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Soybean proteinase inhibitors affect intestinal trypsin activities and amino acid digestibilities in rainbow trout (Oncorhynchus mykiss)
Camp.
Eiochem.
fhysiol.
Vol.
107A,
No.
I, pp.
215-219,
0300-9629/94
1994
$6.00
+ 0.00
0 1993 Pergamon Press Ltd
Printed in Great Britain
Soybean proteinase inhibitors affect intestinal trypsin activities and amino acid digestibilities in rainbow trout (Oncorhynchus mykiss) khild
Krogdahl, Trygve Berg Lea and Jan J. Olli
AKVAFORSK
(Institute of Aquaculture
Research), c/o NLH, N-1430 As, Oslo, Norway
Rainbow trout (Oncorhynchus mykiss), 265 g kept in fresh water, 1 mz surface area tanks, 10 kg per tank were fed diets based on high quality fish meal added 0.00,0.37, 0.74, 1.11 and 1.48% soybean proteinase inhibitors, and chromic oxide as internal marker. After 10 days, 10 fish were dissected to obtain total contents of intestinal segments, and faeces were stripped from the remainder. The inhibitors decreased protein digestibility from 93 to 70%. Cysteine accumulated increasingly in the small intestine with increasing dietary inhibitor level. A linear relationship between intestinal proteolytic activities and protein digestibility was seen. Lipid digestibiity was not significantly affected. Key words: Soybean proteinase
inhibitors; Trypsin activities; Protein digestibility; Oncorhynchus
mykiss. Comp. Biochem. Physiol. 107A, 215-219, 1994.
Introduction Soybean meals are common ingredients in diets for rainbow trout. However, soybeans contain antinutrients which might affect nutrient utilization. Proteinase inhibitors have been considered the most important, and the main goal of soybean processing has been improvement of nutritional quality by inactivation of these protein molecules. In vitro studies of effects of purified inhibitors on proteolytic enzymes of rainbow trout have shown inhibition several times stronger than that of proteolytic enzymes from domestic animals and man (Krogdahl and Holm, 1983). Moreover, proteinase inhibitors have been important tools in investigation of proteolytic enzymes, protein digestion and pancreatic secretion in experimental animals and man (Blow et al., 1969; Blow, 1974; Krogdahl and Holm, 1979; Holm et al., 1988a,b). The present experiment was undertaken to study effects of soybean proteinase inhibitors
Correspondence
to: Ashild Krogadhl. Norwegian College of Veterinary Medicine, Department of Biochemistry, Physiology and Nutrition, P.O. Box 8146 Dep., N-0033 Oslo, Norway. Fax: 472-60-0985. Received 11 March 1993; accepted 14 April 1993
extracted from raw soybeans on proteolytic enzyme activities and protein digestion throughout the digestive tract of rainbow trout. The results were expected to give information on protein digestion and the response of digestive processes to proteinase inhibitors. These inhibitors, being proteins of extremely low digestibilities, might serve as model molecules for proteins of low digestibilities.
Materials and Methods Rainbow trout, average weight 265 g, were used in the experiment. The fish were kept in fresh water in tanks of 1 m2 surface area, 10 kg of fish per tank. The diets were based on high quality Norwegian fish meal (83%), capelin oil (1 1%), and included vitamins and minerals at recommended (NRC, 1981) levels (5%, including starch carrier). Chromic oxide was included in the diet at 1% as internal marker for digestibility evaluation. A mixture of proteinase inhibitors obtained from Sigma (type II, no T-9128) was added to the experimental diets at levels 0.00, 0.37, 0.74, 1.11 and 1.48%, corresponding to the inhibitor activities found in 215
216
khild
Krogdahl et al.
