Study On Screening And Identification Of Probiotics Isolated From Milk And Its Immunoregulation

With probiotic products in recent years gradually being recognized by the public,the market's increasing demand for probiotics,research on probiotics has also been deep and wide.At present,most of the probiotics used in the market come from foreign countries.Therefore,this study separated lactic acid bacteria from fresh cow milk in mountain pastures in Hunan.According to the characteristics of probiotics,they were screened and identified.Then,in vitro experiments were used to screen the high-adhesion strains,and determine their biological classification;finally,through cell experiments,evaluate the immunomodulatory effects of high-adhesion probiotic strains on cells.The specific research content and research results are as follows:(1)The lactic acid bacteria in fresh milk samples collected from Hunan Shaoyang Nanshan Pasture,Wangcheng New Hope Dairy Pasture and Ningxiang Youshi Dairy Pasture were isolated and purified,and 131 lactic acid bacteria were isolated.(2)Perform preliminary screening of acid and bile salt resistant growth on the isolated and purified strains,and then further screen through the simulated gastrointestinal juice test,bacteriostasis test,proteolytic enzyme activity test,leaving strains with potential probiotic characteristics,perform in vitro safety tests including hemolysis and antibiotic sensitivity.Among the 131 strains of lactic acid bacteria,31 strains were screened for acid and bile salt growth,and were rescreened by simulated gastrointestinal fluid test,bacteriostasis test,and proteolytic enzyme activity test,and confirmed that 12 strains of lactic acid bacteria were found in human The survivability and potential probiotic characteristics of the tract,its safety test in vitro results showed no hemolysis and sensitivity to most antibiotics.(3)On the basis of strains with potential probiotic characteristics,carry out research on high adhesion properties,screen the strains through hydrophobicity test and aggregation test,and then evaluate the adhesion of the strains by the adhesion ability of probiotics to Caco-2cells Characteristics,select probiotics with better adhesion ability to inhibit the adhesion of E.coli to Caco-2 cells.The results show that LZ1805,LZ1817,LZ1827,LZ1932,LZ1940 and LZ1949 have good adhesion to Caco-2 cells.Among them,LZ1827 and LZ1949 performed well in the competition,replacement and rejection tests with E.coli.(4)DNA extraction and sequencing of two probiotics with outstanding effects in inhibiting the adhesion of E.coli to Caco-2 cells were carried out,and LZ1827 was identified as Lactobacillus plantarum and LZ1949 as Lactobacillus paracasei.(5)Identify the species of probiotics that will be used for cell-level immunomodulation studies,including co-cultivation of the strain with Caco-2 cells,treatment of LPS-stimulated inflammatory Caco-2 cells by the strain,and strains of Caco-2 cells that have been infected by E.coli were tested for changes in the expression levels of inflammatory cytokines(TNF-?,IL-4,IL-8,and IL-10)in Caco-2 cells.Under the different treatment conditions,the two probiotic strains tested can basically up-regulate the expression level of anti-inflammatory cytokines,and at the same time have a down-regulation effect on pro-inflammatory cytokines,suggesting that the two probiotic strains are resistant to pathogenic infections and inhibit tumors.There are certain potentials for cell growth and alleviation of inflammatory responses,and both probiotics have a positive effect on intestinal immunity attacked by E.coli.