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Trypsin Inhibitor. VI. Effect of Various Heating Periods on the Growth Promoting Value of Soybean Oil Meal for Chicks1
Trypsin Inhibitor. V I . Effect of Various Heating Periods on the Growth Promoting Value of Soybean Oil Meal for Chicks1 RAYMOND BORCHERS, C. W. ACKERSON, AND F. E. MUSSEHL
Departments of Agricultural Chemistry and Poultry Husbandry, Nebraska Agricultural Experiment Station, Lincoln (Received for publication March 1, 1948)
ALTHOUGH the superior value of -**• properly heated soybean oil meal over raw or improperly heated meal for the nutrition of the chick has been recognized since the work of Wilgus et al. (1936) and of Hayward et al. (1937), a precise definition of properly heated soybean oil meal has not been attained. In a recent publication, Clandinin et al. (1947) presented data indicating that soybean flakes autoclaved at 15 lbs. pressure for four to fifteen minutes gave the most satisfactory gains in chicks. These results were contradicted by the findings of Fritz, Kramke and Reed (1947) who reported from tests on turkey poults that the best biological value was obtained when ground raw soybeans were autoclaved at 15 lbs. pressure for 20 to 30 minutes. Westfall and Hauge (1947) stated that the growth efficiency of soybean flour for rats was inversely related to the quantity of trypsin inhibitor present in the soybean flour. Data presented by Borchers et al. (1947) showed that soybean dil meal auto1 Published with the approval of the Director as Paper No. 440, Journal Series, Nebraska Agricultural Experiment Station. Some of these data were submitted in an abstract to the 113th meeting of the American Chemical Society, Chicago, Illinois, April 19-23, 1948.
claved at 15 lbs pressure for four minutes still contained a large portion of the trypsin inhibitor and that autoclaving at 15 lbs. pressure for about 20 minutes was necessary to give complete destruction of the trypsin inhibitor. Although Ham, Sandstedt and Mussehl (1945) assumed that the trypsin inhibitor present in raw soybean oil meal was identical with the growth inhibiting factor, Klose, Hill and Fevold (1946) have pointed out that such an assumption has not been established. It, therefore, seemed of importance to us to obtain data bearing on the relationship between the growth promoting value of soybean oil meal for chicks and the amount of trypsin inhibitor remaining active in the meal after autoclaving for various time periods. For this purpose, seven samples of soybean oil meal were prepared: raw or unheated meal, and samples which were autoclaved at 15 lbs. pressure for 4, 10, 15, 20, 25, and 30 minutes, respectively. The first was chosen as a negative control, the 4, 10, and 15 minute autoclaving periods as representative of samples containing a part of the natural trypsin inhibitor present in soybeans and within the heating period suggested by Clandinin et al. (1947) as satisfactory, and the 20, 25, and
601
602
RAYMOND BORCHERS, C. W. ACKERSON AND F. E. MTJSSEHL
30 minute autoclaving periods as representative of samples containing no trypsin inhibitor and generally recognized as being an adequate heating period. These soybean oil meals were then incorporated into rations and fed to day-old chicks under controlled feed intake to six weeks of age. EXPERIMENTAL The composition of the basal ration used in this study is given in Table 1. TABLE 1.—Base ration Ingredient Yellow corn meal Corn gluten meal Wheat shorts Wheat bran Dehydrated alfalfa meal Meat scraps Sardine meal 1 Salt mix No. 4S Vitamin D carrier2 Dextrin Gum tragacanth Soybean oil meal (raw or heated, see (Table 2) Talc
Parts by weight 24 S 10 10 5 3 3 3 1 10 2 23 1
1 Limestone 60 lbs., iodized NaCl 30 lbs., manganese sulfate 12 oz. 2 200 A.O.A.C. chick units per gm.
The soybean oil meal was prepared in the laboratory from whole soybeans of the Dunfield variety. 2 The soybeans were ground in a feed mill and ether-extracted in a modified Soxhlet apparatus. The meal to be autoclaved was spread in a layer less than 0.5 cm. deep in pans and placed in an autoclave which was connected directly to a 40 lbs. pressure steam line. The pressure in the autoclave was raised to 15 lbs. and the air expelled in less than two minutes when timing was commenced. At the end of the desired heating 2
Supplied by the Department of Agronomy, Nebraska Agricultural Experiment Station, through the courtesy of Mr. W. E. Lyness.
