The Soy Protein Research Committee(Japan)

MOLECULAR CLONING OF OILSEED CYSTATINS AS ANTIVIRAL PROTEINS

Soichi ARAI

Faculty of Agriculture, The University of Tokyo

Rep. Soy Protein Res. Com., Jpn. 14, 1-3, 1993.

Cystatin is a proteinaceous cysteine proteinase inhibitor. We isolated a soybean genomic DNA clone for a cystatin from a soybean genomic library by screening with a cDNA clone for oryzacystatin I. A cDNA clone corresponding to the genomic clone was obtained from a cDNA library of immature soybean seeds using the genomic DNA fragment as a probe. The cDNA clone encodes a 245 amino acid protein. The protein, named soyacystatin, contains the sequence Gln-Val-Val-Ala-Gly conserved among most members of the cystatin superfamily and is similar to other plant cystatins in overall amino acid sequence, especially to oryzacystatin I. Large expression of the soyacystatin mRNA in soybean seeds was observed in an early stage of maturation.

ENHANCEMENT OF THE SMALL INTESTINAL TRANSIT AFTER FEEDING OF SOYBEAN PROTEIN ISOLATE (SPI) IN RATS

Hiroshi HARA, Hideki MORIGAMI and Shuhachi KIRIYAMA

Faculty of Agriculture, Hokkaido University

Rep. Soy Protein Res. Com., Jpn. 14, 4-8, 1993.

Soybean protein isolate (SPI) is known to have different effects from casein on the digestive and absorptive functions in rats. We examined the small intestinal transit speed, which is a factor determining digestive and absorptive rates, by duodenal instillation of unabsorbable radioactive markers in rats fed the diets containing 8% SPI, casein, and no protein. The transit speed was significantly higher in SPI diet group than those in casein and non-protein diet groups. We also compared the small intestinal transit speed between the rats fed SPI and two kinds of SPI hydrolysates: peptic digest of SPI (SPI-large peptide) and SPI-small peptide (Hinute PM). The transit speeds in the groups fed both large and small peptide containing diets were significantly higher than that in the non-protein diet group, and tended to be higher than that in the SPI-fed group.

CHANGES IN FREE AMINO ACIDS AND OLIGOPEPTIDES IN SERUM AFTER ADMINISTRATION OF HINUTE - PM

Yoshiaki NIIYAMA and Sachinobu MANABE

Department of Nutrition, School of Medicine, The University of Tokushima

Rep. Soy Protein Res. Com., Jpn. 14, 9-13, 1933.

Postprandial changes in free amino acids and peptides in portal blood were investigated in rats fed an oligopeptide mixture from SPI (Hinute-PM). After 12 hour fasting, rats (about 300g) were bolus-fed a liquid diet containing 1.5g of Hinute-PM (average chain length 7.8), and then blood samples were collected at intervals from a catheter inserted previously into portal vein. For deproteinization one volume of 3% sulfosalicylic acid was added to two volumes of serum and resultant supernate was used for analyses of free amino acids and oligopeptides. Concentrations of free amino acids and peptides in serum increased with a peak at 30 minutes (from 4.1 É mol/mL for fasting level to 7.9 É mol/mL for peak level and from 1.9 É mol/mL to 2.6 É mol/mL, respectively) and decreased to fasting level within 2 hours. Average chain length of oligopeptides in serum from fasted rats was about 9.5. However, it decreased slightly at 30 minutes after feeding, and then tended to return to fasting values in almost all rats, suggesting that considerable amount of ingested oligopeptides were absorbed as the forms of original or digested oligopeptides.

PREPARATION OF HYPOALLERGENIC SOYBEAN PRODUCTS

Tadashi OGAWA, Hideaki TSUJI and Noriko BANDO

Department of Nutrition, School of Medicine, The University of Tokushima

Rep. Soy Protein Res. Com., Jpn. 14, 14-20, 1993.

A major allergenic protein (Gly m Bd 30K) purified from defatted soybean flakes was identified as soybean seed 34-kDa oil-body-associated protein or P34 vacuolar protein. This protein was shown to have about 30% sequence homology with Der p I, a house dust mite allergen originated from Dermatophagoides pteronyssius. The monoclonal antibodies against Gly m Bd 30K were prepared from mice. Using two distinct monoclonal antibodies, the sandwich enzyme-linked immunosorbent assay system was developed for the selective determination of Gly m Bd 30K. It was shown that the allergen was measured in a range of 5-500 ng/mL in this method.

