Soy Protein Research(Japan)

23-1

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, Suita 565-0871

ABSTRACT
The metabolic syndrome, which clusters insulin resistance, dyslipidemia, and hypertension in each individual is a common basis of type 2 diabetes and atherosclerotic vascular diseases. Accumulation of intra-abdominal visceral adipose tissue triggers the metabolic syndrome. Recent researches have explored adipose tissue is not solely an energy storing tissue but secretes a variety of bioactive substances called adipocytokines. This study was designed to elucidate the role of adipose-expressed genes in the development of the metabolic syndrome, and the significance of soybean protein, which is a major component of classical Japanese diet, on the regulation of the genes in adipocyte. Through the extensive analysis of adipose-expressed genes, we isolated several adipose-specific genes, galectin-12, adiponectin, and aquaporin adipose. Forced expression of galectin-12 induced cellular apoptosis. Although the significance of apoptosis in adipose tissue remains unclear, galectin-12 may work to determine the cell size and the capacity of buffer action against nutritional overload. Adiponectin is a plasma protein having insulin sensitizing activity and modulating vascular functions, but plasma adiponectin levels were decreased in the subjects with body fat accumulation. Promoter analysis revealed that TNF-a decreased and PPARg-ligands increased the expression of adiponectin. Aquaporin adipose, a channel forming integral protein, works as a glycerol channel in adipocyte. The expression of aquaporin adipose decreased in the feeding state, in which glycerol release was suppressed. This suppression was achieved by insulin responsive element in its promoter. In the obese animals, the expression of aquaporin adipose was augmented leading to the enhanced glycerol release from adipocytes. Diet therapy with soybean protein in obese mice reduced body fat and increased plasma adiponectin, resulting in the improvement of glucose tolerance. These results suggested that dysregulation of adipose-specific genes relates to the pathogenesis of the metabolic syndrome. soybean protein may improve the dysregulation and prevent the metabolic syndrome.
Soy Protein Research, Japan 5 1-9, 2002.

Key words : soybean protein, adipocytokine, visceral fat, metabolic syndrome
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23-2

Analysis of the Molecular Mechanisms of Genetic Variations
in Seed Storage Proteins in Soybean

Michiko YOSHINO1, Ken-ichi TSUTSUMI2, Yoshiya SHIMAMOTO1and Akira KANAZAWA1

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

ABSTRACT
The 7S globulin (b-conglycinin), a major component of seed storage proteins in soybean, comprises three subunits, a, a' and b. In order to clarify the molecular mechanisms of gene expression of seed storage proteins, changes in the pattern of DNA-protein interaction during the process of seed maturation were analyzed. We examined the protein-binding on the Box I element, which is located in the upstream of the gene encoding a subunit and is considered to be involved in the transcriptional regulation of the a subunit gene. An increase in the amount of DNA-protein complex was detected using nuclear proteins isolated from seeds with increasing period of seed-maturation (27, 34, and 42 days after flowering) by a gel-shift assay. ince the transcriptional activity of the a subunit gene increased with an increasing period of seed-maturation, these Box I binding proteins were considered to be involved in positive regulation of transcription. Box I-binding proteins that were different from those exist in the seeds were detected in the nuclear extracts from leaves. In addition, CG-2, a gene located in the vicinity of the a subunit gene in a soybean cultivar 'Forrest', was found to share high sequence-homology with the a subunit gene. Deletion of the CG-2 was detected in the genome of a cultivar 'Keburi' in addition to the deletion of a' subunit gene.
Soy Protein Research, Japan 5 10-16, 2002.

Key words : soybean, b-conglycinin, a subunit, DNA-protein interaction, deletion
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23-3

Metabolism of the Precursor of Gly m Bd 28K, a Major Soybean Allergen in Growing Soybean Seeds

Hideaki TSUJI1, Miki HIEMORI1, Masumi KIMOTO1 and Shigeru UTSUMI2

1Faculty of Health and Welfare Science, Okayama Prefectural University, Soja 719-1197
2Graduate School of Agriculture, Kyoto University, Uji 611-0011

ABSTRACT
Gly m Bd 28K is biosynthesized as a preproprotein, which is suggested to be metabolized to form Gly m Bd 28K and its C-terminal peptide (23 kDa peptide) during the growth of soybean seeds. In the present study, Gly m Bd 28K and the tentative 23 kDa peptide were examined in order to elucidate the metabolism of the precursor of Gly m Bd 28K in growing soybean seeds. Monoclonal antibodies (mAbs) recognizing the 23 kDa peptide were prepared. The 23 kDa peptide was detected using an immunoblotting technique with one of the mAbs. N-terminal amino acid sequence of the peptide was shown to be KDTAGSPASYNLYDDKADF. This result shows that the 23 kDa peptide corresponds to sequence 267-473 on the amino acid sequence of the precursor of Gly m Bd 28K. Furthermore, Gly m Bd 28K and the 23 kDa peptide were shown to be localized in the protein bodies in growing soybean seeds by gold colloidal method with the mAb.
Soy Protein Research, Japan 5 17-20, 2002.

