【作者】 任建华；

【导师】 郭顺堂；

【作者基本信息】 中国农业大学 ， 食品科学， 2013， 博士

【摘要】 大豆蛋白是一种优质蛋白资源,在保障我国膳食蛋白质供给方面发挥着重要作用。大豆蛋白及其降解产物具有调节胃肠道健康的功能。本研究室前期以碱性蛋白酶酶解大豆糖蛋白(8-伴大豆球蛋白),并从p-伴大豆球蛋白酶解物中分离大豆糖肽(SGP),且发现SGP具有抑制细菌粘附和调节肠粘膜屏障功能的活性,但是SGP具有抑制粘附作用的机制并不清楚,糖链在抑制粘附中发挥的作用也不明确。本课题在前期研究基础上,分析SGP与宿主细胞的相互作用,评价糖链在抑制粘附中发挥的作用,进一步探讨SGP抑制粘附的机制,为建立SGP的生理活性调控方法和应用技术创新提供理论依据；在明确理论问题的基础上,研究具有维持肠道健康作用的p-伴大豆球蛋白酶解物在模拟体外消化过程中以及在热处理条件下的稳定性,在技术层面上为β-伴大豆球蛋白酶解物作为食源肽类胃肠道调节剂或功能因子添加于各种类型的食品中提供基础。本研究首先采用高碘酸盐氧化的方法制备了糖链破坏的大豆糖肽,并对破坏糖链的大豆糖肽性质进行了分析,结果表明高碘酸盐处理后的SGP中总糖相对含量由15.44%下降至10.93%,样品负电性增强,分子量相对较低的组分含量增加,疏水性相对较强的组分含量增加；高碘酸盐处理后,样品中芳香族氨基酸的暴露状态发生了变化,表明高碘酸盐氧化不仅可以部分破坏糖链,还会引起样品构象的变化。以Caco-2细胞为模型,研究了SGP与Caco-2细胞的相互作用。结果表明,细胞表面Zeta电位值在添加SGP的30min内降低,后在24h内缓慢回复至加入肽前的水平；荧光结合实验结果显示,SGP能够结合于细胞膜表面,增加细胞膜的流动性。细胞膜上的脂筏区域被破坏后,SGP与细胞膜的结合作用明显减弱,表明SGP在细胞膜的脂筏区域与细胞膜发生结合作用。进一步比较SGP和periodate-treated SGP对致病菌粘附肠道细胞的抑制效果。结果表明,periodate-treatedSGP对实验所用Escherichia coli0114、Escherichia coli026、Salmonella enteritidis、S. typhimuriumLT2四株常见的肠道致病菌有一定的抑制粘附的活性。高碘酸盐氧化破坏糖链后对大肠杆菌粘附的抑制效果明显下降,对沙门氏菌粘附的抑制效果则影响不明显；且periodate-treated SGP对细胞膜流动性增加的程度小于完整的SGP。这些结果意味着糖链在大豆糖肽抑制大肠杆菌粘附细胞的作用中发挥了重要作用。β-伴大豆球蛋白酶解物的稳定性是将其应用于食品生产的基础,本研究分析了其在热处理(85℃,30min)条件下的稳定性。结果表明加热会导致β-伴大豆球蛋白酶解物发生可溶性聚集,且聚集程度与溶液浓度有关；较低浓度时(小于200mg/mL),β-伴大豆球蛋白酶解物溶液有良好的耐热性；在较高浓度时,加热会导致β-伴大豆球蛋白解物在胃肠道消化酶的作用下的降解作用。结果表明,在模拟消化过程中,肽段发生了部分酶解物负电性增加,钙敏感性下降。进一步采用体外模拟消化的方式研究了β-伴大豆球蛋白酶降解,但是降解的程度较低,其中10-20kDa组分含量保持相对稳定。亲水性相对较强的组分含量略有减少,疏水性较强的组分略有增加,但整体表面疏水性指数没有显著变化(P<0.05)。说明β-伴大豆球蛋白酶解物中的10-20kDa有效功能组分对胃肠道消化酶的降解有较好的抵抗作用,在消化过程中能保持相对稳定。更多还原

