THE BKCLCGICAL VALUE OF RAW AND HEATED SOYBEAN PROTEEN‘S Thesis ‘or the Degree of M. 5. MICHIGAN STATE UNIVERSITY Suparb Pusobha 1966 THESIS LIBRARY Michigan State University ABSTRACT THE BIOLOGICAL VALUE OF RAW AND HEATED SOYBEAN PROTEINS by Suparb Pusobha Osborne and Mendel found that ground raw soybeans con— tained a protein of low nutritive value. The presence of heat-labile trypsin inhibitor in raw soybeans was a cause of low nutritive value for rats. The destruction of trypsin inhibitor by autoclaving resulted in increased biological value. Alpha protein, the product from soybeans under this study, was autoclaved at 121°C (15 pounds pressure) for four hours. The biological value of raw and heated soybean proteins (alpha protein) was determined by protein digestion and metabolism. Using the growth and maintenance utilization of dietary protein according to Barnes et a1. and the nitrogen balance method by Mitchell, raw soybean protein provided a higher biological value than heated soybean protein. The lower biological value for rats upon feeding heated soybean diet may be caused by poor absorption of protein. The digestibility of protein and lysine has been observed. The results indicate that heated soybean protein was poorly Suparb Pusobha absorbed by rats as compared to raw soybean protein. The low digestibility of lysine in heated soybean protein was also observed. Evans and Butts observed that excessive heat treatment destroyed and inactivated amino acids of soybean meal. The effect of autoclaving on lysine content in heated soy- bean protein was studied. The total lysine content in raw soybean protein is slightly lower than in heated soybean protein. From further observation on the availability of lysine, the results show that raw soybean protein contains more available lysine than heated soybean protein. The excessive heat treatment of soybean protein by autoclaving at 15 pounds pressure for four hours resulted in decreased available lysine which is considered to be the cause of low biological value in rats. THE BIOLOGICAL VALUE OF RAW AND HEATED SOYBEAN PROTEINS BY Suparb Pusobha A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry 1966 ACKNOWLEDGMENTS I have a sincere appreciation and sense of gratitude to Dr. R. J. Evans and Dr. S. L. Bandemer for their en- couragement, assistance, and criticisms. Their invaluable kindness, infinite patience, and helpful technical advice throughout the duration of my present studies can never be adequately acknowledged. Also, I should like to extend my sincere thanks to Dr. R. G. Hansen for his help, encouragement and suggestions. Appreciation is also extended to Dr. E. J. Benne, Dr. R. W. Leucke, and Dr. P. Kindel for the use of laboratory Space, equipment and chemicals. The author is deeply grateful to the Food and Agri- culture Organization of the United Nations and Biochemistry Department for financial support. I wish to thank Dr. K. Lawton and Mr. R. E. Osterbur for their help throughout the duration of fellowships. Cooperation extended by Mr. L. J. Klever, Betty Baltzer, Elizabeth Linden, Lester Hoag, Patricia Gawarecki, John Grieg Gail Shaw, and other colleagues in the laboratory, is highly appreciated. ii INTRODUCTION. . . EXPERIMENTAL. . RESULTS AND DISCUSSION. SUMMARY . . . . . LITERATURE CITED. TABLE OF CONTENTS iii Page 14 41 43 TABLE 10. 11. 12. 15. 14. 15. LIST OF TABLES The Biological Value of Raw and Heated Soybean Proteins for Growing Rats (Experiment I) . . . The Biological Value of Raw and Heated Soybean Proteins for Growing Rats (Experiment II). . . The Biological Value of Raw and Heated Soybean( Proteins for Adult Rats (Experiment III) . . The Biological Value of Raw and Heated Soybean Protein as Measured by Growth Study with Growing Rats . . . . . . . . . . . . . . . . . . . . . The Biological Value of Raw and Heated Soybean Protein as Measured by Growth Study with Growing Rats O O O O O O O O O O O O O I O O O O O O O O The Biological Value of Raw and Heated Soybean Protein as Measured by Growth Study with Adult Rats . . . . . . . . . . . . . . . . . . . . . Weight of Growing Rats Fed Raw and Heated Soy- bean Proteins. . O O O O O O O O O O O I O 0 Weight of Growing Rats Fed Raw and Heated Soyh bean Proteins. . . . . . . . . . . . . . . . . Weight of Adult Rats Fed Raw and Heated Soybean Proteins . . . . . . . . . . . . . . . . . . . Protein Digestibility by Growing Rats. . . . Protein Digestibility by Growing Rats. Protein Digestibility by Adult Rats. . . . . Lysine Digestibility by Growing Rats . . . Lysine Digestibility by Adult Rats . . . . . . The Effect of Heat on Lysine Content . . . iv Page 15 16 17 21 22 25 24 25 26 29 50 51 35 54 57 INTRODUCTION There are numerous sources of plant protein that can be used for human nutrition. Leguminous seeds are known to be a better source of protein than other kinds of seeds, and soybeans are the common legumes. Mendel and Fine (1912) found that soybean nitrogen was well utilized by man and dog. Jones and Devine (1944) reported that soybean contains pro- tein of high nutritive value and offensan excellent means of supplying dietary protein to extend and partially replace protein foods of animal origin. There are now available isolated soybean proteins specially prepared to contain the high value of protein in nutrition; soybean oil meal and soybean protein are examples. Several investigators have re— ported that the use of heat in the preparation of certain foods definitely alters the nutritive value of the protein. Soybean protein has been shown to be low in available methionine (Grau and Kamei 1950). The processing of high- protein foods should be controlled so that the damage to the methionine and other amino acids is minimized. The methods of preparation of soybean protein affect the nutritive value of soybean protein products. Standal (1965) estimated the net protein utilization of several oriental foods from soy— beans. In order to use any source of protein most effectively we need to know the biological value of that food. The biological or nutritive value of a protein as the term was applied originally by Thomas (1919), referred to the utilization by the body of the products of protein di— gestion. The biological value was expressed as the percentage of the absorbed nitrogen which was retained by the body for repair or the construction of nitrogenous tissue. That variation in biological value of protein depends on the amino acid composition and physical nature of protein, was emphasized by Rubner in 1897. A change in distribution of the essential amino acids as a result of different kinds of treatment could alter the biological value of protein. Raw soybean meal has been recognized as an inferior source of dietary protein. Osborne and Mendel (1917) showed that raw soybeans had a low nutritive value for rats, and that cooking them for three hours greatly increased this value. They attributed this increase in nutritive value to an increase in food consumption and to improved nitrogen absorption. Mitchell and Villagas (1925), Mitchell, Simmons and Parson (1921), and Shrewsbury and Bratzler (1955) reported experimental evidence supported by the fact that the raw soybean contains a pro- tein of low nutritive value. Hayward, Steenbock and Bohstedt (1956) concluded from their study that the low nutritive value of the raw soybean diet was not due to unpalatability but was caused by the un- availability of some essential protein fraction, and the appli— cation of heat to the raw soybean made this fraction available for absorption and metabolic use. The importance of amino acid content of the raw soybean meal was involved in the report of Fisher and Johnson (1958), and Booth (1960). They reported that a supplementation of Specific amino acid was effective in overcoming the growth depression of raw soybean meal in chicks and rats. They believed that the unavailability of amino acids in raw soybean meal resulted in the poor growth. Ham and Sandstedt (1944), Bowman (1944), Borchers (1965), and Borchers et a1. (1965) have reported the presence of a trypsin inhibitor in soybean meal. Borchers (1961) discussed in his report that the trypsin inhibitor is the cause of un- availability of some amino acids in raw soybean meal. Growth depression in chicks fed a raw soybean diet has also been studied by Liener (1962). That raw soybean inhibited the proteolytic activity in the small intestine in young chick was observed by Alumot and Nitson (1961). Several investigators believed that the destruction of this enzyme inhibitor by heat accounts for the increase in nutritive value of soybean meal. That cooking increased the digestibility of the soybean oil meal was observed by Shrews- bury and co-workers (1952). Their theory was that heating caused the removal or destruction of certain materials of toxic nature in raw soybeans. Waterman and Johns (1921) also re- ported the increased digestion of phaseolin after heating. However, heating the beans resulted in more than ten percent loss of amino acids and a greater loss under severe heating (Bandemer and Evans 1965). The adverse effect of heat upon the nutritive value of cereal proteins was demonstrated by Morgan and King (1926). The adverse effect of excessive amounts of heat treatment on soybean protein has been reported by Mussehl (1942), Bird and Burkhardt (1945), Evans and McGinnis (1946), Clandinin (1947), Evans, McGinnis and St. John (1947), and Evans and Butts (1949). Upon autoclaving, the lysine content was found to be limiting in roasted peanut meal (McOsker 1962). The effect of excessive heat treatment may have caused the destruction of lysine (Evans and Butts 1948), and low available lysine (Woodham and Dawson 1966). The purpose of this study is to determine the biological value of raw and heated soybean proteins. The observation may have some practical implications in certain countries when there is a shortage of animal protein but plenty of soybean products. Another application is the degree of heat treatment on soybean products in order to obtain the highest biological value of protein in nutrition. EXPERIMENTAL Measurement of protein value of foods by the biological methods according to Mitchell (1942) is determined by protein digestion and metabolism. Protein metabolism is a standard method for the estima— tion of biological value of protein (McCollum, Orent—Keiles and Day 1959). There are two general procedures that can be used, one is the nitrogen balance method and the other is the growth study. The nitrogen balance method for determining the biological value of proteins as described by Mitchell (1924), involves the direct determination of the amount of nitrogen in the feces and in the urine of rats. The biological value of the protein is taken as the percentage of absorbed nitrogen (nitrogen intake minus fecal nitrogen of dietary origin) that is not eliminated in the urine. The formula used by Mitchell (1924), Cahill and Smith (1948), and Boas Fixen (1955) represents the biological value Retained Food N Absorbed Food N x 100' as the ratio Raw and heated soybean proteins1 were used in this study as nitrogen sources for the rat diet. Heated soybean protein was prepared by autoclaving in a shallow pan at 1210C 1Alpha protein obtained from Nutritional Biochemical Corp. (15 pounds pressure) for four hours. The autoclaved sample was allowed to dry at room temperature. For the nitrogen balance method the studies consisted of three experiments. The first and the second experiments were stud- ied with growing rats, weighing 80-110 grams and 140—200 grams, respectively. The third experiment was studied with adult rats, weighing 500-550 grams. In rat