Isolation And Screening Of Enzyme Producing Bacillus Velezensis From Fish-derived And Its Probiotic Effect On Cyprinus Carpio

With the popularization of intensive aquaculture,aquatic animal diseases occur frequently.The abuse of antibiotics and chemical drugs leads to adverse consequences such as drug resistance and pesticide residues of pathogens,which pose a serious threat to people’s health.Probiotics as a substitute of antibiotics have broad application prospects in aquaculture,enzyme-producing probiotics can also secrete a variety of digestive enzymes,digest non starch polysaccharides and other antinutritional factors in feed,and promote intestinal digestion and absorption,so the research and development of enzyme producing probiotics is of great significance to promote the healthy development of aquaculture.In this study,107 strains of bacteria were isolated and purified from the intestines of healthy Cyprinus carpio by designing 12 kinds of culture media,including 3 phyla and 22 genera.Among them,16 strains were capable of producing protease,amylase,cellulase,xylanase andβ-mannanase,and fermentation screening and productionβ-mannanase producing strain HF-14109.According to morphological observation,physiological and biochemical identification,16s r RNA identification and whole gene sequence analysis,the enzyme producing strain HF-14109 was identified as Bacillus velezensis HF-14109.The enzyme producing strain HF-14109 has good antibacterial ability,good tolerance to high temperature,acid and bile salt,sensitive to a variety of antibiotics,and can produce a variety of digestive enzymes,especially the enzyme activity ofβ-mannanase.The optimum temperature and p H are 45℃and6.0,and the optimum temperature and p H are 20℃-50℃,respectively Cu²⁺,Fe³⁺,Zn²⁺,Ba²⁺can activate the enzyme,while Mn²⁺,Mg²⁺,Ca²⁺can inhibit the enzyme;The study of biological and enzymatic characteristics and safety test of B.velezensis HF-14109 showed that the strain could be used as a potential aquatic feed additive.Different concentrations（10~7 CFU/g,10~8 CFU/g,10~9 CFU/g）of enzyme producing probiotics HF-14109 were added to the feed for 30 and 60 days.At the end of the experiment,enzyme activity determination,real-time fluorescence quantitative analysis,tissue HE staining and 16S r RNA detection of intestinal microflora were carried out to explore their effects on growth,antioxidation,serum physiological and biochemical indexes,immune related factors,intestinal tissue morphology,digestive enzyme activity,expression of intestinal tight junction protein gene and intestinal microflora,This study provided theoretical support for the application of enzyme producing Bacillus bailesii from fish in aquatic feed.The results are as follows:（1）Effects of enzyme producing strain HF-14109 on growth performance,serum physiological and biochemical indexes of common carpCompared with the control group,the weight gain rate（WGR）and specific growth rate（SGR）of the treatment group were significantly increased,and the feed conversion rate（FCR）was significantly decreased.Especially,the WGR and SGR of the 10~8 CFU/g treatment group were significantly higher than those of the control group and other treatment groups at 60 days（P<0.05）,and the FCR was significantly lower than those of the control group and other treatment groups（P<0.05）;The levels of glucose（GLO）,alkaline phosphatase（AKP）,high density lipoprotein（HDL）and lysozyme（LZM）in serum of carp were significantly higher than those of control group（P<0.05）;The levels of acid phosphatase（ACP）,aspartate aminotransferase（AST）,alanine aminotransferase（ALT）,total cholesterol（CHO）and triglyceride（TG）in the treatment group were significantly lower than those in the control group（P<0.05）;The contents of total antioxidant capacity（AOC）,glutathione peroxidase（GSH-PX）and the activities of glutathione（GSH）and catalase（CAT）in hepatopancreas of treatment group were significantly increased（P<0.05）,while the contents of malondialdehyde（MDA）were significantly decreased（P<0.05）.（2）Effects of enzyme producing strain HF-14109 on immune related factors in head kidney,kidney,spleen and serum of common carpCompared with the control group,the m RNA relative expression levels of anti-inflammatory cytokine interleukin-10（IL-10）and transforming growth factorβ（TGF-β）in the head kidney,kidney and spleen of the treatment group were significantly higher（P<0.05）.The relative expression of m RNA decreased significantly of proinflammatory cytokine interleukin-1β（IL-1β）、Interleukin-6（IL-6）and tumor necrosis factorα（TNF-α）in cephalic kidney and the relative expression of m RNA decreased significantly of proinflammatory cytokine TNF-αin spleen（P<0.05）.But the m RNA relative expression levels of IL-6 in kidney and IL-1β、IL-6 in spleen were significantly increased（P<0.05）。Enzyme producing probiotics HF-14109 also increasing the secretion of the anti-inflammatory cytokines IL-10 and TGF-βand decreased the secretion of the the pro-inflammatory cytokines TNF-α,IL-1β,IL-6 at the protein level in serum（P<0.05）.（3）Effects of enzyme producing strain HF-14109 on intestinal morphology,digestive enzyme activities,expression of intestinal tight junction protein gene and intestinal microflora of common carpCompared with the control group,the treatment group significantly increased the length of intestinal villi,the thickness of intestinal wall,amylase,lipase,pepsinβ-mannanase and the relative expression of Claudin-2,Occludin and ZO-1 were significantly higher than those in the control group（P<0.05）;Compared with the control group,the abundance of Proteobacteria in the treatment group was significantly increased（P<0.05）.The abundance of fusobacteria and Firmicutes decreased significantly（P<0.05）.At genus level,the abundance of Vibrionaceae spp.,Limnohabitans spp.and Bacillus spp.in the treatment group increased significantly（P<0.05）.The abundance of Aeromonas spp.and Bacteroides spp.decreased significantly（P<0.05）.To sum up,adding appropriate concentration of B.velezensis HF-14109 to feed can promote the growth of carp,improve immunity,enhance antioxidant capacity,serum physiological and biochemical indexes,intestinal digestive enzyme activity,intestinal mucosal barrier function and improve the composition of intestinal flora.