Functional Analysis of a Novel Jasmonate-inducible Cytochrome P450, CYP93A1, in Soybean : the Involvement in Isoflavone Biosynthesis and Induction by a Fungal Elicitor
Hiroyuki OHTA, Hideaki SUEOKA, Genki SUZUKI and Ken-ichiro TAKAMIYA
Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501
Soy Protein Research, Japan, 2(Serial Vol. 20), 1-4, 1999.
ABSTRACT
We have isolated two cDNAs encoding novel jasmonate-inducible proteins from soybean suspension-cultured cells. One of the clone encodes a cytochrome P450 belonging to a new family, CYP93 (CYP93A1). CYP93A1 mRNA was also specifically induced in soybean cells by an elicitor which was drived from cell wall fraction of fungal pathogen, Phytopthora megasperma f. sp glycinea. Furthermore, the mRNA was induced earlier than the accumulation of phytoalexin glyceollin in soybean cotyledon. These facts indicated that the induction of CYP93A1 mRNA is closely associated with the biosynthesis of glyceollin, a major phytoalexin in soybean cells. Thus, we analyzed the activities of (2S)-flavanone 2--hydroxylase and isoflavone synthase using CYP93A1 protein expressed in insect cells. However, no activity was observed. Independently, Schopfer et al also analyzed the function of the CYP93A1, and found that the enzyme is dihydroxypterocarpan 6a-hydroxylase. We screened the genomic gene of CYP93 family using 1.2kb fragment of CYP93A1 cDNA, and obtained two different types of clones corresponding to CYP93A2 and CYP93A3.
Key words : isoflavonoid, cytochrome P450, elicitor, phytoalexin, disease -resistance
Evolutionary Relationship between b-Conglycinin and Glycinin and Attempts to Improve Food Functions of b-Conglycinin Based on the Relationship
Ryouhei SATOH, Nobuyuki MARUYAMA, Motoyasu ADACHI and Shigeru UTSUMI
Research Institute for Food Science, Kyoto University, Uji 611 -0011
Soy Protein Research, Japan, 2(Serial Vol. 20), 5-10, 1999.
ABSTRACT
Soybean proteins consist of two major components, glycinin and b-conglycinin. Both proteins are composed of two domains (glycinin, the acidic and the basic polypeptides; b-conglycinin, N- and C-terminal half molecules). It is believed that both proteins are derived from a duplication of a common ancestral gene. Each domain of both proteins has different hydrophobicity to each other. These suggest that creations of chimeric proteins with novel physicochemical properties between glycinin and b-conglycinin and hexameric b-conglycinin by introduction of the processing site (Asn-Gly) of glycinin and processing may be possible. We constructed Escherichia coli expression systems for b-conglycinin b having the processing site and chimeric proteins 11A-7C, 7N-11B, 11A-7N and 7C-11B between b-conglycinin b (7N and 7C) and glycinin A1aB1b (11A and 11B). The b having the processing site was processed, but did not form a hexamer by itself. Folding abilities of chimeric proteins were lower than those of the original b and A1aB1b. The order of the folding abilities of the chimeric proteins was 11A-7C =7C-11B >11A-7N >>7N-11B (soluble 7N-11B could not be obtained). Only 7C-11B formed oligomers and the others were monomers, although all three chimeras formed similar secondary structures to those of the original proteins. These suggest that b-conglycinin and glycinin followed a different evolutionary mechanism from a common ancester. Physicochemical properties of 11A-7C and 7C-11B were examined. Although the original b exhibited ionic strength-dependency of solubility at pH 7.6 (insoluble <m=0.3), 11A-7C and 7C -11B were soluble at any ionic strengths in analogy with A1aB1b. Both chimeric proteins exhibited better emulsifying abilities than the origina b and A1aB1b did, especially at m=0.1 at pH7.6. These indicate that creation of chimeric proteins having good food functions is possible.
Key words : soybean, b-conglycinin, glycinin, chimera, food functions
New Aspartic Proteinases of Rice and Soybean Origin :
Development and Application to the Modification of Food Proteins
Keiko ABE 1 and Tomiko ASAKURA 2
1 Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113 -8657
2 Laboratory of Food Science, Atomi Junior College, Tokyo 112 -8687
Soy Protein Research, Japan, 2(Serial Vol. 20), 11-15, 1999.
A number of proteinases occur in food plant seeds. These enzymes play a crucial role in the metabolism of seeds by processing newly synthesized proteins into mature forms during ripening and by decomposing storage proteins into amino acids during germination. However, details of plant proteinases are not so well known as those of animal proteinases including pepsin as a digestive enzyme, cathepsins D and E which are involved in the intracellular protein catabolism, and renin as a blood pressure-controlling enzyme. The aspartic proteinases (APs) of plant origin which, structurally different from those of animal, insect and microbial origins, are each characterized by bearing a ca. 100-amino acid insertion in the C-terminal region. A good example can be provided by rice APs (Oryzasins I-IV) and cardon AP which is contained in an extract of this flower and used for milk clotting to make cheese in Portugal. Considering such a background, we carried out experiments of finding out a new AP that may occur in soybean as well. We first screened cDNA libraries constructed from ripening and germinating rice seeds and thus obtained several independent clones encoding APs. These were found to share 60-80% similarity to one another, each conserving the insertion which characterizes to APs of plant origin. Since one of cloned APs, named soy APa, was most different from oryzasin 1, we tried to express the protein by transformation into E. coli, with a satisfactory result.
Key words : aspartic proteinase, soybean, expression
Kinetic Analysis of Freeze Denaturation of Soy Protein
Osato MIYAWAKI
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113 -8657
Soy Protein Research, Japan, 2(Serial Vol. 20), 16-19, 1999.
