Phenotypic, Genotypic And Probiotic Characterization Of Lactic Acid Bacteria Isolated From Chinese Yak Milk Cheeses

There are various kinds of lactic acid bacteria existing in Chinese yak milk cheeses, which is an extremely precious natural microbiology resource. It is a research work of great values that collecting, organizing, preserving and investigating those lactic acid bacteria. This study collected17yak milk cheese samples from Tibet, Yunnan and Xinjiang province, isolated lactic acid bacteria, studied their phenotypic, genotypic and probiotic characterization.The phenotypic characterization study included species identification of isolated lactic acid bacteria with classic taxonomy methods, such as colony morphology, Gram stain and microscopic morphology, primary biochemical and physiological properties, growth capability in different conditions (temperature, pH value, and salt concentration), carbohydrates fermentation patterns, acid producing activity, capability of utilizing citrate and so on. Isolated lactic acid bacteria were assigned to genus level, and preliminary species level. Phenotypic experiment results provided a comprehensive data of lactic acid bacteria. The capability of producing acid and utilizing citrate directly demonstrated lactic acid bacteria’s role in fermentation.The genotypic characterization study included16S rRNA sequenc analysis and randomly amplified polymorphic DNA (RAPD). This study established a rapid identification mode:genus specific PCR→16S rRNA sequence analysis→species specific PCR. Isolated lactic acid bacteria were identified to species level with this mode, phylogenetic tree was constructed based on16S rDNA sequences to confirm species assignment and demonstrate strains’genetic relationship. This study also established a stable RAPD reaction system and amplification program for lactic acid bacteria. Isolated lactic acid bacteria were subjected to this RAPD analysis, NTsys2.10e software was used to perform cluster analysis of strains’RAPD fingerprinting patterns. The cluster result were compared with that of16S rRNA sequence analysis to figure out genetic relations of isolated strains, as well as the correlativity between strains’cluster patterns and origin regions.The probiotic characterization study included bacteriocin, extracellular polysaccharide and folic acid producing capability, cholesterol-reducing ability, antibiotic resistance, tolerance ability to in vivo conditions, adhesion activity. Strains with excellent health care functions and tolerance ability were selected out to be evaluated their potential as probiotic strains.The main results were summarized as follows:(1) The average pH value of yak milk cheese samples collected from Tibet, Yunnan and Xinjiang province were4.31±0.39,4.69±0.29,4.52±0.22respectively, no significant difference between three regions(P=0.176>0.05). The average colony enumeration of samples collected from Tibet, Yunnan and Xinjiang province were4.17±0.43;4.30±0.68;3.9±0.59log CFU/g respectively, no significant difference between three regions (P=0.468>0.05).(2)39strains of lactic acid bacteria were isolated from17yak milk samples. Those39strains were identified to genus level and preliminary to species level with classic taxonomy methods. Among the39strains,35strains were rod shape, assigned to7species:2strains L.buchneri (Y15, Y16),16strains L.casei(T7, Y19, X21, X22, X23, X26, X27, X28, X29, X30, X31, X32, X33, X34, X35, Y36),3strains L.diolivorans (Y18, X24, X25),3strains L.fermentum (T1, T2, T3),3strains L.helveticus (T6, T8, Y9),3strains L.kefiri (Y10, Y12, Y20),5strains L.plantarum (T4, Y13, Y37, X38, X39)。4strains were coccus shape, assigned to2species:1strain Pediococcus acidilactici (T5),3strains Pediococcuspentosaceus (Y11、Y14、 Y17)。(3)6Lactobacillus strains were selected out by acid producing test because they had such a high acid producing capability to meet the standard of starter cultures, and they were X24、X38.X29、X25、X21、X22. Eight strains with excellent ability to utilize citrate were also selected out:T4、T7、Y10、Y13、X25、X28、Y36、Y37. The excellent ability of strains may be developed during their evolution in the plateau climate and long-term dairy-producing practice.(4)39strains were subjected to16S rRNA analysis to identify species with genotypic method. Phylogenetic tree was also constructed based on16S rDNA sequences. The species assignment result with phenotypic method was confirmed and strains’genetic relationship was demonstrated.