Probiotics Bacillus Screening From The Intestine Of Litopenaeus Vannamei And Its Disease Resistance Mechanism

Probiotics having the abilities such as enhancing growth,improving nutrition,activating immune function,and improving disease resistance,probiotics are widely used in the food and feed industries.The application of probiotics in aquaculture not only has the above advantages,but also can effectively reduce the risk of drug residues caused by antibiotic abuse and drug resistance of pathogenic bacteria.Therefore,it has become the focus of related research.In this study,the probiotic Bacillus were isolate from the intestinal tract of Litopenaeus vannamei by the specific method of Bacillus screening.The candidate strains with probiotic function on Litopenaeus vannamei were obtained,after comprehensively evaluated by enzyme production ability,hemolytic test and safety assessment.The promotion effects of obtained probiotics on shrimp growth and disease resistance were determined using feeding experiments.This study could provide a theoretical basis and data reference for the development and utilization of the probiotic resources of Penaeus vannamei.The main findings are as follows:1.Through Bacillus specific screening,microscopic examination and spore staining,33 strains of Bacillus were isolated from the intestinal tract,culture pond water and mud of Penaeus vannamei Using the extracellular enzyme activity(protease,amylase,lipase),hemolysis and safety evaluation as screening indicators,a total of 3 potential probiotic bacterias B5,C6 and C7-1were screened from the intestine of Litopenaeus vannamei.Through comprehensive functional comparison,a strain B5 with high enzymatic activity and non-toxicity was identified.After 16 S r DNA sequencing analysis,it was preliminarily identified as belonging to Firmicutes,Bacillaceae,Oceanobacillus.with 99% similarity to Oceanobacillus picturae.2.The B5 strain was added to the puffed feed at a final concentration of 2 ×108 cfu/g for feeding experiment of 1500 fishes,set Bacillus subtilis(BS)fed at a final concentration of 1~2×108cfu/g as a positive control,a control group was also set without any bacteriological agents The results showed that compared with the control group,the growth performance,immunity level and disease resistance of Litopenaeus vannamei in the B5 feeding group were significantly increased.Compared with the control group,the weight gain rate,feed utilization rate and specific growth rate were increased by 47.1%,30.3% and 22.5% respectively.However,there was no significant difference with the positive control group.The activity levels of serum alkaline phosphatase(AKP),lysozyme(LZM)and superoxide dismutase(SOD)were significantly increased,and transcription levels of Toll,IMD and other immunoregulatory genes were also significantly up-regulated(p <0.05);Challenged by vibrio parahaemolyticus infection for 7 days,the cumulative mortality rate of B5 group was 21 %,significantly lower than the control 56 % and the positive 42.36 %.The results showed that compared with the control group,the growth performance,immune level and disease resistance of prawns in the B5 strain feeding group were significantly increased3.The high-throughput sequencing results of the intestinal flora of Penaeus vannamei showed that compared with the control group,the intestinal microbial richness index(ACE,chao1 and OTUs)and evenness index(Shannon)of B5 group were significantly increased.Evaluating the community composition at family and genus levels,the relative abundance of the harmful bacteria Vibrionaceae and Photobacterium is significantly reduced.