新科斯糖月桂酸酯抑制食源性致病菌的作用机制研究

Neokestose-6-laurate, a novel functional oligosaccharide ester synthesized by the proposer, shows great potential to be developed as a novel promising green nutritional food preservative, due to its functional properties such as emulsification, antimicrobial activity and nutritive function. However, the configuration-active relationship of antibacterial neokestose laurate (NLE) and the inhibitory mechanism of NLE remain unknown. Therefore, the aim of this project is first to synthesize NLE isomers, then to investigate the relationship between antibacterial activity and structure of NLE isomers, and finally to clarify the antibacterial mechanism using Staphylococcus aureus and Escherichia coli as mode bacteria from cellular and molecular level. Three steps will be taken to gain the insight into antibacterial mechanism of NLE: first to study the effect of NLE on bacterial ultrastructure, membrane integrity and permeability by transmission electron microscopy, atomic absorption spectroscopy and fluorescence probe, respectively; then to investigate the effect of NLE on liposome by modeling bacterial membrane; finally to analyze the differentially expressed protein located in the whole cell and the membrane related with the treatment of NLE by dimensional electrophoresis and MALDI-TOF-MS, respectively. In brief, the project would reveal the configuration-active relationship of antibacterial NLE and elucidate the inhibitory mechanism, so as to provide theoretical basis for targeted development of green nutritional food preservative-sugar fatty acid ester.

新科斯糖-6-月桂酸酯是项目申请人前期合成的功能性低聚糖酯，具有乳化、抑菌、营养等特性，有望被开发为新型绿色营养化食品防腐剂。然而新科斯糖月桂酸酯（NLE）构型对抑菌效果的影响及其抑菌机理尚未明确。因此，本项目拟在酶法选择性合成不同构型NLE基础上，以金黄色葡萄球菌（G+细菌）和大肠杆菌（G-细菌）为模式菌株，研究NLE构型与抑菌特性的关系；并通过透射电镜、原子吸收光谱、荧光探针等技术分析NLE对菌体超微结构、细胞膜完整性与渗透性的影响；通过模拟细胞膜分析NLE对细胞膜脂质体的影响；最后通过双向电泳结合MALDI-TOF-MS技术分析NLE处理后细胞总蛋白质与膜蛋白质的表达差异情况，从细胞和分子水平两方面探明NLE对G+和G-细菌的抑菌机理。本研究旨在揭示NLE构型与抑菌特性的关系并阐明其抑菌作用机理，为绿色营养化防腐剂-糖脂肪酸酯的定向开发提供理论依据。

新科斯糖月桂酸酯是功能性低聚糖酯，具有乳化、抑菌、营养等特性，有望被开发为新型绿色营养化食品防腐剂。然而新科斯糖月桂酸酯构型对抑菌效果的影响及其抑菌机理尚未明确。本项目采用酶法以新科斯糖和月桂酸乙烯酯为底物选择性合成了不同构型的新科斯糖月桂酸酯，并进行了高纯度制备及结构鉴定。研究了构型与抑菌活性的关系，筛选了高抑菌活性构型的糖酯。阐明了糖酯对食源性致病菌的抑菌作用机制：碱性磷酸酶实验表明糖酯对指示菌细胞壁有损伤，PBFI探针标记荧光光谱、PI探针标记流式细胞术等实验表明糖酯会导致细胞内钾离子泄漏、破坏细胞膜的渗透性与完整性；透射电镜对菌体超微结构观察结果证实了细胞膜的损伤，并显示胞内物质密度降低；凝胶电泳实验表明糖酯会影响胞内蛋白质表达；进一步采用iTRAQ技术对差异表达的蛋白质进行了筛选，并对相关蛋白质进行了GO功能注释和Pathway代谢通路分析，结果发现主要影响细胞的生物合成功能，胞内含氮化合物代谢等。因此，本项目主要从细胞和分子水平阐明了新科斯糖月桂酸酯的抑菌作用机制，为多功能营养化防腐剂-糖脂肪酸酯的定向开发提供了理论依据。