feeding experiments, raw and heated soybean pro- teins were fed in a basal ration in sufficient amounts to supply ten percent protein. The basal diet was composed of soybean protein 11.4, corn oil 6, Hegsted salt mixture (1941), 4, vitamin fortification mixture1 2, sucrose 50, and corn starch 46.6. The protein of the ration was calculated from the nitro- gen value (N x 6.25). Nitrogen was determined by macro- Kjeldahl method (AOAC 1960). The procedure was as follows: One gram sample was weighed into the digestion flask. To the flask were added one to two grams of copper sulphate, four grams of potassium sulphate and 25 milliliters of concentrated sulphuric acid. The contents were gently swirled to mix and heated on the digestion rack for 2.5 hours. To the cooled '1The vitamin fortification mixture used in the prepara- tion of diet supplies the following vitamins in mg/100 g of diet: vitamin A concentrate (200,000 units per gram), 9.0; vitamin D concentrate (400,000 units per gram), 5.0; alpha tocopherol 10.0; ascorbic acid 90.0; inositol 10.0; choline chloride 150.0; menadione 4.5; p-aminobenzoic acid 10.0; niacin 9.0; riboflavin 2.0; calcium pantothenate 6.0; biotin 0.04; folic acid 0.18; vitamin B12 0.0027. mixture were added 200 milliliters of distilled water and three drops of mineral oil. The flask was swirled to dis- solve all solid material. A smallznmmnnzof zinc granules and 100 milliliterscfif40 percent sodium hydroxide were then added. The mixture was distilled on the distillation unit, and liberated ammonia was captured in a 500 ml-Erlenmeyer flask containing ten milliliters of 0.2N sulphuric acid containing three drops of methyl red as the indicator. The distillation was continued until the volume in the Erlenmeyer flask was about 200 milliliters. The excess sulphuric acid was titrated with 0.1N sodium hydroxide. The percentage of nitrogen was calcu- lated as follows: Since one milliliter of 0.2N sulphuric acid =2‘.81 milligram N, then the percentage of nitrogen ml 0.2N sulphuric acid x 2.81 wt of sample (mgs) x 100 . The rat feeding experiment was conducted as follows: weanling albino male rats were fed a commercial diet for one month until they weighed 80-110 grams. Rats were divided into two groups of eight rats, each housed in individual wire metabolic cages, and each group was equalized as nearly as possible for the initial weight. Food and water were supplied .ad libitum. Body weights were taken at the beginning and the end of each experiment. The animals were fed with the rations under study for an eight day period and the food consumption recorded daily. Feces and urine were collected for the last five days of the experiment. During the collection period the urine of each rat was transferred daily from the collector to collecting bottle and kept in the cold room. At the end of the7collection period the feces were dried at 100°C for 24 hours and ground in a porcelain mortar. The samples were then analyzed for nitrogen and lysine. The total urine col- lections were made up to a suitable volume, from which an aliquot was analyzed for nitrogen and lysine. The amounts of nitrogen in rat feces were determined by micro-Kjeldahl procedure (AOAC 1960) as follows: Ten milli- gram samples were accurately weighed in a small aluminum planchet and carefully transferred into a digestion flask. To the flask were added 0.5 gram of potassium sulphate, 1.0 milliliter of 10 percent copper sulphate solution, 2.0 milli- liters of concentratedsulphuric acid and two glass beads. The contents in the flask were heated on the digestion rack until the solution was clear green. The flask was cooled, three drops of 50 percent hydrogen peroxide were added and digestion was continued for 15 minutes. The cooled mixture was dissolved in a small amount of water placed on the micro— Kjeldahl distillation apparatus, about 5-6 milliliters of 40 percent sodium hydroxide added until the liquid contents were a permanent black and then heated. Liberated ammonia was captured in a 25-ml Erlenmeyer flask containing 20 milli— liters of two percent boric acid and six drops of one percent bromcresol green in alcohol as indicator. The distillation was continued until the volume of the Erlenmeyer flask was approximately 50 milliliters. The distillate was titrated with 0.0145N sulphuric acid. The percentage of nitrogen was calculated as follows: Since one milliliter of 0.0145N sulphuric acid = 0.202 milligram N, then the percentage of ml 0.0145N sulphuric acid x 0.202 wt of sample (mg) x 100 ° nitrogen = The percentage of nitrogen in urine was determined by the macro-Kjeldahl procedure as described previously. Two milliliters of the diluted sample were taken for each de- termination. In order to determine the value of protein for the pro- motion of growth, the general method was the actual measure- ment of weight gained occurring with a given intake of protein. Osborne, Mendel and Ferry's method (1919) relates the growth obtained to the intake of protein, and express the biological value of protein by the ratio: Biological value = Gain in Weight Intake of Protein' Digestibility is considered to be a good index in nutritional value of protein. The digestive efficiency or utilization of dietary protein is measured by the proportion of the intake of nitrogen that is not excreted in the feces. The protein digestibility in percentage was calculated by the formula: N Intake - Fecal N N Intake X 100 ° Protein Digestibility = The decreased protein nutritive value with higher auto- claving temperatures, as studied by Evans and McGinnis (1946) was due to lower methionine availability, although the 10 availability of other amino acids may also have been de— creased. The purpose of this experiment is to determine the effect of heat on lysine content in raw and heated soybean prEteins which might