Soy Protein Research(Japan)

24-1

The Multivariate Functions of Adipose Tissue and the Metabolic Syndrome: The Effects of Soybean Protein on Fat Distribution and Adipocytokines

Yuji MATSUZAWA

Osaka University Graduate School of Medicine, Osaka 565-08711

ABSTRACT
The metabolic syndrome, a cluster of insulin resistance, hypertension and dyslipidemia, is a common basis of Type 2 diabetes and atherosclerotic vascular diseases in industrialized countries. The most common cause of the metabolic syndrome is the accumulation of intra-abdominal visceral fat. Adipose tissue secretes a variety of bioactive substances called adipocytokines. Dysregulation of adipocytokines by overnutrition may play an important role in the development of the metabolic syndrome. Soybeans, a part of the Japanese traditional diet, may protect against the development of the metabolic syndrome through regulation of adipocytokines. This study was aimed to clarify 1) the role of adipocytokines in the development of the metabolic syndrome, and 2) the significance of soybean protein, on the regulation of adipocytokines. Adiponectin is an adipose-specific plasma protein, which we identified in the Human Adipose cDNA Project. Adiponectin possesses the activities to suppress the expression of adhesion molecules in vascular endothelial cells; the expression of scavenger receptor mRNA in macrophages; and growth factor-induced proliferation of vascular smooth muscle cells. Furthermore adiponectin promotes glucose uptake in myocytes. Therefore, adiponectin may play a key role in the development of the metabolic syndrome. In previous studies, we reported dysregulation of adiponectin in subjects with body fat accumulation. In the present study, we investigated 1) the role of adiponectin in the metabolic syndrome using mice lacking the adiponectin gene and mice oveexpressing adiponectin, and also studied 2) the effect of a soy protein diet on adiponectin concentrations. Adiponectin knockout mice exhibited severe insulin resistance when fed a high-fat, high-sucrose diet. The knockout mice also showed severe vascular neointimal thickening from vascular injury. Overexpression of adiponectin in the apoE knockout mice, which is an animal model for atherosclerosis, significantly reduced fatty streak lesions in aorta. In obese KK-Ay mice, body fat weights, plasma cholesterol, triglyceride, free fatty acid, and glucose levels were lower in mice on a calorie-restricted diet containing soy protein isolate. Among the genes related to glucose and fatty acid metabolism, adiponectin mRNA levels in adipose tissue and adiponectin plasma concentrations were elevated in mice on calorie-restricted diets, although there were no significant differences between soy protein and casein protein groups. Our results indicate that adiponectin has anti-atherogenic and anti-diabetic activity in vivo, and a calorie-restricted diet elevates plasma adiponectin concentration in obesity.
Soy Protein Research, Japan 6, 1-10, 2003.

Key words : soy protein, adipocytokines, adiponectin, metabolic syndrome, visceral fat
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24-2

Molecular Mechanisms of Genetic Variations in Seed Storage Proteins and Their Transmission During the Process of Breeding in Soybean

Michiko YOSHINO1, Ken-ichi TSUTSUMI2, Yoshiya SHIMAMOTO1 and Akira KANAZAWA1

1Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589
2
Cryobiosystem Research Center, Faculty of Agriculture, Iwate University, Iwate 020-8550

ABSTRACT
The 7S globulin (β-conglycinin), a major component of seed storage proteins in soybeans, is comprised of three subunits, α, α' and β. In order to reveal genetic bases for variations in 7S globulin, we analyzed the structures of genomic DNA around genes for the proteins. We first constructed restriction maps around genes for each subunit in cultivated soybeans 'Forrest' and 'Keburi'. We next analyzed the structures around genes in various lines of soybean that show genetic variations regarding these subunits. As a result, polymorphisms were detected in these soybean lines with regard to the presence or absence of genes for each subunit as well as the structures around these genes. Large deletions of gene regions were found to be a major cause of genetic variations. We previously found that a gene (CG-2), which is located in the downstream of the α-subunit gene (CG-3) in 'Forrest', shares highsequence homology with the α-subunit gene. Because CG-3 was deleted while CG-2 was present in soybean lines that express α subunit at a low level, the CG-2 was regarded as a second gene for the α subunit. In addition, the deletions of gene regions of the α- and α'-subunit genes were found to be useful genetic variations for producing soybean strains that accumulate 7S globulin at a low level.
Soy Protein Research, Japan 6, 11-17, 2003.

Key words : soybean, β-conglycinin, α subunit, gene expression, deletion
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24-3

Identification and Linkage Analysis of the Genes Expressed in Soybean Seeds

Kyuya HARADA1 and Toyoaki ANAI2

1Faculty of Horticulture, Chiba University, Matsudo 271-8510
2
Faculty of Agriculture, Saga University, Saga 840-8502

ABSTRACT
Map-based cloning is a general method for isolating genes responsible for agriculturally important traits. The use of cDNA markers makes the method more effective. To identify the genes for seed traits including seed quality and quantity of seed constituents, high quality cDNA libraries were made from developing soybean seeds. The 5' partial sequences of 1,500 randomly selected clones were determined and the 3' partial sequences of 233 non-redundant clones were also determined to develop locusspecific cDNA markers by PCR. Ribosomal proteins were most highly expressed and electron transferring proteins had a relatively high expression in the early stage of developing seeds, while storage proteins were highly expressed in the middle stage. Proteinase inhibitors showed relatively high expression in both stages. Phosphoenolpyruvate carboxylase (PEPCase) and acetyl-CoA carboxylase (ACCase) were selected as putative candidate enzymes for protein and lipid contents respectively. Their loci were determined based on RFLP and PCR-RFLP using recombinant inbred lines and compared with the quantitative traits loci (QTLs) for protein and lipid contents, but there was no correspondence among the enzyme loci and the QTLs. Further analyses are necessary to identify QTLs for protein and lipid contents by cDNA mapping and map-based cloning.
Soy Protein Research, Japan 6, 18-21, 2003.

