Relationship of fractions of soybeans and a crystalline soybean trypsin inhibitor to the effects of feeding unheated soybean meal to chicks.

Several experiments were conducted to study the effects of fractions isolated from unheated soybean meal on pancreatic hypertrophy, fat absorption, utili zation of non-fat components of the diet, and growth rate of chicks, to determine whether these measurements were affected by specific components of the meal. Un heated soybean meal was fractionated into a water-insoluble residue, pH 4.4 insoluble proteins, salts insoluble at pH 8, and whey proteins recovered by dialysis and lyophilization of the portion of the meal soluble at pH 4.4. When fed to chicks at levels of 1.3 to 1.9% of the diet, the soybean whey proteins markedly depressed growth rate, caused pancreatic hypertrophy, reduced fat absorption, and lowered metabolizable energy value of the diet. Whey proteins were batch separated on DEAE cellulose into 2 frac tions, one high in hemagglutinin activity and one high in trypsin inhibitor activity. Neither fraction when fed alone affected growth rate, fat absorption or metabolizable energy value of the diet to the same extent as a combination of the 2 fractions. A diet containing 0.6% of a crystalline Kunitz soybean trypsin inhibitor depressed growth rate of chicks and metabolizable energy value of the diet, caused pancreatic hyper trophy, but had no other undesirable effects. These experiments suggest that multiple factors are present in unheated soybean meal which cause the detrimental effects observed. Studies by Brambila et al. ( l ) and Nesheim et al. (2) showed that the absorption of dietary triglycA©rides and dietary free fatty acids was markedly depressed in chicks fed diets containing unheated soy bean meal, being most pronounced up to 3 weeks of age. Chicks 4 weeks of age or older showed little impairment of fat ab sorption. Supplementation of the diet con taining unheated soybean meal with crude or crystalline trypsin preparations (1), or with sodium taurocholate (3) restored fat absorption to normal in 2-week-old chicks. Despite the effectiveness of trypsin supple mentation, trypsin inhibitors per se do not appear to be responsible for poor fat ab sorption as feeding a highly purified tryp sin inhibitor from soybeans did not result in poor fat absorption (2). Reports by several investigators (4â€”6) have indicated that unheated soybean meal decreased chick growth rate, reduced the dietary metabolizable energy value, and produced pancreatic hypertrophy. Some of these effects could be caused by feeding chicks one of the trypsin-chymotrypsin in hibiting proteins in the soybean (2). The objective of the present investiga tion was to study the effects of fractions of unheated soybean meal on pancreatic hypertrophy, decreased fat absorption, de creased utilization of the non-fat portion of the diet, and poor growth of chicks in an attempt to determine whether these effects were caused by specific components of the meal. METHODS AND PROCEDURES Male Rhode Island Red X Barred Plym outh Rock chicks purchased from a com mercial hatchery were fed the basal diet (2) which contained heated soybean meal (50%), soybean oil (15%), and glucose as major components with methionine (0.3%), vitamin and mineral supplements. Received for publication July 6, 1965. 1These studies were supported in part by Public Health Service Research Grant no. AM-8202 from the National Institutes of Health. 2 Present address: Department of Biochemistry, St. Louis University, St. Louis, Missouri. 100 J. NUTRITION,88: '66 by gest on July 8, 2011 jn.nition.org D ow nladed fom FRACTIONATION OF UNHEATED SOYBEAN MEAL 101 When unheated soybean meal was included in the diet it replaced a portion of heated soybean meal. The total amount of soy bean meal was 50% of all diets. Solventextracted dehulled, soybean flakes obtained from a commercial soybean processing plant, at the step prior to removal of the solvent, were used as "unheated" meal in all experiments and for fractionations. The solvent was removed by evaporation into the air at ambient temperature. In experi ments 3 and 4, the heated soybean meal was prepared by autoclaving the raw meal at 107Â°for 30 minutes according to pro cedures described by Renner and Hill (4), while in the remaining experiments com mercially processed, dehulled soybean meal was used. Fat absorption and metabolizable energy (ME) value of diets were measured ac cording to procedures described by Renner and Hill (7) and Hill and Anderson (8). The metabolizable energy values reported in the tables are adjusted to a common de gree of fat absorption using a value of 9.4 kcal/g as gross energy of unabsorbed fat, to allow a comparison of the utiliza tion of non-fat components of the diets under conditions where fat absorption was not equal in all groups of chicks studied. The metabolizable energy values are all corrected to nitrogen equilibrium so that differences in nitrogen retention also do not affect comparisons of ME values of various diets. Fresh pancreas weights were determined immediately after killing the chicks. Pan creas weights given in the tables have been adjusted for differences in body weight among the treatments by analysis of covariance (9). Hemagglutinin activity was determined by the method of Liener (10). Trypsin inhibitor activity of soybean meal and various fractions was determined by measurement of the inhibition of hy drolysis of benzoyl-DL-arginine p-nitroanilide (11) by trypsin. The crystalline trypsin inhibitor3 used in experiment 4 is called the Kunitz inhibitor (12) in this pa^er. Chromatographie analysis of fractions from soybeans and trypsin inhibitor prepa rations was made on diethyl-amino-ethyl (DEAE) cellulose using gradient elution procedures described by Rackis et al. (13). Protein analysis of the column effluent was conducted on individual fractions by the procedure of Lowry (14), or automatically by analyzing column effluent directly by the Lowry method with an autoanalyzer.4 Batch fractionation of soybean whey proteins on DEAE cellulose was conducted as follows: DEAE cellulose (300 g) was suspended in 3 liters of distilled water and the pH adjusted to 7.6 with a saturated solution of KH2PO4. The suspension was poured on a large BA1⁄4chnerfunnel and fil tered. The cellulose was resuspended in 2 liters of solution of 0.034 M sodium chloride in 0.01 M phosphate buffer, pH 7.6. The suspension was filtered, and washed with the sodium chloride-buffer solution twice more. After washing, the DEAE cellulose was left on the filter and 75 g of soybean whey proteins dissolved in 750 ml of 0.034 M sodium chloride in 0.01 M phosphate buffer were added to the DEAE cellulose-packed funnel. The soy bean whey protein was washed into the DEAE cellulose on the funnel with one liter of the 0.034 M sodium chloride-buffer solution. The proteins not retained by the DEAE celluose at this salt concentration were washed from the funnel with three one-liter washings of 0.034 M sodium chlo ride-buffer solution. These proteins were called fraction 1. The receiving flasks were changed and the remainder of the proteins were eluted from the cellulose with three one-liter quantities of 0.5 M sodium chlo ride in 0.01 molar phosphate buffer. These proteins were called fraction 2. The eluted fractions were dialyzed at 4Â°against dis tilled water until barely detectable amounts of chloride ion remained in the dialyzate, and the dialyzate was frozen and lyophilized. This procedure was repeated 4 times to prepare a sufficient quantity for a feeding experiment. The 8 individual preparations were chromatographed on DEAE cellulose. The elution patterns for a given fraction were found to be similar for each preparation. The respective frac tions were combined. 3 Obtained from Nutritional Biochemicals Corpora tion. Cleveland, as 5X crystalline soybean trypsin inhibitor, lot no. 3358. 4 Technicon Chromatography Corporation, Chauncey, New York. by gest on July 8, 2011 jn.nition.org D ow nladed fom 102 J. D. GARLICH AND M. C. NESHEIM