Isolation,Identification And Characteristics Of Bacillus Associated From Intestine Of Marine Fishes

Bacillus is an aerobic or facultative anaerobic gram-positive bacteria that produces spores,which has the advantages of good stability,strong stress resistance and high resurrection rate.Furthermore,bacillus can produce various macromolecules such as protease and amylase to improve the digestive function of the host promoting the absorption of nutrients.Since the 1980s,the aquaculture of marine fish has developed rapidly by the breakthrough of breeding nurture technology of marine fish.However,the frequent occurrence of diseases and other problems have seriously restricted the healthy development of this industry.Therefore,the isolation of probiotics(e.g.,bacillus)from the intestine of marine fish is of great significance for the research of effective micro-ecological agents to improve the disease resistance of marine fish.In this study,isolation,identification and characteristics of bacillus associated by us from intestine of Scomberomorus niphonius,Sebastods schlegelii,Sphyraenus and Scomber japonicus,respectively.Moreover,the application of bacillus in the aquaculture of S.schlegelii and Psetta maxima was also performed.Overall,these results can provide a theoretical foundation for the research of bacillus probiotics and provided a reference basis for its application in the aquaculture of marine fish.1.The isolation,identification and characteristic determination of bacillus from the intestine of marine fishIn this study,a total of 9 strains of bacteria were preliminarily screened by gram identification from the isolated bacteria(i.e.,37 strains)from the intestine of marine fish.Through the analysis of characteristics producing enzymes and the assessment of application for animal safety,1 strain was finally screened and named as YB1.It was then identified as Bacillus cereus based on morphological observation,physiological and biochemical characteristic analysis and 16S rDNA sequencing.The effects of temperature,NaCl content and pH on the growth of B.cereus were also investigated.The results of morphological observation of YB1 showed that it was convex and white with rough surface,irregular edges and folds,and was rod-shaped under microscopic observation.Moreover,YB1 was determined to be positive by gram identification analysis and had the characteristics of producing amylase and protease.Additionally,YB1 was relatively safe to the growth and survival of S.schlegelii and P.maxima within 10~8 CFU/ml exposure.Furthermore,YB1 could survive and grow at the conditions of 15~35℃,pH5~9 and 0~2%of NaCl content,respectively,and best at35℃,pH7 and 0%of NaCl content,respectively.2.The study of application of YB1 in the aquaculture of marine fishIn this study,the application of YB1 in the aquaculture of marine fish was assessed by culture experiments for S.schlegelii and P.maxima juveniles,and respectively fed the diets supplemented with different levels of bacillus(0,10~5,10~6,10~7CFU/mL,diet),named as C,E-L,E-M,E-H.After culture experiments,growth indexes,including weight growth rate(WGR),specific growth rate(SGR)and feed conversion ratio(FCR),were determined.Moreover,the activities of protease,amylase and lipase enzymes in the intestine,malondialdehyde content and the activities of superoxide dismutase and catalase enzymes in the liver of juveniles were also measured.Finally,the histological condition was also observed in the intestine of juveniles.These results are useful for us to evaluate the application of YB1 in the aquaculture of S.schlegelii and P.maxima.These results of culture experiments for S.schlegelii juveniles showed that the WGR and SGR of juveniles were significantly increased at E-L and E-H,respectively,compared to those of the controls.The values of the FCR at E-L,E-M and E-H were lower than the control.These findings suggested that YB1 promoted the growth condition of S.schlegelii juveniles.Additionally,the activities of intestinal amylase and lipase at E-L,E-M and E-H were higher than those of the controls,while protease activity in the intestine at E-L and E-H was higher than the control.These findings demonstrated that YB1 improved the activities of protease,amylase and lipase enzymes in the intestine of S.schlegelii juveniles.Furthermore,MDA content of the liver were lower at E-L,E-M and E-H than the control.The activity of SOD and CAT was higher at E-M and E-H.These findings suggested that YB1 improved SOD and CAT activities in the liver and reduced the level of lipid peroxidation in the liver of S.schlegelii juveniles.Meanwhile,the findings of histological observation showed that the height of intestinal plica,submucosal thickness,muscular thickness and intestinal villus density at E-L,E-M and E-H treatments were higher than those of the controls.The results showed that YB1 could improve the intestinal structure of S.schlegelii juveniles.These results of culture experiments for P.maxima juveniles showed that the WGR and SGR of juveniles were significantly increased at E-H,compared to those of the controls.The values of the FCR at E-L,E-M and E-H were lower than the control.These findings suggested that YB1 promoted the growth condition of P.maxima juveniles.Additionally,the activities of intestinal protease and amylase lipase at E-M were significantly higher than those of the controls,while amylase activity in the intestine at E-L was significantly higher than the control.These findings demonstrated that YB1 improved the activities of protease,amylase and lipase enzymes in the intestine of P.maxima juveniles.Furthermore,MDA content of the liver was significantly lower at E-M than the control.The activities of SOD and CAT were higher at E-L and E-H than those of the controls.These findings suggested that YB1 improved SOD and CAT activities in the liver and reduced the level of lipid peroxidation in the liver of P.maxima juveniles.Meanwhile,the findings of histological observation showed that the height of intestinal plica,submucosal thickness,muscular thickness and intestinal villus density at E-L,E-M and E-H were higher than those of the controls.The results showed that YB1 could improve the intestinal structure of P.maxima juveniles.