- Email: [email protected]
Effects of dietary rice bran trypsin inhibitor on the growth and pancreatic weights of rats
NUTRITION RESEARCH, Vol. 10, pp. 1325-1330,1990 0271-5317/90 $3.00 + .00 Printed in the USA. Copyright (c) 1990 Pergamon Press plc. All rights reserved.
EFFECTS OF DIETARY RICE BRAN TRYPSIN INHIBITOR ON THE GROWTH AND PANCREATIC WEIGHTS OF RATS Misao Tashiro, Ph.D. and Zensuke Maki, Ph.D.
I
Department of Food Science and N u t r i t i o n , Kyoto P r e f e c t u r a l U n i v e r s i t y , Shimogamo, Kyoto 6 0 6 , Japan ABSTRACT Male weanling r a t s were fed a 11.5% c a s e i n basal d i e t or the casein d i e t s c o n t a i n i n g r i c e bran t r y p s i n i n h i b i t o r (RBTI) at l e v e l s of 1.0, 2.0, and 4.0 g/kq d i e t for 3 weeks, and the response of rats to dietary RBTI was examined. Results showed that the body weight gain and food efficiency of the 0.4% RBTI diet group were significantly lower than those of the basal diet group at I wk, but these differences disappeared at 2 wk. Apparent nitrogen digestibility was also lower in the 0.4% RBTI diet group than in the basal when measured on days 3 and 4 after the start of the trial. Concerning pancreas, a significant increase in the weight was observed in rats given diets containing 0.2% and 0.4% RBTI as compared to rats fed the basal diet, and the levels of trypsin and chymotrypsin activities tended to be higher in the RBTI diet groups than in the basal. These findings indicate that RBTI consumption in weanling rats elicits physiological responses such as growth retardation and pancreatic enlargement. However, the deleterious effects of dietary RBTI seem to be ignored when the ingested level of RBTI is low under the conditions used in this study. Key words: Rice bran, Trypsin i n h i b i t o r , Growth depression, P a n c r e a t i c enlargement INTRODUCTION Rice bran i s a v a l u a b l e by-product as a p o t e n t i a l source of food, because its world production can give a n n u a l l y around 4 m i l l i o n tons of p r o t e i n , 5 m i l l i o n tons of edible o i l , 75,000 b i l l i o n c a l o r i e s , plus vitamins and minerals ( I ) . However,r i c e bran a l s o c o n t a i n s a f a i r amount of t r y p s i n i n h i b i t o r (2), being regarded as an a n t i n u t r i t i o n a lf a c t o r {3).
of
The nutritional responses to consumption trypsin, particularly those from legumes,
I p r e s e n t address:
of protein have been
inhibitors extensively
Eastern Osaka Junior College, Osaka 5 9 2 , Japan.
1325
1326
M. TASHIRO and Z. MAKI
s t u d i e d i n s e v e r a l animal s p e c i e s ( 3 , 4 ) . In s p e c i a l , i t is well known t h a t t r y p s i n i n h i b i t o r s from soybean cause growth d e p r e s s i o n and p a n c r e a t i c enlargement when fed to r a t s and the e f f e c t s of the d i e t a r y i n h i b i t o r s have been reviewed r e c e n t l y ( 4 ) . The e f f e c t s of d i e t a r y t r y p s i n i n h i b i t o r s from c e r e a l s , however, have r a r e l y been examined. The purpose of the study was to c l a r i f y the e f f e c t s of f e e d i n g RBTI to weanling r a t s .