Freeze-induced concentration of protein triggered freeze denaturation of soy protein isolate (SPI) in aqueous solution. The SPI denaturation was a rate process strongly dependent on time and temperature. A kinetic model was proposed to describe the freeze denaturation in consideration of freeze-induced concentration of solute, which accelerated the reaction rate substantially. The theoretical model was effective to describe the freeze denaturation process of SPI. The apparent reaction order was 2 suggesting that the reaction cascade of freeze-induced denaturation of SPI is limited by the initial interaction between the two protein molecules. The influence of temperature on SPI denaturation and the protective effect of glucose against denaturation were also effectively explained by the theoretical model.
Key words : soy protein, freeze denaturation, freeze concentration, kinetic model
Deodorization of Soybean Proteins by Enzymatic and Physicochemical Processings
Kuniyo INOUYE
Graduate School of Agriculture, Kyoto University, Kyoto 606 -8502
Soy Protein Research, Japan, 2(Serial Vol. 20), 20-27, 1999.
In order to establish a convenient and effective process for deodorization of soy protein isolate (SPI), various solid absorbents such as polystylene, polymetacrylate, zeolite and charcoal were examined. The efficiency of deodorization was evaluated by measuring hexanal and linoleic acid as well as by a sensory test. The content of hexanal in the SPI solution was decreased in all cases, while the content of linoleic acid was not in any cases. Although a brominated polystyrene absorbent (Sepabeads) and a zeolite absorbent (HSZ) removed hexanal more effectively than charcoal, a considerable amount of hexanal remained (more than one third). On the other hand, a model experiment showed that the absorption ability of these absorbents for hexanal was much higher than the content in the SPI solution. These results indicated a possibility that hexanal in the SPI solution was classified into two states : 1. free or bound on the surface of proteins (removable by absorbents) and 2. bound at the inside of proteins (unremovable). This idea was supported by the evidence that the excess hexanal added to the SPI solution was mostly removed by the absorbents. The chymotryptic digestion of the SPI solution did not promote the removal of hexanal by the absorbents, suggesting that hexanal in state 2 might exist in the similar state even in the digests. Despite the considerable remaining of hexanal after treatment by the absorbents, we succeeded in preparing the SPI solution deodorized well in the sensory test. It strongly suggested that hexanal in state 1 but not in state 2 was related to the soybean odor.
Key words : absorbent, hexanal, deodorization, soybean, soy protein isolate
Preparation and Characterization of Novel Food Materials from Soybean Extract by Heat Treatment
Naofumi KITABATAKE and Yuki FUJITA
Research Institute for Food Science, Kyoto University, Uji 611 -0011
Soy Protein Research, Japan, 2(Serial Vol. 20), 28-32, 1999.
Defatted soybean extract was dialyzed against distilled water at pH7.5. The dialysate was a transparent solution having less beany flavor. Change in the aldehyde level of defatted soybean extract during dialysis was measured with aldehyde dehydrogenase purified from bovine liver mitochondria. Aldehyde level was markedly dropped by 3h dialysis and it became below the measurement limit by the enzyme method after 20h dialysis, and the beany flavor could not be detected after 6h dialysis. Since it is known that aldehyde dehydrogenase is able to oxidize the aldehydes bound on the protein molecules, it seems that such aldehydes should be removed by dialysis of defatted soybean extract against distilled water at pH7.5. Soybean protein was precipitated by acid precipitation method and dissolved by neutralization, and precipitated again. This treatment was repeated three times. The aldehyde level in the neutralized solution was measured. By the first precipitation aldehyde level was lowered remarkably, while the second and third precipitation could not give marked reduction of aldehyde level, suggesting that the bound aldehydes on protein molecules could not be eliminated by the precipitation method. From these results acid precipitation of soybean protein may bind or incorporate aldehydes in the defatted soybean protein extract and such aldehydes before acid precipitation could be removed by dialysis against water at pH7.5.
Key words : soybean extract, soybean flavor, soybean isolate protein, removal of aldehydes
Emulsification Characteristics of Water-soluble Soybean Polysaccharides from Soybean Residue
Hidefumi YOSHII and Takeshi FURUTA
Faculty of Engineering, Tottori University, Tottori 680 -855
Soy Protein Research, Japan, 2(Serial Vol. 20), 33-37, 1999.
Okara (soybean card waste), a by-product of tofu and soybean protein manufacturing, is treated as industrial waste. Maeda has proposed a mean of extracting soybean water-soluble polysaccharides (SSPS) from okara, and has used them as an emulsifier or viscoelastic reagent. SSPS has a high emulsifying ability of d-limonene in the acidic conditions as that of arabia gum. An emulsified solution of ethyl butyrate with arabia gum was very unstable. SSPS could emulsify ethyl butyrate at pH 3-6. The microencapsualtion of flavors by spray drying of an emulsified liquid flavor was investigated. SSPS was found to be a superior emulsifier over gum arabic to retain ethyl butyrate.
Key words : SSPS, spray drying, encapsulation, flavor
Preparation and Characterization of Edible Films from Fish Water Soluble Proteins and Soybean Proteins
Munehiko TANAKA
Faculty of Fisheries, Tokyo University of Fisheries, Tokyo 108 -8477
Soy Protein Research, Japan, 2(Serial Vol. 20), 38-43, 1999.
