Separation Of Growth-stimulating Peptides For Bifidobacterium From Soybean Conglycinin And Effect On Bacterial Community Of Intestine Involved

Soybean storage proteins are composed mainly of two major components, conglycinin and glycinin. Many studies have demonstrated that the enzymatic hydrolysis of conglycinin improved its functional properties and had various physiological activities such as antihyprtensive, immunostimulating and antioxide activities. It is now generally accepted that the human gastrointestinal tract is host to various species of microorganisms and this bacterial plays a significant role in health and disease. Bifidobacteria are gram-positive anaerobic bacteria, which normally inhabit the gut of humans and animals, the presence of a great deal of bifidobacteria has been considered essential to promote intestinal health and to strengthen the local immune response. In this paper, we used pepsin and simulated digestion and fermentation in the animal gastrointestinal tract to hydrolyze the conglycinin in vitro. Then we isolated and identified the soybean conglycinin peptides that selectively stimulate the growth of bifidobacteria in vitro. We also evaluate the in vivo effects of soybean conglycinin peptides on immunity and gut ecolosystem of intestine.1. Effect of growth-stimulating peptides for bifidobacteria from soybean conglycininSeries 1 Separation of conglycinin from soybean and hydrolysis ofconglycinin by pepsinSoybeans were finely ground and defatted with hexane at room temperature. The procedure for fraction of conglycinin was based on the isoelectric precipitation and size exclusion chromartography. The 51-100% saturation fraction was dialysed against water and applied to a Sepharose-CL-6B column. Elution was performed with the phosphate buffer(2.6 mmol/L KH₂PO₄, 32.5 mmol/L K₂HPO₄, 0.4 mol/L NaCl, 10 mmol/L 2-ME, pH 7.6), andthe absorbance of the column effluent was monitored at 280nm. The purification rate was identified by Sodium dodecyl sulfate gel electrophoresis. Finally the purified conglycinin was dialyzed against water. After readjusted to pH7.0 and analyzed for protein concentration by the method of micro-Kjeldahl, the protein solutions were freeze-dried and stored at 4°C. These materials were used as protein samples. Conglycinin was hydrolyzed by pepsin using a 1:30 enzyme: substrate ratio. Enzymatic hydrolysis was performed after acidification to pH1.4 with lmol/L HC1, and mixture of pepsin and conglycinin was incubated for 2 h at 37°C. The reaction was terminated by heating at 70°C for 30 min, and the solution was adjusted to pH 7 with lmol/L NaOH. After centrifugation (20 min, 3000 r/min, 4°C), the supernatant was collected and lyophilized. The nitrogen concentrations of the conglycinin digestion were evaluated by the method of micro-Kjeldahl. The conglycinin was full hydrolyzed with 6 mol/L HC1 at 110°C for 24 h. The solution composed of amino acid composition of conglycinin. The hydrolysates were neutralized and lyophilized. The result showed that the purification rate of conglycinin was 90.13% and the DH of conglycinin hydrolysates was 14.49%. The average length of peptides of the hydrolysates was 7.Series 2 Effect of conglycinin peptides on growth of bifidobacteriaThe basal medium used for a bioassay in vitro was a fully synthetic medium as described by Hassinen. For Growth assays, the assay medium (5mL) was mixed with 0.15mg/mL(nitrogen concentration) samples and inoculated with 100 uL of bacteria culture. The control contained ammonium acetate which has equal nitrogen concentration with samples. Culture was done under anaerobic conditions at 37°C. The extent of growth was measured by the absorbance at 460 nm and total DNA concentration of bifidobacteia after 24h, 48h and 72h of cultivation. The growth experiments were done triplicately. The results of the experiments in vitro conducted with soybean conglycinin hydrolysates on the regulation of the growth activity for Bifidobacterium longum showed that, compared with control, pepsin-treated conglycinin (PTC) could significantly promote the growth of Bifidobacterium longum (P<0.0l), while HCL-full hydrolysis of conglycinin conglycin in (HCL-FH...