with 0, 15, 30, 45 and 60% raw soybeans. The second lowest level, 0.37%, might be found in diets with high inclusion of commercial soybean meal. Water was added to the mixture to obtain a dry matter content of 60%, and the diets were pelleted in a meat grinder. The diets contained 58% crude protein and 18% fat before the water addition. A regression design was used in the experiment. Analysis of regression is most useful for evaluation of dose response relationships and makes replicates unnecessary (Snedecor and Cochran, 1973). Each diet was given to fish in one tank. The fish were adapted to the diets during a 10 day feeding period. Faeces were stripped from the very posterior part of the intestine on days 10 and 14 according to Austreng (1978). Before stripping, 10 fish from each tank were randomly sampled for dissection to obtain contents (total collection) of the small intestine posterior to the pyloric caeca and of the hind gut. Small and large intestinal contents were pooled separately, within the tank. The intestinal samples and faeces were subjected to the following analysis after lyophilization: trypsin activities in saline extracts (1: 10 w/v) using BAPNA as substrate, at 37°C using 10 ~1 extract, 3 ml incubation volume, 20 min incubation time (Kakade et al. 1969), and amino acid content by liquid chromatography. In the faecal samples, fat (micro method according to Zollner and Kirsch, 1962) and fatty acids (gasliquid chromatography) were also measured. The results were evaluated by regression and correlation analysis, using curvelinear and linear models, Y = aX + bX2 + C and Y = aX + C, respectively (Snedecor and Cochran, 1973). Quadratic regression was considered the proper model for the present results based on evaluations of residuals, experience gained through several years of research in the area of nutritional quality of feedstuffs, and in agreement with the discussions of Waterlow (1990) and Young (1990) on the curvelinearity of nutritional adaptations. Both cumulative and fractional absorption of amino acids and protein (amino acid nitrogen x 6.25) in small intestine, large intestine and faeces were calculated. Fractional digestibilities for faeces and large intestine were calculated as the difference between digestibility (cumulative) found in faeces and large intestine, and between large intestine and small intestine, respectively. In the small intestine, fractional and cumulative digestibility were considered to be the same. diets
Table 1. Table 1 also shows the regression of trypsin activities on inhibitor level in the diet. Generally, the trypsin activities seemed to decrease throughout the digestive tract. This was probably due to a continuous autodigestion of the enzymes which takes place throughout the digestive tract. The inhibitors caused significant decreases in trypsin activities in the small intestine as well as in the large intestine and faeces. Inhibition seemed to increase with increasing inhibitor level. The shape of the curve in Fig. 1, indicates that the fish were able to compensate for smaller amounts of inhibitors. A compensation might have occurred by increased enzyme secretion as discussed later. However, reduced enzyme degradation might also add to this effect. In chicks, soybean proteinase inhibitors have been shown to stimulate emptying of the gall bladder (Niess, 198la,b). Bile salts seem to have marked protective effect on digestive enzymes, possibly by stabilizing the protein molecule against autolysis (Green, 1979; Green and Nasset, 1980; review by Krogdahl, 1985). Moreover, it appears likely that the increase in the amount of dietary protein in the intestine might slow down autodigestion of digestive enzymes. Digestibilities of total lipid, the fatty acids C20: 1 and C20: 5, protein, and amino acids calculated on the basis of faecal analysis, are presented in Table 1. Table 1 also presents the results of quadratic regression analysis. Neither total lipid digestion nor digestion of individual fatty acids seemed to be affected by the proteinase inhibitors. The fatty acids C20 : 1 and C20: 5 might be considered representative of fatty acids which show low and high digestibilities, respectively (Olli et al. In press., Krogdahl, 1985). Under the conditions prevailing in the intestines of the fish fed highly digestible ingredients, proteinase inhibitors did not seem to disturb emulsification, lipolysis or micelle formation importantly as judged from the total digestibilities found for total lipid and fatty acids.
o.oa 0.37 0.74 1.11 1.43 +=F
Results and Discussion The effects of proteinase inhibitors on intestinal trypsin activities were as shown in Fig. 1 and
n =LI
o-S1
Lowl ot pretelnaaoInhlbltor,K
Fig. 1. Trypsin activities of faeces (F) and lyophilized contents of small intestine (SI) and large intestine (LI) as function of dietary level of proteinase inhibitors.
217
Soybean inhibitors affect trout trypsin Table 1. Intestinal trypsin activities, digestibilities of lipid, C20: 1, C20: 5, protein (total amino acid nitrogen) and amino acids calculated from faecal excretion, and regressions on dietarv inhibitor level Level of inhibitor 0.00
. .
Trypsm acttvittes, 1.41 SD LI 0.45 F 0.42 Digestibilities (%) Lipid 99 C2O:l 99 c2o:s 99 Protein 93 Asp 90 Thr 93 Ser 89 Glu 95 Pro 91 Gly 85 Ala 94 Cys 84 Val 94 Met 93 Ile 94 Leu 95 Tyr 93 Phe 93 His 92 LYS 96 Arg 93
Estimated regression*
0.74
1.11
1.48
a
b
C
P(mode1)
AOD,,, 1.45 0.95 0.56 0.17 0.23 0.04
0.91 0.20 0.01
0.17 0.00 0.00
0.0 -0.2 -0.7
-0.6 -0.1 0.3
1.43 0.50 0.42
0.064
98 98 99 86 84 86 83 89 87 81 88 74 87 87 87 88 86 86 86 90 87
98 98 99 78 79 81 79 85 83 80 85 68 83 83 82 84 80 81 80 86 84
96 96 98 70 70 66 69 76 74 71 76 56 72 74 70 81 69 69 69 76 74
0.5 0.4 0.3 -3.3 -2.8 2.2 -1.3 -1.1 2.0 2.6 -0.5 -6.2 -1.3 -2.0 -0.7 -8.1 -2.0 -2.1 - 1.3 0.3 0.2
- 1.5
98.8 99.2 99.1 92.8 90.0 92.3 88.9 94.2 90.3 84.4 93.2 83.1 93.9 92.7 93.8 95.2 92.7 92.6 92.0 95.3 92.4
0.051 0.087 0.068 0.003 0.012 0.019 0.011 0.016 0.007 0.061 0.025 0.009 0.012 0.006 0.009 0.013 0.007 0.010 0.006 0.012 0.020
0.37
99 99 99 90 87 91 87 93 90 83 93 78 92 90 92 93 90 90 90 94 91
-1.5 -0.7 -8.3 -7.3 -13.3 -8.1 -7.4 -8.7 -7.8 -7.4 -8.1 -9.1 -7.3 - 10.3 -1.2 -9.6 -9.2 -9.5 -9.2 -8.6
0.210 0.011
*Model = aX + bX* + C. tS1 = small intestine, LI = large intestine, F = faeces.