As a means of effective utilization of fish water soluble proteins (FWSP) which are mostly discarded into waste water of seafood processing plants, the development and characterization of edible films from FWSP of blue marlin meat were investigated. In order to improve the functional properties of FWSP films, the co-use of soybean proteins (SPI) was also studied. The film-forming solutions were prepared from 3%FWSP or SPI solutions at pH10 with 1.5% glycerol, followed by heating at 70oCfor 15min. Transparent edible films were successfully prepared by drying the film-forming solutions at 25oCfor 20h. Tensile strength (TS) of FWSP films and elongation at break (EAB) of SPI films were larger. Larger amount of glycerol caused the decrease of TS and the increase of EAB. FWSP films possessed lower water vapor permeability than SPI films. It was also revealed that FWSP and SPI had to be denatured somehow to unfold the protein structure and the interaction of protein molecules particularly through disulfide linkages was attributed to the formation of films.
Key words : edible film, water soluble proteins, soybean proteins, plasticizer, water vapor permeability
Industrial Applications of Soy Protein Isolate : Effects of Plasticizers on Mechanical Properties
Shuhei SOGOYA and Kiyoshi YAMAUCHI
Faculty of Engineering, Osaka City University, Osaka 558 -8585
Soy Protein Research, Japan, 2(Serial Vol. 20), 44-48, 1999.
Various compounds were examined as plasticizers in the preparation of the films made of soy protein isolate (SPI). Glycerol, sulfolane, tetramethylene sulfoxide and 2, 2'-sulfonyldiethanol were effective to afford flexible films with maximum elongation strength of 80-220 kg/cm2 and elongation of 4-50% in a relative humidity of about 65%. Upon treating with an aqueous formaldehyde, the films were fortified against biodegradation in soil for 2 -3 weeks.
Key words : biodegradation, film, plasticizer, mechanical property, soy protein isolate
Photon Emission Characteristics of Soybean Lipoxygenase in the Presence of Reactive Oxygen Species (X), Hydrogen Donors (Y) and Mediators (Z), and the Application
Yumiko YOSHIKI, Kumi SATAKE, Tetsuo IIDA, Masaaki KAWANE and Kazuyoshi OKUBO
Graduate School of Agricultural Science, Tohoku University, Sendai 981 -8555
Soy Protein Research, Japan, 2(Serial Vol. 20), 49-54, 1999.
The novel screening test by photon emission in the presence of 2% HOOH (X), 5 mM gallic acid (Y, GA) and 2% CH3 CHO (Z, MeCHO) indicated that soybean lipoxygenase (LOX) was as Z rather than Y. Galloyl moiety on C-3 and hydroxyl moity on C-5' of catechin group were effective on the photon emission of LOX in the XYZ system. ÅmPÅn= kÅmXÅnÅmYÅnÅmZ] was observed at low concentration of tert-butyl hydroperoxide (tert-BuOOH) on the photon emission of LOX in the XYZ system. It made clear that photon emission of LOX in the XYZ was not caused by singlet oxygen. Ee (kcal/mol) calculated from emission spectra indicated that the photon emission should be caused by excited triplet carbonyl via O-O bond dissociation. Although photon emission of hemoglobin (Hb), superoxide dismutase (SOD) and horseradish peroxidase (HRP) was observed at some specific hydroperoxide, the photon emission of LOX was observed at HOOH, tert-BuOOH, methylethylketon hydroperoxideÅiMEKOOHÅjand cumene hydroperoxide (cumeneOOH). Indeed, the photon intensity from the oil/GA/LOX system corresponded with peroxidation of linoleic acid and food oil such as salad oil.
Key words : photon emission, chemiluminescence, soybean lipoxygenase, reactive oxygen scavenging activity, scavenger
Research for a New Antioxidative Pathway Activated by Soybean Protein
Asako TAKENAKA
Faculty of Agriculture, Yamagata University, Yamagata 997 -8555
Soy Protein Research, Japan, 2(Serial Vol. 20), 55-58, 1999.
Oxidative stress results in part from the production and accumulation of reactive oxygen species, and is supposed to cause critical damage to DNA, protein and cellular membranes. This study is undertaken to find a new antioxidative pathway which is activated by soybean protein intake in vivo. At first, we fed male Wistar rats diets containing 20% casein, 8% casein and 8% soybean protein as protein sources and examined antioxidative enzyme activities, glutathione contents, and lipoperoxide concentration in the liver. Soybean protein intake did not affect liver antioxidative enzyme activities and liver glutathione contents, while decreased liver lipoperoxide level (estimated as TBARS). These results show a possibility that soybean protein intake can prevent peroxidation. Therefore, we constructed a new experimental system to find a protein concerning for prevention against peroxidation and at the same time activated by soybean protein intake. cDNA library from male Wistar rats was constructed using pYES2 vector and transformed into INVSc1 Saccharomyces cerevisiae. Colonies which can survive on the plate containing 3.3mM paraquat was screened.
Key words : soybean protein, antioxidative pathway
Stability of Soybean Lectin during Food Processing and in Digestive Tract
Yukiko UMEZAWA, Yuhki SATO, Takako NAGANUMA, Tomohisa OGAWA and Koji MURAMOTO
Graduate School of Agricultural Science, Tohoku University, Sendai 981 -8555
Soy Protein Research, Japan, 2(Serial Vol. 20), 59-64, 1999.
Soybean lectin (agglutinin, SBA) is a N-acetylgalactosamine binding protein which can interact with different types of cell and exert various biological activities. This study demonstrated the extent of survival of SBA during food processing and in digestive tract to explore its applicability as a physiological modulator originated from foods. The lectin kept a significant activity even after 2 h at 80oC, and the heat-resistancy improved in the presence of other food components as in soy milk. SBA was stable at neutral or acidic pH, however, its activity was abolished at alkaline pH due to the aggregation. Proteolytic digestion with pancreatin, trypsin and chymotrypsin did not affect on the lectin activity, whereas peptic digestion gradually inactivated SBA to produce the lectin fragments of Mr ~ 4000. ELISA assay of circulating blood in mice which were given SBA by stomach-tube showed that variable but significant amounts of the SBA fragments could across the intestinal barrier. The immunoreactivity level became to be maximal 4 to 6 h after administration of SBA. Gel filtration HPLC analysis of the sera from mice which were orally administered FITC-SBA showed the SBA fragments of Mr ~ 4000 penetrated the intestinal wall and circulated in blood. Furthermore, FITC-SBA and its fragments still remained on the small intestinal mucosa 8 h after administration.