period, the pressure was rapidly brought to zero (less than 30 seconds). Immediately thereafter, the soybean oil meal was spread on paper and allowed to dry at room temperature. The lots of soybean oil meal autoclaved at 15 lbs. pressure for 4, 10, and 15 minutes were found to contain 82, 39, and 15 percent, respectively, as much trypsin inhibitor activity as the original raw meal while the others showed no trypsin inhibitor activity. Trypsin inhibitor determinations were made by the method of Borchers et al. (1947). After mixing of the ration in the proportions indicated in Table 1, omitting the talc, the ration was pulverized in a small laboratorypulverizerpassing through a 1/16" screen. The ration was then moistened with water, 800 ml. per kg. ration, forced through a sausage grinder into coarse threads, and dried in a fan blast at room temperature. The dried ration was then granulated to the proper size in a feed mill, mixed with talc, and formed into 7/32" pellets in an ordinary tablet machine.3 The dextrin and gum tragacanth, included in the ration, act as binders for the pellets and the talc acts as a lubricant in the pelleting process. In each of the two feeding experiments, 48 day-old White Rock cockerel chicks,4 30 to 35 gms. in weight, were placed in electrically heated battery brooders with water and pelleted ration of lot 288, Table 2, available ad libitum. Food consumption was carefully recorded, all scattered pellets being retrieved. On the fifth day, 32 chicks were selected on the basis of best weight gains and distributed 3
Eureka Tablet Machine, F. J. Stokes Machine Company, Philadelphia 20, Pennsylvania. 4 We are indebted to Mr. I. L. Williams, Department of Poultry Husbandry, Nebraska Agricultural Experiment Station, for careful sexing of the chicks.
HEAT PERIODS ON SOYBEAN OIL MEAL
among the four experimental lots, 8 in each. The chicks were placed in individual, electrically-heated compartments with water available ad libitum. For fifteen minutes, three times daily, each chick was allowed access to an individual adjoining compartment where the pelleted ration was available. The amount of food supplied each chick per day was limited approximately to the least amount
603
285. All chicks consumed 700 gm. of ration at the age of 41-45 days when final weighings were made. Lots 350-353 were fed by the regular procedure of this method, consuming 800 gm. at the age of 41-43 days. The results are summarized in Table 2. The results in lots 285-288 indicate that autoclaving soybean oil meal at 15 lbs. pressure for four minutes did not give a significant im-
TABLE 2.—Average weight gains of chicks fed soybean oil meal heated for various periods Experiment 2
Experiment 1
Soybean oil meal autoclaved at 15 lbs. for: minutes Protein (NX 6.25) p.ct. Number of chicks Deaths Food consumption gm. Average gain, gm. ± S.E. t value 1 compared compared compared compared
with with with with
Lot Lot Lot Lot
285 286 287 350
285
286
287
288
350
351
352
353
0 22.6 8 0 700 198.9 ±7.20
4 22.9 8 2 700 216.3 ±3.55
10 23.1 8 0 700 256.1 ±4.62
30 23.0 8 0 700 273.1 ±5.33
15 22.5 8 0 800 256.8 ±5.10
20 22.5 8 0 800 294.0 ±5.83
25 22.6 8 0 800 292.0 ±5.00
30 22.9 8 0 800 293.3 ±4.67
1.926
6.692J 6.208{
8.268t 7.9731 2.392f 5.801J
4.924J
5.238J
1
1 value according to G. W. Snedecor, Statistical Methods, Iowa State College Press, 1940. t Significant at a 5 percent level. % Significant at a 1 percent level.