The proteinase inhibitors affected the protein and amino acid digestibilities markedly, from about 93% to about 70%. Digestibilities of individual amino acids closely paralleled the protein digestibility. Cystine, however, diverged by showing lower digestibilities than the other amino acids. The second degree models fitted the results significantly showing negative trends for all amino acids but glycine (P = 0.06). The reason why cystine digestibility was low, even in the O-inhibitors diet, is not clear. However, cystine in fish meal has shown low digestibilities in several of our recent experiments with both rainbow trout and Atlantic salmon, in contrast to other protein sources. Digestibilities of protein in intestinal contents and faeces are presented in Fig. 2. The figure presents both cumulative and fractional digestibilities. In the small intestine, the digestibility of protein was 65% for the diet without inhibitor and decreased to 31% at the highest inhibitor level. In the large intestine, fractional digestibility was 24% for the diet without inhibitor, increasing to 35% at the highest inclusion level. Fractional digestibilities for faeces, comprising the thyme of the distal l/2 of the large intestine (Austreng, 1978), did not change with inhibitor level. These results indicate that, in the small intestine, the reduction in protein digestibility
caused by proteinase inhibitors may be compensated for by increased absorption in the more distal parts of the intestine. At the lower dietary inhibitor levels the intestine seemed to be able to compensate almost completely. However, at the higher inhibitor levels the compensation was only half of the observed reduction. Proteinase inhibitors affected intestinal digestibilities of amino acids and protein similarly. Cysteine was, again, an exception as can be seen from Fig. 2. In the small intestine, negative cystine digestibilities were found with the diets containing inhibitors. The reduction was approximately 60%. The low cystine digestibilities in the small intestine were compensated for by increased absorption in the more distal parts, as indicated by a 25% increase, in fractional digestibility in the large intestine. The low fractional digestibilities found for faeces, i.e. the distal l/2 of the large intestine, and the lack of effects of inhibitors, may indicate that the large intestine does not play an important role in amino acid absorption. The negative digestibilities seen for cystine in the small intestine, indicates an endogenous supply of cystine-rich compounds to the intestine. Proteolytic enzymes and most soybean proteinase inhibitors are cystine-rich proteins, containing 10 and up to 20% cystine,
218
Ashild Krogdahl er al.
Protein digestibility (%) = 17.2 x trypsin activity (mg/g) + 67, P(mode1) = 0.0 12.
-25
This close relationship between intestinal enzyme activity and protein digestibility shows that inactivation of the proteinase inhibitors is necessary for efficient utilization of soybeans as feed ingredient for salmonids. Conclusions
% 1wr
i
25 0
it
i
-25
% -I
50 II!!!? 75 -50 0.000.370.74 1.111.45
E
-25
+.F
l
1
.LI
25 0 -50 0.000.37 0.74 1.111.43
High dietary levels of soybean proteinase inhibitors reduced intestinal proteolytic activity of protein digestibility markedly. A close relationship between intestinal trypsin activities and protein digestibility was observed. The enzyme inhibition seemed to be partly compensated for by increased enzyme secretion and enhanced absorption of protein in the distal parts of the intestine. Lipid digestion was not affected importantly by the inhibitors.
n =SI
Lava1of pretsttuaa Inhlbtter,% Fig. 2. Digestibilities, cumulative and fractional, of protein and cystine calculated from the protein and cystine concentrations in the small (SI) and large (LI) intestine, and in faeces (F) as function of dietary level of proteinase inhibitors,% of dry diet.
