Biodiversity And Probiotic Properties Of Lactic Acid Bacteria From Traditional Sour Whey In Yunnan

The sour whey is the natural milk coagulant for Yunnan traditional dairy products processing(such as milk cake,milk fan)through natural fermentation.Its unique technology,coupled with the diversity of natural environment in Yunnan,which makes the reserves of lactic acid bacteria is a valuable resources,and it is a natural treasure trove for screening Lactic Acid Bacteria(LAB)with beneficial function.Based on the Illumina MiSeq sequencing platform,the 16 S rRNA sequence of 18 samples of sour whey collected from Dehong,Kunming,Dali and Shangri-La were determined by Paired-End method and compared with SILVA database.The diversity of bacterial community and the bacterial composition of each classification level in sour whey were analyzed.LAB were isolated by traditional culture methods in the acid whey,which were identified and preserved by means of physiological and biochemical tests and gene sequence analysis,and phylogeny trees of lactic acid bacteria were drawn out to explore their evolutionary pathways and genetic relationships.Their probiotic properties were studied including gastrointestinal tolerance,epithelial cell adhesion and food borne pathogens bacteriostatic ability.The results were as follows:1.Diversity of lactic acid bacteriaHigh throughput sequencing indicated that the flora of sour whey of these four districts mainly consisted of 7 phylum and 38 genera.The relative abundance of gene sequence of lactobacillus and lactococcus accounts for 68.98% and 7.73% of total bacterial count in sour whey,which were the dominant genera.Using Simpson and Shannon index to analyze the bacteria diversity of samples,found that Dehong samples microbial diversity were the highest,which was 0.16±0.03,2.30±0.21 respectively;while Shangri-La samples bacterial community diversity were the lowest,which was 0.84±0.12,0.31±0.17 respectively.The bacterial community structure in different areas samples was significant difference(P<0.05).From Dehong Prefecture with low altitude and relatively mild climate,Kunming city and Dali Prefecture with middle and high altitudes,to the Shangri-La with high altitude and relatively cold climate,from the sour whey,it is found that,lactobacillus increased with altitude,while lactococcus gradually decreased with altitude.In addition,in this study,we found that the traditionally fermented sour whey in Yunnan contains small amount of pathogenic bacterium such as Staphylococcus,Enterococcus,Acinetobacter and Achromobacter.2.Isolation and identification of LABTraditional fermented sour whey were separated into 47 strains belongs to 4 genus(Enteroccoccus,Lactobacillus,Pediococcus,Streptococcus),12 species or subspecies,including Enterococcus faecium(22 strains),Lactobacillus fermentum(6 strains),Pediococcus acidilactici(4 strains),Enterococcus durans(3 strains),Lactobacillus reuterii(3 strains),Lactobacillus delbrueckii subsp.bulgaricus(2 strains),Streptococcus gallolyticus(2 strains),Pediococcus pentosaceus(1 strains),Lactobacillus oris(1 strains),Streptococcus macedonicus(1 strains),Lactobacillus pontis(1 strains)and Lactobacillus plantarum(1 strains).Enterococcus faecium was the dominant species of traditional sour whey,held 46.81% of the total isolated LAB.3.Probiotic properties of LAB(1)The lactic acid bacteria isolate strains were cultured for 2h under simulated gastric juice of pH 2.0,and then exposed to simulated small intestinal juice with 0.1% pancreatin and 0.3% bile salt,which had a strong ability to survive for up to 2h.Test 21 strains included L1-6,K2-3,K3-1,D1-2,L5-1,K2-4,D1-1,K1-2,L1-2,D2-2,X1-1,L6-3,X2-2,D2-1,L1-3,L3-1,L5-2,D3-3,D3-2,D3-4 and L2-1 were maintained at a high survival rate,the LAB counts in these strains were greater than 5 log cfu/mL,has good gastrointestinal tolerance,great potential in intestinal colonization.(2)Adhesion ability of HT-29 human colon carcinoma cells showed that the K2-4(Enterococcus faecium)adhesion rate was the strongest,for the 24.12%.Compared with the reference strain Lactobacillus rhamnosus GG,the adhesion ability of 12 strains of LAB isolates was stronger(P <0.05).(3)The antibacterial results showed that foodborne pathogens(Escherichia coli,Salmonella typhimurium,Staphylococcus aureus,Listeria monocytogenes),Lactobacillus plantarum D3-2,Lactobacillus delbrueckii Subsp.bulgaricus D1-1,Pediococcus pentosaceus D2-2,Enterococcus faecium K2-4,L2-1 and L6-3 had a good antibacterial effect(Inhibition zone> 6mm),broad-spectrum antibacterial.In addition,LAB isolated supernatant bacteriostatic effect was better than live bacteria.The diversity of LAB in traditional fermented sour whey was revealed,and the resources of LAB were collected and evaluated for its probiotic properties,which provided scientific basis for the development and utilization of fermented dairy products.