MSU RETURNING MATERIALS: PIace in book drop to LJBRARJES remove this checkout from Jul-zyllln. your record. FINES wiII be charged if book is returned after the date stamped beIow. ’T—Uo JGLO 11 292W STUDIES OF PHYTOHEMAGGLUTININ, THE LECTIN OF PHASEOLUS VULGARIS By DonaId G. Coffey A DISSERTATION Submitted to Michigan State University in partial fuIIfiIIment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1985 ABSTRACT STUDIES OF PHYTOHEMAGGLUTININ, THE LECTIN OF PHASEOLUS VULGARIS By Donald G. Coffey Lectins are toxic heat-stable glycoproteins that depress the protein quality of Phaseolus vulgaris varieties. Studies were designed to address the issue of lectin stability under a variety of conditions. Kidney beans were exposed to controlled soaking and thermal treatments. Analysis of the hemagglultinating activity (HA) indicated that the time required for one log cycle reduction in HA decreased by 50 minutes for each 5.6°C increase in holding temperature. Cooked kidney beans were palatable and showed no detectable HA after six hours at 91°C” .At 82°C, kidney beans were palatable after 11 hours but showed detectable HA up to 14 hours. Phytohemagglutinin was purified by affinity chromatography and exposed to thermal, chemical and enzymatic treatments. At 70°C, the activity of the purified lectin diminished according to the equation: % activity remaining = 102 - 9.87 (hours) Exposure to pH 12.0 sodium hydroxide solution or 5 M urea were the most effective chemical treatments for reducing the HA of the purified lectin corresponding to 65 and 39 % reduction in activity, respectively. Treatment with proteases resulted in 88 - 98 % reductions in HA after Donald G. Coffey three hours. Treatment of the purified lectin with mannosidase resulted in a slight but significant (P < 0.01) reduction in HA but treatment with amylases and neuraminidase had no effect. Hhole beans and bean flours were processed in a commmercial extruder. The HA of extruded products generally decreased with increasing final product temperature and increased barrel pressure enhanced this effect. Soaking kidney beans at pH 12.0 resulted in more rapid inactivation of HA and tenderization of the beans than samples at pH 7.0. Beans soaked at pH 12.0 reached a palatable end point in only 60 % of the time required for control samples at pH 7.0. Of the small seeded Phaseolus vulgaris varieties analyzed, the most promising candidates for breeding for low lectin levels were Nep 2, P766, Carioca, Ica-pijoa, Jalpatagua, Jamapa and Black Turtle Soup. The most promising lines for high digestibility were Carioca, Protop-pi, 8217-111-24 and Sanilac. Electrophoretic analysis was not an adequate tool for screening dry bean lines for HA or digestibility. ACKNOWLEGEMENTS I owe a debt of gratitude to my academic committee members and others who helped me with my Ph.D. program. My thanks to: Dr. M.R. Bennink who always had time to answer questions and draw blood when I was developing the hemagglutination assay. Dr. J.R. Brunner who helped greatly with methods and ideas and was always eager to share his views on economics, politics and science. Dr. G.L. Hosfield who is committed to improving the lives of less fortunate people through agriculture and with whom I shared a productive and enjoyable trip to Guatemala. Dr. P. Markakis who was always encouraging and unselfish with ideas and equipment and whose life and students are dedicated to improving the food situation in many foreign countries. Dr. M.A. Uebersax who“ as my Inajor professor, taught me Inany important lessons about food science, teaching, research, work and life. I consider myself fortunate to have worked and learned from him. Kit and Kate Uebersax who were and are good friends. My fellow students with whom I shared many good and rewarding times (C.S., R.T., S.R., N.S., J.N., A.H.K., J.L., S.R., A.K., A.T., J.D., G.A., B.M., A.B., P.H., M.R., 0.5., T.R., H.I., and T.C.). My family: Joe, Betty, Donna, Denise, Nellie, and Dorothy and all the VanHoogens who always knew I could do it and are finally glad that I did. My wife Susan for everything. I could not have done it without her. ii TABLE OF CONTENTS LIST OF TABLES.......................................... LIST OF FIGURES ......................................... INTRODUCTION............................................. REVIEW OF LITERATURE..................................... Beans in the Diet of Man............................ Nutritional Importance......................... ProteinQOOOOO0.0.00.00...00.0.0.0...00.... Carbohyrate............................... FatOOOOOOOOOOOOOOOOOOO0..OOOOOOOOOOOOOO... Vitaminso.OOOIOOOOOOOOOO0.0...0.0.0.000... Minera1500000000000......OOOOOCIOOOOOOOOO Breeding to Improve Agronomic and Food Quality Characteristics........................ Processing Opportunities for Improved Dry Bean Utilization............................... LeCtinSoocoo00000000000000.0000...000000000000coooo Dafinition Of LectinSOOOOOIOOOOOOOOOOOOOOOOOO. FunCtion Of LectinSOOOOOOOOO0.000000000000.0.. NUtritive va]UeOOOOOOOOOOOOO0.0000000000000... TOXiCity EffeCtSOOOOOO0.0.0.0000...0.0.00.0... Physical and Chemical Properties of PHA....... Amino Acid Compostion.................... Amino ACid SequenceOOOO0.0000000000000.0. SUbUNTt compostionOOOOO0.00IOOOOOOOOOOOOO Molecular Height......................... Carbohydrate Compostion.................. Sedimentation Coefficient................ Partial Specific Volume.................. Frictional Ratio......................... Diffusion Coefficient.................... Isoelectric Point........................ Binding Properties............................ Meta] Bi"dinQOOOOOOOOOOOOOOOO00.0.0000... Carbohydrate and Glycoprotein Binding.... AnaIytiCa] MethOdSOOOOOOOOOOOOOOOOOOOOOOOOO... FraCtionationOOOO0.000000000000COOOOOOOOO Affinity Chromatography.................. iii Page viii ix \Jososoibp 4h 4: H \J Hemagglutinating Activity................ MATERIALS AND METHODSOOOOOOOOOOO0.0.0.0.0000...000...... Experimental outlinEOOOOOO00.000.000.00.0.0.0.0.... Study One: Hemagglutination Assay............ Study Two: PHA Molecular Study............... Study Three: Bean Processing Technology...... Study Four: Breeding Line Study.............. Sample Preparation and Treatment................... source Of BeanSOOOOO0.0.0.000...0.00.00.00.00. Thermal InactivationOOOOOOOOOOOOOOOOOOOOOOOOCO COOkingOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOIOOOOO Affinity Chromatography Purification.......... Thermal Treatment............................. Chemical TreatmentOOOOO..OOOOOOOOOOOOOOOOOOOOO Enzymatic Treatment.’OOOOOOOOOOOOOOOOOOOOOOOO. ExtFUSion.OOOOCOOOOOOOOOOOO0.0.0.0....COO...O. Alka11ne COOkingOOOOO00.00.000.000.0.000000... Ana1ytica] MethOdSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO PrateinIOOOOOOOOOOOOOOOOOOOOOOOOOOOOCOOCOOOOOO Micro-Kje1dah]OO...OOOOOOOOOOOOOOOOOOOOOO Protein Solubility Index................. LOWFYOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO MOiStureOO00.0.0.0...C0.00IOOOOOOOOOOOOOOOOOOO oven DryinQOOOOOOOOOOOOOOOOOO0.0.0.000... Dielectric Moisture Meter................ AShOOOOO0.0.00.00...OOOOOOOOOOOOOOOOOOOOOOOIO. Hunterlab C010r valUESOOOOOOOOOOOOO0.0.0.0.... LeCtin Purification.OOOOOOOOOOOOOOOOO000...... Ammonium Sulphate Precipitation.......... Electrophoretic Separations................... Discontinuous Polyacrylamide Gel E]ectrophoreSiSOOOOOOO00......00.0.0.0... Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis...................... Hemagglutinating Activity..................... Preparation of Bean Extracts............. Preparation of Red Blood Cell Suspension. Hemagg]Utination AssayOOOOOOOIOOOOOOOOOO. In-VltPO DigeStibilityoooooooooooooooooooooooo Sample PreparationOOOOOOOOOOOOOOOOOOOOOO. PFOCEdureOOOIO0.000000000000000.0.0.0.... Texture...0.00.00...OOOOOOOOOOOOOOOOO0.0.0.... Kramer Shear Press....................... Subjective Textural Evaluation........... StatiStica] Ana1y51SOOOOOOOOOOOOOOO000.000.... iv STUDY ONE: THERMAL INACTIVATION OF THE HEMAGGLUTINATING ACTIVITY OF LON TEMPERATURE COOKED KIDNEY BEANS......... IntrOdUCtionCOOOOIO0......00.0.0.0...00.......00... Materia] and MethOdSOOOOOOI.OOOIOIOOOIOOOOOOOOOOOO. Thermal Inactivation.......................... Cooking....................................... Preparation of Bean Extracts.................. Preparation of Red Blood Cell Suspension...... Hemagglutination Assay........................ Resu‘ts and DiSCUSSionOOOOOOOOOO0.0.0.0....00...O. Thermal InaCtivationo.OOOOOOIOOOOOOOOOIOOOOOO Low-temperature Cooking Study................ conC]”SionSOOOOOOOOOO0.0.0.0...OOOOOOOOOOOOOOOOOOO STUDY THO: STABILITY OF PURIFIED PHYTOHEMAGGLUTININ TO THERMAL, CHEMICAL AND ENZYMATIC ABUSE............... IntFOductionOIOOOOOOOOOO0.0.0.0....OOOOOIOOIOOOOOO Materials and Methods............................. PHA PurificationOO0.00000000000000000000...O. Ammonium Sulphate Precipitation......... Affinity Chromatography................. Thermal Inactivation......................... Chemical Treatment........................... Enzymatic Treatment.......................... Hemagglutinating Activity.................... Electrophorectic Separation.................. Discontinuous Polyacrylamide Gel ElectrophoreSiSOOOOOOOIOOOOOOOOOOOOIOOO. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis..................... Results and Discussion............................ Purification of PHA.......................... Hemagglutinating Activity.................... Thermal Treatment....................... Chemical TreatmentOOOOOOOOOOIOOOOOOOOOOO Enzymatic Treatment..................... Electrophoretic Analysis..................... Discontinuous Polyacrylamide Gel EI‘ectrophores‘iSOOOOOO.00......00.0.00... Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis..................... conc1u510n500000coo.000000.000.00.000...000000000 V 51 51 53 53 54 55 56 56 58 63 67 100 STUDY THREE: EFFECT OF THERMAL EXTRUSION AND ALKALI PROCESSING ON THE HEMAGGLUTINATING ACTIVITY OF DRY BEANSOOOOOOOOOOOOOOCOOOCOOOIOOOOOOOCCOOOOOOOOOI0...... IntPOdUCtion...OOOCCOOOOOOOO0.0.00000000000000000 Materials and MethOdSOOOIOOOOOOOOOOOO0.00.0000... Type Of BeanSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Proce551ng OperationOOOOOI.00.000.000.000... EXtrUSionO0.0......OOOOOOOOOOOOOOOOOOOO High pH Soaking and Cooking............ Hemagglutinating Activity................... TextureOOOOOOOOOOOOOOOIOOOOOOOOOOOOOOO0.0... Kramer Shear Press..................... Electrophoretic Separation.................. Discontinuous Polyacrylamide Gel Electrophoresis........................ Sodium Dodecyl Sulphate Polyacrylamide Gel ElectrOphoresis.................... ReSUItS and DiSCUSSionIOOOOOOOOOOOOOOOOOOIIOOOOOO ExtFUSionOOOOO0.0.0.0000...OOOOOOOOOOOOOOOOO Hemagglutinating Activity.............. Alkaline Soaking and Cooking................ Hemagglutinating Activity.............. TextureOO0.0.0000...OOOOOOOOOOOOOOOOOOO Electrophoretic Analysis.................... Alkaline Soaking and Cooking........... EXtPUSionOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. conc‘USionSOOOO0.0.00000000COOOOOOOOOOOOOO0...... STUDY FOUR: BREEDING LINE STUDY...................... IntrOdUCtionOOOOOOOOOOOOIOOOOOOOOOOOOOOOOOOOO0.0. Mater1a15 and MethOdSOOOOOOOOOOOOOOOOIOOOOOOOOOO. source Of BeanSOOOOIOOOOOOCO0.00.00.00.00... MOisture0.0.00.00.00.00000000000000000...... AShOOOOOOO.0......OOOOOOOOOOOOOOOOOOOOO.0... Hunterlab Color Values...................... PreteinOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0... Micro- Kje1dah]OOOOOOOOOOOOOOOOOOOOOOOO In-Vitro Digestibility...................... Sample Preparation..................... ProcedureOOOOOOOOOOOIOOOOOOOOOOOOOOOOOO Hemagglutinating Activity................... ElectrOphoretic Separation.................. Discontinuous Polyacrylamide Gel E'IectrophoresisOOOOOOOOOOOOOO..00.0.... vi 102 102 104 104 104 104 104 106 106 106 106 106 107 107 107 107 113 113 115 120 120 120 120 126 126 127 127 128 128 128 128 128 129 129 129 130 130 130 Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis.................... ReSU]tS and DiSCUSSionOOOOOOOOOOOOOOOOOO000...... Seed COIOrooooooo000000000000000000000000... seed “EightO0.0...OOOOOOOOOOOOOOOOOOOOOOOOO. PrOteinOOOOOOOOOOOOOOOOOOOOCOOOOOOOOOOOOOOOO HemaQQIUtinatIng ActiVitYOOOOOO0.00.00.00.00 Digestibi]ityOOOOOIOOOOOOOOOIOOOOOOOOOOOOOOO Electrophoretic Evaluation.................. Conclusions...................................... SUMMARY AND CONCLUSIONS............................... RECOMMENDATIONS FOR FURTHER RESEARCH.................. LIST OF REFERENCES.................................... vii 131 131 131 133 133 133 135 141 141 147 150 151 10. 