References Almquist H. J., Mecchi E., Kratxer F. H. and Grau G. C. (1942) Soybean protein as a source of amino acids for the chicks. J. Nufr. 24, 385-390. Austreng E. (1978) Digestibility determination in fish using chromic oxide marking and analysis of contents from different segments of the gastrointestinal tract. Aquaculture 55, 255-264.
respectively (Ikenaka et al., 1974). The cystine contents of these molecules are about 10 times higher than found in most proteins. The negative cystine digestibilities that found in the small intestine strongly indicates increased secretion of proteolytic enzymes. Excessive excretion of cystine by feeding raw soybeans has been found in rats, pigs and man (Both et al., 1960; Schneeman and Lyman, 1975; Hahn, 1988b). These findings are in line with the observation of a several-fold increase in the conversion of methionine to cystine in the liver of rats fed soybean proteinase inhibitors (Barnes and Kwong, 1965). Moreover, methionine supplementation seems to improve nutritive value of soybean diets to a greater extent than would be expected from the dietary amino acid composition (Booth et al., 1960; Long et al., 1965; Almquist et al., 1942). From the present experiment, it might appear that the rainbow trout responds much like the rat, pig and man to dietary proteinase inhibitors, and that inhibitors might increase the requirement of sulphur containing amino acids. A correlation between trypsin activity of the small intestine and protein digestibility seemed to exist. Linear correlation analysis showed the following relationship:
Barnes R. H. and Kwong E. (1965) Effect of soybean trypsin inhibitor and penicillin on cystine biosynthesis in the pancreas and its transport as exocrine protein secretion in intestinal tract of the rat. J. Nurr. 86, 245-252. Blow D. M., Birktoft J. J. and Hartley B. S. (1969) Role of a buried acid group in the mechanism of action of chymotrypsin. Nature 221, 337-342. Blow D. M. (1974) Stereochemistry of substrate binding and hydrolysis in the trypsin family of enzymes. In Proteinase Inhibitors. Proc. 2nd Int. Res. Conf., (Edited by Frits H., Tschetsche H.. Greene L. J. and Truscheit E.), pp 473-479. Springer, Berlin. Booth A. N.. Robbins D. J.. Riebelin W. E. and DeEds F. (1960) Effects of raw soybean meal and amino acids on pancreatic hypertrophy in rats. Proc. Sot. exp. Biol. Med. 116, 1067-1073. Green G. (1979) Secretion and intestinal degradation of pancreatic lipase; role of dietary protein and proteinase inhibitors. Fed. Proc. 38, 279. Green G. and Nasset E. S. (1980) Importance of bile in regulation of intraluminal proteolytic enzyme activities in the rat. Gastroentqology 79, 695-702. Holm H., Krogdahl A. and Hansen L. (1988a) High and low inhibitor soybean meals affect human duodenal proteinase activity differently: in vitro comparison of proteinase inhibition. J. Nutr. 118, 521-525.0 Holm H., Hansen L., Krogdahl A. and Florholmen J. (1988b) High and low inhibitor soybean meals affect human duodenal proteinase activity differently: in oiuo comparison with bovine serum albumin. J. Nufr. 118, 515-520. Ikenaka T., Odani S. and Koide T. (1974) Chemical structure and inhibitory activities of soybean proteinase inhibitors. In Proteinase Inhibitors. Rot. 2nd Int. Res. Conf.,
Soybean inhibitors affect trout trypsin (Edited by Frits H., Tschetsche H., Greene L. J. and Truscheit E.) pp. 325-330. Springer, Berlin. Kakade M. L., Simons N. and Liener I. E. (1969) Ana evaluation of natural vs. synthetic substrates for measuring the antitryptic activity of soybean samples. Cereal Cfaem. 40, 518-526. Krogdahl A. (1985) Digestion and absorption of lipids in poultry. .$ Nutr. 115, 675685. Krogdahl A. and Holm H. (1979) Proteinase inhibitorer i soyabsnmer. Niiringsforskning 1, l-l 1. Krogdahl A. and Holm H. (1983) Pancreatic proteinases from man, trout, rat, pig, cow, chicken, mink and fox. Enzyme activities and inhibition by soybean and lima bean proteinase inhibitors. Comp. Biochem. Physiol. 74B, 403409. Long J. I., Hays V. W. and Speer V. C. (1965) Effect of supplemental amino acids on growth and plasma amino acid concentration of pigs. J. Anim. Sci. 24, 894. Niess E. (1981) Untersuchungen zur regulation der gallenblasen und pancreasmtigkeit des huhnes. 1. Mitteilung. Z. Tierphysiol. Tiererniihr. Futtermittelkd. 45, 17-28.
-
Niess E. (1981) Untersuchungen zur regulation der gallenblasen und pancreastatigkeit des huhnes. 2.
