| Probiotics have gradually become the main product to ensure the implementation of the policy of "reducing antibiotics and replacing antibiotics" in aquaculture.Among them,Bacillus has become the most fully researched and most widely used probiotic because of its ability to form stress-resistant spores and a variety of probiotic functions.Indigenous aquatic bacillus can reduce the stress stimulus to aquatic animals,so as to better adapt to the digestive tract of aquatic animals and the environment of aquaculture water,and contribute to the effective performance of its probiotic functions.There is an urgent need to develop aquatic indigenous probiotic Bacillus.The probiotic effect of Bacillus subtilis HGcc-1 from freshwater fish in common carp culture shows that HGcc-1 has no significant effect on the weight gain rate of carp,but adding HGcc-1significantly reduced the level of serum endotoxin,ALT and AST.The serum total complement level of the HGcc-1 supplement group was significantly increased,compared with control group.The results of gut microbiota analysis showed the HGcc-1 supplement diet group significantly increased the abundance of Fusobacterium and Cetobacterium,significantly reduce the abundance of Proteobacteria,,Citrobacter and Aeromonas.The intestinal microbiota of HGcc-1 addition group decreased the content of endotoxin binding protein(LBP)and level of AST of the germ free(GF)zebrafish,and significantly increased the gene expression of complement component 3 and the complement component 4(C3 and C4)of the GF zebrafish.At the same time,the direct interaction between HGcc-1 and GF zebrafish also decrease the ALT and AST levels of the GF zebrafish.Then,five strains of Bacillus velezensis isolated from large yellow croaker(Larimichthys crocea)were analyzed for RAPD strain typing,enzyme production ability and antibacterial ability.Molecular typing showed that T1 and T23 belong to two genotypes,T1 had a strong ability to inhibit Aeromonas veronii,and T23 has a strong ability to secrete digestive enzymes.The growth-promoting and disease-resistance abilities of T1 and T23 were compared.The results showed that T23 promotes the digestion of zebrafish by increasing the activity of lipase and protease,and reduces the feeding metabolic energy to reduce the total metabolic energy,thereby saving energy,and its ability to promote growth and increase feed utilization is stronger;although the T1 group has higher digestive enzymes,but due to the larger standard metabolic energy and the higher feeding metabolic energy,the energy is lost,so its growth-promoting effect is not obvious.In terms of intestinal morphology,both strains significantly increased the length of intestinal villi,but T23 had a stronger ability to maintain the thickness of the intestinal lamina propria.In the challenge test of hm091,T23 had a stronger protection rate for zebrafish than T1.Real-time q PCR detection of genes related to innate immune response showed that T1 has a stronger ability to up-regulate zebrafish gut defbl-1 and Ig T gene expression,T23 significantly improves zebrafish’s antioxidant capacity and reduces inflammation levels,T23 also significantly reduced serum LPS.The indirect effects of gut microbiota induced by T1 and T23 was studied.Real-time q PCR results show that in terms of growth,the gut microbiota induced by the two supplement groups both up-regulated the expression of germ-free zebrafish’s growth-related genes,the effect of gut microbiota induced by T23 up-regulated the expression of myogenic regulator MYOD of the GF zebrafish is more obvious.In terms of disease resistance,the gut microbiota induced by the two addition groups reduced the inflammation level of GF zebrafish,T23 also has a tendency to down-regulate the expression of NF-κBp56 of GF zebrafish.In terms of antioxidant,the effect of gut microbiota induced by T1up-regulating antioxidant-related genes is more obvious than that of T23;finally,the gut microbiota induced by T23 significantly up-regulates the expression of C4 of GF zebrafish.The direct effects of T1 and T23 on zebrafish were further studied.The real-time q PCR results showed that the direct effects only affect the expression of inflammation-related genes in GF zebrafish.T23 inhibits the level of inflammation while T1 increases the level of inflammation,so T1 has a certain negative effect.The antibiotic sensitivity test results showed that T1 was resistant to tetracycline,while T23 is more sensitive to 19 kinds of antibiotics.Based on the above results,considering the effectiveness and risk,T23 has more probiotic properties and is more suitable for aquaculture.In summary,this study screened out two indigenous Bacillus strains that are more suitable for aquatic animals-B.velezensis T23 and B.subtilis HGcc-1.T23 has a good effect of promoting growth,improving gut health,resisting A.veronii and enhancing innate immunity.HGcc-1 has a strong effect of improving gut and liver health and maintaining the homeostasis of gut microbiota. |
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