Key words : soybean agglutinin, lectin, hemagglutination, ELISA, FITC
Development of Assay System for Immunoglobulin Production Regulating Factors Using Mouse Lymphocytes and Its Application
Koji YAMADA 1 , Mikako TAKASUGI 2 , Yuki TAMURA 1 , Yoshiyuki MIYAZAKI 1 , Michihiro SUGANO 3 and Hirofumi TACHIBANA 1
1 Division of Bioresource and Bioenvironmental Sciences, Graduate School Kyushu University, Fukuoka 812 -8581
2 Faculty of Human Life Science, Yamaguchi Prefectural University, Yamaguchi 753 -8502
3 Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, Kumamoto 862 -8502
Soy Protein Research, Japan, 2(Serial Vol. 20), 65-69, 1999.
To clarify the regulatory mechanism of immunoglobulin (Ig) production by food components, we tried to establish the assay system using mouse spleen lymphocytes. At first, we examined the effect of adhesive cells on Ig production by mouse splenocytes and found that adhesive cells markedly enhance Ig production of the lymphocytes. When the spleen lymphocytes were isolated after various adhesive periods to remove adhesive cells, decrease of Ig level was observed with the elongation of adhesive periods and the decreasing rates were varied with Ig classes. The stimulatory effect of adhesive cells was observed most markedly in IgA production within 1 day cultivation, but the effect was observed after 2 to 5 days latent period in IgG and IgM production. When the cells were cultured with anti-cytokine antibodies, IgG production was enhanced in the presence of anti-IL-2 and anti-IL-4 antibodies. These results suggest that cytokines are involved in the class specific enhancement of Ig production by adhesive cells. In addition, Ig production of the lymphocytes was strongly enhanced by the addition of lipopolysaccharide, pokeweed mitogen and phytohemagglutinin, but suppressed by concanavalin A. When the lymphocytes were cultured with 60% ethanol extract of soybean protein and its diluted samples, stimulation of Ig production was observed in the presence of 10 times diluted sample and 10 6 times diluted samples, especially in the absence of adhesive cells.
Key words : mouse splenocytes, immunoglobulin production, lectin, cytokine, soybean protein extract
Effect of Soybean Trypsin Inhibitor on in vivo Digestibility and Oral Immunogenicity of Allergenic Proteins
Noriko NINOMIYA, Chikako YAMADA and Tsukasa MATSUDA
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464 -8601
Soy Protein Research, Japan, 2(Serial Vol. 20), 70-75, 1999.
In the previous report (Ninomiya N et al, Soy Protein Research, Japan, 1, 75 -80, 1998), Kunitz soybean trypsin inhibitor (KSTI) intragastlically (i.g.) administered to mice was shown to remain intact mostly in small intestine and inhibit intestinal trypsin activity, suggesting the lowering of food protein digestibility. In the present study, the inhibitory effect of i.g. administered KSTI on three intestinal proteases, trypsin, chymotrypsin, elastase was investigated and compared with that of egg lysozyme, which induced immune response by i.g. administration. Almost no trypsin activity was detected in the small intestine of the mice, when KSTI was administered. Furthermore, both of chymotrypsin and elastase activities of the mice administered KSTI were also much lower than those of mice given lysozyme or phosphate buffered saline. These results suggested that KSTI inhibited trypsin and markedly reduced the protease activity in the small intestine, and thereby remained intact in large quantity in the small intestine. Furthermore, the effect of the amount of protein administered i.g. on serum antibody response and the protein remaining in the small intestine was also investigated using egg lysozyme as a model antigen. The increase in the amount of protein administered did not increase the amount of intact protein in the small intestine but that of fragments which retained antigenic activity.
Key words : soybean trypsin inhibitor ÅiKSTIÅj, food allergy, digestibility
Effect of Soybean Isoflavone on Bone Metabolism
Kyoko MORITA, Yuko HAMAMATSU, Shinsuke KIDO, Yutaka TAKETANI, Ken-ichi MIYAMOTO and Eiji TAKEDA
School of Medicine, The University of Tokushima, Tokushima 770 -8503
Soy Protein Research, Japan, 2(Serial Vol. 20), 76-82, 1999.
Genistein is an isoflavone abundantly presents in soybeans, and shows a structural similarity to estrogen, which suggests that it may act as a phytoestrogen. Recently, genistein has been shown to have a stimulatory effect on bone formation and an inhibitory effect on osteoclastic bone resorption. In addition, genistein has been reported to be as active as estrogen in maintaining bone mass in ovariectomized (OVX) animals. However, the mechanisms for the action of isoflavone as a phytoestrogen remain unknown. Here, we investigated the effect of genistein on the expression of type I collagen (COL I) alkaline phosphatase (AP), osteopontin (OP) and osteocalcin (OC) genes that have been associated with bone formation in mouse osteoblastic cells (MC3T3-E1 cells) and OVX mice. In MC3T3-E1 cells, genistein as well as estrogen increased the amount of OP mRNA. No significant difference was observed in the levels of COL I, AP and OC mRNAs. In OVX mice, the weight of uterus was significantly decreased compared with the sham-operated control. Estrogen completely restored the weight of uterus in the OVX mice, whereas genistein did not affect. The levels of the four transcripts in the bone were markedly decreased in the OVX mice. Estrogen increased the levels of COL I mRNA, but not AP, OP or OC mRNA. Similar findings were observed in the genistein treated OVX animals. These results indicate that genistein exhibits estrogenic action in bone of OVX animals without estrogenic action in the uterus. Thus, these results suggest that soybean containing genistein may be useful nutritional source in the prevention of osteoporosis.
