late father Mrs. and Mr. Agbo, for their wise advice and encouragement in many ways. I also feel grateful to the Ministry of Research, Republic of Ivory Coast for the opportunity and financial assistance granted to me and USDA program associated with the Department of Crop and Soil Sciences at Michigan State University for supplying the materials and equipment used. I further thank my cousins, Mr. N'Zi Bruno and Mr. N'Zi Remy, and my friends, Mr. Iloki Ignace and Mr. Kraidi Nicolas for their help when I needed it. TABLE OF CONTENTS Page LIST OF TABLES. . . . . . . . . . . . . . . . . . . . vii LIST OF FIGURES . . . . . . . . . . . . . . . . . . . X INTRODUCTION. . . . . . . . . . . . . . . . . . . . . l The World Food Problem. 3 "Protein Problem" . . . . . . . . . . . Possible Solutions to the "Protein Problem" 4 Needs of Vegetable Proteins . . . . . . . . . . . 7 Yield Problem . . . . . . . . . . . . 8 Bean Production and Utilization . . . . . . . 9 Objectives. . . . . . . . . . . . . . . . . . . . l3 5 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . I Nutrition Contribution of Food Legume Seeds to World Hunger. . . . . . . ... . . . . . . . 15 Cooking and Processing. . . . . . . . . . . . . . l7 Moisture Factor . . . . . . . . . . . . . . . 20 Hydration . . . ' . . . . . . . . . . 2] Texture Measurement Means . . . 25 Relationship of Texture Hard- to- cook Beans. . 28 Scanning Electron Microscopy (SEM). . . . . . . . 31 Legume Seed Anatomy . . . . . . . . . . . . . 39 Starch and Viscosity. . . . . . . . . . 43 Indigestible Bean Sugars Problem. . . . . . . 50 Quality Evaluation of Processed Products. . . . . 57 MATERIALS AND METHODS . . . . . . . . . . . . . . . . 61 Sample Source . . . . . . . . . . . . . . . . 61 Seed Coat Separation. . . . . . . . . . . . . . . 51 Grinding of Sample. . . . . . . . . . . . . . . . 52 Moisture. . . . . . . . . . . . . . . . . . . . . 62 Crude Fat . . . . . . . . . . . . . . . . . . . . 52 Ash . . . . . . . . . . . . . . . . . . . . . . . 52 Protein . . . . . . . . . . . . . . . . . . . . . 62 Sugars. . . . . . . . . . . . . . 53 Extraction and Injection. . . . . . . . . . . 63 Methods of Quantitation . . . . . . . . . . . 55 iv Starch. . . . . . . . . . . . . . . . 55 Sample Preparation. . . . . . . . . . . . 66 Buffer and Enzyme Solution. . . . . . 58 0.2 M acetate buffer stock solution . . . 68 0.02 M acetate buffer working solution. . 68 Enzyme solutions. . . . . . . . . . . . . 59 Enzyme Characteristics. . . . . . . . . . . . 59 Hydrolysis. . . . . . . . . . . . . . . . . . 59 Injection . . . . . . . . . . . . . 70 Methods of Quantitation . . . . . . . . . . . 70 Calculations. . . . . . . . . . . . . . . 70 Water Absorption in Beans . . . . . . . . . . . . 71 Canning . . . . . . 71 Texture Measurement Based on Single Bean. . . . . 76 Gelatinization Characteristics. . . . . . . . . 77 The peak viscosity or pasting peak. . . . 77 The viscosity at the end of the heating cycle as sample reaches 95°C.. 79 The viscosity at the end of the 95°C holding cycle . . . . 79 The viscosity at end of the cooling cycle when the paste reaches again 50°C . 79 The viscosity at the end of the 50°C holding cycle . . . . . . . . . . . 80 Scanning Electron Microscopy. . . . . . . . . . . 81 Dehydration . . . . . . . . . . . . . . . . 81 Coating and viewing . . . . . . . . . . . . . 81 Statistical Analysis. . . . . . . . . . . . . . . 32 RESULTS AND DISCUSSION. . . . . . . . . . . . . . . . 83 Proximate Composition . . . . . . . . . . . . . . 83 Sugars. . . , 95 Scanning Electron Microscopy Examination of Dry Beans . . . . . . . . . . . . . . 98 Water Absorption on Soaking . . . . . . . . . . . 116 Pasting Properties. . . . . . . . . . . . . . . . 120 SEM of Soaked Beans . . . . . . . . . . . . . . . 129 SEM Of Processed Beans. . . . . . . . . . . . . . l34 Bean Color. . . . . . . . . . . . . . . . T38 Processed Bean Evaluation . . . . . . . . . . . . 14‘ Moisture content. . . . . . . . . . . . . . . 141 Drained weight. . . . . . . . . . . . . . . . I45 Clump and split . . . . . . . . . . . . . . . 145 Texture . . . . . . . . . . . . . . . . . . 146 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . I53 RECOMMENDATIONS FOR FUTURE RESEARCH . . . . . . . . . 157 Page LITERATURECITED...................159 APPENDIX.......................178 vi Table #WN TO LIST OF TABLES Comparison between microscopes. . . . . . . . . . Legume testa feature characteristics. Bean processing evaluation and calculations Functional and molecular properties associated with pasting characteristics of starches. Percent moisture content (dry basis) of bean cultivars (Sanilac, Nep-Z and San-Fernando) by crop years (l978, 1979 and l980) and seed por- tions (whole bean, cotyledon and seed coat) Percent moisture content (dry basis) Of bean cultivars (Sanilac, Nep-2 and San-Fernando) for three crop years (l978, l979 and l980) by seed portions (whole bean, cotyledon and seed coat) . . . . . . . . Percent protein content (dry basis) Of bean cultivars (Sanilac, Nep-Z and San-Fernandl) by crop years (l978, T979 and l980) and seed portions (whole bean, cotyledon and seed coat). Percent protein content (dry basis) Of bean cultivars (Sanilac, Nep-2 and San-Fernando) for three crop years (l978, 1979 and 1980) by seed portions (whole bean, cotyledon and seed coat). Percent fat content (dry basis) of bean cultivars (Sanilac, Nep-Z and San-Fernando) by crop years (l978, l979 and T980) and seed portions (whole bean, cotyledon and seed coat). . . . . . . . . Percent fat content (dry basis) of bean cultivars (Sanilac, Nep-Z and San-Fernando) for three crop years (T978, 1979 and 1980) by seed ortions (whole bean, cotyledon and seed coat) . vii Page 36 42 74 78 84 85 86 87 88 89 Table Page 11 Percent ash content (dry basis) Of bean cultivars (Sanilac, Nep- 2 and San- Fernando) by crop years (1978,1979 and 1980) and seed portions (whole bean, cotyledon and seed coat). . . . . . . . 9° 12 Percent ash content (dry basis) Of bean cultivars (Sanilac, Nep-2 and San-Fernando) for three crop years (1978, 1979 and 1980) by seed portions (whole bean, cotyledon and seed coat 9‘ 13 Percent starch content (dry basis) of bean culti- vars (Sanilac, Nep-2 and San-Fernando) by crop years (1978, 1979 and 1980) and seed portions (whole bean, cotyledon and seed coat) . . . . . . 92 14 Percent starch content (dry basis) of bean culti- vars (Sanilac, Nep- 2 and San- Fernando) for three crop years (1978,1979 and 1980) by seed portions (whole bean, cotyledon and seed coat) . . . . . . 93 15 Percent sugar (hexose, sucrose, inositol, raf- finose and stachyose) content (dry basis) of bean cultivars (Sanilac, Nep- 2 and San- Fernando) and seed portions (whole bean, cotyledon and seed coat) . . . . . . . . . . . . . . . . . . . . . . 95 16 Percent water absorption of dry beans soaked in ambient temperature water (1: 1, tap water: distilled water) for up to 90 minutes by crop years (1973,1979 and 1980) . . . . . . . . . 117 17 Pasting characteristics Of dry bean portions (whole bean and cotyledon) flours by crOp years (1978,1979 and 1980) and cultivars (Sanilac, Nep- 2 and San- Fernando) in a 400 ml phosphate buffer solution at pH 5.30.. . . . . . . . . 12] 18 Hunter Lab color and color difference values (L, aL, b) of dry and processed bean cultivars (Sani ac, Nep- -2 and San- Fernando) by crop years (1978,1979 and 1980) . . . . . . . . . . . . . . 140 19 Quality parameter Evaluation for processed bean cultivars (Sanilac, Nep- 2 and San- Fernando) by crop years (1973,1979 and 1980).. . . . . . . . 143 20 Texture based on single bean cooking time (minutes) for bean cultivars (Sanilac, Nep- 2 and San- Fernando) . . . . . . . . . . . . . . . . . . ‘52 viii Table Page 1A Percent sugars (hexose, sucrose, inositol, raf- finose and stachyose) content (dry basis) Of bean cultivars (Sanilac, Nep-2 and San-Fernando) for three crOp years (1978, 1979 and 1980) by seed portions (whole bean, cotyledon and seed coat). . . . . . . . . . . . . . . . . . . . . . I73 18 Pasting characteristics of dry bean portidns (whole bean and cotyledon) flours for three crOp years (1978, 1979 and 1980) by cultivars (Sani- lac, Nep-2 and San-Fernando) in a 400 ml phosphate buffer solution at pH 5.30 . . . . . . 182 10 Hunter Lab color and color difference values of dry and processed bean cultivars (Sanilac, Nep-2 and San-Fernando) over three crop years (1978, 1979 and 1980). . . . . . . . . . . . . . 185 10 Processed bean evaluation - three crOp years - (1978, 1979 and 1980) bean samples . . . . . . . 190 ix LIST OF FIGURES Figure l U‘IhMN 05 10 ll 12 13 14 Phylogenetic relationship of several dry edible legumes. . . . . . . . . . . . . . . . . . . . . The principle of scanning electron microscopy. Essential features of SEM. . . . . . . . . Visual Observation of legume seed structure. Structure relationships Of the raffinose family oligosaccharides . . . . . . . . . . . . . Structure of galactopinitol. . . . . . . Flow diagram of sugar analysis . . . . . . . . Flow diagram of starch dispersion, hydrolysis and analysis . . . . . . . . . . . . . . . . Schematic diagram illustrating unit Operations for dry bean processing procedures . . . . . . Flow diagram of sample preparation for SEM . Mean sugar (hexose, sucrose, inositol, raffinose and stachyose) content values over three crop years (1978, 1979 and 1980) for whole bean cultivars (Sanilac, Nap-2 and San-Fernando). Mean sugar (hexose, sucrose, inositol, raffinose and stachyose) content values over three crOp years (1978, 1979 and 1980) for cultivars (Sanilac, Hep-2 and San-Fernando) cotyledons Mean sugar (hexose, sucrose, inositol, raffinose and stachyose) content values over three crop years (1978, 1979 and 1980) for Dean cultivars (Sanilac, Nep-2 and San-Fernando) seed coats Scanning electron micrographs of dry bean seed coat outer (1000x) and inner (1000x) surface structures: A1 = Sanilac coat outer surface; Page 10 32 34 41 53 54 64 67 73 82 99 100 101 Figure 15 16 17 18 19 20 A = Sanilac inner surface; 8 = Nep-Z coat outer 2 . 1 surface; 85 = Nep-2 coat 1nner surface; C1 = San-Fernan 0 coat outer surface; C2 = San-Fer- nando coat inner surface. . . . Scanning electron micrographs of dry bean seed cotyledon outer surface structures (30x and 700x); A] and A2 = Sanilac cotyledon outer sur- face; B and 82 = Nep-2 cotyledon outer surface; C1 and CZ = San-Fernando outer surface. . . . Scanning electron micrographs Of dry bean seed hilum area structures A] = Sanilac hilum area (50x); A = Sanilac micropyle (1000x); B1 = Nep-Z hiIum area(50x); B = Nep-2 micropyle (1000x); C] = San-Fernanfio hilum area (50x); C2 = San-Fernando micropyle (1000x); HC = hypo- cotyl area (50x); H = hilum; M = micropyle. Scanning electron micrographs Of dry bean seed hypocotyl area (1000x) and hilum transverse cross-section (200x) structures A] = Sanilac hypocotyl area; A2 = Sanilac hilum transverse section; 81 = Nep-2 hypocotyl area; 82 = Nep-2 hilum transverse section; C] = San-Fernando hypocotyl area; C2 = San-Fernando hilum trans- verse section . . . . . . . . . . . . . Scanning electron micrographs Of dry bean seed coat cross-section structures (1600x) A] = Sanilac longitudinal cross-section; A2 = Sanilac transverse cross-section; B] = Nep-Z longitudinal cross-section; 82 = Nep-Z transverse cross-section C1 = San-Fernando longitudinal cross-section C2 = San-Fernando transverse section. . . , Scanning electron micrographs of dry bean seed cotyledon inner Side surface structures (30x and 400x). A] and A = Sanilac cotyledon inner side surface; 8] and 2 Nep-2 cotyledon inner side surface; 01 and C San-Fernando cotyledon inner side surface. . . . . . . . . . . . . . . Scanning electron micrographs of 1980 dry Nep-2 bean cotyledon outer and inner side surface structures (30x); A = cotyledon outer side surface; 8 and C = cotyledon inner side surface . xi Page 102 104 106 107 108 110 Figure 21 22 23 24 25 26 27 28 Scanning electron micrographs of dry bean seed cotyledon cross-section structures (1600x). A = Sanilac longitudinal cross-section; A2 = SAnilac transverse cross-section; 81 = Nep-2 longitudinal cross-section; 82 = Nep-2 trans- verse cross-section; C] = San-Fernando cross- section; C2 = San-Fernando transverse cross- section. . . . . . . . . . . . . . . . ' Scanning electron micrographs of dry bean seed cotyledon cross-section structures at lower magnification (400x). A1 = Sanilac longitudinal section; A = Sanilac transverse section; 81 = Hep-2 longitudinal section; 82 = Nap-2 trans- verse section; C] = San-Fernando longitudinal section; C2 = San-Fernando transverse section. Mean percent water absorption pattern Of dry bean cultivars (Sanilac, Nep-2 and San-Fernando) soaked in ambient tem erature water (1:1, tap waterzdistilled water? for up to 90 minutes over three crop years (1978, 1979 and 1980) . . Mean pasting curves Of dry bean flours (whole bean and cotyledon) by cultivars (Sanilac, Nep-Z and San-Fennando) over three crop years (1978, 1979 and 1980). . . . . . . . . . . . . . Mean pasting curves of dr Sanilac bean flours (whole bean and cotyledoni over three crop years (1978, 1979 and 1980). . . . . . . . . . . . . . Mean pasting curves of dr Nep-2 bean flours (whole bean and cotyledoni over three crop years 1978, 1979 and 1980). . . . . . . . . . . . . Mean pasting curves Of dry San-Fernando flours (whole bean and cotyledon) over three crop years (1978, 1979 and 1980). . . . . . . . . . . . Scanning electron micrographs of soaked bean seed coat outer surface (1000x) and cross-sec- tion (1000x) structures A] Sanilac seed coat outer surface structure; A2 Sanilac seed coat cross-section structure; 81 Nep-2 seed coat outer surface structure; 82 Nep-2 seed coat cross-section structure; C1 ‘San-Fernando xii Page 113 115 118 125 126 127 128 Figure 29 30 31 32 33 34 35 seed coat outer surface structure; C = San- Fernando seed coat cross-section structure. Scanning electron micrographs of soaked bean seed hilum area structure; A] = Sanilac hilum area (50x); A2 = Sanilac micropyle (1000x); B1 = Nap-2 hilum area (50x), = Nep- 2 micropyle (1000x); C1 = San- FernandoB hilum area; C2= San— Fernando micropyle area (1000x) . . . . . . Scanning electron micrographs of soaked bean cotyledon structures (400x and 1000x) A] and A2 = Sanilac cotyledon; B1 and B2 = Nep-2 cotyle- don; C] and C2 = San-Fernando cotyledon . . . Scanning electron micrographs of processed Sanilac seed coat outer surface structure; A and B = cuticle and intermembrane outer surface (100x and 400x); C = cuticle cell outer surface (3000x); D = protein intermembrane cells outer surface (3000x); PC protein intermembrane cells; CU = cuticle . . . . . . . . . . . . . . Scanning electron micrographs of processed Nep-2 seed coat outer surface structures; A and B = cuticle and intermembrane surface (100x and 400x); C = cuticle cell outer surface (3000x); D = protein intermembrane cells outer surface (3000x); PC = protein intermembrane cells; CU = cuticle . . . . . . . . . . . . . . . . . . . Scanning electron micrographs of processed San- Fernando seed coat outer surface structures; A and B (100x and 400x) and C and D (3000x) = cuticle cell outer surface; CU = cuticle. . Scanning electron micrographs of processed bean structures; A and A2 Sanilac cotyledon (400x and 2000x);Bc1 and 82 Nep- -2 cotyledon (400x and 2000x); and C2 SF cotyledon (400x and 2000x); RS~= cexpandeg rupture starch granules . Mean Hunter color and color difference values Of dry and processed bean cultivars (Sanilac, Nep-2 and San-Fernando) over three cr0p years (1978, 1979 and 1980) . . . . . . . . . . . xiii Page 130 132 133 135 136 137 139 142 Figure 36 37 38 39 Percent water content for bean cultivars (Sanilac, Nep-Z and San-Fernando) in quality parameter evaluation over three crop years (1978, 1979 and 1980). . . . . . . . . . Drained weight for bean cultivars (Sanilac, Nep-Z and San-Fernando) in canned quality para- meter evaluation Over three crop years (1978, 1979 and 1980) . . . . . . . . ... . . . Mean Kramer Shear Press curves for bean culti- vars (Sanilac, Nep-2 and San-Fernando) over three crop years (1978, 1979 and 1980) . . . Texture for bean cultivars (Sanilac, Nep-Z and San-Fernando) in canned bean quality parameter evaluation over three crop years (1978, 1979 and 1980). . . . . . . . . . . . . . . . . . Percent water absorption pattern of dry bean cultivars (Sanilac, Nep-Z and San-Fernando) soaked in ambient tem erature water (1:1, tap water:distilled water? for up to 90 minutes for crop year 1978 . . . . . . . . . . . . . . . Percent water absorption pattern for dry bean cultivars (Sanilac, Nep-2 and San-Fernando) soaked in ambient tem erature water (1:1, tap waterzdistilled water) for up to 90 minutes for crop year 1979 . . . . . . . . . . . . . Percent water absorption pattern for dry bean cultivars (Sanilac, Nep-Z and San-Fernando) soaked in ambient tem erature water (1:1, tap water:disti11ed water for up to 90 minutes for crop year 1980 . . . . . . . . . . . . . . . Pasting curves Of dry bean flours (whole bean and cotyledon) by cultivars (Sanilac, Nep-2 and San-Fernando) in a 400 m1 phosphate buffer solution at pH 5.30 for crop year 1978 . . . Pasting curves of dry bean flours (whole bean and cotyledon) by cultivars (Sanilac, Nep-Z and San-Fernando) in a 400 ml phosphate buffer solution at pH 5.30 for crop year 1979 xiv Page 144 147 148 149 179 180 181 183 184 Figure B3 Pasting curves Of dry bean flours (whole bean and cotyledon) by cultivars (Sanilac, Nep-2 and San-Fernando) in a 400 ml phosphate buffer solu- tion at pH 5.30 for crop year 1980. . . . Hunter color and color difference values of dry and processed bean cultivars (Sanilac, Nep— 2 and San- Fernando) for crop year 1978.. . . . . . Hunter color and color difference values of dry and processed bean cultivars (Sanilac, Nep-2 and San-Fernando) for crop year 1979. . . . . . . . Hunter color and color difference values of dry and processed bean cultivars (Sanilac, Nep-Z and San-Fernando) for crop year 1980. . . . . . . . . Percent water content of bean cultivars (Sanilac, Nep-2 and San-Fernando) in quality parameter evaluation for crop year 1978 . . . . . . . . . Percent water content for bean cultivars (Sani- lac, Nep-2 and San-Fernando) in dry, soaked and canned beans quality parameters evaluation for crop year 1979. . . . . . . . . . . . . . . . . Percent water content for bean cultivars (Sani- lac, Nep-Z and San-Fernando) in quality parameter evaluation for crop year 1980 . . . . . . . . . . Drained weight for bean cultivars (Sanilac, Nepe2 and San- Fernando) in canned bean quality para- mete; evaluation by crop years (1978,1979 and 1980 O Q 0 O O O O O O O O O I O O I O O Q I O 0 Texture for bean cultivars (Sanilac, Nep- -2 and San- Fernando) in canned quality parameter evaluation for crop year 1978 . . . . . . . Texture for bean cultivars (Sanilac, Nep-2 and San-Fernando) in canned quality parameter evaluation for crop year 1979 . . . . . . . Texture for bean cultivars (Sanilac, Nep-Z and San-Fernando) in canned bean quality parameter evaluation for crop year 1980 . . . . . . . . . XV Page 185 187 188 189 191 192 193 194 195 196 197 INTRODUCTION The World Food Problem The major problems facing the world today are two-fold: one of population; the other of not enough food. Modern medicine and improved sanitation have brought about a spectacular increase in the average life span while the birth rate in many countries remains practically unchanged. The result is the much-discussed population explosion. In developed countries, it is recognized that the difference between birth rate and death rate is small due to birth control and high medical practices while in developing and lesser-developed countries that difference is larger due to the fact that birth rate is highly by lack Of birth control, and also the death rate reduced by improvement Of sanitation and better medication availability. Efforts to check the birth rate and to increase the food supply are severely handicapped by lack Of education, adherence to customs, and lethargy from malnutrition and undernutrition. The problem of population and food is not new; it has confronted man for nearly all the million years that human beings have been on the earth. The present problems differ only in magnitude and intensity from problems that have been faced many times before. Today, one-half billion people out Of four billion (12.5% Of the world population) do not receive close to an adequate quantity of food; moreover, possibly one-half the world is marginally fed._ This non-availability Of food to that portion of the world population, mainly Latin America, Africa and Asia, could be associated to many factors such as drought, infertility of the soils, inadequate farming lands (Sahel, Africa subsa- harian regions), inadequate food production due to the fact that the farmer produces food only to feed his family and not for Commercial purposes; lack Of mechanical means for large production; lack of preservation, transformation and transportation means Of the food; political and economical limitations; low salary leading to inability to buy enough food and food of adequate aesthetic quality; and lack of nutritional information and improvement of quality and yield of the food available. All those factors contribute to the low calorie intake Of the one-half billion of the world population. . Coupled with the general food shortage is the shortage Of specific nutrients. There are six main categories of nutrients that are needed to supply energy, promote growth and repair body tissues and to regulate body processes. These are water, carbohydrates, protein, lipid, vitamins and minerals. Those nutrients are quite present in a wide variety of food but may be limited in some others. By lack of nutritional information and inadequate distribution, one may be exposed to inadequacy Of one of those nutrients leading to different sickness. One such nutrient that has received considerable attention is protein. "Protein Problem" The deficiency in animal protein in the diet deficit regions of the world has been generally associated to poor physical develOpment of adults and even more markedly to the protein deficiency diseases of children. Kwashiorkor and marasmus account for a very high child mortality between weaning and 6 years Of age and often leave the children that survive with stunted physiques and retarded mentality. Different authorities have set different goals for animal protein in the diet. The U.S. Department of Agricul- ture would provide 10 grams of animal protein per person per day, and 10 grams of pulse protein, the latter from increased cultivation and use of leguminous crops and from increased human consumption of the press cake from Oil seeds. Cook (1962) set a goal of 16 grams of animal protein per person per day for the under-nourished countries. Pawley (1963), looking to the year 2000, set a goal of 20 grams of animal protein per person per day with the rest of the requirement from other food sources. In view of the paramount importance Of a complete pro- tein in the diet, particularly for child development, it is not quite easy to accomplish the goals. Four causes Of this "protein problem" have been reported by the Protein- Calorie Advisory Group (PAG) of the United Nations (PAG Statement No. 20, 1973) as: 1) Low wages and income and under-employment or un- employment in rural or urban centers, all Of which limit the purchase of the relatively costly foods that contain protein of good quality. 2) Difficulties associated with the production Of protein-rich food Of animal or plant origin because Of ecological and agricultural limitations with the result that they are usually expensive and relatively short supply. 3) The lack of effective food processing, distribution and marketing system resulting in large loss of food crops. 4) The lack Of knowledge of food values and food preparation for children and specific prejudices against giving some protein foods to young children, especially when they have infectious disease. Possible Solutions to the "Protein Problem" Several approaches have been proposed to solve the pro- tein gap but one should recognize that the situation is very complex. Altschul (1969) mentioned that the conVentional approach to increasing food supply is to increase grain production, and that to increasing protein quality of a diet is to increase animal produc- tion. He proposed two ways beyond conventional means: l) Raise the protein impact Of the major contributor to protein in the diet; 2) develop new nutritious foods to supplement those now available from conventional sources. His views are directed toward synthetic amino acids, vegetable protein mixtures, improved cereal products, protein beverages, texture foods and others. He suggested that the major increase by far in protein supply for the near future will come by increase in total supply of cereal grains and the next will come by increase in conventional protein sources - legumes and animal protein. The United Nations Advisory Committee on the Application Of Science and Technology to development (1968) suggested seven policy directions. 1. Promotion of increased quantity and quality of conventional plant and animal protein sources suitable for direct human consumption. 2. Improvement in the efficiency and scope of both marine and fresh-water fisheries operations. 3. Prevention Of unnecessary losses of proteinaceous foods in field storage, transport and home. 4. Increase in the direct food-use of Oilseeds.and oilseed-protein concentrates by human population. 5. Promotion of production and use of fish-protein concentrate. 6. Increase in the production and use of synthetic amino acids to improve the quality Of protein in cereals and other vegetable sources, and the dEVETOp- ment of the use of other synthetic nutrients. 7. Promotion of the development of single-cell protein for both animal feeding and direct utilization by man. “ Kahn (1981) reported that the world fOOd needs for 1985 are estimated to be 44% greater than that required in 1970, and about 70% of that 1985 estimate will be needed to feed the Third World. His approach to solve the problem is a multisectoral coordination. First, slowing of population growth should be a paramount objective of any developing country's program to alleviate hunger. Second, the choice of strategy that should be undertaken for eradicating poverty asSociated with low protein intake should be left to each government. But it must be recog- nized by planners, economists and policy makers that increasing national income alone will not solve the poverty- hunger problem. Last, he stated that food production yield per acre could be improved in many areas Of the world, even though factors such as climate, water, soil, energy, etc., influence productivity. Of all the suggestions that have been made for solving the protein problem, the one that has received the most attention is the improved production and utilization Of conventional animal and plant protein Sources. Animal products are ideally suited for meeting the protein needs of people through out the world but they are costly, scarce or not utilized fully because of ignorance, taboos or poverty. Needs of Vegetable Protein Vegetable protein which are the basic plant protein sources, typically supply over 80% of dietary protein in developing countries. Cereals and legumes give the largest contributions, and they are utilized in various forms in different regions. Protein content among cereals ranges from 6 to 14%; rice is low and wheat is high among the predominant cereals eaten by humans. Within each variety there is a range in protein content; in wheat alone the range can be 8 to 14%. The quality Of the cereal protein is not as good as that from animals, primarily because the amino acid pattern is deficient in one or more amino acids, principally lysine; others are tryptophan, threonine and methionine. Because of that, attention has been paid to legume protein. Legumes grown in different parts of the world enjoy widespread use, have a high degree of acceptability, relatively low cost and possess good nutritiOnal proper- ties. They are known to be rich sources Of lySine but deficient in sulfur amino acids. This places them as natural complements to cereal based diets. In general, the aim to the solution of the protein problem is the production of protein rich foods within the developing countries or regions. Several indigenous crops which are still unknown to the westernworld need to be investigated in order tO evaluate their nutritional status and incorporate that into local agricultural systems. Grain legumes which fall in that category have become important crops. Yield Problem Carperter (1981) reported that world production of grain legumes relative to that Of cereal grains is falling, mainly because of considerably greater yields obtained with cereal. Yet, governments and international agencies continue to I encourage production of these legumes because they "fix" nitrogen, and because of a belief that they make a special contribution to the diet of people unable to afford high levels of milk and other animal products; In the U.S., it has been shown that average bean yield has a downward trend in the past 20 years suggesting that a barrier to higher yields has been reached in beans. In Michigan, through Michigan Improved Bean and Seed Program, Michigan State University has been made responsible for the development, evaluation and release of new varieties. Hosfield and Uebersax (1980) stated that there is a number Of reasons for yield barriers in crops, but the least common denominator is the variety because variety possesses a certain genetic potential or program prescribed by its genetic complements to produce under a given set of circum- stances. They explained that when circumstances are optimum, the variety will produce at a maximum, and if circumstances change and prevailing conditions limit the genetic potential of a variety, the variety then must be replaced with one that can maximize its genetic potential in terms of productivity. This replacement of variety could be important for the seed quality. Frazier (1978) indicated that some of the highest bean yields being achieved in Michigan wide trials are for beans with tropical germplasm in their pedigrees. Smucker g£_al. (1978) precised that preliminary results from several field experiments suggest that black beans derived from tropical accessions have root systems more tolerant to partial soil compaction and water stress. Bean Production and Utilization Figure 1 shows the family and subfamily Of beans. The papilionaceae sub-family grown in tropical and subtropical regions proyide the most edible beans. The subject of this dissertation is Phaseolus vulgaris L., the common dry bean which is the grain legume consumed in greatest quantity in the world as a whole, and it exempli- fies all the problems associated with these materials. Because of the high nutritional value Of beans, various workers have conducted compositional analyses: investiga- tions generally include protein, fat, moisture, fiber, ash, mineral, vitamin, carbohydrate and texture. lO amazou camp cossou coma mczz :mmn new; camaxom mmaxowzu awn cowmwa .mmssmmp opnwvm xcn Pngm>mm mo awcmcoruumc ovumcmmop>58 ._ mcam?d unaccsocw ommwow> mmmpommmca + » mowgh a .muoom guzzg wuw>oca .mcowmmg mumcmaemu ecu Pmovaogu cw zocw mmmomcowpvama » modemsocxdmwm< 9 .muooe cuss; wuw>oga 3mm m apco .zpco mcowmmc Fmowqocp cw 3ocw ? n / mmmomcwgpmwmmmu uncommocvz AIIIII xpvscwunzm » » xpvsmm mammmc_s=m64 11 Dry beans often require long cooking periods to become eating soft. Hydration of the beans prior to cooking is an important factor in the legume industries. Conventional methods of preparing and cooking dry beans involve soaking overnight in water at room temperature (16-24 hrs) followed by cooking in boiling water for 45 min to 3 hrs. Time of cooking depends on bean variety and storage history. Many investigations have been carried out to solve that problem and it has been suggested that information on chemical composition of specific tissues and localization of chemical constituents in those tissues is a prerequisite for an explanation of physical and chemical changes in bean tissues during mechanical, thermal, chemical and enzymatical treat- ment (Powrie §t_gl,, 1960). Texture is an important quality characteristic of cooked beans. One should also bear in mind that texture of adequate acceptability by consumer is of quite importance in bean production and now to plant breeders. Although there have been numerous attempts to measure objectively the texture of cooked beans, there is very little information on the texture of raw and soaked beans. . Beans have also been associated with excessive intesti- nal gas production known as flatus. Studies have been directed toward elucidating the cause of flatulence in beans. Several indigestible sugars namely stachyose and raffinose have been implicated in the flatus associated 12 with eating cooked dry beans. To study this problem, research has been directed toward: 1. characterizing the specific component. 2. developing physical and chemical means of extrac- ting these components. 3. developing practical cooking or processing tech- niques which may be used to reduce the problem. 4. attempting to identify genetic variants possessing low levels of oligosaccharides which may be used to biologically remove the flatulence factors of beans. Although yield is the most important solution criteria in bean breeding, the breeder is cognizant Of consumer preferences when introducing new cultivars. Consumers have acquired specific preferences for various combinations of visual characteristics of seeds and, beans are generally soaked and must be cooked to make them tender and palatable and to render seed protein nutritionally available before eating (Rockland and Jones, 1974). The use Of modern technology has provided the beans by which food quality evaluations may be'conducted with a high degree of objectivity and precision on small amounts of seed (Hulses _t._l., 1977). However, little information is available as to what characteristics lead to the variability for physical and chemical characters noted in dry edible beans (Hosfield and Uebersax, 1980). 