Screening And Preliminary Application Of Probiotic Bacillus Against Aeromonas Veronii

The rapid development of aquaculture intensification is accompanied by the deterioration of the water environment and the frequent emergence of bacterial diseases,which restricts the production efficiency of breeding and the sustainable development of people and the environment.Bacillus as a common aquatic product micro-ecological preparation is used for aquaculture water purification.Such bacteria produce bacteriocin-like substances,and exhibit a broad-spectrum antibacterial activity,and have a good antibacterial effect against gram-positive bacteria and gram-negative bacteria.Its high producing enzyme properties are also commonly used in industrial production.Bacillus as a micro-ecological preparation has high resistance,so that it has become a trend to replace antibiotics.The development of Bacillus resources in China has considerable prospects.In this study,a suspected Bacillus spp.strain was first screened using a high-temperature water bath.Bacillus spp.was identified by spore staining and Gram staining,biomolecules,and physiological and biochemical characteristics.Antibacterial capacity of the Oxford cup diffusion method was used as a preliminary screening index for Bacillus probiotics.Two strains of Bacillus verruciformis,including 12 strains of aquatic pathogenic bacteria and 10 strains of food-borne pathogens such as Escherichia coli,were screened out.Bacillus velezensis C-11,S-22.Bacillus tested showed high sensitivity to 29 antibiotics,meaning that the two Bacillus species are unlikely to become antibiotic-resistant bacteria.The strains with the highest ratio of cellulose,protein and starch degrading circle were screened to see the activity of extracellular hydrolase;intestine experiments were simulated in vitro at high temperature tolerance,acid and alkali tolerance,artificial intestinal fluid tolerance,and intestinal survival was screened out.Ability of Bacillus.To evaluate whether the selected Bacillus velezensis C-11,S-22 had a beneficial probiotic effect on fish.First,mice were injected with high doses of Bacillus to simulate acute poisoning experiments.No mortality occurred in two strains of spores up to 1×10 ¹⁰ CFU/ml.The safety of these two strains of Bacillus was verified.Bacillus was added into the fish food.The experimental catfish were continuously fed for 5 weeks.Blood samples and organ tissues were taken on the 0th,8th,15th,22d,29d and 36d after feeding to perform immune indicators in serum and organ tissues.Testing.The protective effect of bacillus was evaluated by the acute challenge experiment with Aeromonas versicolor.The phagocytic activity of white blood cells in the test bacillus fed group showed an upward trend.The ELISA test kit was used to detect IgM,IL-1?,IL-10,IFN-?,TNF-?,ACP,AKP,SOD and LZM in the blood.The results showed that Higher levels of IgM,IL-1?,IL-10,IFN-?,and TNF-?were detected in the sera of Bacillus spp.,and the activities of ACP,AKP,SOD,and LZM in the serum were all increased,indicating that Bacillus spp.The humoral immunity of salmon has a good stimulatory effect.Real-time fluorescence quantitative results showed that the two strains of Bacillus spp.increased the transcription levels of IL-1?,IL-10,IFN-?,and TNF-?cytokines in the main immune tissues of carp,and promoted the proinflammatory reaction in the body;Bacillus spp.was found in the experiment.The transcription levels of IL-1?,IL-10,IFN-?,and TNF-?cytokines in carp intestinal tissues showed an upward trend,demonstrating that Bacillus can bind to specific sites of intestinal lymphoid tissues,thereby increasing the host's immune response level..After the 2×LD₅₀ strain of Aeromonas vermicularis TH0426 was used to challenge the squid,the survival rate of the squid fed bacillus was significantly higher than that of the control group,and intestinal tissue sections showed that the intestinal villi was preserved compared to the challenge group.In good condition,the degree of inflammation was lower than that of the challenge group,indicating that Bacillus has a certain degree of immunoprotection as a probiotic.In summary,this experiment screened the best strains through the probiotic and antibacterial ability of Bacillus,and the two strains of Bacillus velezensis C-11 and S-22 have high safety to animals and can improve the immune response level of carp through oral administration.Improve the activity of various types of enzymes in the body,increase the expression level of pro-inflammatory cytokines expressed in the body and the humoral immune response capacity,protect the integrity of the intestine villus of the eel,and reduce the mortality of the cod after infection with Aeromonas veronii.Most importantly,the use of Bacillus velezensis C-11 and S-22 as feeds to promote the growth of freshwater fish and control disease has broad application prospects and practical significance.