STUDIES ON ALLERGENICITY OF SOYBEAN PROTEIN MODIFIED WITH OXIDIZED LIPID

Tsukasa MATSUDA, Tetsuya ISHII, Kazue YAMAMOTO and Ryo NAKAMURA

School of Agriculture, Nagoya University

Rep. Soy Protein Res. Com., Jpn. 14, 21-27,1993.

IgG and IgE antibodies specific for soybean proteins were measured by ELISA for sera collected from several strains of mice fed commercially available non-purified diet, in which soy protein antigens were detected immunologically. Significantly high level of IgG anti-soybean protein was detected in sera of several mice, whereas specific IgE antibody was not; IgG specific for 7S and 11S globulins were detected in the sera of ddY mice, IgG anti-2S globulin was in these of BALB/c and B10.D2 mice, and IgG anti-7S globulin was in that of B10.A mouse. To examine effect of heating with soybean oil on immunological properties of soybean proteins, soybean protein was mixed with soybean oil and heated at 50Åé for 9 days, and reactivity of the mouse serum IgG to the heated protein/oil mixture was measured. Reactivity to the soybean antigens of IgG in the sera of some mice was significantly increased by the heating with soybean oil, suggesting that some new antigenic structures of antigenic compound were formed by reaction of soybean proteins with soybean oil. Therefore, effect of protein modification with lipid peroxidation products was examined on immunological properties of soybean proteins. 4-Hydroxynon-2-enal (HNE), a major degradation product formed by lipid peroxidation was used as a model compound. Soybean 2S, 7S and 11S proteins were incubated with HNE, and HNE-modified proteins were detected by immunoblotting using HNE-specific antibody. HNE-modified 7S and 11S globulins were clearly detected with the specific antibody, whereas no modified protein was detected in 2S globulin. The HNE-modified 7S and 11S proteins mixed with aluminium hydroxide gel (Alum) were intraperitoneally injected into ddY mice, and specific IgG and IgE responses were measured by ELISA. Intact soybean proteins were also injected into another group of mice as a control experiment. Both IgG and IgE antibody responses to HNE-modified 7S globulin were significantly lower than those of intact 7S globulin, suggesting that HNE-modification decreased immunogenicity of soybean 7S globulin.

USE OF SOYBEAN PEPTIDES TO IMPROVE BIOAVAILABILITY OF METHIONINE AND TO REDUCE GASTROINTESTINAL SOY PROTEIN ALLERGY

Misako TANIGUCHI ¹, Mamoru FUJITA ¹, Takami FUSANO ¹,Chinami YANO ¹,Takashi YAMAMOTO ² and Minoru KIMOTO ²

¹ Department of Food and Nutrition, Nakamura Gakuen College
² Fuji Oil Company

Rep. Soy Protein Res. Com., Jpn. 14, 28-33, 1993.

When weanling rats were fed a low methionine diet, liver GGT activity was elevated. In rats fed a 10% SPI or simulated amino acid mixture diet with various levels of methionine, the response in hepatic GGT activity to the added methionine was larger than that in body weight gain, FER, NPU or BV. Using GGT activity as indication of methionine utilization, it was found that methionine utilization from peptide PM was apparently improved in comparison with that from SPI or SP. Though about 40% of glycosides of SPI was removed by treating SPI with glycosidases, methionine utilization could not be improved significantly. Tyrosine aminotransferase activity was not altered by dietary methionine levels. Anti-SPI IgG was produced by oral ingestion of SPI following abdominal administration of SPI to milk suckling rats. However, anti-SPI IgG could not be detected by PM administration. With electron microscopy, absorption pathway of soybean lectin was demonstrated to be similar to that of antibodies following adsorption on coated pit, coated vesicle formation and then transport into blood. Abbreviations: SP, soybean peptide; GGT, É¡-glutamyltransferase; IgG, immunoglobulin G.

REGULATION OF POLYUNSATURATED FATTY ACID METABOLISM AND EICOSANOID PRODUCTION BY SOYBEAN PROTEIN

Michihiro SUGANO and Kazunori KOBA

Faculty of Agriculture, Kyushu University

Rep. Soy Protein Res. Com., Jpn. 14, 34-37, 1993.

To know the mechanism by which dietary soybean protein regulates the activity of ɢ6- desaturase in liver microsomes, the effect of varying dietary proteins, casein, milk whey protein, egg albumin, soybean protein, potato protein and wheat gluten, on the relationship between the ɢ6-desaturase activity, membrane fluidity and lipid composition in liver microsomes was examined. The results indicated that the dietary protein-dependent changes in the liver microsomal cholesterol/phospholipid ratio affected membrane fluidity, and subsequently the activity of ɢ6-desaturase.

