Soy Protein Research(Japan)


19-1

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

Motoko AWAZUHARA, Hiroaki HAYASHI and Toru FUJIWARA

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

Soy Protein Research, JapanÅ@1(Serial Vol. 19), 1 - 5, 1998.

The gene encoding the É¿-subunit of É¿-conglycinin is known to be upregulated by sulfur deficiency in soybean. We previously identified sequences in the É¿-subunit promoter required for upregulation of the gene in response to sulfur deficiency in seeds of transgenic plants. This fragment was also functional in leaves and stems, suggesting that mechanisms of sulfur-deficient control of gene expression are not seed-specific. However, we found that patterns of O-acetyl-L-serine (OAS) accumulation, a metabolite responsible for sulfur-regulated expression of the É¿-subunit gene, differed among organs. Patterns of other metabolites involved in sulfur assimilation also varied. These findings suggest that mechanisms of sulfur-regulated gene expression may be organ specific, though a common cis-regulatory element may be involved.

Key words : É¿-conglycinin, O-acetyl-L-serine, sulfur nutrition, tissue specificity, Glycine max


19-2

Vacuolar Sorting Machinery and Processing Mechanism for Storage Proteins

Ikuko HARA-NISHIMURA, Tomoo SHIMADA, Miwa KUROYANAGI and Kenji YAMADA

Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585

Soy Protein Research, JapanÅ@1(Serial Vol.19), 6 - 12, 1998.

Novel vesicles that accumulate large amounts of proprotein precursors of storage proteins were purified from developing pumpkin seeds and were designated precursor-accumulating (PAC) vesicles. The PAC vesicles mediate the transport of the precursor of a major storage protein (pro2S albumin) to protein-storage vacuoles in the developing seeds. We characterized two homologous proteins from PAC vesicles, a 72-kDa protein (PV72) and an 82-kDa protein (PV82). PV72 and PV82 showed an ability to bind to peptides derived from both an internal propeptide and a C-terminal peptide of pro2S albumin. PV72 was predicted to be a type I integral membrane protein with epidermal growth factor (EGF)-like motifs. These results suggest that PV72 and PV82 are potential sorting receptors for 2S albumin to protein-storage vacuoles. In the next step, we characterized a 100-kDa component (PV100) of the vesicles. Isolated cDNA for PV100 encoded a 97, 310-Da protein that was composed of a hydrophobic signal peptide and the following three domains: an 11-kDa Cys-rich domain with four CxxxC motifs ÅiC, Cys), a 34-kDa Arg/Glu-rich domain composed of six homologous repeats, and a 50-kDa vicilin-like domain. Molecular characterization showed that two Cys-rich peptides, three Arg/Glu-rich peptides and the vicilin-like protein were produced by cleaving Asn-Gln bonds of PV100 by vacuolar processing enzyme (VPE) and that all these proteins had a pyroglutamate at their NH2 terminus. Our findings suggested that VPE was responsible for cleaving Asn-Gln bonds of a single precursor, PV100, to produce multiple seed proteins in the vacuoles of seed cells.

Key words : asparaginyl endopeptidase, cysteine proteinase, vacuolar proteins, vacuolar processing enzyme (VPE), yeast


19-3

Analysis of Physiological Significances and Enzymatic Properties of Aspartic Proteinases Occurring in Food Seeds and Legumes

Keiko ABE 1 and Tomiko ASAKURA 2

1 Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657
2 Atomi Junior College, Tokyo 112-8687

Soy Protein Research, JapanÅ@1(Serial Vol.19), 13 - 18, 1998.

Aspartic proteinases (APs) refer to proteinases that have aspartic acid residues at the active centers. These are widely distributed in animals, plants, and micro-organisms as their intracellular and extracellular enzymes. There are several plant APs besides oryzasin 1 we found in rice 1), and they have been cloned at the protein as well as gene level. Plant APs, structurally different from animal and microbial APs, have a large insertion sequence that accounts for one fourth of the molecular size, though the significance of this interesting insertion for any plant AP remains to be interpreted. We have constructed a system for expression of oryzasin 1 by fusing its pro-form in the downstream of glutathione-S-transferase and have thus succeeded in obtaining an enzymatically active oryzasin 1 as a fusion protein 2). In the present study conducted in the past year, we used this expression system and produced an oryzasin 1 mutant whose insertion sequence had been deleted. Investigating the effect of the deletion on the enzymatic activity, we found that the mutant was activated as well as the wild oryzasin 1 under an optimally acidic pH condition, with the conclusion that the presence of this insertion is not necessary for the AP activity. However, we also observed that during the activation at the acidic pH, the mutant and the wild enzymes behaved differently. This suggests the possibility that the presence of the insertion may be involved in effectuating the processing of the pro-form into the mature AP and/or in stabilizing the mature form in plant tissues. histochemical study using an anti-oryzasin 1 antibody is underway to verify this possibility. 1ÅjEur J Biochem, 232, 77 - 83 (1995). 2ÅjRep Soy Protein Res Com, Jpn, 18, 05-20(1997).

Key words : soybean, rice, aspartic proteinase, expression


19-4

The Structure-function Relationships of Soybean É¿-Conglycinin

Nobuyuki MARUYAMA and Shigeru UTSUMI

Research Institute for Food Science, Kyoto University, Uji 611-0011

Soy Protein Research, JapanÅ@1(Serial Vol.19), 19 - 24, 1998.