MATERIALS
AND METHODS
RBTI was highly purified from the extract of defatted rice bran by heat treatment, ammonium s u l f a t e precipitation, and ionexchange chromatography on CM-Sephadex C-25. The final preparation had 91% c r u d e p r o t e i n a n d 1 mg o f the sample was capable of inhibiting 1 . 2 mg o f c r y s t a l l i n e bovine trypsin {Sigma Chemical Co., Type III), which corresponded to a half of the specific activity of the most purified RBTI(2). A b a s a l d i e t c o n t a i n e d 11.5% c a s e i n (87% crude p r o t e i n , N X 6.25), 6 5 . 5 % corn s t a r c h , 5% s u c r o s e , 4% c e l l u l o s e powder, 6% soybean o i l , 6% s a l t mixture, and 2% v i t a m i n mixture. The b a s a l d i e t was supplemented with t h r e e l e v e l s of RBTI, 0 . 1 % , 0 . 2 % , and 0.4%, a t the expense of corn s t a r c h to o b t a i n the RBTI d i e t s . The d i e t a r y i n g r e d i e n t s were purchased from O r i e n t a l Yeast Co., Tokyo, Japan, except for soybean o i l and s u c r o s e which were o b t a i n e d from Nakarai Chemicals, Kyoto, Japan. Weanling male Wistar r a t s (Shizuoka Laboratory Animal Center, Hamamatsu, Japan), weighing about 50 g, were randomly d e r i d e d i n t o four groups of f i v e animals each and fed one of four experimental diets for 3 weeks, preceded by an a d a p t a t i o n p e r i o d of 4 days. The r a t s were housed i n i n d i v i d u a l m e t a b o l i c cages i n a room with a 12 hr l i g h t - d a r k c y c l e ; temperature and humidity were m a i n t a i n e d at 24C and 5 0 % . W a t e rand food were s u p p l i e d ad l i b i t u m . Food consumption and body weight were recorded d a i l y {between I 0 0 0 and llO0). Feces and u r i n e were c o l l e c t e d a t t h r e e d i f f e r e n t p e r i o d s of two days each d u r i n g the experiment {period I : days 3 and 4; period II: days I0 and I I ; period I l l : days 17 and ] 6 ) . These samples were frozen and s t o r e d a t -20C u n t i l analyzed. At the end of the f e e d i n g p e r i o d , the r a t s were k i l l e d between 1300 and 1600 by d e c a p i t a t i o n a f t e r being f a s t e d for 3 to 6 hours. The l i v e r , k i d n e y s , p a n c r e a s , and s p l e e n of each r a t were d i s s e c t e d o u t , trimmed of f a t , and weighed. P a n c r e a t a were then f r o z e n on dry i c e and s t o r e d a t -20C u n t i l use. The pancreas from each r a t was h o m o g e n i z e d twice with a Potter-Elvehjem h o m o g e n i z e r w i t h 2 ml o f 1 mM HC1 c o n t a i n i n g 20 mM CaCI 9 . After centrifugation of the homogenate, the combined supernatants were diluted t o lO ml w i t h d i s t i l l e d w a t e r a n d lO0 p g of enterokinase {partially purified, S i g m a C h e m i c a l C o . ) i n lO ml of 50 mM T r i s - H C 1 b u f f e r {pH 7 . 5 ) c o n t a i n i n g 0 . 2 M CaC1 o was a d d e d to this solution. After incubation f o r 18 h r a t 5C, altquots were withdrawn from the mixture and used to measure the esterase activities of trypsin and chymotrypsin. This incubation provided
RICE BRAN AND TRYPSIN INHIBITOR
1327
a l l of t h e p o s s i b l e a c t i v i t i e s of t r y p s i n and chymotrypsin.
Nitrogen content was determined b y the micro-Kjeldahl method (5). Trypsin and trypsin inhibitor activities were measured in a PH stat (RTS 622, Radiometer, Copenhagen, Denmark) at 25C under nitrogen gas with 4 mM tosyl-L-arginine methylester HCI in 0.2 M KCI (pH 8). Chymotrypsin activity was also determined with 10 mM acetyl-h-tyrosine ethylester in 0.05 M CaCI 2 (pH 8) One enzyme unit was the activity which would hydrolyze 1 ~mol of substrate per min. Data were analyzed by analysis of variance and Duncan's multiple-range test to determine differences among d i e t g r o u p s . RESULTS Table I shows the data of cumulative three week body weight gain, food intake, and food efficiency, expressed as body weight gain per food consumed. Over the entire experimental period, there were no significant differences in the amount of diet consumed by rats. On the other hand, the body weight gain and food efficiency of the 0.4% RBTI diet group were significantly lower than those of the basal diet group at l wk. However, these differences disappeared at 2 wk.
