Application And Evaluation Of Compound Probiotics In The Culture Of Litopenaeus Vannamei

In order to explore the application effect of compound probiotics in the culture of Litopenaeus vannamei,in this study,the potential probiotics were isolated and identified,and the probiotics with disease-resistant function were selected to form composite probiotics.The effect of the compound probiotics on the anti-vibrio parahaemolyticus infection of Litopenaeus vannamei was investigated and the application effect of the compound probiotics in the culture of the Litopenaeus vannamei was evaluated.The main contents of this study include the following aspects:1.Isolation and identification of potential probioticsIn this study,12 strains of bacteria were isolated from the ponds of shrimp culture,identified by 16 s r RNA method and sequenced on the NCBI website.The results showed that they were all Bacillus bacteria.In the subsequent bacterial culture process,six of them had obvious growth advantages,and three of them did not have hemolytic activity to construct phylogenetic tree.Through phylogenetic tree analysis,we found that No.2,No.9belong to Bacillus licheniformis,No.12 belongs to Bacillus subtilis,and they are named as Bacillus licheniformis BL-2,Bacillus licheniformis BL-9,respectively.Bacillus subtilis BS-12(three other strains of Bacillus subtilis were not identified as hemolytic).In order to verify the safety of the potential probiotics isolated and preserved during the previous experiment,six strains of the bacterias(Bacillus licheniformis BL-2,BL-9,Bacillus subtilis BS-12,lactobacillus LB-1,Pseudomonas pseudomonas PA-1,Pseudoalteromonas flavipulchra,CDM8)were verified.The results of biological safety verification showed that Bacillus licheniformis BL-2,BL-9,Bacillus subtilis BS-12 did not show hemolysis in blood plate culture for 38 hours.In the culture safety verification experiment,the feed containing the six strains of bacteria and the bacterial solution containing the six strains of bacteria were fed into the water respectively.The results showed that the cumulative mortality of Litopenaeus vannamei in the experimental group was not significantly different from that in the control group.In order to verify the probiotics function of these potential probiotics,a bacterial solution of the order of 107CFU/ml was added to the culture water of Litopenaeus vannamei,and after one week of immersion with probiotics,Vibrio parahaemolyticus was used to attack the virus of Litopenaeus vannamei.The cumulative mortality rate of Litopenaeus vannamei was calculated within 132 hours after challenge.The results showed that the mortality of Penaeus prawn was lower than that of the control group.2.Optimization of fermentation medium for a strain of Pseudoalteromonas flavipulchra--CDM8.In the previous research work,a strain of Pseudoalteromonas flavipulchra with Vibrio antagonistic function was isolated from the pond of cultured prawn and named CDM8.In order to improve the fermentation yield,the optimum fermentation conditions of CDM8 were optimized by single factor test and orthogonal experiment.The results showed that the optimum fermentation conditions were as follows: temperature 28 ℃,best salinity is 1%,p H 7.The optimum medium formula was sucrose 20 g / L,tryptone 15 g / L,yeast extract20 g / L,calcium chloride 0.5g / L,magnesium chloride 1g / L.Verify the experimental results,optimized Compared with basic fermentation medium,bacterial culture medium increased 0.69 fermentation efficiency by 48.23%.The results of this experiment not only lay a foundation for the subsequent experiments of shrimp culture,but also provide a reference for the commercial production and application of this bacterium.3.Effect of a compound probiotics on the ability of Litopenaeus vannamei to resist Vibrio parahaemolyticus infectionIn this culture experiments of Litopenaeus vannamei,we explore effect of a compound probiotics on the ability of Litopenaeus vannamei to resist Vibrio parahaemolyticus infection at different experimental stages.one Bacillus licheniformis(BL-9),one Bacillus subtilis(BS-12),and one Pseudoalteromonas flavipulchra(CDM8)were selected to make up a compound probiotics at the same concentration(The concentration of probiotics is 107CFU/ml).The effect of compound probiotics on the Vibrio parahaemolyticus infection of Litopenaeus vannamei was observed by adding the compound probiotics to the culture water of the Litopenaeus vannamei.The experiments was 30 days in all,it included a temporary period(7 days),a probiotics immersion period(15 days),and a Vibrio parahaemolyticus infection period(10 days).The experiment results showed that the addition of compound probiotics in the culture water could significantly increase the total number of culturable bacterias in the water and the intestinal tract of Litopenaeus vannamei(P < 0.05).The cumulative survival rate of shrimp in the experimental group was 73.3±6.8% at the end of the infection experiment.The cumulative survival rate of shrimp was significantly higher than that of the positive control group(25.3±15.43%).The disease-resistant-related gene: heat shock protein 70(Hsp70),β-1,3-glucan binding protein-lipoprotein(βGBP-HDL),Lipopolysaccharide-β-1,3-glucan binding protein(LGBP),Crustin,Immune related enzymes catalase(CAT),was up-regulated in different degrees in the probiotics immersion phase.And all the genes have been up-regulated even more in Vibrio parahaemolyticus infection stage,but expressed in different levels.The results suggested that the compound probiotics with one Bacillus licheniformis(BL-9),one Bacillus subtilis(BS-12),and one Pseudoalteromonas flavipulchra(CDM8)in water could improve the ability of shrimp to resist Vibrio parahaemolyticus infection,The increase of disease resistance of Litopenaeus vannamei may be related to the colonization of probiotics in Litopenaeus vannamei intestinal tract and the expression level of disease resistance related genes and catalase activity in Litopenaeus vannamei.It is hoped that the results of this study can provide reference for the application of compound probiotics in the culture of Litopenaeus vannamei.