consumed by any chick so that each chick was consuming the food of its experimental ration at nearly the same rate. This feeding procedure was developed by Ackerson, Blish and Mussehl (1938), where more details may be found. It soon became apparent that the chicks on the raw soybean oil meal ration, Lot 285, were consuming their food at a markedly slow rate and would not consume 800-900 gms. of ration at 6 weeks of age as is usual in this feeding method. In an effort to increase their rate of food consumption, food was made available ad libitum to all chicks in lots 285-288 after three weeks of age, food consumption of each chick, however, still being limited to the rate of lot
provement over raw soybeans in growth promoting value for chicks to six weeks of age. However, autoclaving for 10 or 30 minutes gave a highly significant improvement over either raw or four minute autoclaved soybean oil meal. Autoclaving for 30 minutes was more effective than for 10 minutes. The difference between the latter two treatments was significant at the 5 percent level. The results in lots 350-353 indicate that autoclaving at 15 lbs. pressure for 20, 25, or 30 minutes gave a highly significant improvement over 15 minutes. However, there was no significant difference in growth on rations containing soybean oil meal autoclaved at 15 lbs. pressure for 20, 25, or 30 minutes.
604
RAYMOND BORCHERS, C. W. ACKERSON AND F. E. MUSSEHL
DISCUSSION The results of this study, summarized in Table 2, definitely indicate that a longer autoclaving period at 15 lbs. pressure is required than indicated by Clandinin et al. (1947). The data conform with the results reported by Fritz et al. (1947). The soybean flakes used in the study of Clandinin et al. (1947) may have been the cause of the variation as indicated by the results of Fritz et al. (1947) who showed that two commercial samples of soybean grits differed markedly in growth value. Another cause of difference in the results reported may be found in the growth period used by Clandinin et al. (1947) who used growth during the second week as a criterion while the data of Fritz et al. (1947) and in this paper were based on a longer growth period. In view of these possible causes of differences in results, it seems desirable to stress the importance of using soybean products of definitely known history in experimental work intended to give data on proper processing of soybeans. Further, it should be stressed that the use of short growth periods may lead to conclusions which do not apply to a more extended growth period simulating more nearly practical conditions. These results suggest, also, that the absence of the trypsin inhibitor in autoclaved soybean oil meal is indicative of adequately autoclaved meal. A trypsin inhibitor test, however, will give no indications of over-heating as a result of autoclaving or dry heating. SUMMARY
Raw and autoclaved soybean oil meal of known history was included in rations
fed to chicks to six weeks of age with measured food intake. The results indicate that autoclaving of soybean oil meal at 15 lbs. pressure must be continued longer than 15 minutes to obtain a meal of optimum growth promoting value for chicks. Further, the results suggest that the absence of the trypsin inhibitor in autoclaved soybean oil meal may be used as a criterion for adequately autoclaved meal. REFERENCES
Ackerson, C. W., M. J. Blish and F. E. Mussehl, 1938. The utilization of food elements by growing chicks. V. Nebr. Agr. Expt. Sta. Res. Bull. 100: 1-8. Borchers, R., C. W. Ackerson and R. M. Sandstedt, 1947. Trypsin Inhibitor, I I I . Arch. Biochem. 12: ' 367-374. Clandinin, D. R., W. W. Cravens, C. A. Elvehjem and J. G. Halpin, 1947. Deficiencies in overheated soybean oil meal. Poultry Sci. 26: 150156. Fritz, J. C , E. H. Kramke and C. A. Reed, 1947. Effect of heat treatment on the biological value of soybeans. Poultry Sci. 26: 657-661. Ham, W. E., R. M. Sandstedt and F. E. Mussehl, 1945. The proteolytic inhibiting substance in the extract from unheated soybean meal and its effect upon growth in chicks. J. Biol. Chem. 161: 635642. Hayward, J. W., J. G. Halpin, C. E. Holmes, G. Bohstedt and E. B. Hart, 1937. Soybean oil meal prepared at different temperatures as a feed for poultry. Poultry Sci. 16: 3-14. Klose, A. A., B. Hill and H. L. Fevold, 1946. Presence of a growth-inhibiting substance in raw soybeans. Proc. Soc. Exptl. Biol. Med. 62: 10-12. Westfall, R. J. and S. M. Hauge, 1947. The protein efficiency of soybean flour as related to its trypsin inhibitor content. Fed. Proc. 6: 423. Wilgus, H. S., L. C. Norris and G. F. Heuser, 1936. Effect of heat on nutritive value of soybean oil meal. Ind. Eng. Chem., ind. ed. 28: 586-588.