11. LIST OF TABLES Amino acid composition of PHA or its subunits as reported in selected studies.................. Statistical summary of effect of frozen storage on the hemagglutinating activity of PHA-P as measured by number of unagglutinated cells................ Statistical summary of effect of thermal treatment on the hemagglutinating activity of PHA-P as measured by the % PHA activity retention......... Statistical summary of effect of chemical treatment on the hemagglutinating activity of PHA-P as measured by the % PHA activity retention......... Statistical summary of effect of enzyme treatment on the hemagglutinating activity of PHA-P as measured by the % PHA activity retention......... Selected extrusion process parameters and product characteristics for extruded dry bean products... Analysis of variance of the hemagglutinating activity of various extruded dry bean products as measured by the % PHA activity retention...... Analyses of variance for heating parameters on the texture and hemagglutinating activity of kidney beanSOOOOOOOOOOOOO0......OOOOOOOOOOOOOOOO. Seed quality characteristics for selected varieties 0f PO vu1gariSOOOOOOOOOOOOOOOIOOOOOOO Statistical summary of the main effects of breeding lines on the % PHA activity, raw digestibility and cooked digestibility of selected small seeded E;_ vulgaris varieties.............................. Analyses of variance ofhemagglutinating activity and digestibilties of P. vulgaris varieties.... viii Page 20 78 80 84 87 105 108 117 132 137 140 LIST OF FIGURES Figure Page 1. Pr0posed isolectin subunit composition........... 25 2. Human erythrocyte glycoprotein oligosaccharide... 30 3. Porcine thyroglobulin oligosaccharide............ 31 4. Agglutination of porcine erythrocytes by commercial Phytohemagglutinin.................... 57 5. Agglutination of porcine erythrocytes by extracts 0f COOKed kidney beanSOOOI...0.00.00.00.00.00.... 59 6. Relative efficiency of thermal treatments for PHA inactivationoo0......OOOOOOOOOOOOOOOOOOO0.0.0.... 61 7. Thermal inactivation of hemagglutinating activity 62 8. Agglutination of porcine erythrocytes by extracts of kidney beans cooked in crock-pots............. 64 9. Relationships among bean texture, cooking condition and hemagglutinating activity for low temperature cooked kidney beans.................. 66 10. Elution of PHA-P from concanavalin A—agarose affinity Chromatography 991 00000000000000.0000... 74 11. Electrophoretic analysis of PHA and PHA-P........ 75 12. Effect of frozen storage on the hemagglutinating aCt1Vity Of PHA-P0000000.00000000000000000IOOOOOO 77 13. Effect of thermal treatment on hemagglutinating aCtiVity 0f PHA-P..0OOOOOOOOOOOOOOOOOOOO...COO... 79 14. Effect of chemical agents on the hemagglutinating aCtiVity Of PHA-POOOOOOOOOOOOOOO0.00.0.0...00.... 82 15. Effect of enzymatic digestion on the hemagglutianting activity of PHA-P............... 86 16. DISC-PAGE analysis of PHA at varying acrylamide ix 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. concentrationSOOOOOCOOOOOOOOOOOOOOOOOOOCOOOOOO0.0 DISC-PAGE analysis of PHA-P eXposed to various Chemical treatmentSOOOOOOOOO.OOOOOOOOOOOOOOOOOOO. DISC-PAGE analysis of PHA-P exposed to various prOteOIytiC enzymeSOOOOIOOOOOOOO0......00.0.00... DISC-PAGE analysis of PHA-P exposed to various enzymatic treatments............................. SDS-PAG electrophoresis of molecular weight StandardSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOCOOCOO... SDS-PAGE analysis of PHA-P exposed to various chemical treatments.............................. SDS-PAGE analysis of PHA-P exposed to various DPOtQOIYtIC enzymeSOOOOOOOOOOOOOOOOOOO000......O. SDS-PAGE analysis of PHA-P exposed to various enzymatic treatmentSOO0.0.0.0000...000......CO... Hemagglutinating activity of extruded whole dry beanSOO00......OOOOOOOOCOOOOOOIOOOO00.0.00...O... Micrographs of selected extruded dry bean prOdUCt500ooooooooooooooooooooooo00000000000ooooo Hemagglutinating activity of extruded bean flourSOIOOOOOOOOOOOOOOOOOOO0.0...OOOOOOOOOOOOOOOO Effect of soak conditions and 76°C heating on the hemagglutinating activity of kidney beans........ Effect of soak conditions and 93°C heating on the hemagglutinating activity of kidney beans........ Effect of pH and thermal treatment on the texture Of COOkEd kidney beanSOOOOIOOOOOOOOIOO0.000...... Electrophoretic analysis of kidney beans cooked at pH 7.0 and pH 12.0..OOOOOOOOOOOOOOOOOOOO000...... SDS-PAGE analysis of extruded bean products (pPOdUCtS 1 - 9)OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. SDS-PAGE analysis of extruded bean products (prOdUCtS 10 - 18)OOOOOOOOOOOOOOOOOOOOOOOOOIOOOOO Protein content and hemagglutinating activity of selected P. vulgaris varieties................... Protein content and hemagglutinating activity of X 90 91 92 93 95 97 98 99 110 111 112 114 116 118 121 122 123 134 35. 36. 37. 38. 39. 40. selected P. vulgaris varieties by bean type...... Digestibility of selected P. vulgaris varieties (1).............................................. Digestibility of selected P. vulgaris varieties (2).............................................. DISC-PAGE analysis of P. vulgaris varieties (1).. DISC-PAGE analysis of P. Vulgaris varieties (2).. SDS-PAGE analysis of P. vulgaris varieties (1)... SDS-PAGE analysis of P. vulgaris varieties (2)... xi 136 138 139 142 143 144 145 INTRODUCTION Legumes play an important role in the diet of man. They are frequently a significant source of protein in the diets of the emerging and lesser devel0ped nations. The importance of food legumes has been emphasized by the Protein Calorie Advisory Group of the United Nations (1974) who stated: "The food legumes are important and economical sources of protein and calories as well as certain vitamins and minerals essential to human nutrition. However, the significant role they play in the diets of many developing countries appears to be limited by their scarcity, caused in great part by their present low yield, their consequent cost, and certain defects in their nutritional and food use qualities." Even though legumes are an important dietary component in many countries, they have not been subjected to research programs as intensive as those devoted to the cereal grains. This is due primarily to the difference in yield between the cereals and legumes, and the increased profitability of cereal crop production for farmers. Legumes are coming under increased scrutiny because their potential dietary impact is so great. One group that is interested in the role of food legumes in deveIOping countries is the United States government. The 0.5. government, through the Foreign Assistance Act, funds a number of coordinated research programs in agricultural deveIOpment in Africa and Central and South America. Title XII of the Act, entitled “Famine Prevention and Freedom from Hunger," provides funding for a number of Collaborative Research Support Programs (CRSP's) that integrate several agriculturally important disciplines in ani effort to investigate crop production and utilization at every level from production through consumption. These disciplines include genetics, plant breeding, entomology, pathology, agronomics, nutrition, food science and sociology. The mission of one of these CRSP's, the Dry Bean/Cowpea CRSP, is to: “...establish active and vigorous collaborative research efforts that will contribute to the alleviation of hunger and malnutrition in develOping countries by improving the availability and utilization of these legumes." (Dry Bean / Cowpea CRSP, 1984 Annual Report, Part 1, Technical Summary). It is projected that this CRSP will directly benefit the rural and urban poor and small farm producers in devel0ping countries by increasing the supply of low cost nutritious legumes. The Dry Bean/Cowpea CRSP is composed of 18 separate projects. One of these projects, ”Improved Biological Utilization and Availablity of Dry Beans," is concerned with improving the quality and quantity of Phaseolus vulgaris varieties in rural settings in Guatemala. This is truly a collaborative project with investigators from five institutions in the 0.5. and the Instituto de Nutricion de Centro America y Panama (INCAP) in Guatemala. The four research areas that this CRSP project addresses are: 1). production, 2). handling and storage, 3). utilization and consumption, and 4). processing and food product development. This dissertation focuses (Hi the evaluation of Phytohemagglutinin (PHA) , the lectin of P. Vulgaris, in raw and prepared dry edible beans. A sensitive PHA assay method was developed and utilized in a series of studies conducted to characterize this antinutritional protein. The first two studies of this dissertation deal with the devel0pment of a hemagglutinating activity assay and 'thee effect of various agents on the stability of purified phytohemagglutinin. This directly relates to the project area of utilization and consumption. The final two studies focus on the bean processing technology and the evaluation of It. vulgaris breeding line germplasm components of this project. This project was performed in conjunction with and partially supported by the Dry Bean/Cowpea CRSP. Finally, this dissertation is presented as a series of four independent research papers preceded by three common chapters. The common chapters are an introduction, a review of the literature and a compilation of the materials and methods used. Each of the research papers is written to conform to the standards and guidelines for publication in the Journal of Food Science. REVIEW OF LITERATURE Beans in the Diet of Man Nutritional Importance The importance of food legumes, especially in the diets of emerging nations, is well established. Legumes have played a significant role in human nutrition since their consumption was initiated 4000 years ago (Bressani and Elias, I977; Gomez Brenes et al., 1975; Elias et al., 1976). Today, (nu: of approximately 13,000 species of legumes, only about 20 are commonly consumed by man, and most of this consumption occurs in the nations of Central America, South America, Africa and Asia. Protein. The most important role of legumes in the diet is as an inexpensive protein source. This is crucial in the developing countries as they are frequently the major source of high quality protein in the diet. The protein quality of legumes is inferior to animal proteins but that problem is obviated by their complimentarity with cereal grains. Methionine is always the first limiting amino acid in legumes (Carpenter, 1981), but they are endowed with relatively high lysine levels. This explains their historic association with cereal grains in adequate, plant—based diets. Because legumes are looked upon as a good source of protein in vegetable based diets, much effort has been spent in investigating the protein quality of legumes and legume/cereal blends. The protein efficiency ratio (PER) of raw and cooked legumes is approximately 0 and 1.2, respectively (Rockland and Radke, 1981). Bressani and Elias (1977) demonstrated an optimization of the apparent PER of legume/corn blends and found the PER was highest when the ratio of legume to corn was 3:7 (weight:weight). Carbohydrate. Although legumes are often considered primarily as a source of protein, carbohydratres are the major component of _P__.