Key words : soybean allergen, Gly m Bd 28K, preproprotein, allergenicity
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23-4

Elucidation of Structure- physicochemical Function Relationships at Subunit Level of Soybean Glycinin by Using Mutant Cultivars

Nobuyuki MARUYAMA and Shigeru UTSUMI

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

ABSTRACT
We purified four mutant glycinins composed of only group I subunits (group I), only group II subunits (group II), only A3B4 subunit (A3B4) and only A5A4B3 subunit (A5A4B3) from mutant soybean cultivars, and compared their structural features and physicochemical functions with those of the wild glycinin (11S) from var. Shirotsurunoko. Thermal stabilities of groups I and II were similar to each other, although that of A3B4 was significantly lower than the others. The order of surface hydrophobicities determined by hydrophobic column chromatography was group I < 11S < A5A4B3 < group II < A3B4. Solubility of 11S was an arithmetic mean of those of groups I and II at low ionic strength (0.08), but that was determined by group I at high ionic strength (m=0.5). Solubility of group II was determined by A5A4B3 at both ionic strengths. The order of emulsifying abilities was group I <11S < A3B4 < group II < A5A4B3. This order did not correlate with those of thermal stabilities and surface hydrophobicities of glycinin species.
Soy Protein Research, Japan 5 21-25, 2002.

Key words : glycinin, emulsifying ability, surface hydrophobicity, solubility, subunit species, soybean
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23-5

Study on Introducing New Physiological Function into Soy Protein by Genetic Engineering

Masaaki YOSHIKAWA, Yuko YAMADA, Nobuyuki MATOBA, Shigeru UTSUMI, Nobuyuki MARUYAMA and Kunihiko ONISHI

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

ABSTRACT
RPLKPW is a highly potent anti-hypertensive peptide obtained by replacing four amino acid residues in ovokinin (2-7) (RADHPF) that is a vasorelaxing peptide isolated from a chymotryptic digest of ovalbumin. n previous study, RPLKPW sequence was introduced into three homologous sites in soybean b-conglycinin a' subunit by site-directed mutagenesis and the mutated a' subunit expressed in Escherichia coli exerted an anti-hypertensive effect in spontaneously hypertensive rats at a dose of 10mg/kg (per os). In this study, we found that RPLKPW was released from only one site among three RPLKPW-sites by in vitro digestion experiment with model peptides around RPLKPW-sites of modified a' subunit. In order to release RPLKPW efficiently, we optimized amino residues around individual RPLKPW-sites. The yields of RPLKPW from 3 RPLKPW-sites were increased markedly. Furthermore, we introduced fourth RPLKPW sequence to the a' subunit. The minimum effective dose of a' subunit containing four RPLKPW was 2.5mg/kg, about one-fourth of that of modified a' subunit that we previously reported. The anti-hypertensive activity of a' subunit containing four RPLKPW was larger than ovalbumin by about eight hundred times.
Soy Protein Research, Japan 5 26-30, 2002.

Key words : ovalbumin, anti-hypertensive peptide, soybean b-conglycinin, spontaneously hypertensive rat, site-directed mutagenesis
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23-6

Salt-tolerant g-Glutamyltranspeptidase: To Utilize Soy Protein as a Source of Umami Taste

Hideyuki SUZUKI

Graduate School of Biostudies, Kyoto University, Kyoto 606-8502

ABSTRACT
g-Glutamyltranspeptidase (GGT) from Bacillus subtilis is an extracellular enzyme that exhibits glutaminase activity. As it is only produced during the mid-stationary phase, a strain overexpressing GGT was generated to obtain sufficient quantities of GGT. The level of GGT activity of this strain increased steadily after the exponential phase, becoming 13-fold higher than that in the parental strain. The recombinant GGT was purified by 107-fold. The enzyme was a heterodimer consisting of one large subunit (45 kDa) and one small subunit (21 kDa). The enzyme was highly salt-tolerant and converted glutamine to glutamic acid effectively even in the presence of 18% NaCl.g
Soy Protein Research, Japan 5 31-35, 2002.

Key words : g-glutamyltranspeptidase, Bacillus subtilis, glutaminase, taste, umami
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23-7

Analysis for Mechanism of Soybean- milk- coagulation by Bacterial Protease and Utilization of the Enzyme to the Food Processing

Masaaki YASUDA1, Megumi KUBA1, Shinjiro TACHIBANA1 and Misako AOYAMA2

1Faculty of Agriculture, University of the Ryukyus, Okinawa 903-0213
2Faculty of Nursing and Nutrition, Siebold University of Nagasaki, Nagasaki 851-2195

ABSTRACT
The coagulation of protein in soybean milk by serine proteinase from Bacillus pumilus and utilization of the enzyme to the food processing were examined. The ratios of precipitated and 4% TCA soluble nitrogen to the total nitrogen increased with increasing reaction time. In order to elucidate the aspect of protein-degradation in soybean milk by this enzyme, slab-SDS-PAGE was carried out. The a'-, a-and b-subunits in b-conglycinin were degraded gradually. The acidic subunit in glycinin was degraded at an initial stage (0.5 min) and was completely degraded by the time the coagulation began (3 min). However, the basic subunit in glycinin still remained even after 30 min. When the enzyme reacted with the denatured glycinin, precipitation in the reaction mixture occurred, while this phenomenon was not observed in the case of denatured b-conglycinin. Therefore, it was considered that degradation of glycinin played a key part in the coagulation of protein in soybean milk. The amount of detectable sulfhydryl group in soybean milk reached the maximum just before the coagulation. The coagula obtained were completely dissolved in the presence of 0.5% SDS solution without 2-mercaptoethanol. Therefore, it is suggested that hydrophobic interaction played an important role in the coagulation process. Soybean protein curd that was prepared by this enzyme with calcium sulfate was firm, and it is expected to be useful for production of tofuyo.
Soy Protein Research, Japan 5 36-40, 2002.

