【作者】 左伟勇；

【导师】 陈伟华；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2005， 博士

【摘要】 大豆种子中贮藏有丰富的蛋白质,其中绝大部分是球蛋白,主要包括大豆球蛋白（Glycinin）和伴大豆球蛋白（Conglycinin）。有研究表明,伴大豆球蛋白水解肽具有特殊的生理活性,能够降低血压,提高免疫力,抗氧化等作用。人和动物肠道内栖息着数量巨大的微生物群体,它们对宿主的健康具有深远的意义,其中双歧杆菌是肠道中重要的生理性细菌之一,具有益生、营养和免疫等功能,对维持肠道菌群的结构及功能起主导作用。本实验通过对伴大豆球蛋白的制备,并模拟机体内环境,通过胃蛋白酶水解伴大豆球蛋白,以研究其水解肽对双歧杆菌增殖的影响,为进一步阐明食物源蛋白活性肽的生理功能,提供实验依据。同时对伴大豆球蛋白胃蛋白酶水解产物中促双歧杆菌增殖肽进行分离,所得到的活性组分应用质谱等方法进行鉴定。分离得到促双歧杆菌增殖肽后,又通过体内研究,了解伴大豆球蛋白促双歧杆菌肽对机体免疫功能及肠道细菌区系的影响,特别是对肠道双歧杆菌的影响。 1.伴大豆球蛋白胃蛋白酶水解肽对双歧杆菌增殖的影响 系列1伴大豆球蛋白的分离纯化及其胃蛋白酶水解肽的制备 大豆粉脱脂后,经过等电点沉淀,硫酸铵分级盐析（51%和100%）得到伴大豆球蛋白粗提品,采用Sepharose-CL-6B柱进一步纯化,洗脱液为磷酸盐缓冲液（2.6mmol/L KH₂PO₄,32.5mmol/L K₂HPO₄,0.4mol/L NaCl,10mmol/L 2-ME,pH7.6）,流速30mL/h,于280nm处进行检测,收集各组分,采用SDS-PAGE电泳鉴定提取物,得到伴大豆球蛋白纯品,应用半微量凯氏定氮法测定其蛋白含量,冻干后备用。取伴大豆球蛋白冻干粉,双蒸水溶解后,调溶液pH值至1.4,按酶底物比1∶30加入胃蛋白酶,37℃水浴条件下水解2h后,在70℃,30min灭活酶并终止反应,同时调pH=7.0,在4℃,以3000rpm,离心20min,取上清液,用半微量凯氏定氮法测定胃酶水解液的含氮量冻干后备用,胃蛋白酶水解物水解度的测定采用茚三酮法。同时取伴大豆球蛋白,加入过量6mol/L盐酸,110℃,水解24h,制得伴大豆球蛋白盐酸彻底水解物。结果表明,经过分离纯化的伴大豆球蛋白纯度为90.13%,可供生理功能的研究,应用更多还原

【Abstract】 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更多还原