Study On Variation Of Lactic Acid Bacteria During Traditional Qula Preparation And Screening And Function Evaluation Of Probiotic Lactobacillus From Qula

Traditional handmade Tibetan Qula cheese made from Yak milk samples from different areas in Qinghai Province are the objects of this study.The nutritional features,microbial flora composition and the characteristic of Lactobacillus were analyzed;while the dynamic changes of various microorganisms and lactic acid bacteria types during the processing of Qula were firstly studied;probiotic Lactobacillus in Qula were firstly screened and the genetic stability of individual probiotic strains were analyzed.Meanwhile,mice models were integrated for comprehensive function evaluation on its immunoregulation and oxidation resistance.It was founded in the study that:(1)A total of 43 traditional handmade yak milk Qula samples that were collected from different areas in Qinghai provinces were collected,among which 11 representative samples were selected on the basis of original area to be analyzed for pH values,water content,ash content,protein and fat content.It was founded by comparing with other cheeses that the protein content in Qula was higher than in other cheeses;Plate count method was adopted to analyze the microbial flora composition of the 43 Qula samples.It was founded by the analysis of lactic acid bacteria,coliform bacteria,mould,yeast,aerobic bacteria,clostridium and bacilli in Quia that,generally,lactic acid bacteria and yeast were dominant bacteria with regarding to quantity:(2)According to the results of colonial morphology and Gram staining,69 representative Lactobacillus strains were isolated from the 43 samples.Physiological and biochemical tests,API 50 CH fermentation examination,16S rRNA gene sequence analysis and recA gene PCR amplification were performed in these Lactobacillus strains.The identification results of the 69 Lactobacillus strains were as follows:there were 2 Lactobacillus sakei strains,1 Lactobacillus diolivorans strain,28 Lactobacillus casei strains,34 Lactobacillus plantarum strains and 4 Lactobacillus kefiri strains;among them L.plantarum and L.casei,accounting for 49%and 41%respectively,were the dominant and second-ranked strains.(3)It was found through the study of dynamic changes of the microbial communities during the preparation of Qula cheese that:lactic acid bacteria,coliform bacteria,aerobic bacteria,mycelial fungi and yeast floras of different levels were detected;lactic acid bacteria and yeast were dominant floras during the fermentation of Qula;lactic acid bacteria strains which participated in the fermentation of Qula contained Leuconostoc mesenteriodes,Enterococcus hirae,Enterococcus mundtii,Lactococcus lactis,Lactobacillus plantarum,Lactobacillus sakei,Lactobacillus buchneri?Lactobacillus diolivorans and Lactobacillus casei;Leuconostoc mesenteriodes was the dominant strain all through the preparation process of Qula.Leuconostoc mesenteriodes?Lactobacillus plantarum.Lactobacillus sakei were original lactic acid bacteria strains in raw milk,played a leading function during the preparation of Qula.(4)Probiotic capacity screening was performed in the 69 isolated Lactobacillus representative strains with screening indexes covering acid-bile tolerance,ability to survive in the simulated gastrointestinal fluid,antimicrobial activity,sensitivity against antibiotics and cell surface hydrophobicity.With the screening,a total of 7 strains with favorable probiotic capacity were founded,including Lactobacillus plantarum 1197,Lactobacillus plantarum 1141,Lactobacillus kefiri1059,Lactobacillus casei1138,Lactobacillus casei1089,Lactobacillus plantarum 1086-1,Lactobacillus casei 1133.Among the 7 strains,L.plantarum1086-1 and L.casei1133 were strains with best probiotic capacity with respect to comprehensive indexes,(5)For the study of 20-generation stability in L.plantarum1086-1 and L.casei1133,viewed form phenotypic characteristics,cellular morphology,viable count,lactic acid production capacity and carbon-source fermentation type could be stably passed on;viewed form fermentation characteristics,both L.plantarum1086-1 and L.caseill33 carried poor stability of fermentation performance which decreased significantly from 15th and 10th generations respectively;viewed form the stability of probiotic characteristics,acid-bile tolerance,the ability to survive in the simulated gastrointestinal fluid,cell surface hydrophobicity and antibiotic resistance of the two strains all could be stably passed on.(6)In vivo BALB/c mice,the immunoregulation function of L.plantarum1086-1 and L.casei1133 were evaluated,indicating that L.plantarum1086-1 and L.casei1133 of different dosages both significantly improve phagocytic activity of macrophages in gut Peyer's patches of mice(P<0.01),while the former shoed no dosage-dependence and the latter is otherwise.High-dosage L.plantarum1086-1 and L.casei1133 bacterial solutions showed no impacts on secretion of IgA in mice,but significantly promoted secretions of IgG and IgM(P<0.05,P<0.01).High-dosage L.plantarum1086-1 and L.casei1133 bacterial solutions both significantly improved serum IL-6,IL-10,IFN-y and TNF-a levels in the mice(P?0.001).L.plantarum1086-1 and L.casei1133 of different dosages both significantly upregulated expression levels of IL-6,IL-10,IFN-? and TNF-amRNA in mice livers(P<0.01).(7)In vivo BALB/c mice,the oxidization-resistance function of L.plantarum1086-1 and L.caseil133 were evaluated,indicating that L.plantarum1086-1 significantly improved serum resistance to oxidization(P<0.01).L.casei1133 showed no significant promotional effect on serum resistance to oxidization but shows an improvement tendency.L.plantarum1086-1 and L.casei1133 of different dosages both significantly upgregulated expression levels of SOD,CAT and GSH-Px mRNA in mice spleens(P<0.05,P<0.01),while the differences among different dosage groups were significant(P<0.05,P<0.01)and there was a feature of dosage dependence.