IHE: .11“, \. , . L» ...: h... r. 3 .3 .2 ‘. 5. ix 0. p: r: A: T, R... : I .mu .2 3 a: ,. Q E g . ..,.. v... T: Q. 3!. n . «C I» .rU r... ...u kn 1a. a; Q. i‘. w. . V. . v” u. ‘. 2» .v I» rflm A. v «J ~ ~ r” T: «T .. . a mi tn“ .. . . y L; h. : ‘ D. 2. F: rm :1 ”H .L. ‘T. .2. E n» 1.: +v n1 ABSTRACT FACTORS IN SOYBEANS INFLUENCING GROWTH, PANCREATIC FUNCTION AND DIGESTION By David J. Schingoethe The effect of age on pancreas size and proteolytic enzyme content in bull calves from birth to one year of age was studied under normal dietary conditions. Pancreas weight, trypsin and chymotrypsin activities increased directly with body weight over the age span studied. The new born calf was the only age group which indicated any marked differences from the mean of all age groups. Pancreas weight and trypsin content were slightly less at this age while chymotrypsin content per mg of pancreas tissue was higher than at all other ages. Weanling rats were used to compare milk protein with soybean protein sources either containing or lacking soy- bean trypsin inhibitor (SBTI) to study factors influencing growth, pancreatic function and intestinal protein diges- tion, Diets containing SBTI caused pancreatic enlargement with corresponding increases in trypsin and chymotrypsin activities. Enzyme activities per mg of pancreas tissue remained constant over all dietary treatments. These same SBTI-containing diets caused increased chymotrypsin [H E David J. Schingoethe activity and stability, decreased trypsin stability, but did not change free trypsin activity in the intestinal con- tents. Intestinal protein digestion, as measured by an in vitro system, was not impaired in the intestinal contents of SBTI-fed rats. Growth rates were depressed by only two of the four SBTI diets indicating that the growth depression exerted by raw or minimally processed soybean products is not caused by SBTI and apparently occurs by some mechanism other than by interference with protein digestion. Raw (unheated) soybean meal was subjected to numerous physical and chemical treatments in efforts to isolate a growth inhibitor fraction which was free of SBTI activity. Each treatment fraction was added to the diet of growing mice and growth inhibition determined by comparing their growth rates to growth rates achieved on an autoclaved soy- bean meal diet. A small molecular weight growth inhibitor was separated from SBTI by ion exclusion chromatography on a Sephadex G-50 column and partially characterized. This growth inhibitor decreased weight gains and feed efficiencies of mice without causing pancreatic enlargement. Evidence of the small size of this growth inhibitor was retardation on a Sephadex G—25 column, removal by dialysis and lack of de- tection by polyacrylamide—gel electrophoresis. Movement to- ward the cathode under high voltage electrOphoresis at pH 3.5 and apparent adsorption on DEAE—cellulose indicated a positively charged compound at that pH. In a; David J. Schingoethe Other factors in soybeans may also inhibit growth. The water-insoluble residue accounted for about 40% of the growth inhibitor activity of raw soybean meal. This growth inhibition could not be removed or destroyed by gastro- intestinal enzyme digestions or by several other solvents tested. Fractions containing SBTI generally caused pancreatic enlargement and usually caused some growth inhibition. [Has FACTORS IN SOYBEANS INFLUENCING GROWTH, PANCREATIC FUNCTION AND.DIGESTION By David J.LSchingoethe A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy and Institute of Nutrition 1969 I?! 5.2 -: :2":/ <2 7 ACKNOWLEDGMENTS. The author wishes to express sincere appreciation to Dr. J. W. Thomas for guidance in conducting this research, and for the constructive criticism in the pre— paration of this thesis. Suggestions and advice from Dr. S. D. Aust regarding experimental techniques are gratefully acknowledged. Appreciation is also extended to Dr. L. J. Boyd, Dr. H. Lillevik and Dr. D. Reinke for serving on his guidance committee and reading this manu- script. Pancreases from cattle were obtained through the courtesy of Keith McMillan. The large soxhlet apparatus used for hexane extracting soybeans was provided by Dr. H. M. Sell. Polyacrylamide-gel electrOphoresis was carried out by Dr. J. E. Wilson who also provided the high voltage electrophorator for the author's use. Appreciation is extended to Dr. C. A. Lassiter for providing a financial assistantship. The author is especially grateful to his wife for the moral support, technical assistance and pre- liminary typing of this manuscript. ii IRE AUTOBIOGRAPHY I was born February 15, l9A2 in Aurora, Illinois, and was raised with two younger brothers on my parents dairy and grain farm in northeastern Illinois. I graduated from Kaneland High School, Maple Park, Illinois in June of 1960. I received the Bachelor of Science degree in Agricultural Science from the University of Illinois in June, 1964, and the Master of Science degree from the Dairy Science Department at the same institution in October, 1965. Research for the M.S. degree was conducted in dairy bio- chemistry under the guidance of Dr. B. L. Larson. I enrolled as a graduate student in the Department of Dairy and Institute of Nutrition at Michigan State University in September, 1965. I am a member of the American Dairy Science Associa— tion, the American Society of Animal Science, Sigma Xi, Alpha Zeta and Gamma Sigma Delta. I am married to the former Darlene Wennlund, a University of Illinois graduate. iii [HE TABLE OF CONTENTS Page LIST OF TABLES o o o o o o o o o o o o 0 Vi LIST OF FIGURES . . . . . . . . . . . . . ix APPENDIX TABLES . . . . . . . . . . . . . x Chapter I. INTRODUCTION . . . . . . . . . . . 1 II 0 LITERATURE REVIEW 0 O O O O O O O O O 3 A. Pancreas size and enzyme activity as affected by species, age and diet . . 3 Prenatal Development . . . . . . . 5 Postnatal Development . . . . . . . 5 Dietary Adaptation . . . . . . . . 8 B. Soyflour in calf milk replacers . . . 9 C. Raw Soybeans and Growth Inhibition . . . ll Trypsin and Chymotrypsin Inhibitors . ll Hemagglutinin . . . . . . . . . . l7 Saponins . . . . . . . . . 20 Pancreatic Enlargement . . . . . . . 21 Loss of Endogenous Nitrogen . . . . . 2“ Metabolic Effects . . . . 26 Impaired Intestinal Digestion and Absorp- tion 0 o o o o o o o o o o o 27 III. MATERIALS AND METHODS . . . . . . . . 3O Experiment I. Effect of Age on Bovine Pancreas Size and Enzyme Activity . . . 3O Tissue Preparation and Homogenization . . 30 Enzyme Assays . . . . . . . . . . 30 Statistical Analyses . . . . . . . 31 Experiment II. Pancreatic Proteolytic Enzymes and Growth of Rats Fed Soybean and Milk Protein Diets . . . . . . . . . . 31 iv Chapter Experiment III. Isolation and Characteriza- tion of Growth Inhibitors from Soybeans . Preparation of Soybean Meal Fractions . . Growth Assay Procedure . . . . . . . Enzyme Inhibitor Assays . . . . . . IV. RESULTS AND DISCUSSION . . . . . . . Experiment I. Effect of Age on Bovine Pancreas Size and Enzyme Activity . . . Experiment II. Pancreatic Proteolytic Enzymes and Growth of Rats Fed Soybean and Milk Protein Diets . . . . . . . . . . Experiment III. Isolation and Characterization of Growth Inhibitors from Soybeans . . . A. Determining the Presence of Digestive Enzyme Inhibitors . . . . . . . General Fractionation Procedure . . Water-Insoluble Residue Studies . . Gastrointestinal Enzyme Digestions . Reasons for Reduced Consumption of RSBM . . . . . . . . Fractionation of Acetone Precipitated Whey Solution . . . . . . Growth Inhibition Due to Crystalline Soybean Trypsin Inhibitor . . . . Acetic Acid Extraction of RSBM . . . Heat Treatments on Different Fractions Effect of Dialysis on Growth Inhibition . . . Effect of Methionine Supplementation . Separation on Sephadex G- 50 . . . Further Characterization of G-50, Fraction III . . . . . . . . Integrated Discussion of Soybean Growth Inhibitor Isolation and Characterization Experiments . . \ CD ’11 WUOIIJ 2: tatim C4HtE V. SUMMARY . . . . . . . . . . . . . REFERENCES I O O C O O 0 O C C O C O O APPENDICES O 0 O O O O O O O 0 O O I O Page 35 35 38 A0 A0 “(5 10“ 113 115 130 1H ht .‘lv Table /LL -5. r 6. 10. 11. l2. l3. 1“. LIST OF TABLES Relative proportion of components of bovine pancreatic Juice (72) . . . . . . . . Pancreatic size in several vertebrate species, calculated from published data . . . . . Physical and chemical prOperties of soybean trypsin inhibitors . . . . . . . . Dietary treatments . . . . . . . . . Composition of diets 2, 3, A and 7 . . Composition of diets . . . . . . . . Pancreas size, trypsin and chymotrypsin activities in Holstein bull calves . . Relationships of pancreas weight, trypsin and chymotrypsin to body weight . . . . . Body weight changes, feed intakes, size and enzyme content of pancreases of rats fed milk or soybean based diet . . . . . Stomach pH and intestinal content trypsin and chymotrypsin activities of rats fed milk or soybean based diets . . . . . . . Protein content and in vitro digestion, and trypsin and chymotrypsin in vitro stabilities Trypsin, chymotrypsin, a-amylase and lipase inhibitor activities in raw soybean meal Response of mice when fed HRSBM, RSBM, or several fractions of RSBM . . . . . Response of mice when fed HRSBM, RSBM, or several fractions of RSBM in Experiment 2 vi Page 12 33 3A 37 Al “3 M6 A8 50 53 57 59 Table Page 15. Solubility of the water-insoluble residue in various solvents . . . . . . . . . . 61 16. Response of mice when fed extraction or digestion preparations of the water-insoluble residue from raw soybean meal . . . . . . 63 17. Efficiency of growth inhibitor extraction from RSBM by several solvents . . . . . . 65 18. Response of mice when fed gastrointestinal enzyme digested fractions of raw soybean meal . . . . . . . . . . . . . . 68 19. Effects of several treatments on feed con— sumption o o o o o o o o o o c o 0 7O 20. Response of mice when fed bentonite-celite, (NH4)2SOq, or heat treated preparations of acetone precipitated whey solution . . . . 72 21. Response of mice when fed bentonite-celite and (NHu)2SOu treated preparations of acetone precipitated whey solution . . . . . . . 75 22. Response of mice to feeding three different levels of crystallized soybean trypsin in- hibitor 0 O O O O O O O O O O o O 77 23. Growth responses to acetic acid treatments of RSBM O O O O I O O O O O O a a 0 8O 24. Growth responses of mice when fed various heat treated preparations of raw soybean meal fractions . . . . . . . . . . . 83 25. Effect of dialysis on SBTI and growth inhibitor activities . . . . . . . . . 85 26. Effect of methionine supplementation on growth rates of mice . . . . . . . . . 87 27. Growth inhibitor assay of fractions of pH 4.4 supernatant separated on a Sephadex G-50 column . . . . . . . . . . . . . 92 28. Growth inhibitor assay of the mild acid hydrolyzed G-50 fraction III and of some other pH 4.4 supernatant fractions . . . . lOl vii Table 29. 30. Page Purification of fraction III growth inhibitor . . . . . . . . . . . 105 Overall mean and range in means of weight gains and growth inhibition achieved on several diets . . . . . . . . . . . 107 viii Jh'e_. «3;; Figure 1. LIST OF FIGURES Page Regression of pancreas weight, trypsin (T) and chymotrypsin (ChT) on body weight . . . 42 Fractionation procedure of raw soybean meal . 55 Chromatography of the pH 4.4 supernatant on a Sephadex G-50 column . . . . . . . . 9O Chromatography of the G-50 fraction III on a Sephadex G-25 column . . . . . . . . 95 High voltage electrOphoresis, pH 3.5, of the G—5O fraction III and the first four peaks separated on a G-25 column . . . . . . 98 High voltage electrophoresis, pH 3. 5, of fraction III and mild acid hydrolyzed fraction III . . . . . . . . . . 103 ix Table II. III. LIST OF APPENDIX TABLES Page Body weight and pancreas size, dry matter, and enzyme content of bull calves from birth to 12 months of age.. . . . . . . . . . . . 131 Amount of test fractions present in 100.3 of 1:881: diet 0 I » O I I O- O O O O I C O 132 Individual body weight gains, pancreas size and feed consumption of rats and mice fed 3 soybean diets for 7 days . . . . . . . . . . . 134 CHAPTER I INTRODUCTION The relatively low cost of many plant source ingre- dients has stimulated interest and research concerning their possible use in milk substitutes for calves as well as humans. Soybeans are a principle source of such ingredients. However, soybeans contain several substances which impair growth, cause pancreatic enlargement, inhibit intestinal protein digestion by pancreatic enzymes, and may inter— fere with intestinal absorption as well as metabolic func- tions of several body organs. Fortunately, these undesir- able factors are heat labile and thus can be readily destroyed or inactivated by heat applied during extraction or processing. However, such heating reduces the solu- bility of the product, which makes the product unacceptable for use in milk substitutes. This is because materials must be soluble or easily suspended to be acceptable in a liquid milk substitute preparation. Thus, the undesirable factors in soybeans must be more adequately characterized so that a more desirable method of destroying or removing them can be developed. The purpose of the research reported herein was to characterize factors in soybeans which depressed growth, 1 and determine if these same factors were responsible for the changes in pancreatic output and function. In addition, the effect of age on pancreas size and proteolytic enzyme content in bull calves from birth to one year of age was studied under normal dietary conditions. I?! E: CHAPTER II LITERATURE REVIEW A. Pancreas Size and Enzyme Activity as Affected by Species, Age, and Diet. The pancreas, in addition to being an endocrine organ, is a major source of enzymes for digesting dietary nutrients. The pancreatic excretory enzymes include the proteases trypsin, chromatrypsin, carboxypeptidases A and B, and elastase, the nucleases-ribonuclease and deoxyribo- nuclease, the carbohydrase amylase and the fat digesting lipase. The relative proportions of these enzymes in pan- creatic Juice are shown in Table l. The proteases are synthesized, stored and secreted in the inactive zymogen forms of trypsinogen, chymotrypsinogen and procarboxypepti- dase A and B. Activation is initiated in the small intes- tine by enterokinase. Pancreatectomy results in decreased amino acid and fat absorption (27). This leads to a sub- sequent loss of body weight and blood lipid concentration together with the development of a fatty liver with impaired function. The relative importance of the pancreas may be dif- ferent in the mature ruminant than in the immature ruminant or monogastric. This is because of the more constant TABLE l.