EFFECTS OF DIETARY PROTEINS ON PLASMA CHOLESTEROL LEVEL AND FECAL STEROID EXCRETION RATES IN VARIOUS SPECIES OF ANIMAL (II)

Shin-ichi HAYASHI, Youichi MIYAZAKI, Hiroko TAKIZAWA and Sanae TERASAKI

Department of Nutrition, The Jikei University School of Medicine

Rep. Soy Protein Res. Com., Jpn. 14, 38-41, 1993.

The hardly digestible high-molecular-weight fraction (HMF) of soy protein was compared with SPI and casein in their feeding effects on fecal steroid excretion rate and plasma cholesterol level of three species of animals, namely rat, mouse and Syrian hamster. In rats and hamster, HMF showed stronger effects than SPI did, compared with casein, to stimulate fecal steroid excretion rate and lower plasma cholesterol level. In mice, on the contrary, HMF and SPI showed a weak and no effect, respectively, on steroid excretion and similar weak effects on plasma cholesterol level. These results were in accordance with the theory that hypocholesterolemic effect of SPI in rats and hamsters, but not mice, is due to the production of undigestible peptides which stimulate steroid excretion. The distribution of 14C-steroids was compared with that of protein in the gastro-intestinal tract of rats after feeding experimental diets containing radioactive cholesterol. The results suggested that HMF stimulates gastro-intestinal transit of steroids and suppresses their absorption, whereas it increases protein contents in the lumen of ileum, cecum and colon. SPI appeared to show similar but weaker effects. These results also supported the above mentioned theory.

BILE ACID-BINDING PEPTIDES FROM DIGESTIVE PRODUCTS OF SOY PROTEIN ÅF THEIR STRUCTURAL AND FUNCTIONAL INVESTIGATIONS

Kimikazu IWAMI, Masahiro KANAYA, Masaru KITAGAWA and Fumio IBUKI

Faculty of Agriculture, Kyoto Prefectural University

Rep. Soy Protein Res. Com., Jpn. 14, 42-48, 1993.

It was tried to isolate bile acid-binding peptides from peptic-pancreatic digests of soy protein. Such peptides were collected by affinity chromatography with glycocholate-linked Sepharose 4B beads. Both SDS electrophoresis and gel filtration revealed that this fraction was composed of several peptides ranging in molecular size from 3K to 10K. A major peak component which had been fractionated by gel filtration, also gave a similar broad band in SDS-PAGE. For this reason, further purification of these peptides was shelved for the time being. Alternatively, very hydrophobic components were first separated from proteins which had partially been purified by repeating isoelectric precipitation at pH 4.6, followed by pepticpancreatic digestion in the usual manner. The digest was passed through another glycocholatelinked Sepharose 4B column. The fraction was desorbed from the column by elution with 0.2% deoxycholate, recovered as a single peak in gel filtration with Bio-Gel P-10 and proved to be homogeneous by reversed phase HPLC. As a result of amino acid analysis, the molecular weight of the peptide containing 41 residues turned out to be 4,210. Estimation of the primary structure and enzymatic or chemical modification in functional aspects are in progress.

EFFECTS OF DIETARY SOYBEAN PROTEIN ON ATP - CITRATE LYASE INDUCTION IN RAT LIVER AND PLASMA HORMONE LEVELS

Nobuko IRITANI ¹, Hiroko HOSOMI ¹,Hitomi FUKUDA ¹ and Takehiko TANAKA ²

¹ Tezukayama Gakuin College
² Osaka University Medical School

Rep. Soy Protein Res. Com., Jpn. 14, 49-52, 1993.

The effects of soybean protein feeding on ATP-citrate lyase induction in rat liver and plasma hormone levels have been investigated. By feeding only carbohydrate without protein to fasted rats, the transcriptional rate was increased threefold, and the mRNA concentration and enzyme induction comparably, to the levels in the carbohydrate/protein diet. It appears that protein feeding is not necessary to the gene expression and enzyme induction of ATP-citrate lyase. One week later, however, the mRNA concentration and enzyme activity were significantly lower in animals fed soybean protein than in those fed casein. The triacylglycerol level in the liver was more greatly decreased by soybean protein than the lipogenic enzyme induction. The results of our present and past experiments suggest the lowering effects of soybean protein on triacylglycerol level besides lipogenic enzyme induction. The plasma triiodothyronine level was significantly decreased by the soybean protein feeding. Moreover, the urinary excretion of norepinephrine tended to be increased. Thus, the turnover of triacylglycerols may be increased by soybean protein feeding.

