The Soy Protein Research Committee(Japan)


17-1

A Cascade Mechanism Responsible for Maturation of Seed Proteins

Ikuko HARA-NISHIMURA and Nagako HIRAIWA

Department of Cell Biology, National Institute for Basic Biology, Okazaki 444

Rep. Soy Protein Res. Com., Jpn. 17, 1-5, 1996.

Vacuolar processing enzymes (VPE) are responsible for maturation of various seed proteins such as glycinins in soybean. The processing enzymes belong to a novel cysteine proteinases with molecular mass of 39 kDa for soybean and 37 kDa for castor bean. Molecular characterization reveals that the enzyme is synthesized on rough endoplasmic reticulum as an inactive precursor with a larger molecular mass. The precursor is transported to vacuoles via dense vesicles together with proproteins of seed proteins and is converted into an active enzyme, after arriving at the vacuoles. This suggests that a cascade of processing is involved in maturation of seed proteins. To examine the mechanism of activation of VPE, we expressed a precursor to castor bean VPE in a pep4 strain of the yeast Saccharomyces cerevisiae. Two VPE proteins with 59 kDa and 46 kDa were detected in the vacuoles of the transformant. They were glycosylated in the yeast cells, although VPE is not glycosylated in plant cells in spite of the presence of two N-linked glycosylation sites. During the growth of the transformant, the level of the 59-kDa VPE increased slightly until a rapid decrease occurred after 9 h. By contrast, the46-kDa VPE appeared simultaneously with the disappearance of the 59-kDa VPE. Vacuolar processing activity increased with the accumulation of the 46-kDa VPE, but not of the 59-kDa VPE. The specific activity of the 46-kDa VPE was a similar level to that of VPE in plant cells.These findings suggest that an inactive VPE precursor synthesized on the endoplasmic reticulum is transported to the vacuoles in the yeast cells and then processed to make an active VPE by self-catalytic proteolysis within the vacuoles.

Key words: asparaginyl endopeptidase, cysteine proteinase, seed protein, vacuolar processing enzyme, yeast


17-2

Expression in Escherichia coli and Crystallization of Constituent Subunits of Soybean beta-Conglycinin

Shigeru UTSUMI

Research Institute for Food Science, Kyoto University, Uji 611

Rep. Soy Protein Res. Com., Jpn. 17, 6-9, 1996.

beta-Conglycinin is one of the most abundant storage proteins in soybean seeds and is composed of three kinds of subunits (alpha,alpha' and beta). The construction of Escherichia coli expression system and crystallization of constituent subunits were attempted to elucidate their structure-function relationships. Nucleotide sequences corresponding to mature polypeptides of cDNAs encoding individual subunits were placed under the control of T7 promoter in an expression vector pET21d. alpha,alpha' and beta subunits were expressed as soluble proteins at the levels of 15, 10 and 20% of total E. coli proteins, respectively. The properties of alpha and beta subunits which were expressed at a higher level were examined. Self-assembly of a and b subunits into trimers was examined and confirmed by sucrose density gradient centrifugation followed by SDS-polyacrylamide gel electrophoresis. The expressed a and b subunits were purified to near homogeneity (90%) by ammonium sulfate fractionation, and Q-Sepharose and hydrophohic column chromatographies. The purified subunits were subjected to crystallization under various conditions by the hanging drop vapor diffusion method. Although a subunit formed only small crystals having the maximum dimension of 0.05 mm, b subunit formed crystals having the size (0.3 x 0.2 x 0.1 mm) suitable for X-ray diffraction. The crystals of b subunit were monoclinic, space group P21, and with unit cell dimensions a =69.2Å&Mac240; b =85.0Å&Mac240; c=79.8Å&Mac240; and Éø = É¡ =90.0Åãand É¿ =110.8Åã

Key words : soybean, beta-conglycinin, expression, crystallization, X-ray analysis


17-3

Molecular Analysis of the Mechanism That Regulates Expression of Soybean Seed Storage Protein Genes in Response to Sulfur Nutrition - Toward a Development of a Novel Strategy for Molecular Breeding of Soybean

Torn FUJIWARA

Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113

Rep. Soy Protein Res. Com., Jpn. 17, 10-13, 1996.

The gene encoding the b subunit of b-conglycinin is known to be upregulated by sulfur deficiency in soybean. The gene is similarly regulated by sulfur nutrition in transgenic petumia and Arabidopsis. These responses are parts of adaptation responses of soybean to maintain levels of nitrogen and sulfur reservoirs in seeds. This implys that the b subunit gene is also regulated by nitrogen nutrition. We found that nitrogen supply also affects expression of the subunit gene in transgenic Arabidopsis. Reduced nitrogen supply reduced the levels of expression from the b subunit gene. Sulfur and nitrogen metabolism in plants interact at the step of cysteine biosynthesis. O-Acetyl-L-serine, a precursor for cysteine biosynthesis, is found accumulated in response to sulfur deficiency and excess nitrogen supply. Application of O-acetyl-L-serine was found to upregulate the accumulation of the b subunit protein.

