高静压诱导柑橘果胶的酶促低酯化机制研究

Pectin is an important natural food additive. Low methoxyl pectin (LM-pectin), with many kinds of physiological function, is being capable of forming physical gels with or without sugar, thus causes more and more attention. Commercial LM-pectin is mainly produced by de-methoxylation of high methoxyl pectin. However, the product may show bad gel property due to pectin degradation coming with de-methoxylation. The rate of de-methoxylation has been shown to be increased after high hydrostatic pressure (HHP) by regulating endogenous pectin methylesterase (PME), while the degradation of pectin is inhibited. To figure out the mechanism of the enzymatic de-methoxylation induced by HHP, the mainly used pectin source citrus peel is chosen as research material. The effect of HHP on citrus pectin de-methoxylation will be studied at different treatment conditions. Furthermore, the action mode and stability of the endogenous PME, being purified before and after HHP, will be studied using enzymatic fingerprinting and automatic potentiometric titration, respectively. And correlation between de-methoxylation and changes of PME catalytic ability will be calculated. Finally, the effect of HHP on PME structure and aggregation will be investigated to explain HHP-induced enzymatic de-methoxylation of citrus pectin, using circular dichroism spectroscopy, fluorescence spectroscopy, electrophoresis, NMR and dynamic light scattering. This study will give further evidences for green and high efficient production of LM-pectin.

果胶是一种重要的天然食品添加剂。低酯果胶由于可在低糖或无糖条件下凝胶且有多种独特的生理功能而被广泛关注，目前主要由高酯果胶脱酯得到，产品常因存在降解而凝胶性差。申请人前期研究发现，高静压技术（HHP）可在抑制果胶降解的条件下调控内源果胶甲基酯酶（PME）促进果胶低酯化。基于此，本项目拟以果胶工业化生产的主要原料-柑橘皮为实验材料，系统研究不同HHP条件下柑橘果胶的低酯化规律；并对HHP处理前后体系中内源PME进行分离纯化，采用酶解图谱和电位滴定法研究HHP处理对PME作用模式和活性的影响，解析PME催化行为的变化与果胶低酯化的相关性；采用圆二色谱、荧光光谱、电泳、NMR和动态光散射法等进一步研究HHP处理前后内源PME结构和聚集形态的变化，从分子水平解释HHP对PME催化行为的影响，提出HHP促进果胶的酶促低酯化的可能机制。项目的研究将为HHP应用于低酯果胶的绿色高效制备提供理论基础。

果胶是一种重要的天然食品添加剂，根据酯化度不同可分为高酯果胶和低酯果胶。低酯果胶由于可在低糖或无糖条件下凝胶且有多种独特的生理功能而被广泛关注，目前主要由高酯果胶脱酯得到，产品常因存在降解而凝胶性差。本项目以柑橘果胶为研究对象，系统研究不同超高压处理（HHP）条件下柑橘果胶的酶促低酯化规律，并将超高压结合酶法制备的低酯果胶与常压酶法和平碱法制备的低酯果胶进行比较研究；分析HHP处理前后体系中PME酶作用模式和活性的影响，解析PME催化行为的变化与果胶低酯化的相关性；研究HHP处理前后PME结构和聚集形态的变化，进一步分析HHP促进果胶的酶促低酯化的可能机制。.研究结果表明，HHP结合酶法果胶低酯化效率显著高于常压酶法和传统碱法，达到相同酯化度~37%时所需时间分别为12 min (HHP结合酶法), 2 h（常压酶法）和20 min（传统碱法）。HHP结合酶法制备的低酯果胶相同浓度条件下粘度高、增稠性更好；钙凝胶离子依赖程度低、相同离子浓度下凝胶强度高；乳化能力较传统碱法低，但贮藏乳化稳定性好。HHP处理后果胶分子的理化性质未发生显著变化，PME酶蛋白的荧光强度稍有降低，但变化不显著，说明HHP处理后果胶和PME酶结构未发生显著变化，推测HHP促进果胶的酶促低酯化是由于高压下酶与底物的高效结合或是压力对反应平衡的正向影响。项目的研究将为HHP应用于低酯果胶的绿色高效制备提供理论基础。