Departments of Agricultural Chemistry and Poultry Husbandry, Nebraska Agricultural Experiment Station, Lincoln (Received for publication March 1, 1948)
ALTHOUGH the superior value of -**• properly heated soybean oil meal over raw or improperly heated meal for the nutrition of the chick has been recognized since the work of Wilgus et al. (1936) and of Hayward et al. (1937), a precise definition of properly heated soybean oil meal has not been attained. In a recent publication, Clandinin et al. (1947) presented data indicating that soybean flakes autoclaved at 15 lbs. pressure for four to fifteen minutes gave the most satisfactory gains in chicks. These results were contradicted by the findings of Fritz, Kramke and Reed (1947) who reported from tests on turkey poults that the best biological value was obtained when ground raw soybeans were autoclaved at 15 lbs. pressure for 20 to 30 minutes. Westfall and Hauge (1947) stated that the growth efficiency of soybean flour for rats was inversely related to the quantity of trypsin inhibitor present in the soybean flour. Data presented by Borchers et al. (1947) showed that soybean dil meal auto1 Published with the approval of the Director as Paper No. 440, Journal Series, Nebraska Agricultural Experiment Station. Some of these data were submitted in an abstract to the 113th meeting of the American Chemical Society, Chicago, Illinois, April 19-23, 1948.
claved at 15 lbs pressure for four minutes still contained a large portion of the trypsin inhibitor and that autoclaving at 15 lbs. pressure for about 20 minutes was necessary to give complete destruction of the trypsin inhibitor. Although Ham, Sandstedt and Mussehl (1945) assumed that the trypsin inhibitor present in raw soybean oil meal was identical with the growth inhibiting factor, Klose, Hill and Fevold (1946) have pointed out that such an assumption has not been established. It, therefore, seemed of importance to us to obtain data bearing on the relationship between the growth promoting value of soybean oil meal for chicks and the amount of trypsin inhibitor remaining active in the meal after autoclaving for various time periods. For this purpose, seven samples of soybean oil meal were prepared: raw or unheated meal, and samples which were autoclaved at 15 lbs. pressure for 4, 10, 15, 20, 25, and 30 minutes, respectively. The first was chosen as a negative control, the 4, 10, and 15 minute autoclaving periods as representative of samples containing a part of the natural trypsin inhibitor present in soybeans and within the heating period suggested by Clandinin et al. (1947) as satisfactory, and the 20, 25, and
601
602
RAYMOND BORCHERS, C. W. ACKERSON AND F. E. MTJSSEHL
30 minute autoclaving periods as representative of samples containing no trypsin inhibitor and generally recognized as being an adequate heating period. These soybean oil meals were then incorporated into rations and fed to day-old chicks under controlled feed intake to six weeks of age. EXPERIMENTAL The composition of the basal ration used in this study is given in Table 1. TABLE 1.—Base ration Ingredient Yellow corn meal Corn gluten meal Wheat shorts Wheat bran Dehydrated alfalfa meal Meat scraps Sardine meal 1 Salt mix No. 4S Vitamin D carrier2 Dextrin Gum tragacanth Soybean oil meal (raw or heated, see (Table 2) Talc
Parts by weight 24 S 10 10 5 3 3 3 1 10 2 23 1
1 Limestone 60 lbs., iodized NaCl 30 lbs., manganese sulfate 12 oz. 2 200 A.O.A.C. chick units per gm.
The soybean oil meal was prepared in the laboratory from whole soybeans of the Dunfield variety. 2 The soybeans were ground in a feed mill and ether-extracted in a modified Soxhlet apparatus. The meal to be autoclaved was spread in a layer less than 0.5 cm. deep in pans and placed in an autoclave which was connected directly to a 40 lbs. pressure steam line. The pressure in the autoclave was raised to 15 lbs. and the air expelled in less than two minutes when timing was commenced. At the end of the desired heating 2
Supplied by the Department of Agronomy, Nebraska Agricultural Experiment Station, through the courtesy of Mr. W. E. Lyness.