Key words : serine proteinase, Bacillus pumilus, soybean-milk-coagulation, soybean protein curd, tofuyo
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23-8

Removal of Phytic Acid from Soy Protein Isolates and Its Effect on Protein Functionality

Miki KATOH, Dong-Hao JIN, Takako NAGANUMA, Tomohisa OGAWA and Koji MURAMOTO

Graduate School of Life Sciences, Tohoku University, Sendai 981-8555

ABSTRACT
Soy protein isolates contain about 2% of phytic acid, which is considered as an antinutrient due to its inhibitory effect on mineral bioavailability. A simple acid extraction method was developed to reduce phytate content. By extracting with 0.6 M HCl, 96% of phytic acid could be removed. The acid extraction significantly changed the gel filtration patterns of soy proteins, however, there was no change in their subunit compositions on SDS-PAGE. The digestibility of soy proteins with pepsin not with trypsin improved upon the extraction. The increase of the surface hydrophobicity of phytate-removed soy proteins resulted in the increase of its emulsifying activity. Moreover, the hydrolysates derived from phytate-removed soy proteins accelerated the calcium transport across the Caco-2 cell monolayer.
Soy Protein Research, Japan 5 41-46, 2002.

Key words : soybean, soy protein, phytic acid, calcium, Caco-2 cell
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23-9

How Does the Radical-scavenging Activity of Soy Protein Food Change during Heating?

Teruyoshi MATOBA

Graduate School of Human Culture, Nara Women's University, Nara 630-8506

ABSTRACT
Soybean contains radical-scavenging activity, which prevents life style-related diseases. Therefore, soy protein foods are the key foods to maintain human health. In this paper, the changes in the radical-scavenging activity of soy protein foods during heating and digestion were determined. Three soy protein fractions (7S fraction, 11S fraction and lipid-including proteins) were fractionated from defatted soybean. These fractions and defatted soybean contain high radical-scavenging activity. The activity did not change during heating. However, enzyme digestion increased the radical-scavenging activity 3-5 times. The activity after digestion was almost same level of those in vegetables in dry weight basis. The isoflavone content did not change during heating or digestion. These results indicate that the increase in the radical-scavenging activity of soy proteins is due to the peptides formed in digestion.
Soy Protein Research, Japan 5 47-50, 2002.

Key words : soybean, soy protein, radical-scavenging activity, isoflavone, peptide
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23-10

Development of a Novel Foam Food Made from Wheat Flour and Soy Protein Isolate

Keiko FUJII

Department of Food Science and Nutrition, Japan Women's University, Tokyo 112-8681

ABSTRACT
The sponge cake was made with rice flour and soy protein isolate (SPI) as main components. In the case of no added SPI, the moisture sorption isotherms of sponge cakes were not affected by aids for foaming. When SPI was added, the equilibrium moisture contents of the cake were affected by the properties of aids owing to the greater moisture sorption isotherm of SPI. The cake made with silk fibroin showed the smallest equilibrium moisture content. It was thought that it reflected the hydrophobicity of silk fibroin. Drying rate of cake with silk fibroin was the smallest. These results suggest that fibroin may form the network to prevent moving of water molecules in the cake. The silk fibroin used was obtained from waste yarn and showed very interesting characters as described above. It may be acceptable as foodstuff.
Soy Protein Research, Japan 5 51-57, 2002.

Key words : sponge cake, rice flour, soy protein isolate, silk fibroin, moisture sorption isotherm
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23-11

A Crucial Role of Soybean Resistant Protein in Increased Fecal Steroid Excretion and Structural Peculiarity of Caught Bile Acids

Kimikazu IWAMI, Naoto FUJII, Tomohiko SUZUKA and Ryuhei KANAMOTO

Graduate School of Agriculture, Kyoto Prefectural University, Kyoto 606-8522

ABSTRACT
Growing male Wistar rats were divided into three groups (n =6), which were fed the respective SPI (+7%cellulose), HMF (no cellulose) and SPI+RP diets with supplementary 0.5% cholesterol over a period of 4 weeks. In the meantime, the rats were examined for fecal acidic or neutral steroid excretion and serum total cholesterol concentration at weekly intervals and for hepatic cholesterol content at the 4th week. There were no significant variations in serum total cholesterol concentration among these three groups during the period of feeding, but the difference in fecal acidic or neutral steroid excretion was significant between both SPI and HMF (or SPI+RP) groups, being inversely proportional to hepatic cholesterol content. These observations support the possibility of RP bringing about improvement in cholesterol level within the body. In this connection, the stereoselectivity of RP for a variety of bile acids was further inspected by equilibrium dialysis. Consequently RP turned out to have a different affinity for DCA, CDCA >> HDCA > TDCA, GCDCA, UDCA, CA in their order. Noteworthily, dihydroxybile acids such as DCA and CDCA were more effectively captured by RP than their amino acid conjugates (TDCA, GCDCA) or other analogs (HDCA, UDCA). Taking into account a serious risk of secondary bile acid in practice, the RP intake favorably serves as a preventive against tumorigenesis in colon.
Soy Protein Research, Japan 5 58-62, 2002.