-—Relative proportion of components of bovine pancreatic Juice (72). Per cent Enzyme or Proenzyme of total Protein Proteolytic Trypsinogen 14 a Chymotrypsinogen 16 B Chymotrypsinogen l6 Procarboxypeptidase A 19 Procarboxypeptidase B 7 Nucleolytic Ribonuclease 2.4 Deoxyribonuclease 1.4 Amylase <2 Lipase very low Unidentified >10 intestinal flow and the pregastric digestion in the rumen. Another consideration is the relationship between pancreas size and body weight. Brody (23) reported that the size of several internal body organs increased with the 0.70 to 0.80 power of body weight, but no data were presented for the pancreas. Prenatal Development Proteolytic activity or zymogen granules were detected in the fetal pancreas after 13 days of gestation in the mouse (104) and chick (99), 50 to 53 days in the pig (125), and 5 to 6 months of pregnancy in the meconium of humans (87). The cytoplasm of mice fetal pancreases was com- pletely filled with zymogen granules by 18 days (104), while maximum amount of proteolytic enzymes occurred at 22 days of development (1 day-old chick) in the chick pancreas (99), and at 60 to 62 days in the pig embryo (125). Postnatal Development Levels of pancreatic protease, lipase, and amylase are low at birth in most species (23, 32, 64, 65, 87, 99, 104, 112, 121, 125, 151). Pancreatic and intestinal enzyme activities, of calves remained fairly constant up to 6 weeks of age (32, 49), although a threefold increase in pancreatic amylase and protease from 7 to 44 days of age was noted by one group of investigators (64). Amylase concentration in pig pancreases increases twentyfold from In is l to 38 days of age (65). Rat pancreatic amylase developed most rapidly during the period from 15 to 20 days of age (112). Trypsin and chymotrypsin content of human pancreases did not increase until about one month post partum (87), resulting in a relatively small increase in proteolytic activity of duodenal contents during this interval (75). Concentration of proteolytic enzymes in the baby pig pan- creas did not increase with age (86), suggesting that pancreas growth may control total enzyme secretion. A limited amount of data on two calves at each of several ages from 1 to 44 days of age indicated that pancreas size ranged from 0.06 to 0.10% of body weight and appeared to increase directly with body weight (64). Table 2 summarizes pancreas size data from several species. Pancreatic Juice secretion in calves increased from 125 m1/24 hr at 3 or 7 days of age to 1300 ml/24 hr at 6 months of age (101). Total trypsin output increased slightly from 3 or 7 days of age to 21 days of age (50). The ratios of chymotrypsin-to-trypsin activities in pancreatic tissue and Juice, and intestinal contents showed marked species difference (46, p. 26). Some of the differences are now known to be due to enzyme substrates used in the assay procedure, other assay conditions (48) and dietary factors (135). Ratios varied from as low as IHI: :- TABLE 2.-—Pancreatic size in several vertebrate species, calculated from published data. Pancreas Size Species Sex Body wt. (kg) (g/kg Body wt.) Reference Bat male 0.00543 2.76 (148) compiled female 0.00541 9.24 by (57) Shrew male 0.00754 13.26 " female 0.00757 14.72 " Lizard male 0.0126 1.66 " female 0.0128 4.06 " Mouse male 0.0141 6.30 " female 0.0159 8.57 " Salamander male 0.0257 0.82 " female 0.0242 1.98 " Frog male 0.0638 1.44 " female 0.0555 1.28 " Rat male 0.03-0.10 7.19 " (30-50 days) female 0.03-0.10 7.60 " Chick 0.205 4.39 (5) (12-14 days) Hedgehog male 0.258 17.55 (148) compiled female 0.389 8.48 by (57) Cat male 2.83 2.08 " female 2.00 2.65 " Dog male 4.86 3.30 " ' female 4.99 4.02 " Both sexes 13.6 1.85 (28) Pig 20: 2.10 (62) 100 1.55 Humans male 56—95 1.16 i .22 (129) female 48-95 1.16 i .32 Calf male 39-62 0.63 (46) aEstimated body weight. b Mean : standard deviation. In as I. 0.14-0.17 in rabbit pancreas Juice (122), using ATEEa and TAMEb for chymotrypsin and trypsin substrates, respectively, to as high as 19.2 in bovine pancreas Juice, using ATEE and BAEEC as substrates (72). Most studies place the chymotrypsin-to-trypsin ratios in the range of 1 to 3; however, ratios of less than one were consistently observed in bovine pancreases (49) and pancreatic Juice (50, 130), while ratios of greater than one were observed in intestinal contents of rats and chicks (47). Dietary Adaptation The pancreas adapts to specific dietary nutrients by altering the proportions of several pancreatic enzymes (31). High starch diets elevated amylase and reduced protease, while high protein diets induced the opposite changes in rat pancreatic tissue or its exocrine secre— tions (6, 7, 52, 63, 100, 118). Amylase represented 25% of total pancreatic proteins on a high starch diet but only 8% on a high protein diet (100). Pancreatic lipase showed no adaptation to dietary fat, but increased on high protein diets (52). Alterations of pancreatic exo- crine enzymes were detected one day after a dietary change, were rapid from 3 to 5 days, and were complete by 7 (137) or 8 days (6, 7). The total pancreatic protein output was aATEE, acetyl tryosine ethyl ester. bTAME, toluene arginine methyl ester. CBAEE, benzoyl arginine ethyl ester. not appreciably altered by dietary changes (7). Chromato- graphy of pancreatic Juice on DEAE cellulose proved that diet modified the amount and not the activity of these enzymes (7). Several studies indicated that dietary alterations modify Chymotrypsinogen systhesis to a greater extent than trypsinogen synthesis (6, 7, 119, 135). Relatively more of the pancreas amino acid supply was diverted to Chymotrypsinogen and less to amylase when whole—egg protein was fed (135). The reverse occurred when casein was given. This may be due to the lower methionine con— tent of casein since the pancreases of rats fed a methionine- deficient diet contained normal amounts of amylase, reduced amounts of chymotrypsin and no trypsin (145). Snook (135, 136) observed ratios of chymotrypsin—to- trypsin activities in rat pancreases of 1.64 on a protein- free diet, 1.93 to 2.04 on a casein-protein diet and 2.74 to 2.86 on a whole-egg protein diet. 'When rats were chan changed from a high—starch to a high—protein diet, incor- poration of Clu-valine into Chymotrypsinogen rose from 14 88 to 266 cpm whereas C activity in trypsinogen increased only from 87 to 116 cpm (119). B. Soyflour in Calf Milk Replacers The increased use of plant proteins in calf milk replacers would appear advantageous for economic reasons. Although some (139, 140, 141) obtained acceptable growth 10 rates in calves fed a mixture of plant and milk protein, growth from plant protein alone usually was unsatisfactory (40, 108, 109, 140). Forty per cent raw soybean meal (RSBM) in calf milk replacer resulted in 100% mortality by 27 to 58 days of age (151). Adding proteolytic enzymes to the replacer did not improve the utilization of soy protein (83, 108). Predigesting fully cooked soyflour with various proteolytic enzyme preparations did not improve its nutri- tive value in calf milk replacers, but acid digestion at pH 4.0 for five hours at 37°C did bring its nutritive value up to almost that of the positive control milk pro- tein group (26). Gorrill and Thomas (49) and Gorrill et a1. (50) studied the effects of soybean and milk protein diets ,On aansrieas troteolitigiaaaymeicQnt-e.n.t.-and lee—22.612.11.92. and on intestinal proteolytic activity in efforts to determine the mechanism whereby soy protein causes poor growth in calves. Pancreatic trypsin and chymotrypsin secretion, as well as activities in the pancreas and intestinal contents were suppressed in calves fed a soyflour milk replacer containing relatively high levels of soybean trypsin inhibitor (SBTI). This response did not occur in calves fed an all-milk replacer nor in those fed a soy— protein-concentrate replacer containing very low levels of SBTI. Since calves lost weight on soyflour diets con— taining significant levels of SBTI (26, 49) they speculated IRE..- 11 that SBTI may be the cause of poor growth rates presumably via its adverse affect on pancreatic response. However, since these calves also had considerable diarrhea (49), one cannot unequivocally say that SBTI is the causative factor of the observed pancreatic and growth responses. C. Raw Soybeans and Growth Inhibition Osborne and Mendel (110) in 1917 reported the improved growth-promoting effect of cooked versus raw soybeans. Despite the numerous studies and improved technology of the past 50 years, the explanation for this observation is still unclear. Several factors have been implicated as causing the growth inhibition associated with raw soybeans and these will be discussed in the following pages. Trypsin and Chymotrypsin Inhibitors Table 3 summarizes some of the physical and chemical pr0perties of several trypsin inhibitors isolated from raw soybeans. Kunitz (77) first crystallized a soybean trypsin inhibitor (CSBTI) in 1946. This inhibitor has been most widely studied and is the one generally available commercially. CSBTI is the only maJor soybean trypsin inhibitor to contain significant amounts of tryptophan (15). F and F 1 3 'crude soybean trypsin inhibitor, also contain tryptophan inhibitors, which were isolated from a commercial but F3 is devoid of tryosine (39). Amino acid analyses and F contain also indicate that inhibitors AA, 1.9S, Fl 3 JHh.‘ p" l2 Aamv Lama Asmav Amav Aaaav Aaaav ASHE .aaav Lama macaamcam III III 0.H III 0.H 0.0 00.H 0.H Apoufibficcfi mE\UmoHpHccfi :Hmdmpo msv zufl>fipom oflmfiomam. III III III III III III III .soa pfiom OCHEm HmcfisbopIo III III .am< III III III .dm< .qm< ofiow ocHEm HmCHEpouIz III III mm.ma oa.ma III III 00.:H am.ma Lav 0:06:00 camoaaaz so H :m0.0 0H~.0 33.0 03.0 III III m20.0 000.0 3E 00m um &H.m .ocofioflmmooo coapocflpxm III III 00.0 III III III 0m~.0 035.0 Aw\HEV 0E3Ho> oflmfioQO Hwfippmm III III 0m.a 0m.m No.2 50.: 00.H 0m.m 30mm .pcmpmcoo coapm»cosfinmm III III m3.ml III m.m| m.3l 3.Nl O.®I AMIUQW HIUHO> JEOV mOHX uaaanoe ofipomocqocoooam ooa.mm oom.ma ooz.aa ooo.am III III oom.:a oos.mm pcwamz amaaaaaoz ma Ha ma.a aa mm Hm Ha Hemmo spamaoaa .mLOpHancfi cfiwahhp cmmnmom no mmauhmaopm HMOfisono new awoamanmII.m mamas [fiat vi ., .._, l3 unusually high levels of half cystine (ll, 39, 70, 153, 154). They contain 34, 26, 14, and 12 half cystine residues per molecule, respectively, while CSBTI contains only four. Rackis et_al. (114, 116) also isolated a trypsin inhibitor designated SBTIA which is identical to 2 Kunitz's (77) CSBTI. Most trypsin inhibitors also inhibit chymotrypsin, but to varying degrees (15, 39, 44, 114, 116, 154). Inhibitors AA (15) and 1.98 (154) are fairly potent chymotrypsin inhibitors, while other trypsin inhibitors are less inhibitory towards chymotrypsin. Gertler §t_al, (44) determined that AA contained 8.0 trypsin-inhibitor units (TIU) and 10.0 chymotrypsin-inhibitor units (ChIU) per milligram of inhibitor as opposed to 6.0 T10 and 0.75 ChIU for CSBTI. The order of trypsin inhibitor potency of soy fractions characterized to date is: CSBTI = A1 = AA = 1.98 > Fl > F3 (39). The approximate order for chymo- trypsin is: 1.98 2 AA 3 Al > CSBTI > Fl >> F3 (39). Most presently known soybean trypsin inhibitors stoichiometrically inhibit trypsin by forming a virtually inseparable complex (51, 82). Sealock and Laskowski, Jr. (132) showed that this was due to a close cystine disul- fide bridge across the active arginine residue which did not allow the trypsin inhibitor molecule to open and release the trypsin once the arginyl residue had been hydrolyzed. Inhibitor AA is noncompetitive (10) while Ina: 14 CSBTI is competitive (51) to the proteolytic and esterolytic activities of trypsin and chymotrypsin on casein, BAEE, and ATEE, respectively. The finding of a trypsin inhibitor in raw soybeans by Ham and Sandstedt (54) in 1944 offered a probable explana- tion to the mechanism whereby heat treatment improved the soybean's nutritional value. Trypsin inhibitor activity is destroyed by autoclaving (54, 150). Further evidence to support this explanation came from observations that the addition of crude trypsin inhibitor to diets containing heated raw soybean mean (HRSBM) reduced the growth rate of chicks (55) and rats (74). Because of these early results, trypsin inhibitors have since received the bulk of atten— tion in attempts to further elucidate the cause of growth depression by raw soybeans. Adding CSBTI to chick (42, 44) and rat (44, 53) diets depressed growth but not to the extent of the depression caused by RSBM diets. Garlich and Neshein (42) batch separated the soybean whey fraction obtained by the pro- cedures of Rackis gtual. (115) into two fractions, one high .inmtrypsin-inhibiting activity and the other high in hemagglutinating activity. Neither fraction when fed alone affected growth rate to thesame extent as a combination of thetho fractions. The whey fraction contains most of the soybean trypsin inhibitors and hemagglutinins, along with other unidentified components (35, 116). Geratz (43) Int: :1 l5 caused growth depression in rats by adding p-aminobenzami- dine, a potent trypsin inhibitor,to their drinking water, but most of the growth depression was attributed to decreased feed intake with only a moderate decrease in efficiency of gain being observed. (”Some suggested that the growth depression associated with trypsin inhibitor could be overcome by adding trypsin to the ration. However, adding crude or crystalline trypsin to a raw soybean meal diet for chicks (22) and rats (20) failed to remove its growth-depressing properties. If the growth depression observed when animals are fed unheated soybean meal is due to interference with intes— tinal tract enzymatic digestion, feeding amino acids instead of intact protein should alleviate the problem. Desikachar and De (30) obtained poor biological values when they fed a papain digest of RSBM to which had been added an aqueous extract of RSBM containing the trypsin inhibitor. Similar results were obtained when crude trypsin inhibitor was added to a casein digest diet for mice (149), or to amino acid diets for chicks (60) and rats (73). Thus, the growth depression mechanism appears to be something other than an inhibition of intestinal enzymatic activity. Since only crude trypsin inhibitor preparations were used in all of these studies the possibility still remains that the causative factor was not trypsin inhibitor. IHL—ie 16 The compensatory capacity of the animal also indi- cates trypsin inhibition is probably not the growth depressant factor in raw soybeans. Even if the soybean trypsin inhibitor tied up more than half of the trypsin in the intestinal tract, the pancreas should be able to compensate for it by secreting extra enzymes. Scow (131) showed no reduction in nitrogen digestion and absorption as measured by fecal excretion in rats having 95% of their pancreatic tissue removed. Reduction in protein digestion was observed only when 99.5% of the pancreatic tissue was removed. Khayambashi and Lyman (73) noted more intestinal protease and more TCA-insoluble nitrogen in the intestinal contents of rats fed SBTI. However, somewhat contradictory to the above is the fact that intestinal proteolysis was almost completely inhibited in chicks up to 3 weeks old fed a raw soybean diet, but increased from the fourth week, reaching normal at six weeks of age (2). Studies with germinated soybeans give additional evidence that trypsin inhibitor is not the growth depres- sant factor. Rats fed germinated seeds grew much better than those fed raw soybeans and had protein efficiency ratios almost equal to those fed autoclaved soybean meal (37). This occurred even though the trypsin inhibitor concentrations remained the same as in the raw soybeans (30)- 17 Because soybeans are more resistant to insect attack than many of the seed grains, insects have been used in some soybean proteolytic inhibitor studies (10, 95). The soybean protein fraction designated Cl inhibits growth and in vitro proteolytic activity in Tribolium larvae, inhibits trypsin and chymotrypsin, and possesses amylase activity. However, the in vitro proteolytic activity of Tribolium larvae was not affected by the trypsin inhibitors CSBTI, AA, lima bean trypsin inhibitor or ovumucoid (16). This indicated that the trypsin—inhibiting component of the Cl protein fraction was not responsible for inhibiting the larval proteinase. Chromatographic separation of the Cl fraction on a calcium phosphate (hydroxyapatite) column showed that the Tribolium proteinase inhibitor is a distinct inhibitor that is free of trypsin- and chymotrypsin- inhibitor and free of amylase (l4). Hemagglutinin In addition to trypsin inhibitors, the hemagglutinin in raw soybeans has been implicated as a growth inhibitor. The presence of hemagglutinating agents in plants was recognized in the 1880's (91). Several groups showed that extracts from various seeds agglutinate red blood cells from some animal species but not the cells from other species (9, 81, 93). Studies concerning a possible role of hemagglutinin in explaining the mechanism of growth depression caused [H 6.: a»! 