Isolation And Application Of Probiotic Lactic Acid Bacteria From Fish

The development of aquaculture has led to the emergence of bacterial diseases of aquatic animal,The abuse of antibiotics has resulted from multiple drug resistance,pollution of water environment and drug residues.In order to develop products that can replace antibiotics probiotics have been alleviated.Therefore,this study isolated and characterized the Probiotic strains of Lactobacillus with good traits from the gut of fish,which will establish a basis for its application to aquaculture.In this research,Lactic acid bacteria was screened obtaining 8 strains that were rescreened by tests of tolerance of bile salts,tolerance of pH,tolerance of protease and tolerance of heat.Two strains with strong anti-resistance were isolated : over 60% of W1 and B4 survived after 3-hour culture in the bile salts of 0.3% concentration;two strains of lactic acid bacteria had a high survival rate under the conditions of acid and alkaline;there was no effect on the survival rate of 2 strains in trypsin resistance test,which could tolerate the temperature of 60 ?,and 70? to certain degree.In sensitivity test of drugs,we found that the two strains showed a certain sensitivity.The safety of two strains of strain was preliminarily confirmed by animal safety test.W1 was identified as Lactobacillus coryate and B4 was Enterococcus faecalis by 16 S gene sequencing and physiological and biochemical tests.In order to verify probiotic effect of isolated lactic acid bacteria in vivo,the bacteria screened were orally immunized with crucian carp for four times and five days for one immunization cycle.We collected serum and organism at 0d,7d,14 d,21d and 28 d.A part of serum was used to test phagocytic activity of leucocytes;the rest were prepared to examine various immune parameters;the drug attack test is an evaluation of the protective effect of lactic acid bacteria.The serum levels of antibodies such as SOD,ALP and IgM were detected by ELISA.The results showed that a higher level of IgM antibody was detected in the serum samples of immunized crucian carp,while the levels of other antibodies showed an upward trend,indicating the enhancement of humoral immunity.The expression of immune related genes showed that W1 and B4 Lactobacillus enhanced the expression of IL-10,IL-1?,TNF-?,IFN-? in the main organs of crucian carp,including liver and kidney.The percentage of leukocyte phagocytosis increased gradually during the immunization process,reaching the maximum after three exempts,and slightly decreased after four exempts.After intraperitoneal injection of Aeromonas hydrophila TPS,W1 and B4 could slow down the infection of pathogenic bacteria and enhance the protection rate.This study is significant for applying lactic acid bacteria to aquaculture industry.