__ vulagaris (55 - 65 % on a dry weight basis (db)). Starch (45 - 60 %) and dietary fiber (15 %) are the major carbohydrate constituents, while sugars contribute only 5 to 8 % to the carbohydrate fraction (Reddy et al. 1984). Sahasrabudhe et al. (1981) and Reddy et al. (1984) determined that the starches of P. vulgaris comprise approximately 53 % (db) of beans. They also reported that these starches contained approximately 30 % amylose with a range of 15 to 38 %. This agrees with results reported in an earlier study of the starch in several legumes (Schoch and Maywald, 1968). These authors reported that the starch content of the beans ranged from 55 - 70 % (db) and amylose was present at a level of about 30 % in the starch fraction. Monosaccharides and some oligosaccharides are present in dry beans but these make a minor contribution to the total carbohydrate. Soluble sugars are present in the range of 5.5 to 8 % (Reddy et al., 1984) and those present in the greatest concentration are sucrose and stachyose with raffinose and verbascose also present. Raffinose and the raffinose containing oligosaccharides constitute from 31 - 76 % of the total sugar fraction in dry beans (Naivikul and D'Appolonia, 1978; Akpapunam and Markakis, 1979). The indigestible residue of dry beans is an important component of the total carbohydrate fraction and makes up from 8 to 9 % crude fiber and about 23 to 25 % indigestible residue. The most prevalent fiber components in I). vulgaris varieties are cellulose, hemicellulose and lignins, but pectins are also present. ASp and Johansson (1981) reported that dry beans contain approximately 13 % water-insoluble fiber and 11 % water-soluble components. In addition to the carbohydrate based indigestible fibers, dry beans contain indigestible proteins that contribute to the indigestible residue (Bressani and Elias, I977; Nolzak et al., 1981a). f_a_t_§_._ Dry P. vulgaris varieties contain only 1 - 4 % and generally less than 2 % (db) fat (Sathe et al., 1981a). The majority (63.3 %) of the fatty acids are unsaturated (Watt and Merrill, 1963). Vitamins. In addition to supplying carbohydrates and proteins, dry beans are a significant source of vitamins (especially folic Acid, thiamin and pyridoxine. Recently, Augustin et al. (1981) published the mean concentrations for a number of water soluble vitamins and the contribution a 175 9 sample would make towards the U.S. Recomended Dietary Allowances (RDA) for a number of P. vulgaris varieties. They reported that the concentration of folic acid ranged from 0.171 to 0.579 mg % and a serving contained on average 33 % of the RDA for this nutrient. Thiamin concentration ranged from 0.86 to 1.14 mg % with a serving containing 30 % of the RDA. Mean values for pyridoxine (vitamin B6) ranged from 0.336 to 0.636 mg % and a serving contained 11 % of the RDA. Dry beans only supply 0.136 to 0.266 mg % representing 6 % of the RDA of riboflavin. Finally, niacin provided from 1.16 to 2.68 mg % or 4 % of the RDA. The fat soluble vitamins are present in dry beans but only in trace amounts. Minerals. Dry beans have a fairly high ash content. Hosfield and Uebersax (1980) reported the ash content for 34 varieties of P. vulgaris and the average of the samples was 3.94 %. Augustin et al. (1981) reported the mineral content of dry beans and the contribution a 175 g serving made towards the U.S. RDA of the following minerals. The overall mean value for phosphorous was 0.46 mg % and a cooked serving provided 26 % of the RDA. The mean value for magnesium was 0.20 mg % and a serving provided 26 % of the RDA. The mean value for copper ranged from 0.69 to 1.20 mg % with the average serving providing 21 % of the RDA. Dry beans are also a good source of iron, providing 3.83 to 7.55 mg % and 19 % of the RDA. Zinc is present at levels of 2.2 to 4.4 mg % and a serving provides 12 % of the RDA. The mean values for calcium ranged from 0.09 to 0.2 % with a serving providing 9 % of the RDA. In addition to these minerals that have established RDAs, dry beans also provide sodium and potassium. The sodium content of dry beans ranges from 4 to 17 mg % and the potassium content is 1.5 mg %. Breeding to Improve Agronomic and Food Quality Characteristics Although legumes are a crucial source of amino acids, especially lysine, in the lesser developed countries their production is far below that of the cereal grains. The most important reason for this is that cereals are much more productive than legumes. Hulse (1977) states that on average, yields of corn, rice and wheat are 2.8, 2.2 and 1.7 tons/hectare compared to a yield of 0.5 ton/hectare for legumes (excluding soybeans). Because of the difference in yields, legumes are less likely to provide a satisfactory economic return to farmers than the higher yielding cereal crops. This is especially evident in Asia where the production ratio of cereals to legumes had increased to 9:1 by 1977 (Hulse, 1977). Since the dietary legume to cereal ratio for optimum protein quality should be approximately 1:2 (Bressani and Elias, 1977), it is readily apparent that legume production should be stimulated. The most effective means to stimulate increased bean production would be to develop higher yielding legume varieties that would be more economically attractive to the small farmers in the developing countries. In addition 1x1 improving yield, there are other areas demanding further study: 1). improved nutrient content, especially total protein and sulphur amino acids; 2). increased digestibility; 3). reduction in antinutritional factors such as enzyme inhibitors and lectins; and 4). reduction in raffinose containing saccharides that may contribute to flatus production. ProceSsing OQPOrtunities for Improved Dry Bean Utilization In the developed nations, beans are generally prepared by commercial food processing operations and consumed as canned beans in sauce. The majority of legumes in the lesser deveTOped nations are grown for home consumption by small farmers. Typically, the dry beans are soaked for a few hours, cooked in an open container and consumed whole or as a mashed bean paste with a ceral grain or tuber. Raw legumes are poorly digested, but adequate heat treatment improves the digestibility significantly (Gomez Brenes et al., 1975; Nolzak et al., 1981a, I981b). However, in many parts of the world the thermal treatment that can be provided for bean preparation in the home setting is not sufficient to inactivate toxic lectins (Coffey et al., 1985) and is often just sufficient to heat and hydrate the beans. Gomez Brenes et al. (1975) reported that peak digestibility and Protein Efficiency Ratios of dry P. vulgaris were obtained after soaking for 8 or 16 hours and cooking at 121°C for 10 to 30 minutes. Heating for longer than this resulted in lower protein quality and decreased available lysine. The effect of gamma irradiation on the nutritional value of _P_. vulgaris was reported by Reddy et al. (1979). The authors found that despite decreasing the protein solubility, the digestibility in all cases increased. However, this irradiation was in combination with autoclaving for 10 minutes. Data on the effect of irradiation on bean protein digestibility in the absence of autoclaving is not presently available. Dehulling is another processing option that would in some cases improve the nutritional character of dry beans. Tannins concentrate in the hulls of colored beans and dehulling would be an efficient way to remove or reduce their levels. Elias et al. (1979) showed the presence of tannins to reduce the digestibilty of cooked beans. This was also more recently shown by Aw and Swanson (1985). Dehulling is already an important bean processing operation for the production of dhal (dehulled Vigna mungo) in India. Soaking in mediums other than water may provide the possibility of improved nutritive value. It is commonly known that lye-treated corn is nutritionalTy better than untreated corn. ‘The high [Ni soak increases the availabilty of niacin and lysine, which is the first limiting amino acid in corn. Soaking beans in water is recommended as it reduces the time necessary for adequate cooking (Molina et al., 1976). Further, Gatfield (1980) reported that trypsin inhibitors were reduced by water soaking prior to cooking. In spite of these reports, the effect of high 10 pH soaking on protein quality of beans has not been reported. This may be an effective approach that will prove advantageous in dry bean preparation from a nutritional perspective. Novel processing operations may allow improved utilization of dry beans. For centuries, the Asian cultures have manipulated soybeans to produce a variety of nutritious food products. Some important soybean based foods are tofu, natto, miso, tempeh and tamari. If dry beans could be prepared by methods such as protein curd production or fermentation, the quality and digestibility may be improved. Swanson and Raysid (1984) described the changes in protein quality in tempeh made from red beans (P. legaris L.) and corn. There was a significant increase in digestibility and PER in the fermented products. Thermal extrusion is another method of processing that has not been widely used in bean processing. High pressure extrusion would be beneficial if the pre-cooked bean products were nutritionally superior to dry beans. It would be expected that the digestibility and protein quality would improve with the heat treatment from extrusion but at this time it is not economically feasible in traditional settings. Milling and fractionation of legumes into flours has recently received increased research effort. Bean flour production would permit the increased use of bean flour fractions in processed food products. Many high starch and high protein bean flour fractions have been produced, studied and incorporated into food products (Bakker-Akema et al., 1967; Schoch and Maywald, I968; Sathe and Salunke, 1981a; Chang and Satterlee, 1979; Lee et al., 1985). These products are nutritionally desirable and have good functional atttributes in selected applications. 11 Lectins Definition of Lectins Boyd and Shapleigh (1954) reported that 57 members of the family Leguminoseae contained blood group specific antibody-like activity. Further, these authors found that protein extracts of Lima beans selectively agglutinated human type A erythrocytes and proposed the existence of a group of plant proteins with the ability to distinguish different blood types. The term “Lectin” (from the Latin Legere, to select or pick out) was selected to describe these proteins. The term lectin has since been used in a generic manner to denote any protein with the ability to agglutinate erythrocytes. Unfortunately many proteins, such as toxins, immunoglobulins and some enzymes, have this ability under certain conditions. Agglutination of erythrocytes is therefore not sufficient to define lectins in a biological context. To further clarify the definition shortcoming, Goldstein et al. (1980) proposed that, "A lectin is a sugar binding protein or glycoprotein of non- immune origin which agglutinates cells and/or precipitates glycoconjugates.“ They also stated that lectins should: I). bear at least two sugar binding sites; 2). agglutinate animal (N‘ plant cells ”00.. omv.e i xvom.m u > "mm aoe.o r xmum.o n > "mm map.o u xnmp.o n > "mm cowuo>wuumcw <2; Low mucwsuomgp Fossmgu mo zucmwuwwmm m>mmemm manor v N 0 li . . VTII... b — |ll1 — - J .o mcsmwn STISO CIEllVNIlfl'lS'OVNfl NI NOLLOFICBH 90'] I SiOVHlXE ’ln 62 oov.mm i xmmm.ou u > zuw>muum mcwpmcwpzpmmmEm; wo cowum>wpuocw Fossmzh 00 cm on 1.. 