EFFECT OF DIETARY SOY PROTEIN ISOLATE ON THE FORMATION OF CHOLESTEROL GALLSTONE IN MICE

Keisuke TSUJI

National Institute of Health and Nutrition

Rep. Soy Protein Res. Com., Jpn. 14, 53-59, 1993.

The interrelationship between dietary protein and cholesterol gallstone formation was studied in ICR-mice. Male mice were fed one of the 10Å`20% protein diets containing 0.5% cholesterol and 0.25% cholate for 4-5 weeks. Animals fed 20% casein diet induced the high incidence of cholesterol gallstone than those fed 10% casein diet. Soy protein isolate (SPI) could not show any changes in gallstone formation. However, SPI suppressed the increment of serum (LDL+VLDL) cholesterol levels and atherogenic index. Fecal excretion of bile acids was increased by SPI feeding. Milk whey isolate showed a significant repress against the increments of total cholesterol concentration in serum and liver, and lithogenic effect by the casein and cholesterol feeding.

METABOLIC CHANGES IN SERUM LIPOPROTEINS INDUCED BY THE ADDITION OF DIETARY EXCESS CYSTINE TO A SOYPROTEIN ISOLATE DIET

Yoritaka AOYAMA, Tomohiro ISHIKAWA, Tetsuji SUZUKI,
Yukiko IWASAKI and Akira YOSHIDA

School of Agriculture, Nagoya University

Rep. Soy Protein Res. Com., Jpn. 14, 60-64, 1993.

The effects of excess cystine added to the diets with casein, egg protein, soybean protein isolate and wheat gluten as protein sources on liver and serum lipids of rats were examined. Liver lipid contents among four groups were not changed when cystine was not added. The addition of excess cystine to the soybean protein isolate diet, but not to the casein, egg protein and wheat gluten, caused lipid accumulation in the liver. The increase in liver lipids was due to an increase in triacylglycerol. In order to know the accumulation of triacylglycerol in the liver, Triton WR 1339 were injected intravenously. The increased rate of triacylglycerol in serum from rats fed the soybean protein isolate diet supplemented with cystine was lowered as compared with that of the soybean protein isolate diet. Both protein and triacylglycerol of VLDL fraction in serum were decreased. Apolipoprotein E was not changed, but apolipoprotein C was decreased. Thus, overall results show that one of the factors for the accumulation of triacylglycerol in the liver induced by the addition of excess cystine to the soybean protein isolate diet might be due to the decreased transport from liver into serum.

EFFECT OF SOY PROTEIN ON RENAL GLOMERULAR LESIONS IN SPONTANEOUSLY DEVELOPED DIABETIC WBN/Kob RATS

Yoshio IKEDA ¹ and Yutaka MORI ²

¹ Department of Preventive Medicine, Center for General Health Care,
Jikei University School of Medicine
² Third Department of Internal Medicine, Jikei University School of Medicine

Rep. Soy Protein Res. Com., Jpn. 14, 65-69, 1993.

To evaluate the effects of chronic protein loading on renal glomerular lesions, 45 diabetic WBN/Kob rats aged 8 months were given 15g per day of 5, 20 and 40% protein diets from different sources for 18 weeks. Codfish, pork and soybean were used as protein source and 9 kinds of diets were used. Urinary albumin excretion (UAE) decreased significantly in all of 5% protein diet-fed groups. UAE in all of 40% protein diet-fed groups increased and the increase in UAE was remarkable in soy protein and fish protein groups. Urinary NAG excretion decreased significantly in all of 5% protein diet-fed groups. In 40% protein diet-fed groups, urinary NAG excretion increased significantly in fish protein and pork protein groups. Morphometrical analysis of renal glomerular lesions showed the average mesangial area increased significantly in all of 40% protein diet-fed groups. These results suggest that an important factor in prevention of development of diabetic nephropathy might be the quantity rather than the quality of dietary proteins.

EFFECTS OF SOY PROTEIN ISOLATE ON SUCROSE - INDUCED FAT ACCUMULATION IN POST - WEANING AND YOUNG RATS

Eiko HARA ¹and Takashi SHIMAZU ²

¹ Imabari Meitoku Junior College
² School of Medicine, Ehime University

Rep. Soy Protein Res. Com., Jpn. 14, 70-75, 1993.