be the cause of low biological value of these proteins. The total lysine content in raw and heated soybean pro- teins was determined by the micro method of Selim (1965). The procedure is as follows: To a 12x100 mm test tube, were added ten milligram sample and one milliliter of 6N hydro- chloric acid. The tube was sealed off with an oxygen flame and incubated in an oven at 110°C for 24 hours. The sealed tube was Opened, the contentswere quantitatively transferred into a 25-ml beaker, and the acid removed by evaporating once to dryness on a steam bath. The dried residue was dis- solved in two milliliters of borate buffer. The aliquot of 0.6 milliliter was pipetted into a 12 ml centrifuge tube, 1 was added and one milliliter of copper phosphate suspension the mixture was well stirred with a glass rod. The centrifuge tube was capped and shaken mechanically for five minutes, then centrifuged for five minutes. One milliliter aliquot of the lCopperphOSphate suspension--To 40 milliliters of sodium phOSphate solution (68.5 gm/1000 ml), were added 20 milliliters of c0pper chloride with swirling. The suspension was centrifuged for five minutes. The precipitate was washed twice by resuSpension in 60 milliliters of borate solution, followed by centrifuging. The washed precipitate was sus- pended in 100 milliliters of borate buffer. Copper phOSphate suspension stored in glass-stoppered flask up to four days gave maximum color with alanine, but the color-producing capacity decreased slightly after 10 days. 11 clear supernatant solution was transferred to a 17x150 mm test tube, 2.5 milligrams of 1-fluoro-2,4-dinitrobenzene (FDNB) in 0.02 milliliter of methanol was added and the mix; ture was mechanically shaken for one hour at 40°C in the dark incubating room. The mixture was acidified by the addition of two milliliter of 2N hydrochloric acid, and hydrogen sulphide was then bubbled through for two minutes. The mix- ture was transferred quantitatively to a 12-ml centrifuge tube, and centrifuged. The supernatant was poured off and the copper sulphide precipitate was washed twice with 1N hydro- chloric acid and the washing combined with the supernatant solution. The hydrogen sulphide was removed from the combined solution by gentle aeration for one minute. The solution was transferred to a 104ml volumetric flask and diluted to volume with 1N hydrochloric acid. e-DNP lysine was extracted from the solution with 10 milliliters peroxide-free ether in a 60 ml dry separatory funnel and the extraction was repeated twice with 5 milliliter portions of ether. The ether was removed from the 1N hydrochloric acid solution by gently bubbling air through for three minutes. The absorbance of the final yellow solution was read at 590 mu in a Beckman Model B Spectrophotometer against a 1N hydrochloric acid blank. The concentration of lysine was calculated by comparing the readings to those of standard solutions of pure crystalline e-DNP lysine in 1N hydrochloric acid. 12 The digestibility of lysine was studied by determining the amount of lysine in rat feces by the micro-method described above. A ten milligram sample of feces was taken for the determination. According to the study of Borchers (1961) the trypsin ink hibitor may be the cause of an unavailability of lysine in raw soybean protein. However, the effect of heat may have re- sulted in the unavailability of lysine in heated soybean pro- tein. The purpose of this experiment is to determine the available lysine content in raw and heated soybean proteins and compare the results to the biological value. The available lysine content in soybean protein was determined by the method of Carpenter and Ellinger (1955) as follows: A 0.55 gram sample was dispersed in eight milli- liters of ten percent (w/v) sodium bicarbonate in a 50-ml Erlenmeyer flask. To the flask was added 0.5 ml of 1-f1uoro— 2,4-dinitrobenzene (FDNB) in 12 milliliters ethanol and it was then shaken for two hours. The ethanol was evaporated on the steam bath. Twenty-four milliliters of 5.5N hydro- chloric acid were added and the contents were autoclaved at 1210C (15 pounds pressure) for six hours. The cooled contents were filtered with washing into a 50 ml volumetric flask and adjusted to volume with distilled water. A five milliliter aliquot was extracted four times with ether, to the solution was added with 1 milliliter glacial acetic acid and adjusted to pH 5.0 with 2N sodium hydroxide and then diluted to 50 15 milliliters. The absorbency was read at 459 mu in a Beckman Model B Spectrophotometer. Repeating the procedure without FDNB gave the blank. The concentration of available lysine was calculated by comparing the reading to 0.1 mg/ml of e-DNP lysine standard in 1N hydrochloric acid. RESULTS AND DISCUSSION The biological values of raw and heated soybean proteins obtained with growing rats are shown in Tables 1 and 2, and with adult rats in Table 5. In comparing young to adult rats the values with young rats were found to be greater than with the adults for both raw and heated soybean proteins. In experiment I, the starting rat weight was 80 to 110 grams and the biological value was found to be 55.79 for raw and 18.25 for the heated soybean proteins; in experiment II, the starting rat weight was 140-200 grams and the biological value was found to be 24.75 for raw and 18.80 for the heated soybean proteins. In experiment III, the starting rat weight was 500—550 grams and the biological value was found to be 8.75 and 5.49 for the raw and heated soybean proteins, respectively. The results indicate that the younger rat has a greater nitrogen requirement for growth and maintenance utilization, as there is a large amount of nitrogen retained in the body. The older rat requires a*less amount of nitrogen for maintenance and very little for growth utilization, so the excess nitrogen above these requirements is excreted into thenurine. The small nitrogen retention results in lowering the biological value, as is seen from the equation for calculating this value. 