重组抗冻肽的食品级乳酸乳球菌构建及其抗冻干胁迫性与作用机制研究

As a leavening agent or functional carrier, lactococcus lactis lyophilized powder has the broad application prospect in the food industry, but lyophilization will cause that a large number of bacterial cell death due to the freezing and drying stresses, which seriously restricts its efficiency of fermentation and large-scale application. By combining the antifreeze characteristic of antifreeze peptides with the advantages of food-grade lactococcus lactis expression system, this project aims to explore the activity and mechanism of the resistance to freeze-drying stresses for the lactococcus lactis with expressing recombinant antifreeze peptides at the molecular level and by integrated application of a variety of analytical methods involving food science, molecular biology, and computer chemistry. Firstly, this project intends to screen highly active recombinant antifreeze peptides by means of the biological expression, the antifreeze activity analysis, and the structural optimization based on theory methods. Further, the intracellular expression vector and the extracellular secretion vector with Nisin resistance gene will be used to construct food-grade genetic engineering of lactococcus lactis, and the antifreeze peptide will be expressed. Finally, this project will study the effect of recombinant antifreeze peptides in lactococcus lactis on the cells activity of resistance to freeze-drying stresses, the cell membrane system, and cell vitrification phase transition. The results will aim to reveal the effect of the expression of antifreeze peptides on the resistance to freeze-drying stresses in lactococcus lactis, and to elucidate the mechanism of action. This research will provide the new technical way and theoretical basis not only for the preparation of antifreeze peptides which can be used in cryogenic food processing as a type of food additives, but also for the research which apply molecule biology control technique to increase of the resistance to freeze-drying stresses of lactic acid bacteria.

乳酸乳球菌冻干菌粉作为一种发酵剂/功能载体在食品工业具有广阔的应用前景，但由于冷冻和干燥胁迫作用而导致冻干菌体细胞大量死亡的问题严重制约了其发酵效率和规模化应用。本项目针对抗冻肽抗冻特性和乳酸乳球菌食品级表达系统优点，综合应用食品科学、分子生物学和计算机化学等方法，从分子调控水平研究重组抗冻肽表达对提高乳酸乳球菌抗冻干胁迫性及其作用机制。本项目拟通过生物表达、抗冻活性分析和结构理论优化，筛选出高活性重组抗冻肽；利用带Nisin抗性基因的胞内和胞外分泌载体构建食品级基因工程乳酸乳球菌，表达抗冻肽；通过乳酸乳球菌基因重组抗冻肽对其抗冻干胁迫性、细胞膜系统与细胞玻璃化相变的影响，揭示抗冻肽胞内或胞外表达对乳酸乳球菌抗冻干胁迫性的作用，阐明其作用机制。本研究将为低温冷链食品加工中的一种新型食品添加剂- - 抗冻肽制备以及采用分子调控手段提高乳酸菌抗冻干胁迫性提供新的技术途径和理论基础。

乳酸乳球菌冻干菌粉作为一种发酵剂/功能载体在食品工业具有广阔的应用前景，但由于冷冻和干燥胁迫作用而导致冻干菌体细胞大量死亡的问题严重制约了其发酵效率和规模化应用。本项目针对抗冻肽抗冻特性和乳酸乳球菌食品级表达系统优点，综合应用食品科学、分子生物学、细胞生物学等方法，从分子调控水平和细胞分子水平，从乳酸菌代谢、细菌膜结构与功能特性、细胞凋亡机制等方面研究重组抗冻肽表达对提高乳酸乳球菌抗冻干胁迫性及其作用机制。项目主要完成了抗冻肽SerD与SF-P在大肠杆菌中的重组表达、分离纯化及活性分析，进一步将抗冻活性较高的SF-P在乳酸乳球菌食品级表达体系中进行重组表达，构建了食品级乳酸菌重组菌株。在此基础上，项目进一步系统分析了重组SF-P抗冻肽在胞内表达与胞外作用下对乳酸乳球菌在冷冻或冻干过程中耐受冷冻胁迫性能的影响。研究结果显示，SF-P在乳酸菌中胞内重组表达有助于提高冷冻胁迫后的菌株发酵活力，减少胞内关键酶活力下降，降低细胞膜的渗透性损伤程度，维持细胞膜的完整性；重组SF-P抗冻肽在乳酸菌中添加后，可以显著提高冷冻处理后菌生长活力，产酸能力和显著减少了乳酸菌胞内的Na+/K+-ATPase、Ca2+-ATPase、Mg2+-ATPase和Ca2+/ Mg2+-ATPase的活力降低，同时明显降低乳酸菌细胞膜通透性，提高细胞膜完整性，降低细胞膜电位，提高了菌液的玻璃化转变温度（数据未发表，暂时未展示）。结果表明SF-P重组抗冻肽主要通过与细胞膜的相互作用，减少细胞膜的低温损伤，降低冷冻过程菌的凋亡或维持菌正常代谢的作用方式来提高其对乳酸菌抗冷冻或冻干胁迫性能的调控。在项目开展过程，还进一步拓展了重组抗冻肽与天然抗冻肽在乳酸菌冻干粉、冷冻面团、速冻馒头等冷冻食品加工过程的应用基础研究，将理论研究和实际应用进行有机结合，提升了理论研究成果的实际应用价值。