can
Values of apparent nitrogen digestibility and nitrogen balance be seen in Table 2. The d i g e s t i b i l i t i e s were s i m i l a r a t
TABLE I Cumulative t h r e e Week Body Weight Gain, F o o d I n t a k e . and Food E f f i c i e n c y f o r Rats fed Experimental D i e t s . [ ' 2
Week(s)
Group
Basal 0.1% RBTI
1§247
~Values Values
33.9 + 24.3 + _
gain
3.6 a b 2 5 " ab 1.4 2.6 b
Food intake ( g )
Food
efficiency
68.4 -+ 5.1 58,7 + 2.3
0.491 0.411
+ 0 017 a. + U.UZ " ' - ~ 9 a_4 b
59.4 • 0 . 9 54.3 • 3.9
0.417 + 0 . 0 2 3 ~ 0 0.398 + 0.022"
RBTI 0.4% RBTI
24.8 -• 2 1 . 9 +_
Basal 0 . 1 % RBTI 0.2% RBTI 0.4% RBTI
60.5 44.0 41.7 40.2
• • • •
8.1 3.8 2.4 5.4
151 124 121 116
• • • •
14 5 3 11
0.395 0.364 0.345 0.341
• • • •
0.020 0.029 0.016 0.016
Basal 0 . 1 % RBTI 0.2% RBTI 0.4% RBTI
84.3 74.3 69.0 62.7
• • • •
10.7 5.4 2.0 4.6
238 211 202 187
• • • •
22 11 4 13
0.351 0.352 0.343 0.335
• • • •
0.017 0.008 0.009 0.005
0.2%
I+2
Body weight ( g )
are means • SEM of 5 rats per group. with differing superscripts are significantly (p
different
1328
M. TASHIRO and Z. MAKI TABLE and Nitrogen
Period
3-4
I:
II:
10-11
III:17-18
Apparent Nitrogen Digestibility Balance for Rats fed Experimental Group
day
day
day
2
Apparent nitrogen digestibility (%)
Nitrogen balance (mg/day)
Basal 0 . 1 % RBTI 0.2% RBTI 0 . 4 % RBTI
93.9 92 8 91 4 90 0
+ ~ ~ ~ _
0 . 2l aa b 1 5a b 0 2b I
104 82.1 83.6 74.2
• II.0 • 6.0 • 4.7 + I0.4
Basal 0 . 1 % RBTI 0 . 2 % RBTI 0.4% RBTI
92.3 92.6 91.2 90.6
+ + + +
0.5 0.2 0.6 1.0
124 93.9 82.7 83.5
+ 15.0 + 9.6 + 6.9 + 15.1
Basal 0,1% RBTI 0 . 2 % RBTI 0 . 4 % RBTI
91.2 90.6 89.9 89.9
+ + + +
0.2 0.5 0.5 1.0
100 118 104 95.6
+ 12.6 + 16.4 + 6.4 + 10.3
~V a l u e s
a r e m e a n s • SEM o f 5 r a t s P e r Values with differing superscripts (p<0.05).
TABLE
and Pancreatic
grouP. are significantly
Basal 0.35 0.1% RBTI 0 . 4 2 0.2% RBTI 0 . 4 6 0.4% RBTI 0 . 5 0
different
Wet Pancreatic Weights Enzyme Activities of Rats fed Experimental
Pancreas(g)/ I00 g Body wt.
+ ~ ~ +
0.02 a 0.039 b 0.02~ 0.03 D
Total units 187 257 235 229
+ ~ ~ ~
Diets.
1,2
Chymotrypsin
Units/ 100g Body w t . 21 35 19 18
i
3
Trypsin Group
1,2 Diets.
138 205 196 206
• • • +
II 27 16 20
Total units 491 629 606 627
• • • +
Units/ 100g Body w t . 92 75 37 23
363 495 505 563
+ ~ + ~
632b 37ab 28 33 b
~Values
are means • SEM of 5 rats per group. Values with differing superscripts are significantly (p<0.05).
different
approximately 90% i n a l l g r o u p s a t t h r e e d i f f e r e n t periods, but the value f o r t h e 0 . 4 % RBTI was s i g n i f i c a n t l y lower than that for the basal at Period I. On t h e o t h e r h a n d , there were no significant differences in nitrogen b a l a n c e among a n y g r o u p s a t a n y periods, t h o u g h a t e n d e n c y f o r t h e 0 . 4 % RBTI d i e t g r o u p t o n i t r o g e n balance less positive than the basal group existed.
RICE BRAN AND TRYPSIN INHIBITOR
1329
Wet pancreatic weights, expressed as percent of body weight, and pancreatic trypsin and chymotrypsin activities are represented in Table 3. Among the organ weights measured, only pancreatic weight responded to the contents of RBTI in the diets. The rats fed the 0.4% RBTI diet yielded the highest pancreatic weight. The second highest weight was shown in the 0 . 2 % RBTI diet group, followed by the 0.1% RBTI and the lowest was the basal. It was also indicated that pancreata of rats fed the 0.2% and 0.4% RBTI diets were significantly heavier than those of rats fed the basal diet. The levels of trypsin and chymotrypsin activities tended to be higher in the RBTI diet groups than in the basal. This tendency was enphasized when the levels were expressed as units per 100 g body weight and this level of chymotrypsin differed significantly between the basal and 0.4% RBTI diet groups. However, there were no significant differences among any groups in each enzyme level expressed as units per g pancreatic weight (data were not shown).