18 by feeding RSBM were initiated by Liener gt_al. (92) when they had difficulty explaining a growth depression result- ing from feeding crude trypsin inhibitor to a diet con- taining a protein hydrolysate. Liener and Pallansch (93) isolated a homogeneous protein in 1952 high in hemagglu- tinin activity. Further purification indicated it has a molecular weight of 96,000 and contains 6-10% glucosamine (146). Lis gt_a1. (96) found four distinct hemagglutinins in soybean oil meal which were separable on DEAE-cellulose columns. The most abundant hemagglutinin is identical with that previously described by Liener and co-workers (93, 146). All four are glycoproteins containing mannose and glucosamine. Hemagglutinins do not appear to be closely associated with the growth inhibiting properties of soybeans. Liener (88) and Stead 33:31. (138) reported that intraperitoneal inJections of hemagglutinin preparations were lethal to young rats. However, this information is of questionable physiological significance for two reasons: (1) hemagglu- tinin is readily inactivated by peptic digestion, even when as few as 12% of its peptide bonds are split (13, 90); therefore, it should be either completely or almost com- pletely inactivated before entering the small intestine; and (2) even if hemagglutinins did survive gastric diges- tion, an intact protein of 96,000 molecular weight would not likely be absorbed from the gut. Thus, Liener (89) Ines; 19 made an optimistic estimation that soyin, the name originally given to soybean hemagglutinin, may account for 50% of the growth inhibition of raw soybean meal. In a later study (146) he found that it decreased rat growth only 25% below that of the positive control group with essentially all of this decrease being attributed to decreased feed intake. Birk and Gertler (13) found that the fraction containing about 50% of the original trypsin inhibitor and about all of the hemagglutinin from raw soy- bean meal only slightly inhibited the growth rates of rats, chicks, and the larvae of Tribolium castaneum. Also, during the purification process of soybean hemagglutinin (93), there was no correlation between toxicity of the fractions and their hemagglutinating activities. The progressive increase in hemagglutinating activity with purification was associated with only a slight increase in toxicity. Hemagglutinin fractions isolated from some other legume species may be more toxic than those from soybeans. Jaffe and Lette (68) found that raw red or black beans (Phaseolus vulgaris) caused a greater reduction in rat growth than several other bean diets tested. The red and black beans were higher in rabbit blood hemagglutinating activity than the other bean varieties, thus implying hemagglutinin as a possible cause of the growth depression. The natal Round Yellow bean (Phaseolus vulgaris) contains I?! E. ’7 20 high hemagglutinating activity; however, DEAE—cellulose fractionation showed that this hemagglutinin was not toxic to rats when injected intraperitoneally (138). Ricin, the caster bean hemagglutinating component, is toxic but the toxicity is probably due to something other than hemagglutinin activity (78, 103, 142). There was a differential loss of hemagglutinating activity and toxicity when ricin was exposed to a variety of reagents (142). Kunitz and McDonald (78) found that the solubility of crystalline ricin resembled the theoretical solubility of a solid solution of two or more components. Further purification employing DEAE-cellulose column chromatography separated ricin into two components (67). The one protein, having a molecular weight of 60,000 was highly toxic to mice. The other protein was the hemagglutinating protein with a molecular weight of 98,000 and possessing little or no toxicity (143). Saponins Saponins are bitter-tasting, foam—producing glyco- sides in which the nonsugar residue (sapogenin) is a triterpenoid alcohol referred to as a soyasapogenol. At least five different Saponins have been isolated which exhibit varying degrees of hemolytic and foam-producing activity (152). High levels of soybean saponin also inhibit the proteolytic activities of trypsin and chymotrypsin (66). Int-1e 21 Because of their characteristic hemolytic activity and interference with proteolytic activity, it was thought that saponins may be factors contributing to the poor nutri- tive value of unheated soybean meal (111). However, Birk gt_al. (12) showed that the hemolytic activity is unaffected by the heat treatment necessary to produce optimum nutri- tive value of soybean meal, indicating that the hemolytic property of saponins has little or no influence on soybean meal nutritive value. The antiproteolytic activity resulted from a nonspecific reaction of saponins with protein and was readily abolished by the presence of dietary proteins (66). . Pancreatic Enlargement Growth depression due to feeding raw soybean meal or its fractions is invariably accompanied by an increase in size of the pancreas in chicks (25, 107, 126) and rats (17, 97, 113). This suggests a mechanism whereby the animal compensates for the reduced tryptic activity in the intestine presumably caused by the inhibitors in the raw meal. Chernick et_al. (25) found that the pancreases of chicks fed unheated soybean meal rations approximated 1% of their body weights, whereas the organ in those fed heated soybean meal or their regular stock ration never exceeded 0.5%. Rackis (113) showed that the extent of pancreatic hypertrophy in rats depended upon the dietary level of RSBM. At a dietary level of 13% RSBM with 26% Int: ralll? I toasted soybean meal as the control, the increment of pancreatic enlargement was 0.07 to 0.14 g/100 g body weight above the control value of 0.79 g/100 g. At a dietary level of 26% raw soybean meal, the pancreas weight increased 0.22 to 0.25 g/100 g body weight above that of the control group. Nesheim §t_gl, (105) claimed that the level of fat in the ration also influenced pancreas size. This pancreatic enlargement is apparently due to an increased size of the acinar cells with no increase in cell numbers (76). ConcentIations of moisture, total lipids, protein, and RNA in the enlarged pancreases of rats were similar to those obtained on heated soybean meal diets, but DNA concentrations were lower. The synthesis of digestive enzymes was higher in chick pancreases adapted to unheated soybean meal diets (124). Histological examinations showed pancreatic hypertrophy to be associated with an accumulation of zymogen granules in the acinar cells (17, 127). An increased secretion of pancreatic enzymes also accompanied pancreatic enlargement (45, 85, 97, 98). Booth gt_al. (17) reported that the pancreas was the only organ affected by RSBM diets, but this may not be entirely true. They found no histological changes in the rat heart, liver, thyroid, testes, Spleen, kidney, adrenals and intestine. However, a closer examination of several liver and kidney enzymes involved in protein metabolism showed that some functions of these organs were affected JH‘: _ ‘ "J‘J'Cm'm :x————_——'— 7 23 (33). RSBM significantly decreased xanthine dehydrogenase, xanthine oxidase, and arginase activity in chicks and rats as compared to HRSBM. Salman gt_al. (124) found a higher incidence of intestinal hemmorrhages in chicks consuming unheated soybean meal. The pancreas rapidly responds to the feeding of raw soybean rations. Chicks showed maximum pancreas enlarge- ment (as percentage of body weight) within 72 hours (126) while rats showed maximum response in 9 days (113). The return to normal size was equally rapid when the raw soy- beans in the ration were replaced by heated beans (126). The pancreas enlargement produced by raw soybeans is apparently associated with its growth depressing effect; however, there is no definite evidence of cause and effect. There is an indication that the pancreas enlargement factor may be associated with soybean hulls (133). When hulls were added to a purified ration, pancreatic enlargement resulted but there was no decrease in weight gains. Although pancreas enlargement often occurs in rats and chicks fed raw soybeans, evidence indicates that this may not be a general occurrence with all species or all ages. Swine may not develop enlarged pancreases when fed RSBM rations (62). The enzyme activities and relative pancreas size were smaller in shoats fed a RSBM ration than in those fed a commercial soybean ration. Calves fed a 50% soybean protein source milk replacer containing 15:2 24 trypsin inhibitor did not have enlarged pancreases (49). The trypsin and chymotrypsin activities of the pancreases and intestinal contents of these calves were less than those from calves fed other diets. However, it should be noted that this diet caused considerable diarrhea which may have counteracted any raw soybean effect (56). Pancreatic enlargement was less in 5 week-old rats than in those 3 or 4 weeks old (113). Pancreases of older chicks were less affected by RSBM diets than those of younger chicks (107) and adult hens were unaffected by the toxic factor (77) in raw soybeans (128). Loss of Endogenous Nitrogen Pancreas enlargement and increased enzyme production by animals fed RSBM suggests that an increased removal of endogenous nitrogen might result (97). Over a period of time, such a loss might increase the animals dietary protein requirements which in turn would account for some of the growth—inhibiting properties of the raw soybeans. The observation by Lepkovsky 32:31. (84) that the proteolytic activity in the feces of rats fed RSBM was much higher than in rats fed heated soybean meal supports the above hypothesis. Haines and Lyman (53) and Khazam- bashi and Lyman (73) observed more protease activity and more TCA-insoluble nitrogen in the intestinal contents of rats fed SBTI or RSBM than in those fed the control diets. The TCA-insoluble protein contained 7 times more essential Inf-4:.- 25 amino acids and 17 times more cystine than that of the con- trols. De Muelenaere (29) observed a similar increase in TCA-insoluble intestinal nitrogen in rats fed SBTI con— centrates which was maintained throughout the entire length of the small intestine for 5 hours. He attributed the increase to excessive pancreatic secretions and intestinal mucosal slough-off. However, since growth rates were not measured, no correlations between his findings and affects on growth are possible. Supplementing diets with methionine, threonine, and valine prevented the growth depression caused by SBTI, but did not prevent the intestinal changes reported above (73). Growth depression due to RSBM was still much more severe than that caused by SBTI when fed at equal trypsin inhibi- tory levels (53), suggesting that while endogenous nitrogen loss could have contributed to growth depression, other factors in raw soybeans are also involved. Kwong gt_al. (80) reasoned that if fecal loss is to explain low nutritive quality, there would have to be either a decreased percentage of nitrogen absorbed as the level of unheated soy flakes in the diet increases or there would have to be a selective failure in the absorption of the limiting amino acid, methionine. They found neither of these situations to exist in rats when unheated soybean flakes were increased from 25 to 75% of the ration. 26 Metabolic Effects Evidence indicates that RSBM or its growth depressing fractions influence the conversion of methionine to cystine (3, 79). There is much more cystine in the intestinal contents of these rats than in the contents of rats fed HRSBM (24, 73). Since crystalline trypsin con- tains 8.7% cystine (4), it follows that the cystine might come from pancreatic trypsin. This high cystine content of trypsin coupled with its enhanced secretion in rats fed RSBM may cause a partial cystine deficiency and thus growth depression. Some evidence of the influence of RSBM of cystine metabolism is based on amino acid utilization studies. Kwong and Barnes (79) found that feeding raw soybeans increased the expiration of C1402 by rats given introperi- toneal inJections of D-L-methionine-2—Clu. The increased metabolism of methionine as measured by CO2 loss did not occur in rats fed RSBM diets supplemented with cystine. 