00—. - All/“1.0V owukumoov NI ' NOLLOFICISH 901 I 83.1.“le F OON .5 mtzmwu 63 Low-temperature Cooking StUdy Monitoring the hemagglutinating activity of beans cooked at low temperature is important for two reasons. First, there is evidence indicating that uncooked beans soaked at a low temperature (29°C) were toxic, even if marginally palatable (Public Health Laboratory Service, 1976). Also, kidney beans caused Efll outbreak of food poisoning even after cooking for 5.5 hours in a crock-pot (Noah et al., 1980). Although the crock-pot cooking temperature was not given, it probably did not exceed 82°C and may have been much lower. Second, beans are also a staple food in many lesser developed regions of the world such as Mexico, Central and South America. In those regions, much higher than sea level, bean cooking water boils at temperatures well below 100°C. For examples, the boiling point of water in Mexico City is 89°C. Thus, the experimental conditions chosen simulated normal crock-pot preparation and high-elevation open-kettle cooking temperatures. The hemagglutinating activity of beans decreased with cooking time (Figure 8). In spite of this, detectable levels of hemagglutinating activity remained even after 11 hours of heating at 82°C. However, a 14 hour cooking time rendered the beans essentially free of PHA activity. Hemagglutinating activity was detected in beans held for five hours or less at 91°C. However, when the sample was held for six hours or more, no hemagglutinating activity was detected. The prescence of detectable levels of hemagglutinating activity following long holding times at low cooking temperatures indicated that the possibility existed for the chronic consumption of low levels of PHA in the diets of consuming p0pulations. It should be noted that these temperatures represent final end point temperatures within the cooking bean mass at each setting. 64 ucmwmz coma op mma mo mE=Fo> x m sue: mmcwuumm Auopmv car; new Auowmv zop um umuuaucoo no: mcwxoou c? umxoou mcmmn xmcurx mo muumspxm an mmuzuosspzsw mcwuson mo cowumcwuapmm< .md ...E \ P0wuuo mcwuocwuzpmmosm: one cowuwucou mcexoou .mtzuxmu coon mcosm mawgmcowumpmm .m mtaawm mEDOI mw or m mw S m . 'Lllb _ H m. Wm C IIZII mm P W. m GIAWII A» - 3 l H A ’ II— 0 WW I O U Mm a S A m-illl ill llllllll ---Il! - TIII. F W. fl: ‘1] N O oz_._.._.mm :0...— V .Au nu , . ufl. Uu V 1 02.5mm 26.. - m _II .0 5 O v v 0 1 w ( O V n - N M m m 3 H a N I. >S>Zo< oz .II..II.I. Fm . 3 .A 33:: o C 00 C W 1 0 Cu 67 rates to duplicate actual methods of home bean preparation. These data indicating presence of hemagglutinating activity in palatable cooked beans have not been demostrated before, although they support the documented toxicity of kidney beans cooked in crock-pots for long periods (Noah et al., 1980). Conclusions The electronic cell counter was a sensitive tool for measuring the hemagglutinating activity of kidney beans. The PHA in cooked kidney beans is usually thought to be inactivated by cooking. However, hemagglutinating activity was found in beans exposed to low temperatures even though the cooking times were greater than 12 hours. These data by design do not indicate the nutritional quality of beans cooked to eating softness at low temperatures but may have important nutritional implications. Further research is needed to evaluate any nutritional or physiological impairment resulting from chronic consumption of bean diets containing low levels of active PHA. STUDY TWO: STABILITY OF PURIFIED PHYTOHEMAGGLUTININ TO THERMAL, CHEMICAL AND ENZYMATIC ABUSE Introduction Phytohemagglutinin (PHA), the lectin of kidney beans, is a toxic carbohydrate-binding glycoprotein. The toxicity of PHA to man and animals is well established (Pusztai et al., 1982; Lorenzsonn and Olson, 1982; King et al., 1980a and 0; Wilson et al., 1980). In addition to its toxicity, this glycoprotein is particularly stable to thermal treatment (Coffey et al., 1985) and therefore is of concern if the beans are not sufficiently cooked, as is sometimes the case when prepared at high elevations or in crock-pot cookers. The toxicity of dry beans is well recognized. Noah et al. (1980) reported seven outbreaks of human food poisoning in Britain that were attributed to undercooked kidney beans. Experimental animals are also sensitive to raw dry beans. Jaffe (1960) was the first to demonstrate the oral and parenteral toxicity of PHA to rats. Since that time, there have been a number of reports concerning the toxicity of a variety of dry beans or their lectins (Kakade and Evans, 1965; Kakade and Evans, 1966; Evans and Bandemer, 1967; Hewitt et al., 1973; Jayne-Williams and Burgess, 1974; Rattray et al., 1974). It is now agreed that the most toxic agents in dry beans are the lectins (Honavar et al., 1962; Jaffe and Vega-Lette, 1968; Liener, 1976; King et al., 1980a and b; Wilson et al., 1980; Pusztai et al., 1981; King et al., 1982; 68 69 Lorenzsonn and Olsen, 1982). The concentration of lectin in beans is correlated to the amount of hemagglutinating activity and red kidney beans have the most potent hemagglutinating activity of the P. vulgaris varieties tested (Jaffe and Vega-Lette, 1968). Since PHA is a glycoprotein with carbohydrate-binding capabiliity, affinity chromatography can be used for purification. Leavitt et al. (1977) successfully used thyroglobulin-Sepharose and Ohtani et al. (1980) used concanavalin A-Sepharose to produce purified PHA. According to Yachnin and Svenson (1972), Miller et al. (1973) and Felsted et al. (1976), PHA. is most probably a family of five tetrameric proteins composed of only two different subunits, a potent erythrocyte agglutinator (E subunit) and a lymphocyte stimulator (L subunit). This paper reports the results of an investigation of the stability of the hemagglutinating activity of purified PHA (PHA-P) to thermal, chemical and enzymatic abuse. Materials and Methods PHA Purification Ammoniuni SUlphate Precipitati0n. Concentrated lectin fractions were precipitated from bean extracts by the method of Jaffe and Hannig (1965). Dark red kidney beans (Michigan Foundation Seed, East Lansing, M1) were used as the source of PHA in this study. One kg of hammermilled bean flour (sieve size # 50) was extracted overnight at 4°C with 5 L of physiological buffered saline. Crude PHA was precipitated from the extract by the addition of ammonium sulphate to 75 % saturation. The precipitate was collected by centrifugation, 70 resuspended in distilled water and precipitated again with 75 % Ammonium Sulphate. 'The precipitate was dialyzed against several changes of distilled water at 4°C, freeze dried and stored frozen (-3°C) for later use. Affinity Chromatography. Phytohemagglutinin (PHA) was purified by concanavalin A-agarose affinity chromatography. For purification, crude PHA was added to con A-agarose (Sigma Chemical Co., St. Louis, MO) and allowed to stand for one hour. The non bound protein was removed from the columns by washing with distilled water and saline at pH 7.2 until absorbance at 280 nm had stabilized. Removal of the bound PHA was accomplished by elution with PBS containing ccuwnethyl-D-mannoside. The sugar was removed from the gel by elution with pH 3.5 Acetate buffer containing 2 M sodium chloride. The ultraviolet absorbance of the eluant was monitored using an ISCO model! UA-2 Ultraviolet Analyzer (Instrumentation Specialties Co Inc., Lincoln, NE). Thermal Inactivati0n Purified PHA (PHA-P) was diluted with PBS (5 mg/mL) and 0.5 mL aliquots were introduced to 6 x 50 mm test tubes. The tubes were placed in either a water bath or oil bath and exposed to the following time/temperature conditions: 2, 4 and 8 hours at 70°C; 2, 4 and 8 hours at 75°C; 2, 4 and 8 hours at 80°C; 2, 4 and 8 hours at 85°C; 1, 2 and 4 hours at 90°C; 15, 30, 45, 60 and 75 minutes at 95°C; and 5, 10, 15, 20, 25 and 30 minutes at 100°C. The samples were withdrawn after treatment, cooled immediately in an ice water bath to 0°C and stored frozen (-3°C) until the hemagglutinating activity and electrophoretic analyses were performed. Three replicates of this experiment were performed. 71 ChemiCal'Treatment PHA-P was again diluted with PBS (5 mg/mL) and 2 mL aliquots were placed in 12 x 175 mm test tubes. The PHA-P was exposed to the following chemical agents: 2 M NaCl; 5 M urea and 5 % mercaptoethanol. In addition to these treatments, the PHA-P was dissolved in PBS adjusted to either pH 12 or pH 3. The PHA-P was held in treatment for times of 30, 60 and 120 minutes and the electrophoretic analyses and the hemagglutinating activity determined immediately following incubation. Three replicates of this experiment were performed. Enzymatic Treatment PHA-P was diluted with PBS (5 mg/mL) and 2 mL aliquots were placed in 12 x 175 mm test tubes. The following enzymes (all from Sigma Chemical Co., St. Louis, M0) were added to the PHA at a level of 0.05 mg/mL: pepsin (3200 units/mg protein); trypsin (14,600 BAEE units/mg protein); Chymotrypsin (58 units/mg protein); peptidase (100 units/g solid); protease (6.0 units/g solid); pancreatin (4 x NF grade); alanine amino-peptidase (6.5 units/mg protein); at -amylase (1900 units/mg protein); £9-amylase (850 units/mg protein); mannosidase (17 units/mg protein); and neuraminidase (19 units/mg protein). In each case the PHA was incubated with the enzyme for one, two and three hours and the digestion inixture assayed for hemagglutinating activity and subjected to electrophoretic analysis immediately. Three replicates of this experiment were performed. Hemagglutinating,ACtivity_ The hemagglutinating activity of the purified and treated lectin was determined using the cell counting method of Coffey et al. (1985). These experiments were conducted in triplicate with duplicate 72 determinations of hemagglutinating activity made on each sample. Electrgphoretic Separations DiScontinuous P01yacrylamide (kfl‘ EleCtrophoresis (DISC-PAGE), DISC-PAGE was used to resolve the component peptide patterns of the treated PHA-P. The method used was that of Davis (1964) but staining was accomplished with 0.04 % Coomassie Brilliant Blue G-250 in 3.5 % perchloric acid overnight. For all DISC-PAGE evaluations, 11 % acrylamide gel concentration was used for the running gel and the proteins were subjected to 1 mA/tube for 10 minutes followed by 3 mA/tube for the remainder of the separation. The gels were destained and stored in 7 % aqeous acetic acid. SodiUm DodeCyl Sulphate Polyacrylamide Gel Electrophoresis”(SDS- £A§§1;_ SOS-PAGE was performed to resolve the component peptides of the treated samples according to their molecular weight. The method used was that of Weber and Osborne (1969). For all SOS-PAGE evaluations, 10 % acrylamide gel concentration was used and the gel tubes were allowed to polymerize for 24 hours before use. The tubes were subjected to 3 mA/tube for 10 minutes followed by 8 mA/tube for the remainder of the separation. After deve10pment, the gels were stored in 7 % acetic acid. A series of molecular weight standards were run along with the bean extracts and treated PHA-P. The protein standards were obtained from Sigma Chemical Co. (St. Louis, MO) used and their molecular weights in daltons were: Phosphorylase B (94,000); Bovin serum albumin (67,000); Ovalbumin (43,000); Carbonic anhydrase (30,000); Soybean trypsin inhibitor (20,000); and oc-Lactalbumin (14,400). 