| Intestinal microorganisms are formed by mutual selection and adaptation in the process of long-term coevolution between host and microorganism.Probiotics,as symbiotic bacteria in intestinal flora,play a very important role in human and animal health.In recent years,the application of probiotics has a good effect on improving feed quality,improving utilization efficiency and promoting animal health.With the increasing market acceptance,probiotics has become a rapidly developing emerging industry.The catalogue of feed additive varieties issued by the Ministry of agriculture and rural areas lists 34 kinds of microbial strains,including 23 kinds of lactic acid bacteria.However,in the face of the continuous expansion of the scale of livestock and poultry breeding industry in China,the deepening of the concept of scientific breeding and the severe situation faced by livestock and poultry diseases,the resources of feedable microbial strains need to be further expanded.At present,lactic acid bacteria from animal intestines are an important resource bank for the breeding of new forage probiotics.Streptococcusalactolyticus(S.Alactolyticus)FGM strain screened from chicken cecum has no adverse reaction to mice,has good fermentation Astragalus and antioxidant properties,and has protective effect on Broilers infected with Escherichia coli(E.coli)O78.It is a candidate probiotic strain.Therefore,this study carried out the study on the biological characteristics,probiotics and the mechanism of adhesion to the host,in order to provide data support for the selection of forage microbial strains,and has guiding significance for the breeding of high adhesion lactic acid bacteria.(1)Study on biological characteristics of the strain:The whole genome sequencing of S.alactolyticus FGM strain has been completed,and the results were uploaded to DDBJ/EMBL/GenBank(access No.jaaxmt000000000),which was 1 701 811 bp,(G+C)mol%content was 40.38%,and 1 768 coding genes.The annotation of coding genes revealed the important characteristics of the strain,of which 151 were related to biological adhesion.It was predicted that 43 amino acids in S.alactolyticus FGM strain were related to the biological characteristics of bile salt tolerance,immune escape,antiphagocytosis,adhesion,anti-oxidation,and stress tolerance,but hemolytic gene wasn’t exist.Phenotypic analysis is also non-hemolytic.It was predicted that the 30 amino acid sequences of S.alactolyticus FGM strain were antibiotic resistance genes,but the results of apparent drug resistance analysis showed that S.alactolyticus FGM strain was only resistant to cotrimoxazole and oxytetracycline which was consistence with the prediction of sul 3 and tet44/B(P)/T.S.alactolyticus FGM strain could survive for more than 4 hours in pH 2 MRS broth and 0.15～0.30%bile salt medium.In addition,crystal violet method showed that S.alactolyticus FGM strain had no biofilm formation,and its self coagulation was significantly lower than that of Lactobacillus reuteri(P<0.05),but there was no significant difference with Lactobacillus acidophilus and Lactobacillus casei(P>0.05).(2)Study on probiotic properties of the strain:microscopic observation and fluorescence labeling method were used to determine the adhesion rate of S.alactolyticus FGM strainto Caco-2 cells(Human clonal colonic adenocarcinoma cells).The results showed that the strain could adhere to Caco-2 cells,and the adhesion rate was was 37.51%at 1.0×109 cfu/mL.The inhibition zone diameter(IZD)of S.alactolyticus FGM strain to E.coli O78 was significantly larger than that of L.acidophilus,L.reuteri and L.casei(P<0.05);The adhesion loss rate of E.coli O78 to Caco-2 competed by S.alactolyticus FGM strain was significantly higher than that of L.acidophilus(P<0.01),significantly lower than that of L.reuteri(P<0.05),and there was no significant difference between S.alactolyticus FGM strain and L.casei(P>0.05);The study of extracellular non targeted metabolomics on S.alactolyticus FGM strain showed that 78 different metabolites were produced after fermentation,including mevalonate,nicotinic acid,acetylcholine,methionine and five short peptides with the difference multiple ≥ 2.The related metabolic pathways were terpene skeleton biosynthesis,nicotinic acid and nicotinamide metabolism,glycerol phospholipid metabolism,cysteine and methionine metabolism biosynthesis of amino acids and cofactors.(3)Identification,expression and antibody preparation of surface protein on the strain:the surface protein of S.alactolyticus FGM strain was extracted by high concentration alkaline lithium chloride method,and the "anchor free" surface protein EF-Tu was isolated;the sequence(1 197 bp)of EF-Tu and amino acid sequence(398)were determined.It has 99.7%homology with EF-Tu of Streptococcus gallolyticus(S.gallolyticus).Its molecular weight is 43.952 kD,isoelectric point is 5.41,no signal peptide,no transmembrane structure,good hydrophilicity and good immunogenicity;the recombinant EF-Tu of S.alactolyticus FGM was obtained by E.coli expression system,and the Rabbit anti EF-Tu polyclonal antibody with titer up to 128 000 was prepared.(4)Functional verification of S.alactolyticus FGM adhesion mediated by surface protein EF-Tu:it was found that the adhesion rate of the strain to Caco-2cells and inhibition of E.coli O78 to Caco-2 cells decreased significantly(P<0.05)after the protein was removed or antigen site was blocked,and the adhesion performance of strain was negatively correlated with the concentration of EF-Tu antibody.Immunofluorescence analysis showed that there were a large number of EF-Tu protein on S.alactolyticus FGM,and EF-Tu was expressed around the membrane of Caco-2 cells adhered to S.alactolyticus FGM.The result of Co-IP test and LC-MS/MS identification technology showed that heat shock protein 90,actin beta,SRY box transcription factor 14 and heat shock protein 60 of Caco-2 cells were more possibly acted with EF-Tu among the interation proteins.In conclusion,S.alactolyticus FGM strain isolated from chicken intestinal can reached the screening conditions for probiotics and has the development prospect as feed probiotics.EF-Tu can mediate the adhesion of S.alactolyticus FGM to intestinal cells,and plays a positive role in the biological process of adhesion and pathogenic bacteria inhibition. |
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