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With the growing rats the retained nitrogen is utilized for the development of body protein and maintenance of the nitrogenous integrity of the tissue whereas in the adult animal the retained nitrogen is utilized for maintenance only. According to Mitchell (1924), increasing the level of nitrogen intake when there is a decline in the absorbed nitrogen that is utilized for maintenance results in a fall in the biological value. The excretion of urine nitrogen on comparable nitrogen intake varied with the body weight of the rat, the larger rat having the larger excretion. This might be another cause of a decrease in the biological value of soybean protein in a comparison between growing and adult rats. Comparing the biological value of raw and heated soybean proteins, it appears that raw soybean protein provides higher nutritive value than heated both with growing and adult rats. The results indicate that prolonged heating of soybean protein in an autoclave at fifteen pounds pressure reduces the nutri- tive value of protein. This finding is in agreement with the study of Parsons (1945), in which the excessive heating of legumes resulted in a loss of nutritive value. She explained that there are two opposite effects produced by moist heat; one, a beneficial effect on the nutritive value of protein which is more apparent during moderate degree of heating; the other, unfavorable which appears to overtake and then surpass 19 the beneficial changes when autoclaving is prolonged or the pressure raised. The results are also supported by the study of Clandinin (1947). In contrast with this study, Everson and Heckert (1944) found that heating for 45 minutes at 15 pounds pressure im- proved the nutritive value of all legumes studied. Evans and McGinnis (1946) observed the nutritive value of raw soybean meal protein was slightly increased by autoclaving for 50 minutes at 150°C. Toasting also increased the biological value of soya extraction meal as studied by Nehring, Bock and Wfinsche (1964). Hayward, Steenbock and Bohstedt (1956) studied the biological value of raw and heated soybean meal at 140- 1500C for 2.5 minutes. They found that the apparent biological values of raw and heated soybean meal were, respectively, 25 and 55 when the rats were fed at 2.89 and 2.87 percent of the nitrogen in the diets. From our experiment the biological values of raw and heated soybean proteins are 54 and 25 when the rats were fed at 1.70 and 1.55 percent of nitrogen in the diets. In the statement of Hayward, Steenbock and Bohstedt (1956), "apparent" is used to designate the fact that the values have not been corrected for endogenous nitrogen accord- ing to the procedure of Mitchell and Villegas (1925) and Mitchell (1924). The t values for Tables 1, 2,1and 5 show that there is no significant difference between the biological values of 20 raw and heated soybean proteins as measured by the nitrogen balance method on growing and adult rats. The short period of time for the rat feeding experiment could be the cause of this finding. The adverse effect of excessive heat treatment which caused the decreased biological value of the protein by the nitrogen balance method partially explains the decreased biological value of the protein in the growth studies. The biological value of raw and heated soybean proteins as measured by the growth of rats are given in Tables 4 and 5 for growing rats, and Table 6 for the adult rats. Rats fed raw soybean protein as a source of nitrogen gave a higher biological value than rats fed heated soybean protein. The t values (Tables 4 and 5) show that there is a great difference between the biological values of raw and heated soybean pro- teins by the growth study on growing rats. The results indi— cate that available lysine in heated soybean protein is deficient for growth and maintenance utilization in growing rats. No significant difference was found by the growth study on adult rats (Table 6) fed raw and heated soybean proteins. An increase in the amount of total food intake by rats fed heated soybean protein resulted in adequacy of available lysine for maintenance. The effect of heat treatment upon the avail— able lysine content will be discussed later. Considering the weight of rats from Tables 7, 8, and 9, rats fed raw soybean protein gained weight. On the other hand, 21 N.¢H u 05Hm> u «no.6: mu am.N on ma mwm.ou NI mm.m mm ma mmfi.dl ml hd.¢ me ea mmowda ohw.dl ml mm.m mm ma Umummm .. mite a mm.m mm NH mmo.NI nu mm.m mm ad mmN.OJ a: m>.m mm OH o o mm.m 06 m m>m.0 m m¢.¢ Nd m mmm.o N ON.m me n o o hm.N mm m emm.o mmm.o m oa.m we m 3mm mNm.o m m>.¢ me e mNm.o m m¢.m am m mm>.o d ON.m me N Omd.o N mm.¢ «w H .>.m meam> Em Em Em Hones: :Hmuoum mmmum>¢ HMUHmoHOHm museummmflfl mRMuCH mxmuCH umm emmnwom usmflmz campoum boom mumm mcHBOHO npfl3 MGDDm £u30u0 ND Umusmmmz mm mcflmuoum cmmnmom Umummm new 3mm mo m5am> HMUHmoHOHm one. d OHQMB 22 HN.OH u msam> u mom.O| ml mm.mH OHH mH Nmm.HI OH: OH.O Om HH mmm.OI ml Om.m mm, MH emm.OI OI mN.OH OOH NH mmO.HI Umumwm mNO.OI ml Nm.HH mNH HH HH0.0 m mm.OH OHH OH HmO.HI OH: m>.m HOH m mmO.¢I mHI O¢.¢ me Q HOm.OI «I wN.MH mNH h ONm.O OH NN.mH HmH O OHH.O O O >0.0H hmH m 3mm OHH.OI NI HM.¢H mMH H mmH.O OH mH.ON OmH m mmm.N MH N¢.m Hm N HmH.OI NI >N.MH mNH H .>.m msam> Em Em Em HmQEsc :kuoum mmmum>¢ HMUHmoHOHm mocwHOMMHO wxmucH mxmusH umm cwmnmom uanm3 chuoum poom mumm mcHBOHO suH3 >U5um nuBOHO xnvpmu5mmm2 mm mchuon cmmflmom Umummm Use 3mm mo msHm> HMUHmoHOHm one .m magma 25 mm.0 n msam> u MN>.HI ONI Hm.HH HHH mH mmm.HI mHI mm.m Hm HH 000.H- HHr 00.0H 00H ‘ 0H 0mm.ou mm>.H 5H mw.m mm NH Umummm OO>.OI ml mH.OH mOH HH mHH.HI HHI mm.m OOH OH m>>.O OH mO.NH mnH m mH>.O m NO.NH «NH m O>¢;OI OI >>.NH mHH n NmH.O N NH.mH HNH O OHM.OI mmH.HI NH: HO.m H» m 3mm mH0.0| mu mn.NH NHH e Hmm.O| ml NN.OH Hm m mmN.H ON >¢.mH HHH N Hem.OI HI m>.OH mm H .