In experiment 1, effects of dietary soy protein isolate (SPI) on sucrose-induced fat accumulation were examined in 3 weeks-old rats (after-weaning rats) and 7 weeks-old rats (young rats). In both groups of rats, the total energy intake did not increase by sucrose drinking. The increased hepatic content of triglycerides (TG) after sucrose-drinking was higher in young rats than in after-weaning rats. In experiment 2, effects of dietary fat on sucrose-induced fat accumulation were examined using after-weaning rats. The rats were fed 6% lard or soy-bean oil as dietary fat for 2 weeks. Dietary lard further augmented the accumulation of liver TG induced by sucrose drinking and attenuated the suppressive effect of SPI on serum TG concentration. These results indicate that saturated fatty acids affect the interaction of sucrose and SPI on fat metabolism, which may contribute to the development of dietary obesity.

EFFECT OF FEEDING SPI TO EXPERIMENTAL DIABETIC RATS

Jong-Hee KIM, Shin-ichiro TAKAHASHI and Tadashi NOGUCHI

Faculty of Agriculture, The University of Tokyo

Rep. Soy Protein Res. Com., Jpn. 14, 76-78, 1993.

A special diet (D-diet) with SPI was designed for experimental diabetic rats. The diet contained less branched chain amino acids and more lysine compared to the casein-based control diet (C-diet). C-diet or D-diet was given to streptozotocin-diabetic rats or the control (untreated) rats. The control rats given C-diet grew better than those fed on D-diet but the diabetic rats lost their body weight on C-diet. When the diabetic rats were given D-diet, the body weight loss was partially prevented. These favorable effects of D-diet on the diabetic rats were supported by the changes in plasma concentrations of IGF-I and IGF-binding proteins.

PROTEIN MALNUTRITION IMPAIRS SUPEROXIDE ANION PRODUCTION AND CANDIDA PHAGOCYTOSIS BY MACROPHAGES

Kazuhito ROKUTAN ¹,Shigetada TESHIMA ¹,Yumiko YAMAMOTO ¹,
Yasuhiro KIDO ¹, Fujiko SHIZUKA ¹,Kyoichi KISHI ¹ and Fusako TERAMOTO ²

¹ Department of Nutrition, School of Medicine, The University of Tokushima
² Kawasaki University of Medical Welfare

Rep. Soy Protein Res. Com., Jpn. 14, 79-82, 1993.

In response to appropriate stimuli, macrophages release a large amount of oxygen radicals through the NADPH oxidase. This "respiratory burst" is essential for their microbicidal activity. Female C3H/He-slc mice at 4 weeks old were allocated to low protein (5% casein or 5% soy protein) or control (20% casein or 20% soy protein) diets for 2 weeks. Superoxide anion (O2-) production, stimulated by phorbol myristate acetate (PMA), in resident peritoneal macrophages was significantly increased in the protein-malnourished groups. These macrophages did not increase the capacity for PMA-stimulated O2- release after treatment with lipopolysaccharide in vitro. Protein malnutrition did not impair the phagocytosis of unopsonized Candida albicans, while there was no increase in the ability of Candida phagocytosis after opsonization. These results suggest that protein malnutrition may impair the O2- release and phagocytosis of macrophages.

EFFECTS OF SOYBEAN PROTEIN ISOLATE (SPI) ON CELLULAR IMMUNE FUNCTIONS OF VOLUNTARILY EXERCISED RATS

Satoru MORIGUCHI, Hitomi MIWA and Yasuo KISHINO

Department of Nutrition, School of Medicine, The University of Tokushima

Rep. Soy Protein Res. Com., Jpn. 14, 83-88, 1993.

The purpose of this study was to determine the effects of soybean protein isolate (SPI) on cellular immune functions of voluntarily exercised rats. Male Fischer rats, 4 weeks old, were fed diets composed of 5, 20 and 40% casein or SPI and subjected daily to voluntary exercise on a wheel mill. At the end of a 7 week-training period, peripheral blood lymphocytes (PBL), splenocytes and alveolar macrophages (AM) were isolated from exercised and sedentary control rats. In 5% protein groups the gain of body weight was apparently higher in casein group than that of SPI group. The difference of body weight gain between casein and SPI groups decreased with increasing protein levels in diets and was not shown in 40% protein group at all. In 5% casein group PBL proliferation with Con A was significantly suppressed by voluntary exercise, but not in 5% SPI group. In addition, rats fed 5% casein diet showed lower proportions of CD4+CD8- and CD4-CD8+ T cells in PBL, which was restored to the level of rats fed 20% casein diet by voluntary exercise. In 40% protein groups the proportion of CD4+CD8- T cells in PBL of both casein and SPI groups was significantly increased by voluntary exercise. Although phagocytosis of AM increased in all casein groups by voluntary exercise, AM from SPI groups did show decreased phagocytosis in AM from 5% protein group rather than the increase of phagocytosis by voluntary exercise. The effects of SPI diets or voluntary exercise on IL-1 production from AM and NK activity of splenocytes were not shown in this experiment. In conclusion, the above results suggest that cellular immune functions in rats fed 5% protein diet are well-maintained in the SPI group compared with that of casein group in case of taking voluntary exercise.

