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Suppressive effect of soybean milk protein on experimentally induced skin tumor in mice
Life Sciences, Vol. 53, pp. 1591-1596 Printed in the USA
Pergamon Press
SUPPRESSIVE EFFECT OF SOYBEAN EXPERIMENTALLY INDUCED SKIN
MILK PROTEIN ON TUMOR IN MICE
Porn-ngarm Limtrakul ~, Maitree Suttajit l, Rie Semura, 2 Katsumasa Shimada 2 and Shigeru Yamamoto2 ~Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand. 2School of Health Sciences and Research Center of Comprehensive Medicine, Faculty of Medicine, University of the Ryukyus, Nishihara, Okinawa, Japan.
(Received in final form September 13, 1993)
Summary
We studied the effect of soybean milk protein (SMP) in a two-stage carcinogenesis experiment on mouse skin. Mice were given soybean protein isolate (SPI) diet or SPI diet supplemented with SMP. After 4 weeks on the diets, the mice were shaved and a tumor initiator was applied. A tumor promoter was then applied twice a week on the same area of the skin throughout the experiment. After 20 weeks on the treatment, the percentage of tumor-bearing mice and the volume of tumor tended to be lower in the mice on the SMP diet than those on the SPI diet. The number of tumor was also significantly lower in the former group as compared to the latter group. There was no difference in growth between mice of the SPI and SMP groups. The results indicate the safety and the anti-carcinogenic effect of SMP in mice.
Soybean milk protein (SMP) which is 7-8% of total soybean protein consists of trypsininhibitor (protease inhibitor), hemaglutinin, goitrogen, lipooxygenase, 13-amylase, protease, phytase and other physiologically active substances (1). Trypsin inhibitor inhibits trypsin and hemaglutinin impairs intestinal mucosal cells and therefore SMP has been considered to be unfavorable "anti-nutrients" (1). However, protease inhibitors occurring in soybeans and from other sources have been identified as chemopreventive agents. Troll (2) observed the anti-breast cancer effect of raw whole soybean in rats. Becker et al. (3) have reported that liver cancer which was observed in 100% mice on 26% casein diet, disappeared when 5% of the casein was substituted with whole soybean protein. Various proteinase inhibitors added in diets (4-6) and water (7) or applied on animal skin (8) have anti-carcinogenic effects. Rats that have cancer are also known to have high serum proteinase inhibitors (9). The anti-carcinogenic effect of various protease inhibitors have been reviewed (10,11). These findings suggest that SMP has anticarcinogenic effect. However during the process of the isolation of soybean protein, defatted soybean was first treated with alkaline solution and then acid solution. SPI is in the precipitate and SMP is in the supernatant of the solution. Therefore it is not clear whether there are similar anticarcinogenic effect in SMP as in raw whole soybean or in extracted specific proteinase inhibitors. 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
1592
Anti-carcinogenicEffect of Soybean Milk Protein
Vol. 53, No. 21, 1993
There have been reports on the unfavorable effects of soybean trypsin inhibitor for health. Major problem is the inhibition of trypsin in intestine (1). Carcinogenic factor has also been reported. Rats maintained on diets containing trypsin inhibitor have been observed to be susceptible to pancreatic cancer (12,13). However, some researchers have reported that such effects can not be observed even with relatively high amount of trypsin inhibitor in man (14), monkey (15), dog (16,17), cow (18), pig (19), mouse (2,6) and rat (20). In this study we tried to find whether there is an anti-tumor effect of SMP within the safe level of intake in mice, for the purpose of finding out proper utilization of SMP which is now practically discarded.
Materials and M e t h o d s
Twenty-eight male ICR mice, 4-5 weeks old, were obtained from the Animal House, Chiang Mai University, Chiang Mai, Thailand. The mice were divided into two groups and fed 20% SPI (donated by Fuji Oil Co., Osaka, Japan) diet (n=13) or 18% SPI diet supplemented with 2% SMP (donated by Fuji Oil Co., Osaka, Japan)(n=15). The SMP was prepared by the following method. Defatted soybean milk (pH 4.5) was centrifuged. The supernatant was neutralized and concentrated 20 times by ultrafiltration (MW=40,000) and then dried by spraydryer. Other components of the diets were: 44.4% a -starch, 22.2% sucrose, 5% soybean oil, 5% mineral mixture (Oriental Yeast Co., Osaka, Japan), 1% vitamin mixture (Oriental Yeast Co., Osaka, Japan), 2% cellulose and 0.4% DL-methionine. Details of the vitamin and mineral mixtures are shown elsewhere (21).