Key words : resistant protein (RP), SPI+RP diet, fecal steroid excretion, equilibrium dialysis, dihydroxybile acid, cancer prevention
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23-12

Correlational Analysis of Allergy and Antigenic N-Glycans Linked to Soybean Storage Glycoproteins

Yoshinobu KIMURA

Faculty of Agriculture, Okayama University, Okayama 700-8530

ABSTRACT
Recently many glycoproteinous allergens (glycoallergens) have been identified from plant food or pollens. In many cases, such glycoallergens bear plant complex type N-glycans, which have b1-2 xylose and/or a1-3 fucose and show strong antigenicity against mammals. However, correlation of intakes of such antigenic N-glycans and allergy symptoms remains to be understood. In this report, the author describes (1) structural analysis of N-glycans linked to the storage glycoprotein in soybean, showing that the plant complex type N-glycans (Man3Xyl1Fuc1-0GlcNAc2; 5%) are linked to some soybean glycoproteins, (2) structural feature of free N-glycans in soybean seedlings, showing that the plant complex type free N-glycan occurs at the concentration of about 100 pmol/g of fresh weight of hypocotyl, and (3) allergenicity of the antigenic N-glycans prepared from soybean glycoproteins from the view point of IgE-binding to the N-glycans, showing that the plant complex type N-glycans can not be major epitope for IgE in sera of pollinosis patients.
Soy Protein Research, Japan 5 63-67, 2002.

Key words : soybean storage glycoprotein, soybean seedling, antigenic N-glycan, IgE binding, inhibition ELISA
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23-13

Development of Practical Method for Screening Patients Allergic to Soybean

Tadashi OGAWA1, Tatsuya MORIYAMA1, Nobuo OTA2and Takaya FUKUI3

1Graduate School of Agriculture, Kyoto University, Uji 611-0011
2Ota Pediatrics and Allergy Clinic, Takamatsu 761-0101
3Fukuyama Medical Laboratory Co., Ltd., Hukuyama 720-0831

ABSTRACT
In previous report (Soy Protein Research, Japan 4 45-51 2001), we demonstrated that the allergic patients sensitized by plant food stuffs such as soybean have specific IgE antibodies recognizing asparagine-N linked oligo-mannose type glycan moieties of Man3GluNAc2 backbone with b1-2 xylose branch or b1-2 xylose and a1-3 fucose branches. Sera of these patients showed high cross-reactivity among various plant food stuffs by radioallergosolvent test (RAST). Accordingly, these patients often gave false positive diagnostic results about various plant food stuffs that do not elicit any allergic symptoms. In order to avoid these false positive RAST diagnoses, especially in case of soybean allergic patients, we tried to develop the improved RAST procedure using a blocking technique of glycan moieties of glyco-proteins immobilized on the allergen screening discs applied to RAST automatic analyses. The improved method demonstrated that the RAST scores of some patients allergic to plant food stuffs are markedly decreased by blocking sugar moieties of glyco-proteins. On the other hand, about half of patients examined did not show significant changes in RAST scores even though using improved method, indicating that they are true patients against tested foodstuffs and can serve as test challengers for an assessment of the hypoallergenic processed foods.
Soy Protein Research, Japan 5 68-72, 2002.

Key words : soybean allergy, food allergy, radioallergosolvent test (RAST), glycan moiety, glyco-protein, hypoallergenic soybean products
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23-14

A Study on the Functions of Dietary Protein on Growth and Differentiation of Neural Stem Cells

Kiwao NAKANO

Nagoya University Bioscience Center, Nagoya 464-8601

ABSTRACT
The effects of systemic administration of tryptophan and its metabolite, kynurenine, were evaluated on growth and differentiation of neural stem cells in the brain of mouse. Daily intraperitoneal injection of kynurenine significantly decreased the number of neural stem cells in the brain of epilepsy-prone EL mouse. In contrast, it had essentially no effect on C57BL/6, C3H/HeN, ddY nor BALB/c mouse. Addition of kynurenine caused a marked increase in the synthesis of nerve growth factor and brain-derived neurotrophic factor in the culture of astrocytes. Together, these results suggest that tryptophan metabolites, especially kynurenine, stimulate the differentiation of neural stem cells to neurons and/or glial cells in the brain of EL mouse through induction of synthesis of neurotrophic factors. These reactions may be associated, in part, with epileptogenesis of EL mouse.
Soy Protein Research, Japan 5 73-75, 2002.

Key words : neural stem cell, NGF, kynurenine, epilepsy, EL mouse
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23-15

Attenuating Effect of Soy Protein and a Semipurified Alcohol Extract of Soy Protein on Glomerular Injury in Spontaneous Hypercholesterolemic Male Imai Rats

Takanobu SAKEMI1, Yuji IKEDA1, Yoshiyuki TOMIYOSHI1and Takehiko UESUGI2

1Saga Medical School, Saga 849-8501
2Research and Development Laboratory, Fujicco Co, Kobe 650-855

ABSTRACT
Soy protein has hypercholesterolemic and estrogenic properties. Because the renoprotective effect of both lipid-lowering agents and estrogen has been reported, we investigated whether soy protein (in the experiment 1) and soy protein's alcohol-extractable components (SPEs;mainly consisting of isoflavones) (in the experiment 2) have an ability to attenuate glomerular injury in male Imai rats in which hypercholesterolemia, focal and segmental glomerulosclerosis (FSGS) and renal insufficiency occur spontaneously. In the experiment 1, soy protein diets resulted in less proteinuria, less hyperlipidemia, less hypoalbuminemia, less renal functional impairment, less glomerular hypertrophy and less renal histological damage as compared with control casein diet. In the experiment 2, male Imai rats were fed a casein-based diet with and without SPE. Group 1 (Cont) was fed standard diet without any additional SPE and groups 2 (SPE-1) and 3 (SPE-2) were fed standard diet supplemented in amounts of 0.05 and 0.10 g/100 g diet of SPE, respectively. Growth rate was not different among three groups throughout the experiment. SPE-supplemented diets resulted in less proteinuria and less hyperlipidemia. The decline in renal function with regard to the levels of blood urea nitrogen and creatinine clearance was less marked in the animals receiving the SPE-supplemented diets. Each SPE-supplemented diet equally induced less glomerular hypertrophy and less renal histological damage as compared with non-supplemented diet. These studies showed a beneficial effect of soy protein diet and a semipurified alcohol-extract of soy protein on glomerular disease.
Soy Protein Research, Japan 5 76-80, 2002.

