益生菌发酵乳冷藏过程低温酸性双重胁迫对益生菌活性的影响机制及保护策略

Probiotics have to endure long-time double stresses from low temperature and high acidity during cold storage of fermented milk. The molecular mechanism of the resulting probiotic activity reduction, thus the decrease or loss of product function is scarcely studied. In this study, probiotics isolated by our research group are used to study their low-temperature growth characteristics, ability of tolerance to low temperature and changing rule of the physiological activity of probiotics under low temperature and acid stresses. Metabonomics methods are used to study change of metabolites under low temperature and acid stresses during cold storage of fermented milk, and its relation to physiological injury of bacteria; proteomics methods are used to study change of protein expression by probiotics, identification and functional classification of differentially expressed proteins and analysis of the metabolic pathways, to verify the important proteins, key enzymes and metabolites related to the physiological injury of bacteria. On the basis of these results, the molecular mechanism will be elucidated with respect to the low temperature and acid stresses induced physiological injury and activity loss of probiotics during cold storage of fermented milk. The probiotic protection strategies to reduce the physiological injury will be studied. These will provide scientific basis for effective improvement of probiotic activity during cold storage of fermented milk, establishment of activity-protection based technical specifications for probiotic fermented milk and definition of the product function. This research has great theoretical and practical significance for enhancing technological levels and product quality of probiotic fermented milk, and standardizing the market of probiotic dairy products in China.

益生菌发酵乳冷藏过程中菌体经受长时间低温和酸的双重胁迫，由此造成的益生菌活性下降致使产品功能性降低甚至丧失的分子机制，目前仍然研究甚少。本研究利用课题组分离筛选的益生菌，研究其低温生长特性、耐低温能力及低温和酸胁迫下益生菌生理活性的变化规律。采用代谢组学方法研究发酵乳冷藏过程中益生菌代谢产物的变化及其与菌体生理损伤的关系；采用蛋白质组学方法研究益生菌蛋白质表达变化，并对差异表达蛋白质进行鉴定、功能分类和代谢途径分析，探明与菌体生理损伤相关的重要蛋白质、关键酶及代谢产物等。在此基础上阐明发酵乳冷藏过程低温和酸胁迫导致益生菌生理损伤及活性降低的分子机制，探讨降低菌体生理损伤的益生菌保护策略，为有效地提高发酵乳冷藏过程益生菌的活性，建立基于活性保护的益生菌发酵乳技术规范及产品功能界定提供科学依据。本研究对于提升益生菌发酵乳技术水平和产品质量，以及规范我国益生菌乳制品市场具有重要的理论和实际意义。

研究从课题组分离的31株乳酸菌中，筛选出益生性植物乳杆菌NMGL2为代表性菌株，明确了其低温生长特性、耐低温能力及低温和酸胁迫下益生菌生理活性的变化规律。采用蛋白质组学方法，确定了NMGL2菌株20个耐低温关键蛋白和10个耐酸关键蛋白，这些蛋白主要负责细胞壁合成、胞外糖脂和糖蛋白合成，F1F0-ATP酶的合成和激活谷氨酸脱酸系统等一系列生理活动，完成酸性和低温环境导致的细胞损伤修复。采用SPME-GC/MS 和UPLC-Q-TOF-MS/MS的方法研究了植物乳杆菌NMGL2发酵乳在4℃下储存21天中代谢产物的变化情况，进一步确证参与耐低温和耐酸的小分子化合物和代谢通路。共检测出20种不同代谢物，酸类7个，醇类4个，醛类2个，酮类4个，酯类3个，差异代谢产物122个，主要涉及嘌呤代谢、有机酸代谢、氨基酸代谢和乳糖代谢。鉴于抗逆代谢涉及的生理活动主要为有机酸和碳水化合物代谢，研究考察了低聚半乳糖对菌株耐受低温、酸性双胁迫的作用，结果显示4%的GOS能有效增加菌株的生长，在发酵乳储存21天后，植物乳杆菌NMGL2的活菌数>8.7 lg cfu/mL。本研究对于提升益生菌发酵乳技术水平 和产品质量，以及规范我国益生菌乳制品市场具有重要的理论和实际意义。研究共发表SCI及CSCD收录科研论文16篇。申请低温和酸耐受菌株相关技术发明专利1项。提出发酵乳冷藏过程中益生菌的保护技术措施1 套。培养研究生6名(其中博士生1 名)，博士后出站1 人。