73 Results and DiScussiOn Purification of PHA Purified phytohemagglutinin (PHA-P) was resolved from saline kidney bean extracts by chromatography on concanavalin A-Sepharose. Following addition of the bean extract, the column was washed with PBS until the absorbance at 280 nm had stabilized. Figure 10 illustrates that a substance with absorbance at 280 nm eluted following incorporation of 0.01 M OC-methyl-D mannoside. This fraction was collected and found to have a high hemagglutinating activity. Several cycles of column charging, elution and regeneration were performed and this same fraction was collected in each cycle, dialyzed and freeze-dried. The tan powder produced was found to contain 87 % protein as determined by micro- kjeldahl analysis (N x 6.25). This protein concentration, although low, is consistent with the fact that PHA is a glycoprotein with 7.8 to 7.9 % carbohydrate (Allen et al., 1969; Ohtani et al., 1980). The protein purified by affinity chromatography was subjected to DISC gel and $05 gel electr0phoresis and compared to a standard PHA. The patterns developed by the electr0phoresed protein (PHA-P) were essentially identical to the pattern of the standard PHA. The results of the electrophoresis are shown in Figure 11. Based on the electr0phoretic patterns and the high hemagglutinating activity data, this protein was designated as a purified phytohemagglutinin. Hemagglutinating Activity Thermal Treatment. The diluted PHA-P was subjected to thermal stress, the hemagglutinating activity determined and the abused protein subjected to electrophoretic analysis. PHA-P is resistant to activity 74 Pom acnmsmouoeoccu apwcwmmm mmocmami< :m_m>o:mu=ou soc» ai ZO_._.D._m _ _ oneuzhmz zp.o wszH. I: 2 p. 40-4 (J I< 1< 1; - 0- ER 20‘ 0 86 Control OC-AITIYIaSE lb -Amylase Neuraminidase °Il-Mannosidase Trypsin Chymotrypsin Pancreatin Pepsin Peptidase ALA-Aminopeptidase Protease FKQHIGD'HMOOW) 4 4 k - l I l l 2 3 TIME (h) Enzyme level = l % substrate level (0.05 mg/mL) Figure l5. Effect of enzymatic digestion on the hemagglutinating activity of PHA-P "100$ > 87 Table 5. Statistical summary of effect of enzymatic treatment on the hemagglutinating activity of PHA-P as measured by the % PHA activity retention MAIN EFFECT MEANS INTERACTION MEANS TIME (h) 1 2 3 ENZYME PEPSIN 7.45 b 6.18 8.21 7.96 a,b TRYPSIN 13.67 a 12.25 16.53 12.17 a CHYMOTR. 17.03 a 30.42 9.79 10.88 a,b PROTEASE 3.47 b 4.50 4.42 1.49 b PEPTIDASE 3.97 b 6.17 3.58 2.17 a,b PANCREATIN 16.75 a 21.67 17.58 11.00 a,b ALA-AM-PEP. 3.66 b 5.58 3.22 2.17 a,b NEURAM. 94.93 c 95.79 96.17 91.23 c “-AMYLASE 96 .09 c 97 .10 94.65 96.51 c 43 -AMYLASE 94.68 c 95.19 95.36 93.50 c MANNOSIDASE 91.69 96.58 88.36 90.14 c CONTROLS 98.99 c 98.79 99.09 99.09 c CHYMOTR. = CHYMOTRYPSIN ALA-AM-PEP. = ALANINE AMINO PEPTIDASE NEURAM. = NEURAMINIDASE Means followed by like letters are not significantly different (P<0.01) ****H***************************************** ** *************** **** ** ********************************************************************** ANALYSIS OF VARIANCE OF ENZYMATIC TREATMENT ON THE HEMAGGLUTINATING ACTIVITY OF PHA-P degrees SOURCE of freedom mean squares ENZYME 11 16,152.56:: TIME 2 246.14** ENZYME X TIME 22 49.08 ERROR 72 22.72 TOTAL 107 1690.52 ** Significant at the 1 % level 88 Incubation with amylases produced no significant. decreases in hemagglutinating activity in this study. Sialic acid is the terminal carbohydrate in the oligosaccharide chain and because of its position, it might be expected to play a role in a recognition event involved with erythrocyte agglutination. Neuraminidase cleaves terminal sialic acids and it was expected that hydrolysis of these residues might cause a decrease in the hemagglutinating activity of PHA-P. A review of the cell means of the main and interaction effects (Table 5) show that there was a slight reduction in the activity but this was not significantly different (P<0.01) than the control. Similarly, (XL—mannosidase cleaves internal mannose residues and would be expected to cause a significant Change in the carbohydrate moiety of PHA. If the carbohydrate residue is important in binding, this would be expected to have a great impact (n1 its binding and therefore hemagglutinating potential. The results of this study when examined by analysis of variance (Table 5) show that there was a slight (non significant (P>0.01), significant (P>0.05)) decrease in hemagglutinating activity of PHA-P treated with OC-mannosidase for three hours. These results of treatment with carbohydrate hydrolyzing enzymes indicate that the carbohydrate moiety is not the sole determinant of hemagglutinating activity for PHA-P. However, since treatment with mannosidase did result in a slight but significant reduction in activity at three hours, the oligosaccharide chain is necessary for full activity. These data do not agree with the results of Ohtani et al. (1980) who reported that hemagglutinating activity did not decrease 89 following digestion with oc-mannosidase. However, their method of visual estimation of hemagglutinating activity probably was not sensitive enough to detect the difference. Electrophoretic Analysis. DISC-PAGE. The results of the DISC-PAGE evaluations are shown in Figures 16 through 19. Figure 16 shows the response of PHA to differing concentrations of acrylamide in the running gel. Based on the results, a concentration of 11 % was chosen for subsequent DISC-PAGE analyses in the study. An evaluation of the PHA-P exposed to a variety of thermal treatments showed no dramatic changes in the protein patterns. It was expected that there may have been some visible changes due to thermal denaturation but this was not observed. The patterns of all the heated samples were essentially identical to the unheated control. The loss of hemagglutinating activity on heating is therefore probably due to unfolding of the peptide chains but not due to more severe destruction. Exposure of the PHA-P to 2 M NaCl, pH 3.0 or 5 M urea has little effect on the gel patterns (Figure 17). Exposure to pH 12.0 however produced some changes. There was a decrease in the number of protein bands with Rm values less than 0.50. For bands with Rm of >O.60 there was no Change. Incubation at pH 12.0 caused a shift in most of the bands although only slightly. This is probably due to the ionization of the amino acids at the high pH causing a shift in the charge/density ratio. Treatment of PHA-P with 5 % mercaptoethanol caused some change in the range of Rm 0.60 to 0.82. In this range there was a lack of resolution, however it seems that there was a concentration in the range 90 8 9 7% 9% ll% l3% (R = relative mobility x l00) Ill Figure 16. DISC-PAGE analysis of PHA at varying acrylamide concentrations 9T mucmEummLp Foumsmcu mzowcm> op ummoaxm au<=m to mwmxpmcm mo on ummonxm a1 op vomoaxm a10.44. There was a major band at 0.44 to 0.48 and a large band at 0.10 in the controls. All proteolytic enzymes were effective in reducing the presence of the 0.10 band and causing other minor changes at the lower Rm values. All of the carbohydrate hydrolyzing enzymes tested had essentially the same patterns as the control PHA-P (Figure 19). It is probable that the amylases had very little effect on the PHA-P structure. This is expected as there is no glucose in the PHA oligosaccharide chain. The effect of neuraminidase on PHA-P pattern is also negligible. It was expected that there may be a large effect on the gel pattern as there are terminal sialic acid residues in the oligosaccharide Chain. However, the effect on the molecular weight after cleaving these residues is negligible and therefore was not observed in the pattern. The mannosidase treated PHA- P showed an increase in the Rm of the 0.50 band to 0.58. This is probably due to cleavage of the mannose residues in the chain, increasing the charge/density ratio of the residual peptide. SOS-PAGE. The Rm values of the molecular weight standards are shown in Figure 20. According to these data, the major band occurred at R of approximately 0.60 which corresponds to a molecular weight of m about 28,000 daltons. Felsted et al. (1981) reported that the molecular weight of the subunits of PHA were 31,700 and 29,900 for the E and L subunits respectively. Since 305 treatment denatures the native PHA-P prior to electrophoresis, it would be expected that the major band would 95 Acomumgucmucoo wowsopzsum a opv mucoucmum pguwmz copzumpoe mo mammsogaocuumFm umh umzz< u~zommo szsm4< 22mmm mzH>om (vOI XI .LH9I3M uv1noa'low 901 mm<4>moxamoza 96 be the average of the subunits. The major band observed at Rm = 0.60 in our study agrees well with the expected results for relative mobility. As observed in the DISC-PAGE evaluations, there was no observable effect of thermal treatment or exposure to pH 3.0. These two treatments were indistinguishable from the control. Treatment at pH 12.0 did cause some changes in the elution profile (Figure 21). There was a lack of the 0.30 band and the presence of stained areas from 0.40 to 0.60 and from 0.73 to 0.94. This indicates an increase in the concentration of lower molecular weight species following this treatment. There was also a new band at 0.05 to 0.08 after treatment. This may be due to condensation of smaller proteins to produce larger species. Addition of urea had little effect also except for the presence of a new band at 0.67. Mercaptoethanol treatment produced gels similar to controls except that there were bands at 0.13, 0.20 and 0.23 following treatment. Digestion with proteolytic enzymes is Characterized by a general lack of high molecular weight components in the gel patterns (Figures 22 and 23). Peptidase is unique in that it caused a broad diffuse staining from 0.68 to 1.0 along with the band at 0.58 and a minor band at 0.12. This indicates reduction in molecular weight of the component peptides. Alanine amino peptidase treatment caused an increase in the number of bands corresponding to higher molecular weight. Digestion with the other proteolytic enzymes yields similar patterns that lack higher molecular weight peptides. Incubation of the PHA-P with amylases yields patterns that are essentially identical to that of the untreated PHA—P. This is expected for the same reason outlined before: there is a lack of glucose in the oligosaccharide chain. Treatment with OC-mannosidase also resulted in a 97 mgcmEuomLu Fouvsmcu maowgm> on ummoaxm ¢1 o» ummoaxm a1ah ozaz< szama Zahaumuz=u Zama>¢h «Ba wmaomeama mml N n 1 ...8338333 Np .em .em 11111 99 mucmEummmp owpmexNEm maowmm> op vmmoaxm m10.F.eoe o>aoapuc u Emv mm<2mozzz<§ mm<4>z0.01) of processing conditions on the 108 Table 7. Analysis of variance of the hemagglutinating activity of various extruded dry bean products as measured by the % PHA activity retention degrees SOURCE of freedom mean squares PRODUCT 17 1693.63** ERROR 36 7.54 TOTAL 53 A 548.36 ** Significant at the 1 % level 109 hemagglutinating activity. Extrusion of whole beans resulted in slight inactivation of hemagglutinating activity. Figure 24 represents the hemagglutinating activity of extruded whole dry beans. Based on a comparison of these extruded products with a standard PHA, whole kidney and black beans have only 82 to 88 % of the activity of native PHA. This level of activity is approximately the level of hemagglutinating activity in the unheated whole Montcalm and Domino pedigrees (Coffey, 1985). In the unheated raw bean, Montcalm has a hemagglutinating activity of 84 % of PHA and Domino has an activity of 82 %. A comparison of the cell means (Figure 24) shows there is no significant difference (P>0.01) between extruded whole kidney beans or whole black beans. These data show that the extrusion processes used were insufficient to cause a significant reduction in hemagglutinating activity of whole kidney and black beans. Pinto beans were incorporated as a low lectin standard and they show an activity of 24 to 26 % of the PHA. This level of ‘1» hemagglutinating activity is significantly less than kidney or black bean products at the 0.01 level. A