Key Words : soy protein, glomerulosclerosis, isoflavones, male rats, estrogen
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23-16

Effect of Soy Protein Intake on High- fat Induced Obesity and Insulin Resistance

Nobuyo TSUNODA, Yuko FUJIKANE, Hyounju KIM, Midori EBATA and Shinji IKEMOTO

Faculty of Pharmaceutical Sciences, Josai University, Sakato 350-0295

ABSTRACT
High-fat diet feeding leads to obesity and insulin resistance. Recently, it was reported that sterol regulatory element-binding protein-1c (SREBP-1c) is involved in this mechanism of high-fat induced obesity and insulin resistance. SREBP-1c is an important factor regulating cholesterol and fatty acid metabolism. Some reports suggested soy protein (SPI) also has the effect of decreasing plasma cholesterol levels. Therefore, SPI may modulate the effect of high-fat on obesity and insulin resistance and/or SREBP-1c expression level. Furthermore, our previous study demonstrated levels of high-fat induced obesity and insulin resistance were different by types of oil. In this study, we investigated whether SPI intake modulates levels of obesity and insulin resistance induced by high-fat diets composed of three different oils (safflower oil, fish oil, palm oil). No significant effects are on body weight, adipose tissue weight and glucose tolerance tests by SPI intake. But insulin tolerance tests deteriorated in mice fed high-safflower oil diets contained SPI (S-SPHF) compared with mice fed high-safflower oil diets contained casein (S-HF). S-SPHF-fed mice had high plasma glucose levels compared with S-HF-fed mice while no difference of plasma insulin levels was observed. These results indicate that S-SPHF-fed mice may produce insulin resistance causing decreased glucose uptake by the peripheral tissues. Furthermore, we found that insulin resistance in S-SPHF-fed mice may be caused by decreased glucose transporter 4 (GLUT4) mRNA in white adipose tissue. But it is remained to clear which tissues (skeletal muscle or adipose tissue or liver) relate to insulin resistance.
Soy Protein Research, Japan 5 81-85, 2002.

Key words : soy protein(SPI), oils, obesity, insulin resistance
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23-17

Study on Search of Repressive Soybean Protein Components to Body Fat Accumulation

Hiroshi KAMISOYAMA1, Shin HASEGAW1and Tohru MOTOKI2

1Graduate School of Science and Technology, Kobe University, Kobe 657-8501
2Nihon Nosan Kogyo K. K., Yokohama 220-8146

ABSTRACT
As a link in the chain of the discovery of physiologically active components of soybean protein in chicks, the present study was conducted to evaluate the effects of soy protein isolate and its enzymatic hydrolysate on body fat deposition in chicks. Firstly, chicks were fed with an 18% casein, soy protein isolate (SPI) or enzymatic hydrolysate of SPI for 3 weeks, and the comparative effects of soybean proteins on growth performance, adipose tissue weight, the triglyceride contents of plasma and liver were examined. SPI and enzymatic hydrolysate of SPI significantly reduced adipose tissue weight as compared with casein. Triglyceride content of liver was significantly lower in the enzymatic hydrolysate of SPI-fed than in the casein-fed chicks. Secondary, an enzymatic hydrolysate of SPI was fractionated with ion-exchange choromatography. Using a hydrophobic resin (HP-21) column, two fractions were obtained. The unadsorbed fraction was rich in hydrophilic amino acids, whereas the fraction eluted from the column with 50% ethanol was rich in hydrophobic amino acids. Chicks were fed with an 18% SPI, an enzymatic hydrolysate of SPI, or its fractions (unadsorbed and ethanol-eluted) for 3 weeks, and the comparative effects of various soybean proteins on growth performance, adipose tissue weight, the triglyceride contents of plasma and liver were examined. The ethanol-eluted fraction significantly reduced adipose tissue weight, whereas significantly elevated the concentration of plasma triglyceride as compared with SPI. From the results obtained above, the mechanisms accounting for the body fat-lowering effect of feeding a soybean protein in chicks were discussed.
Soy Protein Research, Japan 5 86-91, 2002.

Key words : soy protein isolate, adipose tissue, plasma triglyceride concentration, liver triglyceride content, fractions of enzymatic hydrolysate of SPI
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23-18

Effect of Dietary Soybean Protein Isolate Hydrolysate on Oxidation of Dietary Carbohydrate and Fat in Diabetic Obese Mice

Kengo ISHIHARA1, Yoshiko FUKUCHI1, Wataru MIZUNOY2, Kikumi SEGAW1, Miki TAKAHASHI1, Akimori HIRAYAMA2, Yuki OKABE2, Tetsuro SHIBAKUSA2, Tohru FUSHIKI2 and Kyoden YASUMOTO1

1School of Life Studies, Sugiyama Jogakuen University, Nagoya, 464-8662
2Graduate School of Agriculture, Kyoto University, Kyoto 606-8502