219
Mitteilung. Z. Tierphysiol. Tiererniihr. Futtermitrelkd. 45, 2945. Olli J., Krogdahl A. and Ingh T. van den. Nutritive value of soy products varies widely in diets for Atlantic salmon (Salmo salar). Agric. Scund. (in press). Snedecor G. W. and Cochran W. G. (1973) Chapter 6, Regression; Chapter 7, Correlation, pp. 135-171, and 172-198. In Stntistical Methoa!s. 6th ed. Iowa State University Press, Ames. Schneeman B. 0. and Lyman R. L. (1975) Factors involved in the intestinal feed-back regulation of pancreatic enzyme secretion in the rat. Proc. Sot. exp. Biol. Med. 148, 897-903. Waterlow J. (1990) Nutritional adaptation in man: general introduction and concepts. Am. J. Clin. Nutr. 51, 259-263.
Young V. R. (1990) Introduction to the symposium on nutritional adaptations. Am. J. Clin. Nufr. 51, 258. Zollner N. and Kirsch K. (1962) Uber die quantitative Bestimmung von Lipoiden (Mikromethode) mittels der vielen nattirlichen Lipoiden (alle bekannten Plasmalipoiden) gemeinsamen Sulfophosphovanillin-Reaktion. Z. Ges. Med. (Res. Expo. Med.) 135, 545-550.
Eiochem.
fhysiol.
Vol.
107A,
No.
I, pp.
215-219,
0300-9629/94
1994
$6.00
+ 0.00
0 1993 Pergamon Press Ltd
Printed in Great Britain
Soybean proteinase inhibitors affect intestinal trypsin activities and amino acid digestibilities in rainbow trout (Oncorhynchus mykiss) khild
Krogdahl, Trygve Berg Lea and Jan J. Olli
AKVAFORSK
(Institute of Aquaculture
Research), c/o NLH, N-1430 As, Oslo, Norway
Rainbow trout (Oncorhynchus mykiss), 265 g kept in fresh water, 1 mz surface area tanks, 10 kg per tank were fed diets based on high quality fish meal added 0.00,0.37, 0.74, 1.11 and 1.48% soybean proteinase inhibitors, and chromic oxide as internal marker. After 10 days, 10 fish were dissected to obtain total contents of intestinal segments, and faeces were stripped from the remainder. The inhibitors decreased protein digestibility from 93 to 70%. Cysteine accumulated increasingly in the small intestine with increasing dietary inhibitor level. A linear relationship between intestinal proteolytic activities and protein digestibility was seen. Lipid digestibiity was not significantly affected. Key words: Soybean proteinase
inhibitors; Trypsin activities; Protein digestibility; Oncorhynchus
mykiss. Comp. Biochem. Physiol. 107A, 215-219, 1994.
Introduction Soybean meals are common ingredients in diets for rainbow trout. However, soybeans contain antinutrients which might affect nutrient utilization. Proteinase inhibitors have been considered the most important, and the main goal of soybean processing has been improvement of nutritional quality by inactivation of these protein molecules. In vitro studies of effects of purified inhibitors on proteolytic enzymes of rainbow trout have shown inhibition several times stronger than that of proteolytic enzymes from domestic animals and man (Krogdahl and Holm, 1983). Moreover, proteinase inhibitors have been important tools in investigation of proteolytic enzymes, protein digestion and pancreatic secretion in experimental animals and man (Blow et al., 1969; Blow, 1974; Krogdahl and Holm, 1979; Holm et al., 1988a,b). The present experiment was undertaken to study effects of soybean proteinase inhibitors
Correspondence
to: Ashild Krogadhl. Norwegian College of Veterinary Medicine, Department of Biochemistry, Physiology and Nutrition, P.O. Box 8146 Dep., N-0033 Oslo, Norway. Fax: 472-60-0985. Received 11 March 1993; accepted 14 April 1993
extracted from raw soybeans on proteolytic enzyme activities and protein digestion throughout the digestive tract of rainbow trout. The results were expected to give information on protein digestion and the response of digestive processes to proteinase inhibitors. These inhibitors, being proteins of extremely low digestibilities, might serve as model molecules for proteins of low digestibilities.
Materials and Methods Rainbow trout, average weight 265 g, were used in the experiment. The fish were kept in fresh water in tanks of 1 m2 surface area, 10 kg of fish per tank. The diets were based on high quality Norwegian fish meal (83%), capelin oil (1 1%), and included vitamins and minerals at recommended (NRC, 1981) levels (5%, including starch carrier). Chromic oxide was included in the diet at 1% as internal marker for digestibility evaluation. A mixture of proteinase inhibitors obtained from Sigma (type II, no T-9128) was added to the experimental diets at levels 0.00, 0.37, 0.74, 1.11 and 1.48%, corresponding to the inhibitor activities found in 215
216
khild
Krogdahl et al.