Soybean proteins are composed of two major components, É¿-conglycinin and glycinin. Elucidation of the structure-function relationships of soybean proteins is required for development of new food products from soybean proteins. Such relationships of glycinin at subunit and molecular levels have been investigated in detail. However, such a study on É¿-conglycinin has been scarcely carried out. É¿-Conglycinin is a trimeric protein consisting of three subunits Éø, Éø' and É¿. The Éø and Éø' subunits are composed of the extension and the core regions, whereas the É¿ subunit consists of only the core region. We prepared the individual subunits and the deletion mutants lacking the extension regions of the Éø and Éø' subunits using the Escherichia coli expression system and examined their structures and functions. All recombinant proteins appeared to adopt a correct conformation as judged by CD and density gradient centrifugation profiles. Surface hydrophobicity and thermal stability of the É¿-conglycinin constituent subunits are likely to be conferred by their core regions, which exhibit different properties to each other. Solubility, formation of heat-induced soluble aggregates, and emulsifying ability of individual subunits remarkably depend on the extension regions and the carbohydrate moieties in addition to the structural features of the core regions. These facts indicate that we can produce various end products by selecting soybean varieties containing É¿-conglycinins having different subunit compositions.

Key words : soybean, É¿-conglycinin, structure-function relationships


19-5 Studies on Heterogeneity of Soybean Glycinin

Tomohiko MORI

Research Institute for Food Science, Kyoto University, Uji 611-0011

Soy Protein Research, JapanÅ@1(Serial Vol.19), 25 - 30, 1998.

Glycinins isolated from the seeds of four cultivars of soybean were analyzed by column chromatography and gel electrophoresis, in order to obtain the evidence for heterogeneity of glycinin molecular species. Four soybean cultivars, Shirotsurunoko and York as the A4 subunit-containing cultivars and Raiden and Suzuyutaka as the A4 subunit-lacking cultivars, were subjected for analyses of the glycinins. The crude glycinin fraction was prepared from the soybean seeds according to the procedure including the preparation of acetone powder and the cryoprecipitation. The crude glycinin fraction was subjected to chromatographic fractionation by using a column of DEAE-Toyopearl. Significant difference was observed in the elution pattern of column chromatography between the A4-containing and -lacking soybean cultivars. The fraction eluted earlier exhibited single peak and small amount in the case of the A4-containing cultivars, while did two peaks and significant amount in the A4-lacking cultivars. The fraction eluted later exhibited single peak in both the cultivars, however, the fraction comprised 90% and 50% of the total protein in the A4-containing and -lacking cultivars, respectively. SDS-PAGE analysis showed that the fraction eluted later contained all of the constituent subunits of glycinin in both of the cultivars. In contrast, it was found that the fraction eluted earlier in the case of A4-lacking cultivar contained only the A1 and A2 subunits. Further, when the purified glycinin fraction prepared by sucrose density gradient centrifugation was analyzed by the above mentioned column chromatography and gel electrophoresis, it was shown that the fraction eluted earlier in the case of A4-containing cultivar also contained only the A1 and A2 subunits. These results indicate the occurrence of particular glycinin molecular species composed of the limited kinds of the constituent subunits of glycinin in soybean seeds.

Key words : glycinin, soybean proteins, molecular heterogeneity, subunit composition, soybean cultivars


19-6

Functional Analysis of a Bacterial Ice Nucleation-active Factor and Its Application to Freeze Texturing of Food Materials

Michiko WATANABE

Faculty of Education, Tokyo Gakugei University, Tokyo 184-8501

Soy Protein Research, JapanÅ@1(Serial Vol.19), 31 - 35, 1998.

My group isolated an edible ice nucleation-active bacterium, Xanthomonas campestris INXC-1, and clarified that 4-hydroxy-3-nitrophenylacetic acid enhanced the activity. This paper reports a possible mechanism involved in the activity enhancement and its application to freeze texturing of SPI. Cultivation of the bacterial cells in the presence of the activity enhancer resulted in enhancement of their ice-nucleation activity. Both the ice nucleation-active protein and its mRNA were effectively expressed in the bacterial cells cultured in the presence of the enhancer. This indicates that the enhancer stimulated the biosynthesis of the ice nucleation-active protein at the transcription step. The active cells were killed by pressurization before use for freeze texturing. SPI dispersion and its heat-induced gel began to freeze with a slight degree of supercooling in the presence of the pressurized cells and formed ice crystals with a dendrite structure. The frozen samples were dried before setting the resulting textures by steaming if necessary, to form anisotropic textures. SPI showed a weak ice-nucleation activity which gave a similar texture to the resulting product, when freezing was done at -10Åé. Mouth-feel of the textured SPI resembled that of some mushrooms.

Key words : freeze texturing, soybean protein isolate, bacterial ice nucleation


19-7

Chemical Modification of Soy Protein Isolate : Miscibility Control

Kiyoshi YAMAUCHI, Takuya TOKUDA and Yasunori YAMAZOE

Faculty of Engineering, Osaka City University, Osaka 558-8585

Soy Protein Research, JapanÅ@1(Serial Vol.19), 36 - 40, 1998.

Upon mixing with various epoxides including glucidol, styrene oxide and epoxydecane at pH 9.5-10, soy protein isolate (SPI) was hydroxyalkylated, mainly at the lysine residues, to produce SPI - CH2CH(OH)R where R is CH3, CH2OH, C6H5 or n-C8H17. The modified SPI was (i)soluble in water of a wide pH range (5-10) when R is CH2OH and n-C8H17 or (ii)miscible readily with water and organic solvents such as methyl ethyl ketone and dimethylformamide when R is CH3 and C6H5. In the second phase of the study, SPI was esterified by a combination of thionyl chloride and alcohol such as methanol and ethanol. The methylated SPI was very soluble in water to afford a viscous solution while the ethylated SPI was miscible readily with water and most polar organic solvents. The modified SPI was discussed for industrial applications.