Four weeks after the onset of the diets, two stage carcinogenesis experiment on mouse skin was performed. The back of the mice were shaved and carcinogenesis was initiated by a single application of 100/1 g of 7-12 dimethylbenzanthracene (DMBA) dissolved in 0. lml of acetone to the skin of the back. From 1 week after the initiator application, tumor promoter, 2.5 /1 g of 12-0-tetradecanoylphorbol-13-acetate (TPA) dissolved in 0. lml of acetone was applied to the same areas on the mice, twice a week. The hairs which appear on the shaved part were cut by surgical scissors every 2 weeks. The numbers of tumors Imm or more in diameter were counted weekly. Tumor promoting activity was evaluated by the percentage of tumor- bearing mice and the average number and the volume of tumors per mouse. The number of tumors on each animal was recorded once a week by a vernier caliper and the average tumor volume was calculated as [length] x [width] x [height] 7r/6. Food intake and body weight were measured weekly. The data were analyzed by chi-square test and Student's t-test.
Results
Fig. 1 shows the changes in body weight. Final figures of the two groups are shown in parentheses. They were not significantly different (p>0.05). Fig. 2 shows the percentage of tumor-bearing mice. After 20 weeks on the treatment all the mice of SPI group had tumors, while 67% of the animals of the SMP group had tumors.
Vol. 53, No. 21, 1993
Anti-carcinogenic Effect of Soybean Milk Protein
1593
However the values were not significantly different (p>O.05). Fig. 3 shows the number of tumors per mouse. Final figures are shown in parentheses. The number was significantly lower in the whey group than in the SPI group (p<0.05). Fig. 4 shows average volume of tumors per mouse. The final figures are shown in the parentheses. Although the average was much lower in the whey group than in the SPI group, statistical difference was not observed (p>0.05).
46(43.8 ± 4.4)
44,~42-
4o-
,.,'t/t.8±,.,)
--!--
SPl
-.-
SMP
38>, 36 o
m 3432-
30
l ] I p I I ~ I r J r ] J I I I r I I r I 0
5
10 Weeks
15
20
of p r o m o t i o n
Fig. i Changes in body weight. Figures in parentheses indicate means + SD on the 20th week. significant difference was not observed by Student's/'-test (p>0.05).
A
100-
80 ~
60
--IE-
SPI
---0-
SMP
¢.-
-~
40
0
5
10 Weeks
15
20
of p r o m o t i o n
Fig. 2 Percentage o f tumor-bearing mice. The difference was not significant b y chi-square test (p>0.05).
1594
Anti-carcinogenic Effect of Soybean Milk Protein
Vol. 53, No. 21, 1993
10 (8.3 ± 6.0)
•
8 6
---I- SPI -0-
2 7°-"" 6
0"
~
0
5
SMP
±33'1~"
10 15 Weeks of promotion
20
Fig. 3 Number o f tumors/mouse. Figures in parentheses indicate means _+ SD on the 20th wceL Asterisk indicates a significant difference from SPI group by Student's t-test (p>0.05).
200-
~
(190.3±198.1)
150
,oo-t
.,
0
(84.8_+ 84.6)
----0-
SMP
0
0
5
10 15 Weeks of promotion
20
Fig. 4 Average volume o f tumor~mouse. Figure in parentheses indicate means ± SD on the 20th week. A significant difference was not observed b y Student's t -test (p>0.05).
Discussion
The results indicate that 2% SMP in a diet could suppress the skin tumor induced by chemical carcinogens. Our results are consistent with the findings of Troll et al. (4). They observed that rats with chemical carcinogens applied on their back and fed 50% raw soybean diet develop less tumors than those fed casein or roasted soybean diet. Therefore our present results suggest that anti-tumor effect of raw whole soybean is maintained in the SMP even after the various treatments.