period, the pressure was rapidly brought to zero (less than 30 seconds). Immediately thereafter, the soybean oil meal was spread on paper and allowed to dry at room temperature. The lots of soybean oil meal autoclaved at 15 lbs. pressure for 4, 10, and 15 minutes were found to contain 82, 39, and 15 percent, respectively, as much trypsin inhibitor activity as the original raw meal while the others showed no trypsin inhibitor activity. Trypsin inhibitor determinations were made by the method of Borchers et al. (1947). After mixing of the ration in the proportions indicated in Table 1, omitting the talc, the ration was pulverized in a small laboratorypulverizerpassing through a 1/16" screen. The ration was then moistened with water, 800 ml. per kg. ration, forced through a sausage grinder into coarse threads, and dried in a fan blast at room temperature. The dried ration was then granulated to the proper size in a feed mill, mixed with talc, and formed into 7/32" pellets in an ordinary tablet machine.3 The dextrin and gum tragacanth, included in the ration, act as binders for the pellets and the talc acts as a lubricant in the pelleting process. In each of the two feeding experiments, 48 day-old White Rock cockerel chicks,4 30 to 35 gms. in weight, were placed in electrically heated battery brooders with water and pelleted ration of lot 288, Table 2, available ad libitum. Food consumption was carefully recorded, all scattered pellets being retrieved. On the fifth day, 32 chicks were selected on the basis of best weight gains and distributed 3
Eureka Tablet Machine, F. J. Stokes Machine Company, Philadelphia 20, Pennsylvania. 4 We are indebted to Mr. I. L. Williams, Department of Poultry Husbandry, Nebraska Agricultural Experiment Station, for careful sexing of the chicks.
HEAT PERIODS ON SOYBEAN OIL MEAL
among the four experimental lots, 8 in each. The chicks were placed in individual, electrically-heated compartments with water available ad libitum. For fifteen minutes, three times daily, each chick was allowed access to an individual adjoining compartment where the pelleted ration was available. The amount of food supplied each chick per day was limited approximately to the least amount
603
285. All chicks consumed 700 gm. of ration at the age of 41-45 days when final weighings were made. Lots 350-353 were fed by the regular procedure of this method, consuming 800 gm. at the age of 41-43 days. The results are summarized in Table 2. The results in lots 285-288 indicate that autoclaving soybean oil meal at 15 lbs. pressure for four minutes did not give a significant im-
TABLE 2.—Average weight gains of chicks fed soybean oil meal heated for various periods Experiment 2
Experiment 1
Soybean oil meal autoclaved at 15 lbs. for: minutes Protein (NX 6.25) p.ct. Number of chicks Deaths Food consumption gm. Average gain, gm. ± S.E. t value 1 compared compared compared compared
with with with with
Lot Lot Lot Lot
285 286 287 350
285
286
287
288
350
351
352
353
0 22.6 8 0 700 198.9 ±7.20
4 22.9 8 2 700 216.3 ±3.55
10 23.1 8 0 700 256.1 ±4.62
30 23.0 8 0 700 273.1 ±5.33
15 22.5 8 0 800 256.8 ±5.10
20 22.5 8 0 800 294.0 ±5.83
25 22.6 8 0 800 292.0 ±5.00
30 22.9 8 0 800 293.3 ±4.67
1.926
6.692J 6.208{
8.268t 7.9731 2.392f 5.801J
4.924J
5.238J
1
1 value according to G. W. Snedecor, Statistical Methods, Iowa State College Press, 1940. t Significant at a 5 percent level. % Significant at a 1 percent level.