13 Objectives Strains that differ from each other genetically at one locus only are referred to as isolines. Since "Nep-2" was the result of an induced mutation in "San-Fernando" that presumably only affected the color locus in common bean (Moh, 1971), the differences in physico-chemical properties were surprising and prompted this investigation because of the possible impact on cooking quality. Earlier processing evaluations of these strains showed that "San-Fernando" and "Nap-2" differed in the rate of water absorption during soaking and "San-Fernando" required almost 2 times the force as "Hep-2" to shear a sample of beans to the point of deformation using a Kramer Shear Press Instrument. The Objective of this research was to investigate physico-chemical differences noted between the above 150. lines. The dry bean cultivar, Sanilac, was used as a point of reference (standard) for all evaluations. For this purpose experiments were designed to ascertain the: l. Proximate composition of the two isolines and Sanilac (a commercial standard Phaseolus vulgaris L. bean . 2. Possible flatulence sugars for potentiality of reduction of the levels through breeding. 3. Microstructure Of the three cultivars of beans by scanning electron microscope (SEM) and relate differences to texture. 4. Hydration rate of the three varieties and relate that to their microstructures. 14 Physical properties (viscosity) of the flour from the beans and relate that to pasting characteris- tics. Processing quality and texture of the three cultivars. Effect of soaking and cooking on the microstructure of the beans and relate that to texture. LITERATURE REVIEW Nutritional Contribution of Food Legume Seeds to World Hunger Problem Legume seeds are grown and used for food in nearly all the temperate and tropical areas of the world. Rockland and Nishi (1979) mentioned that 12 varieties have commercial importance and legumes require less energy per unit protein production than cereal grains and particularly animal pro- tein. Tobin and Carpenter (1978) in their critical review of the literature concerning protein quality evaluations and other properties of the common beans, Phaseolus vulgaris L., reported PER (Protein Efficiency Ratio) values for varieties and cultivars ranged from lt0 to 2.0. Rockland and Nishi (1979) reported values of 1.2 - 1.4 for authentic strains Of' cooked standard and 1.2 - 1.6 for analogous quick-cooking products prepared from various 3. vulgaris L. Cultivars. Rockland (1978) reported that a maximum PER for quick-cooking beans was observed after cooking for 5 min in boiling water. After cooking fOr 15-30 min, the PER values were slightly lower. Standard-cooked beans, cooked for 5 min, had about the same PER as the quick-cooking beans. There, the time and energy factors are involved in the efficiency of the 15 16 bean protein. Tobin and Carpenter (1978) suggested that extended cooking, especially at higher pressure and tempera- ture, tends to lower nutritional quality. Elias gt 11. (1979) and Rockland and Radke (1981) obtained lower PER values for beans and cook water combined than for drained beans. They suggested that seed-coat tannins in colored beans may prevent complete utilization of legume proteins. Beans, generally, are fairly good sources of riboflavin and vitamin E and others such as thiamin, niacin, vitamin B6,and folacin (Bunnel £3 11., 1965; Harris gt 31., 1950; and Patwardhan, 1962). Guerrant gt 31. (1946), Lamb 33 11. (1946), Lantz (1938) and Shroeder (1971) reported riboflavin retention of 72 - 105% in processed bean. Bunnel _£__l. (1965) and Harris gt 11. (1950) reported vitamin E losses of 70 - 90% in canned beans. Normal bean processing methods may not prevent the formation of hydroperoxides which allow the tocopherol side-chain to be oxidized and further degraded to peroxides which in turn further decom— pose to cleavage to aldehydes and ketones. Riboflavin was higher in steam blanched beans but vitamin E was not affected since vitamin E is not water SolUble (Connie at 11., 1979) using navy beans, Pinks, Pinto and snap beans. As storage time increased, the vitamin retention decreased. Nordstrom and Sistrunk (1977) reported vitamin E loss in canned bean during storage. Bunnel gt a1. (1965) and others (Ames, 1972; Harris gt 11., 1950) reported riboflavin losses 17 of 90% in canned bean during storage. Beans are also source of minerals Such as iron, calcium, phosphorus, magnesium, sodium, potassium, manganese and copper. Augustin gt_gl. (1981) reported retention of those minerals ranging from 38.5% to 103.2% with sodium and calcium being the lowest and highest, respectively. All the above factors are of great interest to plant breeders in the development of a new variety. Cooking and Processing Legume seeds often require long cooking periods to become soft. Cooking is necessary not only to tenderize the seed coat and cotyledon and develop acceptable flavor and texture, but also to make the bean protein nutritionally available. Factors affecting cooking characteristics have been associated with seed coat (Synder, 1936; Gloyer, 1932) and cotyledon (Mattson, 1946). Adams (1975) by relating soaking time and cookability mentioned that the hilum and micropylar areas usually admit water readily, but seed coats differ strikingly in this regard. What he did°not specify was the structural characteristics of the micropyle and seed coat of the beans he used. He stated that both the age of the seed and the genotype of the maternal parent are important regulatory factors. Powrie gt 31. (1960) stated that information of chemical composition of specific tissues and localization, of chemical constituents in those tissues 18 is a prerequisite for an explanation of physical and chemi- cal changes in bean tissues during mechanical, thermal, chemical and enzymatic treatments. The mechanical factors are mainly seed coat breakage and splitting of cotyledons resulting from handling in harvesting and cleaning. Because of temperature, rainfall and other weather conditions during growth, dry beans are subject to cracks, hard seed coats and other problems that can affect processing procedures (Connie gt gl., l979). Splits were affected by bean type, initial moisture content and storage time. Adams and Bedford (1975) stated that, as a general rule, the larger and more irregular shaped seeds are the more sensitive to mechanical abuse than are the smaller, more nearly rounded seeds. Bressani and Elias (1974) reported a minimum of two hours for cooking soaked dry beans (Phaseolus vulgaris L.) at atmospheric pressure. There have been numerous attempts (Esselen and Davis, 1942; Feldberg gt gl., 1956; Dorsey gt gl., 1961) to find a way of lowering the cooking.time for legumes. An important development in the Utilization of whole legumes has been the preparation Of quick-cooking legume products. Steinkraus gt_gl. (1964) reported on a new process for preparation of quick-cooking dehydrated beans by hydrating the dry beans through soaking in water for 15 min, followed by a precooking in steam and coating by dipping in a 20% l_’ 3 (~0- ex 19 sucrose solution at 160°F, then dehydrating. Rockland and Metzler (1967) reported a process for quick-cooking large dry lima beans using an intermittent vacuum treatment for 30 to 60 min in a solution of inorganic salts (sodium chloride, tripolyphosphate, bicarbonate and carbonate), soaking for 6 hours in the same salt solution, rinsing and drying. They indicated that their process facilitated infusion of the salt solution through the hilum and fissures in the hydrophobic outer layer of the seed coat. Wetted by the solution, the inner membrane hydrates rapidly, plasticizing the seed coat and causing it to expand to its maximum dimensions within a few minutes. As a result, cotyledons imbibe the solution rapidly. This causes about 80% reduction in the cooking time. Another method of processing legume seeds involves dehulling of the beans prior to cooking. Practices of dehulling differ from legume to legume and with the same legume in different parts of the world. Although the processing Of legumes for home consump- tion with or without dehulling differs to a considerable extent in different countries and regions, the Similarities outweigh the differences (Aykroyd and Doughty, 1964). Some other methods are fermentation, roasting, parching, agglo- meration and germination, but the important one is the hard- to-cook phenomena. 20 Moisture Factor For processing, the generally desired moisture content in dried beans is in the range of 14 - 18%. USDA (1975) permits moisture content up to 18% without being designated as "high moisture beans". Morris and Wood (1956) reported that beans with a moisture content above 13% deteriorate significantly in texture and flavor after 6 months at 25°C. Beans stored at less than 10% moisture maintained good. quality and stored well even after 24 months. Too low moisture content can also create other processing problems such as not imbibing water normally; bean seed coats becoming brittle, thus being subjected to cracking. But Connie gt gt. (1979) reported that low original moisture level before soaking resulted in higher hydration ratios in all beans used (Pinks, Red kidney, common navy, hite, Pinto, snap beans) except Pinks and Avenger. Bean samples containing 16% initial moisture were firmer in texture after thermal processing. Burr and Morris (1968), Kon (1968), Morris and Wood (1956), Muneta (1964) and Ruiloba (1973) reported that the use of a low storage temperature (4°C) or the practice of storing beans with a low moisture cOntent (8-10%) at a relatively low humidity environment has been shown to minimize the development of a hard-shell condition in legume seeds, including black beans. Adams and Bedford (1975) stated that when various types of beans at about 12.5% moisture level are dropped 10 meters onto a slanting steel 21 surface, they usually will incur sufficient damage to permit detection of genetic differences. Morris (1964) studied the effect of moisture content and temperature during storage on the cooking time of Pinto, Sanilac and lima beans. He found that cooking time increased with storage time, especially at moisture contents above 10%. At 13% moisture content the cooking time after 12 months was three times the initial cooking time. At 10% the cooking time after 12 months was only slightly longer than the initial time. Cooking time also increased with storage temperature, especially at high moisture contents. Barriga-Solorio (1961) reported that at 9.7% moisture 27.8% of Sanilac seed were damaged but at 15.5% moisture damage was reduced to only 5.3%. Muneta (1964) found a correlation of +0.80 between moisture content Of stored dry beans of four cultivars and cooking time. He thought moisture content per se was not the primary factor in determining cooking time and postulated that factor or factors connected with high moisture content resulted in a longer time to breakdown (TTB) phenomena. From all the above views, the speculation Of Muneta (1964) gives space for identification of other factors either chemically or structurally in the understanding of the hard to cook beans phenomenon. Hydration It is well known that when a sample of beans is soaked in water at room temperature, some of the beans do not 22 imbibe water, presumably because the seed coats are imper- meable (Burr, 1973). This condition is referred to as hard-shell or seed-hardness. Hydration of the beans prior to cooking is an important factor in the legume industries. Conventional method of preparing and cooking dry beans involves soaking overnight in water at room temperature (16-24 hours) followed by cooking in boiling water for 45 min to 3 hrs (Oguntunde, 1980). Time of cooking depends On bean variety and storage history. Inadequate soaking lengthens cooking time and affects the cooked bean texture. Longer cooking times lead to the use of more energy than necessary in the processing of beans. Adams (1975) mentioned that hydrating seed to 53 to 57% will insure uniform expansion in the can during the thermal process and insure product tenderness. He specified that a long, soft-water soak will leave the bean more tender or even mushy. Hard water tends to toughen the skins and firm the texture of the cotyledon. He suggested that water containing 25 - 50 ppm calcium is considered optimum. He added that if too-hard water cannot be softened chemically or by ionic exchange resins, the firm effect can be counter- acted by addition of 0.1 to 0.2% of sodium polyphosphate to the soak. I Gloyer (1932), Morris t al. (1950), and Steinkraus t l. (1964) reported a favorable effect of a heat ‘\ 23 treatment on the water absorption of beans which minimized the hard-shell development in the grain. However, other authors (Burr and Morris, 1968) reported that beans that rehydrate as quickly as normal beans usually need a pro- longed cooking time, thus indicating no correlation between water' absorption capacity and cooking time. These findings reveal that, at least in some bean varieties, a higher water absorption capacity (lower hard shell) is not neces- sarily correlated with a shorter cooking time. Molina gt gt. (1976) indicated that heat treatment did not affect the physical appearance of black bean grains (Phaseolus vulgaris L.) but significantly (Ps0.05) decreased the development of the hard-to-cook phenomenon in foods. Powrie gt gt. (1960) reported that the seed coats from soaked navy beans pos- sessed an average moisture level of 76.6%. This high capacity of the seed coat for water uptake suggested the possibility of water migration through the seed coat for the h)‘dration of other bean tissues during the soaking period, Snyder (1936) reported that» for some bean seeds, the entrance of water at ordinary temperature was largely through the micropyle and germinal area). Hamly (1932) sug- gested that fonsome seeds, the micropyle is probably no "‘OI‘G than a hole for exchance of gases. The importance of the hilum in relation to the ripening of the seed and the permeability of the testa in some Papilionaceae was reported by Hyde (1954). He found that 24 the hilum in some seeds performs a function essential for the hardshell condition. But it is not still very clear what anatomical structures in the legume seed are respon- sible for water absorption or the development of the hard- shell condition. According to Powrie gt El- (1960) most of the water, both bound and free, presumably residues in the proteinaceous and cellulosic portions of.the cells. In general, one should recognize that the seed coat, hilum and micropyle together may form an integrated specialized water absorption/removal system. To understand the slow hydration rate and the hard-to- cook phenomenon, various investigations have been carried out. Most of the research and reports have been concentra- ted on the relation of the external structures like hilum, seed coat and micropyle to those characteristics. Little is done on the proteinaceous matrix and the cellulosic portions of the cells. Powrie ££.El- (1960) raised the question of a possible relation between these structures and seed hardness. There is no report on the effect of proteinaceous matrix on the hardness of legume seeds but work done on wheat indicates that there may be such an effect. Scanning electron microscopes has commonly been used in such studies. In general, the primary determinant of wheat hard- ness was shown to be genetically controlled and appeared to relate to factors influencing the degree of compactness of endosperm cell components (Moss gt al., 1973; Greenaway, 25 1969; Stenvert and Kingswood, 1977). Much more work needs to be done on the structural dif- ferences of different varieties of legume seeds in order to be able to Show different water absorption and hardness properties. Texture Measurement Means Texture measuring instruments can be divided into two classes (Voisey, 1971a). a) Special purpose to perform a particular type of test such as tension, compression, shear etc. on either several products, or a specific type of product. D) General purpose to test a wide range of products using a diverse range of methods. To name a few: - Brine flotation test - USDA method (Anon. 1945) - Size of peas (Boggs gt gl., 1942, 1943) - Tenderometer (Martin, 1937) - Texturometer (Lee, 1941) - Penetrometer (Anon., 1938; 80995 gt gl., 1942) - Specific gravity and density (Lee, 1941) i - Alcohol-insoluble solids (Kertesz, 1934) a Total solids (dry matter) (Strasburger, 1933) - Starch (Nielson et al., 1947) - Sugars (Lee, 1941) - Refractive index (Walls, 1936) 9! lie 26 Most of those methods used were not reliable for measur- ing tenderness and maturity of seeds (Makower, 1950). He considered the tenderometer to be probably the best means of determining the maturity of peas. For research, the general purpose type is the logical choice because of operational flexibility. - - Kramer Shear Press (Kramer gt gt., 1951) - Instron Universal Testing Machine (Bourne _t_gl., 1966) . - General Food Texturometer (Friedman _t _l., 1963) - Ottawa Texture Measuring System (Voisey and deMan, 1976) These have basic components common to all such devices: a) a mechanism for deforming the sample b) a system for recording force, deformation and