Key words : soybean, seed, quantitative trait locus (QTL), EST, cDNA marker
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24-4

Chemical Treatment of Plants May Activate Defense Mechanisms and Increase Allergenicity

Tadashi OGAWA1, Tatsuya MORIYAMA1, Noriko NOMURA2 and Koji TAKAHASHI3

1Graduate School of Agriculture, Kyoto University, Kyoto 611-0011
2
Taiyo Chemical Co., Mie 510-0844
3National Agriculture Research Center, Ibaraki 305-8517

ABSTRACT
Many plant allergenic proteins have been isolated and identified as pathogenesisrelated proteins. They are produced when biological, chemical, and physical stresses activate defense mechanisms of plants. These defense proteins are thought to play an important role in pathogen resistance, and some defense-related proteins are significant cross-reactive allergens. Accordingly, the allergenicity of plant foods may depend on the growth conditions of plants or given stress activating defense mechanisms. Using turnips and soybeans, we measured the allergenicity of these model food plants after treatments activating their defense mechanisms to determine whether the allergen content of these plants increase. On immunoblotting profiles, IgE-binding protein bands were found to increase in both turnip and soybean treated chemically, and the allergenicity given as total ELISA values were also increased in the stress-given plant samples. These results demonstrated that activating defense mechanism of plants may increase their allergenicity.
Soy Protein Research, Japan 6, 22-28, 2003.

Key words : plant, soybean, allergen, allergenicity, pathogenesis-related proteins,chemical stress
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24-5

Development of a New Emulsifier with the Conjugation Between Soy Protein and Pectin from Food Waste

Hidefumi YOSHII and Takeshi FURUTA

Faculty of Engineering, Tottori University, Tottori 680-8552

ABSTRACT
Depolymerized citrus pectin has emulsifying properties. It is thought that the presence of a protein moiety in the depolymerized pectin plays a major functional role. In this study, the kinetic characterization of acid hydrolysis was investigated with high-methoxy (HM) pectin and soy protein to obtain water-soluble polysaccharides (WSP). A 6% by weight solution of HM-pectin and soy protein (5:1 w/w) at pH3-5 was incubated at a constant temperature (80oC-120oC) in an autoclave. Acid degradation kinetics of HM-pectin indicated a first-order degradation rate as the viscosity change of WSP. The change of mean emulsion diameter of d-limonene with WSP from HM-pectin and soy protein also showed a first-order rate. Both kinetic activation energies (42 kJ/mol) were obtained at pH 3.0.
Soy Protein Research, Japan 6, 29-33, 2003.

Key words : pectin, hydrolysis, conjugates, emulsifier
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24-6

Antifreeze Protein Improves the Quality of Soy Protein Processed Frozen Food

Hidehisa KAWAHARA, Runa TANAKA and Hitoshi OBATA

Faculty of Engineering, Kansai University, Osaka 564-8680

ABSTRACT
We examined the effect of antifreeze protein (AFP) on the solubilization of soy protein, which is used as food material, under freezing conditions. The soy protein suspension (8 mg protein/mL) was prepared and mixed with AFP to yield its final concentration of 0-100μg/mL. This mixture was frozen at -20oC for 24 h, and then thawed at room temperature. When AFP was added at 5 and 100μg/mL, their solubilization rates were 45% and 48%, respectively. Also, this high level of solubilizing effect was achieved with only one freezing treatment. Furthermore, this solubilizing effect could improve the manufacture of bean curd with different protein contents.
Soy Protein Research, Japan 6, 34-37, 2003.

Key words : antifreeze protein, bean curd, soyprotein
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24-7

Functions of Lecithin in Dough and Bread Supplemented with β-Conglycinin

Reiko URADE

Graduate School of Agriculture, Kyoto University, Uji 611-0011

ABSTRACT
The soy protein, β-conglycinin is known to decrease plasma triacylglycerol and cholesterol levels in humans. Because these effects of β-conglycinin are transient, it is necessary to take it daily to maintain such effects. Bread is a convenient vehicle for introducing β-conglycinin into the daily diet. However, because of its high waterabsorption properties, β-conglycinin increases the water-absorption capacity of flour, alters the rheological properties of dough, and decreases the loaf volume of bread. Studies show the addition of soybean protein isolate or β-conglycinin decrease loaf volume. Strategies to overcome these adverse effects should be developed. We added phosphatidylcholine (PC) to the bread ingredients, in addition to protein isolate and β- conglycinin, and the adverse effect of the proteins on loaf volume was counteracted. Additionally, adding PC showed little effect on the rheological properties of dough, determined with a Reolograph Gel, a Rheoner, and a Microfarinograph. The function of PC could not be replaced by phosphatidylethanolamine, phospatidic acid, or lysophospatidylcholine. The ultrastructure of bread was examined by scanning electron microscopy. The gas cells in the bread containing proteins from soybean were smaller than those in the control bread without soy proteins. In the bread supplemented with soy proteins and PC, the size of the gas cells was similar to those in the control bread. These results may suggest that PC made the cell walls resistant to breakage by gas pressure, thereby maintaining the gas-retaining ability and maintaining the usual loaf volume of the bread.
Soy Protein Research, Japan 6, 38-42, 2003.