Key words : b-conglycinin, O-acetyl- L-serine, sulfur nutrition, nitrogen nutrition, Arabidopsis thaliana


17-4

Lowering the Content of an Allergic Protein Through Efficient Transformation in Soybean Seed

Tetsuo TAKANO 1, Tao LI 2, Yuichi KANEDA 3 and Keisuke KITAMURA 4

1 Asian Natural Environmental Science Center, The University of Tokyo, Tokyo 188
2 Iwate Biotechnology Research Center, Kitakami 024
3 Faculty of Agriculture, Meiji University, Kawasaki 214
4 National Agriculture Research Center, Tsukuba 305

Rep. Soy Protein Res. Com., Jpn. 17, 14-18, 1996

Gly m Bd 30K is a major allergenic protein in soybean seed. To obtain a soybean line lacking Gly m Bd 30K, we introduced plasmids containing the antisense cDNA for Gly m Bd 30K into soybean hypocotyl segments via particle bombardment. At first, we investigated the optimum condition of bombardment using GUS reporter gene. The highest transient GUS expression was observed when 1,300 psi rupture disc was used, the distance between stopping screen and the soybean sample was 9 cm, and 2 shots were made per petri dish. Under this condition, we introduced a plasmid (pBI Bd30KA) which contained antisense cDNA for Gly m Bd 30K driven by CaMV35S promoter, and kanamycin resistant marker gene into hypocotyl segment of soybean. The hypocotyl segments were cultured on the 1/2 L2 agar medium containing 2 mg/ L of thidiazuron (TDZ). Although many shoots regenerated from the segment, which were transferred to the selection medium containing 100 mg/L kanamycin, no transgenic soybean plant was obtained because all the shoots died eventually through selection.

Key words : soybean, transformation, antisense, particle bombardment, allergenic protein


17-5

Soybean Aspartic Proteinases: Cloning, Expression, Functional Analysis and Application

Keiko ABE

Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, 113

Rep. Soy Protein Res. Com., Jpn. 17, 19-22, 1996.

Aspartic proteinases (AP) have deserved note because of their significance of not only physiological roles but also applications for food processing. In this study, I tried to define the presence of APs in soybean. Poly (A) + RNA was prepared from 3-daygerminating soybean seeds to synthesize cDNA. Using RT-PCR with oligoprimers synthesized according to the amino acid sequences commonly conserved in known APs, I identified three cDNA fragments encoding parts of APs. The encoded proteins, named soybean AP a, b, and g, consisted of 102 amino acid residues, containing one of the aspartic acid residues that constitute the active center. In the primary structure, they had 657% identity with one another. Especially, the sequences around the active center including the particular aspartic acid residue are highly conserved. The results of the northern blot analysis demonstrated that AP a, b, and g mRNAs were all expressed as single species of about 2.0 kb in soybean seeds during both ripening and germination. These results suggest that soybean APs play some physiological roles in protein processing and degradation. Practically, they are expected to be used as milk-clotting enzymes for cheese making.

Key words : aspartic proteinase, soybean, milk clotting


17-6

Antioxidant Activity of Designed Peptides Based on the Antioxidative Peptide isolated from Digests of a Soybean Protein

Koji MURAMOTO, Hua-Ming CHEN and Fumio YAMAUCHI

Faculty of Agriculture, Tohoku University, Sendai 981

Rep. Soy Protein Res. Com., Jpn. 17, 23-28, 1996.

Antioxidative activities of 28 synthetic peptides against the peroxidation of linoleic acid in an aqueous system, which were designed based on an antioxidative peptide (Leu-Leu-Pro-His-His) derived from proteolytic digests of a soybean protein, were measured with the ferric thiocyanate method. The deletion of the C-terminal His decreased the activity, whereas the deletion of the N-terminal Leu had no effect. His and Pro in the sequence played important roles in the antioxidative activity and, among the peptides tested, Pro-His-His was the most antioxidative. The activity decreased on substitution of the second His with D-His. Introduction of Tyr to the positions of Pro or His did not increase the activities of the corresponding peptides. Antioxidative peptides showed synergistic effects with nonpeptidic antioxidants as observed in soybean protein hydrolysates. The magnitude of the effects, however, did not correlate with the antioxidative activities of the peptides.

Key words: antioxidative peptide, antioxidant, soybean protein hydrolysate, synergistic effect


17-7

Mechanism of Beany Flavor Formation in Lipoxygenase-Deficient Soybean

Teruyoshi MATOBA 1, Hitoshi TAKAMURA 1 and Keisuke KITAMURA 2

1 Department of Food Science and Nutrition, Nara Women's University, Nara 630
2 National Agriculture Research Center, Tsukuba 305

Rep. Soy Protein Res. Com., Jpn. 17, 29-32, 1996.