ABSTRACT
The effects of a long-term feeding of soy protein isolate hydrolysate (SPI-H) and the amino acid mixture simulating SPI-H (SPI-AA) on the energy expenditure and expiration of 13CO2 from dietary 13C-glucose in a type II diabetic mice, KK-Ay strain were investigated. The mice of 6 wk old were provided free access to a high fat diet (30% of energy from fat) for the initial 4 wk to induce diabetes. The animals were divided into four groups and during the subsequent 4 wk each group of animals was fed a diet containing 35% nitrogen source (SPI-H, casein, SPI-H AA, casein AA), 10% sucrose, and 5% soybean oil on energy intake restricted regimen to 60% of that given during the preceding high fat diet. For the last 3 days on the restricted dietary regimen, the animals were housed in the respiratory metabolic chambers. For the middle 24 h of the metabolic measurement, animals in each group fed a diet that sucrose was replaced by 13C-glucose. Energy expenditure and respiratory quotient were significantly higher in the mice fed SPI-H and SPI-H AA diet than those fed casein and casein AA diet. The expiration rate of 13CO2 from 13C-glucose during the period of initial 12 h was higher in the animals fed the SPI-H diet than those fed the casein diet. The relative weights of gastrocnemius muscle were significantly higher in mice fed SPI-H diet than those fed casein diet. It is concluded that the mice on the SPI-H and SPI-H AA diet can utilize exogenous glucose more readily than those on the casein and casein AA diet, respectively. Furthermore, SPI-H diet caused higher increment in glucose utilization than SPI-H AA diet in diabetic obese mice.
Soy Protein Research, Japan 5 92-98, 2002.

Key words :KK-Ay, 13C-glucose, diabetes, soybean, energy expenditure
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23-19

Studies on Inhibitory Effect of Soy Isoflavones on Release of Allergy-related Mediators from Rat Peritoneal Exudate Cells

Mikako TAKASUGI1, Kazuko SHIMADA1 and Koji YAMADA2

1Faculty of Human Life Science, Yamaguchi Prefectural University, Yamaguchi 753-8502
2Faculty of Agriculture, Kyushu University, Fukuoka 812-8581

ABSTRACT
Effect of soy isoflavones and their derivative equol on release of allergy-related mediators from rat peritoneal exudates cells (PEC) were examined.One hundred mM of soy isoflavones and equol had no effect on histamine release from rat PEC. Though soy isoflavone glycosides, daizin and genistin did not have effect on leukotriene B4(LTB4) release from rat PEC, their aglycon, daizein and genistein inhibit LTB4 release at 100 mM. The inhibitory effect of genistein was greater than that of daizein. One hundred mM of equol inhibited LTB4 release completely, and the effect was dose-dependent.
Soy Protein Research, Japan 5 99-102,2002.

Key words : soy isoflavone, allergy, histamine, leukotriene B4
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23-20

Immunoglobulin Production-regulating Activity of Soy Isoflavones and Environmental Estrogens

Koji YAMADA1, Dal-Ho HAN1, Po-Sheng WU1, Shin YASUDA1, Michihiro SUGANO2 and Hirofumi TACHIBANA1

1Faculty of Agriculture, Kyushu University, Fukuoka 812-8581
2Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, Kumamoto 862-8502

ABSTRACT
We tried the establishment of estrogen-free culture system of mouse spleen lymphocytes and found that 7-days cultivation of the lymphocytes in phenol red-free DMEM medium supplemented with 5% charcol-treated fetal bovine serum allowed us to estimate immunoglobulin production-regulating activity of environmental estrogens. Using the system, we found that soy isoflavones such as daidzein and genistein suppressed IgE production of the lymphocytes at low concentrations around 10-8 M, while enhanced IgE production at high concentrations over 10-4 M. An endogenous estrogen, 17 b-estradiol exerted only IgE production-enhancing activity at high concentrations. These results suggest that IgE production-suppressing and -enhancing activities are regulated by different mechanisms.
Soy Protein Research, Japan 5 103-107, 2002.

Key words : mouse splenocytes, isoflavone, immunoglobulin production
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23-21

Effects of Isoflavone Glucoside and Aglycone on Thrombogenicity as Measured Using apo-/-•LDLR-/-Double Knockout Mice

Junichiro YAMAMOTO1, Yoshinobu IJIRI1, 2, Mayu MURAKAMI1, Mayuko MIURA1 , Tsutomu YAMASHITA1, Masaru HASHIMOTO1, Kazuhiro OIWA3 and Toyoko OKUDA4

1Faculty of Nutrition, Kobe Gakuin University, Kobe 651-2180
2Faculty of Health Science, Hyogo University, Kakogawa 675-0101
3Communications Research Laboratory, Kansai Advanced Research Center, Kobe 651-2492
4Department of Art and Sciences, Osaka Kyoiku University, Kashiwara 582-8582

