Screening Of Clostridium Butyricum And Evaluation Of Its Beneficial Effects In Zebrafish Larvae

With the rapid development of the aquaculture industry,large-scale factory farming become a trend,but disease outbreaks and the increasing scarcity of traditional aquafeed resources have become limiting factors for development.In animal husbandry and aquaculture,the addition of probiotics can prevent and treat diseases and increase Digestibility of feed,improving growth performance,regulating intestinal flora,stimulating immunity,thereby improving animal health performance and economic value.Clostridium butyricum,as an anaerobic probiotic,has been applied and studied for many years in human and animal husbandry,but relatively few in aquaculture.In this study,Clostridium butyricum were screened from soil and chicken feces with the properties of producing digestive enzymes,adhesion,and inhibition of pathogenic bacteria.We evaluated its biological activity and safety in vitro.We explored the effects on growth performance,inhibition of Aeromonas hydrophila,antioxidant,amylase production and immune stimulation in zebrafish larvae.(1)Screening of Clostridium butyricum:The anaerobic culture method was used to efficiently screen by selective medium and PCR with specific primers for Clostridium butyricum.15 strains of Clostridium butyricum were identified by 16S rDNA gene identification.Five strains were selected from the different sample source for the determination of hydrophobicity,self-aggregation,inhibition of Aeromonas hydrophila,amylase production and bacterial growth.The results showed that,comparing with other strains,the hydrophobicity of Clostridium butyricum No.11was 24.12%and the self-agglutination(5h)was 68.30%,proving it has better adhesion.the inhibition zone reached 14.3mm,the amylase transparent circle/colony diameter was 2.45,and the bacterial quantity reached 2.22×10~7cfu/ml culturing in RCM liquid for 24h,the overall performance is better.(2)Biological characteristics of Clostridium butyricum:To evaluate the tolerance of Clostridium butyricum No.11 to gastrointestinal fluid environment,high temperature and oxygen,and whether it contains related pathogenic genes.The experimental results show that it can still grow well in the RCM liquid medium containing 0.3%porcine bile salts.Compared with the control,the survival rate was62.47%.After gastrointestinal fluid treatment,the survival rate exceed 80%,indicating that Clostridium butyricum No.11 can well tolerate the gastrointestinal environment.After being heated in a water bath at a temperature of 70°C for 20 min,compared with the control,the survival rate was 61.23%.Good tolerance to high temperature.After 10 and 30 min of aerobic exposure,the survival rates were both above 55%,indicating that bacteria No.11 was non-lethal to short-term exposure to oxygen and could re-germinate when the growth conditions were suitable.Clostridium butyricum No.11 did not contain the pathogenic gene-Clostridium botulinum E by PCR method.Clostridium butyricum No.11 has good biological tolerance and is suitable for processing,storage and gastrointestinal environment.It does not contain the common pathogenic gene of Clostridium-Clostridium botulinum E,and has good probiotic potential.(3)Based on the in vitro study of Clostridium butyricum No.11,combined with zebrafish,to explore its effects on zebrafish growth performance,disease resistance,antioxidant,amylase production and regulation of immune cytokines.During 5-15 d after birth,C.butyricum was added through culture water,and during 15-40 d,C.butyricum was added by feeding bacteria-containing feed.The results showed that the CB2 group(with a concentration of 10~5cfu/ml in water and a concentration of10~7cfu/g in the feed)after feeding for 40 days,compared with the control group,the zebrafish had increased body weight(P<0.01).In the challenge experiment of Aeromonas aeruginosa,there was no significant difference in mortality between the experimental group and the control group,and the CB2 group had a certain effect of delaying the death time of zebrafish juveniles.The levels of T-AOC andα-amylase in the three experimental groups added with bacteria were significantly increased,and the levels of MDA were significantly decreased,and all groups were significantly difference from control group(P<0.05).The CB2 group increased the relative mRNA expression levels of cytokines TLR2,IL-8 and TNF-α,but had no effect on the relative expression levels of MyD88,IGF-1a and IL-10 mRNA.We speculate,by way of TLR2 signaling pass with MyD88-independent,that the neutrophils were stimulated to secrete IL-8 and TNF-α,stimulating immunity.To sum up,Clostridium butyricum No.11 screened in this study has good properties in amylase production,gastrointestinal fluid resistance,high temperature resistance,etc.In vivo experiments of zebrafish juveniles,Clostridium butyricum can survive in water and feed,does not affect the survival rate of zebrafish juveniles,can improve the growth performance of juvenile fish,and has a tendency to delay death when challenged by Aeromonas hydrophila,and can improve the level of anti-oxidation and amylase production in fish,reducing the content of MDA,increasing the relative expression levels of TLR2,IL-8 and TNF-αmRNA,stimulating the immune system,it has good application value as a probiotic in aquaculture and animal husbandry.