超高压提高NFC柿子汁出汁率的酶促机制研究

Persimmon is one of the important and featured fruits in China, and its cultivation area and yield are ranked first in the world. However, at present, few persimmon products can be found in China. The utilization of the resources is insufficient. Development of not from concentrate (NFC) persimmon juice could meet the growing demands of consumers for minimum process, more nutritious and healthier products. However, the low juice extraction yield due to the high pectin content limits the efficiency of persimmon juice production. Adding pectinase is the traditional method of increasing the extraction yield, which requires a long time treatment and the bitter taste has been found in the juice. Based on the applicant's previous research, high pressure processing (HPP) could significantly increase the persimmon juice extraction yield as the step of pretreatment of persimmon pulp. The aim of this project is to reveal the enzymatic mechanism of juice extraction increased by HPP. The effects of HPP on pectin methylesterase (PME), pectin and rheological properties in both persimmon pulp and “PME-pectin” purification system will be studied, and the correlation of PME with the change of juice extraction yield will be analyzed. Then the mode of action and reaction rate of PME on pectin under HPP will be justified; and together with the effect of HPP on purified PME structure and properties, the enzymatic mechanism of “HPP activated endogenous PME” in persimmon juice extraction increased will be revealed. This project provides a basis for the application of HPP on juice extraction yield increase.

柿子是我国重要特色水果之一，栽培面积和产量均位居世界首位。但目前我国柿子加工产品类型单一，资源利用不充分。非浓缩还原（NFC）柿子汁的开发可满足目前消费者对更少加工、更营养、更健康产品不断增长的需求，但由于果胶含量高带来的较低出汁率制约了柿子汁的高效生产。传统提高出汁率的方法添加外源酶法所需时间长，获得果汁苦味重，品质不高。申请人前期研究发现，采用超高压技术（HPP）预处理柿子浆可以显著提高其出汁率。本项目以探究HPP提高出汁率的酶促机制为目的，拟从柿子浆体系深入到“果胶甲基酯酶（PME）-果胶”纯化体系，系统研究HPP对PME、果胶和流变特性的影响及其与出汁率变化的相关性；分析HPP下PME对果胶酶促反应的作用模式和分子接触；最后通过研究HPP对纯化PME分子结构及特性的影响，揭示HPP提高果汁出汁率的“HPP激活内源PME”机制，为HPP在提高果汁出汁率方面的应用提供依据。

柿子作为我国重要的特色水果之一，具有较高的营养和药用价值。但我国柿子加工产业面临加工程度低，产品类型单一的问题。非浓缩还原（NFC）柿子汁不经过高温浓缩和复原过程，能够更好地保留水果原有的新鲜品质，未来发展空间十分广阔。前期研究发现，柿子存在出汁率低、榨取汁困难的瓶颈。而采用超高压技术（HPP）作为预处理步骤处理柿子浆可显著提高柿子汁的出汁，HPP处理柿子果胶甲酯酶（PME）有激活作用，但超高压对PME的激活机制仍不清楚。基于这一研究基础，本研究对HPP 影响PME的酶促机制进行深入研究。在这项研究中，化学实验与分子动力学模拟相结合，在分子水平上揭示了HPP处理对PME的激活机制。本研究首先对柿子原料、成熟度进行了筛选，通过测定出汁率、PME酶活、多聚半乳糖醛酸酶（PG）活筛选了恭城月柿作为后续研究品种，明确HPP与柿子浆PME的激活呈正相关；同时探究了不同处理时间及处理压力对柿子浆体系的影响。为进一步探究酶促机制，通过分离纯化得到了纯度为81.89%柿子PME，测定其在不同HPP处理条件下结构及空间的变化。研究发现，HPP处理未改变蛋白的一级结构，降低了α -螺旋和β -折叠的占比，增加了不规则卷曲占比，同时缩减了蛋白质体积。进一步对HPP处理下的PME及PME-果胶体系进行了分子动力学模拟。对比常压组，500 MPa 压缩了蛋白总体积13.89%，增加了溶剂和蛋白质之间的接触面积；使得β片层结构内部疏水空间被挤压，酶活性中心亲水狭缝结构被暴露打开，位于活性中心的氨基酸残基（Asp-157、Asp-136 和 Gln-135）在HPP处理下保持稳定，酶与底物之间的亲和力增加，强化降解果胶效果，从而提高了出汁率。本项目查明了HPP提高柿子出汁率的酶促机制，为新型NFC柿子汁的开发提供理论基础，为充分挖掘我国柿子加工资源、避免季节性水果浪费提供研究支持，具有十分重要的成果转化前景。