Isolation Of Probiotics And Evaluation Of Their Usage Possibility For Aquaculture

China is the largest country in aquaculture as the annual production of aquatic products has exceeded 60 million tons in the past five years while shrimp farming is a pillar industry for aquaculture.In order to effectively overcome the diseases susceptible to shrimp and fish,promote the growth,antibiotics are extensively used resulting in the development of antibiotic resistance bacteria(ARBs)and widespread of antibiotic resistance genes(ARGs)among aquaculture which seriously poses a great threat to food safety and public health.The study found that the use of probiotics can significantly reduce antibiotics usage and effectively alleviate the antibiotic resistance problem.Based on the above,we isolated Lactic Acid Bacteria and Bacillus strains from the intestinal microflora of Penaeus vannamei followed by evaluating their antibiotic resistance.And then,evaluating the probiotic characteristics of the sensitive strains to antibiotics.The main contents and results of this study are as follows:1.26 strains of Lactic Acid Bacteria and 16 strains of Bacillus were isolated and purified from the P.vannamei's intestine with the traditional plate culture method.The results of 16 S r RNA identification and phylogenetic tree analysis suggested that the strains of Lactic Acid Bacteria were clustered into 3 subgroups: 4 strains homologous to Lactococcus lactis subsp.Lactis,2 strains homologous to L.taiwanensis,20 strains homologous to L.garvieae and L.lactis.While Bacillus strains were clustered into 7 subgroups: 2 strains homologous to Bacillus megaterium and B.aryabhattai,7 strains homologous to B.stratosphericus,B.pumilus and B.altitudinis,1 strains homologous to B.anthracis and B.cereus,3 strains homologous to B.velezensis,B.subtilis and B.tequilensis,1 strains homologous to B.vietnamensis and B.aquimaris,1 strains homologous to B.circulans,1 strains homologous to B.subterraneus and B.thioparans.2.Analyzing representative isolated strains' resistance to antibiotics from the genotype and phenotype based on the method of PCR as well as minimum inhibitory concentration(MIC).7 strains of lactic acid bacteria showed a single resistance to tetracycline.The resistance spectrum of Bacillus is more diverse.3 strains are sensitive to tetracycline,gentamicin,chloramphenicol and erythromycin,5 strains showed a single resistance to tetracycline,4 strains were double resistant and tolerated tetracycline and erythromycin,1 strain is a triple-resistant strain that can tolerate gentamicin,chloramphenicol and erythromycin.The European Food Safety Authority(EFSA)has not given clear MIC definition values for other antibiotics used in the experiment(chlortetracycline hydrochloride,oxytetracycline,sulfapyridine,ciprofloxacin,tobramycin,acetyl spiraspira).The strains' MIC on chlortetracycline,oxytetracycline,ciprofloxacin and tobramycin ranges from 0.5 to-8 mg/L while 8-64 mg/L for acetyl spiraspira and sulfapyridine.The detection rates of the 18 ARGs corresponding to the antibiotic resistance phenotype in 20 strains were as follows: sulfonamides(95%)>tetracyclines(54.17%)>quinolones(11.67%)>chloramphenicol=macrolide s(10%)>aminoglycosides(6.43%).All strains didn't contain Int I1 gene.3.Bacillus sp.S-R-1,Bacillus sp.J-J-1,Bacillus sp.T-J-1 standarded out because they were absence of quinolones,chloramphenic,macrolides and aminoglycoside's ARGs and sensitive to tetracycline,gentamicin,chloramphenicol,erythromycin.Those three strains' MIC on chlortetracycline,oxytetracycline,ciprofloxacin,tobramycin and sulfapyridine ranges from 0.5 to-4 mg/L while 32,4,4 mg/L on acetyl spiraspira respectively.4.When Bacillus sp.S-R-1,Bacillus sp.J-J-1,Bacillus sp.T-J-1were exposed to p H 2.0 or 0.3 % bile for 3h,the relative survival rate ranging from 79.60 % to 92.11%.All three strains exhibited protease,cellulase,amylase activity and had a good cell-adhesion activity.Bacillus sp.J-J-1 and Bacillus sp.T-J-1 were strongly hydrophobic as their BATH beyond 50%,while Bacillus sp.S-R-1 was moderately hydrophobic.Their auto-aggregation differed from 35% to 50%.These Bacillus strains displayed antagonize pathogens activities to some degree.Bacillus sp.T-J-1 equipped with short lag phase while long stationary phase exhibiting the highest and wildest antimicrobial effect against three aquacultuic pathogenic bacteria including Aeromonas sobria,Vibrio mimicus,V.parahaemolyticus ATCC17802 as well as E.coli.To summarize,Bacillus sp.T-J-1 could be used as potential probiotics for aquaculture especially for shrimp farming.