甘肃和四川省牦牛奶制品中乳酸菌的多样性分析-diversity analysis of lactic acid bacteria in yak milk products in gansu and sichuan provinces.docx

Diversity Analysis of the Lactic Acid Bacteria in Yak Milk Products in Gansu and Sichuan province of China Abstract One hundred and fifty-two samples of raw yak milk and traditional fermented yak milk products were collected from twenty-seven different areas of six counties in Gansu and Sichuan province, China. The lactic acid bacteria (LAB) were isolated and identified from these samples using traditional pure culture method and 16S rDNA sequencing analysis, respectively. Meanwhile, the microbiological biodiversity of some representative samples were analyzed by metagenomics methods, such as PCR-DGGE and 454 pyrophosphate sequencing. The conclusions were showed as follow: The LAB counts in five kinds of yak milk products were ranged from 4.00 lg cfu/mL to 9.15 lg cfu/mL, and most of them were up to 6.00 lg cfu/mL. Five hundred and thirty-three LAB isolates were isolated from these samples by traditional pure culture method using MRS and M17 broth. Five hundred and thirty three isolates were identified to species and/or subspecies by phenotypic characterization and 16S rRNA gene sequence analysis. For the LAB species with very closely related 16S rRNA gene sequence, species-specific PCR, PCR-RFLP and DGGE technologies were used. Meanwhile, the platform for LAB identification by molecular biology methods was established. All the isolates were classified into twenty-four species and/or subspecies, which belonged to six genera (Lactobacillus, Lactococcus, Leuconostoc, Streptococcus, Enterococcus and Weissella). The distributions of the isolates by genus were as follows: 246 strains of lactobacilli (46.15% of total), 156 strains of leuconostoc (29.27%), 67 strains of Streptococci (12.57%), 50 strains of lactococci (9.38%), 11 strains of enterococci (2.64%) and three strains of weissella (0.56%). Meanwhile, according to the results of LAB biodiversity analysis, we can concluded that Lb. helveticus (129 strains, 24.2% of total）and Leuconostoc mesenteroides sub