Key words : isoflavone, genistein, estrogen, osteoporosis, MC3T3 -E1 cells
Anti-osteoporotic Factors Found in the Soybean
Shigeru MATSUZAKI, Takeshi SAGA and Kaoru ICHIMURA
Dokkyo University School of Medicine, Tochigi 321 -0293
Soy Protein Research, Japan, 2(Serial Vol. 20), 83-87, 1999.
Epidemiological studies have shown that incidence of osteoprosis in Japan is much lower than in European and North American countries and that signs of this disease here are not so severe as in these countries. It has been suggested that the Japanese diet rich in products of soybean is beneficial for the prevention of osteoporosis. In the present study, isoflavones such as genistein and daidzein and an extract of soybeans were tested for their preventive effects on bone metabolism in ovariectomized rats. Both genistein and daidzein were effective in lowering urinary excretion of pyridinoline and deoxypyridinoline, markers of bone resorption. An extract of soybeans which contained neither genistein nor daidzein also suppressed bone resorption. This extract, unlike the two isoflavones, increased the uterine weight of ovariectomized rats. These results show that soybeans contain estrogenic factor (s) other than genistein and daidzein which suppress bone resorption in ovariectomized rats. Identification of the anti-osteoporotic factor (s) in soybeans is now under way.
Key words : genistein, daidzein, soybean, ovariectomized rat, osteoporosis
Isoflavones as Modifier of Estrogen Metabolism
Kiyoshi EBIHARA, Taro KISHIDA and Mami BEPPU
Faculty of Agriculture, Ehime University, Matsuyama 790 -8566
Soy Protein Research, Japan, 2(Serial Vol. 20), 88-93, 1999.
The effect of soy isoflavone on the cytochrome P-450 content of the hepatic microsomes and estrogen metabolism were studied. Most of the daidzin and genistin in soybean meal (SBM) were converted into the respective aglycones, daidzein and genistein, by fermentation with Aspergillus awamori. In experiment 1, ddy mice were fed isonitrogenous test diets with one of the following four protein sources for 28 d: casein, SBM, fermented and freeze-dried SBM (FSBM-FD), or methanol-extracted FSBM-FD (FSMB-FD-R). The cytochrome P-450 content was significantly higher in the mice fed the FSBM-FD diet than the respective value in mice fed the other test diets. In experiment 2, ddy mice were fed one of eight diets which contained different levels of aglycone obtained by varying the proportion of FSBM-FD and FSBM-FD-R, for 28 d. The cytochrome P-450 content in hepatic microsomes increased as the dietary level of isoflavonoid aglycones increased, but there was a saturation phenomenon. In experiment 3, ovariectomized C3H-HeJ mice fed one of the following four test diets for 16 days: casein diet (C diet), C+E2 (1mg/kg diet)(E2 diet), E2 diet+genistein (100mg/kg diet)(G100 diet) or E2 diet+geninstein (200mg/kg diet)(G200 diet). G100 and G200 diets did not have effects upon the 2-OHE1 to 16-OHE1 ratio.
Key words : isoflavones, cytochrome P -450, estrogen metabolism
Anti-carcinogenic Effect of Soybean Hypocotyl
Tadashi YASUHARA1, Toshifumi AKIZAWA2, Akiko SUGIMOTO3 and Shaw WATANABE4
1 Junior College of Agriculture, Tokyo University of Agriculture, Tokyo 156-8502
2 Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata 573-0101
3 Institute for Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo 101-0062
4 Faculty of Applied Bio -Science, Tokyo University of Agriculture, Tokyo 156-8502
Soy Protein Research, Japan, 2(Serial Vol. 20), 94-98, 1999.
It is proven that metalloproteinase (MMP) lyses extracellular matrix (ECM). Of all MMPs, membrane type-MMP (MT-MMP) is especially associated with tumor metastasis as well as the formation of nascent vessels. Although new tumor treatment drugs derived from substances with MMP inhibition activity are desired, the problem of side effects caused by candidate inhibitors is still left unsolved. Soybean hypocotyl, the safety of which is long proven, was chosen in search of effective yet safe MMP inhibitors. Soybean is known to contain a number of functional substances including genistein which inhibits the propagation of tumor cells. The study was aimed to isolate unknown substances with MMP inhibition activity in soybean hypocotyl. Soybean hypocotyl was analyzed on HPLC using a reverse-phased column to determine MMP inhibition activity of each fraction. As a result, MMP inhibition activity was found in a 60% CH3CH fraction. Upon comparing the retention time of these two unknown substances and Biochanin A on subsequent HPLC analysis, they were found to be different from isoflavonoids already found in soybean hypocotyl. These substances are now being analyzed for the determination of their structure.
Key words : soybean hypocotyl, metalloproteinaseÅiMMPÅj, MMP inhibitor
Effect of Dietary Protein on Anti-carcinogenesis of Soybean Trypsin Inhibitor and Isoflavone in Rats
Ming-Fu WANG 1 , Tatsushi KOMATSU 2 , Yin-Ching CHAN 1 , Yue-Ching WONG 3 , Li-Wen TSAO 1 , Ching-Lein WU 1 , Cheng-Chung LIN 4 , Sumie SHINJO 5 , Tzu-Hsiu CHEN 2 and Shigeru YAMAMOTO 2
1 Department of Food and Nutrition, Providence University, Taichung, Taiwan
2 School of Medicine, The University of Tokushima, Tokushima 770 -8503
3 Department of Nutrition, Chungshan Medical and Dental College, Taichung, Taiwan
4 Graduate Institute of Veterinary Pathology, National Chung -Hsing University, Taichung, Taiwan
5 College of Education, University of the Ryukyus, Okinawa 902 -0213
Soy Protein Research, Japan, 2(Serial Vol. 20), 99-105, 1999.
