益生菌冰淇淋加工过程冷冻胁迫致菌体损伤机制及控制策略研究

During processing of probiotic ice cream, probiotics added to ice cream mixture will experience periodical temperature decreasing to freezing in the process of aging, freezing and hardening . The mechanism of physiological injury of the probiotic cells caused by the freezing stress is still unclear, and the quality control strategy of ice cream by decreasing such injuries to increase probiotic viability is also less studied. Using probiotic strains isolated in our lab, the present research will be carried out to study the ability of probiotic survival, damage of cell membrane and activity changes of key metabolic enzymes under the freezing stress conditions during ice cream processing. By using metabonomics methods, changes of probiotic metabolites associated with cell physiological damage will be studied in order to determine the most important metabolites. Proteomics methods will be employed to study changes of probiotic protein expression, and the differential proteins expressed will be identified, their functions classified and metabolic pathways analyzed. Further studies will be carried out to elucidate the molecular mechanism of bacterial cell injury caused by freezing stress in ice cream, and to establish the corresponding protection strategy for the probiotics during ice cream processing. These data will provide a scientific basis for effective improvement of functionality and quality of probiotic ice cream, and establishment of its product and technological standards. The results of this study will be of great theoretical and practical importance in enhancing ice cream processing technology of our country, promoting research and development of functional ice cream, and accelerating development of the ice cream industry.

益生菌冰淇淋加工中添加于物料的益生菌经历老化、凝冻和硬化等阶段性降温至冷冻的过程，由此冷冻胁迫导致菌体生理损伤的分子机制及基于降低这种损伤并提高益生菌存活力的冰淇淋品质控制策略，目前仍然研究甚少。本研究利用课题组分离筛选的益生菌，研究其在冰淇淋加工过程冷冻胁迫下的存活能力、菌体细胞膜特性及代谢关键酶活性变化；采用代谢组学方法研究益生菌代谢产物的变化及其与菌体生理损伤的关系，探明与菌体生理损伤相关的重要代谢产物；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。深入研究冰淇淋基质中益生菌冷冻胁迫损伤的分子机制，探讨冰淇淋加工和贮藏过程中益生菌的保护策略，为有效地改善益生菌冰淇淋的功效和品质，建立益生菌冰淇淋产品和技术标准提供科学依据。本项研究对于提升我国冰淇淋加工技术水平、促进功能性冰淇淋产品研发、加快冰淇淋产业发展具有重要的理论和实际意义。

经过4年的努力，项目组超额完成了项目预期目标。发表SCI收录文章9篇，EI收录文章5篇，中文核心期刊收录文章5，科技核心期刊收录文章2篇；获授权发明专利1项；提出冰淇淋基质中益生菌的保护措施1 套。制定益生菌冰淇淋规范化操作规程1 套。培养博士后2人，博士研究生1人，硕士研究生3人。项目成员1人获得高级技术职称。. 从22株耐低温乳酸菌筛选出具有突出的耐受低温、低pH和高胆盐特性植物乳杆菌K25和YW11。发现冰淇淋加工过程的老化、凝冻、硬化和冷藏等低温工艺对益生菌的损伤，确定了益生菌的不同添加方式对菌株损伤的影响。各低温工艺对菌株活力的影响按其大小依次为硬化>老化>凝冻。益生菌以3种不同的添加方式即冻干菌粉、发酵乳、菌株活化后直接接种于浆料，菌株受冰淇淋加工过程低温损伤后，菌株酸耐受能力（pH3.0）分别下降至正常菌株的63.2%，48.2%和38.8%，胆盐耐受能力（0.5%，w/v）分别下降至正常菌株的51.8%，11.9%，10.8%。低温损伤会影响菌株的乳酸、多糖代谢，并影响冰淇淋质构和风味。采用iTRAQ蛋白质组学技术对植物乳杆菌K25在低温条件下的蛋白质表达分析，qRT-PCR对差异蛋白质基因表达验证。结果表明低温条件下，碳水化合物、氨基酸与脂肪酸合成与代谢相关通路受抑制，DAN转录与蛋白质翻译等相关蛋白质表达上调。表明K25在低温条件下采取抑制自身生长的策略，为菌体提供充足的时间以调节胞内代谢通路与蛋白质组成，以适应低温胁迫环境。采用荧光标记法对不同温度条件下菌膜通透性变化进行研究表明，低温条件下菌株细胞膜能够保持的完整性，但胞内的钙离子浓度显著增加。研究采用DIA定量蛋白质组学研究钙离子胁迫下植物乳杆菌K25的蛋白表达，发现钙离子调控植物乳杆菌K25的离子转运、核糖体合成、UMP 的合成、脂肪酸合成、碳源转运和代谢及氨基酸合成。改变MelB蜜二糖转运体、磷酸转移酶系统、α-甘露糖苷酶、甘露糖磷酸转移酶系统IICD、芳基糖苷酶Bgl和糖苷水解酶 GH1、麦芽糖（MalFGK）、DhaK和果糖1, 6-二磷酸酶FBPase的表达量增加，胞外多糖的合成量增加，改变了菌体周边微环境的溶液冰点，保护了菌体免受冰晶损伤。