35 they showed In later experiments using L—methionine—S that methionine was rapidly converted to cystine in the pancreases of rats given CSBTI via stomach tube (3). The increase in expired CO from methionine by rats on RSBM 2 diets was associated with an increased conversion of methionine to cystine for subsequent incorporation into excretory pancreatic protein. In a: 27 Borchers (18, 19) reported an increased requirement for threonine and valine in rats fed RSBM that could not be accounted for by decreased protein digestibility or increased loss via fecal loss of pancreatic enzymes. He, suggested either decreased intestinal synthesis or increased metabolic requirements as possible explanations of this phenomenon (l9). Impaired Intestinal Digestion and Absorption (Decreased protein digestion and amino acid avail— ability are generally accepted as major factors in explain- ing the growth inhibition caused by raw soybeans. Supple- menting a RSBM diet with methionine or cystine increased the growth rates of rats (58, 59, 79) and chicks (44), but not to the rate of those fed HRSBM) This phenomenon should be explainable by either decreased availability of sulfur amino acids or an increased requirement of RSBM diets. Since methionine is the most limiting amino acid in soy protein for rats (80), chicks (134), and poults (94), both possibilities could create a partial sulfur amino acid deficiency. Proteins known to be highly undigestible are often improved by steam cooking with some of the principle changes being a loss of cystine, a corresponding appearance of lanthionine, and an increased susceptibility to enzy- matic hydrolysis (l). Digestibility studies indicate that 28 RSBM contains a fraction which becomes digestible only after heating (80, 85, 106). RSBM liberated more sulfide under pressure cooking than HRSBM (l). Fractionation showed that only the water-insoluble residue liberated sulfide while a similar heating of trypsin inhibitors did not. Melnick et_al, (102) observed a slower release of methionine in the intestinal tract of rats fed raw versus heated soybeans while the release of lysine was not impaired. Trypsin inhibitor AA (see Table 3) but not CSBTI decreased intestinal proteolytic activity in chicks (44). This is consistent with previous studies indicating CSBTI is more susceptible to inactivation by acid or pepsin than inhibitor AA (10, 71). Another group (73) obtained increased protease activity in the intestinal contents of rats fed a crude soybean trypsin inhibitor extract. There is no agreement as to whether the absorption of sulfur amino acids is influenced by heating soybean meal. Early work indicated no difference in the per cent of dietary sulfur which appeared in the feces of rats fed raw or heated soybeans (21, 69). Others claimed Just the opposite for both chicks (36) and rats (80). Part of this discrepancy can be accounted for by differences in techniques used. Sometimes (21) digestibility was esti- mated from the amount of water-insoluble nitrogen in the intestinal tract, which ignores the increased pancreatic secretion associated with unheated soybean meal. A” h: I!" "I . \3 29 Nesheim and co-workers (22, 42, 105) showed that the absorption of dietary triglycerides and dietary free fatty acids was markedly depressed in chicks fed diets con— taining unheated soybean protein. This was most pronounced in chicks up to 3 weeks of age but 4-week-old or older chicks showed little impairment. Supplementing the RSBM diet with trypsin (22) or with sodium taurocholate (40) restored fat absorption to normal in 2-week-old chicks. DeSpite the effectiveness of trypsin supplementation, trypsin inhibitors per se do not appear to be responsible for the poor fat absorption since feeding Kunitz's (77) CSBTI did not result in poor fat absorption (42, 105). The mechanism by which unheated soybean meal depresses fat absorption is unknown. Garlich and Nesheim (42) stated that the decrease is not due to lipase inhibition but sited no data or references. It is not due to a protein or sulfur amino acid deficiency (42). 'H EE I? CHAPTER III MATERIALS AND METHODS Experiment I. Effect of Age on Bovine Pancreas Size and Enzyme Activity Tissue Preparation and Homogenization Pancreases were obtained from 43 Holstein bull calves varying in age from 1 day to 12 months. They were removed as soon as possible after slaughter, placed on ice, dissected free of connective tissue and large blood ves— sels, weighed and frozen until assayed for enzyme activity. Pancreases were homogenized in 0.15 M NaCl containing 0.1% Triton—X-lOOa (48). After centrifugation, samples were diluted to contain 0.3 to 1% tissue for activation of trypsinogen and chymotrypsinogen with a crude enteropepti- dase preparationb at 4°C (48). Enzyme Assays Trypsin and chymotrypsin esterase activities were assayed spectrophotometrically with p-toluensulfonyl-L— arginine methyl ester HClc (TAME) and N-benzoyl-L-tryosine aPurchased from Rohm and Hass, Philadelphia, Penn. bViodenum, produced by Viobin Corp., Monticello, Ill. cPurchased from Cylo Chemical Corp., Los Angeles, Calif. 30 31 c (BTEE) as substrates, respectively (48). ethyl ester Chymotrypsin assays were performed with 4.7% methanol (v/v) in the reaction mixture. Statistical Analyses Animals were grouped by age and the data analyzed for differences among ages using Duncan's multiple rage test (34). Regression analyses were performed on the loglo values of the data to determine the rate of change in pancreas size and its trypsin and chymotrypsin content as functions of body weight (23). The data were fitted in a general regression formula (i.e. Y=aXb) where Y = log Y1, X = log X1, a = intercept and b = s10pe of the regression 1 line. Y is pancreas weight in kg or total pancreatic enzyme content. X1 is the independent variable, body weight in kg. Experiment II. Pancreatic Proteolytic Enzymes and Growth of Rats Fed Soybean and Milk Protein Diets Previous experiments by Gorrill (46) showed that calves grew unsatisfactorily when fed milk replacers con- taining certain soyflours as part of the protein source. These soyflours contained high levels of soybean trypsin inhibitor, implying that SBTI may be a possible growth inhibitor. Feeding a pure soybean trypsin inhibitor to calves seemed unfeasible and too costly. Thus, experiment II was conducted to see if the rat could be used as a test animal in place of the calf. it‘ll: 32 Five or six individually housed female weanling rats were fed each of the seven diets listed in Table 4 for 21 days. The ingredients in diets 2, 3, 4 and 7 are given in Table 5. Diet 2 was formulated as a lactose-free diet since diet 1 caused diarrhea in rats. Diets l, 3, and 5 had been previously fed to calves (46, 49). Diets l, 3, 4, 5 and 6 were fed simultaneously while diets 2 and 7 were fed at a later date. At termination the rats were killed by decapitation. Pancreases were removed, weighed, frozen and stored until analyzed for trypsin and chymotrypsin content as described under experiment I. The small intestine was equally divided into upper and lower sections. Weight of contents from each section and pH of stomach contents were recorded. Intestinal con- tent samples were then frozen and stored until later analysis. A 0.5 to 1 g sample of intestinal contents was used for determining the activities and stabilities of intestinal trypsin and chymotrypsin and in vitro protein digestion. Each sample was diluted to 3 ml with 0.15 N NaCl, then diluted with an equal volume of glycerol and centrifuged at 1300 x g for 30 minutes. One portion of the supernatant was incubated at 37° for 2 hours while the other portion was stored at 4°. Trypsin and chymotrypsin activities were determined in both portions. 18H 5. l f“ J5 _ 33 TABLE 4.--Dietary treatments. Diet Protein SBTI % units/g 1. All milk replacerl 19.2 0 2. Casein replacer 20.5 0 3. Soy protein conc. replacer2 20.6 0 4. All milk--ground soybeans3 24.2 241 5. High soy replacer“ 24.2 243 6. All soy replacer5 24.2 258 7. High inhibitor replacer6 21.3 271 lSkim milk and whey powder. 2Promosoy, Central Soya, Decatur, Indiana; percentage composition (air dried basis): protein, 71; fat, 0.5; fiber, 3.7; ash, 6.3; and carbohydrate, 18.1. 360% of protein supplied by ground soybeans and 40% of protein supplied by skim milk and whey powder. “60% of protein supplied by a 50% crude protein soybean flour, and 40% of protein supplied by skim milk and whey powder. 5100% of protein supplied by a 50% crude protein soybean flour. 6100% of protein supplied by Centex, Central Soya, Decatur, Indiana, a 50% crude protein soybean flour known to contain trypsin inhibitor. “‘11:: 34 TABLE 5.--Composition of diets 2, 3, 4, and 7. Ingredient Diet 2 3 4 7 (s) Casein 20.5 —-- ___ __- All milk replacer --- --- 50.8 --- Promosoyl --- 25.0 --- --- Centexl --— --- --- 43.0 Ground soybeans --- --- 38.2 --- Vitamin-mineral premix2 2.5 4 1.2 2.5 B vitamin complex3 2.0 2 0.9 2.0 Trace mineral salt 2.0 2.0 --- 2.0 DL—methionine --- 0 --- 0.5 Aurofac-lOu 0.25 0.25 0.1 0.25 Corn oil 10.0 --- --- 10.0 Fat premix5 -—- 33.0 ——— --- Glucose monohydrate6 62.75 33.25 8.8 39.75 TOTAL 100.0 100.0 100.0 100.0 lCentral Soya, Decatur, Indiana. 2The premix contained (g); thiamine, 55; menadione, 9.9; vitamin A (30,000 lU/s) + D (2800 IU/g) + E (82 IU/g), 1100; K citrate, 3438; Na28e0 , 0.624; A12(SOu)3-18H20, 300; H 803, 10.5; Na2M04'2H20, 10. ; pyridoxine-HCl, 11.6; NaBr, 20.9; ascorbic acid, 57.2; inositol, 286; folic acid, 1.1; p-aminobenzoic acid, 28.6; biotin, 5.5; vitamin B1 (.1% trituration of cobalamine), 31.4; (kg) K2HP04, 12.48; Mg), 6.24; and cerelose, 21.3. 3Dawes Lab., Inc., Chicago, Illinois, containing (g/lb) riboflavin, 2; pantothenic acid, 4; niacin, 9; and choline chloride, 90. ”American Cyanamide Co., Princeton, N. J., containing 10 mg aureomycin 1 lb. 530% fat premix (mixture of dried whey and fat), supplied by milk specialties, Inc., Dundee, Illinois. 6Cerelose, Corn Products Company, Argo, Illinois. 35 In vitro protein digestion was calculated from the change in protein during incubation of intestinal contents for 2 hours at 37°. Nonprotein nitrogen was removed by precipitating protein with 4 volumes of 10% trichloroacetic acid. Precipitates were washed two times with acetone and once with ether before redissolving in 5 volumes of 0.1 N NaOH. Protein was determined spectrophotometrically by methods of Waddell (147) and Tombs §t_a1. (144). Experiment III. Isolation and Characteriza- tion of Growth Inhibitors from Soybeans Preparation of Soybean Meal Fractions Raw soybean meal (RSBM) was prepared by grinding soy- beans in a Wiley mill using a 2 mm mesh screen and defatting the soybean meal with hexane in a soxhlet apparatus. The solvent was removed by evaporation under a hood at room temperature after which the defatted meal was finely ground in a Wiley mill to facilitate more complete extraction in later steps. Heated soybean meal (HRSBM) was prepared by auto- claving the raw meal at 101° (4 lbs steam pressure) according to procedures described by Renner and Hill (120). The raw meal was adJusted to about 20% moisture with dis— tilled H O and held overnight in a closed container at 4°. 2 It was then spread in layers 1/4 to 3/8 inch thick in metal trays for autoclaving. After autoclaving the meal was air 36 dried at room temperature, finely ground in a Wiley mill and stored until needed. The following procedure was used for preparing the various soybean meal fractions. A weighed quantity of RSBM was subJected to the treatment under consideration. Details of these various treatments are given in the results section as they apply to specific trials. The fractions desired for growth inhibition testing were lyophilized to dryness in a Virtisa shelf-type lyophilizer, weighed and stored in air tight containers at room tempera- ture until needed for growth assays. Growth Assay Procedure The test diet composition is shown in Table 6. The soybean meal source was HRSBM except for the RSBM diet, in which case it was RSBM. The soybean meal fraction being tested replaced part of the HRSBM in the diet. The extent of this replacement was equivalent to the quantity of original RSBM represented by the material being tested. This was determined by the weight of 1y0philized sample from a known quantity of starting RSBM. Small adJustments were made for material lost during the isolation procedure. The amounts of each fraction added are shown in Appendix Table II. Preliminary experiments indicated that weanling mice gave the same type of growth response as weanling rats; aVirtis Research Equipment, New York. 37 TABLE 6.--Composition of diets. Ingredient Amount (g) Salt mixl 4.0 Vitamin mix2 2.2 Corn oil 5.0 Solka floc3 1.5 Cerelose 37.3 Soybean meal sourceu’5 50.0 100.0 lGeneral Biochemicals Corporation, Chagrin Falls,. Ohio. Wesson modification of Osborne-Mendel Formula containing (%): CaCO Ca3 (PO )2, 14.9; CuSOu 5H20, 0.039; SePOu 4H2 0, 1.47? MgSOu u, 9.; MnSOu, 0.02; K2A1(SOu) 24H20, 0.1009; K01, 12.0; KI, 0.005; KH2POu, 31.0; NaC , 10. 5; NaF, 0.057. 2Nutritional Biochemicals Corp., Cleveland, Vitamin diet fortification mixture containing (g/100 lbs diet): vitamin A (200,000 IU/g), 4.5; vitamin D (400,000 IU/g), 0.25; atocopherol, 5.0; ascorbic acid, 45.0; inositol, 5.0; choline chloride 76.0; menadione, 2.25; p-amino- benzoic acid, 5.0; niacin, 4.5; riboflavin, 1.0; pyrido- xine-HCl, 1.0; thiamine-H01, 1.0; calcium pantothenate, 3.0 (mg/100 lbs. diet): biotin, 20; folic acid, 90; and vitamin B-l2, 1.35. 3a-Cellulose, Brown Co., Gorham, New Hampshire. “Soybean meal is HRSBM in all cases except for the RSBM diet. 5Additions of soybean meal fractions replace part of the HRSBM. 4H& 3" 38 however, the growth response was more rapid and more pro- nounced. Details of this comparison are given in Appendix Table III. Differences due to dietary treatment were detectable within three days. Thus 21 day—old mice were used in all the growth studies since they required con- siderably less feed and thus a smaller quantity of fractions prepared from SBM. Males were used except in two trials when females were used. Mice receiving the same diet were housed in the same wire meshed cage. Usually 5 to 7 mice were used per treatment but the number ranged from 3 to 8 depending on the amount of SBM preparation and the resulting feed mixed._ Growth studies continued four days or longer if there was sufficient quantities of feed. Beginning and terminating weights as well as daily weights from the third day on were recorded. On the final day mice were killed by decapitation and the pancreas removed, weighed and frozen for later enzyme analyses. Feed intake for each treatment group was estimated by weighing feed initially and after termination of the experiment. Enzyme Inhibitor Assays Trypsin and chymotrypsin inhibitor activities of soy— bean meal and various fractions were determined by measure— ment of the inhibition of hydrolysis of TAME by trypsin and hydrolysis of BTEE by chymotrypsin, respectively. Inhibi- tor samples were sufficiently diluted to insure that the assay mixture was not saturated by inhibitor. 