Hexanal is the key compound for beany flavor in soybean products. This volatile aldehyde is formed from linoleic acid via hydroperoxide by the actions of lipoxygenase and hydroperoxide lyase. Soybean seeds contain three lipoxygenase isozymes (L-1, L-2 and L-3). A mutant which lacks all three isozymes was recently developed. However, this mutant still produces hexanal. In this study, the mechanism of the hexanal formation in the mutant soybean seeds was investigated. First, soybean seeds were separated to soluble and membrane fractions by ultracentrifugation. Then, linoleic acid or its 13-hydroperoxide was incubated with these fractions, and the formation of 13-hydroperoxide and hexanal was determined. Both soluble and membrane fractions of normal soybean seed converted linoleic acid to its 13-hydroperoxide and hexanal, and converted 13-hydroperoxide to hexanal. In mutant soybean seed, only soluble fraction produced linoleic acid 13-hydroperoxide and hexanal. Membrane fraction of mutant soybean seed did not form 13-hydroperoxide and hexanal, while the activity converting 13-hydroperoxide to hexanal was high. These results suggest that a new lipoxygenase isozyme exists in the soluble fraction of the mutant soybean seed lacking L-1, L-2 and L-3.

Key words: soybean, lipoxygenase, linoleic acid, hexanal, beany flavor


17-8

Active Oxygen-Radical Scavenging Activity of Soybean Proteins

Husniza HUSSAIN, Masaaki KAWANE, Yumiko YOSHIKI and Kazuyoshi OKUBO

Faculty of Agriculture, Tohoku University, Sendai 981

Rep. Soy Protein Res. Com., Jpn. 17, 33-39, 1996.

Active oxygen species (X) can cause damage to biomolecules including protein and DNA. Previously, we have reported that catalytic species (Y) such as flavonoids as natural radical scavengers exhibited very weak light emission (chemiluminescence; CL) in the presence of X and receptive species (Z) such as acetaldehyde and DDMP saponin and proposed that the system of XYZ is a scavenging mechanism for active oxygen species in accordance with the CL intensity [P] = k [X][Y] [Z] ; k, photon constant. In this study, soybean protein fractions involved in the XYZ system were investigated. It could be concluded that the isolated protein from soybean whey fraction acted as Z in the XYZ system due to the proportionate P against [X, H202] , the O2- scavenging activity increased with the addition of gallic acid as Y, and that pro-oxidative characteristic changed into antioxidative with Y through the ferric thiocyanate method. The isolated protein might be lipoxygenase on the basis of SDS-PAGE profile.

Key words : active oxygen, radical scavengers, chemiluminescence, XYZ system, lipoxygenase


17-9

Improvement of the Functional Properties of Soy Protein by the Treatment with Protein Deamidase from Germinating Wheat Seed

Akio KATO 1, Ranajit Kumar SHAHA 2 and Naotoshi MATSUDOMI 1

1 Faculty of Agriculture, Yamaguchi University, Yamaguchi 753
2 Department of Biochemistry, Rajshahi University, Bangladesh

Rep. Soy Protein Res. Com., Jpn. 17, 40-44, 1996.

An enzyme catalyzing the deamidation of seed storage proteins was found in germinating wheat grains and was partially purified by salting out, DEAE-cellulose column chromatography and gel filtration on a Sephadex G-50 column. Wheat seeds were soaked in water for 6 h and then germinated in the dark at 25à¡ for 5 days. The deamidase activity was reached to maximum with the germination for 48 h and then the activity was greatly decreased with subsequent germination. The functional properties of wheat gluten were improved by the crude protein deamidase, while those of soy protein were not. This suggests that the deamidase derived from wheat seed acts on only wheat gluten, indicating the specificity for substrate. Further purification and the construction of cDNA library are in progress.

Key words: protein deamidase, wheat gluten, emulsifying properties, foaming properties


17-10

Strategy for Determination of Cysteine Residues Involved in Disulfide Bridge Formation During Thermal Gelation of Glycinin

Tomohiko MORI

Research Institute for Food Science, Kyoto University, Uji 611

Rep. Soy Protein Res. Com., Jpn. 17, 45-50, 1996.

Fractionation of intermediary subunits of glycinin for preparing each highly purified intermediary subunit which provides single molecular species of glycinin through reconstitution procedure was performed. Partially purified glycinin was prepared by column chromatography and subjected to chromatography using Mono Q HR 10/10 column connecting to HPLC system. Fourteen peaks were observed in the elution pattern, nine of which were shown to be the intermediary subunits by SDS-PAGE. The late-eluted fraction which contained peaks corresponding to A3B4 type of intermediary subunits was subjected to the chromatography. The first peak in the elution pattern was shown to contain mainly the A3B4 and then subjected further to the chromatography. Single peak was observed in the elution pattern. This peak was shown to contain the A3B4 intermediary subunit in high purity from analyses by SDS-PAGE and N-terminal amino acid sequencing. On the other hand, application of lysyl endopeptidase for peptide fragment analysis was investigated. a-Lactalbumin was subjected in this investigation. The a-lactalbumin was reacted with transglutaminase, where polymerization of the protein occurs through formation of isopeptide bonds between glutamine and lysine residues. The native and the polymerized a-lactalbumin was digested by lysyl endopeptidase and then the peptide fragment analyses were performed. The lysine and glutamine residues involved in the polymerization of the a-lactalbumin could be determined. From these results, it seems possible to determine cysteine residues involved in disulfide bridge formation during thermal gelation of glycinin, by using the reconstituted A3B4 pseudoglycinin for thermal gelation and lysyl endopeptidase for peptide fragment analysis.