DISCUSSION The present data demonstrate that RBTI consumption in weanling rats elicits Physiological responses such as growth retardation and pancreatic enlargement. However, a significant growth depression was observed in only rats fed the diet containing 0.4% RBTI for 1 week. Although both diets containing 0.2% and 0.4% RBTI induced a significant pancreatic enlargement after the 3-wk feeding, no significant effect on pancreas was observed in rats receiving the 0.1% RBTI diet. Therefore, the deleterious effects of dietary RBTI seem to be ignored when the ingested level of RBTI is low under the conditions used in this study. Similar results were shown by Mitchell et al. (6), who reported that a casein-based diet containing about 0.1% trypsin inhibitor from opaque-2 corn does not cause any growth depression or pancreatic enlargement, when fed to weanling rats for 4 weeks. In their case, however, it should be noted that the trypsin inhibitor was purified by affinity chromatography, which may have produced a modified inhibitor. We have already reported that weanling rats fed a rice bran protein concentrate with trypsin inhibitor activity grew slower than rats fed the heated protein concentrate and have suggested that the trypsin inhibitor in the concentrate accounts for the growth retardation (7). However, our previous finding is not completely compatible with that in the present study, since the trypsin inhibitor activity of the diet containing the raw concentrate only corresponds to that of the 0.1% RBTI diet which did not induce a significant growth retardation in this study. Therefore, we presume that the degree of growth retardation depends on the quality and/or quantity of a dietary protein in diet. It is also probable t h a t RBTI i s n o t t h e s o l e f a c t o r contributing to the deleterious effects of the rice bran protein concentrate fed to rats. In the study concerning soybean flour, Kakade et al. (8) have also postulated that trypsin inhibitor is responsible for only 40% o f t h e d e l e t e r i o u s effects of raw soybean flour.
1330
M. TASHIRO and Z. MAKI
It has been proposed that pancreatic enzyme secretion in rat is regulated by a feedback mechanism, which can explain a stimulatory effect of dietary trypsin inhibitor on secretion (9). Pancreatic enlargement is also believed to be caused by the stimulatory effect. Probably, the pancreatic enlargement observed in this study can be interpreted by the above hypothesis. On the other hand, growth depression is supposed to occur as a result of the endogenous nitrogen loss produced by over secretion of pancreatic enzymes in addition to the incomplete intestinal proteolysis of dietary proteins (4). Our present data seem to support this idea. However, long-term feeding studies should also be necessary to understand the overall effects of dietary RBTI.
REFERENCES 1.
B a r b e r S. R i c e b r a n a s a p o t e n t i a l source of food. In: F u j i m a k i M, l w a i K, M i t s u d a H, M o r i t a Y, e d s . Proc. Cong F o o d S c i T e c h , T o k y o : K o d a n s h a , 1 9 7 9 : 1 1 2 - 2 1 .
2.
Tashiro M, Maki trypsin inhibitor 25: 255-64.
3.
Liener Toxic Press,
4.
Gallaher D, S c h n e e m a n BO. N u t r i t i o n a l and metabolic response to plant inhibitors of digestive enzymes. In: Friedman M, ed. Nutritional and Toxicological Significance of Enzyme Inhibitors i n F o o d s , New Y o r k : P l e n u m , 1 9 8 6 : 1 6 7 - 8 4 .
5.
H o r w i t z U, Uashington,
6.
Mitchell HL, Parrisb DB, C o r m e y M, U a s s o m CE. E f f e c t of corn trypsin inhibitor on growth of rats. J Agric F o o d Chem 1976; 24: 1254-5.
7.
Maki Z, T a s h i r o M. Nutritional significance concentrate with trypsin inhibitor activity. Vitaminol 1983; 29: 293-302.
8.
Kakade ML, Hoffa D, Liener IE. inhibitors to the deleterious effects to rats. J N u t r 1973; 103: 1 7 7 2 - 8 .
9.
G r e e n GM, Lyman RL. Feedback regulation of pancreatic enzyme secretion as a mechanism for trypsin inhibitor-induced hypersecretion in rats. P r o c S o c Exp B i o l Med 1 9 7 2 ; 1 4 0 : 6 - 1 2 .
Z. Purification from rice bran.
IE, K a k a d e ML. Constituents of 1980: 7-71.
ed. Official D . C . : AOAC,
and characterization d Nutr Sci Vitaminol
Protease inhibitors. Plant Foodstuffs,
Methods of 1970: 858.
Accepted for publication July 11, 1990.
