Study On The Antibacterial Properties Of Bacillus Velezensis FIO1408 And Its Probiotic Mechanism On Scophthalmus Maximus

In the past decade,the development of turbot（Scophthalmus maximus）aquaculture industry in China has stagnated.In order to expand the economic benefits brought by turbot culture,it is crucial to select suitable feed probiotic additives.Based on this,this paper investigated the biological characteristics and applications of a deep-sea-derived Bacillus sp.FIO1408,analyzed the physiological characteristics,safety and antagonistic ability of strain FIO1408 against pathogenic bacteria,explored the antibacterial mechanism and other functional properties of the strain at the genomic level,and finally comprehensively analyzed the effects of the FIO1408 on water quality,growth performance,intestinal digestive enzymes,non-specific immune enzymes,antioxidant enzyme activity and the intestinal microecologicay in turbot culture.The main results of the study are as follows:1.Study on biological characterization of strain FIO1408:By using common pathogenic bacteria of turbot as the indicator bacteria for antagonism experiments,the results show that strain FIO1408 was antagonistic to Listonella anguillarum,Vibrio parahaemolyticus,Aeromonas hydrophila,E.anguillarum,E.tarda and E.piscicida,and the inhibition zone reached 14.67±0.03,8.55±0.59,12.26±0.12,15.20±0.31,11.45±0.07,and 14.02±0.17 mm,respectively.The strain FIO1408 was preliminarily identified as Bacillus velezensis by bacterial morphology,physiological and biochemical characteristics and 16S rDNA sequence analysis.Meanwhile,The strain FIO1408 did not show hemolysis during growth in blood agar medium,indicating that the strain has certain safety.In the growth test,the strain could grow in the range of temperature 10～50℃,salinity 0～50‰,pH 3～11 and enter the logarithmic growth period at 4 h and reached the growth peak at 18 h.2.Study on strain FIO1408 genome:The genome of B.velezensis FIO1408consists of a 4,137,639 bp circular chromosome and two 16,439 bp and 24,472 bp plasmids,containing 4,364 coding genes.Based on the genome-wide phylogenetic analysis,the taxonomic status of strain FIO14108 was further confirmed.The genome of strain FIO1408 was also functionally annotated,and 220 and 114 genes related to secondary metabolite synthesis were annotated in the KEGG and COG databases,respectively.In addition,11 secondary metabolite gene clusters with antimicrobial activity as well as one novel gene cluster were also identified,including three lipopeptides,three polyketides,three bacteriocins,one siderophore,one dipeptide and one unknown type compound.By comparative genomic analysis,the FIO1408genome contained the most unique genes（265 genes）compared to B.velezensis FZB42^T,KACC 13105,and NRRL B-41580,mainly related to functions such as resistance to phagosomes,protein hydrolysis,environmental viability,and genetic stability.mainly associated with functions such as resistance to phagosomes,protein hydrolysis,environmental viability and genetic stability.3.Study on the probiotic mechanism of strain FIO1408 on turbot:In vivo probiotic experiments were conducted on turbot by feeding feed supplemented with B.velezensis FIO1408（5.03×10~7 CFU/g）,and the results showed that the use of FIO1408 could effectively reduce the concentration of NO₂^--N in the aquaculture pond during the closure period;The activity of digestive enzymes in the intestine of turbot in the experimental group with the addition of B.velezensis FIO1408 increased to a certain extent,and the weight gain rate of turbot reached 89.88%,which was significantly higher than the counterpart of the control group;The antioxidant enzyme activity of turbot in the experimental group was higher than that of the control group during normal recirculating water culture（at 16 d and 48 d）,but weaker than that of the control group in some organs at 32 d,probably due to the effect of closed culture;the non-specific immune enzyme activity of turbot in the experimental group was higher than that of the control group at the first and middle stages of culture（at 16 and32 d）,but there was no significant difference at the later stage of the experiment（at 48d）.The effect of FIO1408 on turbot intestinal microorganisms was investigated using16S rDNA amplicon technique,and it was found that the intestinal flora of the experimental and control groups were significantly differentiated at 32 and 48 d.The results of CCA and Spearman correlation analysis showed that the main dominant bacteria in the gut of the experimental group,ASV1（Pseudomonas sp.）were positively correlated with LPS activity.It was also found that the relative abundance of potential probiotics Pseudomonas sp.and Lactobacillus sp.in the intestinal tract of turbot in the experimental group increased significantly under the effect of B.velezensis FIO1408,indicating that FIO1408 could improve the intestinal microecology of turbot.In this study,the characteristics and probiotic mechanism of B.velezensis FIO1408 were investigated in order to provide a theoretical basis for the proper use of this probiotic.