39 The presence of amylase or lipase inhibitors was studied by comparing the effects of whey solutions from HRSBM and RSBM on amylase and lipase activities. Amylase activity was determined by the method of Bernfield (8) wherein the reducing groups liberated from starch are measured by the reduction of 3, 5-dinitrosalicylic acid. Absorbancy was measured at 540 mu. Lipase activity was determined by the rate of hydrolysis of Tween 20a as measured by potentiometric titration. The assay procedure was as follows. To the reaction tube was added 4.0 ml 0.05 M sodium acetate, pH 8.2, 0.5 ml lipase substrate (20 ml 0.2 M sodium acetate, 10 m1 Tween 20 and 20 ml H2O) before adJusting to pH 8.2 with NaOH. Then 0.5 ml enzyme solution (goat lipase plus HRSBM or RSBM extract) was added and the sample titrated to pH 8.2 with .0188 N NaOH using a pH stat to measure the rate of base addition. One unit of activity is equal to 1 micromole of acid produced per minute at 25°. aAtlas Chemical Industries, Inc., Wilmington, Del. In his CHAPTER IV RESULTS AND DISCUSSION Experiment I. Effect of Age on Bovine Pancreas Size and Enzyme Activity Pancreas size, trypsin and chymotrypsin activities for bull calves are summarized in Table 7. All age groups except the one day—old calf had about the same pancreas size and enzyme activity on a body weight basis. The pancreas size and trypsin content of the one day-old group were less than the overall mean for all groups (P < 0.01). The most notable difference in the one day-old calf was the higher chymotrypsin activity per mg of pancreas dry matter. Only at this age did the chymotrypsin to trypsin ratio exceed 1.0. This is in agreement with previous studies which indicated that dietary alterations modify chymotryp- sinogen synthesis to a greater extent than trypsinogen synthesis (6, 7, 119, 135) and confirmed Gorrill's observa— tion (49) that ruminants generally have a lower chymotrypsin-to-trypsin ratio than most monogastrics. The regressions of pancreas weight, trypsin and chymotrypsin activities on body weight are illustrated in Figure l. The regression and correlation coefficients are listed in Table 8. All ages were weighted equally to 40 41 .mamsficm mo gonads on» mpcmmopaop mommnpcopma :0 LmQESZ .Azmv .cmoe 000 no moppo ULmUCMpm m m . pmop 00:00 00000035 0.200csa mean: .00.0 v 0 .qu000000 >00c00000cw0m no: 000 000000 0600 050 an 003oa0o0 Cezaoo mEmm map 20 mcmms 0 00.0 000 00.0 000 00.0 00.0 0Mm 00.0 000 00.0 000 00.0 00.0 0002 000.0 000 00.0 000000 00.0 0000.0 0.000 000 000000 00 000.0 000 00.0 000000 00.0 0000.0 0.000 000 000005 00 000.0 0000 00.0 00000 00.0 0000.0 0.000 A00 000005 00 000.0 000 00.0 000000 00.0 0000.0 0.000 000 000005 0 000.0 000 00.0 000000 00.0 0000.0 0.000 000 000005 0 0.0.0 000 00.0 000000 00.0 000.0 0.000 000 000005 0 000.0 000 00.0 00000 00.0 000.0 0.000 000 000005 0- 000.0 000 00.0 000000 00.0 00000.0 0.000 000 000005 0 000.0 000 00.0 000000 00.: 00000.0 0.00 A00 000005 0 000.0 000 00.0 0000 00.0 0000.0 0.00 0100 000 000 0 003 0000 wx\00 0000 03 moon .ocma 03 anon .och B wx\mpfics wE\mpH:: wx\mpfic: wE\mp0cs 000 00000000 000000002000 000>000< 0000000 03 00000000 0wwm 000 .mm>000 00:0 :Houmaom :0 moaufi>auom Camazpposhco 0:0 Camazpp .0000 mmopocmmll.0 mqm2mu20 00202022000 .p2w003 0202 .m020020> 02m©2mam©20 020 00 x 220 manmfi2m> 020020200 020 00 w .m2m23 02x0 u wa mwm ©002m003 0000200 2020 202002 00200> 008020 00:00>0©20 002200 20 vmmmm 00.0 00.0 0 00.0 00.0 0.08 00 00 0 0m.o om.o H om.a mo.m .08 NH op m mm.o 0m.o 0 00.0 :0.m .08 m0 Op 22202 200202008020 00020202 mw.o om.o H mo.H mm.m . m.oE NH 0» m mm.o mm.o H m0.a m©.m .08 NH on m 00.0 00.0 0 00.0 00.0 .08 NH 00 20202 2002020 00020202 mm.o 00.0 0 00.0 mm.m| m.o8 ma on m mm.o 00.0 0 mo.a Hm.ml .08 NH 0p m 00.0 000.0 0 00.0 00.0- 00 00 00 00000 p2w003 00m2o20m 2 p 00 00000000000 00000000000 000000000 0 0000000> 20000002200 20000m2mmm .22w0m3 0202 o» 200202008020 020 200202» .u2w0m3 00020202 20 02020200000mmll.m m0m¢e 4H0: uu remove any variation due to differences in sample size. Since the newborn calf was the only age group which appeared to possibly deviate significantly from the mean, calculations were made both including and excluding this age group. Calculations were also made using individual, unweighted data on animals two to 12 months of age. The results were not significantly different from the weighted values although variances were greater. Pancreas weight, trypsin and chymotrypsin content of the pancreas were directly related to body weight as indicated by regression coefficients which were not signifi- cantly different from 1.0 (p > 0.05). This was consistent with previous calf data (6“), but different from the growth responses of several other internal body organs (23). The present data have one exception in that pancreatic chymotrypsin content may increase at a faster rate than body weight from two to 12 months of age. During this age span total pancreas chymotrypsin activity increased some- what faster than did body weight (p < 0.2). When Gorrill's data (49) on calves from one to six weeks of age were included with these results the following response in pancreatic chymotrypsin activity was observed. At birth the calf pancreas contained high levels of chymotrypsin which rapidly dropped within a week, remained low until about two months of age, then steadily increased, and eventually leveled off to a fairly constant level as [Pi is 45 expressed per unit of body weight. The period of lower chymotrypsin activity corresponded to the milk feeding period and thus indicated a dietary adaptation in chymotrypsin synthesis. Experiment II. Pancreatic Proteolytic Enzymes and Growth of Rats Fed Soybean and Milk Protein Diets Data on live weight gain, feed intake, size and enzyme content of the pancreas when rats were fed seven different diets are shown in Table 9. 0f the four soybean- source diets containing soybean trypsin inhibitor (no. “-7) only two(no. 5 and 6) reduced weight gains significantly (P < 0.005) below that of the no. 3 non-inhibitor soy protein concentrate. Pancreases of rats fed these same four diets (no. u to 7) were significantly enlarged (P < 0.005), averaging 620 mg/100 g body wt compared to M30 mg/100 g body wt for those from the non—inhibitor diet groups. Pancreatic trypsin and chymotrypsin activity per animal likewise increased (P < 0.005) but activity per g wet pancreas tissue remained unchanged by diet. Pancreas size and proteolytic enzyme content were the same on all diets devoid of trypsin inhibitor activity (no. 1 to 3) regardless of protein source, milk or soybean. The unsatis— factory weight gain by rats fed the all milk replacer was apparently due to diarrhea which was probably caused by the high lactose content of the diet. Gains on the casein diet, I?! E: M6 .pcmEpmozp on moo cmzp pmnpmh mocmzo an wcfimflzm mcmoE coozpon mocopoMMHo w mo zpaaabmbopm : .cmoe mcp ho Loppo oomocmpmm .mo.o v m .mcme psoEumoLp cmmzpmb oocopmmpfio 02m .Azmv ammo mwcmp mHQHuHSE m.:docsa mcflm: Ho.o v m pm pcmoomMHp haucmoHMchHm poc mpm udahompmdzm mEMm spas mcmofia .m.c mo.ov .m.c o.ov w.m.c moo.ov moo.ov moo.ov zosfim> m x m:.o oam Hmm OOH mm m: m.o za.o m m o~.m mmwma mmma mmqs can mods am.mH mom.m nonfipflccfi emu: .N ma.m mmwmfi mmaa gamma gs: mwmo 9mm.MH 900.: sow HH< .m m:.m pmomMH omaa pmmmm mam pmmzm p«m.ma omH.m sow swam .m :m.m Hmsfl oqga mam 0mm Nam mm.:H mmo.m mcwmosom m pm pm 9 n + xHHe Hfla .3 om.m QMMWMH Dawn Dams: mum 9mm: pm:.:H mms.m .ocoo cflmpocd mom .m . iJ a u . . c mmm . an m ammo Hrna bomb am: com: 25 HH oma m H o m . W J c J a c E . ms m pmumoa mwom boom «mm ham: 9mm ma HUmo m xafi HH< a AHMEHcm A»: box AHmEHcm Auz pm: An: moon \mpflcsv m\muficzv \mpacsv m\mofic:v mooa\mev Ammo\wv Ammo\wv B Hapoe .ocoo fiance .0200 9:0 zpfi>fipo< cfimamppoEmLo muH>Hpo< cflwdzpe mNHm oXMuca mmcmco o . some o3 zoom 9 Ho mmmoocmm Lo xHHE ooh womb no mommmpoCMQ mo ocmpcoo mammcm ocm mNHm .moxMucfi comm .mpofio comma :mmnzom .mmmcmno pnmfimz moomll.m mqm 0.10) by dietary treatments, averaging 137 and 171 units/animal on the SBTI (no. u to 7) and non—SBTI diets (no. 1 to 3), respectively. However, chymotrypsin activity was 5 times higher (767 vs 147 units/ animal) on the SBTI diets. This indicated that either (a) the SBTI-containing diets inhibited trypsin secretion, or (b) trypsin secretion was increased in the same proportion as chymotrypsin but the enzyme assay procedure used measured only trypsin which was not bound to SBTI. The increase in both trypsin and chymotrypsin in the pancreas was evidence against the first possibility. If the second situation were .m manmb .2 ouOCuood mwmv . .m magma .m mucouooe comm .u3.o v m .mcmmE pcmsomopp cmmgpop mocopmmmap o: 148 7 m .Anmv omen omcmn oaafioasa m.:moczm mcfimz Ho.o v m pm unmatddfic maozw:flmflcwflm no: mom ooficomcmasm oEmm oz» cpfiz mamas:H moc.0v J.m.c Ho.ov :osaw> m . . c xxJ :.®HH 3.9H : o mic Hm.m mo.mao m.mnm o.ama w.ao 2mm.: LOpHoagcfi :mH: .w mw.o u:.umo m.mm: 3.99m D.w:H m.a~ m.os om.m mom afiq .w Ho.m me.imo 0.xMH m.mm m.moH 3.0m m.am pm:.: how am“: .m om.: m:.umn m.amd o.MHH o.awa m. m H.Qm omw.: mcmoo%om . + xHHE HH< .: no.0 om.mza N.m: w.o> o.mma m.pn H.3m 2mm.: .ocoo sapwood mom .m mm.o so.aaa 3.0ma $.2ms F.3mm no.0 cammmo .m wu.a Hom.mma m.a~ H.0m m.z:a :.~m 3.0m Hom.m xHHE HH< .H AHMEHCM AHmEH2m \muficsv Amocoucoo mxmoficzv \mofi:5v Ampcvn:00 m\wowzdv Lozoq Load: pmzoq Load: Hmpob cofipomm Hooch :oHpoom. 9 a :QMEoom pofio ecu zpfi>fipo< :HmdmpuoEmno >pfi>fiuo< :Hmazpe mucmpCOO HwCfipmprH .mpmfic comma cmmnsom no xfifie ewe mums co mpcmpcoo Amadpmmpcfi ca mmfipfi>fipom :Hmascp05230 ucm cfimdch new ma sumacpm--.OH mqm 0.10), but tended to correlate with total proteolytic activity in the intestinal contents (see Table 11) and indicated that intestinal protein digestion was not limiting growth. These results confirmed observations by Scow (131) that the pancreas has a tremendous compensatory capacity and therefore can compensate for SBTI by secreting extra enzymes. Trypsin stability decreased in intestinal contents of rats fed SBTI diets (P < 0.005), averaging 0.88 on In 5;. 5() TABLE ll.--Protein content and in vitro digestion, and trypsin and chymotrypsin in vitro stabilities. Protein In vitro enz me stability Interaction Nonincubated in vitro Tr\ sin Chymo- conc. digestion Jp trypsin (mg/s) (ms/s) Diet X intestinal section 1. All milk - upper 30.7 3.20 0.90 0.89 - lower 22.1 1.61 1.01 0.80 - total 25.u 1.75 0.99a3 0.82bC — total (mg/animal) 2. Casein - total 22.0 5.86 0.99a 0.76c - total (mg/animal) 6.83 3. Soy protein conc. — upper “ .2 11.86 1.05 0.85 - lower 23.3 -2.1U 0.96 0.80 - total 37.7 2.0a 0.98a 0.83bc - total (mg/animal) “.26 u. A.M. + soybeans - upper 38.9 5.6“ 0.80 0.76 - lower 22.? 1.01 0.90 0.82 - total 26.9 2.12 0.88bC 0.80bc - total (mg/animal) 10.53 5. High soy — upper 27.9 h.U9 0.79 0.92 . - lower 18.9 -1.09 0.80 0.91 - total 23.0 1.99 0.80c 0.91ab - total (mg/animal) 7.Uh 6. A11 soy - upper 50.7 11.2“ 0.91 0.86 - lower 38.1 -°.1U 0.91 0.92 - total 92.9 2.0a 0.92ab 0.92ab - total (mg/animal) “.26 7. High inhibitor - total 30.9 u.33 0.91:ab 0.98ab - total (mg/animal) 11.38 P value“ n.s. 0 n.s. <0.005 <0.025 1Protein digested during a two hour incubation of diluted intestinal contents at 37°. 2Ratio of enzyme activity of incubated-to-nonincubated intestinal contents. 3Means of total contents in the same column followed by the same superscript are not significantly different at P < 0.05 using Duncan's multiple range test (39). “See footnote h, Table 9. JHE: 51 diets A to 7 as opposed to 0.99 on non-SBTI diets (no. 1 to 3). The opposite occurred with chymotrypsin stabilities which averaged 0.90 and 0.80 (P < 0.005), respectively. This reflected the fact that SBTI binding reduced the amount of trypsin available for substrate hydrolysis (51, 82). As a result, chymotrypsin was degraded less rapidly especially in the lower half of the small intestine and thus appeared to be more stable on SBTI diets. Since growth of rats was reduced on only two (no. 5 and 6) of the four (no. A to 7) SBTI-containing diets, it appeared as though growth depression was not caused by SBTI. The increased pancreas size and accompanying increased trypsin and chymotrypsin synthesis and secretion associated with all of these SBTI—containing diets indicated a possible compensatory mechanism for combating SBTI effects, but appeared to be unrelated to growth depression. In vitro protein digestion in intestinal contents was unrelated to growth of rats. Thus, impaired protein diges- tion can also be eliminated from consideration as a mechanism by which soyflour diets 5 and 6 depressed growth. Experiment III. Isolation and Characteri- zation of Growth Inhibitors from Soybeans Experiment III was initiated in an attempt to isolate a factor(s) from raw soybean meal (RSBM) which would inhibit growth but be devoid of SBTI activity since the results of Experiment 11 indicated that SBTI does not I?! E 52 cause the growth depression which results from feeding raw or minimally processed soybean products. This isolation procedure involved numerous extraneous experiments which were all related in some way to the isolation and characterization of growth inhibitory factors in soybeans. These experiments are described in the following pages. A. Determining the Presence of Digestive Enzyme Inhibitors The results of trypsin, chymotrypsin, amylase and lipase inhibitor assays on raw soybean meal are shown in Table 12. Each value represents the mean of four deter- minations. High levels of trypsin and chymotrypsin inhibitors were present as expected. No d—amylase inhibi- tion was detected by comparing the effects of RSBM vs HRSBM extracts on amylase activity. In fact, a-amylase activity was increased in the presence of RSBM extract confirming the presence of previously reported (14) water—soluble amylase. Thus, if an amylase inhibitor is present in soybeans, it cannot be detected by this method. Lipase assays indicated that a trace of lipase inhibitor may be present in soybeans. More stringent assay condi- tions would be needed to ascertain if this apparent inhibition is real. However, even then it is unlikely that such inhibition would be sufficient to appreciably influence digestion or absorption. [H E: 53 TABLE l2.—-Trypsin, chymotrypsin, d-amylase and lipase inhibitor activities in raw soybean meal. Inhibitor Activity (units/g RSBM) . 1 Tryps1n 13,000 Chymotrypsin 2,5002 d-Amylase 03 Lipase A“ 1One unit equals inhibition of hydrolysis of l pmole TAME/minute. 2One unit equals inhibition of hydrolysis of l umole BTEE/minute. 3 One unit equals reducing groups/minute. “One unit equals minute. inhibition inhibition of liberation of 1 umole of 1 umole acid produced/ 3!...“ JHE: 5A B. General Fractionation Procedure Unheated soybean meal was fractionated as illustrated in Figure 2 to determine whether any of the biological activities could be concentrated in a single component. This procedure is essentially the same as that used by Garlich and Nesheim (A2) and Rackis §£_al. (115), thus providing a check with some previously reported results. Fractionation of unheated soybean meal by this procedure yielded the following distribution of dry matter: water insoluble residue 50.8%, pH A.A insoluble proteins 18.8%, pH 8.0 insoluble material 0.8% and total solids in the whey solution (pH 8.0 supernatant) 29.6%. After dialysis and lyophilization, the whey proteins represented A.9% of the original meal extracted. Two other fractions were also tested in Experiment 1: (a) acetone precipitated whey solution (APWS) and (b) whey solution heated at 60° for 5 minutes (H6OWS). The former was prepared as described by Garlich and Nesheim (A2) in which two volumes of cold acetone were added to undialyzed whey solution. A small amount of saturated NaCl was added to induce flocculation of the protein. The precipitate was suspended in distilled water and dialyzed against distilled water at A° for A8 hours. Any insoluble material remaining after dialysis was discarded before lyophilizing the dialyzed solution. The H6 WS fraction was prepared by heating 0 JHE: 55 Raw soybean meal Q/ Residue (lyophilized) Extract with distilled water at room temperature, using a water—to-meal ratio of 10 to 1 (w/w). Re-extract residue with 5 to 1 ratio. Water extract; acidify ‘ to pH A.A with 6 N HCl. Let stand atlfl’ for 12 hours. Acid-insoluble proteins (not washed; lyophilized) Whey solution- adjust to pH 0.0 with 6 N NaOH. Let stand at A0 for 2 hours. ~17 precipitated salts (not washed; lyophilized) Soybean whey solution Dialyzed, l/lyophilized Soybean whey protein Figure 2. Fractionation procedure of raw soybean meal. 121E: 56 undialyzed whey solution in 200 ml volumes to 60° for five minutes then rapidly cooling in an ice bath. The results of Experiment 1 are shown in Table 13. The loss of weight by mice fed the water—insoluble residue indicated that (l) the growth inhibitor was water insoluble or (2) the extraction was incomplete. Other investigators have noted reduced weight gains and feed efficiencies when the water insoluble residue was fed to rats (117) or chicks (A2); however, the reductions were not as marked as shown here. In this experiment the RSBM was not finely ground after hexane extraction and apparently incomplete extraction was the most probable cause of the marked growth reduction by this group especially since the growth inhibi— tor(s) may not be readily soluble. The RSBM was finely ground before use in all subsequent experiments. Growth rates were reduced slightly in all the other fractions tested, with the weight reduction being most pronounced in mice fed derivatives of the pH 8.0 supernatant (diets 3, 7 and 8). Heating the whey solution 5 minutes at 600 did not remove or destroy the growth inhibitor activity but did destroy considerable amounts of SBTI. Much of the growth reduction on the RSBM and residue diets was due to low feed intake as indicated by the marked reduction in per cent growth inhibition when corrected for feed intake. .However, no group consumed as much feed as the positive control HRSBM. Mice fed diets containing higher levels of JHE: 57 .l... (E... . . .cmmE ocp no Loggo ppmpcmpm .Azmv 5 .mo.o v m .pcopmumfip mapcmOHchmHm no: mam Cezaoo was» CH Loppoa 0500 mg» mp pozoaaom mopsmfim A0000 2000: so 0000 03\0000 0000 00 0H00 02..0000 20000 so 0000 .mHmEHcm no LmoEsc mucommudop mfimozpcopma CH Lonesz 0 m .0000 20000 co 000000 0000\0000 0000 co 00000H 0000 0 0000000000 003000 0000 000 uzv u coaquHSC0 zpzoaw pcmo Low : ummp mmcmu maqfipasE m.c00csa wcfim: .00H x m 000000 0000 an 0000>00 0000 000003 N .0000 000 000 0000 0000 H X 50.0 00.0 0H 00.0 510 J . . . 