C h i b a H, 5th Int. of a 1979;
In: Liener IE, ed. New Y o r k : Academic
Analysis
of
AOAC,
llth
of a rice J Nutr
Contribution of unheated
ed.,
bran Sci
of trypsin soybeans fed
EFFECTS OF DIETARY RICE BRAN TRYPSIN INHIBITOR ON THE GROWTH AND PANCREATIC WEIGHTS OF RATS Misao Tashiro, Ph.D. and Zensuke Maki, Ph.D.
I
Department of Food Science and N u t r i t i o n , Kyoto P r e f e c t u r a l U n i v e r s i t y , Shimogamo, Kyoto 6 0 6 , Japan ABSTRACT Male weanling r a t s were fed a 11.5% c a s e i n basal d i e t or the casein d i e t s c o n t a i n i n g r i c e bran t r y p s i n i n h i b i t o r (RBTI) at l e v e l s of 1.0, 2.0, and 4.0 g/kq d i e t for 3 weeks, and the response of rats to dietary RBTI was examined. Results showed that the body weight gain and food efficiency of the 0.4% RBTI diet group were significantly lower than those of the basal diet group at I wk, but these differences disappeared at 2 wk. Apparent nitrogen digestibility was also lower in the 0.4% RBTI diet group than in the basal when measured on days 3 and 4 after the start of the trial. Concerning pancreas, a significant increase in the weight was observed in rats given diets containing 0.2% and 0.4% RBTI as compared to rats fed the basal diet, and the levels of trypsin and chymotrypsin activities tended to be higher in the RBTI diet groups than in the basal. These findings indicate that RBTI consumption in weanling rats elicits physiological responses such as growth retardation and pancreatic enlargement. However, the deleterious effects of dietary RBTI seem to be ignored when the ingested level of RBTI is low under the conditions used in this study. Key words: Rice bran, Trypsin i n h i b i t o r , Growth depression, P a n c r e a t i c enlargement INTRODUCTION Rice bran i s a v a l u a b l e by-product as a p o t e n t i a l source of food, because its world production can give a n n u a l l y around 4 m i l l i o n tons of p r o t e i n , 5 m i l l i o n tons of edible o i l , 75,000 b i l l i o n c a l o r i e s , plus vitamins and minerals ( I ) . However,r i c e bran a l s o c o n t a i n s a f a i r amount of t r y p s i n i n h i b i t o r (2), being regarded as an a n t i n u t r i t i o n a lf a c t o r {3).
of
The nutritional responses to consumption trypsin, particularly those from legumes,
I p r e s e n t address:
of protein have been
inhibitors extensively
Eastern Osaka Junior College, Osaka 5 9 2 , Japan.
1325
1326
M. TASHIRO and Z. MAKI
s t u d i e d i n s e v e r a l animal s p e c i e s ( 3 , 4 ) . In s p e c i a l , i t is well known t h a t t r y p s i n i n h i b i t o r s from soybean cause growth d e p r e s s i o n and p a n c r e a t i c enlargement when fed to r a t s and the e f f e c t s of the d i e t a r y i n h i b i t o r s have been reviewed r e c e n t l y ( 4 ) . The e f f e c t s of d i e t a r y t r y p s i n i n h i b i t o r s from c e r e a l s , however, have r a r e l y been examined. The purpose of the study was to c l a r i f y the e f f e c t s of f e e d i n g RBTI to weanling r a t s .
