可溶性膳食纤维对多酚抑制淀粉消化的影响研究

The velocity of starch digestion affects postprandial blood glucose level, highly related with human health. α-Amylase is the key enzyme of starch digestion. Polyphenols have been reported to retard starch digestion through the enzyme inhibition. However, the effects of other food components on the digestion inhibition by polyphenols are still unknown. Recently, we’ve found that soluble dietary fibers (SDF) are able to reduce amylase inhibition by polyphenols when without affecting the viscosity of digestion system significantly. However, the mechanism needs to be further investigated. The inhibition of amylase by polyphenols have been reported to result from binding interactions between them; therefore, SDF is supposed to influence the interactions between polyphenols and amylase. The project aims to study the mechanism of the effects of SDF on polyphenol-amylase interactions and the resultant influences on starch digestion. For this aim, the experiments of interactions and starch digestion in vitro are to be performed by use of dialysis, enzyme inhibition, spectroscopy, thermodynamics and STD-NMR methods, in terms of different mixing order (polyphenols/amylase/SDF), polyphenol-amylase interaction types and SDF structural properties. By doing this, the parameters of polyphenol-SDF interactions, polyphenol-amylase interactions (in the absence and presence of SDF) and starch digestion can be determined. Through analysis of the parameters, the affecting manner of SDF on polyphenol-amylase interactions is to be illustrated, and the affecting rule of SDF-polyphenol interactions on polyphenol-amylase interactions can be obtained, as well as the structure-function relationship of SDF with the effects. In addition, how SDF influence the inhibition of starch digestion by polyphenols can be elucidated, providing novel theoretical basis for the study of effects of food components on starch digestion.

淀粉消化速率影响餐后血糖水平，与健康密切相关。α-淀粉酶是淀粉消化的关键酶，研究表明多酚可通过抑制该酶活性抑制淀粉消化，而膳食中其他组分对多酚抑制淀粉消化的影响仍不清楚。申请者近期发现可溶性膳食纤维（SDF）在不显著改变消化体系黏度时能减弱多酚对淀粉酶的抑制作用，但机理不明。多酚对酶的抑制由两者相互（结合）作用引起，因此推测SDF可影响多酚-淀粉酶相互作用。本项目拟针对多酚/SDF/酶不同混合顺序、多酚-酶不同互作类型及SDF不同结构特性，通过体外互作和消化，采用透析、酶抑制、光谱学、热力学及STD-NMR分析法，测定多酚-SDF互作参数、在SDF影响下多酚-酶互作参数的变化以及淀粉消化参数，阐释SDF影响多酚-淀粉酶互作的机制，分析SDF-多酚互作对多酚-酶互作的影响规律，揭示SDF与该影响的构效关系，阐明SDF影响多酚抑制淀粉消化的途径，为探究膳食组分对淀粉消化的影响提供新的理论依据。

膳食多酚可通过抑制淀粉消化酶的活性，延缓淀粉消化速率，调控餐后血糖水平。膳食中其他成分是影响多酚对酶抑制效果的重要因素，本项目探究了可溶性膳食纤维（SDF）对多酚抑制淀粉消化酶的影响及机制。首先，完善了多酚对淀粉消化酶（α-淀粉酶及α-葡萄糖苷酶）抑制作用的构效关系，通过消化分析、抑制动力学、多光谱学、热力学、分子对接等分析技术，发现没食子酰基和咖啡酰基是多酚发挥酶抑制作用的两个关键基团，两基团可进入酶活性中心，与酶关键催化氨基酸残基发生氢键及π-π共轭相互作用；进而，探究了SDF与多酚之间的相互作用，选取6不同分子结构特征（电荷、侧链结构、分子量）的SDF及茶多酚EGCG，通过透析、NMR、FTIR、XRD等分析技术，发现SDF主要通过氢键与EGCG发生吸附作用，且吸附作用强弱与SDF的分子结构有关；最后，阐明了SDF对多酚抑制淀粉消化酶的影响，通过消化分析、抑制动力学、多光谱学、微观学等分析技术，发现在SDF、EGCG、α-葡萄糖苷酶的三者体系中，在不影响消化体系黏度时，3种阿拉伯木聚糖（低、中、高黏度）和多聚半乳糖醛酸均能显著降低EGCG对酶的抑制作用，而2种β-葡聚糖（在6种SDF中具有最高黏度）几乎不影响EGCG对酶的抑制能力，这表明SDF对EGCG酶抑制能力的影响与自身黏度无关。尽管阿拉伯木聚糖和多聚半乳糖醛酸能减弱EGCG对酶的竞争性抑制作用，但它们维持了EGCG对酶的荧光淬灭效果（未减弱EGCG与酶的π-π共轭），另外，阿拉伯木聚糖和多聚半乳糖醛酸能显著降低EGCG-酶复合物的粒径大小和浊度，这表明在SDF、EGCG、酶的三者体系中，形成了SDF-EGCG-酶的三者复合物，而非减少了与酶结合的EGCG的分子数。结合第二部分研究内容，在三者复合物中，SDF与EGCG之间的氢键互作影响了EGCG与酶活性中心的氢键互作，从而减弱了酶抑制作用，该减弱作用源于阿拉伯木聚糖的侧链为其吸附捕获EGCG提供了更多机会、多聚半乳糖醛酸中羧基的极性增强了其与EGCG的氢键互作，而由于β-葡聚糖与EGCG之间的相互作用较弱，其并未显著影响EGCG对酶的抑制能力。围绕以上研究内容，在基金资助下发表SCI论文12篇，本项目为解析可溶性膳食纤维对多酚与淀粉消化酶相互作用的影响提供了技术支持，同时为综合分析膳食成分对淀粉消化的影响提供了理论依据。