time c) a test cell to hold the sample (Voisey, 1971b). The test cell should subject the sample to appropriate forces to determine the textural characteristics of interest. All these instruments measure texture based on the response of multi-bean sample. - The shear press was first used by Kramer and Asmlid (1953) to test peas. Other scientists have used the same instrument to measure texture of vegetables (Ang gt gl., 1963) and beans (Rockland, 1964; Sanchez and Woskow, 1964; Hoff and Nelson, 1964; Rockland, 1966; Luh t 1., 1975; and Quast and de Silva, 1977a, 1977b). According to Voisey 27 and Nonnecke (1973) the Kramer Shear Press is not entirely suitable for use in a production envirOnment because safety features and fully automatic operation are not incorporated. The advantages associated with the instrument is that the force gauge, cell and shearing blades come as separate attachments which can be easily transported for calibra- tion and it is suitable for testing a wide range of foods. The InstronUniversal Testing Machine measures compres- sive and tensile properties of materials and it has become one of the most widely used texture measuring instrument because the working parts of most texture measuring devices used in the food industry can be used in that machine (Sefa-Dédeh, 1979). Several scientists have used also the instrument on different types of food products (Bourne gt gl., 1966; LaBelle _t _l., 1969; Lee, 1970; Voisey and Larmond, 1971). The Ottawa Texture Measuring System (0.T.M.S.) was designed for testing foods to combine the advantages of Kramer Shear Press and Instron machines and to eliminate some disadvantages (Voisey and deMan, 1976). A wide range of test cells and attachments can be used in this machine depending on the food being tested. Other investigators have measured texture based on the response of single beans (Malcom gt gl., 1956; Ismael, 1976; Steinkraus gt gl., 1964; Burr and Morris, 1968; Molina gt gl., 1976; Bourne, 1972; Ige, 1977). The methods 28 used include a penetrometer, alcohol-insoluble solids, brine flotation and starch measurement. Recently, Hudson (1982), at the USDA Regional Plant Introduction Station at Washington State University, has developed a new method for measuring cooking based on the response of single beans. His apparatus consists of 100 test points in a 10 x 10 array. Each point has a cup holding a single bean seed. The method is as described in the Materials and Methods of this dissertation and it is based on time to breakdown (TTB) system with each individual best looking bean seeds. Sensory analysis and objective measurement are also used to conduct research into textural characteristics and for production quality control but using taste panels is cumbersome, time consuming, expensive and requires suffi- cient quantity of sample. Relationship_of Texture with_Hard-to-cook Beans Texture expresses three parameters (Adams, 1975): a) Firmness: measured by the force required to pene- trate a substance. Perceived on first bitei b) Gumminess: measured by the force required to disintegrate a substance. Perceived during chewing. c) Adhesiveness: measured by force required to remove the material from the mouth. Perceived during chewing. 29 These parameters could well be evaluated with reasonable accuracy by a sensory panel and also measured quantitatively by quite a variety of methods as discussed above. Those methods have been used by various investigators to study Some basic quality characteristics of beans. _ Powrie _t__l. (1960) raised the question of possible relation between the seed structure and hardness. Rockland and Jones (1974) suggested that the separation of Dean cells during cooking may be related to the transportation or removal of divalent cations, particularly calcium and magnesium, from bridge positions within the pectinaceous matrix of the middle lamella. They also showed that there is no breakdown of the cell wall of cooked bean. Rather they found that cooked or partially cooked bean cells separated readily along the surface of individual intact cell walls. Short heat treatment loosened the intercel- lular matrix of the middle lamella sufficiently to allow separation of individual cells without rupture of cell walls. Other scientists (Linehan _t _l., 1969; Huges gt gt., 1975) attributed the hard-to-cook phenomena to the solubili- zation and diffusion of starch from cells while plant tissues are being cooked. According to Hahn gt gt. (1977), starch granules in lima beans maintained slight birefringence after cooking. Varriano-Martson and de Omona (l979) indi- cated that many of the starch granules of black beans 3O (Phaseolus vulgaris L.) exhibited some birefringence even after long cooking periods. Voisey (1971) working on the measurement of baked bean texture found that large differen- ces in texture are evident from year to year and between recipe used. It was also suggested that development of hard shells can be the product of a chemical or enzymatic process in the seed (Burr _t _l., 1968; Kon, 1968; Morris and Wood, 1956; Muneta, 1964; Ruiloba, 1973). Mattson (1946) reported that the cookability of different dry pea varieties is related to their contents Of phytic acid content and calcium. He suggested that when the phytic acid content is low, the pectin in the middle lamella formed insoluble calcium and magnesium pectates, causing the poor cooking quality. . In general there is no clear understanding at the present time of the mechanism that governs the water uptake in beans and the factors that affect the tenderness or softness of the canned product. From that point, knowledge of the chemical composition and characteristics of some major constituents in beans, and also of the physical and structural properties of the seeds and flours is necessary to help in the understanding of the hard-to-cook phenomena. That could also be effectively used in breeding to improve yield of food and feed associated with quality of the grain. Scanning Electron Microscopy (SEM) The concept of a SEM is credited to Knoll (1935) who suggested that characteristics of a sample surface could be observed by focusing a scanning electron beam on the surface and recording the emitted current as a function of beam position. Unlike the TEM, which uses ultra-thin sections, the SEM samples would not be sectioned at all (Rasmussen and Hooper, 1974). The first functional SEM was constructed by von Ardenne (1938) based on Knoll's concept but the first commercial instrument became available in 1965. Today's models provide images with resolution limits of 6 - 10 nm. Buono (1982) stated the principle of SEM as: l. A concentrated beam of electrons is focused on the surface of a sample (Figure 2A). Electron beam size vary from 15 to 10,000 n and energy from 1 to 40 Kv. 2. The electron beam penetrates the sample to depths of 0.2 to 2 um, depending on the energy of the incident electrons and the atomic number (2) or density of the sample. The incident beam electrons are called the primary electron beam. Multiple scatterings of the primary electrons by the beam interaction with the atoms of the sample surface causes the beam to widen in diameter as it penetrates into the sample (Figure 28). The width of this scattering is on the order of 0.2 to l um. The combination of lateral beam spreading with depth produces an interaction volume within the sample. 3. The interaction of the primary electrons with the atoms of the sample produces a variety of signals including secondary electrons, backscattering electrons, Auger electrons, X-rays and light (Figure 2C . 4. The signals generated within the interaction volume have different escape depths which can be used to tailor the analysis to specific regions of the 32 28 ELECTRON BEAM PENETRATING 2A CONCENTRATED BEAM THE SAMPLE -' - - OF ELECTRONS Penetration Depth 20 SIGNALS GENERATED 20 81911111.: rnoouccn Secondary Electrons Anger Electron Clorocteriettc x-roye Continuum x-roye Photons Electron been induced current Specimen current Tronelnitted Electron Figure 2. The Principle of Scanning Electron Microscopy. Source: Buono (1982) 33 sample by judiciously choosing the output signal and by varying the energy of the primary beam (Figure 20). The escape depths for secondary electron is 1 to 10 nm, the depth of backscattered electron is 0.1 to 1 pm and the escape depth for X-rays is 0.5 to 2 pm. Based on those principles, the SEM is rapidly becoming necessary to plant studies along with light microscopy (LM) and Transmission Electron Microscopy (TEM). A basic differ- ence between SEM and other microscopes is the use of scanning coils to drive the beam in Y direction while being deflected in the X direction and the detection and display of low energy secondary electrons (Rasmussen and Hooper, 1974). SEM technology is progressing toward greater system automation to the point of using stepper motor stages that provides images and analyses of selected areas on a large sample without the presence of the operator (Buono, 1982). The essential features of the SEM are shown in Figure 3. The instrument contains: (1) an electron gun which provides an electron beam capable of being accelerated from 1 to 40 Kv; (2) a system of magnetic lenses to provide a means of focusing a tiny spot of electrons from the sourceon the specimen; (3) a specimen stage for holding the sample; (4) a scan generator to provide a means of scanning the spot of electrons across the specimen; (5) an electron collector coupled with a photomultiplier and amplifier to provide a means of detecting the response from the specimen; and (6) a cathode ray tube (CRT) display System capable of being 34 agate; 6"" high voltage A no de mm W :1: "" —Aperturc ,___1 _, Condenser | Sean Generator . . Aperture ' , a Z Condenser 2 Deflection a o a o O , . C 81" Visual Coils _]Grld O t 0 t o , . . "'1 —e.porture 7 '/ i . % Condenser 3 1.10m A A A a V/I///// Aperture Specimen 7... Collector _. 250v Light Scintillator Gm“ Photomultlplier 12.511V and Head Amplifier Figure 3. Essential Features of SEM. Source: Mee, cited by_Kasse1 and Shih, 1976 35 scanned in register with the incident scan. SEMS are practical tools that have-moved out of the laboratory into the production and quality control areas. They have become easier to Operate and more cost effective in providing information about surface and subsurface properties of microelectronic devises. In early 1970, Hearle (1972) provided a comparison information between optical, direct electron and Scanning Electron Microscopy (Table 1). The advantages of SEM given are: a) great depth of focus b) the possibility of direct observation of the external form of real objects, such as complex fracture surfaces, at high magnification thus avoiding the necessity to make thin replicas for use in direct transmission electron microscopy c) the ability to switch over a wide range of magni- fication, so as to zoom down to fine detail on some part identified in position on the whole object d) the ease of operation, and the large space available for dynamic experiments on the specimen e) the use; besides the secondary electrons, of scat- tered primary electrons, light emission, X-ray emission, current in the emission, and many other responses to generate an image and obtain useful information about the specimen. 36 .32 emcee we a_=o eats: FPmEm Ezzum> ems“ mum> wowpamg co Admgu xpco mpomwwpcm op «Pomep .umppwxm mu we» Mucuommmmpmm no: mwx mumcmcoe RAE: N.ov < N is: S < S 1...: o: m 2: .Nmmp .3.w .mFLem: "moczom .vmcwpcmwca one mommucw>ummwc mmcmguo cm>o mommaca>v<« saw; 93o, . umou «mcmmmmuoca , cow mpneppm>m mower mm.zpco Pmcmwm gasocm macep gmaocm mosey 36r> mo upmrd , woman .ammgmp PPmEm mpampmm>m 1 umwewnoe on :mo pan Ezsom> appmam: smgvummcm> ucme=oEP>cm . . commmwe -mcmgu coe _ mmmcxowca Ezwuwe «gown» Eaewxme 1 mowpamc go Fem; mowpqmc co Fem; maze upwuemcm> mpwpmmcm> new emcee . kxmmm Ammo appezm: cowumcmgmca 1 cmewomnm «xcme meow cmsuo 1 we» we» coppoeceewu - mmx mm» cowuompwmc 1 mm» mm» cowmmVEmcmgu . muoz «cow; Loom mzoom eo genes as: m.ov < m . E: —.o _mwumam . as: opv < cop e: ~.o emppvxm 1 e: N.o e: m xmmm . cowuspommm cocuompm uomgwo coeuompw mcwccmum Peuwuao .Azmmv xaoumoco_e cocuom—m mcwcceom ucm :ocuome powcmu .qu_uao cmmzumn :owueEcomcw compceaeou .F mpame 37 f) the information can be processed in various ways to present images in different forms. Some of the disadvantages are: a high cost CT lack of high resolution 0 ) ) ) the vacuum environment of the specimen d) inability to show up internal detail visible in optical microscopy e) lack of color response Because of the advantages enumerated above, scientists have used SEM in many areas (biology, medicine, horticul- ture, food science, microbiology...etc.) to understand basic principles involved in many materials and products. Many cereal scientists abandoned light microscopy techniques because SEM samples could be prepared easily. In the food area, although microsc0py has been used for many years, research articles on food microstructure attemp- ting to relate structural and functional characteristics of food have become common only within the past 10 — 15 years. As mentioned by Hansen and Flink (1976), the structure of individual food components, as well as of the finished product, plays an important role in determining appearance, flavor, rheological properties and keeping qualities. To study these interrelationships, the microscope in its various modes is being used to allow visualization Of the different heterogeneities in the structure of food systems. 38 On the use Of cereals and Oilseeds for food, SEM has been used extensively (Pomeranz and Sachs, 1972; Stanley _t gt,, 1976; Sullins and Rooney, 1974; Wolf, 1970). Hall and Sayre (1971a) used SEM to show the surface characteris- tics of starch granules from tender white, pinto and lima beans. They also worked on root and tuber starches, cereal starches, legumes starches and other foods and reported much information on the sizes, shapes and surface details of these starches (Hall and Sayre, 1969; 1970a, 1970b). Wolf and Baker (1972) used to investigate the cotyledon interior of water-soaked soybeans and revealed that protein bodies of 1 - lO/lin diameter exhibited a covering spongy network. McEwen gt gt. (1974) used SEM to examine the cotyledon and seed coat of faba bean seed. They revealed that there was no discontinuity in the thick seed coat. Cross section Of the seed coat showed characteristic palisade, parenchyma, tracheid, and hour-glass cells, similar to those of other legumes. Hahn ££.El- (1977) used SEM to characterize intracellular configurational changes of starchgranules during gelatinization of standard and quick-cooking lima bean cotyledon. Rockland and Jones (1974) studied the effect Of cooking on water-soaked and salt water-soaked beans. Other studies on foods using SEM include those of Vix _t gt. (1971) on cottonseed, Van Hofsten (1972), Gill and Tung (1976), Stanley gt gt. (1976) on rapeseed. 0n food starches, reports were published from Hood gt gt. (1974), 39 Shetty _t _t. (1974), da Silva and Luh (1978), Dronzek gt gt. (1972), Robutti gt gt. (1973), 9111 and Dronzek (1973), Crozet (1977), Agbo t 1. (1979), Saio gt gt. (1977), Bernardin and Kasarda (1973), Orth _t _t, (1973), Watson and Dikeman (1977), Badi gt gt. (1976), and Chabot gt gt. (1976). The food items involved were cereals, roots or tubers, legumes and banana. Other food systems such as meat and related products and dairy products have also been studied with SEM (Schaller and Powrie, 1971, 1972; Stanley and Geissinger, 1972; Jones gt gt., 1977; Buma and Henstra, 1971; Eino, 1974; Kalab and Emmons, 1974; Stanley and Emmons, 1977). The above reports are few among the numerous reports on the use of SEM in food research. That technique which is progressing toward greater system automation is presently used extensively and it will continue to be so because of the advantages it has over other microscopy techniques. But in general, the key to obtaining useful information from the microscopes is the Scientistfls-ability to recognize which microscope, if any, to use (Varriano- Marston, 1981). Legume Segd Anatomy Corner (1951) in his classification of leguminous seeds specified that legume seeds are generally of medium or large size, more or less compressed and exalbuminous, with 40 large embryo and a hard, dry, generally smooth testa (Figure 4). He indicated that two microscopic characteris- tics distinguished the leguminous testa: 1) the external palisade, developed from the outer epidermis of the outer integument, and 2) the hour-glass cells, developed from the outer hypodermis of the outer integument and, in some cases, from its inner epidermis. He stated that any microscopic particle with these features is apparently identifiable as leguminous. The different features of the testa found in various legume genera and species are: cuticle, palisade, hypodermal hour-glass cells, meSOphyll, inner hypodermal hour-glass cells, vascular bundles and subhilar tissue (Corner, 1951; Chowdhury and Buth, 1970). The position and characteristics of these features are reported in Table 2. The seed coat surrounds two cotyledons which are the most important compo- nent of the bean seed. They are composed of parenchyma cells containing starch granules embedded in protein matrix (Bagley t 1., 1953; Harris gtgt.,1975;0pik, 1966,1968; Powrie _t _t., 1960; Yatsu, 1965) and vascular bundles (Opik, 1966) and protein bodies forming the protein matrix. Many scientists report that textural characteristics and nutri- tive value of processed bean presumably are influenced to a large extent by the size and shape of cells, dimensions of the cell walls and the localization of chemical constituents in the cotyledon. These factors and a few definitive 41 HICROPYLE . . O fiuaflhe oobflcn'tfll . 