Isoflavone (IF) and trypsin inhibitor (TI) are identified as anti-carcinogenic substances of soybean. We have shown such effects of soybean whey (rich source of TI) in mice and rats. However, when we switched dietary protein from soybean to casein, the effect was not observed. We hypothesized that the effects may be due to the combination of TI and IF and this study was designed to clarify it. IF was supplied by embryo bud (EB, rich source of IF). Five diets with different combination of TI and IF were prepared. They were; Casein diet (TI, 0 U/100g diet ; IF, 0mg/100g diet), SPI diet (TI, 422 ; IF, 55), SPI+TI inactivated SP diet (TI, 310 ; IF, 81), SPI +TI active SP diet (TI, 1310 ; IF, 81), and SPI+TI inactivated SP+EB diet (TI, 290 ; IF, 97). Incidences of the mammary tumor were 95, 75, 80, 55 and 30% in the Casein, SPI, SPI+TI inactivated SP, SPI+TI active SP and SPI+TI inactivated SP+EB group, respectively. The results support our hypothesis that anti-mammary tumor effect was due to the combination of TI and IF.
Key words : trypsin inhibitor, isoflavone, mammary tumor, rat
Effects of Dietary Soy Protein Levels and Amino Acid Supplement on Cytokine Productivity in Macrophages from Nephritic Rats
Kazumi YAGASAKI, Junko NAGATA and Yutaka MIURA
Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 183 -8509
Soy Protein Research, Japan, 2(Serial Vol. 20), 106-111, 1999.
The effect of dietary soy protein isolate (SPI) on cytokine productivity in macrophages was studied using nephritc rats. Nephritis was induced in rats by injecting nephrotoxic serum into tail vein. The animals showed proteinuria, hypoalbuminemia and hyperlipidemia 3, 7 and 14 days after the injection. Under these conditions, the productivity of tumor necrosis factor-a (TNF-a) in resident peritoneal macrophages was found to be higher in nephritc rats than in normal ones, indicating a stimulation of TNF-a productivity in the nephritic state. To examine the effects of SPI on the stimulated productivity of TNF-a, nephritic rats were fed either a 20% casein diet (20C), 20% SPI diet (20S), 8.5% SPI diet (8.5S), or 8.5S supplemented with 0.3% L-methionine (8.5SM) for 5 or 10 days. The feeding of 20S for 10 days, as compared with that of 20C, was demonstrated to significantly suppress the stimulation of TNF-a productivity by 73%, suggesting an anti-inflammatory action of SPI in the nephritic state.
Key words : cytokine, macrophage, nephritis, soy protein isolate, tumor necrosis factor-a (TNF-a)
Effect of Soy Protein Isolate on Muscle Atrophy under Simulated WeightlessnessÅ@by Suspension Hypokinesia / Hypodynamia in Rats
Osamu TADA and Hidehiko YOKOGOSHI
Graduate School of Nutritional and Environmental Sciences, The University of Shizuoka, Shizuoka 422-8526
Soy Protein Research, Japan, 2(Serial Vol. 20), 112-117, 1999.
Under the microgravity condition like the universe, it is known that various nutritional and physiological changes in the body are induced. Especially in the aspect of nutrition, muscle atrophy is characteristic accompanying the weightlessness. In this study, we examined the ameliorative effect on muscle atrophy caused by suspension hypokinesia using soy protein isolate (SPI) as the protein source as compared with casein. Male rats of the Wistar strain (8 weeks old) were divided into two groups, suspended with suspension harness, and were fed 20%casein diet or 20%SPI diet for 10 days. The body weights of the suspended rats fed casein or SPI decreased similarly. The weights of the gastrocnemius and soleus muscles in the suspended rats were significantly decreased, however the degree of the decrease of the weight of gastrocnemius muscle of rats fed SPI diet was smaller than that of rats fed casein diet. The activity of serum creatine kinase of rats fed SPI diet was also smaller than that of rats fed casein diet. The activity of muscle protein-degrading enzymes such as calpain and proteasome in gastrocnemius muscles were almost the same in both test diet groups, but the SPI diet tended to decrease the calpain activity. Therefore, it is suggested that SPI diet might ameliorate the muscle atrophy through the reduction of the activities of calpain in gastrocnemius muscles and of creatine kinase in blood.
Key words : hypokinesia / hypodynamia, muscle atrophy, calpain, proteasome, soy protein isolate
Effect of Soy Protein Isolate on Uncoupling Protein in Brown Adipose Tissue in the Experiment of Body Weight Reduction by Energy Restriction Using OLETF Rats
Yutaka MORI 1 , Masayuki SAITOH 2 , Naoyuki KUROKAWA 3 , Hideaki KOMIYA 3 and Yoshio IKEDA 4
1 National Higashi Utsunomiya Hospital, Utsunomiya 329 -1104
2 Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060 -0818
3 Faculty of Education, Utsunomiya University, Utsunomiya 321 -8505
4 Center for General Health Care, The Jikei University School of Medine, Tokyo 105 -8461
Soy Protein Research, Japan, 2(Serial Vol. 20), 118-124, 1999.
