基于多组学技术的原料乳冷藏过程中微生物群落演替驱动机制研究

High quality of raw milk is the premise of good dairy products. Ningxia is the national dairy advantage region which was determined by The Ministry of agriculture and is also the important high quality milk source base of china. Refrigeration can effectively reduce microbial propagation speed. But, in low temperature, raw milk and milk products also can be deteriorated by the microorganisms growing slowly and the enzyme they produce. In recent years, researchers in the world pay more attention to analysis the biodiversity, characte and detecte the dominant spoilage strains in raw milk. However, scholars did not do indepth research on microbial ecology on the changes of milk components leading to the microbial community succession and corruption mechanism in raw milk. Using the techniques of metagenomics based on SMRT with dependent cultivation methods, the microorganism community succession will be studied. Using the techniques of proteinomics based on iTRAQ and metabolomics based on UPLC-Q-TOF-MS, the relationship among microorganism community succession, nutrition, metabolism and the environmental conditions will be studied. Through building the gene interactions network, the protein interaction network and the metabolic networkthe, associating the multiple omics, the inner driving mechanism of microbial community succession on raw milk during refrigeration will be constructed. The study aims to provide an indepth theory basis for the quality control of raw milk, and it will provide a theoretical reference for the renovation of the refrigerated technology.

优质原料乳是生产高品质乳制品的前提，宁夏是农业部确定的全国奶牛优势区，是重要的优质奶源基地。冷藏可以有效降低原料乳中微生物的生长。但其缓慢生长及产生的酶类物质仍会造成原料乳及乳制品的品质劣变。目前国内外原料乳微生物的研究着眼于生物多样性、腐败菌及嗜冷菌的特性研究及检测，对冷藏过程中乳成分变化导致的微生物群落演替及其腐败机制的相关生态学领域未做深入研究。项目拟采用基于SMRT的宏基因组学技术结合选择培养法研究原料乳冷藏过程中微生物群落演替规律，采用基于iTRAQ的宏蛋白质组学技术、采用基于UPLC-Q-TOF-MS的代谢组学技术研究群落演替与营养、代谢组份及环境条件变化的关系，研究基因、蛋白质及其分子间相互作用，通过构建基因互作网络、蛋白质互作网络、代谢网络，经多组学关联，揭示微生物群落演替的内在驱动机制。为原料乳冷藏的质量控制层次的提供深理论依据，为冷藏技术创新提供理论参考。

冷藏可以有效降低原料乳中微生物的生长，却无法完全避免最终变质。冷藏过程中微生物改变乳环境使得乳代谢物变化的同时乳代谢物又驱动着微生物的定向演替，造成原料乳品质随时间的延长逐渐劣变直至腐败。目前国内外原料乳微生物的研究着眼于生物多样性、腐败菌及嗜冷菌的特性研究及检测，对冷藏过程中乳成分变化导致的微生物群落演替及其腐败机制的相关生态学领域未做深入研究。本项目针对冷藏6天的原料乳，采用Illumina测序平台进行的宏基因组学分析，采用UHPLC-Q-TOF MS进行代谢组学分析，采用Lable-free进行宏蛋白质组学分析，在此基础上进行多组学关联分析，从而探究冷藏原料乳微生物群落演替的机制。具体研究结果如下：随冷藏时间的延长，微生物由不动杆菌属、链球菌属、无浆体属向黄杆菌属、假单胞菌属、乳球菌属逐渐演替。冷藏过程中鉴定的341个微生物蛋白质，多为3d以后产生，主要来源于假单胞菌属、梭菌属、乳球菌属、不动杆菌属、分歧菌属以及乳杆菌属。乳代谢物主要分为脂质及类脂分子代谢物、氨基酸及其类似物、有机酸、碳水化合物及其结合物、核苷酸和维生素6类。代谢物与微生物关联分析发现，冷藏2天内，显著性差异代谢物种类较少，且与菌群的关联性较弱；冷藏3 d ~ 4 d，显著性差异代谢物种类增多，参与的代谢途径增多，且与菌群的相关性复杂。脂类与不动杆菌属呈显著正相关；V C与黄杆菌属呈显著正相关；氨基酸类、碳水化合物类物质与小丰度菌属呈正相关性，乳酸与乳球菌属的相关性＜假单胞菌属、链球菌属、肠球菌属、肉杆菌属、环丝菌属。宏蛋白质组学和代谢组学关联分析发现，1 d/0 d的差异微生物蛋白质和差异代谢物并未涉及相同的通路，而随着冷藏时间的推移，共同代谢通路增加。比较差异微生物蛋白质和差异代谢物的相关性发现，5种蛋白质与超过30种以上差异表达代谢物的相关性较强，可能是原料乳中代谢物差异表达的关键蛋白。