1. u CI. 0. . a. O. “a .11 ‘I e too ."oo do I ~ .. s . . .. f... .. .k '0 E.‘ ‘a-Oe - ”one.“ I e‘. O... A... \N o . I edm.‘oo-"Oo-JI III ’I O 'o. e Q on no... do I o. o e. 0. fl 9 o O E o o o o no 0.. not fitcnfo ‘ .h- .e-.. o e no. a e o a . n e o c o h I. a e o o e creel. .‘eoou whoa. cunt. u" so a oe-oeuoo Do a e.- u e. -.o- .o l?- ‘01. .ho. .* "Hm. MuNIo: bl . one o o a D . u o o e .o. 5.9-0.4 w! e .u... . .. 3......) “1.26...“ v .0 ‘99., o on oil ..I - .0.- nl e- cboflOnr e I 0 e 0.31on . o. lo 0 . - .El- f I r... . . o. o . . o o o e no ore-Vooo S.oeeecah&o‘ouO\ o. e o o n . "e‘owodoav . e 0 t o oo . . oo". o l— v: T 0 c 0 P Y. " EPICOTYL ”ILUH COTYLEDON -" SEED COAT Visual observation of legume seed structure. Figure 4. 42 Aceopv spam use eczeozoco AoeaFv spam new easeozoeo Aoea_v seam one stseozoeo Aoempv eosm oca xtaeezogo mumou.ooom oz» wo Looceoeoc one o3» o>og Zoe poo mmoqucoo; opmcmm o>os mesomoF meow movooom segue: oco morooom o» mowooom ooomPpoo ow uxo: eocw mo_go> ooogm- coxop gopoo ogvse peace; oco ooogm sopomoggfil coxop Louzo ocooom canoe oco spooem- m_Foo _Fx;a0moz mFFoo mmopmlcoo: mppmu moomrpma Apmmpv Locgou spooEm oco ewe»- Loxop gonzo pmcwm oFopuou mococomom owpmrgopooeogu cowuwmom weapoou .movumwcopoosoco oczuoom momma esomoo .N opooh 43 answers have been obtained to relate the ultra-structure of legume seeds to the textural characteristics of the processed product. Further research in this area is warranted. Starch and Viscosity Starch is one of the most abundant naturally occurring organic compounds. It is found in almost all plant tis- sues, where it functions as a source of reserve energy, which can be utilized gradually through enzyme actions. Starch accumulates to high concentration (20-70%) in the roots, tubers, fruits and seeds of many plants. Chemically, starches from all sources are carbohydrates, polymers of a-D-glucopyranose units linked primarily by 1,4- and 1,6-glucosidic bonds. Each glucose unit contains one primary and two secondary hydroxyl groups which are respon- sible for the hydrophilic properties of the starch. Starch is made up of two types of polymer chains: amylose, the linear fraction (1,4 bonds) and amylopectin, the branched chain fraction (1,4 bonds and 1,6 bonds). The-ratio of amylose and amylopectin in starches alSo provides Some indication of their origin. The exact structural rela- tionship between the two components in the starch grain is not known, but the molecules are linked together to form starch granule by hydrogen bonds (Hahn, 1969; Wurzburg, 1968). 44 Starch granules are insoluble in cold water. In a humid environment, starches will take up moisture, but the swelling which occurs is reversible. When starch granules in water are heated past a critical temperature, in the range 60 - 70°C (gelatinization temperature, characteristic of a specific starch), the hydrogen bonds which hold the granules together begin to weaken, allowing the granules to swell. SinCe the gelatinization begins at the hilum, the first indication of swelling is the loss of birefringence. Then, as the amylose fraction is dissolved and leached out, the granules begin to take in water and clarity and the vis- cosity of the slurry begins to increase. Eventually, the granules, having become completely hydrated, may collapse and break down. The resulting starch "paste" is composed of granule fragments and molecules in solution. The visco- sity of the paste decreases as a result of this granule breakdown. 0n cooling the paste usually increases in viscosity and decreases in clarity. This results from retrogradation of the linear (amylase) molecules,_a process whereby the amylose molecules tend to form rigid gels by hydrogen bonding. The degree of retrogradation is dependent on amylose content and the degree to which the amylose has been solubilized during the heating cycle. Cross-bonded starches have their granule structures reinforced by covalent bonds and the granule shows less tendency to swell and retrograde (Wurzburg, 1968; Leach, 1965; Smith, 1964; 45 Collison, 1968; Mazurs _£._l-: 1957; Hoseney gt gt., 1978). Effective control and utilization of retrogradation is necessary to obtain food products of good quality after transport and storage. Since each raw starch has its own characteristic viscosity/temperature profile as a result of its particular granular composition and structure, some degree of control can be achieved simple by selection of the correct starch (Sanderson, 1981). The suitability Of starch, natural or modified, for a specific use depends on the functional properties. The functional properties would include, for example, ease of cooking (temperature, and stirring energy to cook through the swelling region), thickening power (final viscosity of the paste after cooking and cooling, as a function of I concentration), and stability (resistance to thinning resulting from stirring, pH or temperature change). Because of the variety of changes which may occur in starch paste during processing, many different methods have been developed for following these changes in the paste and estimating the functional properties of the starch from them. These methods fall into two categories, those invol- ving direct microscopic observation such as monitoring granule swelling, loss of birefringence or staining reactions (Collison, 1968; McMaster, 1964), and those involving measurement of phySical properties such as swelling power, solubilization, sedimentation rate or 46 viscosity (Collison, 1968; Schoch, 1964; Smith, 1964). The most useful methods measure the viscosity of the paste continuously during the standardized-cooking and cooling cycle which stimulates a wide variety of processing condi- tions (Smith, 1964). The most common instrument used for this purpose is the Brabender Amylograph. This machine continuously measures the viscosity of starch pastes and flours while they are stirred and heated at a constant rate, held at 95°C for 30 minutes and held at 30°C for 30 to 60 minutes (these methods have also been developed for relating the viscosity data to the functional properties of the starch) (Mazurs gt gt., 1957). These kinds of data are available for most food starches such as cereal, roots, tubers, banana and legumes (Mazurs gt gt., 1957; Kite ._£._l-: 1957; Hahn gt gt., 1977; Carson, 1972; Berry _t gt., l979; Rodriguez-Sosa and Gonzales, 1975; Agbo gt gt., 1979). Legume starches which are of interest in this disser- tation have been studied by several scientists for their incorporation into bread, cookies and other product formu- lations. Hall and Sayre (1971a), Kawamura gt gt. (1955), Lineback and Ke‘(l975), McEwen gt gt. (1974), Rockland and Jones (1974) found good agreement among various legume starch granules of the same species. They reported that legume starch granules were ellipsoid, kidney-shaped, or irregularly swollen, with an elongated hilum and smooth 47 surface with no evidence of fissures. The Sizes were deter- mined as diameter width and length ranged from 12 to 36 um and 12 to 40 um for navy bean and from 16 to 18 um and 16 to 48 pm for pinto bean respectively. Brabender hot-paste viscosity pattern of various starches appears to be determined not only by the extent Of swelling of the starch granules and the resistance of the swollen granules to dissolution by heat and fragmentation by shear (Lineback and Ke, 1975), but also by the presence Of soluble starch, which is leached from the granule struc- ture (Allen gt gt., 1977; Miller gt gt., 1973) and the interaction or cohesiveness between the swollen granules (Leach, 1965). Schoch and Maywald (1968) classified the viscosity patterns of "thick-boiling" starches into four types. Type A: High-swelling starches, e.g., potato, tapioca, -the waxy cereals, and ionic starch derivatives. The granules of these starches swell enor- mously when cooked in water, and the internal bonding forces become tenuous and fragile toward shear. Hence the Brabender shows a high pasting peak followed by rapid and major thinning during cooking. Type B: Moderate-swelling starches, e.g., normal cereal starches. Because the granules do not swell excessively to become fragile, these Type C: Type D: 48 starches show a lower pasting peak and much less thinning during cooking. Restricted-swelling starches, especially chemically cross-bonded products. Cross- linkages within the granule markedly reduce swelling and solubilization, and stabilize the swollen granule against mechanical fragmenta- tion. Hence the Brabender curve shows no pasting peak, but rather a very high viscosity which remains constant or else increases during cooking. Starches with highly restricted swelling, especially "high-amylose" corn starches con- taining 55 - 70% linear fraction. BecauSe of the internal rigidity imparted by the high content of associated linear molecules, the -granules of the starches do not swell suffi- ciently to give a viscous paste when cooked in water at normal concentrations. Hence, the amount of starch must be increased two- or threefold to give a significant hot-paste viscosity of Type C. However, such high- amylose starches give a Type A or B viscosity pattern when cooked in media which cause greater granule swelling, e.g., 0.1 N sodium hydroxide. 49 The above two scientists found that navy bean, lentil, yellow pea and garbanzo gave Type C Brabender curves. Mung bean starch showed a mixed viscosity pattern - Type C at low concentration and Type B at high concentration. Navikul and D'Appolonia (1979) studying navy beans, pinto beans, faba beans and mung beans reported that the amylogram curves of the legume starches showed higher initial pasting temperature and higher viscosity than did wheat starch, which would indicate a higher resistance to swelling and rupture. NO peak viscosity during the hold period at 95°C occurred with any of the legume starches, indicating that the paste was relatively stable and that the granules did not rupture during stirring, which is not the case with wheat starch. Lineback and Ke (1975) reported that the Brabender hot-paste viscosity patterns for chick pea starch at concentrations of 5, 6, 7, 8 and 9% are similar to those reported by Schoch and Maywald (1968). They also indicated that the Brabender hot-paste viscosity patterns for horse bean starch are virtually identical-to those obtained for chick pea starch at the same concentration. The gelatini- zation temperature ranges were 63.5 - 65 - 69W3for chick pea and 61 - 6315 - 70°C for horse bean starches. Vose (1977) working on smooth-seeded field peas reported that pasting curves of smooth pea starch showed restricted- swelling characteristics similar to those shown by chemi- cally cross-linked starches. Retrogradation of the cooked 50 pastes resulted in rigid, Opaque, friable gel with a firmer texture than corn gels. The gels occurred in either acidic or basic solutions, demonstrating characteristic differences when compared with corn or wheat pastes. Because of the importance of legume seed in food supplementation and new product development, the pasting characteristics and functional properties of several other varieties and species of beans still need to be investi- gated. Indigestible Bean Sugars Problem Although legume seeds have high nutritional value among vegetable proteins, they contain a considerable amount of dligosaccharides which have been implicated as factors responsible for flatulence (Steggerda, 1968; Steggerda and Dimmick, 1968; Hellendoorn, 1969). Flatulence is a common complaint even among healthy individuals and is one of the most common causes of abdominal discomfort. Flatus generation has been associated with the absence of a- galactosidase activity in the upper intestinal tract of humans and animals and the fermentation process of the oligosaccharides by microorganisms with production of gas. It has been attributed also to swallowed air, inhibition of intestinal anhydrase, production of carbon dioxide from pancreatic bicarbonate (Rockland, 1969). Rackis t l. (1970) reported that the flatus produced by fermentation of 51 dietary carbohydrate contained in the lower intestine may cause nausea, dyspepsia, constipation, cramps, diarrhea and discomfort. He specified that the flatus-forming factor is mainly found in the low molecular weight carbohydrate fractions of legumes which contain primary sucrose, raf- finose and stachyose. Takana gt gt. (1973) and Cerning _t _t. (1975) reported that legumes contain appreciable amounts of a-galactosides of sucrose, particularly raffi- nose, stachyose and verbascose. Akpapunam and Markakis (l979), Cerning-Beroard and Filiatre (1976, Navikul and D'Appolonia (1978), Olson gt gt. (1975), Rackis (1975) found appreciable amounts of those oligosaccharides in mature seed such as beans - California small white, Great Northern, navy, pinto, kidney, soy, faba, lima, field, and mung; peas, cowpeas; chick peas; pigeon peas; horse gram; lentils; and lupines. Tanusi (1972) reported that mung bean, broad bean and smooth pea have small amounts of ajucose; reducing sugar ranges from 0.06 to 0.10% and nonreducing sugar from 5 to 20% in broad Dean (1. faba), mung bean (Phaseolus aureus) and kidney bean (E, vulgaris). Navikul and D'Appolonia (1978) showed that the total sugar content is higher in all legume flours than in wheat flour. The legume flours contain high levels of sucrose, stachyose and verbascose. but navy and pinto bean flours contain small amounts of verbascose. Reddy and Salunkhe (1980) presented the chemical structure Of those sugars containing 52 a-galactosido-glucose and a—galactosidO-galactose bonds as nonreducing sugars shown in Figure 5. AWhen those sugars are ingested by humans, two enzymes (invertase and a-galactosidase) are required for complete hydrolysis. Gitzelmann and Auricchio (1965) explained that because the human gastrointestinal tract does not possess an a-galac- tosidase enzyme and because mammalian invertase is an a-glucosidase (Reddy and Salunkhe, 1980), the metabolic fate of raffinose family sugars is uncertain. Schweizer gt gt. (1978), working with twelve mature leguminous seed crops, reported in their study using 70% ethanol extraction procedure and gas chromatography tech- niques, a new compound pinitol (3-O-methy1-D-chiro-inositol) and three isomers Of the new disaccharide, a-D-galacto- pyranosyl-pinitol, for which the name galactopinitol was proposed. The chemical structures are presented in Figure 6. These sugars isolated from soya beans (Glycine max) were also identified in chick peas (Cicer aristinum L.), lentils (Lens esculenta) and beans (Phaseolus vulgaris L.). Their amounts ranged from 0.2 to 0.9% and 0.03 to 0.8% of dry weight respectively. Schweizer _t _t. (1978) stated that being a-galactosides, the new disaccharides must be taken into account when considering flatulence problems. Wursch (1977) proved the galactopinitols being indigestible to human intestinal enzymes. Schweizer gt gt. (1978) specified that it is probable that gas chromatography peaks 53 .owmp .u.o .wzxcspmm use .m.z .xvumm "muezom .mooweogooomommro z—VEoe omocwmwoe o5“ mo movsmoowquoe weapoaepm .m ocamwe arm. I a I t I a~f3I340 lol .0 I31: 1.20.1013 loo-31440.... alyotwbels IJ to RI... act-lo .lQiaNelaiq—JG in! .0 Ia01.~ld¢01 al .0 l?o1..|.4<¢ l at .0 pi e um 02.53% 10 :o :u . accloilo oauloiaizts 1391?. 3131.74: 1m 16 umozigc GALACTOPINITOL 1 O on OH OH OH 0H a ’ “-0- “LACTOPYIANOSTL— PIIITOL 54 MR OF GALACTOPI NITOL cu, OH o H '50“ OH OH . F1gure 6. Stnucture of ga1actopinit01. Source: Schweizer ££.11-9 1978. 55 from non-fermented soya bean identified as melibiose in the literature were in fact a galactOpinitol. Although the incidence of flatulence in humans is unpredictable - depending on the psychological and physical state of the subjects and the type of diet. oligosaccharides are generally considered undesirable and many attempts have been made by food scientists to treat beans or their products to remove or degrade them. Mital and Steinkraus (l975) attempted to degrade oligosaccharides with lactic acid bacteria. Sugimoto and Van Buren (1970) used commer- cial B-galactosidase to hydrolyze oligosaccharides to their component sugars. Thanamunkul gt 31. (1976) and Smiley _£‘_l. (1976) used B-galactosidase in a hollow fiber reactor to hydrolyze oligosaccharides with varying degree of success. Rackis gt_al, (1970) and Steggerda (l967), working on experiment 1_ vivo and in vitro, suggested that antibodies and certain phenolic acids can inhibit flatus activity. Many workers have investigated the removal of_oligo- saccharides during soaking and germination (East 35 al., 1972; Hand. 1967; Iyengar and Kulkarni. 