| Bacillus pumilus SE5 is a dominant bacterium in the gut of fast growing grouper Epinephelus coioides and exhibited in vitro antagonistic activity against several fish pathogens.Our research group observed that dietary administration of viable or heat-inactivated B.pumilus SE5 could modulate the intestinal microbiota,improve the immune response and the growth performance and feed utilization in marine fish.For the commercial realization and economic viability of Bacillus pumilus SE5,the fermentation medium,fermentation process and spray-drying process of B.pumilus SE5 were optimized in this study.The main results are as follows:1.The response surface analysis and single-facter test were used to optimize the fermentation medium and fermentation condition of B.pumilus SE5.The optimized medium was:wheat bran10 g/L,soybean power 38 g/L,NaCl 6 g/L and the predicted optimal number of viable bacterium was 3.71×109 CFU/mL,the actual average number is 3.61×109 CFU/mL.The optimized fermentation condition was:filling volume 50 mL/250 mL,inoculum 5%,temperature 28?,initial pH 6.4.Using the orthogonal experiment test,the inorganic salts that influence the spore formation of SE5 were determined,and the optimized inorganic salt combination was:CaCO3 0.3 g/L,MgSO4·7H2O 1.5 g/L,MnSO4·H2O 0.4 g/L.In summary,the fermentation medium and fermentation condition of B.pumilus SE5 were:wheat bran 10 g/L,soybean power 38 g/L,NaCl 6 g/L,CaCO3 0.3 g/L,MgSO4·7H2O 1.5 g/L,MnSO4·H2O 0.4 g/L;filling volume 50 mL/250 mL,inoculum 5%,temperature 28?,initial pH 6.4,shaking speed150 r/min.According to the fermentation process,three repeated experiments were carried out and the results showed that the average viable bacterial number was 6.29×109 CFU/mL and no spore formation after 24 h of fermentation,while the average viable bacterium and spore number were 3.21×109 CFU/mL and 1.60×109 CFU/mL for 48 h,respectively.2.5 L fermenter experiment showed that the level of dissolved oxygen has significant effect on the growth and spore formation of B.pumilus SE5,the best level of dissolved oxygen was40%.No significant effect on the growth of B.pumilus SE5 was obtained under the control of pH during fermentation.The condition of B.pumilus SE5 in the 5 L fermenter was:ventilation volume 2.4 L/min,tank pressure 0.05 Mpa,temperature 28?,natural pH,stirring speed 200r/min from 0 to 4 h,dissolved oxygen level 40%from 4-24 h.Under the fermentation condition,the average viable bacterium and spore number were 5.28×109 CFU/mL and 5.20×109 CFU/mL after 24 h of fermentation,respectively.3.The spray drying test of SE5 fermentation?containing 10%maltodextrin?was carried out at a feed rate of 18.38 mL/min and atomization pressure of 0.3 Mpa.The properties such as viable count,product yield,moisture content,density,pH and tatal sugar contents of the spray-dried powder were determined,and the parameters of powder were viable count 3.725×1011CFU/g,product yield 74.47%,moisture content 2.26%,density 0.4255 g/cm3,pH 6.91,total sugar 3.18%.The artificial gastric tolerance test showed that the survival rates of powder SE5 in artificial gastric juices at pH 3.0 and 4.0 were higher than that in artificial gastric juice at pH 2.0for 2 h,and the viable bacterial number were 7.83×1010 CFU/g and 11.0×1010 CFU/g,respectively.The powder SE5 exhibited strong tolerance to artificial intestinal juices with different concentrations of bile salt,and the viable bacterial number of SE5 was higher than 1010CFU/g in artificial intestinal juices containing 0.1%-0.5%bile salt after 3 h of incubation. |
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