ABSTRACT
Prevention of life style-related diseases, in which thrombogenesis is deeply involved, is the currently important social problem. Diet is undoubtedly one of the important risk factors of the diseases. Therefore, prevention of thrombogenesis is beneficial for that of the diseases. To prevent, prothrombotic state has to be assessed. Currently used multifactorial tests seem not to have succeeded. As thrombus is the result of the interaction among blood cells, blood vessels and blood flow, we established a sensitive and reproducible animal model of thrombogenesis for assessing the effect of diets on thrombotic state. In the model thrombus is formed by He-Ne laser irradiation in the carotid arteries of genetically atherogenic mice deficient both in apolipoprotein E (apoE-/-) and low density lipoprotein receptor (LDLR-/-). We have demonstrated using this model that genistein-rich isoflavone aglycone prevents high fat diet-induced prothrombotic states. However, purity of the isoflavone preparation used in this study was low (30%). Therefore, the results were not conclusive. The aim of the present study was to clarify the antithrombotic effect of isoflavone and to compare the effects of isoflavone forms, glucoside (daidzin and glycitin rich) and aglycone (daidzein and glycitein rich) , using the preparation with high purity (glucoside 80% ;aglycone 90%). The male mice were raised with commercial solid diet until 6 weeks of age and the test diet feeding started from 6 until 14 weeks of age. Isoflavones were supplemented to the basic high fat diet (40% in energy;ontrol diet) to be 25 mg/kg diet (glucoside), 13 mg/kg diet (aglycone equivalent to 25 mg/kg diet of glucoside in molar concentration)and 650 mg/kg diet (aglycone). Thrombus was formed in the left carotid artery by He-Ne laser irradiation in the presence of Evans blue. He-Ne laser-induced thrombosis method (in vivo) revealed that aglycones showed antithrombotic effect at 13 mg/kg diet (P=0.031) and at 650 mg/kg diet (P=0.007) but not glucoside (P =0.349). Shear-induced platelet reactivity test (in vitro)showed the antiaggregatory tendency in aglycone at 650 mg/kg diet (P=0.064). The results confirmed that daidzein and/or glycitein have antithrombotic effect, but not daidzin and/or glycitin. The antithrombotic effect may be partly due to antiaggregatory effect. Deficiency of antithrombotic effect in glucoside might be due to its lower absorption in the intestine.
Soy Protein Research, Japan 5 108-116, 2002.

Key words : thrombosis, atherosclerosis, isoflavone, polyphenol, platelets
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23-22

Studies on Bone-protective Function of Isoflavone via the Mevalonate Metabolic Pathway

Hidemichi EBISAWA and Yasuko KOSHIHARA

Bone Research Group, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015

ABSTRACT
The purpose of the present study was to elucidate the mechanism on bone protective function of isoflavone in relation to mevalonate-geranylgeranylpyrophosphate (GG-PP) metabolic pathway and signal transduction. We examined the effects of dietary isoflavone on mevalonate and GG-PP production in ovariectomized (OVX) aged-rats in experiment-1 and the effects of daidzein and genistein treatment on Rho-A level in rat osteosarcoma cells (UMR-106) in experiment-2. In experiment-1, rats fed isoflavone supplemented diet (OVX-IF) showed 1) relatively lower biochemical markers of bone metabolism, 2) higher urinary mevalonate and relatively higher serum GG-PP than those of control (OVX-CA). In experiment-2, 3) daidzein or genistein treatment caused apparently lower Rho-A level in particulate and soluble fraction and lower ratios of particulate to soluble fraction (P/S ratio) than those of control. 4) Mevalonate caused higher P/S ratio than that of control, although diminished Rho-A level was observed. 5) Diminished Rho-A level by genistein treatment was rescued by co-treatment with mevalonate or GG-PP. These results suggest that daidzein and genistein inhibits the expression and activation of Rho-A by mevalonate-GG-PP metabolic pathways in osteoblast. But, in the present study, dietary isoflavone did not reduce the mevalonate and GG-PP production in rats since aged female rats had wide variation of responsibility to dietary isoflavone.
Soy Protein Research, Japan 5 117-121, 2002.

Key words :isoflavone, daidzein, genistein, mevalonate, Rho-A
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23-23

Preventive Effects of Dietary Isoflavone on Age-associated Modulation of Lipid Metabolism and Antioxidative System in Rats

Kyoichi OSADA, Wakako TSURUGASAKI, Yuki KAWAKAMI and Shingo NAKAMURA

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

ABSTRACT
Male Sprague-Dawley rats, 4 weeks (immature) or 8 months (mature) of age, were given one of purified diets with or without 0.15% isoflavone-aglycone (IFA) to know the regulative effects of dietary isoflavone on modulations of antioxidative function and lipid metabolism with aging. Dietary IFA inhibited the age-related lowering of the activities of antioxidative enzymes such as superoxide dismutase, glutathione peroxidase and catalase in both red blood cells and liver. Moreover, the increase of levels of lipid hydroperoxides such as phospholipid hydroperoxides in both serum and liver was lowered by consumption of IFA. Dietary IFA also regulated the activities of hepatic key enzymes of lipid metabolism including cholesterol synthesis, cholesterol catabolism and linoleic acid desaturation in both immature and mature rats. Dietary IFA seems to have potent regulative function against the modulation of antioxidative system with aging among each regulative function of dietary components. Oxidative damage and deleterious perturbation of metabolism in biological systems is one of the triggers of aging process and degenerative diseases such as atherosclerosis and cancer. Therefore, a part of degenerative diseases caused by modulations of antioxidative function and lipid metabolism with aging may be prevented by consumption of IFA.
Soy Protein Research, Japan 5 122-128, 2002.