1977; Kawamura, 1966; Kim t al., 1973; Ku gt al., 1976; Rao and Balavady, l978; Reddy t 1., 1980). Applications of ultrafiltra- tion techniques in the removal of oligosaccharides had also been used (Omosaiyre gt al., l978). 56 Bean breeders are also attempting to identify genetic variants possessing low levels of oligasaccharides which may be used to biologically remove the flatulence factors of beans. Hymowitz _t al. (l972) have indicated that the removal of oligosaccharides by plant breeding does not look promising. But because removal of oligosaccharides by l) soaking results in loss of water soluble vitamin, minerals and digestible sugars, 2) germination shows altera- tion of the carbohydrate content of the seeds (Bond and Glass, 1963; Linko _tf_l., 1960; Dubois S£.il-: l956l, 3) chemicals need economic feasibility and approval for human use, the general potential for eliminating flatulence factors still lies in both biological and technological areas. Bean breeders can therefore still include that type of research in their programs. The quantitative determination of oligosaccharides has been the subject of many investigations using either paper chromatography, thin layer chromatography, column chro- matography or gel filtration (Kawamura, 1967a, 1967b; Hardinge 33 al., l965; De Stafanis and Gonte, l§68). Delente and Ladenburg (l972) used gas-liquid chromatography to quantitate oligosaccharides in defatted soybean meal. Kim gt 31. (l973) employed liquid chromatography for the rapid determination of monosaccharides, disaccharides, stachyose and raffinose in soybeans, but absolute values for the quantity of the different sugars were not obtained 57 by this method. Takana t l. (l975) used an analytical procedure based on thiobarbituric acid reaction (Percheron, l962) to determine sucrose, raffinose and stachyose in whole legume seeds. All of these methods are time consuming and require some skill and experience to produce reliable results. Newly developed column packings for HPLC in the recent years have greatly simplified sugar analysis in foods and other plant materials. Quality Evaluation of Processed Products In the early history of breeding, the plant breeder who was the cereal breeder and particularly the wheat-breeder himself made his selections on the basis of agronomic performance and disease resistance. The main quality test that he used was the "chewing test", usually applied out in the plots after hand threshing the grain of two or three heads. To an experienced person, the chewing test gave all the quality information needed for early generation selec- tion. The pressure applied by the jaws to crack the wheat gave a good, albeit subjective, measure of grain hardness. As the crushed grain was masticated in the mouth into a dough, the starch and other solubles, due to salivary amylase, were gradually dissolved and swallowed, or spit out, depending on the number of samples tested. Experience showed that the size of the remaining gluten ball was indi- cative of both protein content and gluten quality (Bushuk, 58 l982). Gradually, as the cereal Chemist's knowledge of the fundamentals of milling and baking quality expanded, screening of varieties for quality became much more SOphis- ticated. Today as many as 26 different quality tests are applied to material in the final stages of development (Bushuk, l982). Those tests are applied now not only to cereals but to many other food materials such as tubers, roots and legumes. Legume quality evaluation has been of interest in the recent years by bean breeders because of its role as a high protein source and supplement to cereal products. Adams and Bedford (1975) stated that the bases for selection of improved breeding lines require additional expenditures to conduct the various quality tests on the canned product besides the quality tests on the dry seeds. They mentioned the more subjective evaluations of quality: l) Wholeness - the tendency of the legume seed to remain whole throughout the processing operations — not to break apart, burst, or disintegrate." 2) Consistency - the fluid is slightly viscous and clear, not cloudy or grainy; it separates or drains readily‘from the beans. 3) Freedom from defects - no extraneous material, loose skins, or mashed beans. 4) Flavor d must be scored by a taste panel. 59 5) Color - pigments in the seed coat that escape detection in the dry seed may impart an off-color to the cooked product. 6) Texture - the measure of firmness, gumminess and adhesiveness evaluated with reasonable accuracy by a sensory panel and by machines such as Kramer Shear Press, Instron and Ottawa Texture Measuring System. Adams and Bedford (l975) stated also that there are no comprehensive studies on the source and nature of variation in the quality factors referred to above. From limited studies of special factors such as texture, and on the basis of experience, the assumption has emerged that at least a portion of the variability observed among lines dépends on genetic differences although it is clear that the length of processing, the temperature of soak, the hardness of the water, and the character of the added fluid all play an important role. The above two authors recognize that, in practice, in order to assure acceptance of'a new selection by growers, the breeder generally will find it necessary to compromise between the best level of quality possible in a particular program and the levels of disease resistance and agronomic performance that must be maintained. They are right to say that there is no escape from that because consumer choice for a food product is primarily good aesthetic appearance and qualities of the prepared food except that now he or she becomes more aware of nutritional 60 evaluation of the food. Hence quality evaluation of processed foods becomes progressively important to plant breeders in order to meet the consumer requests and provide nutritious foods with great acceptability. MATERIALS AND METHODS Sample Source Dry beans (Phaseolus vulgaris L.) used in this study were: Sanilac, a standard commercial bean; Nep-Z (Nuclear Experimental Project-2), a mutant; and San-Fernando, a tropical genotype (Phaseolus vulgaris L.). They were obtained from Saginaw Valley Bean and Sugar Beet Research Farm near Saginaw, Michigan, through the Department of Crop and Soil Science, Michigan State University, East Lansing, 'by the-courtesy of Dr. G.L. Hosfield. They were delivered in paper bags to the Food Science Building and stored at room temperature in the laboratory where the experiments were carried out. Seed Coat Separation A certain amount of the seed from each type of bean was immersed in warm tap water for about S-minutes. Coats were then carefully removed because they were still attached to the cotyledOn outer sides. The soaking period was short in order to avoid diffusion of cell constituents such as sugars into the water. Each separated fraction was left at room temperature for three days to dry. 61 62 Grinding of Sample Cyclose Sample Mill, U.D. Corporation, was used to grind into flour, whole seed, cotyledon (coatless) and coat from each type of bean. Moisture Moisture was determined by oven drying the sample at 80 i 2°C until weight remained constant in order to avoid browning or caramelization reactions taking place when drying at l30°C for l hr (Hosfield and Uebersax, l979). Crude Fat Crude fat was determined using A.0.A.C. method (l975) based on extracting the fat from the bean flour with petro- leum ether in a Goldfisch extractor. Ash The A.0.A.C. method (l975) was used for ash determina- tion. The method involved oxidizing all organic matter in a weighed sample by incineration at 550°C until the ash was gray-whihaand determining the weight of the ash remaining. Protein The Kjeldahl semi-micro-method based upon the determi- nation of the amount of reduced nitrogen present was used. 63 The method consisted of: first, the wet oxidation of the sample and the conversion of protein nitrogen into ammonium sulfate; second, the decomposition of the ammonium sulfate with 10 N sodium hydroxide and the distillation of the ammonia evolved into saturated boric acid solution, and finally, the determination of the nitrogen content by titra- ting the ammonia with standard hydrochloric acid, 0.1 N. £19111: Extraction and Injection The simple and oligosaccharide sugars (glucose, sucrose, inositol, raffinose and stachyose) commonly found in beans (Phaseolus vulgaris L.) were determined using High Pressure Liquid Chromatography (HPLC). The method consisted of: first, an extraction of the sugars in a 80% ethanolzwater (v/v) using a technique developed in_our laboratory (Figure 7); second, precipi- tation and centrifugation of protein materials by a solution of lead acetate (l0%, w/v); third, precipitation of excess lead in the extract using an oxalic acid solution (10%, w/v), and removal of the lead oxalate by centrifugation fourth, bringing-up of extract to volume in a 25 ml volu- metric flask; fifth, filtration of the prepared sample extract through Waters Associates Sep-Pak C18 cartridges having the same separation properties as the separation column C18 where the polar compounds elute before the (54 .mwmzpmcm mezm mo Emgmmmu 30pm xmcau llllllv xumnaom mac IIIIIIIIIV cum: cu aooncH ounao :Ho> Ha mm a" sea: uoaaauoum o : sad: msauo> cuuonwu on means 2623* an .2253“ .335 can: ofiacxo undounv owuhaucu zohuam as N and you oouoam N mawwnom «N accaawmomsm o acoaaflmo 3m 0 oJMMQom :mm ooow as :«l m omsuauacoo Adv :« no cocoouq vac Handgun now ousfifllm Amy H. CH 66¢ ounaooo uuoH a ouwdnox acaaanuona 9 am .a«. n to» 2mm coca a. ousuauacoo a. ouaoxfiz o~a1um .aa. ma you coca a. gang yoga: a“ oxagm accusao man an ad on< n acavucuomzm H. N 22 a. l o + n + u AHv nu ea convoys can Hoaagao uom H. n can a osuaaom L AH. .N mgzmwu 65 non-polar ones. Finally, the clear extract sample was injected onto the C18 carbohydrate column (30 cm x 3.0 mm 1.0. plates/column N/A 3000, Waters Associates, Milford, MA) for quantitation. The elution solvent was filtered acetonitrilezwater (70:30, v/v, pH = 4.0) with a pump rate of 2.0 ml/min. Thirty microliter samples of the extracts were injected using a 100 ul pressure-lock microsyringe into a model U6K injector which allows the sample to be put on a bypassed injection part (no pressure exists in the bypassed state). After injection of the sample into the U6K injector loop, the bypass valve on the Waters Associ- ates pump system Model 6000A was then switched to introduce- the sample to the pressurized column held at room tempera- ture. The refractive index detector Waters Associates Model R40l was used. Methods of Quantitation External standard and repetitive injection techniques (Waters Associates l980) were used. 'They consisted of: first, preparing a mixed standard solution containing a known amount (l mg/ml) of sugars to be quantitated; second, injecting a sample of the standard solution to find out the retention times of each individual known sugar appearing on the peaks of a chromatogram through a Data Module computer- ized machine (Waters Associates, Milford, MA); third, the retention times of the sugars were put in calibration into 66 the computer Data Module according to the techniques of Waters Associates (l980); fourth, the unknown samples injected onto the HPLC column were then quantitated by comparison with the standard sugars put in calibration previously. _ The percentage of the individual sugar extracted from the beans used (Sanilac, Nep-Z and San-Fernando) were cal- culated acCording to the following formula. Percentage sugar = Amount shown on data original sample module (mg/ml) , value (ml) x 100 sample weight (mg) Starch A new technique using HPLC was developed (Figure 8) and used in this determination. The technique consisted of a) solubilization of the sample; b) hydrolysis of the sample with amyloglucosidase enzyme to obtain glucose mole- cules; c) injection of the hydrolysis product to the HPLC. The amount of glucose obtained is relative to the amount of starch hydrolyzed. Sample Preparation Reagents sodium hydroxide (NaOH) 0.50 N acetic acid 0.50 N 67 Starch Pellet (el.000 g) Disperse with 0.50 N NaOH (l0 ml) Neutralize with 0.50 N acetic acid (l0 ml) Neutral colloidal suspensions Let stand few min. (not necessary) Starch- ucose assay 1 Add 3 ml amyloglucosidase (5 mg/ml) Incubate 30 min/55° 20c continuous shaking RemoVe tubes Let cool (room temperature) Filter through amberlite Injection to HPLC Figure 8. Flow diagram of starch dispersion, hydrolysis and analysis. "1 68 The samples were the dry residue pellets obtained from extraction techniques developed in this same laboratory. 10 ml of 0.50 N sodium hydroxide was added to the centri- fuge tube containing the pellet (l.000 9). With a rod, the pellets were colloidally dispersed into the 10 ml Na0H solutions (0.50 N) by continuous crushing and stirring. After all the pellet was completely dispersed, l0 ml of acetic acid solution (0.50 N) were added into the tube to neutralize the sodium hydroxide used for dispersion. The neutral colloidal suspensions were allowed to stand for a few minutes in order to allow unsolubilized particles, mainly cellulose, and pectic substances, to settle before using.the sample for injection (this step is not obligatory). Buffer and Enzyme Solutions 0.2 M Acetate Buffer Stogk Solution 0.2 M acid: 12 ml glacial acetic acid was trans- ferred to l000 ml volumetric flask and diluted to volume with distilled water. 0.2 M Sodium Acetate: 16.408 9 of anhydrous sodium acetate were dissolved in 800 ml of distilled water, transferred to a l000 ml volumetric flask and diluted to volume. Stock Solution: Three volumes of 0.2 M sodium acetate with two volumes of 0.2 M acetic acid were mixed. Volumetric flasks were used. The pH was 69 4.9 (theoretically 4.92). The solution was stored in a well-stoppered colored bottle under refrigera- tion; it could be stable for at least a month. 0.02 M Acetate Buffer Working Solution To one volume of the 0.2 M acetate buffer stock nine volumes of distilled water were added and shaken to mix. This dilution was carried out using pipette and volumetric flask and prepared each time immediately before each analysis. Enzyme Solutions 5 mg/ml amyloglucosidase (500 mg/lOO ml, w/v) solutions were prepared in 0.02 M acetate buffer working solution. Enzyme Characteristics Amyloglucosidase (glucoamylase l,4-a-D-glucan gluco- hydrolase; EC 3.2.l.3 from rhizopus genus mold 10,000 units/ 9 solids). The enzyme was purchased from St. Louis, MO. Hydrolysis 1 ml of sample was mixed with glucosidase (5 mg/ml) solution in were placed in a water bath at 55 Sigma Chemical Company, 3 ml of the enzyme amylo- a test tube. The samples 2°C (optimum temperature |+ for amyloglucosidase) and continuously shaken for 30 min. At the end of the time period, the test tubes were removed from the water bath and filtered through a Sep-pak to 70 remove the acetate (Lester, 1980) prior to the injection to the HPLC column. Injection Injections proceeded as described above for the intro- duction of sugars onto the C carbohydrate column. 18 Methods of Quantitation Same asin the above quantitation of sugars with excep- tion that only glucose was used as standard solution. Calculation The percentage of starch was calculated according to the following formula: Percent starch Amount shown on data Original module (mg/ml) x 10xvolume (ml) X 100 X 0-9 sample weight (mg) where: 0.9 (factor to account for the water gained during hydrolysis) lO (equation correction factor) Triplicate determinations were performed on samples for all above methods. 71 Water Absorption in Beans The hydration properties of beans were determined by soaking a l0 9 sample of dry beans (Sanilac, Nep-Z and San-Fernando) contained in cylindrical grids in tapzdis- tilled water (l/l) at room temperature for 0, l5, 30, 45, 60, 75, and 90 minutes. After soaking, the cylindrical grids were removed from the water and allowed to stand on laboratory paper towel in order to remove surface water. The beans were then weighed and the increase in weight taken as the amount of water absorbed. Triplicate deter- minations were performed. Nater content was calculated using the following formula (Hosfield and Uebersax, 1980). 100 _IWt° 0f SOIIdS (9) in dry beans \ Wt-i(9) of soaked beans )x l00 Canning Beans used for canning evaluations were adjusted rapidly to l6.0 : 0.02% moisture content in a controlled air circu- lating humidity chamber prior to soaking and processing. This was done in order to eliminate any effect differential seed moisture might have on cotyledonary tenderization during soaking and cooking and to insure that each sample lot of beans contained a constant level of total solids (TS). 