Isolation,Identification And Safety Assessment Of A Strain Of Lactococcus Lactis And Molecular Mechanism Of Biological Effects On Two Species Of Freshwater Fish

The abuse of antibiotics pollutes the ecological environment and leads to the emergence of drug-resistant bacteria.It is urgent to find safe and effective substitutes.The research of probiotics replacing antibiotics is more and more in-depth,a large number of studies have proved the effectiveness and safety of probiotics.Probiotics have been applied to all aspects of production and life,and are often used in aquaculture to prevent and treat related bacterial diseases.Lactococcus lactis is one of the commonly used probiotics,which is recognized as a safe food microorganism.Physiological and biochemical studies of probiotic strains,and histological analysis of the effects of probiotics on the organism all contribute to the scientific exploitation of probiotics.In this paper,a strain of bacteria with strong bacteriostasis against Streptococcus agalactiae,Vibrio parahaemolyticus and Escherichia coli was isolated from the intestine of healthy tilapia,and was identified by 16 S gene analysis.After testing for acid and alkali resistance,bile salt resistance and artificial gastrointestinal juice,drug resistance and hemolytic activity,this strain was sent to the National Culture Collection for preservation.Then use this strain to feed mice and tilapia to analyze its toxicological effects and growth effects on the tilapia body.Finally,from the perspective of intestinal flora diversity and spleen transcriptome,the molecular mechanism of this strain's biological effects on zebrafish and tilapia was discussed.The main results are as follows.In the Oxford cup method,the diameter of the inhibition zone for S.agalactiae was 10 mm(negative: 8 mm).The survival rate was 38.03% in the acid solution with p H4.The survival rate was 10.95% in the artificial gastrointestinal fluid.There was no hemolysis and strong drug resistance.The 16 S sequence identification results showed that the strain was Lactococcus lactis,and the strain number was KUST48.The results of 28d chronic attack and 7d acute attack experiments showed that there was no mortality in mice,normal diet,water intake,normal physiological activities,and no signs of toxicity.The organ ratios of heart,liver,spleen,lung and kidney were not significantly different from those of the control group(P>0.05),and the sections of heart,liver,spleen,lung and kidney were normal with clear cell structure,no cell necrosis,no inflammatory cell infiltration and no bruising.Tilapia was fed with feed mixed Lactococcus lactis KUST48 for 30 days.Compared with the control group,the weight and length of the experimental group increased slightly,and there was no significant difference(P>0.05).The above results indicate that this strain is safe and can be used as a probiotic for fish.Lactococcus lactis KUST48 was used to treat zebrafish infected with Streptococcus agalactiae.The diversity analysis of the intestinal flora showed 1022029 effective sequences were obtained,with an average length of 418.26 bp.The abundance of Streptococcus agalactiae in the treatment group injected with Lactococcus lactis KUST48 was significantly lower than that of the Streptococcus agalactiae group(0.01<p<0.05),and the diversity of intestinal flora in the treatment group approached the blank group.Then,Lactococcus lactis KUST48 was used to treat tilapia infected with Streptococcus agalactiae.The transcriptomics analysis of tilapia spleen response demonstrated the top10 differentially expressed genes and29 immune-related differences.The immune-related differentially expressed genes include:Antigen processing and presentation,Toll-like receptor signaling pathway,Cell adhesion,Complement and coagulation cascades,Hematopoietic cell lineage,Leukocyte transendothelial migration,Pattern recognition receptors.In this study,we obtained a strain of L.lactis which can live in the body and inhibit the growth of S.agalactiae.This strain can affect the growth of the body and is non-toxic to the body.We also analyzed the impact of this strain on the body from the perspective of intestinal flora diversity and spleen transcriptome,which provides theoretical guidance for the subsequent development and utilization of this strain.