CLINICAL APPLICATION OF SOY PROTEIN FOR HYPERLIPIDEMIC SUBJECTS

Haruo NAKAMURA and Emiko MIYAJIMA

First Department of Medicine, National Defense Medical College

Rep. Soy Protein Res. Com., Jpn. 14, 89-91, 1993.

Since we have tried several clinical applications of soy protein for clinical and normal subjects, minimum dose of protein was found to be 20g per day or greater to decrease low density lipoprotein (LDL) cholesterol. However, the mechanism of the LDL reduction is still unknown. In this trial, three subjects were investigated the changes of HMG-CoA reductase activity in their blood lymphocytes. Their LDL cholesterol tended to decrease after the ingestion of soy protein. Concomitantly, HMG-CoA reductase activities in lympohocytes were found to be lowered. The present data indicate that soy protein accelerates LDL catabolism through enhanced LDL receptor activities resulting in the inhibition of HMG-CoA reductase activities.

EFFECTS OF SOY PROTEIN DIET ON ENERGY METABOLISM IN CIRRHOTIC PATIENTS - AN EVALUATION BY INDIRECT CALORIMETRY -

Masahiko KATO, Tetsuya HIRAOKA, Yoshiyuki MIWA, Takashi YOSHIDA,
Hisataka MORIWAKI and Yasutoshi MUTO

First Department of Internal Medicine, Gifu University School of Medicine

Rep. Soy Protein Res. Com., Jpn. 14, 92-97, 1993.

A study was conducted to investigate the effect of soy protein diet on energy metabolism in 6 hospitalized patients with liver cirrhosis. They were fed a control diet which provided 2100Å`2200 kcal and 70Å`80 g protein for the first seven day period (control period). The protein composition was 50% animal protein (AP) and 50% vegetable protein (VP). For the next seven days (test period) they were fed an isocaloric, isonitrogenous diet, in which protein consisted of 50% VP and 50% soy protein (SP). Indirect calorimetry was performed before and after breakfast on the last day of each period. Respiratory quotient (RQ), energy expenditure (EE) and relative composition of energy source of the EE were analysed. After breakfast in the test period, RQ increased significantly (pÅÉ0.05) and EE/basal metabolic rate (BMR) did not change. In addition, percentage glucose oxidation rate was increased, while percentage fat oxidation rate was reduced. In conclusion, these results suggest that soy protein diet is useful for the cirrhotic patients, particularly for those with glucose intolerance (diabetic tendency).

EFFECT OF SOY PROTEIN DIET ON LIPID TRANSFER PROTEIN ACTIVITY IN PATIENTS WITH ISCHEMIC HEART DISEASE

Hiromitsu TAMACHI ¹, Tomoki IWAMOTO ¹, Koji NAKAYAMA ¹, Yoshiko NAKAYAMA ¹, Yuichi FUSEGAWA ¹, Yutaka SHIINA ¹,Yuichiro GOTO ¹,Hiroaki ISHII ²,Honami FUJII ² and Miho HIDA ²

¹ School of Medicine, Tokai University
² Unit of Nutrition in Hospital, School of Medicine, Tokai University

Rep. Soy Protein Res. Com., Jpn. 14, 98-103, 1993.

The effects of substituting soy protein for protein in the basal diet on lipid metabolism were investigated in ten male patients with ischemic heart disease. Administration of the diet substituted with 10-15g of soy protein for 10 days resulted in a tendency to lower levels of TG, VLDL and Apo B/Apo A-I ratios, and also to increase levels of HDL2-C/HDL3-C ratios and Apo A-I. No changes in LTP activities were observed. These results suggest the possibility that 12g of soy protein intake improves TG metabolism in patients with ischemic heart disease.

CHARACTERIZAION OF GLYCOLIPID IN SOY PROTEIN ISOLATE

Seiichi HOMMA and Masatsune MURATA

Department of Nutrition and Food Science, Ochanomizu University

Rep. Soy Protein Res. Com., Jpn. 14, 104-107, 1993.