Key words : β-conglycinin, soybean protein isolate, bread, dough, phosphatidylcholine
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24-8

Genetically Engineered Bacillus subtilis Produces Soybean-Milk-Coagulating Enzyme

Masaaki YASUDA1, Naoko KUBA1 and Hiroshi TAKAGI2

1Faculty of Agriculture, University of the Ryukyus, Okinawa 903-0213 2Faculty of Biotechnology, Fukui Prefectural University, Fukui 910-1195 Bacillus subtilis

ABSTRACT
Soybean-milk-coagulating enzyme (SMCE) is important for coagulating soybean protein, and the technology for its effective production is needed. In this study, we investigated the expression of SMCE gene in genetically engineered Bacillus subtilis. The SMCE structural gene containing the putative promoter and terminator regions were isolated from B. pumilusby PCR using the primers, each of which contains the BamHI or XbaI site. The unique amplified band was digested with BamHI and XbaI, and then ligated to the BamHI and XbaI sites of plasmid pHY300PLK. The resultant plasmid (pNK) was introduced into Bacillus subtilis DB 403 that was a proteasedeficient strain by electroporation, and three transformants containing pNK were obtained. When the recombinant strains ( B. subtilisDB 403 (pNK)) were cultured on a LB agar plate containing skimmed milk, all strains were shown to form haloes. The band corresponding to SMCE from B. pumilusand SMCE activities were observed in the culture fluid from the recombinant B. subtilisDB403 (pNK). These results showed that the SMCE gene was expressed and the active enzyme was produced in B. subtilis.
Soy Protein Research, Japan 6, 43-47, 2003.

Key words : soybean-milk-coagulating enzyme, Bacillus pumilus, genetic engineering, Bacillus subtilis
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24-9

Enhanced Function of Calcium Absorption by Phytate-Removed and Deamidated Soybean Proteins

Hitomi KUMAGAI

College of Bioresource Sciences, Nihon University, Fujisawa 252-8510

ABSTRACT
Calcium is an important mineral for the formation of bones and teeth. Currently, calcium intake of Japanese is nearly half that of Westerners, and does not meet the recommended level. The amount of calcium absorbed in the intestine is about 20-50% of intake. Therefore, increasing calcium absorption as well as intake is important for achieving optimal calcium status. Casein phosphopeptide (CPP) and calcium citrate malate (CCM) enhance calcium absorption, and are used in foods for specified health purposes. However, the use of these substances in foods is limited because of their low molecular weight, and CPP has a milky flavor, and CCM a sour taste. Soybeans are a traditional part of the Japanese diet and the isoflavones are known to inhibit calcium resorption in bones. However, phytate in soybeans renders calcium insoluble, and inhibits calcium absorption by the body. If one could enhance calcium absorption from soybeans, it would expand their use and decrease the calcium inadequacy in Japanese. We showed previously that the removal of phytate and the deamidation of acid amide from soybean proteins improves their calcium binding, and that about 70% of the total acid amide can be deamidated by ion-exchange resins without any detectable peptide-bond hydrolysis. In this study, we evaluated the calcium-binding properties of phytate-removed and deamidated soybean proteins (PrDS) during hydrolysis by digestive enzymes, and examined the enhancement effect of calcium absorption by PrDS on bone formation. The results indicate that PrDS retained the high calcium binding properties even after hydrolysis by digestive enzymes, and enhanced calcium absorption from the intestines.
Soy Protein Research, Japan 6, 48-52, 2003.

Key words : calcium absorption, phytate removal, deamidation, soybean protein, hydrolysis
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24-10

Comprehensive Evaluation of Soy Protein Functions by DNA Microarray Analysis

Keiko ABE and Ichiro MATSUMOTO

Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657

ABSTRACT
Feeding rats with soy protein isolate (SPI) for a short term (2 weeks) decreased serum cholesterol and neutral lipid to a similar degree as feeding for a longer term (8 weeks). To better understand the molecular mechanism behind this phenomenon, we carried out a comprehensive analysis of the resulting gene expression profiles, with the expectation that it would contribute to our in-depth understanding of SPI functionalities. Male Wistar rats aged 6 and 12 weeks were dichotomized and fed an SPI or casein diet (control) for 2 weeks. Liver samples were obtained from rats in each diet group and a cRNA sample was prepared for use in DNA microarray analysis. The SPI feeding significantly up-regulated 138 and down-regulated 218 genes in the 6-week old rats, and significantly up-regulated 87 and down-regulated 248 genes in the 12-week old rats. Down-regulated genes included those for enzymes involved in fatty acid anabolism, the same genes down-regulated in longer term SPI feeding (mentioned above), as well as genes for enzymes involved in cholesterol catabolism, which were not down-regulated in the longer-term feeding studies. The present study suggests the possibility that the use of DNA microarray analysis is effective as a method for evaluating physiological functions of food proteins in general as well as those of SPI in particular.
Soy Protein Research, Japan 6, 53-56, 2003.

Key words : soy protein isolate, functions of food proteins, DNA microarray, comprehensive analysis
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24-11

Phytomedical Effect of Soy Protein and Soy Isoflavone on Hypercholesterolemia and/or Atheroscelerosis : Its Molecular Pathophysiological Mechanism in Monkeys

Shin NAKAMURA1, Fusako MITSUNAGA1, Maki OKADA1, Aram JOENG1, Takashi HAYASHI2, 3, Rosario M. PEREZ3, Romualdo L. CARRASCO3, and Hiroshi NAKAGAWA2, 3.