Key words : chemical modification, esterification, glucidol, styrene oxide, epoxydecane, soy protein isolate


19-8

Deodorization of Soybean Proteins by Physicochemical Processing

Kuniyo INOUYE

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

Soy Protein Research, JapanÅ@1(Serial Vol.19), 41 - 45, 1998.

Deodorization of soy protein isolate (SPI) was examined by treating the isolate by various absorbents such as polystyrene, polymetacrylate, zeolite, and charcoal. The SPI solution treated by the absorbents was smelled by 14 persons, and hexanal in the solution was determined. With increase in the hydrophobicity of the absorbents, the soybean ordor and hexanal of the absorbent-treated solution decreased. Brominated polystyrene beads were the most effective absorbent when they were used after soaking in ethanol. The hexanal content was decreased to 1/5 of that of the untreated solution, and nobody could recognize the soybean odor of the solution.

Key words : absorbent, hexanal, soybean, deodorization, soy protein isolate


19-9

Microbial Transformation of Soybean Germ Components to Antioxidative Isoflavonoids

Akio MIMURA and Shinichi YAZAKI

Department of Applied Chemistry and Biotechnology, Yamanashi University, Kofu 400-8511

Soy Protein Research, JapanÅ@1(Serial Vol.19), 46 - 51, 1998.

Soybean is rich in isoflavonoid glycosides such as daidzin and genistin, especially in the part of germ. During the fermentation by microorganisms to produce traditional fermented foods such as miso (soybean paste), soy sauce, natto and tempeh, these isoflavonoid glycosides can be hydrolyzed to aglycon isoflavonoids, and further transformed to biologically active compounds. Aspergillus niger IFO 4414 was cultivated in the medium composed of soybean germ extract rich in isoflavonoid glycosides, and it was observed that antioxidative activity, superoxide dismutase (SOD) like activity and anti-tumor promoting activity were increased remarkably during the fermentation. In the previous paper, we reported that one of the transformed products from daidzin was 4', 7, 8-trihydroxyisoflavone (8-hydroxydaidzein) with potent antioxidative and anti-UVB activities. From the chromatographic experiments, it was considered that in the fermented broth there have been produced some other transformed isoflavonoids. These observations indicate the possibility of production of the isoflavonoids with potent antioxidative, SOD like and anti-tumor promoting activities.

Key words : soybean isoflavonoid, microbial transformation, antioxidant, SOD like activity, anti-tumor promoter


19-10

Reactive Oxygen Scavenging Activity of Soybean by Photon Emission : Especially Screening Test of Reactive Oxygen Scavenging Components and Photon Emission Characteristics of Soybean Lipoxygenase

Yumiko YOSHIKI, Yuan HU, Tetsuo IIDA, Masaaki KAWANE and Kazuyoshi OKUBO

Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555

Soy Protein Research, JapanÅ@1(Serial Vol.19), 52 - 57, 1998.

The novel screening test by photon emission in the presence of 2% hydrogen peroxide ÅiXÅjand 5 mM gallic acid ÅiYÅjindicated that a typical Z components was either of iron phosphateÅiIIÅj or ÅiIIIÅjinsoluble in water and that the spectral maxima were 380, 515, 678 and 777 nm. Also the test indicated bases of nucleic acid, some compounds of ketone, amide and carbonic acid, iron chelate and vitamins. Root and root hair of sprouted soybean in the presence of X and Y emitted photon of spectral maxim 507 nm as Z. Soybean lipoxygenase emitted photon of spectral maxim 519 nm in the presence of t- butyl peroxide and 690 nm in the presence of butyl peroxide and gallic acid.

Key words : photon emission, chemiluminescence, soybean lipoxygenase, reactive oxygen scavenging activity


19-11

ÉGStudy on the Antioxidant Activity of Soybean Proteins in Emulsion Systems

Kazuo MIYASHITA

Faculty of Fisheries, Hokkaido University, Hakodate 041-8611

Soy Protein Research, JapanÅ@1(Serial Vol.19), 58 - 62, 1998.

This study was aimed at evaluating the antioxidant activity of various proteins on the triacylglycerolÅiTGÅjoxidation in emulsion. Of all proteins examined, soybean protein hydrolysates showed the most remarkable inhibitory effect and this activity increased with increasing degree of hydrolysis. However, the activity of other proteins was dependent on TG species. The antioxidant activity of water soluble proteins may be explained by their scavenging effect of free radicals and their adsorption ability on the emulsified TGs.

Key words : antioxidant activity, triacylglycerol, soybean protein hydrolysates, radical scavenger, adsorption effect


19-12

Inhibition of Calcium Carbonate Crystallization by Soybean Protein Hydrolysate and Its Promotion Effect on Calcium Absorption

Dong Hao JIN, Yasuko SUZUKI, Takako NAGANUMA, Tomohisa OGAWA and Koji MURAMOTO

Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555

Soy Protein Research, JapanÅ@1(Serial Vol.19), 63 - 68, 1998.