Soy protein isolate (SPI) was extracted with 87% ethanol and chromatographed on an Iatrobeads column. Glycolipid was found to be eluted with acetone and methanol. Since the methanol eluate was found to contain glycolipid and phospholipid, the methanol eluate was adjusted to pH 1.4 with HCl to form precipitates. The supernatant was revealed to consist of glycolipid mostly, and it was combined with glycolipid in the acetone eluate. The glycolipid was separated by silica gel TLC with various kinds of developing solvent mixtures. Sugar was converted into alditol acetate and determined by GLC. Ten glycolipids were separated, and the major sugars were found to be galactose and glucose with minor sugars of mannose and rhamnose. According to the molar ratio of sugar to fatty acid, a few kinds of glycero-type of glycolipid were speculated to be present.

PHYSIOLOGICAL FUNCTION OF SOYBEAN GLYCOSIDES, ESPECIALLY DDMP SAPONIN

Kazuyoshi OKUBO

Faculty of Agriculture, Tohoku University

Rep. Soy Protein Res. Com., Jpn. 14, 108-111, 1993.

Relative to other foodstuffs, soybean seeds contain high levels of four classes of compounds with demonstrated anticancer activity: glycosides, phytosterols, protease inhibitors, and phytic acid. Soybean seeds contain about 2% glycosides, which are composed of several kinds of soyasaponins and isoflavonoids. In this report, we reinvestigated the composition of group B saponin, and the structures of saponins containing DDMP named soyasaponins Éøg, Éøa, É¿g, É¿a, É¡g and É¡a are described. Recently, we have reported five kinds of DDMP (2, 3-dihydro-2, 5-dihydroxy-6-methyl-4H-pyrane-4-one) saponins as intact constituents of soybean seeds, named soyasaponin Éøg, É¿g, É¿a, É¡g and É¡a. DDMP saponins showed the same UV spectrum with a maximum absorption at 292 nm due to DDMP moiety. Using this character, DDMP saponin composition in leguminous seeds was investigated. Hypocotyl was extracted with 70% ethanol contained 0.01% EDTA and analyzed by HPLC, directly. As a result, DDMP saponins, especially É¿g, were widely distributed in legumes, and legumenous seeds were classified into six types (IÅ`VI) by DDMP saponin composition. This classification almost agrees with genus or subgenus of leguminosae, and consequently DDMP saponins may be useful as chemotaxonomical marker. New DDMP saponins from Phaseolus angulatris, Dolichos lablab and Phaseolus coccineus were isolated and the structures were elucidated by FAB-MS, 1H-NMR and 13C-NMR, respectively, such as soyasaponin Éøa. Furthermore we have found that the DDMP moiety was unstable by oxygen and ferric ion, and reacted with FeCl3 into brown color formation. Using this reaction, we investigated distribution of DDMP saponins in fine structure of leguminous seeds, especially soybean seeds. FeCl3 saturated in 70% ethanol solution was sprayed on seeds after cutting longitudinally and horizontally. The DDMP saponins distributed just hypocotyl, especially epicotyl parts of leguminous seeds, except soybean seeds, in which they distributed not only hypocotyl part but also cotyledon. DDMP saponins existed peripheral cells of mid vein and lateral vein. This result suggested that DDMP saponins related to germination and difference of plant body against active oxygen, virus and microorganism etc.

STUDIES ON MECHANICAL AND TEXTURAL PROPERTIES OF THERMALLY INDUCED SOY PROTEIN GELS

Tomohiko MORI

Research Institute for Food Science, Kyoto University

Rep. Soy Protein Res. Com., Jpn. 14, 112-116, 1993.

Heat-set gels were prepared from 11S globulins of various soybean cultivars at protein concentrations of 18 to 20%. The gels were measured for mechanical parameters by means of a compression-decompression test. Textural properties of the gels were evaluated in terms of three-dimensional representation of the gels through factor analysis of instrumental data and calculation of factor scores. The data were composed of the values taken for 16 variables: CW, RS, CM, and F at small, medium, and large compression levels and at rupture for the gel samples examined. The results demonstrated that textural properties of the 11S globulin gels can be characterized by their positions in the three-dimensional diagram in which textural attributes involved were hardness, toughness, elasticity, and fracturability. Differences in textural characteristics among soy 11S globulin gels, gels from other food proteins, and gel-like foods were evaluated by their positions in the diagram prepared through factor analysis of instrumental data of those samples.