Effect of soy protein isolate (SPI) on the mitochondrial uncoupling protein (UCP) was examined in the experiment of body weight reduction by energy restriction using a genetically obese-hyperglycemic OLETF rat. Obese OLETF rats whose body weights were over 600g were fed with 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 decrease in plasma levels of leptin and FFA was significantly greater in SPI-diet group. There was no significant difference in UCP1 mRNA levels in brown adipose tissue (BAT) between casein-diet and SPI-diet groups. Concerning the cellularity of subcutaneous and retroperitoneal adipose tissues, the average size of white adipocytes in SPI-diet group was significantly smaller than in casein-diet group. In the experiment of body weight reduction by energy restriction using obese animal model, it was unclear whether the mitochondrial UCP in BAT contributed to anti-obesity effect of SPI. The decrease in plasma leptin and FFA levels in SPI-diet group might be caused by the transformation into small-sized adipocytes.
Key words : Otsuka Long Evans Tokushima Fatty (OLETF) rats, soy protein isolate (SPI), brown adipose tissue (BAT), uncoupling protein (UCP), cellularity of adipose tissue
Study on a Low Molecular Weight Peptide Derived from Soybean Protein Having Hypocholesterolemic Activity
Masaaki YOSHIKAWA, Taichi YAMAMOTO and Yasuyuki TAKENAKA
Research Institute for Food Science, Kyoto University, Uji 611 -0011
Soy Protein Research, Japan, 2(Serial Vol. 20), 125-128, 1999.
We measured hypocholesterolemic activity of peptides in mice. During the evaluation of hypocholesterolemic activity of Leu -Pro -Leu -Pro -Arg, a complement C3a agonist peptide, we found that a fragment peptide of soybean glycinin Leu-Pro-Tyr-Pro-Arg also exhibited hypocholesterolemic activity despite the absence of C3a agonist activity. We noticed that these peptides had structural homology to enterostatin Val-Pro-Asp-Pro-Arg, which is known as an anorexigenic peptide derived from procolipase. Therefore, we examined whether enterostatin had hypocholesterolemic effect. It was active at a dose of 50mg/kg/day. As well as enterostatin, Leu-Pro-Tyr-Pro-Arg also lowered serum triglyceride after oral administration and suppressed food intake after intracerebroventricular administration in mice fed with high fat diet.
Key words : soybean, peptide, cholesterol, hypocholesterolemic, enterostatin, food intake, triglyceride
Characteristics of Soybean Resistant Protein in Nutritional-physiological Aspects and Approach to the Elucidation of Mode of Bile Acid-binding
Kimikazu IWAMI, Naoyuki AZUMA, Hitoshi SUDA, Hiroyuki IWASAKI and Ryuhei KANAMOTO
Kyoto Prefectural University School of Agriculture, Kyoto 606 -8522
Soy Protein Research, Japan, 2(Serial Vol. 20), 129-137, 1999.
Six groups of azoxymethane treated mature Fischer rats (n=10) were fed the respective diets different in protein sources such as milk casein, soybean high-molecular-weight fraction (HMF), yolk protein, wheat gluten and codfish meat, which had been supplemented as a cancer promoter with 0.2%deoxycholic acid (DCA) except for a DCA-unfed casein group. All rats were inspected for tumor incidence in the colon at 6 weeks' intervals between the 10th and 34th weeks by the use of a bronchus fiberscope. Colonic tumorigenesis was perceived at week 22 only in the DCA-fed casein group and at week 28 in five dietary groups except the DCA-unfed casein group, and at week 34 in all the groups among which both soybean HMF and yolk protein groups ranked in tumor incidence as inferior to the DCA-unfed casein group. When plasma steroid or lipid concentration was plotted against tumor incidence at week 28 or 34, positive correlations were found at both weeks between plasma bile acid concentration and tumor incidence. Soybean HMF and yolk protein had something in common with each other in antitumorigenic aspects, which can be accounted for by their excellent capacities of binding bile acids. In another experiment, the feces from casein-fed or HMF-fed rats were analyzed for their nitrogen and steroid contents or amino acid composition in order to ascertain this assumption. Fecal nitrogen excretion (g/g feces in weight) increased about 3.6-fold a few days after exchange of casein for HMF in the diet: there was a positive correlation between fecal nitrogen and bile acid excretions. As a result of HPLC, many kinds of polypeptides were detected in the water-extracts of feces from HMF-fed rats much more than from casein-fed rats, but their individual isolation and amino acid sequencing remained further investigated.
Key words : soybean HMF, yolk protein, tumor incidence, fiberscope, plasma bile acids, fecal nitrogen excretion, resistant protein, fecal amino acid composition
The Suppressing Mechanism of Soy Protein against Atherosclerosis Development in Apolipoprotein E-deficient Mice
Yasuyuki TSUDA, Miwa KUMASHIRO, Mei-Chu HUNG, Masao SATO and Katsumi IMAIZUMI
Division of Bioresource and Bioenvironmental Sciences, Graduate School, Kyushu University, Fukuoka 812 -8581
Soy Protein Research, Japan, 2(Serial Vol. 20), 138-142, 1999.
In the first experiment, male apolipoprotein (apo) E-deficient mice were fed a diet containing casein, soy protein isolate (SPI) or ethanol extracted SPI (EE-SPI) for 9 weeks. The diet containing SPI or EE-SPI, compared with casein contained diet, reduced atherosclerotic lesion development in the aorta, suggesting that antiatherogenic effects of dietary SPI attribute to the proteins. In the second experiment, male and female apoE-deficient mice were fed a diet containing casein, SPI or rice protein isolate (RPI) for 9 weeks. The latter two proteins contained a comparable amount of arginine. Both SPI and RPI diet, compared with the casein diet, lowered the atherosclerotic lesion development in the aorta and aortic root. There were no dietary effects on the serum cholesterol level. These results suggest that the arginine content in SPI might exert these beneficial effects through arterial wall.
