低频低强度超声波促进植物乳杆菌生物转化酚酸的代谢机理研究

Phenolic acids are an important phenolic family in fruits and vegetables. During fermentation, phenolic acids can be metabolized by Lactobacillus plantarum to generate various phenolic acid derivatives. Some of the derivatives are important flavor and bioactive compounds, contributing to the enhancement of quality and nutrition of fermented fruits and vegetables. However, the growth and metabolism of Lactobacillus plantarum can be inhibited by phenolic acids. Our previous studies found that low-intensity power ultrasound had positive influence on the metabolism of phenolic acids by Lactobacillus plantarum, whereas the details about the effect of ultrasound on phenolic acid metabolism are unknown. Phenolic acids.are mainly metabolized by Lactobacillus plantarum through phenolic acid decarboxylases and reductases. Meanwhile, the stress response and energy metabolism of Lactobacillus plantarum can affect phenolic acid metabolism profoundly. Therefore, this project aims at investigating the influence of low-intensity power ultrasound on phenolic acid metabolism, stress response and energy metabolism of Lactobacillus plantarum. Proteomic analysis will also be performed to study protein synthesis during ultrasound-assisted fermentation of Lactobacillus plantarum. Moreover, the expression of genes relating to phenolic acid metabolism under sonication will be studied. Overall, this project can reveal.the biochemical and molecular mechanism about ultrasonic enhancement of phenolic acid metabolism by Lactobacillus plantarum.

酚酸是果蔬中重要的酚类物质。植物乳杆菌发酵可将果蔬中酚酸转化为一系列功能性和风味酚酸衍生物，提高发酵果蔬制品的营养和品质。但是酚酸对植物乳杆菌生长和代谢具有一定的抑制作用。本课题组研究发现，低频低强度超声波处理提高了植物乳杆菌生物转化酚酸的效率，但是超声波对微生物代谢酚酸的影响机理尚不清楚。酚酸脱羧和还原途径是植物乳杆菌生物转化酚酸的主要途径，同时，该途径受到菌体应激调控和能量代谢途径的影响。因此，本项目拟研究超声波对植物乳杆菌脱羧和还原酚酸、应激调控和能量代谢三条途径的影响。在此基础上，进一步研究超声波辅助植物乳杆菌发酵过程中蛋白质组变化，以及与酚酸代谢相关基因的表达情况，从而阐明低频低强度超声波促进植物乳杆菌生物转化酚酸生成功能性及风味衍生物的生化和分子代谢机理，为定向调控该代谢过程提供理论基础。

适宜的低频超声波处理对微生物发酵具有积极促进作用。本项目将低频低场强超声波应用于植物乳杆菌发酵果蔬汁过程，重点研究了超声波对植物乳杆菌发酵生物转化多酚（以酚酸为主）的影响机制。项目首先分析了在植物乳杆菌发酵苹果汁和草莓汁过程中超声波对微生物生长和代谢的影响，研究表明在苹果汁环境下，超声对糖、有机酸和多酚等物质的代谢具有显著影响。在此基础上，项目重点分析了植物乳杆菌发酵苹果汁过程中，超声作用阶段（延滞期、对数期和稳定期）和超声强度对微生物生长、糖代谢、酸代谢和多酚代谢的影响，结果表明延滞期和对数期超声可强化绿原酸水解为咖啡酸、原花青素B2的衍化和没食子酸的脱羧，相关性分析则显示有机酸、氨基酸的代谢与多种酚类物质的衍化高度相关，表明超声可通过改善微生物代谢有机酸和氨基酸，进而强化植物乳杆菌生物转化多酚类物质。通过对延滞期和对数期超声处理的植物乳杆菌进行转录组学分析，发现超声对糖酵解途径、磷酸戊糖途径、能量代谢等多条途径上的基因表达具有显著影响，同时，延滞期58.3 W/L超声处理下，padA(羟基肉桂酸类酚酸脱羧酶)的转录抑制因子padR表达上调，对数期93.6 W/L超声处理下padA基因下调，说明两个时期超声处理均抑制了羟基肉桂酸类酚酸的脱羧反应。对数期超声处理下，单宁酶tanL和与没食子酸脱羧酶活性有关的UbiD基因表达上调可促进植物乳杆菌将单宁转化生成没食子酸，并且进一步将没食子酸转化为焦棓酸。除此之外，编码酯酶的lp_2953表达上调促进绿原酸等具有酯键的酚类物质水解，释放出更多香豆酸、阿魏酸和咖啡酸等小分子酚酸。此外，蛋白质组学分析发现超声辅助发酵显著影响微生物蛋白质的表达，延滞期超声处理下显著富集的代谢通路为萜类化合物的生物合成，而对数期超声下显著富集代谢通路有核糖体、嘌呤和嘧啶代谢、同源重组、氧化磷酸化和RNA降解，后期需要进一步建立蛋白组、转录组数据和多酚衍化的关联机制。总体上，本项目研究为低频超声波在果蔬汁发酵领域的应用奠定了理论基础。