00 . 00 H5 000 00HH 0 0000 0 00000 000 0 A00 00000000 0003 m 0 :0 00 000 0000.0 0000.H 000a 050.0 A00 .m.3 .000 0000000 .5 5 0 0 000.0 000.0 0050 000.0 A00 .000 0.0 mg .0 00 0H 50 0050.0 0055.0 00000 005.0 A00 .000 0.0 :0 .0 05 00H 000 050.0- 00H.H 000HH 0000.0. A00 0000000 .0 0m 00 000 0000.0 0000.0 0000H 000.0 100 coflpsflom 0003 .0000 .m 00 000 0000 000.0- 0005.0 005 0000.0. A00 2000 .m -u- -1- 0 00.0 000.0 DNHH 0000.0 0A50 2000: .H 000000 000 00H0 03 mmuoophoo a m“ w\mufics anon 00 R we >0p\w 0 cofipanfincH Luzopo Hemm m ocmwmwmmm oz mmonocmm cfimw 93 HpcoEpmwpe upmumfio ho mcoauomum Hmpo>wm no .zmmm .Emmm .zmmmm com Ems: moae mo mmcoammmll.MH mqm<9 J'HE: 58 SBTI tended to have enlarged pancreases, except for the RSBM diet group. Starvation was probably an overriding factor on that diet. Since only solubilized SBTI can be measured by the inhibitor assay used, SBTI values for water-insoluble residue diets are always low. Experiment 1 was repeated using finely ground RSBM E which enabled more efficient water extraction. The results I (Experiment 2) are shown in Table 1A. Extraction of RSBM J.- was more complete; however, almost half of the growth . 1 inhibitor activity remained in the water-insoluble residue. In the next fractionation step most of the growth inhibitor activity remained in the pH A.A supernatant. However, if SBTI activity can serve as an indication of "cleanness" of separation between precipitate and supernatant, growth inhibitor activity was not completely separated at this step. Similarly, in the next step (pH 8.0 precipitation) growth inhibitor activity was equally divided between the two fractions while SBTI was concentrated in the supernatant. This experiment indicated that growth inhibitor activity present in the pH 8.0 supernatant was not dialyzable; however, experiments reported later cast some doubt on that observation. 0. Water-Insoluble Residue Studies Almost half of the growth inhibitor activity remained in the water-insoluble fraction indicating that water extraction may not be the most effective method for removing JHE: 59 .ia. ..aia, .ma 00000 .mH0>H000Qm0L .wlm 0000C0oou 00m0|m .0000 0300 00m 0000 CommH X 00.0 00.0 0 00.0 . 5:0 00 00 0000 00.0 005.0 00 0000.0 A50 0000 0003 .0000 .00 00 00 0000 00.0 0000.0 00 0000.0 A50 00000 0.0 00 .00 00 00 0000 00.0 0000.0 05 000.0 A50 00000 0.0 00 .00 n: nu- 0 00.0 000.0 00 000.0 A50 0000: .0 0 0C0E000mxm K 00.0 00.0 0 00.0 510 00 00 0 0000.0 00.0 00 0000.0 000 000 0.0 00 .0 00 00 00 0000.0 00.0 00 0005.0 A00 000 0.0 00 .0 00 m0 050 000.0 00.0 00 000.0 000 0000000 .00 00 N00 0000 000.0- 05.0 05 000.0- A00 0000 .m n. In- 0 000.0 00.0 00 0000.0 0000 20000 .0 0 0C0E000mxm 0000000 000 0000 03 000000000 0 mm w\m0HC5 0000 00 0 wE >00\m 0000000000 003000 0000 0000MWMM000 03 00000000 0000 03 H000000000. 0000000 .m 0C0Eap0axm CH zmmm 00 0C0000000 0000>0m 00 .mem .zmmm: 00% C033 00HE mo 0mcoqmmmnl.0H mqm 0.10) compared to the HRSBM diet. Extracting the residue with water, 0.15 N NaCl, or digestion with pepsin or papain did not remove or destroy the factor(s) responsible for reduced growth and reduced feed efficiency. Growth rates of mice fed re-extracted residue fractions were less (P < 0.005) than those of mice fed extract fractions. Pancreas size was not affected by any of the treatments. 3. Raw soybean meal extraction studies.--In con- Junction with extraction experiments on the water-insoluble residue, similar extractions of RSBM were also conducted to determine if water is actually the most efficient solvent In La: Q‘ ~11? . . 4 . I - .‘k; .osoflmmp oaosaomcfilsmomz mamsvm .mmm 63 m .oaosaomCH mmz mHQEmm moch 2mmmm poc oasoom .ma maome azao>fipomommp .wnm mmuOCpoou mommlm .msme xam ewe boas comma x No.0 mo.o m ma.o elm J lull . o _ c a o . 3m pa w unwed 0 am o @m 9mm 0 Amv sag Chanda mmm mm a H 03m mmm.o mm.o mm mom.o Amv chSm enmamg ..mmm .mm mm mm a--- pmom.o Ho.o mm pmmm.o Amv pad cflmama ..mmm .sm 0 o mma a m.o sm.o ow mmm.o Amv smasm :Hmama ..mmm .mm III . . . . . . . mm a: m mama 0 mm 0 am 2mm: 0 . Amv use Homz mom mm o 0 com mmm.o mm.o mm wam.o Amv stSm Homz ..mmm .zm mm om m--- opmsa.o wo.o 9m pmmm.o Amv sag 0mm ..mmm .mm o o osa mmm.o No.0 Hm mam.o Amv smasm mm m..mmm .mm :m m: com spam .0 H©.o mm pmsz.o Amv msefimmm .Hm mm mHH comm emo.ou mo.o mm oma.ou Amv Emmm .m nu- nu: o amm.o ms.o was wmam.o mAmV 2mmmz .H mwxwpflfi LCM pmHU p3 monomppoo & mw w\mpfics moon do R ME mmo\m cofipfinficcH spzono Hemm m ocmwmwucm p: mmmpocma cfimm .pz Hucmsummse samumao .Hmme cmmnmom 3mm Eomm ozofimmp maozaomcfiubmpmz can do mCOHombmompo coapmomao so coapomppxo com con: ooHE uo omcoommmll.ma mqm 0.10) than 0.15 N NaCl and 1% Triton- X-100 in extracting the growth inhibitor from RSBM. Chloroform-methanol extraction did not remove the factor(s) causing reduced feed consumption. In fact, intake was markedly reduced on all CHCl -MeOH extracted RSBM diets, 3 particularly diets 37 and 38. Intake was somewhat higher on diet 39 (1.3 g/day vs 3.2 g/day per animal on diet 1) so the pH 10 CHCl3—Me0H extraction was repeated (diet Al). Intake was still low and the extraction efficiency of growth inhibitor activity still below that of water alone. 65 his}? . .maosfiomcfi mm: mHoEmm mocfim >Mmmm poc oazoom .ma magma .mao>fipomammb .mlm mmpOCpoom mommlm .mzmo Lsom om“ mpmfiaa x mo.o No.0 2 00.0 wlm mm mma m--- oam.ou ms.o mm oam.ou Amv OH ma .H: om Hma min: onmo.on :m.o Hm omw.ol Amv 0H ma .mm om mmm m--- ozm.ou mo.o om eHo.Hu Amv 5 ma .sm NH mmfl m--- opma.ou 05.0 mm oam.on Amv m mm .mm "pm 2mmm oopomppxm zoo: maomo mm mm min: w:H.o mm.o pm omm.o Amv 2mmm o.pxm QQHIXICOpHLB .2: on we will omma.o mm.o om osm.o Amv Emma ompompoxm Homz .m: H: mm m--- msH.o ms.o Hm swam.o Amy 2mmm empomppxm 0mm .Hm 3m :ma oomm onwa.ou om.o om omm.on Amv Emmm .m nu- -u- o mom.o mm.o 3m owms.o mime gammz .H mxmoCH Loo omfio oz mmuooppoo R MR w\mpfic: hoop no a ma >m6\m coaanHSCH cpzopm Hemm mmocwwmwmmm oz mmmpocmm cfimw .63 pcospmmpe mpmumao \ .muco>aom Hmho>om an 2mmm Eopm coapomppxm nonwnfincfi npzopw mo mocoHOHmmmul.~H mqm0mmenu wcoEm Umgmano 00030000 000 new .0 pd000000d30 £003 mQSOLm 000 0000 000:0 .m0 00909 .000>0pomammp .0|m mmpocw000 00m 0 mum .0000 0:00 000 0000 comm H x 00.0 00.0 0 00.0 010 mm 000 000 wmmo.01 00m.o :00 w0:.ol mflmv LmQSm mHU cfimazpe .mm 00 00 00 0000.0 00000.0 00 0000.0 000 000 000 0000000 .00 00 00 0000 00000.0 000000.0 00 000.0 000 00000 000 000000 .00 00 00 000 0000.0 00000.0 00 00000.0 000 000 000 000000 .00 . I . . . Q m % . 00 000 0000 0000 0 000000 0 000 0000 0 0000 00 00 00 0000 00 00 00 00 000 0000.0 0000.0 00 00000.0 .000 000 000 0000000 .00 mm 00 0000 00.0 00.0 000 00.0 000 00000 no ohm so omumowfiu cflmqmm .mm 00 00 000 00.0 0000.0 00 0000.0 000 000 00000000 000000 .00 00 000 0000 000 0- 0000.0 000 0000.0- 0000 2000 .0 00 000 0000 000.0: 000000.0 00 000.0- 000 2000 .0 nu- nu- 0 00000.0 000000.0 000 0000.0 0000 00000 .0 -u- -uu 0 000.0 000.0 00 0000.0 0000 20000 .0 000000 000 0000 03 00000000 0 m0 m\mpfics moon 00 a we zmo\m £000090£CH guzohc 09mm mmocmwwwppm 93 00000200 chw 03 00:08pmmhe ammpmfio .0008 2009000 300 00 020000000 omummeU 050000 00000000C000000m com con: 0005 00 omcoammmll.ma mqm0000Qmmp .01m mopOCpoow 00m .mmmo 0:00 mom 00m 0000 £00m H FIN X 00.0 00.0 a 00.0 0:0 0m 00 000 00000.0 00000.0 00000 00000.0 000 00000 000000 .000 00 .00 00 00 000 00000.0 0000.0 0000 000000.0 000 00000 000000 .000 0 .00 00 00 000 000.0 0000.0 0000 000.0 000 00000 000000 .000 0 .00 0m 0m 0mm :m.o m0.o 00 mm.o pcmpmc00d50 000 0 00 0000 000 000000-000000000 .00 0 0 000 0000.0 0000.0 00000 0000.0 000 00000 000000020 000 .00 00 00 0000 0000.0 000.0 00000 000.0 000 000 000000020 0000 .00 00 00 0 00000.0 000.0 0000 0000.0 000 00000 000000020 0000 .00 0m 0m 0000 00000.0 0000.0 0000 00000.0 000 0300 .00 n.. nu- 0 000.0 000.0 000 000.0 0000 20000 .0 0000000 000 0000 03 0 000000000 0 mm m\wpfics moon go 0 we zmo\m coaufioHECH £03000 Hemm mmocmwmwmwm p: mmopocmm chm .03 HpcmEummsB mpmpofio .COHpsaom 0023 Umpmp0afiompa . 0coumom mo mCOHumpmompo 000000» p00: go .0omm00mzv .0000oonouHCOpcmo 00% C033 0005 mo omcoommmll.om m0m¢e 1th 73 amount of the growth inhibitor activity was apparently lost or destroyed prior to this step. Of the growth inhibitor activity present in the APWS, most of it appeared to be precipitated by 50% (NHH)2SOu saturation, but this sample was lost and therefore not tested. Veri- fication of these results had to await completion of the next experiment. Although the five minute heated sample gave inconclusive results, the one and ten minute heated samples indicated that the growth inhibitor, when in solution at approximately neutral pH, was not destroyed by heating, but one-half of the SBTI activity was destroyed. The bentonite-celite treatment results were inconclusive, but there was no evidence to indicate preferential adsorp- tion of trypsin inhibitor over growth inhibitor. Pancreas enlargement appeared to be directly related to dietary SBTI activity since removal or denaturation by bentonite- celite adsorption or by heat treatments resulted in a concomitant reduction in pancreas size relative to body weight. Experiments A and 5 were conducted to further clarify the effects of the treatments tested in Experiment 3. Experiment 4 repeated the 50% (NHu)2SOu treatments and the bentonite-celite treatment with an additional step. Five hundred ml of APWS was stirred with 8.0 g of bentonite- celite (1:1, w/w) overnight at 4°, centrifuged and the precipitate discarded. The supernatant was brought to 75% ' JtRf-h “0|." WILM. ‘; " JHL—iz 74 (NHu)280u saturation by adding 232 g (NHu)ZSOu to use ml of solution. After centrifugation, the precipitate was dissolved in distilled water. Precipitate and super- natant solutions were both dialyzed against distilled water and lyophilized. In Experiment 5 APWS was divided into three fractions. 25% and 80% (NHM)2SO,4 precipitates, and 80% (NHu)2SOu supernatant. The intent was to con- centrate SBTI in the 80% (NHu)2SOu precipitate. The results of Experiments 4 and 5 are shown in Table 21. Weight gains in Experiment 4 were based on the first four days growth even though the experiment con- tinued for six days. This was because some groups were short of feed the sixth day which interferred with the dietary effects on weight gains. The growth inhibitor and SBTI remaining in the supernatant of the bentonite-celite treated APWS were concentrated in the 75% (NHu)2SOu satura- tion precipitate (diet 33, Table 21) which partially con- firmed the results of Honovar gg_al. (127). However, con- siderable growth inhibitor activity and SBTI were either destroyed or retained on the bentonite-celite. The 50% (NHA)2804 saturation treatment did not completely separate growth inhibitor activity or SBTI into one fraction but tended to concentrate both, particularly SBTI, in the (NHu)2SOu precipitate. Experiment 5 indicated that most SBTI precipitated between 25% and 80% (NHA)ZSOA saturation while the factor(s) responsible for most of the growth Vlflfl '. ' ‘ JH': 75 .MH 00909 .zam>flpooammg .0Im mmuOCp000 mom 0:0 .0 pcmefipmoxm 20 0000 0300 p0000 000 000 0chw pnw003 .mzmp x00 00% 000 0000 comm0 00.0 00.0 0 00.0 0M0 00 00 000 000.0 00.0 00 000.0 000 00000 000000020 000 .00 00 00 000 0000.0 00.0 000 000.0 000 000 000000020 000 .00 0 0 00 000.0 00.0 000 000.0 000 000 000000020 000 .00 II. In: 0 000.0 00.0 000 0000.0 000 20000 .0 m 020800mmwm 00.0 00.0 0 00.0 0M0 00 00 000 00.0 00.0 000 00.0 000 000 0 0 0 000000020 .000:.0000 .00 0 0 0 00.0 00.0 00 00.0 000 00000 0 0 0 000000020 .000u.0c00 .00 00 00 000 00.0 0000.0 00000 000.0 000 000 000000020 000 .00 00 00 000 00.0 0000.0 0000 000.0 000 00000 000000020 000 .00 00 00 000 00.0 0000.0 00000 000.0 000 0300 .00 1:. an- 0 00.0 00000.0 00000 0000.0 0000 20000 .0 0 pcosfigumwm 0000000 000 0000 02 000000000 0 m0 w\mpfics hoop mo 0 we zmo\w soapHQHSCH £03000 09mm mocmwmwuum .93 mmmpocmm camw .pz Hpcmsummub hungoam .c000300 awn: ompMpaafiomha 0200000 mo 02000000Q00Q Uopmmpp om Azrzv new muaaoolouficoucmn vow cm£3 man mo mmcoammmll.am mqm¢9 Jht: 76 inhibition remained in the 80% (NHu)2SOu supernatant. Pancreases of mice fed the higher SBTI containing diets tended to be larger (as per cent of body weight) but the trend was not as definite as in some other experiments. Growth rates of mice on the 80% (NHu)2SOu precipitate and supernatant diets indicated that a partial separation of growth inhibitor activity from SBTI had been achieved. However, these results were inconsistent with the 50% (NHH)2SOH and bentonite-celite-(NHu)2SOu results. This prompted the conclusion that the differential (NHu)2SOu precipitation techniques used did not separate the biological activities sufficiently for satisfactory progress. 