Diversity Analysis Of Lactic Acid Bacteria In Yak Milk Products In Gansu And Sichuan Provinces

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 and16S rDNA sequencing analysis, respectively. Meanwhile, the microbiological biodiversity of some representative samples were analyzed by metagenomics methods, such as PCR-DGGE and454pyrophosphate sequencing. The conclusions were showed as follow:The LAB counts in five kinds of yak milk products were ranged from4.00lg cfu/mL to9.15lg cfu/mL, and most of them were up to6.00lg cfu/mL. Five hundred and thirty-three LAB isolates were isolated from these samples by traditional pure culture method using MRS and M17broth.Five hundred and thirty three isolates were identified to species and/or subspecies by phenotypic characterization and16S rRNA gene sequence analysis. For the LAB species with very closely related16S 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:246strains of lactobacilli (46.15%of total),156strains of leuconostoc (29.27%),67strains of Streptococci (12.57%),50strains of lactococci (9.38%),11strains 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 (129strains,24.2%of total) and Leuconostoc mesenteroides subsp. mesenteroides (110strains,20.64%) could be considered as the predominant LAB species in traditional fermented yak milk products in Gansu and Sichuan provinces. The composition of LAB in different types of yak milk products in different regions expressed differences. Lb. helveticus was the dominant bacteria in kurut, qula and whey samples in Gansu province. However. Streptococcus thermophilus and Lb. helveticus are the predominant LAB in kurut and qula in Sichuan province, respectively. The16S rRNA gene sequence database of LAB in yak milk products in Gansu and Sichuan povinces were established. The16S rRNA gene sequences of533LAB strains were deposited to GenBank database with the accession numbers as HM058517-HM058580, HM058582-HM0059029, HM217942-HM217944, HM217948, HM217953, HM217956, HM217962, HM217964, HM217967, HM217971-HM217975, M217984-HM217986, HM217994-HM217999.Microbial diversity in ten representative qula samples from Sichuan province were analyzed using metagenomic method PCR-DGGE. The result showed that a complex microflora including more than nine kinds of LAB species(Lb. helveticus, Lb. plantarum, Lb. fermentum, Lb. paralimentarius, Lb. sanfranciscensis, Leu. citreum, Lb. curvatus, E. durans and Lac. lactis) was exised in these sampls.Microbial diversity in seven representative kurut samples collected from Gansu and Sichuan province were comprehensively examined by high-throughput parallel454Pyrosequencing technology. The results showed that an abundant and complex microflora was presented in these kurut samples. The kurut microbiota was far more diverse than previous studies, and thirty seven genera species belonging to six phyla(Firmicutes, Actinobacteria, Bacteroidetes, Deinococcus-Thermus, Proteobacteria, Environmental samples) were identified after16S rDNA sequencing analysis. Firmicutes(Lactobacillus, Streptococcus, Lactococcus, Leuconostoc) possessed the highest ratio in these kurut samples. Moreover, the main bacterial composition of these kut samples in Gansu and Sichuan province expressed obvious regional differences. These data gave us the opportunity to renew the understanding of the microbial diversity in kurut samples.The pure culture method and metagenomics methods all confirmed that the microorganisms in traditional fermented yak milk products were diverse and LAB could be considered as the predominant bacteria, among which Lactobacillus was the preponderant genus. The three methods used for microbial diversity analysis have their specific advantag Traditional method can only analyse the culturable bacteria, and its advantage was the obtained pure culture could be used for further starter culture selection. PCR-DGGE can intuitively reflect the the composition of the dominant microorganisms in these samples.454Pyrosequencing analysis enables us to analyze an increased number of samples at a time, to obtain more reads in a single run and to perform indepth analyses for studies of comparative microbial ecology.This paper firstly systematically analyzed the microbial composition in raw yak milk and various traditional fermented yak milk products in Gansu and Sichuan province of China. These obtained LAB pure cultures maybe a valuable source for selection of probiotics and starter cultures in dairy industry. Meanwhile, these results provided the basic data for further industrilization of traditional fermented dairy products.