1Primate Research Institute, Kyoto University, Inuyama 484-8506
2
Ina Research Inc., Nagano 399-4501
3
INA Research Philippines, Philippines. Research, Japan 6, 57-62, 2003.

ABSTRACT
Soy protein (β-Conglycinine) and soy isoflavone (Soyaflavone HG) are known to regulate hypercholesterolemia and its mediated atherosclerosis. In the current study we examined the phytomedical effect of β-Conglycinine and Soyaflavone using an established monkey hypercholesterolemia/atherosclerosis model, in normal male, female and ovariectomized (OVX)-female monkeys. The molecular mechanism of the phyto-medicial function of the soy products was investigated by analyses of blood lipid markers (total Chol, LDL-C, HDL-C, GOT, GPT, Glu, CPK, TG, Apo-Al, Apo-B, and Apo-E). To elucidate the genetic mechanism, gene expression profiling analyses of an inflammatory and thrombotic gene (TF) and lipid metabolic genes (LDL-R, VLDL-R, HMG-CoA, SR) were performed by RT-PCR. β-Conglycinine and Soyaflavone both suppressed upregulated TF gene expression in hypercholesterolemia, suggesting a new mechanism of these soy products in hypercholesterolemia/atherosclerosis.
Soy Protein Research, Japan 6, 57-62, 2003.

Key words : β-conglycinine, soyaflavone, hypercholesterolemia, monkey model, gene expression profiling
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24-12

Studies on Soy Proteins Stimulating Cholesterol Efflux Driven by the Intestinal ABCA1 Transporter

Ryuichiro SATO

Graduate School of Agricultural and Life Sciences. The University of Tokyo, Tokyo 113-8657

ABSTRACT
Cholesterol efflux from human cultured intestinal (Caco-2) cells was studied. ABCA1, which stimulates cholesterol efflux in a manner similar to HDL from various cells, was expressed in differentiated Caco-2 cells. A nuclear receptor, Liver X Receptor(LXR), was also expressed in these cells. ABCA1 gene expression was augmented by treatment of cells with an LXR agonist. [3H]cholesterol efflux to the apical or basolateral side of Caco-2 cells was measured after induction of ABCA1 expression by the LXR agonist. Cholesterol efflux to the basolateral side was increased by the agonist or the addition of apo A-1, which is involved in formation of HDL by ABCA1, to the culture medium, suggesting that ABCA1 particpates in HDL production on this side. Cholesterol efflux to the apical side was increased by the agonist or the addition of bile acid, not apo A-1. This indicates that certain transporters including ABCG5 and G8 induced by LXR stimulate cholesterol efflux to the apical side in a bile acid-dependent manner. We speculate that some peptide molecules digested from soy proteins as well as bile acids might stimulate cholesterol efflux from the intestinal epithelial cells. Alternatively, it is possible that some peptides in the small intestinal tract directly enhance the transport activity of transporters involved in cholesterol efflux. To find out functional soy peptides reducing cholesterol absorption (increasing cholesterol efflux), several kinds of screening are now being carried out.
Soy Protein Research, Japan 6, 63-66, 2003.

Key words : ABCA1, ABCG5, ABCG8, cholesterol efflux, LXR
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24-13

Studies on Suppressive Effect of Soy Protein on LPS-induced Hepatitis

Kimio SUGIYAMA, Ena ATSUMI, Yasuhiko SHIMADA and Tatsuya MORITA

Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529

ABSTRACT
We investigated the effect of dietary protein on lipopolysaccharide (LPS) +Dgalactosamine (GalN)-induced hepatitis by measuring plasma alanine aminotransferase and aspartate aminotransferase activities as hepatitis markers in rats. When rats were fed diets containing 25% casein or an equivalent amount of other proteins (purified whole egg protein, soy protein isolate (SPI) or wheat gluten) for 10 days and then injected with LPS+GalN, hepatitis was most suppressed in rats fed an SPI diet. The hepatitis-suppressive effect of SPI could not solely be ascribed to impurities included in SPI. When L-Methionine was added to the SPI diet to make it comparable to that of the casein diet, hepatitis was significantly intensified, suggesting that the hepatitis-suppressing effect of SPI might be attributable, at least in part, to the low methionine content of the protein. LPS is thought to induce apoptosis of liver cells through the action of TNF-α, which is produced by macrophages in response to LPS, and leading to hepatitis. Thus, we investigated the effects of an SPI diet on plasma TNF-α concentration and apoptosis of liver cells. The SPI diet did not suppress LPS+GalN-induced enhancement of plasma TNF-α concentration as compared with the casein diet, but it significantly suppressed apoptosis of liver cells. On the other hand, the SPI diet significantly suppressed TNF-α+GalN-induced hepatitis compared to the casein diet, suggesting that SPI might elicit its protective effect against LPS+GalN-induced hepatitis by suppressing the action of TNF-α. Our results suggest that SPI might be effective in preventing certain types of hepatitis that are mediated by TNF-α.
Soy Protein Research, Japan 6, 67-72, 2003.