Key words: glycine, gelation, disulfide bonding, reconstitution, peptide fragment analysis


17-11

Influence of Peptide Chain Length on Absorption of Soybean Hydrolysate in Rat Small Intestine

Hwal CHUN, Masaya SASAKI, Tomoyuki TSUJIKAWA, Yoshihide FUJIYAMA and Tadao BAMBA

Second Department of Internal Medicine, Shiga University of Medical Science, Otsu 520-21

Rep. Soy Protein Res. Com., Jpn. 17, 51-54, 1996.

Influence of peptide chain length on the absorption of soybean hydrolysate was investigated by the intestinal perfusion of the rat. Two types of soybean hydrolysate, one is small peptide (SP, average peptide chain length: 3.2) and the other is large peptide (LP, average peptide chain length: 5.2), were prepared for this experiment. Krebs-Ringer phosphate buffer (pH 7.0) which contains 0.5% of either of the two types of soybean hydrolysate was perfused in 15-cm of rat jejunum for 70 min. After the perfusion,, samples were collected and the amounts of amino acids were measured by the amino acid analyzer (Hitachi L-8500) after the hydrolysis and the amount of absorption of each amino acid was determined. Absorption, amounts of glycine, histidine, arginine, phenylalanine, and proline in SP were significantly greater than those in LP. Total amount of peptide absorption of SP was significantly greater than that of LP. It is concluded that the amount of absorption of SP was greater than that of LP in the intestinal perfusion model of rat small intestine.

Key words : soybean hydrolysate, perfusion, absorption, average peptide chain length


17-12

Species Difference in the Effect of Soybean Protein to Stimulate Fecal Steroid Excretion

Shin-ichi HAYASHI and Hiroko TAKIZAWA

Department of Biochemistry (II), The Jikei University School of Medicine, Tokyo 105

Rep. Soy Protein Res. Com., Jpn. 17, 55-58, 1996.

We have shown previously that, compared with casein, soy protein isolate (SPI) stimulates fecal steroid excretion and lowers plasma cholesterol level in rats, but not in mice. In order to understand the exact reason why SPI fails to stimulate fecal steroid excretion, rats and mice were fed synthetic diet containing either 125I-labeled casein or similarly labeled SPI and killed 3 h later. Radioactivity and its acid precipitability were determined in contents of 7 consecutive segments of gastrointestinal tract. In both rats and mice fed radioactive SPI, marked accumulation of radioactivity was observed in the contents of ileum and cecum, whereas no such accumulation was observed in rats and mice fed radio-labeled casein, indicating that SPI is digested and absorbed much more slowly than casein in both rats and mice. In lower ileum, where most steroids are absorbed, nearly 4 times more acid-insoluble peptides were accumulated in rats than in mice, both fed radioactive SPI. It is likely, therefore, that in mice SPI is digested differently from digestion, in rats, so that steroid binding proteolytic intermediates are not produced so much as in rats.

Key words: soy protein isolate (SPI), fecal steroid excretion, plasma cholesterol level, protein digestibility, mice


17-13

Studies on the Allergenicity of Soy Products - Assay Method Using Cultured Cell Line -

Rintaro YAMANISHI, Hideaki TSUJI, Noriko BANDO and Tadashi OGAWA

School of Medicine, The University of Tokushima, Tokushima 770

Rep. Soy Protein Res. Com., Jpn. 17, 59-65, 1996.

Soybeans treated with proteases were investigated on their antigenicity and allergenicity.Based on the ability to proteolyze Gly m Bd 30K, a major soybean allergenic protein, two of eight proteases tested, Proleather and Protease N, were selected to produce the hypoallergenic soybean. When more than 250 units Proleather or 5,000 units Protease N for 1 g soybean were used, not only the antigenicity for a mouse anti-Gly m Bd 30K monoclonal antibody but also the allergenicity for patients' sera were significantly attenuated. In in vitro assay system to measure the allergenicity attributable to Gly m Bd 30K, extracts from protease-treated soybeans showed significantly weaker allergenicity than that from intact soybeans. These results suggest that soybeans treated with an adequate protease could be supplied for soybean-sensitive patients as a hypoallergenic foodstuff.

Key words : soybean, allergenicity, Gly m Bd 30K, protease


17-14

Immunological Response and Tolerance to Dietary Soybean Protein

Tsukasa MATSUDA, Kenji YAMAGUCHI and Ryo NAKAMURA

School of Agricultural Sciences, Nagoya University, Nagoya 464-01

Rep. Soy Protein Res. Com., Jpn. 17, 66-70, 1996.

