Biodiversity Analysis And Disease Resistance Of The Intestinal Microorganism In Apostichopus Japonicus

Apostichopus japonicus,which belongs to the Echinodermata Holothuroidea animal,is rich in nutrition and active substances,and regarded as the first in the "eight most precious marine products".In recent years,with the increasing of the demand on the Apostichopus japonicus in the market,the breeding industry in Fujian province has springing up rapidly,gradually forming the trend of "South breeding Apostichopus japonicus".Because of the huge difference to northern culture environment,micro ecological environment of Fujian Apostichopus japonicus has regional characteristics.This makes it different from the culture characteristics of North Apostichopus japonicus,especially in disease control.At present,there are few studies on the microecology of Apostichopus japonicus in Fujian,the prevention and control of disease is backward.Therefore,it is urgent to develop a new targeted microecological preparation.In this paper,the 16S rDNA strain identification and UHPLC-MS/MS technology was used to study the intestinal microecological environment of the Apostichopus japonicus in the sea area of Fujian Province,and analyzed the diversity of the intestinal microorganism of Apostichopus japonicus.Meanwhile,the disease resistance of the endogenous epiphytic probiotics of Apostichopus japonicus was studied,which provided important theoretical support for improving the industrial foundation,developing new microecological preparations and functional probiotics.1.In this study,Fujian cage farming Apostichopus japonicus was taken as a sample,divided into the health group and the disease group,95 dominant strains were isolated and purified from the intestinal tract of 2 groups of the Apostichopus japonicus.Identification of dominant strains based on colony morphology and 16S rDNA sequence homology comparison method.It was found that the dominant strains were composed of 11 bacteria,respectively as Bacillus,Pseudoalteromonas,Pseudomonas,Oceanimonas/Marinomonas,Acinetobacter,Shewanella,Marinobacterium,Microbacterium,Pseudidiomarina,Vibrio,Oceanisphaera.Analysis of microbial community structure showed that in the healthy group,the abundance of Bacillus and Pseudomonas is the highest,36.96%and 17.39%,respectively;the abundance of Pseudomonas and Bacillus was the highest in the disease group,which was 30.61%and 28.57%,respectively.It was found that the abundance of Pseudoalteromonas and Acinetobacter genera were higher while that of Bacillus genus was lower in diseased group than those in healthy group.It is one of the possible causes of the body surface lesion of Apostichopus japonicus.2.Taking 9 typical pathogenic strains as indicator bacteria,and the Oxford cup method was used as a bacteriostasis test in vitro to determine the antibacterial ability of probiotics.Two probiotic strains with wide antimicrobial spectrum and high bacteriostatic ability were screened out,which were B.amyloliquefaciens YD1 and B.subtilis YK5.Extraction of lipopeptides from strains YD1 and YK5,Fengycin,Iturin,Surfactin standard as the control,using thin layer chromatography(TLC)method for identification of lipopeptide compounds,initially identified metabolites of two strains containing a kind or a variety of lipopeptide substances in Fengycin,Iturin,and Surfactin.3.Finally,the bacteriostatic ability of lipopeptide compounds extracted from strain YD1 and YK5 was determined.The results showed that,starting from 9 h,the concentration of bacteria suspension showed a significant difference compared with the blank control group,indicating that the antimicrobial effect of lipopeptide metabolites was obvious.Using ultra performance liquid chromatography tandem mass spectrometry(UHPLC-MS/MS),C12-C17 Surfactin and C13-C18 Iturin A C13-C19 and Fengycin A as the target compound.The lipopeptide compounds extracted from strains YD1 and YK5 were detected.The results showed that the chromatographic peaks corresponding to the target compounds appeared.It is indicated that strains YD1 and YK5 can produce lipopeptide compounds which have bacteriostasis effect during the metabolic process.