Studying The Microbial Diversity In Raw Milk By Biological Methods

Both milk and dairy products are rich in nutrition, in people’s diet structure has an important position and role. At the same time, it also provide a good medium for the growth of microorganisms. The raw milk is the key to influence the quality and safety of dairy products, the quality must be controlled from the source control of raw milk.Therefore, it is necessary to establish a rapid and effective method for analyzing its bacterial species and floras in raw milk.In order to analysis bacterial communities in raw milk, raw milk from Shandong and Ningxia were chosen as objects of research. The methods of the isolation and culture of bacteria,physiological and biochemical characteristics analysis,the extract of isolated strain DNA- PCR amplification-16SrDNA molecular sequencing technology were used to analyse the bacterial species, diversity and floras of culturable bacteria from raw milk by Microbiological and molecular biological methods. Analysis of the 16 s rDNA sequence results show that:34 strains of culturable bacteria species of A, B, C three raw milk samples from Shandong in June were identified mainly to Acinetobacter sp.and Klebsiella sp. which accounted for 26.5% and 14.7% respectively of the total number of bacteria.21 strains of culturable bacteria species of D, E, F three raw milk samples from Ningxia in October were identified mostly to Lactococcus lactis subsp.lactis and Acinetobacter sp.respectively, accounted for 33.3% of total bacteria and 14.3%.9 culturable bacteria strains of raw milk sample A in October from Shandong were identified mainly to Pseudomonas aeruginosa accounted for 22.2% of the total bacteria.Since most of the bacteria can not be identified by cultivating, so the diversity of bacteria in raw milk can not really reflected by means of pure culture.It is difficult to extract genome DNA directly from raw milk due to the amount of bacteria in raw milk is less.So the genome DNA of bacterium was extracted from raw milk which were concentrated by straightforward filtration and the 16S rDNAs of the extracted genomic DNA were amplified using bacterial universal primers 27F and 1492R,PCR products were ligated into the T Vectors and transformed into Escherichia coli DH5α, Colony PCR amplification of 16S rDNA fragment in the positive transformant, amplified ribosomal DNA restriction analysis(ARDRA) patterns which used enzyme digestion(Msp I and HaeⅢ)of cloned PCR16S rDNA gene sequnces, respectively, their fingerprints were analyzed, representative strains were send to sequence the 16S rDNA sequence. Analysis of the 16S rDNA sequence results show that:The clone library of raw milk samples A from Shandong in October mainly to Lactic acid bacterium accounted for 25% of the total clones.The clone library of raw milk samples D from Ningxia in October mostly to Lactococcus sp. accounted for 62.5% of the total clones.Using 16S rDNA-ARDRA to establish clone library methods in the study of bacterial diversity also has limitations, so we can combine the traditional culture with 16S rDNA-ARDRA clone library to better understand the population structure and diversity of bacteria in raw milk. Through the bacterial species identification in raw milk showed that raw milk has a variety of bacterial.From the pathogenic point of view, all strains are opportunistic pathogens or spoilage, no virulent pathogens.