(5)39strains were subjected to RAPD fingerprinting analysis. Except for strains X23, Y36and Y37, the fingerprinting band of strains were clustered to different groups according to their species; this result corresponded well with that of16S rDNA sequences analysis. The cluster analysis also showed excellent correlative relationship between group patterns and origin regions. It is proposed that RAPD method showed excellent efficiency in taxonomy and genomic relationship analysis.(6)31strains were tested to produce bacteriocin. Bacteriocin produced by strain Y13had the antagonism action against5pathogenic reference strains:Bacillus cereus ATCC10876、Escherichia coli O]57:H7、Listeria monocytogens、Citrobacter freundii ATCC8090、Salmonella Typhimurium ATCC13311. Strain X29and X30had the antagonism action against4pathogenic reference strains:Bacillus cereus ATCC10876, Citrobacter freundii ATCC8090, Salmonella Typhimurium ATCC13311, Enterobacter cloacae ATCC23355. All39strains had no antagonism action against Streptococcus thermophiles which was a widely used strain in dairy industry. All strains were suitable for use with Streptococcus thermophiles in dairy fermentation.(7) Nine strains were selected out because they have capability to produce extracellular polysaccharide:T3. T4、T5、T7、T8、Y1K、Y14、Y17、X29. The yield of extracellular polysaccharide was tested with phenol-sulphuric acid method. Strain gave the highest yield result193.72mg/L, which was a relatively low level by comparison with literature report.(8) Nine strains were selected out because they have capability to produce folic acid:T3、T7、T8、X23、X25、X26、X27、X29、Y37. Strain Y37gave the highest yield result85.98±5.09ng/ml. X25ranked in second place, amounted to81.17+2.08ng/ml, which was a very high level by comparison with literature report17-1OOng/ml.(9) Strains Y15. X25and X27were selected out because they had excellent capability to remove cholesterol from MRS-CHOL medium, the removal percentages were amounted to45.59%,50.01%and53.18%, which was a very high level by comparison with literature report. (10) The antibiotic resistance of39strains were tested with disc diffusion method. Among39strains,74.4%,82.1%and82.1%were sensitive to ampicillin; chloramphenicol and erythromycin respectively,100%,97.4%,92.3%and82.05%were resistant to nalidixic acid, streptomycin, vancomycin and neomycin.(11) Strains Y15, X25and X27were selected out because they have excellent tolerance ability to in vivo conditions, as well as excellent adhesion activity.(12) By summarizing all experiment data, Strains Y15, X25and X27were selected out as potential probiotic strains. Strain Y15(L.buchneri) had the ability to produce extracellular polysaccharide (50.26±1.25mg/L), reducing cholesterol (removing percentage45.59%), resistance to nalidixic acid, streptomycin and vancomycin. Strain X25(L.diolivorans) had the ability to produce forlic acid (81.17±2.08ng/mL), reducing cholesterol (removing percentage50.01%), resistance to nalidixic acid, streptomycin, tetracycline and vancomycin. Strain X27(L.casei)had the ability to produce extracellular polysaccharide (38.62±1.21mg/L), produce folic acid (77.83±2.52ng/mL), reducing cholesterol (removing percentage53.8%), resistance to neomycin, nalidixic acid, streptomycin and vancomycin, producing bacteriocin which showed antagonism action against pathogenic reference strains Bacillus cereus ATCC10876, Staphylococcus aureus and Escherichia coli O157:H7.The creativeness and innovation of this study:(1) This study investigated yak milk samples from a broad area, Tibet, Yunnan and Xinjiang province, which covers1/3-1/4of our country’s area. It is the most comprehensive and systematic fundamental and application research work on lactic acid bacteria from yak milk cheeses.(2) This study established a rapid identification mode:genus specific PCR→16S rRNA sequence analysis→species specific PCR, which is suitable for massive identification work of natural lactic acid bacteria, promoting efficiency and precision.(3) This study established a RAPD reaction system and amplification program with excellent reproducibility and stability for lactic acid bacteria. The result of this method corresponded well with that of16S rRNA sequence analysis. It is efficient to figure out genetic relations of isolated strains, as well as the correlativity between strains’cluster patterns and origin regions. (4) This study selected out three potential probiotic strains with excellent health care function and tolerance capability.