Study On The Selection Of Lactic Acid Bacteria In Traditionally Fermented Xinjiang Cheese And Analysis Of Anti-aging Effects Of Selected Strains

Xinjiang milk knot is also known as Xinjiang cheese. It is a special kind of traditionally fermented dairy product made by Xinjiang local residents which has already became an indispensable food for the local people in Xinjiang. The production method of Xinjiang cheese mainly depends on natural fermentation, and it is the natural fermentation process that makes these fermented products have a high value in terms of nutrition, taste and microflora diversity. Therefore, studying the microbial resources in traditionally fermented cheese products is very necessary. In this study, we took the samples of cheese products collected from five different regions in Xinjiang as research objects, and we used the method of combining the traditional cultivation method and 16S rDNA molecular biology techniques to research the potential probiotic bacteria in these cheese samples. In addition, we have established the Caenorhabditis elegans(C.elegans) model to the further selection of effective probiotic strains. Then we conducted the transcriptomics analysis to figure out the mechanisms of health span enhancing activities of C.elegans which fed Lactobacillus strains. Below are the results we got from this study: 1 In this study, we have collected 5 cheese samples from 5 different areas of Xinjiang. We obtained the microflora and composition information of these cheese samples collected from Xinjiang through high-throughput sequencing method, and investigated the microflora composition by using the biological statistics analysis method. The predominant bacteria in five cheese samples are Streptococcus, Lactobacillus, Lactococcus and Pseudomonas. Among them, Lactobacillus is a predominant strain in four other samples besides sample RS1.1. In addition, the bacterial diversity of sample RS1.5 is most abundant among five samples, while the classification of bacteria in sample RS1.1 is relatively single, and its abundance is the lowest. 2 We have obtained cultivable microorganisms through traditional isolation and identification method, that are:Lactobacillus rhamnosus R4, Lactobacillus helveticus S4, Enterococcus hirae H4, K4-8, K4-9 and R5, Streptococcus salivarius K4-7, Bacillus amyloliquefaciences B3, B5, B6, B9, R2, R7, K3-2, H3,12, M3, X2, X4 and Y2. It is interesting to note that Lactobacillus and Bacillus amyloliquefaciences strains were appear in the samples of cheeses simultaneously, which indicated that these two strains can be coexist in the fermentation process.3 Strains of L. rhamnosus R4, L. helveticus S4, E. hirae H4、K4-8、K4-9 and R5 were all showed good characteristics of acid and bile salt tolerance and the tolerance in simulative gastrointestinal juice. Among them, the antimicrobial activity, hydrophobicity and co-aggregation abilities of L.rhamnosus R4 were particularly significant. In addition, L.rhamnosus R4 also showed a better ability of antioxidation, degradation of cholesterol and triglycerides. Therefore, R4 can be used as an excellent potential probiotic strain.4 We established a Caenorhabditis elegans (C.elegans) animal model for the selection of probiotic strains. Firstly, we used liquid cultivation assay to preliminarily determine the optimum action concentration of Lactic acid bacteria (LAB) strains and Escherichia coli OP50 (E.coli OP50) on nematodes. The results showed that the nematodes can be clearly observed and counted for their numbers when the concentration of LAB and E. coli OP50 was 108 CFU/mL. The lifespan, brood sizes, motility, resistance to heat and oxidative stresses were determined after feeding 6 different LAB strains, and OP50 was used as control. Compared with the control group (E.coli OP50),6 LAB strains exhibited different influences on the phenotype characteristics of nematodes. Among them, L.rhamnosus R4 showed a significant effect on extending lifespan, and enhanced effects on brood sizes and mobility. R4 strain also performed a good resistance in both heat and oxidative stress environments. Moreover, the rising of enzymatic activities of SOD and CAT and the declining of MDA content as well as illustrated its possible mechanism. Results obtained in this research laid a theoretic foundation for the further study of lactic acid bacteria.5 The results of Real-time fluorescent quantitative PCR showed that L.rhamnosus R4 strain has significantly increased the gene expression level of sod-3, hsp-16.2, ctl-1 and daf-16, which indicated that, under the condition of oxidative stress, L.rhamnosus R4 not only stimulated the expression of free radical scavenging gene of sod-3 in nematodes, but also significantly increased the expression of heat shock protein HSP-16.2. Moreover, the expression of daf-2 and sir-2.1 significantly decreased in nematodes after feeding L.rhamnosus R4, which demonstrated that the lifespan extending effects of L.rhamnosus R4 on nematodes may depend on Insulin (Insulin/IGF-1) signaling pathway.6 The results of transcriptome sequencing showed that there was a total of 196 genes’ expression level has changed (including 48 up-regulated genes and 158 down-regulated genes) after feeding L.rhamnosus R4 strain to the nematodes. L.rhamnosus R4 has significantly stimulated the encoding genes of key enzymes in the metabolic pathways related to endocrine system, signal transduction and immune system. There are 4 groups of metabolic pathways which have more significant differences, and they were respectively: Insulin signaling pathway, FOXO signaling pathway, MAPK signaling pathway, and PI3K Akt signaling pathway. Among them, in insulin signaling pathway, there was an encoding gene of key enzyme has an up-regulated expression, and four encoding genes of key enzymes have a down-regulated expression in Insulin signaling pathway. Due to the insulin/IGF-1 signaling pathway plays a vitally important regulating role in human body, our research has provided a significant theoretical basis for developing L.rhamnosus R4 into anti-aging related microbial agent in the future.