Immune response of infant mice to fed soybean proteins was investigated to get information on oral-immunogenic properties of soybean proteins. The 5 strains of inbred mice (A/J, BALB/ c, C57BL/6, C3H/He, DBA/2) of 3 week-old were fed a purified diet containing the defatted soybean flour (50% by weight) as a protein source. Blood was collected individually from each mouse before and after the soybean protein feeding. Measurement of serum antibodies specific for major soybean proteins such as 2S, 7S and 11S fractions indicated that weak but definite antibody response was induced in A/J mice by the feeding with the defatted soybean-based diet. To estimate oral-tolerance these mice were received the i. p. injection of crude soybean protein with Freund's adjuvant and serum antibody response was examined. The marked IgG response to 7S fraction was induced for all the 5 strains, whereas no or very weak IgG response to 2S and 11S fractions was induced for C3H/He and C57BL/6 strains, suggesting that oral tolerance to the 2S and 11S fraction was induced in a strain-dependent manner. Furthermore, no IgE response to 11S fraction was induced for all the 5 strains, and C3H/He and C57BL/6 mice showed IgE response to neither of the three soybean protein fractions.

Key words : soybean allergy, antibody response, oral immunogenicity, oral tolerance


17-15

Effect of a Diet with Soy Protein Isolate as the Principal Source of Nitrogen to Rats with Galactosamine-Induced Hepatitis

Tadashi NOGUCHI and Tomomi HIDAKA

Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113

Rep. Soy Protein Res. Com., Jpn. 17, 71-76, 1996.

A diet for rats with galactosamine-induced hepatitis was formulated using soy protein isolate (SPI) as the primary source of dietary nitrogen. The diet was formulated as that of high branched-chain amino acids and low methionine contents. The validity of the diet was assessed by plasma concentrations of glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), total insulin-like growth factor-I (IGE-I), IGE-binding proteins and particularly dirIGF-I which has been elucidated to correlate well wit the status of protein nutrition. The rats injected wit galactosamine showed increased plasma concentration of GOT and GPT. The rats given casein (control) diet showed a significantly decreased plasma IGF-I concentration by galactosamine injection. However, in the case of the rats given SPI diet, there was no difference in the IGF-I concentration between the rats injected with galactosamine and those not injected with it. Other indices also gave favorable results to the protective effect of SPI diet on galactosamine-induced hepatitis rats. We concluded that SPI diet has an enough possibility to suppress the deteriorative effect of galactosamine to rats.

Key words: insulin-like growth factor-I, insulin-like growth factor-binding proteins, galactosamine-induced hepatitis, SPI diet, high branched-chain amino acid diet


17-16

Alleviation of Tumorigenesis in Rat Colon by Soy Protein (HMF) Intake

Kimikazu IWAMI, Naoyuki ADZUMA, Keiko MACHIDA, Tohru SAEKI and Ryuhei KANAMOTO

Faculty of Agriculture, Kyoto Prefectural University, Kyoto 606

Rep. Soy Protein Res. Com., Jpn. 17, 77-83, 1996.

HMF, a high-molecular weight 'peptide (insoluble)' fraction from the digestive product of soy protein, was examined for its anti-tumorigenic effect in rat colon. Male Fischer-344 rats with 3 shots of azoxymethane (15 mg/kg BW, once a week) were fed over a period of 39 weeks on 21% HMF- or 15% casein-based diets with or without supplemental 0.2% deoxycholic acid (DCA). Twelve weeks later, five rats from each group were sacrificed to excise the colon, of which the mucosal surface was microscopically observed for abberant crypt foci (ACF). As a result, the DCA administration was found to cause a considerable decrease iii ACF in the casein-fed group, but not iii the HMF-fed group. During the feeding period, fecal bile acids were much more excreted in the latter than in the former. This implies that a harmful action of DCA must have been effectively masked by unabsorbed leavings of digested HMF. At the end of 39 weeks' feeding, the colons were taken out of all the rats and subjected to tumorigenic inspection. Among the animals without DCA loading, tumor was observed for only one of the casein-fed group. With respect to the DCA-loaded animals, tumor incidence was 30% in the HMF-fed group (n 9) relative to 60% in the casein-fed group (n= 10). The total tumor number was one-sixth as much in the former as in the latter and the average size of tumors (mm in major axis) was 2.0.1 in the former against 4.7.4 in the latter. It thus seems reasonable to consider that HMF has some alleviative effect on azoxymethane-induced colonic tumorigenesis.

Key word: colonic tumor, azoxymethane, deoxycholic acid, abberant crypt foci, anti-tumorigenic HMF


17-17

Effect of Okinawan Yose-Tofu on Skin Tumor in Mice

Sumie SHINJO 1, Yuko MIYAGI 1, Chika MIYAGI 1, Megumi KUBA 1, Mika TOUMA 1, Ming-Fu WANG 2, Kiyoharu TAKAMATSU 3, Takashi YAMAMOTO 1, Liu ASATO' and Shigeru YAMAMOTO 1

1 Research Center of Comprehensive Medicine. University of the Ryukyus, Okinawa 903-01
2 Department of Food and Nutrition, Providence University, Taichung, Taiwan
3 Applied Research Institute, Fuji Oil Co., Izumisano 593

Rep. Soy Protein Res. Com., Jpn. 17, 84-88, 1996.