Vol. 53, No. 21, 1993
Anti-carclnogenic Effect of Soybean Milk Protein
1595
The addition of SMP did not impair normal growth and development as evidenced by body weight gain and food intake, although trypsin inhibitor of SNIP is regarded as an unfavorable substance and discarded as a waste product of SPI processing. Troll et al. (2) have shown that the growth of rats fed 50% raw soybean diet is similar as those fed a commercial diet. Clair et al. (6) have also reported that mice fed diets containing 0.5% pure proteinase inhibitor obtained from soybean for 65 days had similar body weight gain in comparison to those fed a commercial diet and had no hypertrophy or cancer in pancreas. No unfavorable effects of trypsin inhibitor have been reported in man (14), monkey (15), dog (16,17), cow (18) and pig (19). Even in the research of Gumbmann (13) who tried to show the carcinogenic effect of trypsin inhibitor on pancreas, body weight changes of mice fed diets containing 0, 0.1, 0.2, 0.3 or 0.6% trypsin inhibitor were similar throughout the experimental period (15 months). Based on these results we can speculate that SMP can be beneficial as an anti-carcinogenic food or medicine. We are further studying the effect of SMP on histopathlogy of various internal organs.
References
1) I.R. LIENER, Am. J. Clin. Nutr. 1..A_1281-298(1962). 2) W. TROLL, R. WIESNER, C.J. SHELLABARGER, S. HOLTZMAN and J. P. STONE, Carcinogenesis 1469-472 (1980). 3) E E BECKER, Carcinogenesis 21213-1214 (1981). 4) W. TROLL, S. BELMAN, R. WlESNER and C. J. SHELLABARGER, Biologica Functions of Proteinases, in Z. Holzer, and H. Tschesche (Eds.), 165-170, Springer-Verlag, Berlin (1979). 5) J. YAVELOW, T.H. FINLA¥, A.R. KENNEDY and W. TROLL, Cancer Research 4._~3 2454s-2459s (1983). 6) W.H. CLAIR, P.C. BILLINGS, J.A. CAREW, K, McGANDY, P. NEWBERN and A.R. KENNEDY, Cancer Research 5 0 5 8 0 - 5 8 6 (1990). 7) J.G. CORASANTI, G.H. HOBIKA and G. MARKUS, Science 2161020-1021 (1982). 8) W. TROLL, A. KLASSEN andA.JANOFF, Science 1691211-1213 (1970). 9) Y. KONDO and N. OHSAWA, Cancer Research 4..~21549-1554 (1982). 10) W. TROLL, K. FRENKEL and R. WIESNER, J.N.C.I. 7 1245-1250 (1984). l l ) EP. SCHELP and P. PONGPAEW, Int. J. Epidemiol. 1_.~7287-292 (1988). 12) E.E. MaGUINNESS, R.G. MORGAN, D.A. LEVISION, D.L. FRAPE, D. HOPWOOD and K.G. WORMSLEY, Scand. J. Gastroenterol. 1 5 4 9 7 - 5 0 2 (1980). 13) M.R. GUMBMANN, W.L. SPANGLER, G.M. DUGAN, J.J. RACK/S, and I.E. LIENER, Qual. Plant. Plant Foods Hum. Nutr. 3__55 275-314 (1985). 14) D.E FLAVIN, Vet. Hum. Toxicol. 2._4_4 25-28 (1982). 15) L.M. AUSMAN, J.P. HARWOOD, N.W. KING, P.K. SEHGAL, R.J. NICOLOSI, M.D. HEGSTED, I.E. LIENER, D. DONATUCCI and J. TARCZA, J. Nutr. 115 1691-1701 (1985). 16) J.R. PATTERN, E.A. RICHARDS and J. WHEELER, Proc. Soc. Exp. Biol. Med. 1 3 7 5 8 (1971). 17) J.R. PATTERN, E.A. RICHARDS and .1. WHEELER, Life Sci. 1_.9_0145-150 (1971). 18) A.D.L. GORRILL, and J.W. THOMAS, J. Nutr. 9._2_2 215-223 (1967). 19) R.D. HOOKS, V.W. HAYS, V.C. SPEER and J.E McCALL, Fed. Proc. Am. Soc. Exp. Biol. 2 4 8 9 4 (1965)
1596
Anti-carcinogenicEffect of SoybeanMilk Protein
Vol. 53, No. 21, 1993
20) K. KISHI, S. TERAI, S. OOGURI, F. SHIZUKA, S. YAMAMOTO and G. INOUE, Nutr. Sci. Soy Prot. 7_ 47-52 (1986) (in Japanese). 21) S. YAMAMOTO, T. KOHRIN, M. MORI, L. ASATO, S. SHINJO, J. NAGAMINE and M.E WANG, J. Nutr. Biochcm. 3_ 279-284 (1992).