with 0, 15, 30, 45 and 60% raw soybeans. The second lowest level, 0.37%, might be found in diets with high inclusion of commercial soybean meal. Water was added to the mixture to obtain a dry matter content of 60%, and the diets were pelleted in a meat grinder. The diets contained 58% crude protein and 18% fat before the water addition. A regression design was used in the experiment. Analysis of regression is most useful for evaluation of dose response relationships and makes replicates unnecessary (Snedecor and Cochran, 1973). Each diet was given to fish in one tank. The fish were adapted to the diets during a 10 day feeding period. Faeces were stripped from the very posterior part of the intestine on days 10 and 14 according to Austreng (1978). Before stripping, 10 fish from each tank were randomly sampled for dissection to obtain contents (total collection) of the small intestine posterior to the pyloric caeca and of the hind gut. Small and large intestinal contents were pooled separately, within the tank. The intestinal samples and faeces were subjected to the following analysis after lyophilization: trypsin activities in saline extracts (1: 10 w/v) using BAPNA as substrate, at 37°C using 10 ~1 extract, 3 ml incubation volume, 20 min incubation time (Kakade et al. 1969), and amino acid content by liquid chromatography. In the faecal samples, fat (micro method according to Zollner and Kirsch, 1962) and fatty acids (gasliquid chromatography) were also measured. The results were evaluated by regression and correlation analysis, using curvelinear and linear models, Y = aX + bX2 + C and Y = aX + C, respectively (Snedecor and Cochran, 1973). Quadratic regression was considered the proper model for the present results based on evaluations of residuals, experience gained through several years of research in the area of nutritional quality of feedstuffs, and in agreement with the discussions of Waterlow (1990) and Young (1990) on the curvelinearity of nutritional adaptations. Both cumulative and fractional absorption of amino acids and protein (amino acid nitrogen x 6.25) in small intestine, large intestine and faeces were calculated. Fractional digestibilities for faeces and large intestine were calculated as the difference between digestibility (cumulative) found in faeces and large intestine, and between large intestine and small intestine, respectively. In the small intestine, fractional and cumulative digestibility were considered to be the same. diets
Table 1. Table 1 also shows the regression of trypsin activities on inhibitor level in the diet. Generally, the trypsin activities seemed to decrease throughout the digestive tract. This was probably due to a continuous autodigestion of the enzymes which takes place throughout the digestive tract. The inhibitors caused significant decreases in trypsin activities in the small intestine as well as in the large intestine and faeces. Inhibition seemed to increase with increasing inhibitor level. The shape of the curve in Fig. 1, indicates that the fish were able to compensate for smaller amounts of inhibitors. A compensation might have occurred by increased enzyme secretion as discussed later. However, reduced enzyme degradation might also add to this effect. In chicks, soybean proteinase inhibitors have been shown to stimulate emptying of the gall bladder (Niess, 198la,b). Bile salts seem to have marked protective effect on digestive enzymes, possibly by stabilizing the protein molecule against autolysis (Green, 1979; Green and Nasset, 1980; review by Krogdahl, 1985). Moreover, it appears likely that the increase in the amount of dietary protein in the intestine might slow down autodigestion of digestive enzymes. Digestibilities of total lipid, the fatty acids C20: 1 and C20: 5, protein, and amino acids calculated on the basis of faecal analysis, are presented in Table 1. Table 1 also presents the results of quadratic regression analysis. Neither total lipid digestion nor digestion of individual fatty acids seemed to be affected by the proteinase inhibitors. The fatty acids C20 : 1 and C20: 5 might be considered representative of fatty acids which show low and high digestibilities, respectively (Olli et al. In press., Krogdahl, 1985). Under the conditions prevailing in the intestines of the fish fed highly digestible ingredients, proteinase inhibitors did not seem to disturb emulsification, lipolysis or micelle formation importantly as judged from the total digestibilities found for total lipid and fatty acids.
o.oa 0.37 0.74 1.11 1.43 +=F
Results and Discussion The effects of proteinase inhibitors on intestinal trypsin activities were as shown in Fig. 1 and
n =LI
o-S1
Lowl ot pretelnaaoInhlbltor,K
Fig. 1. Trypsin activities of faeces (F) and lyophilized contents of small intestine (SI) and large intestine (LI) as function of dietary level of proteinase inhibitors.
217
Soybean inhibitors affect trout trypsin Table 1. Intestinal trypsin activities, digestibilities of lipid, C20: 1, C20: 5, protein (total amino acid nitrogen) and amino acids calculated from faecal excretion, and regressions on dietarv inhibitor level Level of inhibitor 0.00
. .