comparison of the micrographs of these extruded products (see Figure 25) shows granular material incorporated in the extruded pellets of whole beans. The presence of these granules represents uncooked portions of cotyledon. The hemagglutinating activity of dry bean flours show large changes in the hemagglutinating activity with extrusion. For products one, two and three in Figure 26, there is a strong trend in hemagglutinating activity reduction with increasing temperature, at least over the range from 116 to 133°C. One likely reason that there was such a dramatic effect of temperature in this series is that there were fairly high TlO 80 ‘ f; 60 E p. o < < :I: a. a: 20 0 3:15;: 5:35;: 15 16 l7 l8 11 T2 13 14 Il— _ _ KIDNEY BLACK PINTO Figure 24. Hemagglutinating activity of extruded whole dry beans Ill mausuomq :mwn Ame vmuamuxm umuum—wm mo mzmmsmomuwz .mN acsmmm 112 % PHA ACTIVITY KIDNEY BLACK Figure 26. Hemagglutinating activity of extruded bean flours 113 operating pressures in the barrel. Likewise, for products five, six and seven there is a strong trend in reduction (H"the hemagglutinating activity when temperature of the final product increases from 148° to 182°C. No trend was observed with the hemagglutinating activity and barrel pressure in this series. For black bean flour, there seems to In: a relationship between the final product temperature, barrel temperature and hemagglutinating activity. Product eight had a value of 43 %, nine had a value of 48 % and 10 had a value of 37 %. This does not correSpond to the final product temperature as product eight had a hemagglutinating activity between products nine and 10. However, in the course of the extrusion runs, the barrel temperature dr0pped to 100°C for product eight whereas it was 190° and 200°C for samples nine and 10 respectively. For the two samples produced at the higher barrel temperature, decreasing hemagglutinating activity corresponded to increasing final product temperatures. Alkaline Soakingpand C00king Hemagglutinating, Activity. Earlier’ work has shown a: dramatic effect in the hemagglutinating activity when PHA-P was held at pH 12.0 (Coffey, 1985). A similar response is observed in the hemagglutinating activity of whole kidney beans that are soaked and cooked at pH 12.0. As shown in Figure 27, at 76°C, there is a large reduction in the hemagglutinating activity of beans held for eight hours at pH 12.0 versus those at pH 7.0. Cooking for eight hours at pH 12.0 resulted in a retention of only 20 % of the PHA activity versus 97 % for the samples at pH 7.0. There is no significant difference (P>0.01) among samples held for two or four hours at either pH level. ll4 76°C p—H 12 >. 1: 2 p. o < ' < :1: n. 32 TIME (11) Figure 27. Effect of soak conditions and 76°C heating on the hemagglutinating activity of kidney beans 115 At 93°C, the situation is similar in that there is greater retention of hemagglutinating activity at pH 7.0 than at pH 12.0 after a two hour cooking time (Figure 28). For samples held two hours at pH 7.0, there was a 53 % retention of hemagglutinating activity. Samples held at (Hi 12.0 for two hours had no remaining activity. All samples held for four or eight hours at both temperatures had no remaining activity either. The analysis of variance for the effect of pH and temperature on hemagglutinating activity on kidney beans is summarized in Table 8. There are significant effects (P>0.01) due to temperature, pH and time. In addition, there are significant differences (P>0.05) for the interactions of temperature x time and pH x time. TextUre. An analysis of the cooked bean texture was performed and the results are summarized in Figure 29. At both temperatures tested, the texture of the high pH beans was softer than those soaked and cooked at neutral pH. For the beans cooked at 76°C, there was a linear correlation of shear force to 1cooking time for samples at both pH levels. At pH 7.0, the correlation coefficient of the data points and the regression equation describing the line were: r = -O.995 Y = 1140 - 55.2 (cooking time in hours) At pH 12.0 the following values were obtained: r = -0.999 Y = 883 - 62.9 (cooking time in hours) These values indicate that from two to eight hours of cooking time at this temperature, the beans soften more rapidly at pH 12.0 than pH 7.0 and at any particular time the pH 12.0 beans will be softer than ll6 % PHA ACTIVITY TIME (h) Figure 28. Effect of soak conditions and 93°C heating on the hemagglutinating activity of kidney beans 117 Table 8. Analyses of variance for heating parameters on the texture and hemagglutinating activty of kidney beans. ANALYSIS OF VARIANCE 0F EFFECT OF HEATING PARAMETERS ON TEXTURE OF KIDNEY BEANS degrees SOURCE of freedom mean squares 15* TEMPERATURE 1 11,630,000.00** pH 1 1,343,005.44** TIME 2 2,371,175.69** TEMPERATURE X pH 1 86,240.11** TEMPERATURE X TIME 2 23,590.58 pH X TIME 2 1096.53 ERROR 26 704.42 TOTAL 35 82,000.44 Significant at the 0.01 % level ANALYSIS OF VARIANCE 0F HEATING PARAMETERS ON THE HEMAGGLUTINATING ACTIVITY OF KIDNEY BEANS degrees - SOURCE of freedom ‘ mean squares ** TEMPERATURE 1 49,424.70** pH 1 4323.06** TIME 2 3111.30 TEMPERATURE X pH 1 171.17* TEMPERATURE X TIME 2 1156.61* pH X TIME 2 1189.68 ERROR 26 241.04 TOTAL '35_‘ 2031.46 ** Significant at the 1 % level * Significant at the 5 % level lOOO 500 4* FORCE/100 g BEANS Figure 29. 118 PM 12.0, 76°C pH 7.0, 93°C TIME (II) Effect of pH and thermal treatment on the texture of cooked kidney beans 119 those at pH 7.0. At a cooking temperature of 93°C, the same relationship was observed. At every time tested, the (Hi 12.0 beans were softer than those at pH 7.0. At this temperature however, there was no linear or logarithmic correlation of shear force to cooking time. Figure 29 indicates that at eight hours, both samples are approaching a minimum shear value. Coffey et al. (1985) reported the minimum palatability for cooked kidney beans was 250 lbs/100 g bean.. Using this figure and ‘the regression equations for 76°C, beans cooked at (Hi 12.0 would reach minimum palatability at: Time (250 - 883)/-62.9 = 10.06 hours For samples cooked at pH 7.0, the time required is: Time = (250 - 1140)/-55.2 = 16.12 hours To summarize the 76°C data, soaking and cooking beans at pH 12.0 speeds the softening of the seeds and decreases the time required to reach minimum palatability by approximately six hours or by 62.4 1. For 94°C cooking, the minimum palatability can be estimated from Figure 29. At pH 12.0, a shear value of 250 lbs would be expected at approximately 3.5 hours. At pH 7.0, it takes approximately six hours. To summarize the data at 94°C, soaking and cooking beans at pH 12.0 speeds the softening of seeds and decreases the time required to reach minimum palatability by approximately 2.5 hours or by 58.3 %. The strong relationship between pH and texture and pH and residual hemagglutinating activity suggest that alkali treatment of beans may be a possible approach for processing to improve the nutritional value of dry beans. A comparison of thése results with those of the 120 hemagglutinating activity determinations show that at 76°C (Figure 27), eight hours at pH 12.0 was sufficient to reduce the % PHA activity by approximately 80 % whereas at pH 7.0 for the same time, there was no reduction in activity. For beans cooked at 94°C, this argument becomes relatively unimportant because the hemagglutinating activity is reduced to zero before the beans reach a palatable texture (Figure 28). Electrophoretic Analyses Alkaline Soaking and Cookingg The SOS-PAGE gels of the beans soaked and cooked at pH 12.0 are shown in Figure 30. ‘There .are differences for both two and eight hour cooking times for the pH 12.0 beans. For beans held at (Ni 7.0, the patterns were the same as the control bean extracts and were essentially unchanged during the heating period. For pH 12.0 samples cooked two hours, there were major bands at 0.22, 0.30, 0.42, 0.54, 0.66 and 0.82. For beans held for eight hours at this pH, there was one major band at 0.20 and very faint bands at 0.32, 0.49, 0.59 and 0.69. This indicates that the proteins are being digested and only some of the larger ones (lower Rm values) are remaining. ExtruSion. The results of the SOS-PAGE analysis of the extruded products is illustrated in Figures 31 and 32. According to the patterns produced, there was no easily identified change or correlation with decreasing hemagglutinating activity. C0nclusion There is considerable variation in the effect of extrusion on dry beans due to changes in the processing parameters. For bean flours, the l21 ES;:-;-:.i-’;§:‘555§5333523;=§§ 9 2 pH 7 pH 12 pH 12 COOK 8 h COOK 2 h COOK 8 h (Rm = relative mobility x 100) Figure 30. Electrophoretic analysis of kidney beans cooked at pH 7 and pH 12 (SOS-PAGE) 122 Am 1 P mpusuosav mausuomn coma vmvzspxm to mwmz—mmm mw0.01) than a retention of 44.50 % following extrusion at 171°C and 500 psi. The most effective extrusion parameters for bean flours are relatively high pressure (900 - 1200 psi) and temperatures of 270 - 300°C. Extrusion of whole beans under the conditions employed in this study (150 - 185°C, 700 - 1200 psi) did not result in efficient reduction in hemagglutinating activity. Extruded whole kidney beans and whole black beans showed the presence of uncooked cotyledon particles and retained from 82 - 88 % PHA activity. There was no apparent correlation of hemagglutinating. activity with process conditions for whole bean extrusion. Soaking and cooking kidney beans in alkaline media reduces the hemagglutinating activity more effectively than the same thermal treatment at neutral pH. At 76°C, samples treated at pH 12 retained 20 % PHA activity while samples at pH 7.0 retained 97 %. At 93°C, samples treated at pH 12.0 retained no detectable activity whereas those treated at pH 7.0 retained 53 % of the original activity. In addition to the increased inactivation of PHA activity, high pH treatment results in significantly softer beans. The regression equation describing the texture of beans cooked at pH 7.0 and 76°C is: v = 1140 - 55.2 (hours at 76°C) The regression equation for beans cooked at pH 12.0 and 76°C is: 125 Y = 883 - 62.9 (hours at 76°C) The time required to reach an acceptable tenderness based on a minimum palatability end point was reduced when beans were cooked at a pH of 12.0. Beans cooked at pH 12.0 required only 60 % of the time needed for those cooked at pH 7.0 to reach the same textural end point. These are important considerations for dry bean preparation. A persistent problem in Guatemala and other Central American nations is the development of the hard-to-cook phenomenon in dry beans. This is the condition where dry beans fail to hydrate and soften during cooking. The resultant hard-to-cook beans are difficult to eat and therefore represent a dietary and economic loss for the consumers in those areas (R. Bressani, personal communication). If a simple treatment preventing or ameliorating the hard-to-cook phenomenon could be developed, it would represent a significant improvement in the quality of the diet of those consumers. The results of this study indicate that alkaline soaking and cooking may represent a practical approach to solving this problem. STUDY FOUR: BREEDING LINE STUDY Introduction Dry edible beans are an important food crop, especially in the lesser developed countries of Central America, South America, Africa and Asia. They play an important role as a source of high lysine protein to complement grain based diets. Because of this, bean production is being encouraged in the rural areas of many lesser developed countries. One way to increase production of dry beans is to introduce higher yielding cultivars to small farmers in those countries. However, dry bean yields in the U.S. have remained constant for the last 20 years, suggesting that a "yield plateau" has been reached. If a yield plateau has been reached, cultivar improvement would be an important method for overcoming it (Hosfield and Uebersax, 1980). Cultivar improvement is necessary if we are to increase the productivity of dry Phaseolus vulgaris species for human food. Many factors must be considered when breeding to improve legumes. Bressani and Elias (1977) reported that the factor of greatest importance for the selection of food crops was crop productivity. 