Key words : lipopolysaccharide, hepatitis, soy protein, tumor necrosis factor-α
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24-14

Production of Hypotensive Peptide, SVY, from 7S Globulin of Soybean Protein and Its Physiological Functions

Toshiro MATSUI

Faculty of Agriculture, Kyushu University Fukuoka 812-8581

ABSTRACT
To clarify the latent hypotensive effect of soybean proteins, we measured production of angiotensin I-converting enzyme (ACE) inhibitors from 7S globulin, and characterized peptides responsible for an in vivo depressor effect. Soybean protein was hydrolyzed by pepsin, chymotrypsin, and trypsin, and the prepared hydrolysate (F-1) with a peptide length of 7.2 showed an ACE inhibitory activity with an IC50 value of 0.286 mg/mL. The F-1 also contained 44.2% di- & tri-peptides (IC50: 0.358 mg/mL), which could be absorbed intact through the intestinal tract. From the di-/tri- peptide fraction, Val-Tyr (VY), Ile-Val-Tyr (IVY), and Ser-Val-Tyr (SVY) (IC50: 1.7 mM) were isolated and identified. A single oral administration rat study of the fraction revealed that it had an ability to lower blood pressure, in which a 0.3 g/kg dose in an 18 wk old spontaneously hypertensive rat resulted in a significant reduction of systolic blood pressure of 19.7 mmHg at 6 h. Useful information on the hypotensive effect of soybean proteins was obtained by an intestinal membrane transporting study using the Ussing system. Results from the rat small intestinal transport experiment indicate that F-1 and 18 di- and tri-peptides, including SVY and IVY with ACE inhibitory activity, were absorbed intact through the membrane. Phe-Ala (FA) was the most absorbable di-peptide with a permeation rate of 164.9 nmol/cm2/30 min/mL.
Soy Protein Research, Japan 6, 73-77, 2003.

Key words : ACE inhibitory peptide, hypertension, soybean, gastrointestinal protease, absorption
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24-15

Comprehensive Evaluation of Soy Protein Digests as Sources of Bioactive Peptides

Masaaki YOSHIKAWA, Yasuyuki TAKENAKA, Takahiro TSURUKI, Kunihiko ONISHI, Naomi DOYAMA, Nobuyuki MARUYAMA and Shigeru UTSUMI

Graduate School of Agriculture, Kyoto University, Uji 611-0011

ABSTRACT
Yield of soymetide-13 (MITLAIPVNKPGR), an immunostimulating peptide, from a recombinant β-conglycinin α' subunit after trypsin digestion was 15%. Yield of soymetide-13 from a recombinant β-conglycinin α' subunit fortified with 4 mol/mol of RPLKPW, a potent hypotensive peptide, by site-directed mutagenesis was 17%. Yields of soymetide-13 and DPR, a hypocholesterolemic/bile-secreting peptide, from a recombinant β-conglycinin α' subunit fortified with 3 mol/mol of DPR were 14% and 40%, respectively. Thus, it was concluded that RPLKPW and DPR sequences introduced into the β-conglycinin α' subunit did not interfere with release of soymetide-13 by the action of trypsin.
Soy Protein Research, Japan 6, 78-82, 2003.

Key words : β-conglycinin, immunostimulating peptide, site-directed mutagenesis, blood pressure, cholesterol, trypsin
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24-16

Effect of Water-Soluble Soybean Peptide Fractions on Liver and Serum Triglyceride Concentrations in Rats

Takayuki KURAYAMA, Masanobu SAKONO and Nobuhiro FUKUDA

Faculty of Agriculture, Miyazaki University, Miyazaki 889-2192

ABSTRACT
Effects of various peptides prepared by enzymatic hydrolysis of soy protein isolate (SPI) on liver and serum triglyceride (TG) were compared with those of SPI alone or casein in Sprague-Dawley rats fed diets with and without cholesterol. In experiment 1, the liver triglyceride-lowering effect of SPI, HD1 (a low molecular peptide fraction with residual high molecular peptides), and LD3 (a low molecular peptide fraction) was evident, compared to casein. In addition, SPI and LD3 exhibited a potent hypotriglyceridemic effect in rats fed diets with and without cholesterol; the extent of this lowering effect being greater in LD3 than in SPI, suggesting the effectiveness of LD3 in lowering serum and liver triglyceride concentrations. In experiment 2 with no dietary cholesterol, the effect of increasing amounts of LD3 on liver and serum triglyceride was examined, and we found that there was a dose-dependent reduction in the concentrations of triglyceride with increasing amounts of LD3 in the diet. In experiment 3, also with no added cholesterol in the diet, other fractions such as HMF (a high molecular peptide fraction in HD1) and D5 (an acid-soluble low molecular peptide fraction) were compared with SPI, HD1 and LD3; the D5 fraction exhibited a potent serum and liver cholesterol lowering activity; the extent of reduction appeared to be greater in D3 than in LD3. HMF had no lowering effect. In experiment 4, the effect of F5 (a highly water soluble low molecular peptide) was examined in rats fed diets free of cholesterol, and we found that there is a greater reduction in the concentration of serum and liver triglycerides compared to SPI and LD3. The results of these four experiments suggest that the water-soluble low molecular fraction prepared from SPI is a useful dietary component to prevent and/or ameliorate hypertriglyceridemia and fatty liver.
Soy Protein Research, Japan 6, 83-87, 2003.

