宏基因组学探究原料乳冷藏过程菌群变化规律

Metagenomics approach to explore the changes of bacterial community in raw milk during refrigerated storage

为探究原料乳4℃冷藏期间细菌群落的变化规律,该研究使用Illumina Hiseq测序平台,对原料乳冷藏期间微生物变化及功能注释进行宏基因组学分析。结果表明:随着冷藏时间的延长,原料乳中的微生物在数量和种群构成上均发生了显著改变。冷藏72 h期间,优势菌群由不动杆菌属、链球菌属、无浆体属和梭菌属向黄杆菌属、假单胞菌属和乳球菌属逐渐演替。功能注释结果显示:复制重组修复、翻译/核糖体结构与生物发生、细胞壁/膜的生物发生、脂质代谢在冷藏前期相对丰度较高。氨基酸、碳水化合物的转运与代谢在冷藏后期相对丰度较高。其中脂质代谢与不动杆菌属显著相关(P<0.001),氨基酸代谢、碳水化合物代谢与假单胞菌属显著相关(P<0.01)。研究表明冷藏原料乳中不动杆菌属及假单胞菌属对乳成分影响较大。通过控制冷藏原料乳有害微生物繁殖,能够维持乳成分稳定。研究结果可为生鲜乳保藏、液态乳灭菌控制、奶酪加工等提供理论依据。

Refrigeration storage is one of the important methods to inhibit the reproduction of microorganisms in raw milk.But the psychrophilic bacteria still grow during this time,particularly on synthesizing phospholipids at the relatively low storage temperature.Meanwhile,these psychrophilic bacteria hydrolyze protein and fat can cause discoloration,odor,viscosity increase,gelation,rancidity,and other defects phenomena in the raw milk.So far,there are few reports on the changes of the flora in the raw milk during cold storage.In this study,an IlluminaHiseq sequencing platform was established to conduct metagenomic analysis of bacteria in the raw milk,in order to investigate the bacterial community succession and functional characteristics of raw milk with the extension of refrigeration time.During 72 hours of storage,the number of culturable microorganisms observably increased in the raw milk,including the number of colonies,psychrophilic bacteria,lactic acid bacteria,and coliform bacteria.Taxonomy annotation indicated that there was a significant effect for the microbial succession of raw milk during cold storage.At the genus level,the dominant flora gradually evolved from Acinetobacter,Streptococcus,Anaplasmaand and Clostridium to Flavobacterium,Pseudomonasand Lactococcus.At the species level,it evolved from Acinetobacter sp.TTH0-4,Anaplasmaphagocytophilum,Streptococcus pneumoniae,and Streptococcus mutans to Flavobacteriumfrigidarium,Pseudomonas fluorescensand Lactococcuspiscium.There were few reports on Acinetobacter sp.TTH0-4,Flavobacteriumfrigidarium,and Lactococcuspiscium.The results of COG functional annotation showed that the L(replication,recombination and repair),J(translation,ribosomal structure and biogenesis),M(cell wall/membrane/envelope biogenesis)had relatively higher abundance in the early stage of refrigeration(24 h),suggesting that the raw milk microbes were more resistant to the variable milk environment during this period.The relative abundance of E(Amino acid transport and metabolism)and G(Carbohydrate transport and metabolism)were higher in the late stage of refrigeration(72 h),indicating that the utilization of carbon and nitrogen sources by microbes increased at this stage.The results of correlation analysis between dominant microorganisms and main functions show that Acinetobacter was significantly related to the lipid metabolism.Combining with its characteristics of accumulating fatty acids,it infers that Acinetobacter had a greater impact on the composition of raw milk lipids.However,Pseudomonas was significantly related to the carbohydrate metabolism and amino acid metabolism,where to produce protease and lipase,and thereby pose a great influence on the decomposition of raw milk protein and the formation of flavor.In addition,the characteristics of Lactococcus,decomposing lactose to produce acid and inhibit bacteria,were closely related to the microbial control of raw milk and chess processing.The finding can provide new ideas for the research of raw milk microorganisms in the preservation of raw milk,sterilization control of liquid milk,and processing of dairy products.Further in-depth study of this subject can be conducted in the correlation analysis between metabolomics and metagenomics,and thereby to discover the mechanism of quality changes in the raw milk.