Screening Of Probiotic Bacillus From Intestine Of Chinese Mitten Crab (Eriocheir Sinensis) And Study On Its Growth Character And Application Effect

At present, probiotics used in aquaculture were mostly from terrestrial animals rather than aquatic animals, and so far no one has studied the effect of dietary Bacillus on Eriocheir sinensis, especially the bacterial strain is screened from the intestine of itself.Bacillus from intestine of Chinese mitten crab (Eriocheir sinensis) were screened according to their tolerance of high temperature, low pH, enzyme producing features and activity of inhibition against pathogens. The results indicated that Bacillus ESB3 had the most activity of inhibition against Aeromonas hydrophila CL99920 and good capability of producing protease and amylase. Dealt with initial pH 4.0 in Nutrient Broth medium and water-bath of 90?C for 10 minutes, its survival rate was (33.98±5.51) % and (68.54±8.28) %, respectively. Not only was it safe for the crab when they were challenged by the immersion concentration of ESB3 below 108 CFU/mL, but also it could enhance the resistance of crab against A. hydrophila CL99920 when the immersion concentration of ESB3 was 107 CFU/mL. Morphological physiological and biochemical tests initially confirmed that ESB3 was Bacillus licheniformis. Those suggested ESB3 possessed the development potential as a probiotic of E. sinensis.LB Medium was used as culture for cell multiplication of ESB3. The effects of culture time, initial pH, temperature, salinity and rotation speed were studied. The results showed: the optimal time, initial pH, temperature, salinity, volume of liquid medium and inoculation volume were 20 h, 7.8, 30 ?C, 1.5%, 190r/min, 30mL/250mL and 9% respectively. Compositions of culture media were optimized through single factor test and orthogonal experiment of L9 (34). The results were as follows: tragantine (2%), tryptone (2%), K2HPO4 (0.3%), and under those optimal conditions, the number of ESB3 reached 11.09×108 CFU/ml.In order to reveal the potential effect of Bacillus licheniformis ESB3 on growth, digestive enzyme activity of intestine, microflora of intestine and immunity of Chinese Mitten crab (Eriocheir sinensis), a 60-day feeding experiment was conducted by feeding control diet (non-supplemented probiotics), diets supplemented living ESB3 (doses of 1.3×107 CFU/g, 1.4×108 CFU/g, 0.9×109 CFU/g) and diet supplemented heat-killed ESB (dose of 1.4×108 CFU/g). Living ESB3 of medium dose and low dose could significantly increase weight gain rate, the activity protease and amylase (P<0.05), and the former was a little higher than the latter (P>0.05). Living ESB3 of high dose could only significantly increase the activity of protease (P<0.05). Living probiotics could also enhance the immunity of crab, which showed the increasing tendency with the increasing doses of probiotics in diet basically. Living ESB3 of high dose could significantly increase Total Haemocyte Count (THC), the activity of respiratory burst (O2 - ), Phenoloxidase (PO), lysozyme (LSZ), acid phosphatase (ACP), alkaline phosphatase (ALP)(P<0.05), and had no effect on the activity of superoxide dismutase (SOD) (P>0.05). Living ESB3 of medium dose could significantly increase THC, activity of PO, LSZ, ACP and ALP (P<0.05) and had no effect on O2-, SOD (P>0.05). Living ESB3 of low dose could only significantly increase activity of ALP (P<0.05). O2-, activity of PO and ALP of high dose group were higher than those of medium dose group (P<0.05). THC, activity of O2-, PO, LSZ, ACP and ALP of high dose group were higher than those of low dose group (P<0.05). Living ESB3 had no effect on Aerobic bacteria, Anaerobic formers and E.coli, but could significantly increase the number of Bacillus and decrease the number of Aeromonas (P<0.05). High dose of living ESB3 worked a little more well than the other dose, but the difference was not significant (P>0.05). Heat-killed ESB3 could significantly increase LSZ, ACP and ALP (P<0.05), but it had no effect on growth, protease activity, amylase activity, O2-, PO, SOD and intestinal microflora. Furthermore, living ESB3 could enhance the resistant of Eriocheir sinensis to A. hydrophila infection to a certain extent, and heat-killed ESB3 was not obvious beneficial to Eriocheir sinensis.In conclusion, suitable doses of living Bacillus licheniformis ESB3 could promote growth and enhance immune response of Eriocheir sinensis. It was worth further studying and exploiting.