高压均质抑制NFC桃汁多酚氧化酶及酶促褐变的机制研究

As the consumers are demanding more safe and nutritious food products, NFC juice (Non From concentrate) is gaining popularity due to its closer to the taste, flavor and nutrition of fresh fruit. NFC peach juice should be an important product to promote the development of peach industry in Lijiang, Yunnan. Enzymatic browning is mainly attributed to the action of polyphenol oxidase (PPO), which contributes to oxidization of phenolic compounds in the presence of molecular oxygen. Enzymatic browning decreased the commercial quality, organoleptic acceptance and nutritional value of the product. Generally, there are two forms of PPOs existed in plant, including soluble PPO (sPPO) and membrane-bound PPO (mPPO). While mPPO is either weakly or strongly bound to the thylakoid membrane and it shows more stability towards processing. Thus mPPO is a potential detrimental factor for browning in juice during storage. High pressure homogenization (HPH) is a non-thermal technology achieving continuous processing for liquid food, which can inhibit the Maillard reaction and enzymatic browning in the juice. Previous studies showed that the inactivation effects of PPO in juice and buffer were significant different by HPH. In this study, mechanism of HPH on enzymatic browning and changes of related factors in NFC peach juice will be investigated. Effects of HPH on the activity and structure of mPPO and sPPO will be analyzed, especially the release and inactivation of mPPO in NFC peach juice after HPH; The changes of phenolic substrate and browning product in NFC peach juice after HPH also will be analyzed; The PPO-pectin simulation system will be built to explore the interaction between PPO and pectin, and the effect of pectin on activity and structure of sPPO and mPPO will be analyzed. The present study will reveal the effect of HPH on PPO activity and structure, and the inhibition mechanism of enzymatic browning in NFC peach juice after HPH, and provide the basis for application of non-thermal technology in NFC juice industry, which have important theoretical and practical significance.

NFC桃汁品质接近新鲜水果，是促进丽江雪桃产业发展的重要加工产品。但NFC果汁加工中易发生多酚氧化酶(PPO)引起的酶促褐变，使品质降低。果蔬中普遍存在游离态(sPPO)和膜结合态(mPPO)两种PPO，mPPO不易被钝化，会在贮藏期引发褐变。高压均质(HPH)是一种可实现连续加工的非热加工方式，能有效抑制褐变，但对果汁和粗酶液中PPO钝化效果差异显著。本项目以雪桃为原料，采用HPH作为加工手段，分析HPH对NFC桃汁中mPPO和sPPO活性和结构的影响，重点研究HPH处理后mPPO的释放和钝化；明确sPPO和mPPO主要内源性酚类底物和对应褐变产物，建立HPH处理后酚类底物和褐变产物相关性;基于PPO-果胶模拟体系，探讨PPO和果胶的相互作用机制，研究HPH处理后果胶变化对PPO活性和结构的影响。最终阐明HPH对sPPO和mPPO活性和结构的作用，揭示HPH抑制NFC桃汁酶促褐变机制。

NFC桃汁品质接近新鲜水果，是促进丽江雪桃产业发展的重要加工产品。果蔬中普遍存在游离态（sPPO）和膜结合态（mPPO）两种多酚氧化酶（PPO），因此NFC桃汁加工过程中易发生由此引起的酶促褐变，使品质降低。高压均质（HPH）是一种可实现连续加工的非热加工方式，能有效抑制褐变，但对果汁和粗酶液中PPO钝化效果差异显著。本项目经硫酸铵分级沉淀、柱层析分离纯化后从雪桃中获得sPPO和mPPO，分析其生物信息学，发现两者的亚细胞定位和跨膜结构不同；利用同源建模构建其三维结构进行解析，发现sPPO结构相对松散，而mPPO则是稳定的椭球形结构。HPH和HPP等非热加工手段对sPPO构象的影响要强于mPPO，从而对sPPO具有更强的钝化效果。低压会激活PPO活性，而高压则会钝化PPO，分子动力学模拟结果也发现200 MPa HPP处理后PPO铜结合区的变化有利于催化作用，而400 MPa以上处理后酶蛋白铜结合区的变化和loop区域的强烈运动均不利于催化作用。另外，sPPO的结构相对松散，其loop区域离主链较远，从而导致高压对sPPO的loop区域具有显著的失稳作用；而mPPO的内外膜结构可以通过跨膜螺旋紧密连接，形成了高度紧凑的椭圆形结构，因而高压对其三维结构没有显著影响。果胶对PPO活性具有显著的抑制作用，但HPH可破坏果胶的主链，对其有碎片化作用，会影响果胶对PPO的抑制作用。HPH还会改变果汁中酚类物质的含量和组成，游离态、酯化态、结合态三种状态多酚含量在较高压力下均显著提高，HPH可能会促进酚类底物与PPO的催化作用。因此，通过本项目研究发现，HPH通过抑制PPO活性、改变酚类底物状态和组成、对果胶碎片化处理等多种途径抑制NFC桃汁的酶促褐变。