Pergamon Press
SUPPRESSIVE EFFECT OF SOYBEAN EXPERIMENTALLY INDUCED SKIN
MILK PROTEIN ON TUMOR IN MICE
Porn-ngarm Limtrakul ~, Maitree Suttajit l, Rie Semura, 2 Katsumasa Shimada 2 and Shigeru Yamamoto2 ~Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand. 2School of Health Sciences and Research Center of Comprehensive Medicine, Faculty of Medicine, University of the Ryukyus, Nishihara, Okinawa, Japan.
(Received in final form September 13, 1993)
Summary
We studied the effect of soybean milk protein (SMP) in a two-stage carcinogenesis experiment on mouse skin. Mice were given soybean protein isolate (SPI) diet or SPI diet supplemented with SMP. After 4 weeks on the diets, the mice were shaved and a tumor initiator was applied. A tumor promoter was then applied twice a week on the same area of the skin throughout the experiment. After 20 weeks on the treatment, the percentage of tumor-bearing mice and the volume of tumor tended to be lower in the mice on the SMP diet than those on the SPI diet. The number of tumor was also significantly lower in the former group as compared to the latter group. There was no difference in growth between mice of the SPI and SMP groups. The results indicate the safety and the anti-carcinogenic effect of SMP in mice.
Soybean milk protein (SMP) which is 7-8% of total soybean protein consists of trypsininhibitor (protease inhibitor), hemaglutinin, goitrogen, lipooxygenase, 13-amylase, protease, phytase and other physiologically active substances (1). Trypsin inhibitor inhibits trypsin and hemaglutinin impairs intestinal mucosal cells and therefore SMP has been considered to be unfavorable "anti-nutrients" (1). However, protease inhibitors occurring in soybeans and from other sources have been identified as chemopreventive agents. Troll (2) observed the anti-breast cancer effect of raw whole soybean in rats. Becker et al. (3) have reported that liver cancer which was observed in 100% mice on 26% casein diet, disappeared when 5% of the casein was substituted with whole soybean protein. Various proteinase inhibitors added in diets (4-6) and water (7) or applied on animal skin (8) have anti-carcinogenic effects. Rats that have cancer are also known to have high serum proteinase inhibitors (9). The anti-carcinogenic effect of various protease inhibitors have been reviewed (10,11). These findings suggest that SMP has anticarcinogenic effect. However during the process of the isolation of soybean protein, defatted soybean was first treated with alkaline solution and then acid solution. SPI is in the precipitate and SMP is in the supernatant of the solution. Therefore it is not clear whether there are similar anticarcinogenic effect in SMP as in raw whole soybean or in extracted specific proteinase inhibitors. 0024-3205/93 $6.00 + .00 Copyright © 1993 Pergamon Press Ltd All rights reserved.
1592
Anti-carcinogenicEffect of Soybean Milk Protein
Vol. 53, No. 21, 1993
There have been reports on the unfavorable effects of soybean trypsin inhibitor for health. Major problem is the inhibition of trypsin in intestine (1). Carcinogenic factor has also been reported. Rats maintained on diets containing trypsin inhibitor have been observed to be susceptible to pancreatic cancer (12,13). However, some researchers have reported that such effects can not be observed even with relatively high amount of trypsin inhibitor in man (14), monkey (15), dog (16,17), cow (18), pig (19), mouse (2,6) and rat (20). In this study we tried to find whether there is an anti-tumor effect of SMP within the safe level of intake in mice, for the purpose of finding out proper utilization of SMP which is now practically discarded.
Materials and M e t h o d s
Twenty-eight male ICR mice, 4-5 weeks old, were obtained from the Animal House, Chiang Mai University, Chiang Mai, Thailand. The mice were divided into two groups and fed 20% SPI (donated by Fuji Oil Co., Osaka, Japan) diet (n=13) or 18% SPI diet supplemented with 2% SMP (donated by Fuji Oil Co., Osaka, Japan)(n=15). The SMP was prepared by the following method. Defatted soybean milk (pH 4.5) was centrifuged. The supernatant was neutralized and concentrated 20 times by ultrafiltration (MW=40,000) and then dried by spraydryer. Other components of the diets were: 44.4% a -starch, 22.2% sucrose, 5% soybean oil, 5% mineral mixture (Oriental Yeast Co., Osaka, Japan), 1% vitamin mixture (Oriental Yeast Co., Osaka, Japan), 2% cellulose and 0.4% DL-methionine. Details of the vitamin and mineral mixtures are shown elsewhere (21).