Many investigations into mineral bioavailability are concerned with improving the dietary supply of minerals. Casein phosphopeptide ÅiCPPÅjis known to enhance the passive calcium absorption from the small intestine by inhibiting the precipitation of calcium phosphate and increasing the concentration of soluble calcium. We found that acidic peptides could also inhibit the crystal growth of supersaturated calcium carbonate solution. In this study, we hydrolyzed soybean protein, in which Glu/Gln and Asp/Asn comprise 35% of total amino acid residues, using several proteases and investigated the inhibitory activity of the hydrolysates on the crystal growth. Each hydrolysates showed different strength of the activity, suggesting the key role of the structure of peptides for the activity. The potency of a soybean protein hydrolysate was less than one tenth of that of a tryptic digest of casein, however, it increased over four fold by treating the soybean protein hydrolysate with glutaminase. The magnitude corresponded to the conversion rate of Gln residues to Glu residues. Furthermore, the potency increased by the addition of some food constituents, such as sodium chloride, citric acid, lactose, into the reaction mixture.

Key words : soybean protein hydrolysate, calcium, glutamic acid, glutaminase


19-13

Effect of the Protein Chemical Nature of É¿-Conglycinin Subunits ÅiÉø', Éø and É¿Åj on Their Immunological Activity

Tadashi OGAWA 1 , Kaeko MUROTA 2 and Noriko BANDO 2

1 Research Institute for Food Science, Kyoto University, Uji611-0011
2 School of Medicine, The University of Tokushima, Tokushima 770-8503

Soy Protein Research, JapanÅ@1(Serial Vol.19), 69 - 74, 1998.

IgE antibodies from soybean-sensitive patients with atopic dermatitis are shown to recognize only Éø- subunit of É¿-conglycinin, while the primary structure of these subunits ÅiÉø', Éø and É¿Åjis highly homologous to each other. Present work was carried out to examine the relationship between the protein structure of these subunits and their immunological activities. First, we tried to prepare the monoclonal antibodies against each subunit to establish a way for selective and quantitive determination of subunits. However, all the monoclonal antibodies raised against Éø'- , Éø- or É¿-subunit were shown to be cross-reactive to each other. We also examined the susceptibility of three subunits to various proteinases as an index to evaluate the antigenicity. Éø' -and Éø-Subunits were rapidly hydrolyzed by both pepsin and pancreatin. É¿-Subunit was shown to be resistant against pepsin digestion, but not to pancreatin. These findings indicate that the susceptibility of the subunits to proteinase digestion does not correspond to their immunological activity.

Key words : É¿-conglycinin, Éø-subunit, allergen, digestion, stability


19-14

In vitro / in vivo Digestibility and Oral Immunogenicity of Soybean Protein

Noriko NINOMIYA, Aiko ICHIBA and Tsukasa MATSUDA

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601

Soy Protein Research, JapanÅ@1(Serial Vol.19), 75 - 80, 1998.

Digestibility and oral immunogenicity of Kunitz soybean trypsin inhibitor ÅiKSTIÅjwere investigated, and compared with those of several other food proteins. Soy protein isolate ÅiSPIÅj was digested with simulated gastric fluid, and the protein digestibility was analyzed by SDS - PAGE and Western blot using anti-KSTI antibody. To analyze the digestibility of KSTI more in detail, purified KSTI was digested in vitro with pepsin, chymotrypsin and trypsin, or administered intragastrically to mice, and the in vitro and in vivo digested samples recovered from the test tubes and the digestive tract of mice were analyzed by SDS-PAGE and Western blot analyses. The results showed that KSTI was markedly stable and resistant against protease digestion even in vivo, as compared with some other proteins. However, repetitive intragastric administration of KSTI to mice did not induce serum antibody response against KSTI, suggesting that digestion - resistance of KSTI was not necessarily correlated to the ability for oral sensitization. Effect of the digestion - resistance on the ability for provocation of allergic symptoms remains to be investigated.

Key words : soybean trypsin inhibitor, food allergy, digestibility


19-15

Studies on Anti-allergic Effect of Soybean Peptides: Immunoregulatory Activity of Soy Protein and Dietary Fats

Koji YAMADA 1 , Shin - ichi YUNOKI 1 , Ken - ichi OKURA 1 , Shihoko KAKU 1 ,Michiko NONAKA 1 , Hirofumi TACHIBANA 1 and Michihiro SUGANO 2

1 Faculty of Agriculture, Kyushu University, Fukuoka 812-8581
2 Faculty of Human Life Sciences, Prefectural University of Kumamoto, Kumamoto 862-8502

Soy Protein Research, JapanÅ@1(Serial Vol.19), 81 - 85, 1998.

It has been shown that soybean protein and peptides inhibit the release of LTB4 from rat peritoneal exudate cells ÅiPECÅj. In the present study, effect of dietry fats and soybean protein on the production of LTB4 and immunoglobulin was examined to estimate their immunoregulatory activity. Sprague-Dawley rats were fed purified diets containing safflower, borage or perilla oil at the 10% level and casein or soybean protein at the 20% level for 3 weeks. Though soy protein decreased LTB4 releasing activity of PEC as strong as borage oil, the effect was weaker than that of perilla oil. In the rats fed soy protein, decrease of arachidonic acid level was detected in PEC total lipids, suggesting that it decreased LTB4 releasing activity through the interference in lipid metabolism. In immunoglobulin production, soy protein enhanced immunoglobulin production of mesenteric lymph node lymphocytes more strongly than splenocytes, especially IgA production of the former. These results suggest that soy protein exerts anti-allergic effect and stimulates immune function by the activation of the gut immune system.

Key words : soybean protein, dietary fats, LTB4 release, immunoglobulin production.


19-16

Mechanism of Elevation in Plasma Glucagon/Insulin Ratio by Soybean Protein Intake

Shin HASEGAWA, Kohichiro KANO and Tohru MOTOKI

Graduate School of Science and Technology, Kobe University, Kobe 657-0011

Soy Protein Research, JapanÅ@1(Serial Vol.19), 86 - 90, 1998.