MATERIALS
AND METHODS
RBTI was highly purified from the extract of defatted rice bran by heat treatment, ammonium s u l f a t e precipitation, and ionexchange chromatography on CM-Sephadex C-25. The final preparation had 91% c r u d e p r o t e i n a n d 1 mg o f the sample was capable of inhibiting 1 . 2 mg o f c r y s t a l l i n e bovine trypsin {Sigma Chemical Co., Type III), which corresponded to a half of the specific activity of the most purified RBTI(2). A b a s a l d i e t c o n t a i n e d 11.5% c a s e i n (87% crude p r o t e i n , N X 6.25), 6 5 . 5 % corn s t a r c h , 5% s u c r o s e , 4% c e l l u l o s e powder, 6% soybean o i l , 6% s a l t mixture, and 2% v i t a m i n mixture. The b a s a l d i e t was supplemented with t h r e e l e v e l s of RBTI, 0 . 1 % , 0 . 2 % , and 0.4%, a t the expense of corn s t a r c h to o b t a i n the RBTI d i e t s . The d i e t a r y i n g r e d i e n t s were purchased from O r i e n t a l Yeast Co., Tokyo, Japan, except for soybean o i l and s u c r o s e which were o b t a i n e d from Nakarai Chemicals, Kyoto, Japan. Weanling male Wistar r a t s (Shizuoka Laboratory Animal Center, Hamamatsu, Japan), weighing about 50 g, were randomly d e r i d e d i n t o four groups of f i v e animals each and fed one of four experimental diets for 3 weeks, preceded by an a d a p t a t i o n p e r i o d of 4 days. The r a t s were housed i n i n d i v i d u a l m e t a b o l i c cages i n a room with a 12 hr l i g h t - d a r k c y c l e ; temperature and humidity were m a i n t a i n e d at 24C and 5 0 % . W a t e rand food were s u p p l i e d ad l i b i t u m . Food consumption and body weight were recorded d a i l y {between I 0 0 0 and llO0). Feces and u r i n e were c o l l e c t e d a t t h r e e d i f f e r e n t p e r i o d s of two days each d u r i n g the experiment {period I : days 3 and 4; period II: days I0 and I I ; period I l l : days 17 and ] 6 ) . These samples were frozen and s t o r e d a t -20C u n t i l analyzed. At the end of the f e e d i n g p e r i o d , the r a t s were k i l l e d between 1300 and 1600 by d e c a p i t a t i o n a f t e r being f a s t e d for 3 to 6 hours. The l i v e r , k i d n e y s , p a n c r e a s , and s p l e e n of each r a t were d i s s e c t e d o u t , trimmed of f a t , and weighed. P a n c r e a t a were then f r o z e n on dry i c e and s t o r e d a t -20C u n t i l use. The pancreas from each r a t was h o m o g e n i z e d twice with a Potter-Elvehjem h o m o g e n i z e r w i t h 2 ml o f 1 mM HC1 c o n t a i n i n g 20 mM CaCI 9 . After centrifugation of the homogenate, the combined supernatants were diluted t o lO ml w i t h d i s t i l l e d w a t e r a n d lO0 p g of enterokinase {partially purified, S i g m a C h e m i c a l C o . ) i n lO ml of 50 mM T r i s - H C 1 b u f f e r {pH 7 . 5 ) c o n t a i n i n g 0 . 2 M CaC1 o was a d d e d to this solution. After incubation f o r 18 h r a t 5C, altquots were withdrawn from the mixture and used to measure the esterase activities of trypsin and chymotrypsin. This incubation provided
RICE BRAN AND TRYPSIN INHIBITOR
1327
a l l of t h e p o s s i b l e a c t i v i t i e s of t r y p s i n and chymotrypsin.
Nitrogen content was determined b y the micro-Kjeldahl method (5). Trypsin and trypsin inhibitor activities were measured in a PH stat (RTS 622, Radiometer, Copenhagen, Denmark) at 25C under nitrogen gas with 4 mM tosyl-L-arginine methylester HCI in 0.2 M KCI (pH 8). Chymotrypsin activity was also determined with 10 mM acetyl-h-tyrosine ethylester in 0.05 M CaCI 2 (pH 8) One enzyme unit was the activity which would hydrolyze 1 ~mol of substrate per min. Data were analyzed by analysis of variance and Duncan's multiple-range test to determine differences among d i e t g r o u p s . RESULTS Table I shows the data of cumulative three week body weight gain, food intake, and food efficiency, expressed as body weight gain per food consumed. Over the entire experimental period, there were no significant differences in the amount of diet consumed by rats. On the other hand, the body weight gain and food efficiency of the 0.4% RBTI diet group were significantly lower than those of the basal diet group at l wk. However, these differences disappeared at 2 wk.