72 The soaking and processing procedures developed by Hosfield and Uebersax (l980) were applied in this process (Figure 9). The parameters considered in the evaluation of the beans used in this experiment were: color of dry and canned beans by Hunter colorimeter; drained weight by decanting contents of cans on a number 8 mesh sieve, rinsing in ZlOC tap water to remove adhering brine and draining for 2 minutes on the sieve positioned at a 15° angle; texture using a Kramer Shear Press (KSP) fitted with a standard multiblade shear compression cell No. C338 (Food Technology Corp., Reston, VA). One hundred grams of washed processed beans were placed in the compression cell and force was applied until blades passed through the bean sample while the instrument was set at range lo (300 lb of force full scale). Water content of canned beans (final moisture) was determined from 100 gm texture samples. These were OVen dried at 800 i 2°C to a constant weight (Table 3). _ Subjective bean quality evaluation was made on contents of all processed cans while beans were drained on the mesh screens. The degree of packing (clumping) was rated on a 3 point scale. ‘Dverall bean appearance was evaluated to measure the suitability of beans to commercial processing. Criteria included examining beans for loose or free coats ("free skins"), individual bean integrity, and fluid con- sistency (Table 3). 73 .mmgzcmuoca mcwmmuuoga ccma zen go» mcowumgmao are: EnemmVU ovumsmcum .m mgzmwu mz¢n owShw 74 Table 3. Bean processing evaluation and calculations. Character Description Soaking Hydration Coefficient Ratio of wt of soaked beans (9) wt of dry beans (9) Water content (%) 100- ,wt of solids (g) in dry beans ‘ wt (9) of soaked beans xlOQ Canning Objective Drained weight (9) Weight of rinsed beans drained for 2 minutes on a number 8 mesh screen (0.239 cm) positioned at a l5° angle. One determination per can was made. Texture (Kg force/lOO g) Determined by placing 100 g of washed processed beans into a standard shear-compression cell of a Kramer Shear Press and applying force with a dynamic hydraulic system. Values repor- ted indicate the Kg force required to sheat loo 9 of beans. _ Three determinations per can were made. Water content (%) Determined by oven drying each texture evaluation at 80: 2°C until weight remained constant. %:=initial wt - dry wt initial wt x 100 Subjective Degree of packing (l-3) Extent of packing (clumping) of beans in can. I = no clumping; 2 = bean clumping but easily decanted from can; 3 = beans clumped or packed solidly in bottom of can. One determination per can was made. 75 Table 3. (cont'd.). Overall Appearance (l-S) Evaluation for general suita- bility of commercial processing made on each can. Criteria included examination for loose (free) seed coats (split), bean integrity, and brine con- sistency. Low values indicate poor appearance; high values indicate excellent appearance. Source: Hosfield, G.L. and Uebersax, M.A. l980. 76 All data were subjected to an analysis of variance appropriate to a completely randomized design. Duplicate readings were taken on all cans for texture and final moisture content. Texture Measurement Based on Single Bean To accomplish this, some samples of our bean varieties under investigation were sent to Dr. L.N. Hudson at the Regional Plant Introduction Station, Washington State Uni- versity, Pullman, Washington. The tests were performed using the apparatus developed at that station. The apparatus consists of lOO tests points in a 10 x lO array. Each point has a cup holding a single bean seed. Resting on each bean is‘a 1.47 mm diameter pin attached to the end of a length of tubing. The tube contains enough No. 8 shot so each of the lOO pins bears 90.6 grams on each bean. The cooking vessel contains about 180 liters of water heated by steam-coils to 93.3°C. A Taylor controller keeps the water at a very constant temperature (:_O;25°C). When the water is at the prescribed temperature, the testing device is put in the water and the time recorded as "Beans in". Simultaneously a timer is started and in due course, as each bean reaches the proper state of "cook" the pin penetrates and the time is noted in the square corresponding to the test point. 77 The conditions of the test were: a) the moisture content of the seeds was adjusted by humidifying the seeds for 6 hrs; b) the beans were scarified by clipping away a small portion of the pericarp on the edge of the seed opposite the hilum and then c) soaked for l2 hours using distilled water kept at room temperature. Tests have shown that with the apparatus, significant differences can be shown with ten seeds per lot. In this study, the test was performed four times using best looking bean seeds (twice with seed coat intact and twice with seed coat partially removed). The results were reported as time to breakdown (TTB) in minutes with standard deviation and _coefficient of variation. Gelatinization Characteristics A sample of 50 g of whole bean and cotyledon flour (adjusted to-l4% moisture) from each type of bean was used with 400 ml of phosphate buffer, pH 5.30 to make the slurry ready for heating. The functional properties of the flours were evaluated according to the AACC methods using Braben- der Amylograph (1969). The method of graphic analysis for functional properties are as follows (Table 4) (Mazurs t 1.,1957; Kite 3331.,1957): The Peak Viscosity or Pasting Peak This is the highest viscosity which is reached during the gelatinization of the starch. The temperature where 78 mew—Fezm eecmepcwee ee uceuxe zuwewmw; mpecegw Xpwewmeg erzcegw mopeeepee Leecw_ we cowpeeegmegmem :ewueNPPVezpem ye eegmee ece xpwpwmegm epecego mCTFPezm ereceLm we ucepxm mcwppezm mefizceem we epem xeemweweee mcweexewcu Le gezee mcecexewsh mecm 0.... mUCMHMwmwm mcwpeee :e xeeeuuem eeexeoe eeeeae mee_eeeem mcwxeee :e mmecxewzu Eeswxez memxeee we ewem .nmmp ..p u meanez ”meeeem . he eeeoev eeem ea acwepe; Levee xpwmeemp> Pee?» Am e» a maewee ea eeeedev eeem ea eeee_e; mewgee zuwmeem_> cm memcegu Le ee u mucmee ee :emaegv mcwpeee mewgee hummeemw> cw emeegecH Au ea < meceee we :ewmegv mepeze m:_e_e; oemm use mcwuee; mevgee Azupmeemw> Eeewxes mcvgeeeg gepwev memeeze xuwmeemr> A< ucweev xeee xpvmeemv> A< newee e» Levee :ewmemv uemm e» eeuee: ces; xuwmeemw> cw emeegec. we mama mewugeeege Lepzeepez mewugeeegm Pecewaeczu we mewumwge em» a one m=_pmaa 59*: eeueeeemme me_ugeeege ge_=ee_es wee 5 If Aeeewsgeueo appeucespgeexuv mewueeeege enmee It: .mecegeem e=e_ee==g .V eNQeK 79 the viscosity begins to increase, and the rate of increase are also considered. Together these three factors indicate the ease of cooking and the pasting peak provides an esti- mation of the power requirements for stirring the starch paste during gelatinization. Some starches do not have a distinct peak. The visco- sity simply increases during heating and tends to remain Icelatively constant during the holding cycle at 95°C. Tlie Viscosity at the End of the Heating Cycle as Sample Reaches 95°C This gives an indication of stability during cooking when related to peak viscosity. A sharp dr0p in viscosity from the viscosity peak indicates granule fragility and 501 ubilization. Me Viscosity at the End of the 95°C Holding Cycle This indicates the degree of fragility or stability of the: hot paste. A drop suggests additional breakdown of granules or solubilization due to stirring. QID£L_jLiscosity at the End of the Cooling Cycle, When the EEEEEjggReaches Again 50°C T‘his is a measure of the thickening or "set-back" of the paste when cooling. It arises from retrogradation of tnle linear molecules and is a serious obstacle during pro- CeSsing. 8D The Viscosity at the End of the 50°C Holding Cycle This indicates the stability of the paste to stirring in the form in which it will most likely be used by the industry. It is a good indication of granule rigidity and resistance to shear. The actual viscosity at this point may also be considered as a measure of thickening power or thickening efficiency of starch. Table 4 summarizes the relationships between the Bra- bender viscosity curves and the functional properties. These functional properties are the basis for determining the usefulness of a good starch. Table 4 also indicates the molecular) events which are believed to be responsible for the observed changes (Mazurs‘gt. _a__]_., 1957; Carson, 1972). Medcalf and Gilles (l9v66) and Schoch and Maywald (T968) also described the terminology used to express the amylogram results. The temperature of initial pasting for this work is defined as_ the temperature at which the viscosity curve starts rising during the heating period. Perpendicular r"iS‘ing temperature is the temperature at which the C-shape curve starts rising and a peak reading is taken'just at the top of that first curving point. The normal peak height is taken at the. second curving point of the C-shape curve. I" the cases where a definite sharp peak is not obtained, "0 Value is given because height values at 95°C are repor- ted. The peak after l5 min is the viscosity of the sample after 15 min holding period at 95°C. The peak drop is the 81 height obtained at the drop point of the curve during the cooling period. The height is the viscosity of the sample after it has cooled to 25°C. Peak on cooling is the height of the curve obtained during the l5 min cooling period. After 15 min at 25°C is the height reached after the IS min cooling period. Scanning Electron Microscopy Dehydration Bean samples (dry, soaked, canned) were freeze-dried iri a freeze dryer Unitrap II (Virtis, Gardiner, NY, 12525), 4 'to 8 hrs respectively. Coating and Viewing Dried beans were dry-fractured by hand or using blade to "open" tissues and cells, mounted on stubs with colloidal firigaer-polish and coated with an approximately 20 nm layer 0f gold using a sputter coater. The coated samples were Viewed and photographed in a Philips. Super III Scanning EleCtron Microscope (SEM) at an acceleration voltage of 15 KV VVi th a Polaroid P/N film type 665. 82 Freeze-Drying Dry-fracturing l Coating 1 Viewing Figure 10. , Flow diagram of sample preparation for SEM. Statistical Analysis 'The "Statistical Package for Social Science" computer PY‘Og rams described by Nie _e__t_ §_l_. (1975) for use on the CDC 65()() computer operated by Michigan State University Computer Lat>c>t~atory was used to assist statistical analyses. hflultivariate analyses of variance and covariance were dete rmined using Subprogram ANOVA. Mean values were repor-' ted after roundings. Single classification analyses of var-i ance, Tukey mean separations were determined using subprogram ONENAY. - ‘rukey separations were presented such that treatments “hiCh were not significantly different '(pso.05) were indi- catE3letely randomized design. Mean squares with significant F ‘Piitios were used to determine significant probability ‘EVei of p 0.05. RESULTS AND DISCUSSION Proximate Composition The proximate composition of the three dry bean cultivar, Sanilac, Nap-2 and San-Fernando over three crop years is shown in Tables 5, 6, 7, 8, 9, 10, ll, 12, l3 and l4. Moi sture content was higher in the 1978 beans than in the other two crop years (Table 5). Few significant dif- ferences were obtained within the crop years among the three cultivars of whole bean. The combined result indi- cated no significant differences among the cultivars for the th ree portions.of the seed. The seeds have similar high crude protein contents as obtained in other Phaseolus vulgaris L. beans (RUth 9}. ELL-a 19793 Pedro and Ladermiro, l979; Shalini _e__t_ 21.. 1968; “we"! ‘i ro __t __l_., l979). There were- no significant dif- ferences in the whole bean and seed coat protein for the three cultivars over the three crop years except San-Fer- nando Which showed significant differences for the 1979 and 1980 beans (Table 7). In cotyledon alone no singificant diffarenas were observed among the varieties except Nep-Z and Sanilac cotyledons which DOSSESSEd significant differ-g ences in the 1978 and l98O beans,°respectively. The 83 84 .meewpgee eeew ece meme» eege :_;pw3 ece mee>wppee ucose.fimo.oaev meecegemewe aceeewwcmvmicec.euecee caepee cw mgeuuep exwe .se>wupze\cewueee eeem\meueewpeeg me m u c eo.m em.m em.o em.“ em.o ee.e wo.m em.m em.m um em.m no.0 em.e e~.e em.e e~.o em.w em.m e~.m Nieez ee.m em.m em.e em.o em.o em.m em.w em.u e~.m ee—Fcem peeu ueeu peeu eeem :eeepzpee epezz eeem :eeepxeeu epegz emem :eeepzueu epegz mceweeee eeem .i mcewegee eeem . meewugee eeew mee>eu_=e omm— . mum, weep mgee> eego .mpeee eeem eee :eempapee .eeee epegzv mcewugee eeem eee Aommp ece asap .wumpv meme» eege an fleececsemicem eee N-eez .eepweemv mge>_epee ceee ee Amwmee xsev uceueee egeumFee peeegee .m epeee 85 Table 6. Percent moisture content (dry basis) of bean cultivars (Sanilac, Nep-2, San-Fernando) for three crop years (l978, 1979 and l980) by seed portions (whole bean, cotyledon and seed coat). Seed Portions Cultivars Whole Cotyledon Seed Coat . a b c Sanilac 7.3 6.5 6.8 Nep-2 7.2a 7.2b 5.7c SF 7 7.2a 7.2b 7.3c n = 9 (3 replicates/seed portion/cultivar x 3 years). Like letters in column denote non-significant differences (p60.05) among cultivars and within seed portions. 86 .mcewagee eeem ece meme» eege cespvz .eee mse>wuyee meeEe Amo.omev meeeegeewwe pceewwwem_mcec.epecee easpee cw meeupep ex_e .Aeueewpeeg\mcewueencw m x ge>wupee\:evwgee eeem\meueewpemg my m n : em 0 em mm eo om em.“ eN.mN em.mm em.e e_.mm eo..m em em.m ee.~m ep.wm em.m em.em ep.om em.o em.- ep.mm mieez em.m em.nm em.~m em.“ em.m~ em.FN em.m ew.em eo.m~ eeppcem ueeu peeu peeu eeem :eeepxpeu e_e;2 eeem :eeepxueu eFesz eeem eeeepzpeu open: L e e so we ego to me 1, mac?» on em m I.ceep e e {Mil _ e» e e m mge>wupeu ommp _ mnmp weep meme» eegu ii . .Apeee eeem use :eeepxuee .ceee epegzv meewugee eeem ece Aomop ece mmep .mumpv meme» eese an Aeeeeeseu-cem ece mieez .eepwcemv,mge>wupae come we Amwmen Anew peeueee cpmuege ueeegee .5 epeep 87 Table 8. Percent protein content (dry basis) of bean cultivars (Sanilac, Nep-2 and San-Fernando) for three crop years (l978, 1979 and l980) by seed portions (whole bean, cotyledon and seed coat). Seed Portions Cultivars Nhole Cotyledon Seed Coat Sanilac 24.0a 25.8b 7.7d Nep-2 23.8a 23.3c 7.5d SF 23.8a 24.7bc 5.7d n = 9 (3 replicates/seed portion/cultivar x 3 years). Like letters denote nonsignificant differences (p20.05 among cultivars and within seed portions. 88 .mcewpgee eeem eee meme» cwzpvz .ece mee>wppee meeEe Amo.o~ev meeceeewewe uceewewcmwmcec_eae=ee caepee :p mgeuueP exwe .Age>wu~:e\cemugee emem\meueewpeeg my m u c em.o eF.F e~._ ee.o em.~ em.~ ee~.e em.. e~._ um em.e oe._ oN.P ee.e ee._ om.P oe.e ee._ ee.P N-eoz em.c ep.~ eP.P ee.o ee._ ee._ em.o en.~ em.~ eercem eooe eooe ii eooe eeem ceeepxpeu egos: eeem ceeepxueu epecz eeem :eeepzueu mpecz mceepeee eeem meevugee eeem meewpeee eeem _ mee>_epsu ommF axe? weep mgee> eegu Aueee eeem eee :eeepzuee .eeee epegzv meewpgee eeem ece Aommp eee mum, .mNaPV meme» eege an Aeeceeseeicem ece Nieez .eeppcemv mge>eupee seen we “memee xgev ueeucee we; eceeeee .m efiee» 89 Table lO. Percent fat content (dry basis) of bean cultivars (Sanilac, Nep-2 and San-Fernando) for three crop years (l978, 1979 and 1980) by seed portions (whole bean, cotyledon and seed coat). Seed Portions Cultivars Whole Cotyledon Seed Coat Sanilac 1.3ab 1.4C ’ 0.5d Nap-2 1.4b 1.7C 0.5d SF 1.2a 1.5C 0.4d n = 9 (e replicates/seed portion/cultivar x 3 years). Like letters in column denote nonsignificant differences (p>0.05) among cultivars and within seed portions. 9D .mcewugee eeem.ece meme» eege segue: .ece mge>wupee meeEe Amo.onev meecegeewwe ucee_mwcmwmcec euecme caepee cw mgeeeep exwe Age>wu~:e\eewugee emem\meeeewpeeg my m u : mm m em e ee e eh m ep e eF e mm m em m 2” e um . . . . . . . . . lam em m ee e em e wk e eeo e no m ee m. e_ e em e N z o o o o o o o o u m ep 0 ee e we e mm o em m ep e mp o eo e em m eeppc m Heeu peeu peeu eeem ceeerueu epezz eeem ceeepaueu. apes: eeem eeeepxuee epenz mceeugee eeem mcewugee eeem meewugee eeem _ mee>wppee ommfi _ mmmp weep mgee> eegu .Apeee eeem ece :eeepzuee .ceee epezzv mcemugee eeem ece Aommp ere mnmp .wmmpv meme» eege an Aeececgeeicem ece Nunez .empwcemv mge>wppee seen we Am_mee agev uceueeo :me uceegee ._~ epeep 9l Table 12. Percent ash content (dry basis) of bean cultivars (Sanilac, Nep-Z and San-Fernando) for three crop years (l978, l979 and 1980) by seed portions (whole bean, cotyledon and seed coat). Seed Portions Cultivars Nhole Cotyledon Seed Coat Sanilac 4 7a 4.2C 5 3d Nep-Z 4 2b 4.2c 5 1e SF 4 2b 4.1c 3 8f n = 9 (3 replicates/seed portion/cultivar x 3 years) Like numbers in column denote nonsignificant differences (p%0.05) among cultivars and within seed portions. 92 .mcewpgee ween ece meme» eeee segue: .ece mge>wapee mceEe Amo.o»ev meecegemewe uceewwecmwmcee_eeecee caepee cw meeupep exre .Amge>wupee\cewpeee eeem\mepeew_eeg my m u : eeeee en.~e em.~e em.e em.me eo.oe eeN.m em.me eo.om mm em.m ee.~e em.~m e~.m em.om em.—e em.m e~.ne eem.me Nieez meet» eo.ne em.me eeegh ee.em eo.~m ep.e . eo.Fe ee.me eepmcem ueee peeu ueeu . eeem :eeepxueu epesz eeem :eeepzueu apes: eeem :eee_zueu open: m e meerLee eeem mcewucee eeem _ eevpgee e em mee>_w_:u emmp _ asap weep mgee> eesu .Aeeee eeem ece ceeepzuee .ceee epegzv mcewugee eeem ece Aommp ecu asap .wNmPV mgeez eege an Aeeeecgeuicem ece Nieez .eepweemv mge>eupee came ee Amwmee xgev ueepcee cegeum aceegee .mp epoch 93 Table 14. Percent starch content (dry basis) of bean cultivars (Sanilac, Nep-2 and San-Fernando) for three crop years (1978, 1979 and 1980) by seed portions (whole bean, cotyledon and seed coat). Seed Portions Cultivars Hhole Cotyledon Seed Coat Sanilac 47.5a 42.3b 1.5C Nep-2 42.8a 45.7b 5.2d SF 48.1a 45.5b 2.7Cd 27 (3 replicates/seed portion/cultivar x 3 injections years) n: x 3 Like letters in column denote nonsignificant difference (p>0.05) among cultivars and within seed portions. 