Key words : isoflavone, aging, lipid metabolism, antioxidant, rat
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23-24

Dietary Treatment for Obesity Using Supplementary Food Made of Soy Protein:
Evaluation of the Usefulness by the Newly Developed Meal Test

Junko NABIKI1, Takeshi MIYAWAKI1, Tomomi ADACHI1, Keiko AOKI1, Tomomi INOUE1, Yuko SASHIRO1, Nayuta MORI1, Kyoko YAMAOKA1, Miwako YOSHIMURA1, Takamitsu NAKANO2 and Yutaka HARANO1

1Koshien University College of Nutrition, Takarazuka 665-0006
2Japan Immunoresearch Laboratoies, Takasaki 540-0021

ABSTRACT
For obese subjects who were unable to reduce body weight on the regular dietary therapy, 300-350 kcal supplementary lunch or alternatively breakfast and 150 kcal subsidiary dish made of soybean protein added to dinner were advised to take in a total 1, 200-1, 500 kcal per day. This energy is 200-300 kcal less than the estimated daily energy expenditure in the regular daily life. After at least 3 months, 3-5 kg weight reduction and 4-8 cm decrease of west size and improvement of metabolic parameters including triglyceride (TG), cholesterol (chol), Apo-B and insulin levels have been observed in 44% of the subjects. No reduction of body weight was noted in 21% because of possible failure to reduce other food than soybean supplement. In 35%, they could not continue supplementary diet because of taste and cloying. In successful group, basal level of TG, Apo-B and LDL-chol as well as 1 h insulin, 2 h chol and Apo-B at 1 and 2 h all reduced after dietary treatment. By PAGE analyses, elevation of VLDL at 1 h after the test meal significantly reduced following soybean supplementation. In conclusion, diet treatment using supplementary food made of soybean protein was effective in half of the subjects for the efficient weight reduction in the obese subjects who had not responded to the regular dietary guideline.
Soy Protein Research, Japan 5 129-133, 2002.

Key words : soybean protein, obesity, insulin resistance, meal test, dyslipidemia
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23-25

Effect on Bone Density of Long-term Clinical Administration of Soy Isoflavone Tablet

Kazuhiko KATO1, Hiromi SUEMITSU1, Li FUANG2 and Gang YOUNG2

1Kato Clinic, Komae 201-0012
2China-Japan Friendship Hospital, Beijin

ABSTRACT
The health benefits of soybean isoflavones gather recently much concern. While soy isoflavone is recognized as phytoestrogen that may affect bone metabolism, there were few clinical experiments reported. We examined preliminary clinical trial (Experiment 1) by 5 postmenopausal women who were outpatients of Kato Clinic in Komae, Japan. Each subject was given detailed informed consent, and agreed experimental protocol. Bone density, osteocalcin and bone-type alkaline phosphatase were measured 3 times (at start, 6 months and 1 year) with ingestion of 10 Isofla tablets/day that contained 10 mg soy isoflavone in total. As a result, bone density was maintained and osteocalcin and bone-type alkaline phosphatase were increased significantly. Other blood profiles were not changed but some improvement in motion was observed. In experiment 2, 20 Chinese outpatients in Beijing, China who were diagnosed as osteoporosis, and did not take any osteoporosis curative medicine such as calcareous preparation participated in this experiment. They were divided into two groups that matched age and gender, then were administered 10 tablets/day, which contained 2 mg isoflavone / tablet or placebo tablet. After 6-month ingestion, lumber spinal bone density was increased in Isofla group (initial, 1.011 ± 0.069 ; 6 month, 1.040 ± 0.068 ; change=3.07%), while slightly decreased in placebo group (initial, 1.074 ± 0.081 ; 6 month, 1.056 ± 0.082 ; change=-1.69%), although it had no significance. Plasma Ca and P were not changed from initial values and only alkaline phosphatase tended to increase in Isofla group. These data obtained from just 6 months to 1-year ingestion suggested that soy isoflavone might improve bone maintenance. To confirm these data, long-term follow-up is necessary.
Soy Protein Research, Japan 5 134-137, 2002.

Key words : soy isoflavone, phytoestrogen, bone metabolism, bone density
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23-26

Effects of Soy-protein-fortified Foods on Reducing Risk Factors for Cardiovascular Diseases in Japanese Elderly Women

Tomo KANDA1, Masari AMANO1, Miwa TAKAMATSU1, Miki SAGARA2 and Yukio YAMORI3

1Kyoto University Graduate School of Human and Environmental Studies, Kyoto 606-8501
2Research Institute for Promotion Development, Kyoto 606-8501
3WHO Collaborating Center for Primary Prevention of Cardiovascular Diseases, Kyoto 606-8501

ABSTRACT
Although Japanese have the longest life expectancy at birth in the world, numbers of dementia (vascular type, Alzheimer type) and bedridden disability are increasing due to the rapid increase in elderly population. There are accumulating evidence that soy protein as well as isoflavones reduce risk factors for cardiovascular diseases in relation to lowering blood pressure (BP) and total cholesterol (T-CHO) levels. However, it is not yet tested in a double blind design that soybean diets do reduce cardiovascular risk factors especially in elderly female population. In this study, we investigated whether soy-protein-fortified diets reduce cardiovascular risk factors in Japanese elderly women by means of double blind trial. Our results revealed that urinary isoflavone excretion in 24-hour urine collection was significantly increased in soy-protein-fortified group (P < 0.01). We also observed significant reduction in T-CHO, LDL-CHO and systolic blood pressure (SBP) in soy-protein-fortified group whereas placebo group did not show significant difference in a 4-week period. Our study suggests that dietary intake of soy protein over 25 g/day containing over 50 mg of isoflavones per day could prevent cardiovascular diseases by reducing BP, T-CHO and LDL cholesterol levels even in postmenopausal elderly women. Also soy-protein-fortified products have potential benefits for the primary prevention for cardiovascular as well as lifestyle-related diseases through improving eating habits.
Soy Protein Research, Japan 5 138-143, 2002.

Key words : elderly female, soy protein, isoflavones, cholesterol, blood pressure, cardiovascular diseases
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