Key words : apolipoprotein E-deficient mice, atherosclerosis, cholesterol, casein, soy protein isolate, rice protein isolate, arginine
Effect of Soy Protein on the Pressor Response in Hyperlipemia
Takemichi KANAZAWA
Medical Corporation Fuyokai Murakami Hospotal, Aomori 030-0811
Soy Protein Research, Japan, 2(Serial Vol. 20), 143-149, 1999.
The 719 cases with great hypercholesterolemia over 300mg/100mL were selected from 61,730 cases in health examination, and blood pressure was measured. Percents of the cases with high blood pressure were 11.1%. The percent was very low, compared to the percents of hypertension observed in general health examination. Therefore, the following experiment was carried out to investigate the responsiveness to pressor substance using cholesterol-fed rabbit. The pressor increases due to physiological saline injection were 2~4 mm in normal rabbits, 15 ~18 mmHg in 3 mg norepinephrine injection and 26~34 mmHg in 6 mg one. On the otherhand, the pressure increases were 2~5 mmHg, 12~16 mmHg and 16~24 mmHg, respectively, in 1% cholesterol-fed rabbits for 6 weeks. Next, in 1% cholesterol fed rabbits with soy protein, the pressor responses recoverd between normal rabbits and 1% cholesterol-fed rabbits. Plasma concentration of endothelin-1 was lower in cholesterol-fed rabbits than normal rabbits, although no difference was found in the concentration of norepinephrine. It is concluded that great hypercholesterolemia shows low response to norepinephrine. It may relate to get low concentration of endothelin-1. Soy protein can protect the hyporesponse on epinephrire due to hypercholesterolemia.
Key words : soy protein, hypercholesterolemia, hyporesponse, blood pressure, norepinephrine, endothelin-1
Soy Protein Further Reduced Serum Cholesterol Level in Hypercholesterolemic Patients Treated with HMG-CoA Reductase Inhibitor
Masako WAKI 1 , Motoo TSUSHIMA 2 and Naoko YAMASHITA 2
1 Shimada Municipal Hospital, Shimada 427-8502
2 National cardiovascular center, Suita 565-8565
Soy Protein Research, Japan, 2(Serial Vol. 20), 150-154, 1999.
Hypocholesterolemic effect of soy protein has been known. Recently, 3-hydroxy-3-methyl glutaryl ÅiHMGÅj-CoA reductase inhibitors has been widely used as the first drug to control serum cholesterol level. However, a part of patients, such as familial hypercholesterolemia, can not reach the target cholesterol level with HMG-CoA reductase inhibitors alone. We conducted this study to know whether a soy protein product (Proleena, Fuji Oil Co. ; SP) could further reduce the serum cholesterol level in such cases, comparing to the effect and tolerability of cholestyramine. The subjects were six patients who were free from cardiovascular diseases and still hypercholesterolemic with a HMG-CoA reductase inhibitor for more than 3 mo. They were treated with 9 g of SP or cholestyramine (9g of Questran) 3 times a day for 8-12 wk. After 4 wk of washout, the other regimen was introduced. With SP treatment, serum total cholesterol (TC ; mg/100mL) level changed from 295±48 (mean±SD) to 283±72, HDL-C (mg/100 mL) from 69.5±12.5 to 70.5±17.0, triglyceride (mg/100mL) from 82.7±21.8 to 81.0±29.3, and non HDL-C (TCÅ|HDL-C ; mg/100mL) from 225±57.1 to 212±76. TC and LDL-C levels in two subjects were reached to the sufficient level (TC : 273->213, 266->217 ; LDL-C : 178->125, 181->137). All the subjects tolerated 8-12 wk SP regimen, although three could accomplish cholestyramine administration. These data indicated the clinical usefulness of SP as a part of pharmacological strategies in some severely hypercholesterolemic patients.
Key words : soy protein, hypercholesterolemia, HMG -CoA reductase inhibitor, cholestyramine
Time-lag Analysis of Interrelationships between Dietary Plant Protein Intake and Breast Cancer Mortality in Japanese Women
Keisuke TSUJI 1 , Emiko HARASHIMA 2 , Asako MIURA 2 and Yasue NAKAGAWA 2
1 School of Humanities for Environmental 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, Japan, 2(Serial Vol. 20), 155-160, 1999.
The daily intake of dietary protein by Japanese people was collected during a 50-year period from the result of National Nutrition Survey. In order to study the time-lag effect of dietary protein on breast cancer, the age-adjusted mortality from breast cancer (MBC) of Japanese females and the protein intake were determined by a simple correlation coefficient and a time-series correlation coefficient. Total protein intake increased gradually from 1947 to 1970 and after 1973 reached to around 80 g/d. Animal protein increased 4.4 times in 50 years, while the plant protein decreased to 26%. Coefficients of correlation of total protein and animal protein intakes was positively correlated with MBC. While plant protein intake was negatively correlated with MBC. Each of these coefficients of correlation increased and reached to a maximum value with increasing time lag. Maximum positive correlation with total protein intake was obtained after 23-year delay, while animal protein after 22-year delay. Similar tendencies were observed with meat, egg and milk proteins, and the maximum correlation were found within 25 to 33-year delay. The maximum negative correlation between plant protein intake and MBC was found with a 15-year delay. The protein intake from cereals showed the highest correlation among all plant protein. It is suggested that the increase of animal protein intake and the decrease of plant protein intake relate to MBC accompanied with the long-term effect.
Key words : protein intake, breast cancer mortality, time -lag, correlation coefficient, Japanese female