can
Values of apparent nitrogen digestibility and nitrogen balance be seen in Table 2. The d i g e s t i b i l i t i e s were s i m i l a r a t
TABLE I Cumulative t h r e e Week Body Weight Gain, F o o d I n t a k e . and Food E f f i c i e n c y f o r Rats fed Experimental D i e t s . [ ' 2
Week(s)
Group
Basal 0.1% RBTI
1§247
~Values Values
33.9 + 24.3 + _
gain
3.6 a b 2 5 " ab 1.4 2.6 b
Food intake ( g )
Food
efficiency
68.4 -+ 5.1 58,7 + 2.3
0.491 0.411
+ 0 017 a. + U.UZ " ' - ~ 9 a_4 b
59.4 • 0 . 9 54.3 • 3.9
0.417 + 0 . 0 2 3 ~ 0 0.398 + 0.022"
RBTI 0.4% RBTI
24.8 -• 2 1 . 9 +_
Basal 0 . 1 % RBTI 0.2% RBTI 0.4% RBTI
60.5 44.0 41.7 40.2
• • • •
8.1 3.8 2.4 5.4
151 124 121 116
• • • •
14 5 3 11
0.395 0.364 0.345 0.341
• • • •
0.020 0.029 0.016 0.016
Basal 0 . 1 % RBTI 0.2% RBTI 0.4% RBTI
84.3 74.3 69.0 62.7
• • • •
10.7 5.4 2.0 4.6
238 211 202 187
• • • •
22 11 4 13
0.351 0.352 0.343 0.335
• • • •
0.017 0.008 0.009 0.005
0.2%
I+2
Body weight ( g )
are means • SEM of 5 rats per group. with differing superscripts are significantly (p
different
1328
M. TASHIRO and Z. MAKI TABLE and Nitrogen
Period
3-4
I:
II:
10-11
III:17-18
Apparent Nitrogen Digestibility Balance for Rats fed Experimental Group
day
day
day
2
Apparent nitrogen digestibility (%)
Nitrogen balance (mg/day)
Basal 0 . 1 % RBTI 0.2% RBTI 0 . 4 % RBTI
93.9 92 8 91 4 90 0
+ ~ ~ ~ _
0 . 2l aa b 1 5a b 0 2b I
104 82.1 83.6 74.2
• II.0 • 6.0 • 4.7 + I0.4
Basal 0 . 1 % RBTI 0 . 2 % RBTI 0.4% RBTI
92.3 92.6 91.2 90.6
+ + + +
0.5 0.2 0.6 1.0
124 93.9 82.7 83.5
+ 15.0 + 9.6 + 6.9 + 15.1
Basal 0,1% RBTI 0 . 2 % RBTI 0 . 4 % RBTI
91.2 90.6 89.9 89.9
+ + + +
0.2 0.5 0.5 1.0
100 118 104 95.6
+ 12.6 + 16.4 + 6.4 + 10.3
~V a l u e s
a r e m e a n s • SEM o f 5 r a t s P e r Values with differing superscripts (p<0.05).
TABLE
and Pancreatic
grouP. are significantly
Basal 0.35 0.1% RBTI 0 . 4 2 0.2% RBTI 0 . 4 6 0.4% RBTI 0 . 5 0
different
Wet Pancreatic Weights Enzyme Activities of Rats fed Experimental
Pancreas(g)/ I00 g Body wt.
+ ~ ~ +
0.02 a 0.039 b 0.02~ 0.03 D
Total units 187 257 235 229
+ ~ ~ ~
Diets.
1,2
Chymotrypsin
Units/ 100g Body w t . 21 35 19 18
i
3
Trypsin Group
1,2 Diets.
138 205 196 206
• • • +
II 27 16 20
Total units 491 629 606 627
• • • +
Units/ 100g Body w t . 92 75 37 23
363 495 505 563
+ ~ + ~
632b 37ab 28 33 b
~Values
are means • SEM of 5 rats per group. Values with differing superscripts are significantly (p<0.05).
different
approximately 90% i n a l l g r o u p s a t t h r e e d i f f e r e n t periods, but the value f o r t h e 0 . 4 % RBTI was s i g n i f i c a n t l y lower than that for the basal at Period I. On t h e o t h e r h a n d , there were no significant differences in nitrogen b a l a n c e among a n y g r o u p s a t a n y periods, t h o u g h a t e n d e n c y f o r t h e 0 . 4 % RBTI d i e t g r o u p t o n i t r o g e n balance less positive than the basal group existed.
RICE BRAN AND TRYPSIN INHIBITOR
1329
Wet pancreatic weights, expressed as percent of body weight, and pancreatic trypsin and chymotrypsin activities are represented in Table 3. Among the organ weights measured, only pancreatic weight responded to the contents of RBTI in the diets. The rats fed the 0.4% RBTI diet yielded the highest pancreatic weight. The second highest weight was shown in the 0 . 2 % RBTI diet group, followed by the 0.1% RBTI and the lowest was the basal. It was also indicated that pancreata of rats fed the 0.2% and 0.4% RBTI diets were significantly heavier than those of rats fed the basal diet. The levels of trypsin and chymotrypsin activities tended to be higher in the RBTI diet groups than in the basal. This tendency was enphasized when the levels were expressed as units per 100 g body weight and this level of chymotrypsin differed significantly between the basal and 0.4% RBTI diet groups. However, there were no significant differences among any groups in each enzyme level expressed as units per g pancreatic weight (data were not shown).