94 combined three crop years results showed significant dif- ferences among the cultivars for cotyledon protein content ranging from 23.3% (Nep-2) to 25.8 (Sanilac (Table 8). Fat content constitutes a relatively small amount of the overall bean composition like in most low fat legumes (Table 9). There were no significant differences in the whole seed fat content among the cultivars except that San-Fernando showed a slight difference in the 1979 beans. Significant differences in fat were obtained in the cotyle- don from the 1979 and l980 beans but not in those of 1978 (Table 9). Seed coat fat content was less than one percent. Nep-2 and San-Fernando showed no significant differences in their seed coat fat content for the year I978. Sanilac and Nep-2 showed no significant differences for l980 beans (Table 9). Combined fat content from the three crop years results indicated significant differences in the whole bean among the cultivars but not in the beans cotyledons and seed coats (Table l0). Ash content falls into the range reported for legumes (Phaseolus vulgaris L.) by Ruth gt gt. (l979), Pedro and Ladermiro (l979) (Table 8). The ash content did not show a significant difference between the two isolines 1978 and l980 whole bean samples (Table ll). Within the years, significant differences were obtained for cotyledons and seed coats among the three cultivars. The combined percent ash content revealed no significant differences between the 95 two isolines in whole beans and cotyledons however, in the seed coat a significant difference was obtained (Table 12). The starch content in Table 13 is similar to other legumes Phaseolus vulgaris L. reported by Navikul and d'Appolonia (1978), Cerning and Aliette (1979), Roberto and Eidiomar (1971), and Theravuthi _t gt. (1974). There was no significant differences between the two isolines for the three seed portions (whole, cotyledon and seed coat) for the three crop years (Table 14), except San-Fernando which showed some difference in the cotyledon with the 1980 beans. In general, the statistical analysis of the proximate composition revealed, except for fat content, no signifi- cant differences for the protein, ash and starch contents in the whole beans among the three cultivars although dif- ferences were obtained in some cotyledon and seed coats. In this case, we can not rely on the proximate composition to explain the textural behavior differences of the beans used in the experiment. Suoars Sugars commonly found in legumes (Phaseolug_vulgaris L.) are reported in Table 15A, B, and C. Hexose represents a mixture of D-glucose and D-galactose, and probably 0- fructose because in this study, these three reducing sugars appear at the same retention time from a standard "cocktail" 96 .mee.usee soon c.zu.3 ecu ms->—u_:u ace-a Amm.o»e. «ouceco~u_u acne-vvcuvucec oueeuo esapeu c. ugouuo— use; o—nauuoueeeez - oz— Aouaewpeog\nee—uuune. n x g->.w_au\=evugee euenxmeuuu._eec my a u e oz cn.~ u¢.~ ez on. an. oz on. on. eu. un.~ na~.~ 9.. ea._ um.— um ez oo.~ ac." ez ez oz ez 9.. an. e~. ue._ ao.~ up. en.— a... Nieez oz em.m e~.~ _ez em. on. ez an. no. e~. u~.~ oo.— uez no. em. oo—vcom eea. coo» eoee .e ez em.~ o~.~ ez am. am. oz o~. u e. e». am.~ eo.~ um.— em.— em.~ um ez ee.n so.~ ez ee. ec. .—. men. a N. on. nn.n so.— on.— no.p em.— wieoz ez e~.~ ae.~ ez em. on. ep. no. as. em. ao.~ no.p uo.~ a... e~.p u-__=om «so. e.o» mote .e ez e~.. ~¢.p ez em. on. eep. no. ac. eon. em.~ eo.~ u~. no.» um. um oz 5... .m.. ex 5.. on. oz 9.. oc. e~. oe.~ on.~ e_. em. on. ~-eoz ez a... a... ez an. on. u—. so. an. em. nm.~ -—.~ um. am. am. ue__cam asap can» menu .< eoeu ueeu ueeu useu ueeu econ gees—xaeu o—ez: eoom seem—aueu opez: eoem cove—hueu open: comm coco—xueu «_ezz eoom coco—aueu o—ezz ce.eeee eoom co.»eee eoum eeeecee comm cowecee econ sewage; eeem emewgeoem owes—cue: _ea.mee_ emeceem emexoz .Aomo— use use. .m~o_v see» note an uoeu econ ecu coco—xuee .eeea o_ezxv meeeugee some can .euceecomieom we. ~-eoz .e~_.:-m. uc~>.u_=u coon ee An.uun Le. ucoueeu Aoaeazuoua we. once—ueag .peu_mee. .ouecuea .onexozv Leos“ accuse; .m— u-aah 97 mixture of these sugars. It has previously been reported that anomeric forms of reducing sugars such as D-glucose and D-galactose are not resolved in HPLC (Palmer, 1975; Eileen _t__l,, 1979). Two flatulent sugars raffinose and stachyose, which are of concern to consumers, were eluted. The stachyose content was higher as compared to raffinose. In general, no significant differences for each sugar were shown in the whole bean, cotyledon and seed coat of the three cultivars for the three crop years (Appendix Table 1A). Hexose and sucrose did not show any significant differences in the whole bean and cotyledon for the crop years 1978 and 1979 for the three cultivars. The combined results were similar although they differ in the 1980 bean samples (Tables 15A, B, and Appendix Table 1A). Higher hexose contents ranging from 1.7 to 2.3% were obtained in 1979 whole beans (Table 15B) and lower values in those of 1978 (Table 15A). ~Stachyose showed its highest content (2.4%, 2.7% and 3.0%) in 1980 whole beans (Table 15C) and lowest (1.4%, 1.4% and 1.5%) in 1978 beans (Table 15A). Nep-2 and Sanilac had the highest (3.0%) and lowest (1.4%) stachyose values respectively (Table 15C and 15A). The 1980 beans revealed no detectable amount of raffinose in the entire beans (Table 15C). The inositol content was less than 0.7% in the beans used and, in the three crop years combined results, it did not show any significant difference at the three portions among the isolines (Appendix Table 1A). The 98 seed coat present no detectable raffinose and stachyose. Combined results are presented in Figures 11, 12 and 13. All the results obtained on sugars fall in the range of values reported in legumes by Navikul and d'Appolonia (1978), Quemener and Mercier (1980), Cerning gt_ 1. (1975), Akpapunam and Markakis (1979), and Munehiko _t_gl. (l975). >The variation in the content of the individual sugars from year to year could be related to soil conditions, weather, nutrient availability. Nep-2,which showed no detectable amount of raffinose in the 1980 whole beans, possessed the highest value of stachyose (Table 15C). This implies a compensation process which could be important to plant breeders in the selection of cultivars leading to elimination of flatulent factors in beans. Scanning Electron Microscopy Examination of Dry_Beans Examination of the seed coat outer surface showed both Sanilac and Nep-2 to possess a highly rough and wrinkled outer surface with pores and sinking holes while San-Fer- nando revealed only a highly rough and convoluted structure (Figure 14A], B1, C1). The coat inner surface views showed Sanilac and Nep-2 with similar structure, wide "hills" and narrow "valleys" (Figure 14A2, 32, C2). The seed coat of Sanilac was not difficult to remove compared to the two isolines Nep-2 and San-Fernando. A N... .Aeececcemucem eee mieez .eeFPcemv mce>wppze came epezz Lee Aomm— ece enmp .mnm—V meme» eece mecca ce>e meepe> uceucee Aemezzeeam ece emecwmmeg .Peuemeew .emegeem .emexezv mcemem :eez .FP ecumeu $02.35 meg-"5: 40562. $058 $05! v ’91 O 60.0 -18 99 oezwupze seen Lee Acwmp ece mum, .wnmpv meme meepe> Heeucee Aemexzeeam ece emecrmeeg .peuwmecm .emeseem .emexezv mgeaem :eez .mceeepzuee Meececsmuieem eee eese megs» ce>e .N_ ecemve $052.5 meg-e3. .5502. $988 $9.! 824229.23 «we aim: U 9.3.23 a liq «& ‘ - 9?: O‘Q'Q'Q‘ «fiflfifi O'O'O'O' O .0 '0'0'9' of ° . ’O'O'. .0. . co.» lDl .aeee eeem eeeeegeeteem eee eege megs“ Le>e ~-eez .eerwcomv mse>_u_:e :eee Lee Aome ece mnm— .wmmrv meme me=Pe> pceucee Aemezzeeum eee emeewmweg .Feuwmecw .emeceem .emexezv gemem ceez .m_ weave; mm0>10 coco mgmm> aogu mgmm> aogu we?» xmom ovcmcgmu-cmm ~-amz ompvcmm Lm>wupao :mmm . wuugmumz amp .Pupv .AommF use mkmp .wmmpv mgmmx gogo xn mmuacwe om ow a: Low Loam: umpppam Loam; mgaumgmasmu pcmwnsm cw cmxmom.mcmmn Lu we Fcowpasomnm Loam: “smegma .op mpnm» 118 . cm mmmy .mmm— mgmmx aogu omega Lm>o mmuzcre om o» a: so; Aswan: empppumpwwmwwmw amp .Pnpv sown: mgspmgmasmp ucowasm cw umxmom Aoucmcgwuucmm can w-awz .ompwcmmv mgm>mppsu :mmn xgn yo cgmuama comuagomnm Lean; ucmogma cam: m2... om 8 2. 8 on ov on 0.... o. e . o. ‘o‘olIU'aI‘ \‘1 .oum ..1\ o i.\\niqu-.-.I-I... .\ L On «We oozaaxmaéfi 1.11 - nllll¢1\\\\. . Cc mm «dun m . H 1 L on v i u - a 93.2% s .. om m a 3 G . 2. .mm mcsmmm 119 to give a higher water uptake on soaking is not applicable in this study because less than 10% moisture content were found in the beans under investigation. This implies Sanilac's easy water absorption through the seed coat and micropyle compared to Nep-Z and San-Fernando which possess a sticky protein membrane on the cotyledon outer surface preventing the easy translocation of water between the seed coat and the cotyledon during the hydration process (Figure 153], C1). The hypothesis presented on cowpeas by Sefa- Dedeh (1978) and soybean by Mayer and Poljakoff—Mayber (1975) indicating that highest amount of water absorbed is related to high protein content is not supported here because the three cultivars used in this study did not have any significant difference in their protein content but there were differences in their water content absorption rates during soaking. If protein is the chief component absorbing water in seeds as reported by Mayer and Poljakoff- Mayber (1975), the absorption quality of the protein may be a factor to consider in the hydration of seeds. The firm packing of the parenchyma cells in San-Fernando bean could also be suggested to delay the water absorption through the entire cotyledon. 120 Pasting Properties Tables 17A, B, and C and Appendix Table 18 show the Brabender Visco/amylograph pasting properties of the three cultivars. The flours from the three bean cultivars (Sanilac, Nap-2 and San-Fernando) have Type C pasting curves with no definite peak which is commonly observed in cereals. This particular shape is commonly found in legumes (Colonna and Mercier, 1979; Lineback and Ke, 1975; Navikul and d'Appo- lonia, 1979; Suzuki gt al., 1981; Schoch and Maywald, 1968; Vose, 1980). All three cultivars had high initial pasting temperature 72.8°c, 72.3°c and 77.3°c for Sanilac, Nep-2 and San-Fernando whole flours, respectively (Appendix Table 18). The highest value for San-Fernando indicates the slow swelling of the starch granules of this cultivar. Higher values were also obtained with cotyledon flours with 70.60C, TS.3°C and 75.80C over the three crop years for Sanilac, Nep-2 and San-Fernando, respectively (Appendix Table 13). While Nep-2 initial pasting temperature is similar to that of Sanilac in the whole bean flour, it is most similar to that of San-Fernando in the cotyledon flour alone. There was no difference in the initial pasting temperature within the crop years between the whole bean and cotyledon flours among the individual cultivar. From the whole bean flours over the three crop years study, 121 9:2: mmozumdmm a. .08 mm 08 93 con 2. 2h 8. o: «m ee at. 3 $268 53 I own I men _m 0% 2. I I I 2. m... $023 ooz u “mam 2.90%“. 0.8 38 man. "News 93 we use 035... 932.. fig “22 .2 .22 52 .5... cut: t. 2565225.. .352. .zEz. 3m 2. >e_wooe> .om.m In an cowp:_0m cwmwso mangamoza PE cow m cw Aouzmcgmm -cmm vcm «Iamz .umprcmmv mgc>wu_:o use Aommp new mnm— .wmmpv memo» coco An mesopw Acovazuou tam coma mpogzv mcovucoa same age we muwpmvgmpumcmgu mcpummm I< .np wpnmh 122 9:2: mmozwmém a. 00¢ ONO Own II II I... Ow. cm In I. I ”N Om mm0_.—UOU 3m mom 0% I. l I om on I I I t. mm 2055 OOZ> NIQMZ Ohm CNN nos 00¢ owe. mm o? 0: SN mm 9‘ mm mm mmmzooo 0.... mm... 0%. 8m 8n :. 8m 8. 9. a me mm 8 90:3 O<£Z ommwemo. one“; 93 come ohm...” "Name 93 Fag—hilum o p ma: ozwrmwm 05w? cute game .2 53.. 33.. 23.. 5:2 .5 5465223.. .32... 45:2. .3 z. :_moom_> In .A.e.peoev .NF e_eee 123 Ampoflpw>\cowppoa @omm\mopw0fiaamp NV N II mpHCD powcmnwpmm mtz: mwozumdmm u. 03. 03 mmv 0mm n¢~ :. o- 0m 00. mm 3 t. mm mmmzooo own omv 9c I. I .I ov. no I I l 2. ~m 20:3 ooz> NIsz 0mm can com 0%.. com on o.~ oo. om. mm on on ma mmmaooo owv nmm Own I I I nnm ON. 00. mm nv m» Nn 20:3 qu49eqm . «58 II 93.. 2e: 3.... 93 Eamauufi. 0L mum. mamas; .55.. .3 0.3 $5 uzz. .25. 93 05.3 23.2.. aura game .2 game x5e x3: 5:: .5 5385225.. .35.. 43.22. _:m z. >tmoom_> .A.e.peoev V" .n— mpnmh 124 Nep-2 showed the highest viscosity curves and San-Fernando the lowest with the Sanilac curve intermediate (Figure 24 and Appendix Figures BlAl’ 82A1 and 83A,). With the coatless (cotyledon) bean flours, Sanilac demonstrated higher curves followed by Nep-2 and then San-Fernando A and B A ) l l 2 l except in the l980 flour samples where Sanilac showed a (Figures 24, A2 and Appendix Figures B lower curve only during the heating period (Appendix Figure B3A2). Considering the individual cultivar, viscosity curves_appear to be higher after removal of the seed coat in the 1978 and 1979 Sanilac and San-Fernando bean flours (Figures 25, 27 and Appendix Figures BlAZ and 82A2)' In the year l980 beans, this increase did not occur with Sanilac (Appendix Figure 83A ). Inversely, Nap—2 showed. a loss in its viscosity strength after removal of the seed coat (Figures 24A2, 26 and Appendix Figures 81A and 82A ). 2 The l979 Nep-Z beans which presented a low water absorption in rate pattern (Appendix Figure A2) revealed the highest whole bean flour viscosity of all materials evaluated (Appendix Figures 81A], 82A] and B3AI). This behavior was not explainable on the basis of structural tepography, but it could be suggested that the starch granules, unable to pick up water in the cold stage, absorbed the water to a large extent during the heating period before they rupture and that made the slurry more viscous. The same behavior can not be related to starch content because the Nep-2 beans 125 TEMPERATURE ('0) 25 42.5 60 77.5 95 mo ' ' 1 ' 95 77.5 60 42.5 25—25 ' A1 WHOLE BEAN FLOUR 800 " 2’. Pnsmuc‘ CURVES . 'KWEL. E I Z / ’- \ D 600 n / / fl: / /s .0 '0. III / . 0' §4a3- /’ ,u‘ III-I 0' m 3‘: D200 - ° I; 30 45 50 75 90 I05 IZO TIME (MIN) ' TEMPERATURE ('0) '00 0 25 42.5 80 77.5 95 95 77.5 60 42.5 25—25 I O I I r I I I O I‘2 BEAN comeoou FLOUR coo - PASTING CURVES BRABENDER uwn‘s ‘ 8 8 o l I § O I 75 90 IOS I20 Figure 24. Mean pasting curves of dr bean portions (whole bean and cotyledonI flours by culti- vars (Sanilac, Nep-Z and San-Fernando) over three crop years (1978, l979 and 1980). 126 .Aommp vac asap .mumpv memo» nogu owes» Lm>o menopm Acoumpzuou use camp wpoczv mcowpcog :mmn umpwcmm mew mo mm>sau mcwumma :mmz 2:5 mg... 8. no. 8 2. oo 3 on. n. 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ON. 00. om on om n¢ on mu . a e o .u. 3 llllllmUWVl n comm“ u£¥. \\ mm xxx 3.x . \\ M. \ zoau...?..oo . 1 000 N H“ s 1 com # n I n n I u n P 08— nNIInu 93 ee 2L. 8 we met. em Que nu 8.0 mm2h4mmmzmh. mm>m=o 022.93 130...."— z Lepeazp .Age>wu~=e\m:eu x :ee\mmpesem N x m—eEem\meueeppeeg NV w u c um.m- gm.m mp.¢~ em.o e~.o- em.~_ eeeecseuucem _e.kp em.e_ ee.om e_.PP em.c ew.em N-eez P~.op :m.ep $5.0m eo.Fp em.o e¢.oo ee_reem eeep eee> eeee .m< no.m- so.m em.mp. em.o ep.o- em.~_ eeeeeeeu-:em we.k_ no.5, em.ee on.e~ ep.e e~._e N-eez wo.~_ mm.¢_ e~.om em.o~ em.o eo.mo eepweem aka, ewe» eoee .N< . . . . I . . e emcee Ice _N m we e em up we 0 em 0 e“ e_ e u m xw.ep he.m m~.me ee._P ep.e- eN.oe N-eez xm.e_ w¢.e m~.om m~.w e—.o- eo.¢c eefiweem JD AM A 42 AM A II seem mceem eemmeeeca mceem ago mum, cam» comm .P< . Pmoeou zwupae eeee eemmeeege ece Age we Aee .em .40 meape> meeceeemewe cepee ece eepee nee Lopez: .wF epeeh 141 cultivars have the highest values above 50 (Hunter readings) while that of San-Fernando is low and below 20 (Tables 18A1, A2, A3 and Appendix Table 1C, and Figure 35 and Appendix Figures C1, C2 and C3). The "L" values are higher in dry seeds than in processed ones except in the 1978 San-Fernando beans where the reverse was obtained (Table 18A and Appendix Figure C1). Greenness or redness represented by "a" values is very low in dry seeds but high in processed ones. Contrary, a reverse‘process is observed with "b" values which denote the yellowness or blueness (except in 1978 SF beans) (Figure 35 and Appendix Figure C1 and CZ)“ This implies that when "L" values decrease the "aL" values increase respectively in the seeds during processing. This is expected as a result of browning during the thermal proces- sing as reported by Uebersax and Bedford (1980). Through the overall study, dry and processed San-Fernando beans showed significant differences of "L" and "bL" values when compared to the other two cultivars (Tables 18A], A2, A3 and Appendix Table 1C). Processed Bean Evaluation Moisture Content During the canning process water contents reach the optimum after the hot soaking step in each individual bean cultivar (Tables 19A, B, C and Figure 36 and Appendix Table 10 and Figures D], D and D3). 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