Four weeks after the onset of the diets, two stage carcinogenesis experiment on mouse skin was performed. The back of the mice were shaved and carcinogenesis was initiated by a single application of 100/1 g of 7-12 dimethylbenzanthracene (DMBA) dissolved in 0. lml of acetone to the skin of the back. From 1 week after the initiator application, tumor promoter, 2.5 /1 g of 12-0-tetradecanoylphorbol-13-acetate (TPA) dissolved in 0. lml of acetone was applied to the same areas on the mice, twice a week. The hairs which appear on the shaved part were cut by surgical scissors every 2 weeks. The numbers of tumors Imm or more in diameter were counted weekly. Tumor promoting activity was evaluated by the percentage of tumor- bearing mice and the average number and the volume of tumors per mouse. The number of tumors on each animal was recorded once a week by a vernier caliper and the average tumor volume was calculated as [length] x [width] x [height] 7r/6. Food intake and body weight were measured weekly. The data were analyzed by chi-square test and Student's t-test.
Results
Fig. 1 shows the changes in body weight. Final figures of the two groups are shown in parentheses. They were not significantly different (p>0.05). Fig. 2 shows the percentage of tumor-bearing mice. After 20 weeks on the treatment all the mice of SPI group had tumors, while 67% of the animals of the SMP group had tumors.
Vol. 53, No. 21, 1993
Anti-carcinogenic Effect of Soybean Milk Protein
1593
However the values were not significantly different (p>O.05). Fig. 3 shows the number of tumors per mouse. Final figures are shown in parentheses. The number was significantly lower in the whey group than in the SPI group (p<0.05). Fig. 4 shows average volume of tumors per mouse. The final figures are shown in the parentheses. Although the average was much lower in the whey group than in the SPI group, statistical difference was not observed (p>0.05).
46(43.8 ± 4.4)
44,~42-
4o-
,.,'t/t.8±,.,)
--!--
SPl
-.-
SMP
38>, 36 o
m 3432-
30
l ] I p I I ~ I r J r ] J I I I r I I r I 0
5
10 Weeks
15
20
of p r o m o t i o n
Fig. i Changes in body weight. Figures in parentheses indicate means + SD on the 20th week. significant difference was not observed by Student's/'-test (p>0.05).
A
100-
80 ~
60
--IE-
SPI
---0-
SMP
¢.-
-~
40
0
5
10 Weeks
15
20
of p r o m o t i o n
Fig. 2 Percentage o f tumor-bearing mice. The difference was not significant b y chi-square test (p>0.05).
1594
Anti-carcinogenic Effect of Soybean Milk Protein
Vol. 53, No. 21, 1993
10 (8.3 ± 6.0)
•
8 6
---I- SPI -0-
2 7°-"" 6
0"
~
0
5
SMP
±33'1~"
10 15 Weeks of promotion
20
Fig. 3 Number o f tumors/mouse. Figures in parentheses indicate means _+ SD on the 20th wceL Asterisk indicates a significant difference from SPI group by Student's t-test (p>0.05).
200-
~
(190.3±198.1)
150
,oo-t
.,
0
(84.8_+ 84.6)
----0-
SMP
0
0
5
10 15 Weeks of promotion
20
Fig. 4 Average volume o f tumor~mouse. Figure in parentheses indicate means ± SD on the 20th week. A significant difference was not observed b y Student's t -test (p>0.05).
Discussion
The results indicate that 2% SMP in a diet could suppress the skin tumor induced by chemical carcinogens. Our results are consistent with the findings of Troll et al. (4). They observed that rats with chemical carcinogens applied on their back and fed 50% raw soybean diet develop less tumors than those fed casein or roasted soybean diet. Therefore our present results suggest that anti-tumor effect of raw whole soybean is maintained in the SMP even after the various treatments.