USABILITY OF SPI AND ITS HYDROLYSATE AS AN AGENT TO ENTRAP LIQUID LIPIDS

Shuji ADACHI, Tatsuji ISHIGURO and Ryuichi MATSUNO

Faculty of Agriculture, Kyoto University

Rep. Soy Protein Res. Com., Jpn. 14, 117-121, 1993.

Rapid drying of liquid lipid emulsified with a solution of an entrapping agent can allow the encapsulation of lipid droplets into the powdery matrix of the entrapping agent. One of the advantages added by the entrapment is retardation of lipid oxidation due to the diffusional barrier of the matrix against oxygen. The possibility in use of SPI and soybean peptide as an entrapping agent for liquid lipid entrapment was studied from viewpoints of their effects on the diffusion of oxygen and on the oxidation kinetics of a lipid. The diffusion coefficients Dapp of oxygen through pullulan films containing SPI or soybean peptide were measured. They were in an order of 10Ü¢9 m2/s, which was much larger than the Dapp through pullulan film containing neither. This indicates that the addition of SPI or soybean peptide to pullulan gives unfavorable effect on the retardation of lipid oxidation. An addition of soybean peptide to linoleic acid (LA) retarded its oxidation to some extent, while an addition of SPI did not. Soybean peptide was supposed to have a suppressive effect on the initiation step of LA oxidation. Oxidation progresses of LA entrapped into powdery matrixes of pullulan itself or pullulan containing soybean peptide were simulated by solving numerically the mass balance equations in terms of oxygen and LA within the powdery matrixes under some conditions. The simulations suggested that a finding of an entrapping agent where the Dapp is in an order of 10-13 m2/s or less is inevitable for retardation of lipid oxidation by the diffusional resistance of a matrix against oxygen.

VACUOLAR PROCESSING ENZYME RESPONSIBLE FOR PROPROTEIN PROCESSING OF VARIOUS SEED PROTEINS

Ikuko HARA-NISHIMURA

Department of Cell Biology, National Institute for Basic Biology

Rep. Soy Protein Res. Com., Jpn. 14, 122-126, 1993.

Proprotein precursors of seed proteins are transported from endoplasmic reticulum to the dense vesicles, and then targeted to the vacuoles, where they are processed proteolytically to their mature forms by a vacuolar processing enzyme. However, the processing mechanism in plant vacuoles is very obscure. We purified a vacuolar processing enzyme from castor bean endosperm and characterized the enzymatic properties. The pH optimum for the purified vacuolar processing enzyme was at 5.5, coinciding with the pH of the vacuolar sap. The result supports the localization of the enzyme in vacuoles of maturing seeds. Characterization of the purified processing enzyme showed that a single enzyme is responsible for processing many vacuolar proteins with a large variability of molecular structure. How can it recognize the numerous variety of processing sites? Analyses of hydropathy and surface probability of precursor of 2S albumin, one of major seed proteins, revealed that post-translational processing occurs at C-terminal side of two asparagine residues which are located in the hydrophilic regions. These findings suggest that a vacuolar processing enzyme can recognize exposed asparagine residues on the molecular surface of precursor protein and cleave the peptide bond on the C-terminal side of each asparagine residue to produce mature protein in the vacuoles. We also examined the distribution of the enzyme in different tissues of various plants. The processing enzyme was found in cotyledons of pumpkin and soybean, as well as in endosperm of castor bean and rice, and low-level processing activity was also detected in leaves of spinach. These results suggest that the proprotein-processing machinery is widely distributed in vacuoles of various plant tissues.

N - GLYCOSYLATION OF SOYBEAN GLYCININ BY PROTEIN ENGINEERING

Shigeru UTSUMI and Makoto KITO

Research Institute for Food Science, Kyoto University

Rep. Soy Protein Res. Com., Jpn. 14, 127-130, 1993.

Glycinin is one of the dominant storage proteins of soybean seeds. Glycosylation of glycinin would improve its functional properties with respect to hydrophilicity. We constructed various cDNAs which encode specific variants containing potential N-glycosylation sites into each variable region of the A1aB1b proglycinin subunit. Potential sites were introduced by site directed mutagenesis to form modified proglycinins IIN, IIIN1, IIIN2, IVN and VN. Expression plasmids carrying the modified cDNAs were constructed and expressed in yeast. All of the modified proglycinins except IIIN2 were N-glycosylated. IIN and IVN were efficiently N-glycosylated, although IVN was limited-proteolyzed. N-glycosylation did not cause secretion of proglycinin from the yeast cell.

The soy protein protein reserch(Japan)