Trypsm acttvittes, 1.41 SD LI 0.45 F 0.42 Digestibilities (%) Lipid 99 C2O:l 99 c2o:s 99 Protein 93 Asp 90 Thr 93 Ser 89 Glu 95 Pro 91 Gly 85 Ala 94 Cys 84 Val 94 Met 93 Ile 94 Leu 95 Tyr 93 Phe 93 His 92 LYS 96 Arg 93
Estimated regression*
0.74
1.11
1.48
a
b
C
P(mode1)
AOD,,, 1.45 0.95 0.56 0.17 0.23 0.04
0.91 0.20 0.01
0.17 0.00 0.00
0.0 -0.2 -0.7
-0.6 -0.1 0.3
1.43 0.50 0.42
0.064
98 98 99 86 84 86 83 89 87 81 88 74 87 87 87 88 86 86 86 90 87
98 98 99 78 79 81 79 85 83 80 85 68 83 83 82 84 80 81 80 86 84
96 96 98 70 70 66 69 76 74 71 76 56 72 74 70 81 69 69 69 76 74
0.5 0.4 0.3 -3.3 -2.8 2.2 -1.3 -1.1 2.0 2.6 -0.5 -6.2 -1.3 -2.0 -0.7 -8.1 -2.0 -2.1 - 1.3 0.3 0.2
- 1.5
98.8 99.2 99.1 92.8 90.0 92.3 88.9 94.2 90.3 84.4 93.2 83.1 93.9 92.7 93.8 95.2 92.7 92.6 92.0 95.3 92.4
0.051 0.087 0.068 0.003 0.012 0.019 0.011 0.016 0.007 0.061 0.025 0.009 0.012 0.006 0.009 0.013 0.007 0.010 0.006 0.012 0.020
0.37
99 99 99 90 87 91 87 93 90 83 93 78 92 90 92 93 90 90 90 94 91
-1.5 -0.7 -8.3 -7.3 -13.3 -8.1 -7.4 -8.7 -7.8 -7.4 -8.1 -9.1 -7.3 - 10.3 -1.2 -9.6 -9.2 -9.5 -9.2 -8.6
0.210 0.011
*Model = aX + bX* + C. tS1 = small intestine, LI = large intestine, F = faeces.
The proteinase inhibitors affected the protein and amino acid digestibilities markedly, from about 93% to about 70%. Digestibilities of individual amino acids closely paralleled the protein digestibility. Cystine, however, diverged by showing lower digestibilities than the other amino acids. The second degree models fitted the results significantly showing negative trends for all amino acids but glycine (P = 0.06). The reason why cystine digestibility was low, even in the O-inhibitors diet, is not clear. However, cystine in fish meal has shown low digestibilities in several of our recent experiments with both rainbow trout and Atlantic salmon, in contrast to other protein sources. Digestibilities of protein in intestinal contents and faeces are presented in Fig. 2. The figure presents both cumulative and fractional digestibilities. In the small intestine, the digestibility of protein was 65% for the diet without inhibitor and decreased to 31% at the highest inhibitor level. In the large intestine, fractional digestibility was 24% for the diet without inhibitor, increasing to 35% at the highest inclusion level. Fractional digestibilities for faeces, comprising the thyme of the distal l/2 of the large intestine (Austreng, 1978), did not change with inhibitor level. These results indicate that, in the small intestine, the reduction in protein digestibility
caused by proteinase inhibitors may be compensated for by increased absorption in the more distal parts of the intestine. At the lower dietary inhibitor levels the intestine seemed to be able to compensate almost completely. However, at the higher inhibitor levels the compensation was only half of the observed reduction. Proteinase inhibitors affected intestinal digestibilities of amino acids and protein similarly. Cysteine was, again, an exception as can be seen from Fig. 2. In the small intestine, negative cystine digestibilities were found with the diets containing inhibitors. The reduction was approximately 60%. The low cystine digestibilities in the small intestine were compensated for by increased absorption in the more distal parts, as indicated by a 25% increase, in fractional digestibility in the large intestine. The low fractional digestibilities found for faeces, i.e. the distal l/2 of the large intestine, and the lack of effects of inhibitors, may indicate that the large intestine does not play an important role in amino acid absorption. The negative digestibilities seen for cystine in the small intestine, indicates an endogenous supply of cystine-rich compounds to the intestine. Proteolytic enzymes and most soybean proteinase inhibitors are cystine-rich proteins, containing 10 and up to 20% cystine,
218
Ashild Krogdahl er al.
Protein digestibility (%) = 17.2 x trypsin activity (mg/g) + 67, P(mode1) = 0.0 12.
-25
This close relationship between intestinal enzyme activity and protein digestibility shows that inactivation of the proteinase inhibitors is necessary for efficient utilization of soybeans as feed ingredient for salmonids. Conclusions
% 1wr
i
25 0
it
i
-25
% -I
50 II!!!? 75 -50 0.000.370.74 1.111.45
E
-25
+.F
l
1
.LI
25 0 -50 0.000.37 0.74 1.111.43
High dietary levels of soybean proteinase inhibitors reduced intestinal proteolytic activity of protein digestibility markedly. A close relationship between intestinal trypsin activities and protein digestibility was observed. The enzyme inhibition seemed to be partly compensated for by increased enzyme secretion and enhanced absorption of protein in the distal parts of the intestine. Lipid digestion was not affected importantly by the inhibitors.
n =SI
Lava1of pretsttuaa Inhlbtter,% Fig. 2. Digestibilities, cumulative and fractional, of protein and cystine calculated from the protein and cystine concentrations in the small (SI) and large (LI) intestine, and in faeces (F) as function of dietary level of proteinase inhibitors,% of dry diet.