>.m wsHm> Em Em Em umnEse chuoum mmmnm>¢ HMUHmoHOHm mocmnmmme mxmucH meucH umm cmmnwom uanmz chuoum noon mpg 0.33. :33 >026 susono ha pmusmmwz mm meHmuoum cmmnmom pmummm new 3mm mo 09Hm> HMUHmoHOHm one .O mHnme .mmHmEmm HHmEm mo mcmmE cmmBuwQ mUGmUHMHcmHO* Om.> 05Hm> u OI Om Om OO OH NI NHH HHH OO OH OI 5O NO OH HH *OH.H H.O.NI OI nOH OHH OO OH pmummm H mO OO ON NH nI Om OOH OO HH HI HO Om mO OH O OOH OOH OH O O NO mO NH O N HHH NHH OH H O 5O HO ON O *O#.H.HVO.N O HO OO OH O 3mm O OOH OOH OH H O HHH OOH HO O H OHH OOH OH N N Om Om HH H mocwummeO Em (Em Bm Em HwnEdc :Hmuoum uanm3 mnemHOMMHO mcHUcm mcHuumum mxmucH umm cmmflhom mmmnm>¢ uzmHmS uSmHmS uanmS Ooom mchuoum ammnmom Umumwm new 3mm Umm mumm mcHBOHO mo usmHmz .5 mHnma 25 .mmHmEmm HHmEm mo mcmmE cmm3umn mocmoHMHcmfim* H0.m u msam> u OI OHN HNN OHH OH OHI NOH NbH OO HH ml OHH NOH mm OH OI NON OON OOH NH *m.O H.O.>I Owummm OI OOH HHH ONH HH m NOH OOH OHH OH OHI HhH wOH HOH m OHI HNH NHH OH O HI OOH NmH ONH s OH NNN nHN HOH O O OHN OHN hOH O *m.O H 0.0 NI OOH OOH OOH H 3mm OH NHN NON OmH O OH OOH OHH HO N NI OOH OOH ONH H mocmnwmmHO .Em Em Em Em HmQEdc :Hmuoum uanmB wocmumNMHU meHOcm meHunmum mxmucH umm cmmflwom mmmum>< uanmB uanm3. uanOS voom maHmuoum cmmnmom Omumwm cam 3mm Omm mumm mCHBOHO mo usmHmz .O mHQma .mmHmEmm HHmEm mo memmE cwm3umn mocmonHcmHO* OO.O n msHm> u ONI NOO NNO HHH OH OHI OhN NON HO HH HHI OOO HHO OOH OH *O.O.fl H.OI 5H ONO OOO OO NH Umummm . OI ONO HOO OOH HH HHI OHO HNO OOH OH OH NwO NOO OOH O O OHO OOO HNH O OI OHO OnO OHH n N OOO OOO HNH O NHI OOO ONO He O *0.0 H.H.NI OI OOO OOO NHH H 3mm OI OOO OOO HO O ON OOO OHO HHH N HI HHO OHO OO H mocmummmHU Em Em Em Em MODES: aHmuoum uanm3 moamHmmeO mCHpcm mcHHHMum mxmucH umm GMOQ>OO mmmnm>¢ uanUB uanmBV uanmS poom mCHmuoum emmnmom Omummm use 3mm Umm mumm uHDUO mo uanmS .O mHQMB 27 rats fed heated soybean protein lost weight.’ The average weight change of rats fed raw and heated soybean proteins was found to be 2.9 and -2.9 in experiment I, 5.6 and —7.5 in experiment II, and —2.4 and -5.4 in experiment III, respectively. The t values (Tables 7 and 8) show that there is a significant difference between the change in weight of growing rats fed raw and heated soybean proteins but no sig- nificant difference for adult rats fed these same proteins. The results of this finding are comparable to the study of Barnes (1946) in which the poorly heated soyflour supported a maximum protein gain that was lower than the crude soyflour. The results are also substantiated by the growth study of Bird and Buckhard (1945). They compared the nutritive value of meals processed by autoclaving soyflakes for varying lengths of time at twenty pounds pressure. Their graphs showed that flakes processed from four to fifteen minutes gave the most satisfactory gains. From Tables 7, 8, and 9, the total food intake of rats fed heated soybean protein was considerably decreased from the intake of those on raw soybean protein. A low feeding value in chicks fed heated solvent extracted soybean flakes was shown in a report of Clandinin (1947). The greater food in- take of rats fed raw soybean protein is in agreement with the observation of Heckler (1965), that dehulled soybean flour enhances the appetite of rats. 28 The growth inhibition of rats fed heated soybean protein may have been cuased by several possible reasons: a lower available methionine (Evans and McGinnis 1946); the destruc- tion or inactivation of essential amino acids (Evans and Butts 1948); a lower lysine and methionine (Clandinin 1947); a limit- ing of lysine, threonine and tyrosine (McOsker 1962); and a decreased available lysine (Woodham and Dawson 1966). From the nitrogen balance method the low biological value of heated soybean protein may have been caused by decreased digestibility. The decreased digestibility might have been caused by damage to some amino acid in soybean protein. The results in Tables 10, 11, and 12 indicate that nitrogen was less absorbed in the animals fed heated soybean protein com- pared to those fed raw soybean protein for both growing and adult rats. The smaller nitrogen absorption of heated soybean protein may have been caused by the unavailability of certain amino acids which will be discussed later. The decreased nitrogen digestibility from this finding is confirmed by the study of Evans, McGinnis and St. John (1947). They observed that autoclaving raw soybean oil meal for 50 minutes at 1000C increased digestion by the chick but autoclaving at 1500C for 60 minutes decreased protein digestion. Mitchell (1949) found that during the heating process the digestibility and the biological value of six different kinds of seed and flour was decreased. 29 Table 10. Protein Digestibility by Growing Rats Percent Average- Soybean Rat Food N Fecal N digesti- digesti- protein number mg mg bility bility % 1 696.6 128.5 81.58 2 852.5 110.0 86.79 5 1056.4 170.6 85.54 Raw 4 764.6 51.7 95.24 88.28 5 815.5 62.5 92.56 6 475.7 56.8 88.06 7 852.5 75.5 91.20 8 715.6 64.0 91.05 9 620.4 94.7 84.74 10 604.9 126 6 79.08 11 542.9 105.7 80.55 12 556.7 56.7 84.10 Heated 13 542 9 178.8 67.07 78°91 14 666 9 155.5 77.01 15 589.4 114.2 80.22 16 465.5 100.1 78.49 50‘ Table 11. Protein Digestibility by Growing Rats Apparent Average Soybean Rat Food N Fecal N fiigesti- digesti— protein number mg mg bility % bility % 1 2125.8 211.5 90.05 2 866.5 167.9 80.62 5 5228.1 540.5 89.46 4 2295.7 208.6 90.91 Raw 5 2667.4 280.0 89.50 88°08 6 5075.2 520.9 89.56 7 2125.8 287.7 86.45 8 715.5 150.9 81.65 9 1566.5 254.1 85.78 10 1706.1 544.5 79.82 11 1907.7 465.0 75.65 Heated 12 1644.1 441 5 