The present study was conducted to elucidate the mechanism of elevation in plasma glucagon/insulin ratio by soybean protein intake in chicks. Firstly, chicks were fed purified diets containing two types of protein Åisoy protein isolate and caseinÅj, and the effects of dietary protein types on growth performance, concentrations of plasma free fatty acid, glucose, glucagon and insulin were examined. Soybean protein intake had tendencies to reduce the body fat accumulation , and to raise the concentrations of plasma free fatty acid, glucose and glucagon, and glucagon/insulin ratio. Secondly, chicks were administered intravenously with glucagon-like peptide-1ÅiGLP-1Åj. GLP-1 administration raised the concentrations of plasma free fatty acid, glucose and glucagon, and glucagon/insulin ratio. Finally, chicken adipocytes in primary culture were used to characterize the effects of GLP-1 on lipolysis. GLP-1 did not affect lipolysis in chicken adipocytes. And, BALB/c mice were received the intraperitoneal injection of GLP-1, and the marked serum antibody response was induced for all mice. Further the conjugaton of the spleens from the immuned mice and mouse myeloma cells are in progress to obtain monoclonal antibodies specific for chicken GLP-1.

Key words : soybean protein diet, plasma glucagon/insulin ratio, glucagon-like peptide - 1, lipolysis, chicken, body fat accumulation


19-17

Preparation of Subfractions with Characteristic Amino Acid Composition from ÅgHinuteÅh: A Peptide Product Prepared from SPI

Tadashi NOGUCHI and Tomomi HIDAKA

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

Soy Protein Research, JapanÅ@1(Serial Vol.19), 91 - 95, 1998.

A peptide preparation Hinute from SPI was subfractionated with activated charcoal, HP20 or reverse phase chromatography. Using an activated charcoal column, three fractions were obtained. The unadsorbed fraction contained lower phenylalanine and tyrosine ÅiPheÅ{TyrÅj compared with Hinute. The fraction eluted from the column with 50% acetone also contained lower PheÅ{Tyr. The third fraction eluted from the column with 1 N NH4OH was rich in Phe Å{Tyr. The ratio of branched chain amino acids ÅiBCAAÅj/ÅiPheÅ{TyrÅjwas approximately 2 in Hinute and 4 in the unadsorbed fraction, 3 in acetone - eluted fraction and 1.5 in ammonia - eluted fraction. Subfractionation of Hinute with a HP20 column also gave a preparation with BCAA/ÅiPheÅ{TyrÅjratio of 5. These preparations will be conveniently used for preparation of diets with characteristic amino acid composition, particularly those with various BCAA/ÅiPheÅ{ TyrÅjratios.

Key words : diabetes, hepatitis, soybean peptide, soybean protein hydrolysate, Hinute


19-18

Effect of Soy Protein Isolate on Body Weight Reduction by Energy Restriction in OLETF Rats

Yutaka MORI 1 , Toshiaki AOYAMA 2 , Kensuke FUKUI 2 , Naoyuki KUROKAWA 3 ,Hideaki KOMIYA 3 and Yoshio IKEDA 4

1 National Higashi - Utsunomiya Hospital, Utsunomiya 329-1193
2 Novelty Materials Research Institute, Fuji Oil Co., Ltd., Izumisano 598-8540
3 Faculity of Education, Utsunomiya University, Utsunomiya 321-8505
4 Center for General Health Care, The Jikei University School of Medicine, Tokyo 105-8461

Soy Protein Research, JapanÅ@1(Serial Vol.19), 96 - 101, 1998.

Effect of soy protein isolateÅiSPIÅj on the body weight reduction by energy restriction was studied in a genetically obese-hyperglycemic Otsuka Long Evans Tokushima FattyÅiOLETFÅjrat, characterized by intra-abdominal fat accumulation. Obese OLETF rats aged 25 weeks were fed the energy-restricted diets containing SPI or casein as protein source for 3 weeks. Body weight reduction was significantly greater and body fat content was significantly decreased in SPI diet-group compared with those in casein diet-group, although body protein content was significantly higher in SPI diet-group. Moreover, the decreases in plasma levels of leptin, total cholesterol and free fatty acid were significantly greater in SPI diet-group. In the experiment of body weight reduction by energy restriction using obese animal model, SPI was suggested to have more favorable effect on the amelioration of obesity and insulin resistance than casein.

Key words : Otsuka Long Evans Tokushima FattyÅiOLETFÅj rats, soy protein isolateÅiSPIÅj,
intra - abdominal fat accumulation, brown adipose tissue ÅiBATÅj, uncoupling protein ÅiUCPÅj


19-19

Supplementation of L-Arginine to Casein-based Diet Alleviates Atherosclerosis Development in Apolipoprotein E-deficient Mice

Wei - Hua NI, Tomoyasu TSUDA, Masao SATO, Koji NAGAO and Katsumi IMAIZUMI

Faculty of Agriculture, Kyushu University, Fukuoka 812-8581

Soy Protein Research, JapanÅ@1(Serial Vol.19), 102 - 105, 1998.

Apolipoprotein E-deficient mice were fed on diet containing soy protein isolate ÅiSPIÅj, casein and 0.92% L-arginine supplemented casein for 15 wk. Increasing L-arginine content in casein diet to the same level as SPI contained diet significantly reduced lesion size by 20%, compared with casein contained diet. Mice received arginine, compared with those fed casein, had significant reduction of liver thiobarbituric acid-reactive substances and higher level of liver Éø-tocopherol, and tended to have lower serum autoantibody titers against oxidized LDL and higher level of serum NO2/NO3. These results suggest that antiatherogenic properties of dietary SPI at least are mediated through increasing formation of NO and preventing lipid from oxidation in arterial wall.