Vol. 53, No. 21, 1993
Anti-carclnogenic Effect of Soybean Milk Protein
1595
The addition of SMP did not impair normal growth and development as evidenced by body weight gain and food intake, although trypsin inhibitor of SNIP is regarded as an unfavorable substance and discarded as a waste product of SPI processing. Troll et al. (2) have shown that the growth of rats fed 50% raw soybean diet is similar as those fed a commercial diet. Clair et al. (6) have also reported that mice fed diets containing 0.5% pure proteinase inhibitor obtained from soybean for 65 days had similar body weight gain in comparison to those fed a commercial diet and had no hypertrophy or cancer in pancreas. No unfavorable effects of trypsin inhibitor have been reported in man (14), monkey (15), dog (16,17), cow (18) and pig (19). Even in the research of Gumbmann (13) who tried to show the carcinogenic effect of trypsin inhibitor on pancreas, body weight changes of mice fed diets containing 0, 0.1, 0.2, 0.3 or 0.6% trypsin inhibitor were similar throughout the experimental period (15 months). Based on these results we can speculate that SMP can be beneficial as an anti-carcinogenic food or medicine. We are further studying the effect of SMP on histopathlogy of various internal organs.
References
1) I.R. LIENER, Am. J. Clin. Nutr. 1..A_1281-298(1962). 2) W. TROLL, R. WIESNER, C.J. SHELLABARGER, S. HOLTZMAN and J. P. STONE, Carcinogenesis 1469-472 (1980). 3) E E BECKER, Carcinogenesis 21213-1214 (1981). 4) W. TROLL, S. BELMAN, R. WlESNER and C. J. SHELLABARGER, Biologica Functions of Proteinases, in Z. Holzer, and H. Tschesche (Eds.), 165-170, Springer-Verlag, Berlin (1979). 5) J. YAVELOW, T.H. FINLA¥, A.R. KENNEDY and W. TROLL, Cancer Research 4._~3 2454s-2459s (1983). 6) W.H. CLAIR, P.C. BILLINGS, J.A. CAREW, K, McGANDY, P. NEWBERN and A.R. KENNEDY, Cancer Research 5 0 5 8 0 - 5 8 6 (1990). 7) J.G. CORASANTI, G.H. HOBIKA and G. MARKUS, Science 2161020-1021 (1982). 8) W. TROLL, A. KLASSEN andA.JANOFF, Science 1691211-1213 (1970). 9) Y. KONDO and N. OHSAWA, Cancer Research 4..~21549-1554 (1982). 10) W. TROLL, K. FRENKEL and R. WIESNER, J.N.C.I. 7 1245-1250 (1984). l l ) EP. SCHELP and P. PONGPAEW, Int. J. Epidemiol. 1_.~7287-292 (1988). 12) E.E. MaGUINNESS, R.G. MORGAN, D.A. LEVISION, D.L. FRAPE, D. HOPWOOD and K.G. WORMSLEY, Scand. J. Gastroenterol. 1 5 4 9 7 - 5 0 2 (1980). 13) M.R. GUMBMANN, W.L. SPANGLER, G.M. DUGAN, J.J. RACK/S, and I.E. LIENER, Qual. Plant. Plant Foods Hum. Nutr. 3__55 275-314 (1985). 14) D.E FLAVIN, Vet. Hum. Toxicol. 2._4_4 25-28 (1982). 15) L.M. AUSMAN, J.P. HARWOOD, N.W. KING, P.K. SEHGAL, R.J. NICOLOSI, M.D. HEGSTED, I.E. LIENER, D. DONATUCCI and J. TARCZA, J. Nutr. 115 1691-1701 (1985). 16) J.R. PATTERN, E.A. RICHARDS and J. WHEELER, Proc. Soc. Exp. Biol. Med. 1 3 7 5 8 (1971). 17) J.R. PATTERN, E.A. RICHARDS and .1. WHEELER, Life Sci. 1_.9_0145-150 (1971). 18) A.D.L. GORRILL, and J.W. THOMAS, J. Nutr. 9._2_2 215-223 (1967). 19) R.D. HOOKS, V.W. HAYS, V.C. SPEER and J.E McCALL, Fed. Proc. Am. Soc. Exp. Biol. 2 4 8 9 4 (1965)
1596
Anti-carcinogenicEffect of SoybeanMilk Protein
Vol. 53, No. 21, 1993
20) K. KISHI, S. TERAI, S. OOGURI, F. SHIZUKA, S. YAMAMOTO and G. INOUE, Nutr. Sci. Soy Prot. 7_ 47-52 (1986) (in Japanese). 21) S. YAMAMOTO, T. KOHRIN, M. MORI, L. ASATO, S. SHINJO, J. NAGAMINE and M.E WANG, J. Nutr. Biochcm. 3_ 279-284 (1992).