'They reported this however, as a function of production per unit of arable land, nutritional importance of the cr0p and the acceptability of the crap to consumers and processors. It was expressed as follows: Productivity = Yield x Nutritive Value x Technological Value Obviously, the first and most important emphasis must be given to developing higher yielding varieties. However, these high yielders must 126 127 be agriculturally and nutritionally adequate. One important aspect of nutritional adequacy is digestibility and P. 'vulgaris varieties are known to have digestibilities in the range of 60 to 80 % depending on the cultivar and method of preparation (Wolzak et al., 1981a and b). If a dry bean could be produced that had significantly higher digestibility than is presently available, it would be an important agricultural and nutritional contribution. Another nutritional factor in dry beans that should be considered is the concentration of lectins. These toxic glycoproteins depress the nutritive and protein quality of the beans. Lectins are resistant to heating (Coffey et al., 1985; Gomez Brenes et al., 1975) and if this component could be bred out of the seed, it might represent an improvement in nutritional quality of dry beans. This study reports the results of an investigation of the digestibility, hemagglutinating activity and selected food quality attributes for 16 pedigrees of P. vulgaris recently produced at the small seeded dry bean nursery at Michigan State University. Materials and Methods Source of Beans The P. vulggris varieties used in this study' were produced at Michigan State University as part of the international dry bean quality nursery. The seed was produced in four row plots 4.9 m long Spaced 50.8 cm apart. Standard practices for fertilizer and herbicide application were followed and pods were harvested manually from the middle two rows of individual plots. The pods were threshed by hand and the seeds stored at room temperature. The seed pedigrees produced for this study 128 were: 800242, Sanilac, 8217-111-24, Nep 2, BTS, MSU-61380, 8217-V111, Jalpatagua-72, San Fernando, Ica-pijao, Jamapa, FF4—13-M-M-M-M, Protop- pi, Carioca, P766, and Mexico 12-1. Moisture Approximately 5 g of bean flour were weighed onto previously dried and tared crucibles and dried to a constant weight at 80°C for 24 hours in an air oven. Percent moisture was determined by weight loss on a fresh weight basis (AACC Method 44-15). % Moisture = (Moisture Loss (9)/Sample Fresh Weight (g)) x 100 1331 Dried samples obtained from the above moisture determination were placed in a muffle furnace at 525°C for 24 hours. The uniform white ash was cooled in a dessicator and weighed at room temperature. Percent ash was determined on a dry weight basis (AACC Method 08-01). Percent Ash = (Residue Weight (g)/Sample Dry Weight (9)) x 100 Hunterlab Color Values The Hunterlab Model 025-2 Color/Difference Meter (Hunter Associates Laboratories, Reston, VA) standardized with a white tile (L = 95.35, aL = -0.6, °L = +0.4) was used to evaluate the color of dry bean varieties. A 100 9 sample of dry beans was placed in an optically inert glass cup and covered with an inverted white lined can to keep out extraneous light. Protein Micro-Kjeldahl. Approximately 30 mg of bean flour were weighed and analyzed by the standard micro-kjeldahl procedure. Percent nitrogen obtained was then multiplied by 6.25 to obtain % protein (AACC Method 46‘13). 129 In-Vitro Digestibility Sample Preparation. Samples used included a lot of 16 dry bean varieties which were assayed for protein digestibility raw and after cooking. Raw beans were soaked in distilled water (water/bean, 3:1, v/w) for 18 hours at 5°C. Soaked beans were cooked in the autoclave (15 psi, 121°C) for 30 minutes. Cooked beans were dried in a forced draft oven at 60°C for 18 hours, ground in a Udy cyclone mill and analyzed for protein content by the micro-kjeldahl method. Procedure. The in-vitro digestibility of the samples was assessed by measuring the extent to which the pH of the protein suspension dropped when treated with a multi-enzyme system (all enzymes were obtained from Sigma Chemical Co., St. Louis, MO) including trypsin (porcine pancreatic, Type IX), Chymotrypsin (bovine: pancreatic, ‘Type II), peptidase (porcine intestinal) and Stréptomyces griséus prOtEaSe (Type XIV) as described by Wolzak et al. (1981a and b). An amount of sample containing 250 mg of crude protein was weighed into a 100 mL beaker and 40 mL of distilled water were added. The protein suspensions were placed in the refrigerator for two hours before analysis. The enzyme solutions were freshly prepared before each series of tests. The multi-enzyme solution contained 22,704 BAEE units of trypsin, 186 units of chymotyrypsin and 0.052 units of peptidase per mL. This solution was adjusted to pH 8.00 with 0.1 N NaOH and/or HCl while stirring in a 37°C water bath. The protease solution contained 7.95 mg/mL. Both were distributed into tubes containing the amount needed for each assay (4 mL) and placed in an ice bath until used. Each sample was placed in a 37°C water bath and adjusted to pH 8.00 with 0.1 N NaOH and/or HCl, while stirred constantly with a magnetic 130 stirrer. The multi-enzyme solution was then added. The pH drop was measured with an Orion Research Inicroprocessor ionalyzer/901 (Orion Research, Cambridge, MA). At exactly 10 minutes of digestion, the bacterial protease solution was added and water of a 55°C water bath circulated to the sample. The pH at 15 minutes of total digestion was recorded and the in-vitro digestibility calculated according to the formula proposed by Wolzak et al. (1981b) for P. vulgaris: In-vitro digestibility = 289.677 - 32.811 (pH 15 min) Sodium caseinate was used as a reference sample with each series of tests. Duplicate determinations of each sample were performed. Hemagglutinating Activity The hemagglutinating activities of the bean varieties studied were determined by the method of Coffey et al. (1985). All experiments were performed in triplicate with duplicate determinations made on each sample. Electrophoretic Separations Discontinuous 'POIyacrylamide (RH Electrophoresis (DISC-PAGE)L DISC-PAGE was used to resolve the component peptide patterns of the treated PHA-P. The method used was that of Davis (1964) but staining was accomplished with 0.04 % Coomassie Brilliant Blue G-250 in 3.5 % perchloric acid overnight. For all DISC-PAGE evaluations, 11 % acrylamide gel concentration was used for the running gel and the proteins were subjected to 1 InA/tube for ‘10 ininutes followed by 3 mA/tube for the remainder of the separation. The gels were destained and stored in 7 % aqeous acetic acid. 131 Sodimn Dodecyl Sulphate P01yacrylamide Gel EflectrophOresis (SDS- BAEEL; SOS-PAGE was performed to resolve the component peptides of the treated samples according to their molecular weight. The method used was that of Weber and Osborne (1969). For all SOS-PAGE evaluations, 10 % acrylamide gel concentration was used and the gel tubes were allowed to polymerize for 24 hours before use. The tubes were subjected to 3 mA/tube for 10 minutes followed by 8 mA/tube for the remainder of the separation. After deve10pment, the gels were stored in 7 % acetic acid. 11 series of molecular weight standards were run along with the bean extracts and treated PHA-P. The protein standards were obtained from Sigma Chemical Co. (St. Louis, MO) and their molecular weights in daltons were: PhOSphorylase B (94,000); Bovin serum albumin (67,000); Ovalbumin (43,000); Carbonic anhydrase (30,000); Soybean trypsin inhibitor (20,000); and OCmLactalbumin (14,400). Results and Discussion Seed Color The HunterLab 1., a and t) color values are summarized in Table 9. Of the 16 varieties tested, four of them (numbers one through four) were white beans. Samples one, two and four looked like typical navy beans however number four was darker than the first two. Sample three was the lightest of the four white beans but had a typical kidney bean shape. Samples five through 12 were all of the black turtle soup commercial class designation and all were much darker (lower L values) and had more red (+aL) and blue ('bL) characteristics than the white navy types. One of the samples of undefined designation, P 766, looked very much like a 132 Table 9. Seed quality characteristics for selected varieties of P. vulgaris com ' SEED HEAL , SEED CLAss COLOR HT. 100 MOIS PROT MOIS ASH PEDIGREE DESIG L 0L 11L (9) 1. z z s SANILAC NAVY 63.65 7.90 0.3 15.35 10.3 20.80 8.51 '4.09 8217-111-24 00F 65.45 7.05 2.15 16.09. 9.9 26.68 8.56 3.98 HEP-2 NAVY 60.25 8.65 1.25 15.14 10.1 21.31 8.58 4.19 8T5 BTS 16.85 46.85 -55.75 18.71 ' 10.7 18.62 9.02 3.85 "SO-61380 BTS 15.55 50.6 -61.2 18.20 10.8 22.15 9.11 4.14 8217-V111 8T5 16.05 48.7 -58.9 20.98 10.5 23.03 9.54 4.01 JALPATAQUA- 8T5 15.85 49.6 -59.9 21.72 10.5 26.10 9.55 4.29 72 ' FERNANDO ICA-PIJAO 8T5 15.6 50.3 o60.85 18.10 10.7 19.95 8.69 4.13 FF4-l3— BTS 15.75 49.95 -60.35 19.72 11.0 20.92 9.09 3.75 M-H-M-M PROTOP-PI PINTO 24.55 32.25 ~33.05 27.05 10.0 24.87 8.95 3.89 12-1 133 brown colored black turtle soup bean. Protop-pi and Carioca are both of the pinto designation however they are very different in color. Protop- pi is a dark bean with a nfinimum of tan mottling on a dark surface. Carioca has some green-brown streaks on a tan surface and is therefore a fairly light bean. Mexico 12-1 is another bean of undefined designation and has a tan coat similar to that of the Carioca base coat. Seed Weight All the seeds produced for this study had average seed weights between 15.35 g and 27.05 g per 100 seeds (Table 9). This is expected as they were derived from the small seeded nursery. For comparison, large kidney bean breeding lines have a seed weight of approximately 60 g per 100 seeds (Hosfield and Uebersax, 1980). Protein The seeds investigated had protein contents ranging from a low of 18.9 % for the pinto type Carioca to a high of 26.68 % for an undefined white bean 8217-111-24 (Table 9). These data are in agreement with protein values reported for P.' vulgaris varieties (Hosfield and Uebersax, 1980; Agbo, 1982). Hemagglutinating Activity_ 0f the pedigrees selected, three have fairly high hemagglutinating activity. They are Protop-pi (94 78), 800242 (83 z) and FF4-13-M-M-M-M (80.5 1»). The hemagglutinating activity for all lines is shown in Figure 33 and it ranges from a high of 94 % to a low of 3 %. If it becomes important to breed dry beans for low hemagglutinating activity, a number of these beans would serve as good parents as they contain less than 50 % PHA activity. These low lectin varieties are: Sanilac, San Fernando, Mexico 12-1, Black Turtle Soup, Jamapa, Jalpatagua, Ica-Pijao, 134 % PHA ACTIVITY l'Zl ODIXBW OONVNHBd NVS DVTINVS 72‘ 111'ZlZ8 8 3 3 8 I l I I J I Q 1 z dEN 99Ld VDOIHVD I OVPld-VDI vnov1va1vr vavwvr 818 :1'3‘1 fmtmom —'I u I l ‘1 Ln 0 m o m N N .— ,... D NIBlOHd % vul aris Protein content and hemagglutinating activity of selected P. varieties Figure 33. 