consumed by any chick so that each chick was consuming the food of its experimental ration at nearly the same rate. This feeding procedure was developed by Ackerson, Blish and Mussehl (1938), where more details may be found. It soon became apparent that the chicks on the raw soybean oil meal ration, Lot 285, were consuming their food at a markedly slow rate and would not consume 800-900 gms. of ration at 6 weeks of age as is usual in this feeding method. In an effort to increase their rate of food consumption, food was made available ad libitum to all chicks in lots 285-288 after three weeks of age, food consumption of each chick, however, still being limited to the rate of lot
provement over raw soybeans in growth promoting value for chicks to six weeks of age. However, autoclaving for 10 or 30 minutes gave a highly significant improvement over either raw or four minute autoclaved soybean oil meal. Autoclaving for 30 minutes was more effective than for 10 minutes. The difference between the latter two treatments was significant at the 5 percent level. The results in lots 350-353 indicate that autoclaving at 15 lbs. pressure for 20, 25, or 30 minutes gave a highly significant improvement over 15 minutes. However, there was no significant difference in growth on rations containing soybean oil meal autoclaved at 15 lbs. pressure for 20, 25, or 30 minutes.
604
RAYMOND BORCHERS, C. W. ACKERSON AND F. E. MUSSEHL
DISCUSSION The results of this study, summarized in Table 2, definitely indicate that a longer autoclaving period at 15 lbs. pressure is required than indicated by Clandinin et al. (1947). The data conform with the results reported by Fritz et al. (1947). The soybean flakes used in the study of Clandinin et al. (1947) may have been the cause of the variation as indicated by the results of Fritz et al. (1947) who showed that two commercial samples of soybean grits differed markedly in growth value. Another cause of difference in the results reported may be found in the growth period used by Clandinin et al. (1947) who used growth during the second week as a criterion while the data of Fritz et al. (1947) and in this paper were based on a longer growth period. In view of these possible causes of differences in results, it seems desirable to stress the importance of using soybean products of definitely known history in experimental work intended to give data on proper processing of soybeans. Further, it should be stressed that the use of short growth periods may lead to conclusions which do not apply to a more extended growth period simulating more nearly practical conditions. These results suggest, also, that the absence of the trypsin inhibitor in autoclaved soybean oil meal is indicative of adequately autoclaved meal. A trypsin inhibitor test, however, will give no indications of over-heating as a result of autoclaving or dry heating. SUMMARY
Raw and autoclaved soybean oil meal of known history was included in rations
fed to chicks to six weeks of age with measured food intake. The results indicate that autoclaving of soybean oil meal at 15 lbs. pressure must be continued longer than 15 minutes to obtain a meal of optimum growth promoting value for chicks. Further, the results suggest that the absence of the trypsin inhibitor in autoclaved soybean oil meal may be used as a criterion for adequately autoclaved meal. REFERENCES
Ackerson, C. W., M. J. Blish and F. E. Mussehl, 1938. The utilization of food elements by growing chicks. V. Nebr. Agr. Expt. Sta. Res. Bull. 100: 1-8. Borchers, R., C. W. Ackerson and R. M. Sandstedt, 1947. Trypsin Inhibitor, I I I . Arch. Biochem. 12: ' 367-374. Clandinin, D. R., W. W. Cravens, C. A. Elvehjem and J. G. Halpin, 1947. Deficiencies in overheated soybean oil meal. Poultry Sci. 26: 150156. Fritz, J. C , E. H. Kramke and C. A. Reed, 1947. Effect of heat treatment on the biological value of soybeans. Poultry Sci. 26: 657-661. Ham, W. E., R. M. Sandstedt and F. E. Mussehl, 1945. The proteolytic inhibiting substance in the extract from unheated soybean meal and its effect upon growth in chicks. J. Biol. Chem. 161: 635642. Hayward, J. W., J. G. Halpin, C. E. Holmes, G. Bohstedt and E. B. Hart, 1937. Soybean oil meal prepared at different temperatures as a feed for poultry. Poultry Sci. 16: 3-14. Klose, A. A., B. Hill and H. L. Fevold, 1946. Presence of a growth-inhibiting substance in raw soybeans. Proc. Soc. Exptl. Biol. Med. 62: 10-12. Westfall, R. J. and S. M. Hauge, 1947. The protein efficiency of soybean flour as related to its trypsin inhibitor content. Fed. Proc. 6: 423. Wilgus, H. S., L. C. Norris and G. F. Heuser, 1936. Effect of heat on nutritive value of soybean oil meal. Ind. Eng. Chem., ind. ed. 28: 586-588.