Key words : soy protein, soy peptide, triglyceride, water soluble, low molecular
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24-17

Isolation and Application of Physiologically Active Peptides from Soybean Whey and Okara Proteins

Masami YONEKURA and Ayako TANAKA

School of Agriculture, Ibaraki University, Ibaraki 300-0393

ABSTRACT
Peptides with inhibitory effects on angiotensin I-converting enzyme (ACE) were isolated from thermolysin digests of soybean whey proteins; basic 7S globulin; and the "Hinute DC5" soybean peptide preparation. The amino acid sequence of the inhibitory factor from the whey protein digest was Leu-Ala-Pro and the IC50 value of the peptide for ACE from rabbit lung was 0.13μM. Isolation of the inhibitory peptide from the basic 7S globulin digest was attempted, however the sequence of the peptides has not been clarified. Thirteen kinds of ACE inhibitory peptides from "Hinute DC5" were isolated. Among these peptides, dipeptides such as Lys-Tyr and Ala-Phe showed comparatively high inhibitory activity for ACE. and their IC50 values were 25μM and 97μM, respectively. Our results suggest that the thermolysin digests of soybean whey proteins, basic 7S globulin and "Hinute DC5" may be suitable materials for the development of a functional food with antihypertensive activity.
Soy Protein Research, Japan 6, 88-93, 2003.

Key words : soybean, peptide, angiotensin I-converting enzyme inhibitor, whey protein, basic 7S globulin
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24-18

Identification of Amino Acid Sequence Responsible for the Satiety Effect of Soybean β-Conglycinin

Hiroshi HARA and Takashi NISHI

Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589

ABSTRACT
Peptides derived from dietary protein promote cholecystokinin (CCK) secretion via a putative receptor on the mucosal cells of the small intestine, and soybean β- conglycinin peptone suppresses food intake in rats via CCK secretion. The aim of the present study was to identify the amino acid sequence (peptide fragment) responsible for CCK secretion. We found that the β-conglycinin β-subunit 51-63 (β51-63, VRIRLLQRFNKRS) has the strongest binding activity to the brush-border membrane proteins of the rat jejunum. Duodenal infusion of the synthetic peptide β51-63 reduced food intake for 1 hr (satiety effect), which was completely abolished by devazepide, a CCKA-receptor antagonist. Also, duodenal infusion of β51-63 induced marked increase in portal plasma CCK concentration. We conclude that soybean β- conglycinin includes the sequence that strongly stimulates CCK secretion by binding to the brush-border membrane of the small intestine.
Soy Protein Research, Japan 6, 94-99, 2003.

Key words : soybean b-conglycinin, satiety effect, cholecystokinin, brush-border membrane, rats
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24-19

Regeneration of Neural Circuit: Role of Soy-Protein

Tatsuhiro HISATSUNE

Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562

ABSTRACT
We studied the effect of dietary soy protein on brain circuit regeneration in nestinenhanced green fluorescent protein (nestin-GFP) mice. The mice were fed either a control (MF) or a soy protein-rich (SPR) diet for 14 days, and were supplied with bromodeoxyuridine (BrdU) during the last 10 days. The mice were killed and their brains dissected. Cerebral slices (40 micron) were cut by cryostat, and each slice was stained with both anti-GFP and anti-BrdU. The numbers of nestin-positive cells per 1 mm3 in the cerebral cortices of the MF and SP groups were 503 ± 70 and 569 ± 90, respectively. In addition, the frequencies for nestin-positive dividing cells per total dividing cells were 50% and 66% in the MF and SP groups, respectively. These results suggest that soy protein promotes the generation of neural precursor cells in the cerebral neocortex, thought to be the center of intelligence, and enhances the regeneration of neural circuits in the murine cerebral cortex.
Soy Protein Research, Japan 6, 100-103, 2003.

Key words : soyprotein, regeneration, neural circuit, cerebral cortex
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24-20

Immunological Modulative Activity of Soybean Saponins: Suppressive Effect of Soybean Saponins on High Affinity IgE Receptor Expression

Hirofumi TACHIBANA, Yoshinori FUJIMURA, Satomi YANO and Koji YAMADA

Faculty of Agriculture, Kyushu University, Fukuoka 812-8581

ABSTRACT
We examined the effect of soybean saponins on the cell surface expression of high affinity IgE receptor (FcεRI) in human basophilic KU812 cells. Flow cytometric analysis showed that soybean saponin decreased the cell surface expression of FcεRI. FcεRI is a tetrameric structure comprising of one α chain, one β chain, and two γ chains. The level of mRNA of each subunit in KU812 cells was not reduced by treatment with the saponin. Furthermore, immunoblot analysis revealed that total cellular expression of the FcεRI α and γ chains were not affected by treatment with the saponin. However, the saponin treatment inhibited the FcεRI cross-linking-induced histamine release. The cross-linkage of FcεRI causes the activation of basophils, which leads to the secretion of inflammatory mediators including histamine. These results suggest that soybean saponins can negatively regulate basophil activation through the suppression of FcεRI expression.
Soy Protein Research, Japan 6, 104-107, 2003.