We studied the effect of Okinawan yose-tofu on the prevention of the chemically induced skin tumor. Male ICR hairless mice (Hr-/Kud) were given milk casein diet or Okinawan yose-tofu diet. After a week on the experimental diets, the mice were applied tumor initiator on their back. Then tumor promoter was applied twice a week on the same area of the skin throughout the experiment. The percentage of tumor-bearing mice and the average volume of a tumor after 18 weeks on the treatment were lower in the yose-tofu group than in the casein group. The result indicates the anticarcinogenic effect of Okinawan yose-tofu.

Key words : Okinawan yose-tofu. skin tumor, anticarcinogen, hairless mice


17-18

Dietary Lipids and Proteins Influence Learning and Memory in the SAMP8 Strain of Mice

Masanori HOSOKAWA 1, Makiko UMEZAWA 2, Bashudeb CHAKROBORTY 1 and Keiichi HIGUCHI 1

1 Chest Disease Research Institute, Kyoto University, Kyoto 606-01
2 Department of Nutrition, Koshien University. Takarazuka 665

Rep. Soy Protein Res. Com., Jpn. 17, 89-93, 1996.

The SAMP8 mouse is a model of age-related deficit in learning and memory ability. A semipurified diet supplemented with soy protein isolate (SPI) and either with safflower oil or with perilla oil was fed to SAMP8 mouse dams and their pups. The offspring (males) at 28 weeks of age were used for behavioral test and measurement of serum lipids. The learning and memory abilities of the two dietary groups were tested with the Sidman active avoidance task. The group given perilla oil showed much greater improvement in learning than did the group fed safflower oil. Observations of the diurnal rhythm of spontaneous motor activity (SMA) indicated that the mice in the safflower oil group tended to be more active than those in the perilla oil group in both right and dark periods. Serum cholesterol level in perilla oil group was half of that in safflower oil group. In experiment B, a semipurified diet supplemented with perilla oil and either with SPI or casein was fed to SAMP8 males of 10 months of age for 4 weeks. Two dietary groups were tested the degree of senescence and serum lipid contents. The degree of senescence of the SPI group was significantly lower than in the casein group. Although exploration behavior was not different between both groups, escaping behavior was stronger in the SPI group than in the casein group. Serum lipid contents were not different between both groups. These results indicate that in the SAMP8 mouse, the dietary lipids and proteins influence learning and memory abilities.

Key words : Senescence-Accelerated Mouse, dietary oil, dietary protein, Sidman active avoidance, spontaneous motor activity, serum lipids


17-19

Effects of Soybean Peptide on Suppression of Body Fat Accumulation during Endurance Swimming in Mice

Kengo ISHIHARA, Keitaro MATSUMOTO, Ryouhei UOHASHI and Tohru FUSHIKI

Faculty of Agriculture, Kyoto University, Kyoto 606

Rep. Soy Protein Res. Com., Jpn. 17, 94-97, 1996.

Suppression of body fat accumulation of mice chronically administered soybean peptide was investigated. Mice were fed with commercial chow and 5% soybean digestion peptide in the water during endurance swimming every 2 days for 5 weeks. The mass of the epididymal fat pads and the peripheral fat were significantly smaller than control. Sedentary mice did not show any difference in mass from control. Amino acid mixture which simulated the soybean peptide gave the same suppression of body fat accumulation when it was administered to the mouse for 5 weeks. These results suggested that soybean peptide would decrease in body fat during exercise training with its characteristic amino acid composition. Lysine and arginine contents, which are markedly high in the soybean peptide, may not be relevant to it.

Key words: body fat content, endurance swimming, soybean peptide


17-20

Effects of Dietary Fatty Acids and Proteins on Glucose Tolerance and Insulin Receptor Gene Expression in Wistar Fatty (NIDDM) Rats

Nobuko IRITANI, Tomomi SUGIMOTO, Hitomi FUKUDA and Masumi KOMIYA

Tezukayama Gakuin College, Sakai 590-01

Rep. Soy Protein Res. Com., Jpn. 17. 98-102, 1996.