9. Growth Inhibition Due to Crystalline Soybean Trypsin Inhibitor Crystalline soybean trypsin inhibitora was added to diets for mice at three levels to ascertain its affects on growth rates. This study was conducted simultaneously with Experiment 5 (see Table 21). The results of this experiment are shown in Table 22. Growth rates were reduced (P < 0.05) only by the two highest levels of SBTI in experiment a, but were not reduced (P > 0.05) by the highest SBTI level in experiment b. The growth inhibitory effect of crystalline SBTI was aSoybean trypsin inhibitor, five times crystallized, Nutritional Biochemicals Corp., Cleveland, Ohio. 77 . . “humanly .ma canoe «mao>fipooomop .wum meOCpoom mom sum .mfim>flpoodmmp .9 wow m mpcoefipmaxo :fl mmmo o>Hm new xflm pom powu commH x mo.o :o.o o oo.o 5 Im om ma 0mm oa.o m~.o Hoa Hm.o Amv Hemm x 3 .mm I- I. o Hm.o mo.o mm mo.o Amv 2mmmm .H . o pcoeflpodxm x II In an No.0 mo.o m 00.0 sum m: m: 0mm pmfi.o mm.o zHH pm:.o Asv Hemm x x .mm mm mm 03H pwmfi.o me.o OOH pmzm.o Amv Hemm .sm 3: a: cam QHH.O os.o 30H 903.0vaHemm + paaqommflqzzv sow .mm ma ma 03H pmsfl.o ea.o mHH pmH©.o Amv saasommflszzv sow .mm II II o wom.o m~.o HHH momm.o mAmV Emmm: .H m ocoEHLonm :mxmocH pom ooflo o3 popomppoo & m& w\muH:3 zoos mo & we mmo\m socmfloficem coflpfioflncH cpzopo Hemm m Comm o3 mwopocmm cfimo .px pcmEpmmLB mpmpofia .LOpHpHSCH :quzpp cmmomom ommfiaampmmgo mo mao>ma ocmgomaflo oopnp wcflooow 0p moss co mmcoammmnn.mm mamas use: 78 somewhat additive since adding an equal number of SBTI units to diet 52 more than doubled growth inhibitor activity (diet 53). However, increasing the SBTI activity to four times the level present in diets 52 and 54 did not quadruple growth inhibition. The pancreas was not enlarged (P > 0.05) by any of the SBTI-containing diets. Diet 55 was repeated at a later date (experiment b). Growth rates and feed efficiencies were reduced slightly (P > 0.05) and pancreases were enlarged slightly (P > 0.05). Thus it appears that SBTI may cause some growth depression, but the amount of depression is not nearly as great as that caused by RSBM. H. Acetic Acid Extraction of RSBM In previous experiments there was some difficulty in lyophilizing some of the undialyzed fractions to com- plete dryness. The material was often sticky, sometimes almost caramely, most likely because of the high sugar content of this material. In attempt to eliminate one step in the fractionation procedure a preliminary experi- ment was performed in which RSBM was extracted with 20% acetic acid instead of the usual water extraction followed by acidification. This resulted in a lyophilized super- natant product which was not sticky and caused essentially 100% growth inhibition. However, some uncertainty as to the cause of growth inhibition remained because (a) even after lyophilization both the supernatant and precipitate www- ‘ 79 fractions had strong acetic acid odors, and (b) the mice ate very little of the precipitate diet. Thus an experiment was conducted to determine the effect of acetic acid on growth of mice. This was determined by extracting 75 g of HRSBM with one liter of 20% acetic acid four hours, centrifuging and lyophilizing the supernatant. Several other treatments to the acetic acid extract of RSBM were also tested in this experiment. These included the effects of (a) dialysis, (b) heating at «3.35:4 * - both acidic and neutral pH and (c) adsorption on DEAE— cellulose.a The dialyzed, neutralized sample was pre— pared by dialysis of the acetic acid supernatant followed by neutralization, and then heating in a boiling water bath for one minute. The DEAE—cellulose adsorption was determined by stirring 50 g DEAE-cellulose with 880 ml acetic acid extract (from 75 g RSBM) for four hours, centrifuging and testing the supernatant for growth inhibitor activity. A loss of growth inhibitor activity was assumed to indicate adsorption on the DEAE-cellulose.' The amount of the ion exchange resin used was based on amounts used by Garlich and Nesheim (M2). The responses by mice when fed these preparations involving acetic acid extraction of RSBM are shown in Table 23. Statistical analyses were performed on only the aDiethylaminoethyl cellulose, Cellex-D, Calbiochem, Los Angeles, Calif. lat-l: .9... .. Cay-.53 .UHom oHumom mpcmmopdop oHuoQOmL .mlm mmpOCpoom mommlm .mzmp snow pom pmHo commH x mH Hm me o om.o No.0 oMH.o Hwy poo o 0.10), but trypsin inhibitor was reduced by the process. Most, if not all of the growth inhibitor was retained on DEAE-cellulose (diets 76 < 73 at P > 0.10 and 76 > 69 at P < 0.10) which agreed with results previously reported by others (U2). I. Heat Treatments on Different Fractions Autoclaving RSBM or any of its fractions destroys the growth inhibitory activity of the fraction; however, heating dry RSBM or heating a solution containing the RSBM fraction may not destroy the growth inhibitory activity (109). This was verified in several experiments ‘which involved heating dry RSBM or heating RSBM fractions under different conditions of time and pH. A lyophilized Vikki-f"- h.-g“ M [H E: 82 pH 4.“ supernatant sample was also autoclaved and fed to mice as a check on the heat-lability of this fraction. The results of this experiment are shown in Table 2“. Many of these treatments were also reported in other sec- tions of Experiment 3; therefore, no statistical indica- tions are shown in Table 24. Dry heating RSBM did not improve growth rates of mice confirming the results of Osborne and Mendel (110). Autoclaving the pH “.4 super- natant destroyed the growth inhibiting as well as its trypsin inhibiting ability of RSBM (diet 71). Heating the pH A.A supernatant solution in 15 ml quantities in test tubes placed in a boiling water bath for one or five minutes also destroyed the sample's growth inhibiting activity and about three-fourths of its trypsin inhibiting capability (diets 62 and 63). This indicated that the growth inhibitor was destroyed by mild acid hydrolysis. However, similar heating of an acetic acid extract of RSBM (diet 7U) destroyed little if any growth inhibitor activity. Thus the lability of the growth inhibitor(s) under mild acid hydrolysis situations remains uncertain. The growth inhibitor activity of a RSBM fraction in solution at neutral pH was not destroyed by this type of heat treat- ment even when continued for as long as 10 minutes (diets 8, 18, 19, 20 and 75). Pancreas weights, expressed as per cent of body weight, decreased (except on diet 74) slightly ,0. [.71 [HE-l: 83 . .... 0.. up? I .nmcH5t0000 002 o .mH mHomB .zHo>HpoQOms .mlm monogaoom mom mum .mHmogp chp no puma mpHSmos on» :H osoczome pmumHH Ome cpm mucoEpmopp zsmpoHp ommcp mo mam: H 0m 00 050 Hm.0 H0.0 HHH 05.0 H00 .cHe 0H 000000 .0m 5m 50 050 00.0 05.0 00 00.0 Hmv .cHe m 00pmtm .0H mm 00 0:0 0H.0 00.0 00 Hm.0 Hmv .cHe H 060000 .0H mm 0m. 0mHH 00.0 00.0 00H H5.0 H00 .m.3 0.000 0000000 .NH 00 H5 00H HH.0 00.0 mHH 00.0 H00 .cHe 0 0000mm .0 0m 00 0H0 0H.0 00.0 0MH 00.0 Hmv .tHom 50:3 .HmHa .0 mm mm 00H MH.0 05.0 00 mm.0 H50 .cHE H 000: ..HmHn ..0002 .05 0: m0 0HH 00.0 55.0 H0 00.0 H50 .cHe H 00000: .05 mm 00 000 50.0 00.0 05 00.0 HmHv tmasm 0Hom OHHmoa .00 5 5 0 0H.0 00.0 05 50.0 H00 tm>mHoosza .H5 0 0 000 mm.0 50.0 05 H0.0 H50 .cHe 0 06000: .m0 m m 050 00.0 mm.0 50 00.0 H50 .cHe H cmpmm: .N0 00 00 00mm 00.0 05.0 H0 0m.0 H50 tmazm 0.0 00 .H0 m: 0HH 0--- 0H.0u 05.0 00 mH.0n msz 2000 00000: 5H0 .0m wamch tom stHe 03 popooppoo 5 m5 w\mpHcs moon mo we zmo\w COHpHancH spzopo Hemm mzocwwwwpmm p3 mmmpocmm chw p3 HpcoEpwmpB mamumHQ .mCOHpompm Hmos cmmomom 30H mo mcoHumprmHQ Umpmmpp Hams m30Hpm> pom cocz mOHE mo momcoamom szopull.zm mqm 0.10) probably in response to the decrease in SBTI in the test fraction. J. Effect of Dialysis on Growth Inhibition Several eXperiments were conducted to determine the effects of dialysis on growth inhibitor activity of various fractions. The fractions dialyzed were (a) the 20% acetic acid extract of RSBM, (b) the pH 8.0 supernatant (soybean whey solution) and (c) the acetone precipitated whey solu- tion. The results reported in Table 25 are the means obtained from several experiments most of which are more completely reported in previous tables. One-half of the growth inhibitor activity was removed or destroyed by dialysis. More than half was removed by dialysis of the acetic acid and pH 8.0 supernatants (55% and 62%, respec— tively), but less than half (35%) was removed by dialysis of the acetone precipitated whey solution. This may merely reflect normal variation in response but may also mean that acetone causes some destruction of growth inhibitor activity. Some growth inhibitor activity may also be destroyed with time in the acetic acid solution. Trypsin inhibitor was too large (14,000 to 2u,000 molecular weight, Table 3) to be removed by dialysis; however, “0% of the SBTI activity present in the acetic acid supernatant was not present after dialysis. This probably indicated acid must": 1H0: 85 TABLE 25.-—Effect of dialysis on SBTI and growth inhibitor activities. Growth Fraction SBTI Inhibitor (units/g 2 diet) (units ) l Acetic acid super (15) 460 55 Dialyzed acetic acid super (8) 270 25 Whey solution (1“) 2620 82 Dialyzed whey soln (1“) 2110 31 Acetone ppt'd whey soln (7) 870 50 Dialyzed APWS (12) 860 32 1Number in parenthesis represents number of animals. 2One unit equals 1% growth inhibition corrected for feed intake as described under footnote 4, Table 13. 1 ___—_‘Q' 86 denaturation especially since total SBTI activity was lower in the acetic acid supernatant than in either the whey solution or acetone precipitated whey solution. K. Effect of Methionine Supplementation D-L Methionine was added at the expense of cerelose to both the HRSBM and the RSBM diets at a level of 0.5% of the total diet to determine its effects on growth rates. The results shown in Table 26 indicate that growth rates on the RSBM diet were increased only slightly (P > 0.05). The major response to methionine supplementation on this diet was increased feed consumption with no decrease in intake corrected growth inhibition. Methionine supplemen- tation actually decreased growth rates (P > 0.05) on the HRSBM diet, but this was partially attributed to decreased feed consumption. L. Separation on Sephadex G-50. The results of experiments previously reported, indicated that the factor(s) responsible for much of the growth inhibitor activity of RSBM may be of small molecu- lar weight. The best evidence for this conclusion was the removal of half of the growth inhibitor activity by dialysis. The lack of distinct separation at the various steps in the general fractionation scheme (section B) or in the (NHu)2sou and bentonite-celite treatments (section F), and the inability to eliminate the growth inhibition nu: 87 If‘?‘ .MH memB .zHo>Huoonop .5nm moHOCpoom mom 5|m .0500 m 000 msmH0H x mo.o no.0 : mo.o 5Im m5 00 0000 m0.0 m00.0 00H 000.0 H00 005 50.0 + 2000 .05 00 mHH 0000 00.0: 000.0 00 00H.0u H50 2000 .m 0H mm 0 00.0 000.0 00 005.0 H00 005 Hm.0 + 20000 .55 III Ian 0 mm.o oom.o OOH 0mm.o mH5V sawm: .H m oxmch pow mpme p3 monomppoo 5 m5 w\much moon mo 5 we zwm\w coHpHszcH cpzopo Hamm mmocwwwwmmm p3 mmopocmm chw .pz HpcoEpmoLB mumpmHQ .mOHE mo mopMp cpzopw co COHpMHCoEoHQQBm ocHCOchmE mo pomQMMII.wm mqm 0.10) than those of mice fed the positive control diet (HRSBM). Therefore, although hemagglutinin assays were not per— formed, the lack of growth inhibition in the fraction where they should appear indicated that they were not involved in 41-10: ..I JHE: 90 Figure 3.--Chromatography of the pH A.A supernatant on a Sephadex G-50 column. Column dimensions: 8.02 x 109 cm. Sample: 950 ml of pH A.A supernatant from RSBM. Eluting buffer: distilled water. Flow rate: 9 ml per minute. Protein (O—b), SBTI (t—'), SBChTI (A—A). 91 “Stméfqrun) ILWOHS r4 :4 a) w) i— ? ‘9 l I j (Tm/31TH“) IQgS p O O O O O O O :r m N H O I 1 I I '. \ O 1 m H \ L8 ‘ r—l Q O b ‘t-l H \ E ~° \ . O , H O . $0 ON C . fl 0 ./ "" A“' ‘ H3 < 1:1 o -O \O -O L Ln id cl ui ‘ d l. H O (rm/8w) uteqoad Fraction number .LHt-i: A" 92 i. . I: (LII! thir‘s.¢l..flfuifvt .IE ‘1' .MH mHnt .mHm>HpoQOmp .5Im mononuoom mom 5-0 .0000 m>Hm pom muoHQH X 00.0 00.0 0 00.0 510 00 50 0 00H.0 00.0 00 000.0 H50 HHH eoH00000 .00.0 .00 --- III 0 0Hm.0 00.0 00 000.0 H00 20000 .H x 00.0 00.0 5 00.0 5I0 m: 0: 000m 05H.0 0055.0 000H hH0.0 H50 HH :oH000am .00u0 .H0 . . . a l . NH 0H 5H 000 0 O0H5 0 0mHH 000 0 H50 H 00H000am 00 0 00 00 00H 000m 0H0.0- 000.0 005 000.0- H50 00000 0.0 :0 .05 00 mHH 0000 000.0- 000.0 0000 00H.0u H5vzmmm .m nun nu: 0 000.0 000.0 000H 000.0 0H50 50000 .H 00000H ao0 5 00H0 03 mmuomppoo 5 m w\mpHc: hpoo mo 5 we zap\m coHpHancH Luzopo Hemm mzocmwmwmmm p3 mmopocmm CHmw .p3 HpcmEummpB madamHQ .cE:Hoo omuo xopmnoom 0 co ompmmmqmm ucmpmcpmazm 3.: ma mo mCOHuompm mo mammm MOUHDHECH npSOLOII.5m mamHuomeop .5im monocuoom omm5um .mm ooHp now 0000 oops» .mmwo XHm pom mpoHQ H x 00.0 00.0 0 00.0 5:0 0HHH 0oHoo0cH .00.0 50 50 --- 0HH.0 000.0 050 000.0 H50 oothocoss 0Ho0 0HH2 .00 00 00 0 00H.0 --- In- 000.0 H00 HHH eloo0am .00:0 .00 0 0 00Hm 050.0 050.0 000H 005.0 H00 HH 0 H mcoHoo000 .00.0 .00 50 50 0000 oH0.0 0000.0 000 000.0 H00 00000 0.0 00 .05 00 00H 0000 00H.0- 000.0 000 0Hm.0- H00 2000 .m nun --- 0 000.0 000.0 000 0000.0 0H50 20000 .H 000000H noH 00H0 oz popooppoo 0 m5 w\mpHc: moon mo 5 we zmp\m COHpHoanH cpzopo Hemm mzocmwwwmwm p3 mmopocmm chw .uz Hpcospmmne humpoHQ Locpo oEom mo new HHH coHpomum omio oonHonozn oHom oHHE .mCOHpompm ucmpmcnmasm 2.: mm on» no momma nopHnHQCH nuzohoin.mm mqm w cutim wOD. OUOO- O O (+) (+) l 2 Figure 6H".High voltage electrophoresis, pH 3.5, of (a) fraction III and (b) mild acid hydrolyzed fraction III. (1) ninhydrin stain, (2) periodate-benzidine stain. ‘5‘- . WV.»- “fir-fig“ ~ [HE-.3 104 soybeans (152) but probably do not affect growth rates of animals (12, 66). No hemolytic activity was detected in any of the samples tested. Table 29 summarizes the extent of purification of the fraction III growth inhibitor. When growth inhibitor activity is expressed on a dry matter basis, the purifi- cation is only four-fold since the G—50 fraction III still contains a considerable amount of water and acid soluble small MW compounds. However, when activity is expressed on a protein basis, which will be the appropriate compari- son if final purification shows it to be a protein, then purification is more than 28-fold. The water-insoluble residue accounts for nearly 40% of the protein present in RSBM, the pH 4.4 precipitate is essentially pure protein and more than 60% of the protein in the pH 4.4 super- natant is accounted for by G-50 fractions I and II. N. Integrated Discussion of Soybean Growth Inhibitor Isolation and Characterization Experiments Mice responded rapidly to the dietary treatments. The average daily gain after three days on a diet was essentially the same as after four to six days. Additional days on a diet did provide a slight reduction in variance within the dietary groups. Four mice were used per dietary treatment in early experiments, but to reduce the standard error, five to seven were used whenever sufficient test material was available in later experiments. " 7. wow“. ' "~.'-A flu“ 105 ..p ..-...a... - . _ .. 