Dietary Administration Of Bacillus Subtilis HAINUP40 Enhances Growth, Digestive Enzyme Activities And Innate Immune Responses Of Tilapia, Oreochromis Niloticus

Probiotics could enhance the growth performance, immune response of aquaculture animals. Because they were safe and environmental friendly, they have became a powerful alternative to antibiotics. Before the use of probiotics in aquaculture, the probiotic properties need to be evaluated in vitro, and the effect of the dietary administration of probiotics on the growth performance and innate immune of the host need to be evaluated in vivo. Preliminary studies of our lab have isolated a Bacillus subtilis strain (named HAINUP40) from the aquatic environment, which could inhibit Streptococcus agalactiae growth, compete with, exclude and replace S. agalactiae in tilapia mucus in vitro, and was safe to tilapia. The objective of this study was to evaluate the probiotic properties of B. subtilis HAINUP40 in vitro and in tilapia and to determine its effect on growth performance, innate immune parameters of tilapia. Our result will provide data for its application in the culture of tilapia.The investigated probiotic properties include tolerance to simulated gastrointestinal stress, auto-aggregation, cell surface hydrophobicity and extracellular enzyme production. After 4 hours in simulated gastric fluid (SGF) at pH=2.0, pH=3.0, pH=4.0 and small intestinal fluid (SIF) at pH=6.8, HAINUP40 bacterial cell number showed little or no decrease. B. subtilis HAINUP40 revealed strong auto-aggregation property (34.6-87.0%) after 24h incubation period. HAINUP40 exhibited a significant cell surface hydrophobicity in xylene (28.81%) and chloroform (41.33%). HAINUP40 was able to produce extracellular protease and amylase. The diameter of proteolysis circle of the cell free supernatants of HAINUP40 was 18.6±0.3mm, the diameter of starch hydrolysis circle of the cell free supernatants of HAINUP40 was 15.7±0.2mm.By adding HAINUP40 and/or Lactic Acid Bacteria in tilapia feed, the effect of the dietary administration of probiotics on the growth performance and innate immune of tilapia were evaluated. Experiment divided into four groups:H40 Group, S4 Group, MIX Group, CK Group, each group has four replicates, each replicates has twenty tilapia. The H40 Group were fed HAINUP40 diet (2.5×108cfu/g), the S4 Group were fed Lactobacillus casei S4 diet (2.5×108cfu/g), the MIX Group were fed diet with both HAINUP40 and Lactobacillus casei S4 (2.5×108cfu/g, HAINUP40:S4=1:1), the CK Group were fed Control diet. The growth performance, innate immune parameters were examined at 2,4 and 8 weeks of post-feeding.Results showed that tilapia fed a diet containing probiotics demonstrated increased final body weight, percent weight gain (PWG), and specific growth rate (SGR) compared to the control group. The final body length, and specific growth rate (SGR) of the H40 Group and MIX Group were significantly higher compared to the control group (P<0.05). The percent weight gain (PWG) of the H40 Group was significantly higher (P<0.05). Protease and amylase activity in the digestive tract was significantly higher (P<0.05) in H40 Group after 4 and 8 weeks. Respiratory bursts and serum lysozyme activity of fish fed HAINUP40 diets were significantly higher (P <0.05) than the control diet group after 2 weeks of feeding. Total antioxidant capacity (T-AOC) and serum superoxide dismutase (SOD) of the experimental group were significantly higher (P<0.05) than the control group after 8 weeks of feeding. But serum alkaline phosphatase, serum total protein and serum albumin were not significant different between the experimental group and control group (P>0.05).In conclusion, B. bacillus HAINUP40 displayed most properties of a probiotic, including secreted extracellular enzymes, resistance to pH, gastric juice, and bile salts in conditions mimicking the gastrointestinal tract. The oral administration of B. subtilis HAINUP40 could enhance the growth performance and digestive enzyme activities and improve immunity in tilapia. Our results indicated that Bacillus subtilis HAINUP40 is a good candidate strain of probiotic strains. Our research will provide data for the application of HAINUP40 in the culture of tilapia.