To investigate the effects of dietary fatty acids and proteins on glucose tolerance, insulin secretion and insulin receptor gene expression, the Wistar fatty rats (genetically obese, noninsulin dependent diabetes mellitus) and their lean littermates (8 wk old) were fed a casein or soybean protein diet containing 9% hydrogenated fat (plus 1% corn oil), 10% corn or fish oil for 3 wk. In fatty rats fed polyunsaturated fat, the plasma insulin concentrations were significantly higher than in those fed hydrogenated fat, particularly in the soybean protein group (similarly in glucose tolerance test). On the contrary, the insulin contents in pancreas were lowered in the rats. The insulin receptor mRNA concentrations were significantly higher in rats fed soybean protein/hydrogenated fat than in those fed casein, regardless of genotype, whereas the increment was lowered by dietary polyunsaturated fat. The insulin receptor gene expression appeared to he suppressed by down regulation due to the high insulin secretion. Thus, dietary soybean protein may help to reduce the diabetic insulin resistance, but not in the presence of polyunsaturated fatty acids because of stimulation of insulin secretion.

Key words : Wistar fatty rat, NIDDM, insulin receptor gene expression, insulin, glucose tolerance, dietary fatty acids


17-21

Effect of Soy Protein Isolate on Sucrose- or Lard-Induced Fat Accumulation in Rats Fed for Long Period

Eiko HARA 1, Yasutake SHIMIZU 2, Meiko HAMAI 2 and Takashi SHIMAZU 2

1 Imabari Meitoku Junior College, Imabari 794
2 School of Medicine, Ehime University, Ehime 791-02

Rep. Soy Protein Res. Com., Jpn. 17, 103-107, 1996.

Effects of dietary soy protein isolate (SPI) on sucrose- or lard-induced fat accumulation were examined in rats. Thirty percent sucrose diet feeding for 8 weeks increased the liver weight and the liver triglyceride (TG) content significantly, but SPI feeding suppressed the augumentation completely. On the other hand, 14% lard diet also increased liver TG content remarkably, but SPI feeding did not suppress the augumentation. Thermogenic activity of the interscapular brown adipose tissue (BAT) was examined by measuring the uncoupling protein (UCP) content in the mitochondria by means of immunoblotting. In sucrose fed rats, the UCP content increased by SPI feeding additionally. In lard fed rats, the UCP content was increased remarkably, but SPI feeding did not increase the UCP content any longer. In conclusion, SPI feeding worked suppressively for sucrose-induced fat accumulation, but not for lard-induced fat accumulation.

Key words: brown adipose tissue, uncoupling protein, sucrose feeding, fat accumulation


17-22

Effect of Soy Protein Isolate on Fat Distribution and Glucose Tolerance in Genetically Obese - Hyperglycemic OLETF Rats

Yutaka MORI 1, Shoichi HATA 2, Yuichi MURAKAWA 1, Shuichi KATOH 2 and Yoshio IKEDA 3

1 Department of Internal Medicine, Higashi-Utsunomiya Hospital, Tochigi 329-11
2 Third Department of Internal Medicine, The Jikei University School of Medicine, Tokyo 105
3 Center for General Health Care, The Jikei University School of Medicine, Tokyo 105

Rep. Soy Protein Res. Com., Jpn. 17, 108-113, 1996.

Effects of dietary soy protein isolate (SPI) on fat distribution and glucose tolerance were examined in genetically obese-hyperglycemic OLETF rats which were characterized by mesenteric fat accumulation. Twenty male OLETF rats aged 4 weeks were randomly divided into two groups which were given SPI-diet (n=10) or casein-diet (n=10) and observed until 32 weeks of age. There were no significant differences in food intakes and body weights between two groups. The oral glucose tolerance test at 12 weeks of age showed that the plasma glucose level at 120 minutes after glucose load in SPI-diet group decreased significantly compared with that in casein-diet group, while there was no significant difference in insulin response. As for fat distribution, the tissue weights of epididymal and abdominal subcutaneous fats in SPI-diet group decreased significantly compared with those in casein-diet group. The interscapular brown adipose tissue (BAT) weight in SPI-diet group increased significantly compared with that in casein-diet group. Thus, SPI was more effective in suppression of body fat accumulation, probably due to the enhancement of BAT thermogenesis.

Key words: soy protein isolate, OLETF rats, fat distribution, glucose tolerance, visceral fat accumulation


17-23

Effects of Dietary Soybean Protein on Arterial Lesions in Apolipoprotein E-Deficient Mice

Masanobu SAKONO, Toshihiko FUKUYAMA Wei-Hua NI and Katsumi IMAIZUMI

Faculty of Agriculture, Kyushu University, Fukuoka 812-81

Rep. Soy Protein Res. Com., Jpn. 17, 114-117, 1996.

Homozygous apo E-deficient mice were fed atherogenic diet containing either soybean protein or casein for 6 wk. The serum cholesterol level of soybean protein-fed mutant was higher than that of casein-fed mutant. Atherogenic diet resulted in foam cell lesions in the aortic root, and the extent was greater in casein-fed mice than in soybean protein-fed mice.