0.1.4.40? .mH oHomB .0 ouonpoom noon: UoQHHommo 00 mxmch 0000 now Umpomnnoo COHpHoncH £03000 0H mesvo pHcs mco H 50 00.00 00.0 HHH coH0o0c0 .00.0 00000000 H5 00.mm 00.: pompmcmeSm 0.: mm mm 00.5 00.m pomppxo Hmumz 00H 00.0 00.H 2000 :Hopopd SQ 0H0H5 5 05H00H00 0\H00Hcs 50H>H00< co0H0H00H 003oh0 eoH00000 .0o0H0H00H 002o00 HHH 0oH00000 Ho 0oH00oHHH000::.00 00000 [P1502 106 The per cent growth inhibition corrected for feed intake was used as the index of growth inhibitor activity in order to correct for differences in weight gains from one experiment to the next and to correct for inequalities in feed consumption. The correction for differences in feed consumption on the test versus the control diet was i usually small except for mice fed the RSBM diet. Consump- tion was consistently low on the RSBM diet and consequently calculated growth inhibition was less precise on that diet mums 4* - since it was somewhat confounded with starvation effects. The data summarized in Table 30 illustrate the overall mean and ranges of means for both average daily gains and growth inhibitor activities achieved on several diets which were tested at different times during these trials. This summary illustrates very good precision and repeatability with this growth assay. The range was more for actual weight gains among trials than when gains were eXpressed as a per- centage of that particular trial control group. Much of the variation in rate of gains among experiments was due to changes in environmental conditions. For instance, weight gains on all groups were proportionately lower in feeding;trials conducted during hot, humid summer weather. The results of these experiments indicate that there may be two or more factors in RSBM which inhibit growth. 1310 two primary factors are in the water-insoluble residue axujin the G-50 fraction III. Trypsin inhibitors and other IHE: will - «a to i ..V. .mHmHHp mo poneszH 00 op mm 00 H5.0 o0 H0.0 H0.0 H00 03 0.000 0co00o< 00 o0 00 H0 05.0 o0 00.0 H0.0 H00 0Ho0 5003 0005H0H0 0: op mm m: m5.0 OH 00.0 00.0 Hmv mschmp mezHomcHIHopmz 00 oo 00 m5 0H.0: o0 00.0- H0.0- H00 2000 --- --- 0H.H o0 00.0 00.0 HHNHV 20000 7 m 0x00cH 0o0 ompompmoo 0 mmo\w Cde Cde mwcmm HHmno>o mmcmm HH090>0 Hamspmmne znmpmHQ coH0H0H00H 003o00 0H00 03 0o 00002 no 02002 .mpmHo Hmpo>mm so Um>oH£om COHpHoanH gpsopw 0cm mchw panms mo mcmme CH mmcmu ocm 000E HHmLm>OII.0m mqmdB 108 factors may also affect growth, but the extent of their effect on growth is not as well substantiated. The growth-inhibition attributed to the water- insoluble residue may be caused by the same factor present in the soluble portion. In such a case, the growth inhibitory factor would be a relatively insoluble compound. The poor efficiency of water extraction and the incomplete separation of growth inhibitor at several precipitation steps may indicate such a situation. However, if the growth inhibition attributed to the water—insoluble resi- due is merely a reflection of incomplete dissolving, re- extracting or extending the extraction time should reduce the growth inhibitor activity of the more completely extracted residue. This did not occur. Digestion by gastrointestinal enzymes also failed to remove or destroy the growth inhibiting properties of the water-insoluble residue. Thus it appears that the residue may be (or contain) a growth inhibitor completely separate from the inhibitors in the soluble portion of RSBM. The water—insoluble residue may accomplish its growth inhibiting effects via its indigestible and insoluble prOperties. Decreased protein digestion is generally accepted as a major factor in explaining the growth inhibi- tion caused by raw soybeans. Digestibility studies indi— cate that RSBM contains a fraction which becomes digestible only after heating (80, 85, 106). The steam cooking J'Hhs 109 necessary to bring about such a response causes a loss in cystine with a corresponding appearance of lanthionine and liberated sulfide as well as increased susceptibility to enzymatic hydrolysis (l). Fractionation of RSBM showed that these properties were attributed to the water- insoluble residue (1). There is considerable evidence to indicate that a small molecular weight compound accounts for much of the growth inhibition caused by RSBM. Retardation on Sephadex- G-50 and G—25 columns, loss of activity by dialysis and lack of detection by polyacrylamide-gel electrophoresis all strongly indicate a small molecule. Incomplete separation of growth inhibitor into either precipitate or super- natant in many of the fractionation steps also indicates that a small molecule may be involved. Movement in an electric field and adsorption on DEAE-cellulose indicate that the molecule carries a charge, which is most likely positive. Therefore, it may be reasonable to assume that this small, charged molecule can readily become trapped by or attached to larger protein molecules and in this way be incompletely separated by a particular chemical treatment. Such a situation may eXplain the partial growth inhibitor activity of the pH 4.4 and pH 8.0 precipitates and the inconsistent separation of growth inhibitor activity by (NH4)2SO4 fractionation. Other research teams have also observed this slight reduction in growth rate of - : “~0J1-f‘f." IHE: Li.1ll.l lllu 4.} . .4Iril Illulmvlr ... 110 rats (117) or chicks (42) fed diets containing pH 4.4 and pH 8.0 precipitated fractions, but tended to ignore this phenomena since they were trying to correlate growth inhi— bition with trypsin inhibitor activity. The attachment of a small molecule to a larger protein may also explain the growth inhibition attributed to trypsin inhibitors, even five times crystallized SBTI. Eldridge et_al.(35) detected 6 to 13 bands on polyacrylamide- gel electrophoresis from each of 9 commercial SBTI samples. This indicates that crystallinity is no guarantee of purity and may also account for the disagreement‘in literature over the growth-inhibiting properties of SBTI. Pancreas enlargement appeared to be more closely related to dietary SBTI activity than to growth inhibition. In experiment II, this correlation between pancreas enlarge- ment and SBTI activity was very obvious. Although there were discrepancies in some of the mouse growth assays, pancreas enlargement was usually closely related to SBTI activity. One of the best illustrations of this was the second Sephadex G-50 separation experiment. In that experiment, the SBTI—containing diet was the only one which caused pancreatic enlargement. The diet causing half of the growth inhibition (fraction III) caused no pancreas enlargement. On the other hand, pancreases were not sig- nificantly enlarged when crystalline SBTI was added to diets; however, the level may have been too low to have a marked effect. 1 . ;_..'o:‘ Ff' mamas- - It’ll-.1 111 Differences in actual pancreas size attributed to dietary treatments were generally only moderate and sta- tistically significant differences usually occurred only when pancreas size was corrected to a constant body weight. This moderate effect on pancreas size may be attributed to the short duration of the mouse growth trials since Rackis (113) found that rats required nine days to reach optimum pancreas enlargement. When growth trials were continued for 21 days as with the rats in Experiment II, the actual pancreas size of SBTI-fed rats was enlarged. These results do not necessarily imply a cause and effect relationship between SBTI and pancreas enlargement. Several factors have been implicated in pancreatic enlarge- ment including the level of fat in the diet (105), soybean hulls (133) and trypsin inhibitors (43). However, the results of these experiments indicate that pancreatic enlargement may be associated with SBTI activity but not associated with growth inhibitor activity. The cause of reduced consumption of RSBM diets was explored but not ascertained. Limited attempts to extract a factor responsible for reducing feed consumption were unsuccessful. Almost any treatment to RSBM restored feed intake essentially to that of the HRSBM-fed group. Wada §£_§l. (146) suggested that hemagglutinins elicit their growth inhibiting properties by reducing feed consumption. Feed consumption of mice fed G-50 fraction I, the fraction “ "(Jug fiw ‘04:... . Jul-1: AK 112 most likely to contain hemagglutinins, was slightly less than intakes of the HRSBM—fed group, and in this manor was in agreement with Wada's claims. However, no factor(s) was isolated which sufficiently accounted for all of the reduced intake on the RSBM diet. ” 35"“.131" J'Ht: CHAPTER V SUMMARY M419“ Pancreas weight, trypsin and chymotrypsin contents increased directly with body weight in bull calves from birth to one year of age. The new born calf was the only age group which indicated any marked differences from the i mean of all age groups. Pancreas weight and trypsin con— tent were slightly less at this age while chymotrypsin content per mg of pancreas tissue was higher than at all other ages studied. Diets containing soybean trypsin inhibitor (SBTI) caused pancreatic hypertrophy in rats with corresponding increases in trypsin and chymotrypsin activities. Enzyme activities per mg of pancreas tissue remained constant. These same SBTI-containing diets caused increased chymo— trypsin activity and stability, decreased trypsin stability but did not change free trypsin activity in the intestinal contents. Intestinal protein digestion, as measured by an in vitro system, was not impaired in the intestinal contents of the SBTI—fed rats. Growth rates were depressed on only two of the four SBTI diets indicated that the growth depression exerted by raw or minimally processed soybean products is not caused by SBTI and apparently occurs by 113 .1"th 114 some mechanism other than by interference with protein digestion. Rat growth data and literature data indicated that SBTI and growth inhibition activities in soy preparations were not the same compound. Therefore, attempts were made to separate these two factors by partition and precipita— tion techniques after establishing a growth assay using mice. A small molecular weight growth inhibitor present in unheated soybean meal was separated from SBTI by ion exclusion chromatography on a Sephadex G-50 column and partially characterized. This growth inhibitor decreased weight gains and feed efficiencies of mice without causing pancreatic enlargement. Further evidence of the small size of this growth inhibitor was its retardation on a Sephadex G—25 column, removal by dialysis and lack of detection by polyacrylamide-gel electrophoresis. Movement toward the cathode under high voltage electrophoresis at pH 3.5 and apparent adsorption on DEAE-cellulose indicated a positively charged compound at that pH. Other factors in soybeans may also inhibit growth. The water—insoluble residue accounted for about 40% of the growth inhibitor activity of raw soybean meal. This growth inhibition could not be removed or destroyed by gastro- intestinal enzyme digestions or by several other solvents tested. Fractions containing SBTI generally caused pan- creatic enlargement and had some growth depressing activity. lHEi REFERENCES Ins: 10. REFERENCES Almquist, H. J., H. L. Christensen, and S. Maurer. 1966. 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Biochem., 62:141. \' .LJ‘. -:’ P" IH E1: APPENDICES “an“, [Hts 131 APPENDIX TABLE I.—-Rmdy weight “n1 punsrcas S13t, dry mxiLvr, ini nn:yme canivnt if ball .1 calxu s {Ywnn b11W15 1L? :. Hunltns 04’ age. Age and Pancreas calf no. Body wt wt cry matter Trypsin Chymotrypsin (kg) (g) (2) (units/mg UH) 1 day old 1 29.0 16.0 71.9 4.7: 8.42 2 34.0 70.0 :c u 3.07 a :3 3 44.9 23.“ 24.“J 5.20 8 87 4 33.6 19 0 5.0 5.30 4.32 5 33.6 14 5 23.4 4.40 8.90 2 months 175 73.5 4..0 34.0 3.82 2.02 176 76.2 t7 3 32.9 6.97 3.75 177 69.0 “a D 81.b 3.70 1.57 178 68. td.0 :’3.0 5.92 3 27 185 74.3 43 U 03.9 5.27 3.55 4 months 3 1 3 137.9 112.0 2‘.4 5.39 4.65 170 140.0 94.0 23.9 4.71 2.98 171 146.1 121.0 22.4 4.74 4.05 172 135.2 94.0 I” 1 2.91 2.65 174 129.7 93.0 22 4 3.66 3.18 5 months 162 152.4 09 0 3 2 4 73 4.17 6 months 154 133.7 150.0 3 o 0.46 4.63 155 198.7 174.0 1 4 4.66 3.04 157 163.7 175.0 -4 6 5.64 4. 5 7 months 143 222.3 6“ 0 22.0 3.41 2.90 144 206.4 16: 0 24.8 5.69 4.97 145 191.9 15 .0 15.3 7.01 5.55 146 173.7 l4t.0 21.5 3.64 3.?4 147 175.5 149.0 22.1 3.27 2.80 8 months 135 130.5 123.0 22.9 5.13 3.17 136 240.4 186 0 23.1 {.13 4.08 137 235.0 130.0 23.7 5.02 4.43 138 225 9 111.0 22.1 3.82 3.22 142 225.9 174.3 23.3 5.07 4.90 148 217.7 143.0 23.0 5.25 4.66 10 months 123 276.7 197.0 22.3 4.71 4.32 124 224.5 245.0 2 .9 3.73 3.53 127 298.5 200.0 22 8 5.21 3.95 132 299.4 222.0 24.1 9.63 10.09 134 278.5 236.0 22.6 8.3- 7.87 11 months 117 306.2 290. 118 316.2 226. 119 313.0 < 12 months .18 6.56 CJOO r): "‘I‘u \OD—‘CF Lukfixl m0.) 0.; .09 \ \0 J1 101 344.7 238.0 23.5 4.00 4.'9 102 328.4 245.0 24.2 6.06 4.91 104 357.4 277.0 22.8 6.’7 5.52 107 382.8 314.0 19.6 5.90 4.21 109 289.4 225.0 21.8 6.19 4.72 J‘H E.- 132 APPENDIX TABLE II.--Amount of test fractions present in 100 g of test diet. Diet . Amount No FraCtIO“ (g/lOOg diet) 1. HRSBM (positive control) 50.0 2. RSBM (negative control) 50.0 3. Dialyzed whey solution 4.5 4. Water-insoluble residue 24.1 5. pH 4.4 ppt 8.1 6. pH 8.0 ppt 0.4 7. Acetone ppt'd whey solution 2.4 8. H50 whey solution 2.2 9. H60 whey solution 2.2 10. Water—insoluble residue 25.0 11. Dialyzed whey solution 2.9 12. Acetone ppt'd whey solution3 2.2 13. APWS - 100% (NHu)2SOu super 0.4 14. APWS - 100% (NHu)QSOu ppt 0.9 15. APWS - 50% (NHu)280u super 0.8 17. APWS — Bentonite-celite super 0.7 18. APWS - heated l min 1.4 19. APWS - heated 5 min 1.3 20. APWS — heated 10 min 1.3 21. Water-insoluble residue“ 22.0 22. Residue - water super 0.8 23. Residue - water ppt 21.9 24. Residue - 0.15 N NaCl super 0.7 25. Residue — 0.15 N NaCl super 21.5 26. Residue - pepsin super 0.9 27. Residue — pepsin ppt 21.5 28. Residue - papain super 1.9 29. Residue — papain ppt 21.0 30. APWS - 50% (NH4)2SOu super 0.6 31. APWS - 50% (NHu)2SOuppt 0.4 32. APWS - bentonite-celite (NH4)280u super 0.6 33. APWS - bentonite-celite (NHu)2804 ppt 0.6 34. Dialyzed whey solutionu 1.7 35. Acetone ppt'd whey solution” 0.8 36. Vacuum heated RSBM 50.0 37. Dry CHCl -MeOH extracted RSBM 50.0 38. pH 2 CHC -MeOH extracted RSBM 50.0 39. pH 10 CHC 3-MeOH extracted RSBM 50.0 40. Lipase extracted RSBM 46.0 41. pH 10 CHCl3-MeOH extracted RSBM 22.6 42. Lipase extracted RSBM 18.8 42 — b. Lipase digested RSBM — ppt 33.0 43. 0.15 N NaCl extracted RSBM 25.5 44. 1% Triton-X-lOO extracted RSBM 22.6 45. Amylase digested RSBM - ppt 33.0 ' 1' .m-m ‘-- .- .3 - v.. . . h-‘d"v -_ ___—fl. J‘Hh: be" 133 APPENDIX TABLE II.-— Continued. Diet No. Fraction Amount (g/lOOg diet) 46. Pepsin digested RSBM - ppt 47. Trypsin digested RSBM - ppt 48. Water-insoluble residue 49. Lipase digested RSBM - super 50. Undialyzed whey solution (pH 8.0 super) 51. APWS - 25% (NHu)2 sou ppt 52. APWS - 80% (NHu ) 280u ppt 53. APWS - 80% (NHu)QSOu ppt 5 x crystalline SBTI 54. 5 x crystalline SBTI 55. 5 x crystalline SBTI, 4 x 56. APWS — 80% (NHu )2 so super 57. Amylase digested 2R8814 - super 58. Pepsin digested RSBM — super 59. Trypsin digested RSBM - super 60. pH 4.4 super 61. pH 4.4 super (conc) 62. pH 4.4 super (conc) — heated 1 min super 63. pH 4.4 super.(conc) - heated 5 min super 64. Undialyzed whey solution (pH 8.0 super) 65. Dialyzed whey solution 66. APWS (undialyzed ppt) 67. APWS - super 68. 20% acetic acid treated RSBM — ppt 69. 20% acetic acid treated RSBM - super 70. Water-insoluble residue6 71. Autoclaved pH 4.4 super 72. Dialyzed acetic acid super 73. 20% acetic acid extract of HRSBM 74. 20% acetic acid super, heated 1 min 75. Neutralized, dialyzed acetic acid super, heated l min. 76. Acetic acid super, DEAE-cellulose super 77. D-L-methionine (HRSBM + met) 78. D—L—methionine (RSBM + met) 79. pH 4.4 super 80. G-50 fraction I 81. G-50 fraction II 82. G-50 fraction III 88. 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