References Almquist H. J., Mecchi E., Kratxer F. H. and Grau G. C. (1942) Soybean protein as a source of amino acids for the chicks. J. Nufr. 24, 385-390. Austreng E. (1978) Digestibility determination in fish using chromic oxide marking and analysis of contents from different segments of the gastrointestinal tract. Aquaculture 55, 255-264.
respectively (Ikenaka et al., 1974). The cystine contents of these molecules are about 10 times higher than found in most proteins. The negative cystine digestibilities that found in the small intestine strongly indicates increased secretion of proteolytic enzymes. Excessive excretion of cystine by feeding raw soybeans has been found in rats, pigs and man (Both et al., 1960; Schneeman and Lyman, 1975; Hahn, 1988b). These findings are in line with the observation of a several-fold increase in the conversion of methionine to cystine in the liver of rats fed soybean proteinase inhibitors (Barnes and Kwong, 1965). Moreover, methionine supplementation seems to improve nutritive value of soybean diets to a greater extent than would be expected from the dietary amino acid composition (Booth et al., 1960; Long et al., 1965; Almquist et al., 1942). From the present experiment, it might appear that the rainbow trout responds much like the rat, pig and man to dietary proteinase inhibitors, and that inhibitors might increase the requirement of sulphur containing amino acids. A correlation between trypsin activity of the small intestine and protein digestibility seemed to exist. Linear correlation analysis showed the following relationship:
Barnes R. H. and Kwong E. (1965) Effect of soybean trypsin inhibitor and penicillin on cystine biosynthesis in the pancreas and its transport as exocrine protein secretion in intestinal tract of the rat. J. Nurr. 86, 245-252. Blow D. M., Birktoft J. J. and Hartley B. S. (1969) Role of a buried acid group in the mechanism of action of chymotrypsin. Nature 221, 337-342. Blow D. M. (1974) Stereochemistry of substrate binding and hydrolysis in the trypsin family of enzymes. In Proteinase Inhibitors. Proc. 2nd Int. Res. Conf., (Edited by Frits H., Tschetsche H.. Greene L. J. and Truscheit E.), pp 473-479. Springer, Berlin. Booth A. N.. Robbins D. J.. Riebelin W. E. and DeEds F. (1960) Effects of raw soybean meal and amino acids on pancreatic hypertrophy in rats. Proc. Sot. exp. Biol. Med. 116, 1067-1073. Green G. (1979) Secretion and intestinal degradation of pancreatic lipase; role of dietary protein and proteinase inhibitors. Fed. Proc. 38, 279. Green G. and Nasset E. S. (1980) Importance of bile in regulation of intraluminal proteolytic enzyme activities in the rat. Gastroentqology 79, 695-702. Holm H., Krogdahl A. and Hansen L. (1988a) High and low inhibitor soybean meals affect human duodenal proteinase activity differently: in vitro comparison of proteinase inhibition. J. Nutr. 118, 521-525.0 Holm H., Hansen L., Krogdahl A. and Florholmen J. (1988b) High and low inhibitor soybean meals affect human duodenal proteinase activity differently: in oiuo comparison with bovine serum albumin. J. Nufr. 118, 515-520. Ikenaka T., Odani S. and Koide T. (1974) Chemical structure and inhibitory activities of soybean proteinase inhibitors. In Proteinase Inhibitors. Rot. 2nd Int. Res. Conf.,
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Niess E. (1981) Untersuchungen zur regulation der gallenblasen und pancreastatigkeit des huhnes. 2.
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Mitteilung. Z. Tierphysiol. Tiererniihr. Futtermitrelkd. 45, 2945. Olli J., Krogdahl A. and Ingh T. van den. Nutritive value of soy products varies widely in diets for Atlantic salmon (Salmo salar). Agric. Scund. (in press). Snedecor G. W. and Cochran W. G. (1973) Chapter 6, Regression; Chapter 7, Correlation, pp. 135-171, and 172-198. In Stntistical Methoa!s. 6th ed. Iowa State University Press, Ames. Schneeman B. 0. and Lyman R. L. (1975) Factors involved in the intestinal feed-back regulation of pancreatic enzyme secretion in the rat. Proc. Sot. exp. Biol. Med. 148, 897-903. Waterlow J. (1990) Nutritional adaptation in man: general introduction and concepts. Am. J. Clin. Nutr. 51, 259-263.
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