Key words: apo E-deficient mice, atherosclerosis, soybean protein, casein, foam cells


17-24

Effects of Soy Protein Isolate on Fasting and Postprandial Lipemia in Normolipidemic Subjects

Hideki SHIGE, Kenji HIGASHI, Toshitsugu ISHIKAWA, Michio SUZUKAWA, Takeshi YAMASHITA, Koji TOMIYASU, Hiroshi YOSHIDA, Hiroshi HOSOAI, Toshimitsu ITO, Kei NAKAJIMA, Makoto AYAORI, Atsushi YONEMURA, Emiko MIYAJIMA, Miyuki NOMI and Haruo NAKAMURA

First Department of Internal Medicine, National Defense Medical College, Tokorozawa 359

Rep. Soy Protein Res. Com., Jpn. 17, 118-124, 1996.

Postprandial lipemia has recently been considered to be atherogenic. We have previously reported that soybean protein decreased the postprandial level of remnant lipoprotein in normolipidemic subjects, whereas casein increased it. To elucidate the effect of soy bean protein on fasting and postprandial plasma apolipoproteins, oral fat load tests were performed in normolipidemic male subjects. Eleven normolipidemic male subjects were assigned to a diet containing either 20 g/day soy protein isolate (SPI) or casein for three weeks in a crossover design. Both diets consisted of ordinary foods and compositions added with 20 g/day of SPI or casein. Forty g/m2 of cow milk fat were administered before and after three weeks of the experimental diets. Fasting plasma apolipoprotein concentrations did not change during both SPI and casein diet. Remnant like particles-cholesterol (RLP-C) response area was significantly increased by casein diet, whereas it was decreased by SPI diet. ApoE response area tended to decrease after SPI diet compared to casein diet. These combined results suggest that SPI diet favorably alters a postprandial lipemia in normolipidemic subjects.

Key words: soybean protein, postprandial lipemia, apolipoprotein


17-25

Effect of Soy Protein Formula on Hypercholesterolemia

Kazuhiko KATO 1 and Keiji MIYATA 2

1 Kato Clinic, Komae 201
2 Applied Research Institute, Fuji Oil Co., Izumisano 598

Rep. Soy Protein Res. Com., Jpn. 17, 125-128, 1996.

It is well known that soy protein decreases cholesterol in blood. Recently, soy protein containing formula useful for hyperlipidemia is available in Japan. Fifty eight patients with hyperlipidemia were fed soy protein formula (6 pieces of biscuit and a cup of soup ) once a day as a substitute for breakfast, lunch or supper for 2 weeks. Blood lipids were examined before and after 2 weeks of feeding soy protein formula. Plasma total cholesterol was 244.8 30.2 mg/dL and 232. 4 36.7 mg /dL before and after 2 weeks of feeding, respectively. The decrease in total cholesterol was statistically significant (t = 3.94, P < 0.001). It is concluded that the soy protein formula is one of the treatments for hypercholesterolemia.

Key words: soy protein formula, hypercholesterolemia, Koresukkiri


17-26

Effect of Soy Protein on Serum Lipid, Platelet Aggregation and Hemostatic Markers and Progression of Atherosclerosis in Hyperlipidemic Patients

Masako WAKI, Motoo TSUSHIMA, Naoko YAMASHITA, Kayoko RYOHMOTO, Hideki KOH, Ryohya KOMATSU, Yasushi HARA and Tatsuo MATSUYAMA

National Cardiovascular Center, Suita 565

Rep. Soy Protein Res. Com., Jpn. 17, 129-134, 1996.

Platelet aggregation and hemostatic markers play an important role in atherosclerosis. Soy protein has been known to be antiatherogenic and it has mainly been explained by its hypocholesterolemic effect. Whether soy protein affects platelet function and hemostatic markers has not been well known. This study conducted to compare the effect of soy protein intake to low fat milk on serum lipids, platelet function and coagulation and fibrinolysis system in hypercholesterolemic subjects. Fifteen middle aged female patients with mild hypercholesterolemia were examined. The patients were randomly assigned to take firstly soy protein (12 g/ day) or low fat milk (protein 14 g/day, energy 160 kcal). After a month with first food., the patients took the other food for the second month. Blood sampling was performed monthly before and after the study. Serum lipids, apoproteins, platelet aggregation, coagulation and fibrinolysis markers were examined. Total cholesterol in serum was not changed significantly with soy protein and low fat milk intake (mârD; 2445 mg/dL at baseline, 2484 mg/dL with soy protein and 2505 mg/dL with milk). Serum levels of triglyceride, HDL-cholesterol, Apo A1, A2, B, C2, C3 and E, platelet aggregation induced by ATP, hemostatic markers were not significantly changed either, except for plasminogen activator inhibitor 1 (PAI-1) and D-dimer. Percent change in PAI-1 level estimated after one month intake of soy protein and its baseline level showed a negative relationship, although it did not reach the significance.There was no such tendency with milk intake. Change in D-dimer with soy protein intake was significantly lower than that with milk intake (-6.6% vs +14.2%, respectively, P< 0.05). These results indicate that compared to low fat milk intake soy protein may have a favorable effect on hemostatic system in the process of atherosclerosis.

Key words: soy protein, milk, platelet aggregation, plasminogen activator inhibitor-1 (PAI-1), D-dimer, atherosclerosis


The soy protein protein reserch(Japan)