Key words : apolipoprotein E-deficient mice, L-arginine, atherosclerosis, casein, cholesterol, L-methionine, nitric oxide, soy protein isolate


19-20

Effect of Soy Protein Isolate on Cytokine Productivity in Macrophages from Rats

Kazumi YAGASAKI, Wataru KOMATSU and Yutaka MIURA

Faculty of Agriculture, Tokyo Noko University, Tokyo 183-8509

Soy Protein Research, Japan1(Serial Vol.19), 106 - 110, 1998.

The effect of soy protein isolate ÅiSPIÅj on cytokine productivity in macrophages was studied in both normal and hepatoma-bearing rats, using casein as the control. When normal rats were fed a 20% casein diet Åi20CÅj or 20% SPI diet Åi20SÅj for 14 days, productivity of tumor necrosis factor-Éø ÅiTNF-ÉøÅj, interleukin-1 ÅiIL-1Åjand nitric oxideÅiNOÅj in resident peritoneal macrophages from the 20C-fed rats was similar to that from the 20S-fed ones. In contrast, productivity of TNF-Éø, IL-1 and NO was significantly lower in the 20S-fed animals than in the 20C-fed ones when each diet was fed to hepatoma-bearing rats for 14 days. Since cytokine productivity in macrophages is several times higher in the hepatoma-bearing state than in the normal state, SPI may be regarded as a depressant of enhanced cytokine production. There appears to be a marginal possibilty that a difference in arginine/lysine ratio between two protein sources is involved in the regulation of cytokine productivity.

Key words : cytokines, interleukin-1, macrophages, soy protein isolate, tumor necrosis factor


19-21

Utilization as Antitumorigenic Foodstuffs of Undigested High-molecular-weight Fraction ÅiHMFÅjand Curd Refuse ÅiOkaraÅjfrom Soy Protein

Kimikazu IWAMI, Naoyuki AZUMA, Hitoshi SUDA, Hiroyuki IWASAKI, Natsuko YAMAGATA, Tohru SAEKI and Ryuhei KANAMOTO

Faculty of Agriculture, Kyoto Prefectural University, Kyoto 606-8522

Soy Protein Research, JapanÅ@1(Serial Vol.19), 111 - 116, 1998.

Fischer rats under resectional operation on ileum and jejunum were taken as rat models devoid and possessed of active bile acid transporter, respectively, and examined for azoxymethane-induced colonic tumorigenesis. The rats were fed for 39 weeks 20% casein diets supplemented with 0.2% deoxycholate, and then the individual colons were excised to locate the tumors and to count their number. Supplementation of the diets with deoxycholate was necessary to actualize the experimental design. Ileal resection caused a considerable increase in tumor number compared to jejunal resection, indicating that such a secondary bile acid may well serve as an important risk factor for colonic tumorigenesis. Subsequently, azoxymethane-treated F-344 rats weighing about 300 g were fed 0.2% deoxycholate-supplemented diets including HMF, okara, gluten, codfish protein, yolk protein or casein at a 10% level. At 16, 22 or 28 weeks, tumor development in the colon was inspected by the use of a bronchial endoscope. As a result of endoscopic observation Åiat 28 weeksÅj, one-half the casein-fed group proved to bear tumorÅisÅj but only a rat did so in the HMF- or okara-fed group.

Key words : ileal resection, deoxycholate, colonic tumor, soybean curd refuseÅiokaraÅj, undigested high-molecular-weight fraction ÅiHMFÅj


19-22

Effect of Soybean Trypsin Inhibitor and Isoflavone on Chemically Induced Mammary Tumor in Rats

Ming-Fu WANG 1 , Li-Wen TSAO 1 , Yin-Ching CHAN 1 , Shun-Cheng WANG 2 , Jiunn-Wang LIAO 2 , Shu-Ying CHUNG 3 , Yue-Ching WONG 4 , Sumie SHINJO 5 , Kiyoharu TAKAMATSU 6 , Takashi YAMAMOTO 6 and Shigeru YAMAMOTO 7

1Department of Food and Nutrition, Providence University, Taichung, Taiwan
2Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Taichung, Taiwan
3Department of Nursing, Chungtai Institute of Health Sciences and Technology, Taichung, Taiwan
4Department of Nutrition, Chungshan Medical and Dental College, Taichung, Taiwan
5College of Education, University of the Ryukyus, Nishihara 903-0213
6Novelty Materials Institute, Fuji Oil Co., Izumisano 598-8540
7School of Medicine, The University of Tokushima, Tokushima 770-8503

Soy Protein Research, JapanÅ@1(Serial Vol.19), 117 - 123, 1998.

Many researchers have reported the anti-carcinogenic effects of soybean. Isoflavone and trypsin inhibitor are identified as the effective substances. We have also shown such effects of soybean whey Åirich source of trypsin inhibitorÅj in chemically induced skin-tumor mice and hepatoma rats. Last year we switched dietary protein from soybean to casein and were not successful to observe such effects of soybean whey and embryo bud ÅiEB, rich source of isoflavoneÅj on skin tumor in mice. In this study we tried to confirm such results in rats induced mammary tumor by chemical carcinogen. We fed control, whey or EB diet to female SD strain rats. Control diet contained 30% casein Åiisoflavone-free, trypsin inhibitor-freeÅj, EB diet contained 27% casein plus 3% EB Åiisoflavone 18.6 mg/100 g, trypsin inhibitor-freeÅj, and whey diet contained 27.5% casein plus 2.5% whey Åiisoflavone 0.5 mg/100 g, trypsin inhibitor 1,440 U/100 gÅj for 18 weeks. Rats were administered chemical carcinogen orally at the first week of the study, sacrificed at 18th week and incidence, number and weight of mammary tumor were measured. The result showed no difference in the mammary tumor among the three dietary groups, suggesting that soybean whey and isoflavone are effective to prevent cancer when soybean protein is used but not when casein is used.