75.15 77°29 15 1555.5' 577.5 75.45 14 1025.7 265.5 74.06 15 2171.4 547.4 74.79 51 Table 12. Protein Digestibility by Adult Rats Apparent Average Soybean Rat Food N Fecal N digesti— digesti— protein number mg mg bility % bility % 1 1725.1 281.6 85.68 2 2474.9 286.7 88.42 5 1655.5 500.5 81.62 4 1975.5 249.1 87.59 Raw 5 1281.1 144.4 88.75 85°42 6 2098.8 522.5 84.64 7 2045.7 558.1 85.46 8 1925.2 450.9 77.59 9 2062.8 595.5 80.95 10 1575.5 198.4 87.59 11 1628.6 508.5 81.07 Heated 12 1511.9 598.5 75.66 81'42 15 1752.8 268.0 84.71 14 1559.9 278.4 79.22 15 1856.9 245 9 86.76 52 A report of Clandinin (1947) showed that heating solvent extract soybean flakes in an autoclave at 15 pounds pressure for four hours resulted in a low feeding value for the chick. Supplementation with lysine and methionine sustained growth as with properly heated soybean oil meal. The result of this finding might be due to decreased biological availability of lysine in the meal which had been autoclaved for four hours at 15 pounds pressure. He also showed that the lysine in over- heated meal was less available to the chick than in properly heated meal. Evans and Butts (1948) reported that autoclaving caused an inactivation of lysine which renders it unavailable to enzymatic digestion in vitro. Lysine was determined by microbiological assay with Leuconostoc mesenteroides. The digestibility of lysine by rats from this study is presented in Tables 15 and 14. Raw soybean protein was di- gested better than heated soybean protein by growing rats, but there is a slightly greater digestion of heated soybean protein by adult rats. The results are comparable to the study of Nitson and Alumot (1964). They observed that younger chicks suffered from inhibition of the proteolytic activity longer than older chicks. Evans and McGinnis (1948) explained in their report that the drastic autoclaving procedure reduced the nutritive value of soybean oil meal in two ways; one is a partial destruction of cystine and lysine and the other is a decreased digestibility of lysine. 55 Table 15. Lysine Digestibility by Growing Rats Food Fecal Percent Average Soybean Rat lysine lysine ~Digesti- digesti- protein number mg mg bility bility % 1 562.5 120.8 66.68 2 147.9 62.1 58.01 5 551.0 112.5 79.62 Raw 4 591.5 82.1 79.05 74.41 5 455.5 105.5 76.85 6 524.9 109.4 79.16 7 562.5 66.9 81.54 8 128.8 45.7 64.52 9 282.8 101.9 65.97 10 508 0 115.7 62.45 11 544.4 165.0 52.09 Heated 54.55 12 296.8 146.6 50.61 15 277 2 158.2 50.14 14 184.8 100.6 45.56 15 592.0 215.7 45.48 54 Table 14. Lysine Digestibility by Adult Rats _ * _‘— Food Fecal Percent Average Soybean Rat lysine lysine Digesti- digesti- protein number mg mg bility bility % 1 268.8 76.1 71.69 2 599.4 95.0 76.21- 5 254.8 86.7 65.97 Raw 4 516.5 70.6 77.69 72.75 5 211.0 45.5 79.58 6 544.7 115.8 66.41 7 556.4 95.1 71.72 547.2 92 5 75.42 9 572 4 105.4 72.25 10 286 0 55.9 81.15 11 294.0 75.2 74.42 Heated 12 276.4 98.5 64.54 74°25 15 522.0 61.6 80 87 14 245.2 75.6 69.98 15 545.2 74.1 78.41 55 In contrast with this finding, raw soybean oil meal was an inferior source of dietary protein for the rat as well as other Species, as was reviewed by Borchers (1947), Liener (1950i and Griswold (1951). They failed to develOp a satis- factory explanation for the slow rate of gain on raw soybean oil meal when compared with properly heated soybean oil meal. Hayward, Steenbock and Bohstedt (1956) observed that the apparent digestibility of raw soybean oil meal and that heated at 140-1500C for 2.5 minutes was 80 and 81, reSpectively, when rats were fed with 2.89 and 2.87 percent of nitrogen value. The apparent digestibility of raw and heated soybean proteins from our experiments (Tables 10, 11, and 12) is 88 and 78 for growing rats and 85 and 81 for adult rats when fed 1.70 and 1.55 percent of nitrogen value, respectively. Evans and McGinnis (1948) observed that steaming apparently in- creased digestibility of the soybean oil meal protein, prob- ably by a destruction of the trypsin inhibitor. Borchers (1958) found that certain other amino acids in addition to methionine were less available in raw soybeans. In a further study, Borchers (1965) assumed that raw soybean growth inhibi— tory factor was acting as a block to threonine and valine catabolism. A recent study by Khayambashi and Lyman (1966) reported that the action of soybean trypsin inhibitor caused a loss of methionine, threonine, and valine from rats fed raw soybean meal. An extracted and purified preparation of this toxic substance from raw soybean flour was fed to rats by 56 Liener and Pallansch (1952) and they reported on the possible relationship of the toxic substance to the poor nutritive value of raw soybeans. In order to achieve the promotion of growth on rats the soybean protein was heated by autoclaving as described above. The results of heat treatment on the lysine composition of soybean protein are presented in Table 15. The lysine content was calculated based on 16 percent nitrogen. The total lysine content of raw and heated soybean proteins was 5.57 and 5.88 percent, respectively. The lysine content of heated soybean protein as compared to that raw soybean protein is Slightly higher. The multi—step procedure might be the cause of this finding. The results compare with the study of Eldred and ‘ Rodney (1946). They reported that chemical analysis of heated casein detected no decrease in the amount of lysine present. The change produced by heat appeared to be in the arrangement or unavailability of amino acids, that might be the cause of lower biological value. Bandemer and Evans (1965) determined the lysine content in raw and heated soybean meal by ion exchange chromatography and found that the percentage of lysine in raw and heated soybean meal to be 5.6 and 4.1, respectively. From the observation of Greaves, Morgan and Loveen (1958), they found that when lysine was added to the heated casein the biological value was definitely raised from 57 to 64. It must be concluded that lysine of the heated casein had been in- activated in some way and the lysine was the first amino acid 57 Table 15. The Effect of Heat on Lysine Content Percent Percent Percent Soybean Percent tOtal available available lysine protein nitrogen lysine lysine total lysine Raw 14.58 5.57 4.92 88.55 Heated 14.26 5.88 4.42 75.17 58 damaged when casein was heated at 1400C for 50 minutes. The lysine content was found to be the limiting factor in roasted as well as autoclaved peanut meal (McOsker 1962). Reisen (1947) found that lysine, arginine and tryptophansuffered partial destruction when soybean oil meal was autoclaved for four hours and 15 pounds pressure and about 50 percent of the lysine was destroyed. Evans and Butts (1948) observed that 40 percent of the lysine in soybean oil meal was destroyed by autoclaving at 1500C for 60 minutes. The lysine content was determined by microbiological method using Leuconostoc mesenteroides. Further study on the available lysine, shown in Table 15, indicate that raw soybean protein gave a greater biological value than heated soybean protein. The percentages of avail- able lysine based on the total lysine in the raw and heated soybean proteins were 88.5 and 75.2, respectively. A decrease in available lysine content might be the cause of deficiency for growing rats fed heated soybean protein. The results are corroborated with the study of Clandinin (1947). They showed that the lysine in overheated meal was less available to the chick than in properly heated meal. They found that there was no loss in total nitrogen, thus, the decrease in the amount of free amino nitrogen may be due to the formation of new anhydride linkage involving the e-amino group of lysine and available hydroxyl groups. The decrease in available lysine is confirmed by the observation of Woodham and Dawson (1966) 59 that the rapid destruction of trypsin inhibitor in the moist heated samples caused a decrease in available lysine. It was proposed by Greaves, Morgan and Loveen (1958) that the heat damage to a protein depends not only on the temperature and time of heating but also on the molecular structure of the protein. Harris and Mattill (1940) suggested that possibly heat treatment brings about combinations between the e-amino groups of lysine with free carboxyl, imino or hydroxyl groups, and that such linkages may not be broken by acid hydrolysis. It is assumed that the form in which lysine is liberated from such linkages by acid hydrolysis is not utilized by micro- organism. Denaturation by altering configuration might thus reduce both biological value and digestibility by damag- ing essential amino acids. The observation of Reisen (1947) indicate that excessive heat treatment resulted in further change which makes the meal less readily attacked by proteo- lytic enzymes. Evans and Butts (1948) found that autoclaving causes inactivation of lysine. There are two types of heat inactivation of lysine that apparently take place: one, a destruction of the lysine and the other, a binding of the lysine in some form which is not liberated by digestion in vivo or by hydrolysis in vitro but liberated by acid hydrolysis. They also explained that lysine combines with some other groups of the other amino acids of the protein. Probably the free carboxyl groups combine with the free amino groups, to form enzyme-resistant linkages. Further explanation by Lea 40 (1960) that lysine residues in the proteins of heat—processed foods whose e-amino groups are bound to other groups and so are unable to react, are likely also to be nutritionally un- available. At least two types of deterioration of available lysine can occur, the oxidative type predominating during mild heating, and non oxidative type during severe heating. Carpenter (1960) indicated that samples which had been heated and had shown decreased nutritional value in feeding tests also showed lower available lysine values by the chemical analysis. SUMMARY The biological value of raw and heated soybean proteins was studied by rat feeding experiments. Raw soybean protein gave a higher biological value than'heated soybean protein. The results with the nitrogen balance method were supported by the growth study. The low biological value of heated soy- bean protein appears to be due to the excessive heat-treatment which caused inactivation or destruction of essential amino acids in soybean protein. The digestibility of nitrogen and lysine by rats receiv— ing raw and heated soybean protein was observed. It is sug- gested that the low absorption of nitrogen and lysine in heated soybean protein may be due to the unavailable essential amino acids. It is postulated that the unavailability of lysine is a major factor in the low biological value of heated soybean protein. Further study by chemical means on the available lysine content in raw and heated soybean protein showed that raw soybean protein contains more available lysine than heated soybean protein. The results of this study Show that it was the adverse effect of excessive heat treatment which caused the low biological value of soybean protein. 41 LITERATURE CITED Alumot, E. and Z. Nitson. The influence of soybean anti- trypsin on the intestinal proteolysis of the chick. J. Nutr. 7_Z>_, 71 (1961). ’ Bandemer, S. L. and R. J. Evans. The amino acid composition of seeds. J. Agr. Food Chem. 11, 154 (1965). Barnes, Richard H., Mary J. Bates, and Jean E. Maack. The growth and maintenance utilization of dietary protein. J. Nutr. 52, 555 (1946). Bird, H. R. and G. J. Burkhardt. Factors affecting the nutritive value of soybean oil meal and soybean for chickens. Univ. of Md. Bull. 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