Key words : soyasaponin, FcεRI, histamine, basophils
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24-21

Function of Lipid Metabolism and Antioxidative Activity of Dietary Soybean Isoflavone Aglycone: Differences in Regulative Function between Male and Female Rats

Kyoichi OSADA, Yuki KAWAKAMI and Tomoko KIYOSAWA

Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561

ABSTRACT
The regulative effects of aglycone (a soy isoflavone) on lipid metabolism and antioxidative function in male vs. female rats were examined in young and old Sprague-Dawley rats. Immature (4 wk) and mature (8 mo) male and female rats were fed either a diet containing a 0.1% isoflavone-aglycone rich powder or an isoflavonefree diet for 24 days. Hepatic antioxidative activities were higher in rats fed isoflavone vs. isoflavone-free diets in both sexes. Feeding isoflavones resulted in lower levels of lipid peroxide in immature male rats fed isoflavones, and decreased liver and serum peroxide concentrations in mature rats of both sexes. Liver and serum triglyceride concentrations were lower in all rats fed isoflavones, especially in the mature females. Tissue cholesterol concentrations were also lower in all rats fed isoflavones. Thus, dietary aglycone may enhance the biological antioxidative system when fed over the long-term, regardless of age or sex. Based on our observations, the regulation of lipid metabolism by dietary isoflavone may be more effective in female than in male rats. The residence time of isoflavone-metabolites derived from dietary soy isoflavones may be a factor in our observations.
Soy Protein Research, Japan 6, 108-115, 2003.

Key words : isoflavone, aglycon, lipid metabolism, antioxidation, sex, rat
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24-22

Study of Daidzein on Bone Metabolism: Individual Variability in Aging Rats

Hidemichi EBISAWA1, 2 and Yasuko KOSHIHARA1

1Bone research group, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015
2
School of Human Life Sciences, Showa Women's University, Tokyo 154-8533

ABSTRACT
Soy isoflavones have received increasing attention because of their potential role in the prevention of osteoporosis. However, we previously observed no response in bone mineral density (BMD) in some aging rats fed isoflavones. Thus, in the present study, we investigated individual variability in bone metabolism of rats in response to dietary daidzein. Ovariectomized (OVX) 4-mo and 24-mo old rats were fed 13 g of a diet supplemented with no (OVX-CA), 0.44% (OVX-DL) or 0.88% (OVX-DH) daidzein for 12 wk. Rats fed OVX-DH had lower femoral BMD and higher bone metabolic markers than OVX-CA fed rats, although wide individual variation in BMD was observed in the 24-mo old rats. ER-alpha, ER-beta, HMG-CoA reductase, BMP-2 and FT-alpha mRNA expressions in the femurs of the 24-mo old rats were measured by the RT-PCR method. Lower mRNA expression of HMG-CoA reductase was observed in the OVX-DH group compared to the OVX-CA group. When comparing characteristics of the 24-mo old rats fed OVX-DH, we found that rats with the highest BMD (BMD-H) had lower mRNA expression for HMG-CoA reductase and ER-alpha than rats with the lowest BMD (BMD-L). This suggests that dietary daidzein may inhibit HMG-CoA reductase mRNA expression. HMG-CoA reductase and ER-alpha expressions may partially explain the individual variability in response to dietary daidzein on bone metabolism.
Soy Protein Research, Japan 6, 116-121, 2003.

Key words : daidzein, bone metabolism, gene expression, ovariectomized rat
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24-23

Effects of Soybean Isoflavones on the Proliferation and Invasion of Hepatoma Cells

Kazumi YAGASAKI, Maki TAKABATAKE and Yutaka MIURA

Department of Applied Biological Science, Tokyo Noko University, Tokyo 183-8509

ABSTRACT
Effects of soybean isoflavones, genistein and daidzein, on the proliferation and invasion of a rat ascites hepatoma cell line of AH109A were studied in vitro and ex vivo. Genistein and daidzein dose-dependently suppressed the proliferation and invasion of AH109A in culture, although the suppressive effect of genistein was more prominent than that of daidzein. Sera from rats orally given genistein (5 mg/100 g body weight) 2-3 hours before blood collection most strongly suppressed the proliferation and invasion of hepatoma cells. Genistein resulted in cell cycle arrest at the G2/M phase in AH109A cells, suggesting cell cycle arrest may be one mechanism for the inhibition of AH109A proliferation by genistein. Hepatoma cells pre-cultured with hypoxanthine (HX) and xanthine oxidase (XO) showed a highly invasive capacity. Genistein suppressed the reactive oxygen species (ROS)-potentiated invasive capacity by simultaneously treating AH109A cells with HX, XO and genistein, suggesting that the antioxidative property of genistein may be involved in its anti-invasive action.
Soy Protein Research, Japan 6, 122-127, 2003.

Key words : soybean isoflavones, genistein, hepatoma, proliferation, invasion
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24-24

Osteogenic Effect of Genistein on Cultured Bone Marrow Cells

Noriko OKUMURA1, Takafumi YOSHIKAWA*1, Jin IIDA2, Kunio ICHIJIMA1and Yoshinori TAKAKURA2

Departments of Pathology1 and Orthopaedic Surgery2, Nara Medical University, Nara 634-8521

ABSTRACT
The effect of genistein, a soybean isoflavone, on new bone formation by bone marrow cells from mature rats and humans was examined. Bone marrow cells were collected from the femoral diaphysis of 7-week-old Fisher rats, cultured in MEM containing fetal calf serum and then cultured with or without the addition of dexamethasone to the bone-forming medium. Genistein was added at concentrations of 10-5, 10-6, 10-7 or 10-8 M. Bone formation was examined 2 weeks after culture. After informed consent was obtained from a 55-year-old woman with lumbar spondylosis deformans, bone marrow cells were collected from her ilium for culture by the same process, and bone formation investigated. In both rats and humans, when dexamethasone was added to the bone-forming medium, genistein (10-7 M and 10-8 M) caused a significant increase in the levels of calcium, alkaline phosphatase, and DNA compared with cells not cultured in genistein. In conclusion, genistein was found to promote bone formation at physiological concentrations across species, and thus may be useful as a bone formation-promoting factor.
Soy Protein Research, Japan 6, 128-134, 2003.