Key words : trypsin inhibitor, isoflavone, mammary tumor, rat


19-23

Inhibitory Effect of Soybean Hypocotyls on Tumor Promotion: Effect of Dietary Hypocotyls on N-Nitroso-N-Methylurea-Induced Rat Tumorigenesis

Masazumi TAKESHITA, Yukihiro ZAIZEN, Noritaka MATSUO and Komei SHIRABE

Department of Biochemistry, Oita Medical University, Oita 879-5593

Soy Protein Research, JapanÅ@1(Serial Vol.19), 124 - 128, 1998.

We reported an inhibitory effect of the extract of soybean hypocotyls on tumor promotion examined with Epstein-Barr virus activating assay and mice skin tumor promoting test. In the present study, the effect of feeding the soybean hypocotyls on chemically induced carcinoma in rats was examined. In this experiment, rats were fed casein diet, soy protein isolate diet, 1.5% soybean hypocotyl diet or 5% soybean hypocotyl diet. Carcinogen was injected via tail veins of the rats at ages of 9 weeks and 29 weeks. Tumors were observed macroscopically at 42 weeks of age. One tumor was detected in one of 23 rats in casein group, and tumors were detected in 4 of 19 rats in soy protein isolate group, whereas in 1.5% and 5% hypocotyl groups, tumors were detected in 2 of 23 rats, and in 2 of 24 rats, respectively. Analysis of cumulative palpable tumor incidence indicated at 51 weeks of age tumor incidence of soy protein and hypocotyl diet groups was less than casein diet group. In the four diet groups tumors appeared least rapidly in 5% hypocotyl group.

Key words : soybean hypocotyl, chemically - induced tumor, isoflavone, rat, tumor promotion


19-24

Anti-carcinogenic Effect of Isoflavonoids in Soybeans

Hoyoku NISHINO

Department of Biochemistry, Kyoto Prefectural University of Medicine, Kyoto 602-8566

Soy Protein Research, JapanÅ@1(Serial Vol.19), 129 - 132, 1998.

In the previous study, we showed that genistein, one of the isoflavonoids found in soybean, inhibited the proliferation of prostate cancer DU145 cells. In addition to prostate cancer cells, various tumor cell lines were also proven recently to be sensitive to genistein. In the present study, we found that genistein induced morphological differentiation, and apoptosis in rat neuroblastoma B104 cells. In the previous study, we showed that oral administration of isoflavonoid-rich soybean hypocotyl powder resulted in suppression of spontaneous liver carcinogenesis in C3H/He male mice. Since various substances in soybean hypocotyl co-existed with isoflavonoids may have anti-carcinogenic activity and/or potentiate the anti-carcinogenic activity of isoflavonoids, studies on these soybean constituents seem to be valuable. In this context, we have started to assess biological activities of these substances. In the present study, we evaluated the anti-carcinogenic activities of soybean constituent-related compounds, i.e., myo-inositol and tocotrienol, and found that these substances were effective to suppress spontaneous liver carcinogenesis in C3H/He male mice, and also 4-nitroquinoline 1-oxide-initiated, and glycerol-promoted lung tumorigenesis in ddY male mice.

Key words : isoflavonoids, genistein, myo-inositol, tocotrienol, anti-carcinogenic activity


19-25

Administration of a Soy Protein Formula Product to Diabetic Patients

Kazuhiko KATO 1 and Toshiaki AOYAMA 2

1 Kato Clinic, Komae 201-0012
2 Novelty Materials Research Institute, Fuji Oil Co., Izumisano 598-8540

Soy Protein Research, JapanÅ@1(Serial Vol.19), 133 - 137, 1998.

Effectiveness of a soy protein formula product named ÅgKoresukkiriÅhÅi6 pieces of biscuit and a cup of soupÅjwas examined in diabetic patients. Thirty-three patients were divided into 2 groups, a drug ÅigliclazideÅjonly group and a drug with ÅgKoresukkiriÅhgroup. The treatments were continued more than one month. Food intake and blood biochemistry were examined before and after the treatment. Average energy intake was not different before and after ÅgKoresukkiriÅhtreatment. However, variability in energy intake among the subjects was reduced and eating habit was improved. Concentrations of serum Hb-A1C, glucose and cholesterol were tended to reduce by both treatments, and Hb-A1C and glucose were significantly decreased by the combination of the drug and ÅgKoresukkiriÅhÅiPÅÉ0.05Åj. Serum triglyceride concentration was reduced significantly by the combination of drug and ÅgKoresukkiriÅh, but was tended to increase in drug only group. Above results suggest beneficial effects on clinical condition and eating habit of diabetic patients without altering energy intake.

Key words : soy protein formula, diabetes, Koresukkiri


19-26

Long-term Effect of Soy Protein on Serum Lipids, Platelet Aggregation, Hemostatic Markers and Early Atherosclerosis in Hypercholesterolemic Patients : Comparison to Low-fat Milk

Masako WAKI, Motoo TSUSHIMA, Naoko YAMASHITA, Masaaki SUZUKI and Hideki KOH

1 Shimada Municipal Hospital, Shimada 427-8502
2 National Cardiovascular Center, Suita 565-8565

Soy Protein Research, JapanÅ@1(Serial Vol.19), 138 - 142, 1998.