【Abstract】 Soybean protein is the quality resources of food protein, and plays an important role in protein supply in the daily diet for people in China. Soybean protein and its derivatives could regulate intestinal function. Soybean glycoprotein (β-conglycinin) was hydrolyzed using alcalase2.4L and soybean glycopeptides (SGP) was seperated from β-conglycinin hydrolysate in previous study. SGP could inhibit the adhesion of pathogens on human intestinal cells, but the mechanism of this effect and role of carbohydrate chain are still unclear. Based on these results, interactions between SGP and host cells and the role of carbohydrate chain in inhibiting adhesion were examined in this study. The mechanism of inhibiting adhesion was also analyzed in this study. The study provides theoretical foundation for regulation of SGP biological activity and application of the technical innovation. On the basis of theoretical research, the in vitro digestibility and thermal stability of β-conglycinin hydrolysates were also evaluated in this study, which could supply the technical foundation for the application of β-conglycinin hydrolysates as functional component for intestinal health in food industry.The carbohydrate chain in SGP was destroyed partly by periodate treatment and properties of periodate-treated SGP were also examined in this study. Results showed that the carbohydrate content in SGP decreased from15.44%to10.93%after periodate treatment. The electronegativity, content of peptides with relatively low molecular weight and the content of component with relatively high hydrophobicity were increased in periodate-treated SGP. Moreover, the exposure of the aromatic amino acids in periodate-treated SGP changed. These results indicated that periodate treatment could destroy part of sugar chain and led to conformation changes in SGP.The changes in Zeta potential and cell membrane fluidity of Caco-2cells with or without SGP and the binding interaction between SGP and cell membrane were examined using a Caco-2cell model. Results showed that the cell surface Zeta potential decreased after30min of incubation with SGP and then slowly restored to the level of Caco-2cells alone within24h. Fluorescent experimental results showed that SGP could bind to the cell membrane, and increase the membrane fluidity of Caco-2cells. The extent of binding decreased when the lipid rafts in the cell membrane were destroyed, indicating that SGP bound to the cell membrane on lipid rafts,The inhibitory effects of SGP and periodate-treated the SGP on the pathogenic bacteria adhesion intestinal cells were compared. Results showed that SGP presented inhibition effect on the bacterial adhesion to the host cell. The inhibition effect of periodate-treated the SGP on Escherichia coli adhesion were significantly lower than SGP, but there was no significant differenece between the inhibition effects of SGP and periodate-treated the SGP on Salmonella adhesion. Moreover, the increasing extent of membrane fluidity of Caco-2cells affected by periodate-treated the SGP was lower than SGP. These results suggested that carbohydrate chain in SGP played an important role in inhibiting Escherichia coli adhesion to intestinal cells.The stability of P-conglycinin hydrolysates was foundation of using it in food processing. The stability of soybean β-conglycinin hydrolysates in heat treatment (85℃,30min) was studied. Results indicated that heat treatment caused β-conglycinin hydrolysates soluble aggregation and the aggregation degree related to sample concentration. β-Conglycinin hydrolysates solution had good heat resistance at lower concentration (<200mg/mL), while heat treatment induced electronegativity increased and calcium sensitivity decreased at higher concentration. The digestibility in vitro of P-conglycinin hydrolysates was also studied. The results showed that β-conglycinin hydrolysates were degraded through in vitro digestion, but the degree of degradation was relatively low. The content of peptides with molecular weights from10kDa to20kDa remained stable during digestion in vitro. The proportion of highly hydrophobic peptides decreased through in vitro digestion, but no significant change in the surface hydrophobicity indices of digestion products was observed (P<0.05). These results indicate that peptides with molecular weights from10kDa to20kDa in the β-conglycinin hydrolysates resisted digestion by pepsin and trypsin and they remained stable during in vitro digestion.更多还原