Key words : genistein, osteogenesis, marrow, dexamethasone
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24-25

Effect of Soy Isoflavones on Premenstrual Syndrome

Naoko ISHIWATA*1, Sayo UESUGI2 and Mariko UEHARA2

1Department of Domestic Science, Atomi Junior College, Tokyo 112-8687
2
Faculty of Applied Bio-Science, Tokyo University of Agriculture, Tokyo 156-8502

ABSTRACT
The purpose of this study was to evaluate the efficacy of soybean isoflavones (IF) in reducing of premenstrual syndrome (PMS). Two hundred forty-two young women aged 18-21 yrs, voluntarily participated in the intervention study. Premenstrual symptoms affected daily life activities in 86.0% of all subjects. Major symptoms were sleepiness, abdominal pain, backache, breast fullness and mental irritability. The severity of PMS was related to previous experience with dietary restriction for losing weight and menstrual pain. Fifty six women with premenstrual symptoms were identified by questionnaire and participated in the randomized, double-blind, crossover intervention trial with IF and placebo for 8-menstrual cycles. Physical symptoms of PMS were significantly reduced in the 40 mg IF/day group compared with the placebo group ( P< 0.05). Headache was reduced the most. Psychological symptoms were not improved as much as physical symptoms. Individual personality influenced the severity of premenstrual symptoms. Significant associations were observed between openness and mental irritability; agreeableness and acne and psychological symptoms; and conscientiousness and headache (all P< 0.05). Our results suggest IF supplements may be an effective treatment for the reduction of physical symptoms of PMS.
Soy Protein Research, Japan 6, 135-139, 2003.

Key words : premenstrual syndrome, soybean isoflavones randomized placebo-controlled trials, personality
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24-26

Effects of Soy and Soy Products on Bone Mineral Density, Bone Composition and Fatigue in Japanese Collegiate American Football Players

Takako HIROTA1, Kaori KOSIYAMA1, Naoko IMAI1, Tomoko AOE1 and Hirosuke KATAOKA2

1Research Laboratory, Tsuji Academy of Nutrition, Osaka 530-0021
2Osaka University Graduate School of Dental Medicine, Osaka 565-0871

ABSTRACT
The effects of daily intake of soy and soy products on bone mineral density, body composition and self-reported fatigue after 16 weeks of physical training were examined in Japanese collegiate American football players. Forty-six football players aged 19-25 years in a national university and 105 male students aged 18-27 years in a nutrition academy (as controls) were recruited. Additionally, football players were sub-divided into "almond" and "no supplement" groups, where the "almond" subjects were supplemented with 60 g of almonds per day during the 16 weeks of training prior to the fatigue testing. Almonds were selected because of their rich vitamin E content. Bone mineral densities (BMD) and body composition were measured by dual energy X-ray absorptiometry (DEX). Self-reported fatigue was measured through Profile of Mood State (POMS) and "subjective fatigue symptoms" questionnaires established by the Japan Society for Occupational Health. BMDs of total body, Lumbar Spine (L2-L4) and proximal femur were significantly higher in the football players than in the control group. BMDs of the proximal femurs of the football players were significantly higher in those with high (≥3 times/wk) intakes of soy and soy products than in those with low intakes. "Subjective fatigue symptoms" during 16 wk of physical training were significantly lower in the football players with high (≥3 times/wk) intakes of soy and soy products who were supplemented with almonds, compared to unsupplemented players. Our data suggest that higher intakes of soy and soy products may promote higher accumulation of bone mineral density and, with almond consumption, may decrease fatigue after heavy training in collegiate football players.
Soy Protein Research, Japan 6, 140-146, 2003.

Key words : bone mineral density, vitamin E, fatigue, soy and soy products, almond, antioxidants
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24-27

Modulating Effects of Soy Protein Isolate and Soy Protein Hydrolysate on Human Brain Function

Eiko HATAKEYAMA1, Masato YAMAGUCHI1, Koji MURAMOTO2, Go ITO2, Yutaka MOTOHASHI3 and Shigekazu HIGUCHI3

1Kansei Fukushi Research Center, Tohoku Fukushi University, Sendai 981-8522
2
Graduate School of Life Science, Tohoku University, Sendai 981-8555
3
Akita University School of Medicine, Akita 010-8543

ABSTRACT
We evaluated modulating effects of soy protein isolate and soy protein hydrolysate on human brain functions by cerebral blood flow and electroencephalography, both noninvasive measurements. Oxy-Hb concentration and cerebral blood flow at the forehead increased significantly while performing assigned tasks and decreased while listening to music when the subjects ingested soy protein and peptides. In the electroencephalogram, a significant increase in amplitude of all the frequency bands was observed in the peptide group. Specifically, there were significant increases in the amplitudes of theta, alpha-2 and beta-1 frequency bands after the ingestion of peptides ( P<0.05). These results suggest ingestion of soy protein and peptides has effects on brain activity. Additionally, salivary cortisol, a stress hormone, was significantly lower after performing the tasks in the soy protein ( P<0.01) and peptide ( P<0.05) groups compared with the placebo group. This suggests soy protein and peptides may decrease stress. Further research is required to clarify these points as well as to identify the active components contained in soy proteins.
Soy Protein Research, Japan 6, 147-152, 2003.

Key words : soy protein and peptide, brain function, task, cerebral blood flow, electroencephalogram
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