The long - term effects of soy protein intake on serum lipids, platelet aggregation, hemostatic markers, and early atherosclerosis of carotid arteries and lower abdominal aorta were compared to those of low - fat milk. Fifteen middle - aged hypercholesterolemic females, randomly assigned to take 9 g/day of soy protein or 400 mL/day of low - fat milk, were examined at baseline and after 18 months . Nine subjects with soy protein did not show any significant changes in serum levels of cholesterol ÅimeanÅ}SD, 248Å}37 mg/100 mL at baseline and 240Å}33 mg/100 mL after 18 monthsÅj, triglyceride, HDL - cholesterol, apoproteins AI, AII, B, CII, CIIIand E, platelet aggregation induced by ADP and collagen, or hemostatic markers Åifibrinogen, PAI-1, D-dimer, plasminogen, AT-III, É¿TG and PF4Åj. Six subjects with low - fat milk also showed no significant changes in these markers except total cholesterol level which was slightly increased Åi250.0Å}16.5 mg/100 mL at baseline to 270.2Å}25.1 mg/100 mL after 18 monthsÅj. Arterial calcification ÅiACVÅjand wall thickening and volume ÅiAWVÅjin lower abdominal aorta were estimated by computed tomography at baseline and one year of the study. In soy protein group, ACV did not progress during the study and the increase in AWV was smaller than that in low - fat milk group, although the difference between the groups was not reached the significance. Annual increase in intima-media complex thickening ÅiIMTÅjon common carotid arteries estimated by ultrasound sonograghy was smaller in the soy protein group ÅiÅ|0.02Å}0.02 mmÅjthan that in low-fat milk group Åi0.01Å}0.01 mmÅj. These findings suggest that the progression of early atherosclerosis might be modulated by soy protein intake in hypercholesterolemic patients.

Key words : soy protein, milk, atherosclerosis, carotid artery, intima-media complex thickness



19-27

Time-lag Analysis of Interrelationships between Dietary Animal and Plant Protein Intakes and Colon Cancer Mortality in Japanese

Keisuke TSUJI 1 , Emiko HARASHIMA 2 , Asako MIURA 2 and Yasue NAKAGAWA 2

1 School of Humanity for Environment Policy and Technology, Himeji Institute of Technology, Himeji 670-0092
2 Faculty of Human Life Sciences, Jissen Women's University, Hino 191-8510

Soy Protein Research, JapanÅ@1(Serial Vol.19), 143 - 150, 1998.

The daily intake of protein ÅiPÅjwas evaluated from data collected by the National Nutrition Survey in Japan over a period of 49 years since 1947. The interrelationships between total dietary P or individual P sources and the mortality from colon cancer ÅiMCCÅjwere determined by a simple correlation coefficient and a time - series correlation coefficient. Total and animal P intakes per capita increased, especially the latter increased about 4.5 times. Plant P decreased from 53 g to 37 g. As MCC increased year by year, animal and plant Ps showed positive and negative simple correlation with MCC, respectively. A time - series analysis indicates that MCC of male Japanese has a significantly positive correlation with the intake of total P after a 17 - yearsÅfdelay. Similar tendencies were observed with animal, meat, egg and milk Ps. On the contrary, plant and cereal Ps showed the negative maximum correlation with MCC after 20 - 32 yearsÅfdelay. Different sources of P may influence the risk of colon cancer after a long delay of years.

Key words : protein intake, colon cancer mortality, correlation coefficient, time - lag, Japanese



19-28

Influence of Nutritional Education Program for Mother on ChildrenÅfs Dietary Patterns

Seiki NAMBU 1 , Ikuko MATSUI 1 and Susumu HATTORI 2

1 The 3rd Division, Hyogo Institiue of Clinical Research, Akashi 673-8558
2 Division of Public Health, Zohda Public Primary School, Nagao 761-0900

Soy Protein Research, JapanÅ@1(Serial Vol.19), 151 - 157, 1998.

Westernization of the Japanese diet has significantly contributed to the improvement of nutritional conditions. However, it is also one of the most important factors that have caused health problems in Japanese children. The purpose of this study is to clarify exactly in what way the nutritional education program aimed at mothers can help promote the Japanese childrenÅfs health promotion. A. The study on the actual nutritional intake by Japanese mothers showed a remarkable decrease in the consumption of traditional Japanese style food. 1. When comparing the data of the rural and those of urban areas, it has been confirmed that the intake of rice in urban areas has decreased while the intake of bread has increased. 2. When comparisons were taken between two groups of housewives, group 1, 45 years of age or younger, and group 2, 46 years of age or older, the above mentioned results were conspicuous even in the rural districts. Another point that we clearly observed was that housewives who were 45 years of age or younger living in urban areas had a higher intake of meat and lower intake of fish and soy beans foods, in comparison to housewives who were 46 years of age or older. B. It has been substantiated that 38% of the mothers who received the nutritional education program attempted to make use of what they learned from the program. It has especially been observed that the recent intake of green and yellow vegetables has increased and the frequency of overweight people has decreased. C. The validity of this nutritional education program to Japanese mothers will be evaluated using the results from childrenÅfs health status as indicators Åie.g. body build indices, thickness of subcutaneous fat, serum lipid and serum insulinÅj. Additionally, diet-information as indicators shown in Table 5.

Key words : nutritional education, indicators for health promotion, school - children, Japanese traditional diet, maternal diet behavior


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