135 Carioca, P 766 and Nep-2. It is also fortunate that a number of these have tropical bean germplasm in their pedigrees (San Fernando, Mexico 12-1, Jamapa, Jalpatagua). These would be expected to perform well in tropical climates where many of the people rely on dry beans for a significant part of their diet. Within classes (Figure 34, Table 10) there is wide variation in hemagglutinating activity. For white beans, it ranges from a high of 84 % for 800242 to a low of 2.9 % for Nep-2. There is a similar wide range of values for black turtle soup type beans. Pedigree FF4-13-M-M-M-M has a high value of 81 % PHA activity, and the lowest value is 5 % for the Ica-Pijao pedigree. The pedigree that had the highest hemagglutinating activity was the pinto designation Protop-Pi with a value of 94 %. This is interesting as pintos generally have low concentrations of toxic lectins and low hemagglutinating activity. It should be noted here that even though this pedigree had a high hemagglutinating activity, it does not indicate the absolute toxicity of the bean. Digestibilty Based on the digestibility data (Table 10), the most digestible beans after cooking were: Carioca (78.02 %); Protop-pi (77.95 %); 8217- 111-24 and Sanilac (both 76.32 %); and Nep-2 (75.78 %). These digestibility results are in agreement with the published values for dry bean digestibility (Wolzak et al., 1981a and b; Bressani and Elias, 1977). According to the data in Figures 35 and 36, there is a large difference in digestibility between raw and cooked samples, as expected. According to the analyses of variance (Table 11), there are significant effects due to breeding line for digestibility of the raw and cooked bean varieties tested. AJso, the digestibilities of white 136 % PHA ACTIVITY O O C :0 <3‘ (\1 l I 9928 I v3018v3 UDF l'ZL ODIXBN PINTO Id' d0108d OVPId' V31 I V08VlleVF Tl Vdevr I S18 OGNVNHHd NVS BLACK WHITE 5 10‘ 5 ol N D laIElOHd % vul aris Protein content and hemagglutinating activity of selected P. varieties by bean type Figure 34. 137 Table 10. Statistical summary of the main effects of breeding line on the % PHA activity, raw digestibility and cooked digestibility of selected small seeded P. vulgaris varieties CELL MEANS FOR THE HEMAGGLUTINATING ACTIVITY AND DIGESTIBILITY OF RAW AND COOKED DRY BEANS BREEDING % PHA DIGESTIBILITY LINES ACTIVITY RAN COOKED 800242 17.82 c,d,e 66.58 c 73.60 c,d SANILAC 43.83 68.55 a,b 75.60 b,c 8217-111-24 65.20 a,b 69.57 a 76.33 a,b NEP-2 9.25 e 67.05 b,c 75.79 b,c BLACK TURTLE SOUP 22.47 c,d 60.54 71.11 e MSU-61380 73.30 a,b 63.53 d 72.69 d,e 8217-V111-32 67.44 a,b 62.82 d 71.43 e JALPATAGUA-72 17.43 c,d,e 63.87 d 71.77 d,e SAN FERNANDO 24.61 c,d 65.61 c 71.96 d,e ICA-PIJAO 15.12 c,d,e 66.62 c 71.23 e JAMAPA 21.79 c,d,e 63.08 d 73.10 d,e FF4-13-M-M-M-M 77.16 a 66.72 c 72.18 d,e PROTOP-PI 94.81 69.24 a 77.94 a CARIOCA 14.82 c,d,e 68.57 a,b 78.02 8 P766 11.88 d,e 66.71 c 72.13 d,e MEXICO 12-1 21.19 c,d,e 65.97 c 72.93 d,e DOMINO 26.16 c ---------- MONTCALM 61.38 b ---------- Values followed by like letters are not significantly different (P>0.01) 138 $18 OVPId'VDI RAN ZE'llLA'1128 ZL'VDDVIVdTVP OONVNH33 NVS 991 d D LIJ M O O U N'N‘N'H'El'bdd 082l9'DSN 1‘21 ODIXBW VdVWVP 202008 2 d3N DVTINVS 92'111'1128 Id'dOlOHd VOOIHVD O [\ (z) AlIlISIlSBSIG 60 Digestibility of selected P. vulgaris varieties (l) Figure 35. 139 $18 26'llA'1128 RAH VdVNVP 08819'DSN Zl'VDOVlVdTVP OGNVNHBJ NVS C) LlJ >4 0 O U ['21 ODIXHW 202008 OVPId'VOI 991 d W'N'N'N'El'fidd 2 d3N DVTINVS VDOIHVD Id'dOlOUd VZ’lll'llZB O N to (z) 1111181183810 Digestibility of selected P. vulgaris varieties (2) Figure 36. 140 Table 11. Analyses of variance of hemagglutinating activity and digestibilities of P. vulgaris varieties ANALYSIS OF VARIANCE OF HEMAGGLUTINATING ACTIVITY OF BREEDING LINES degrees SOURCE of freedom mean squares *9: BREEDING LINE 17 1493.93 ERROR 18 33.14 TOTAL 35 742.66 ** Significant at 1 % level ANALYSIS OF VARIANCE 0F DIGESTIBILITY 0F RAN BEANS IN BREEDING LINES degrees SOURCE. of freedom mean squares ** BREEDING LINE 15 13.24 ERROR 16 0.52 TOTAL 31 6.67 ** Significant at 1 % level ANALYSIS OF VARIANCE 0F DIGESTIBILITY OF COOKED BEANS IN BREEDING LINES degrees SOURCE of freedom A mean squares ** BREEDING LINE 15 11.04 ERROR 16 0.92 TOTAL 31 . 5.82 ** Significant at 1 % level 141 beans 'Hi general are higher than those (n: colored beans. The lower digestibility of colored beans than white beans has been reported (Aw and Swanson, 1985; Wolzak et al., 1981a and b; Bressani and Elias, 1977) and is presumed to be due to the higher concentration of tannins in the seed coat. The tannins presumably bind to soluble proteins and reduce their digestion. These results support that hypothesis. No correlation between digestibility of either raw or cooked beans with protein content or hemagglutinating activity was observed in this sampling of P. vulgaris varieties. However, if lines were to be chosen on the basis of digestibilty for further breeding studies, Sanilac, Nep- 2 and 8217-111-24 would be most promising among white navy beans, and Protop-pi and Carioca would be most promising among pintos. ElectrophOretic Evaantion All lines were subjected to DISC-PAGE and SDS-PAGE and the results are shown in Figures 37 - 40. Based on these results, there doesn't seem to be any correlation with the major bands shown on the gels with either digestibility or hemagglutinating activity. Conclusions The dry beans produced in this study have acceptable food quality characteristics. The color, protein content and digestibilities of the varieties are appr0priate for further consideration in breeding programs. A number of the varieties tested had low hemagglutinating activities and based on this characteristic would be most promising. The low lectin lines were Nep 2, P766, Carioca, Ica-pijao, Jalpatagua, Jamapa, Black Turtle Soup, Mexico 12-1- and San Fernando. 142 mm— mm om vw mm on mm v0 mm mm mm em om me on on an - Pam AFV mmwumwcm> mwgmmA2> .a $0 mwmzpmcm mwwuach em Fm, cs cm um mm pm mm mm «a an en su up Em om— em mm mm mm on mm no em me mm mm Em map em am we em mm we om pcm mnp V “up vm hm 8' mm mm EmlllL, .nm mcammm Em 1|: O 01 6'01 9"" Q 40 uc em on zJ mpcumpa mpumwcm> . ANV m o E”: mpmc n .3522: 3.5 22:8 Aoop x . an. a: mop- mm _m nap mm a mm— _. amp om mm 8. 3 III a I I on I a. m. 8 I! ma 2 2 2 a II I- : .l. . ....|____ 8 II III. 2 II I m cm e as WM me III we 3 3 cm .2 2 S ow I I . I um I vs I o I om : mm mm mm lullu mm mm co co mm 2 S 3 a 3 2 cm cm I m I am I . WW I me Ill 2 . 2.. . m m 3 a. I z "_ l a. I 21.11 N. (1'- : I ll. 8 a I 8 I I 1 I I 8 .1 I I a a . mm mm me m¢ cc 1 mm Nm mu m... I 2 S I a I , z I I mm mm mm m I I I z 8 8 8 I 2 I an on mm a n on Em 2 Em 2 2 2 I - Emll. EC 1 Em Em Em I Em .IL 144 A—V mmwummLm> wwmeF3> .m mo mwmhwmcm ww_pmfimm 1 EV om c~ ON Em mm m” =;u|llll .mm mgamwm “Np pm mm —m Nu pm me Em_ pm we ow ms mm —s «m w— Em mo— llll zq mrmmm~=> .m mo mpm»_mcm mumm-mom .OE mm=m_m hoop x aum_vnoe m>wpmpmm u say mm— mm— em— 0223: N... I I 3 I 3 I 3 I w B I. 3 l1! 3 I m S g I 3 I Nu : . _k we I we mm I an em om Fm mm mm J cm ON 2 S Em Em Em Emrlll 146 The digestibilties of the varieties varied, with some beans having relatively high values. The most promising candidates for breeding for the digestibility trait would be Carioca, Protop-pi, 8217-111—24, Sanilac and Nep 2. Electrophoretic analyses of extracts of the beans involved in this study were not conclusive. The patterns produced by DISC-PAGE and SDS- PAGE did not correlate with digestibilty, hemagglutinating activity or protein content. Based on these results, the use of electrophoretic techniques would not be an adequate screening procedure for evaluating newly produced bean varieties for hemagglutinating activity or digestibility. SUMMARY AND CONCLUSIONS The electronic cell counter was a sensitive tool for measuring the hemagglutinating activity of kidney beans. The PHA in cooked kidney beans, usually thought to be inactivated by cooking was found in beans exposed to low temperature cooking at times greater than 12 hours. Further research is needed to evaluate any nutritional or physiological impairment resulting from chronic consumption of bean diets containing low levels of active PHA. Purified PHA was stable to freezing and retained full activity for seven months when stored at -3°C. The thermal inactivation of PHA was rapid at temperatures of 80°C or greater and can be described at 70°C by the regression equation : % PHA Activity = 102.18 - 9.87 (hours at 70°C) Purified PHA was also strongly affected by chemical treatment. The most effective chemical agents for reducing the hemagglutinating activity of PHA-P were urea and a pH 12.0 sodium hydroxide solution, corresponding to 39 and 65 % reduction, respectively after three hours of treatment. Purified PHA was inactivated by proteolytic enzymes. Treatment with carbohydrate hydrolyzing enzymes was not effective for inactivating the hemagglutinating activity of PHA-P. 0f the the carbohydrate hydrolyzing enzymes tested, CK.-mannosidase caused only a slight reduction in the hemagglutinating activity. This indicates that the oligosaccharide chain was not the major determinant of hemagglutinating 147 148 activity in PHA. Decreased hemagglutinating activity of purified PHA may correspond to decreased enteral toxicity of’ this lectin. If hemagglutinating activity is correlated to toxicity, high pH treatment of beans may be a possible approach for investigating new processing treatments and procedures. There was considerable variation in the effect of extrusion on dry beans due to changes in the processing parameters. The hemagglutinating activity of bean flours decreased with increased product temperature and internal barrel pressure enhanced this effect. 'The Inost effective extrusion parameters for bean flours were relatively high pressure (900 - 1200 psi) and temperatures of 270 - 300°C. Extrusion of whole beans under the conditions employed in this study (150 - 185°C, 700 - 1200 psi) did not result in efficient reduction in hemagglutinating activity. There was no apparent correlation of hemagglutinating activity with process conditions for whole bean extrusion. Soaking and cooking kidney beans in alkaline media reduces the hemagglutinating activity much more effectively than the same thermal treatment at neutral pH. In addition to the increased inactivation of PHA activity, high pH treatment results in significantly softer beans after cooking. The regression equation describing the texture of beans cooked at pH7.0 and 76°C is: Y = 1140 - 55.2 (hours at 76°C) The regression equation for beans cooked at pH 12.0 and 76°C is: v = 883 - 62.9 (hours at 76°C) Beans cooked at pH 12.0 required only 60 % of the time needed for those cooked at pH 7.0 to reach the same textural end point. 149 The dry beans investigated in Study Four had acceptable food quality characteristics. The color, protein content and digestibilities of the varieties were appropriate for further consideration in breeding programs. The most promising low lectin lines were Nep 2, P766, Carioca, Ica-pijao, Jalpatagua, Jamapa, Black Turtle Soup, Mexico 12—1- and San Fernando. ‘The most promising candidates for breeding for the digestibility trait would be Carioca, Protop-pi, 8217-111-24, Sanilac and Nep 2. Electrophoretic analyses of extracts of the beans involved in this study did not correlate with digestibilty, hemagglutinating activity or protein content. The use of electrophoretic techniques would not be an adequate screening procedure for evaluating newly produced bean varieties for hemagglutinating activity, digestibility or protein content. RECOMMENDATIONS FOR FURTHER RESEARCH There are a number of further studies that could be pursued that relate to bean proteins and the improvement in nutritional quality of legumes: 1). It is suggested that a feeding trial be undertaken to determine the effect of denatured PHA on laboratory animals. 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