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Structure-Physicochemical Function Relationships of Soybean-Conglycinin Constituent Subunits:

Abstract:

Conglycinin, one of the dominant storage proteins of soybean, has a trimeric structure, being composed of three subunits , and . The and ' subunits contain the extension regions in addition to the core regions common to all subunits, which are N-glycosylated. Physicochemical functions of recombinant nonglycosylated individual subunits and deletion mutants (c and 'c) lacking the extension regions of the and ' subunits were examined at pH 7.6 and 3.7 at low ( = 0.08) and high ( = 0.5) ionic strengths. Although individual recombinant subunits exhibited different properties at all conditions, there were some consistencies. Surface hydrophobicities and thermal stabilities of the individual subunits were likely to be conferred by their core regions, and the carbohydrate moieties did not contribute to these properties at any conditions examined here. Solubility at = 0.08, heat-induced association, and emulsifying ability remarkably depended on the extension regions and the carbohydrate moieties in addition to the structural features of the core regions. These findings indicate that various end products could be produced by the selection of soybean varieties containing -conglycinin with different subunit compositions and suggest a direction for a principle of soybean breeding.

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

Kinetic Analysis of Freeze Denaturation of Soy Protein:

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.

Deodorization of Soybean Proteins by Enzymatic and Physicochemical Processings:

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.

Preparation and Characterization of Novel Food Materials from Soybean Extract by Heat Treatment:

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.

Emulsification Characteristics of Water-soluble Soybean Polysaccharides from Soybean Residue:

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.

Development of Assay System for Immunoglobulin Production Regulating Factors Using Mouse Lymphocytes and Its Application:

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.

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.

Anti-osteoporotic Factors Found in the Soybean:

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.

Characteristics of Soybean Resistant Protein in Nutritional-physiological Aspects and Approach to the Elucidation of Mode of Bile Acid-binding:

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.

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.

Soy Protein Further Reduced Serum Cholesterol Level in Hypercholesterolemic Patients Treated with HMG-CoA Reductase Inhibitor:

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.

The Nutritional Role of Soybeans:

From the nutritional standpoint, soybean contributes significantly as a source of supplementary and complementary protein as well as a source of calories and nitrogen. Well processed soy protein based foods have a high digestibility value with an excellent profile of amino acids and can serve as the major or sole source of the protein intake. The value of soy protein isolate as judged by Protein Quality Evaluation based on the FAO/WHO Protein Digestibility-Corrected Amino Acid Score Procedure is essentially equivalent to values of animal origin proteins. Soy protein products are capable of meeting long-term essential amino acid and protein needs of children and adults. Low in saturated fat, soybean oil is an excellent source of essential fatty acids, linoleic (C18:2 n6) (50%) and linolenic (C18:3 n3) (7%) as well as oleic acid (24%) and tocopherol (Vitamin E). This targets its use as a high quality vegetable oil with cholesterol lowering function. A moderate consumption with the recommended dietary intake level will provide optimum benefits.

Figure 1. Electrophoretic pattern of soybean proteins. Left lane. Molecular weight standards; wright lane. Water soluble soybean seed proteins (2).

Soybean proteins consist of discrete group of proteins which cover a broad range of molecular sizes (fig.1). The major portion of total soy proteins consists of glycinin (11S) and b-conglycinin (7S) proteins. Glycinin has an estimated molecular weight of about 350,000 and is composed of at least six nonindentical subunits. Each of these subunits has an acidic polypeptide linked to a basic polypeptide by a single disulfide bond. b-conglycinin (MW 180,000) also consists of at least four nonindentical subunits (a´, a , b and g). Both proteins are heterogenous .

Application of soybean proteins is limited by the presence of biologicali active components, such as protease inhibitors and lectin (SBA), in spite of the fact that tripsin inhibitors (TIs) have higher levels of essential amino-acids than storage proteins. Protease inhibitors are classified in two major types: Kunitz (KTI) and Bowman-Birk (BBI) inhibitor. Depending on variety, soybeans contain not more than three KTIs and 5-12 isoforms of BBI. During processing soy protein products for human consumption, thermal treatment is employed in order to inactivate antinutritional factors and to improve flavor quality. Generally, live steam treatment at 100°C for 20-30 minutes provides an adequate margin of safety to ensure maximal nutritional benefit (3).

Figure 2. Phytic acid interactions with minerals and proteins. a. Interactions with Ca 2+;
b. Interactions with proteins at low pH; c. Interactions with proteins at pH 5 - 10.

Soyban Phytic Acid:

The presence of phytate in soybeans is well documented. Phytic acid is myo-inositol, phosphorylated on all of its 6 hydroxyl groups. The term phytate designates calcium /magnesium salt of phytic acid . At neutral pH and at pHs commonly encountered in foods, phytic acid is negatively charged. Therefore, it is very reactive with many positively charged groups such as cations and proteins. Cations can bind to one or two phosphate groups of phytic acid molecule (fig. 2a), while protein interactions with phytic acid depend on pH (fig. 2 b,c).

Because of these properties phytic acid has been considered as an antinutrient. In high concentrations, it can reduce the bioavailability of minerals and proteins, and it can complex with ions-cofactors of enzymes, which are necessary for optimal enzymatic activities. However, in small concentrations in nutrition phytic acid may also have some beneficial effects. These include: slowing the rate of starch digestibility and lowering the blood glucose response, controlling dental caries, suppression of colonic cancer (colonic bacteria have been shown to produce oxygen radicals in appreciable amounts, dietary phytic acid might suppress oxidant damage to intestinal epithelium and neighboring cells) and improving the oxygen-providing ability of red blood cells .

Fig.a b Influence of the various auxinoid concentrations on the growth dynamic of the soybean calli cultures during one subcultivation.