DISCUSSION The present data demonstrate that RBTI consumption in weanling rats elicits Physiological responses such as growth retardation and pancreatic enlargement. However, a significant growth depression was observed in only rats fed the diet containing 0.4% RBTI for 1 week. Although both diets containing 0.2% and 0.4% RBTI induced a significant pancreatic enlargement after the 3-wk feeding, no significant effect on pancreas was observed in rats receiving the 0.1% RBTI diet. Therefore, the deleterious effects of dietary RBTI seem to be ignored when the ingested level of RBTI is low under the conditions used in this study. Similar results were shown by Mitchell et al. (6), who reported that a casein-based diet containing about 0.1% trypsin inhibitor from opaque-2 corn does not cause any growth depression or pancreatic enlargement, when fed to weanling rats for 4 weeks. In their case, however, it should be noted that the trypsin inhibitor was purified by affinity chromatography, which may have produced a modified inhibitor. We have already reported that weanling rats fed a rice bran protein concentrate with trypsin inhibitor activity grew slower than rats fed the heated protein concentrate and have suggested that the trypsin inhibitor in the concentrate accounts for the growth retardation (7). However, our previous finding is not completely compatible with that in the present study, since the trypsin inhibitor activity of the diet containing the raw concentrate only corresponds to that of the 0.1% RBTI diet which did not induce a significant growth retardation in this study. Therefore, we presume that the degree of growth retardation depends on the quality and/or quantity of a dietary protein in diet. It is also probable t h a t RBTI i s n o t t h e s o l e f a c t o r contributing to the deleterious effects of the rice bran protein concentrate fed to rats. In the study concerning soybean flour, Kakade et al. (8) have also postulated that trypsin inhibitor is responsible for only 40% o f t h e d e l e t e r i o u s effects of raw soybean flour.
1330
M. TASHIRO and Z. MAKI
It has been proposed that pancreatic enzyme secretion in rat is regulated by a feedback mechanism, which can explain a stimulatory effect of dietary trypsin inhibitor on secretion (9). Pancreatic enlargement is also believed to be caused by the stimulatory effect. Probably, the pancreatic enlargement observed in this study can be interpreted by the above hypothesis. On the other hand, growth depression is supposed to occur as a result of the endogenous nitrogen loss produced by over secretion of pancreatic enzymes in addition to the incomplete intestinal proteolysis of dietary proteins (4). Our present data seem to support this idea. However, long-term feeding studies should also be necessary to understand the overall effects of dietary RBTI.
REFERENCES 1.
B a r b e r S. R i c e b r a n a s a p o t e n t i a l source of food. In: F u j i m a k i M, l w a i K, M i t s u d a H, M o r i t a Y, e d s . Proc. Cong F o o d S c i T e c h , T o k y o : K o d a n s h a , 1 9 7 9 : 1 1 2 - 2 1 .
2.
Tashiro M, Maki trypsin inhibitor 25: 255-64.
3.
Liener Toxic Press,
4.
Gallaher D, S c h n e e m a n BO. N u t r i t i o n a l and metabolic response to plant inhibitors of digestive enzymes. In: Friedman M, ed. Nutritional and Toxicological Significance of Enzyme Inhibitors i n F o o d s , New Y o r k : P l e n u m , 1 9 8 6 : 1 6 7 - 8 4 .
5.
H o r w i t z U, Uashington,
6.
Mitchell HL, Parrisb DB, C o r m e y M, U a s s o m CE. E f f e c t of corn trypsin inhibitor on growth of rats. J Agric F o o d Chem 1976; 24: 1254-5.
7.
Maki Z, T a s h i r o M. Nutritional significance concentrate with trypsin inhibitor activity. Vitaminol 1983; 29: 293-302.
8.
Kakade ML, Hoffa D, Liener IE. inhibitors to the deleterious effects to rats. J N u t r 1973; 103: 1 7 7 2 - 8 .
9.
G r e e n GM, Lyman RL. Feedback regulation of pancreatic enzyme secretion as a mechanism for trypsin inhibitor-induced hypersecretion in rats. P r o c S o c Exp B i o l Med 1 9 7 2 ; 1 4 0 : 6 - 1 2 .
Z. Purification from rice bran.
IE, K a k a d e ML. Constituents of 1980: 7-71.
ed. Official D . C . : AOAC,
and characterization d Nutr Sci Vitaminol
Protease inhibitors. Plant Foodstuffs,
Methods of 1970: 858.
Accepted for publication July 11, 1990.
C h i b a H, 5th Int. of a 1979;
In: Liener IE, ed. New Y o r k : Academic
Analysis
of
AOAC,
llth
of a rice J Nutr
Contribution of unheated
ed.,
bran Sci
of trypsin soybeans fed