High Cell Density Culture Of Saccharomyces Boulardii And Preparation Of Microcapsules

Saccharomyces boulardii is the only yeast probiotic,which can decompose enterotoxin,improve the intestinal immune function and promot digestion.On clinic,it is mainly used in treating enteritis,indigestion and dysbacteriosis.In addition,active dry yeast of S.boulardii has been widely used as a feed additive with effective results in many countries.To increase the biomass of S.boulardii and improve the tolerance to the digestive juices,we attempted to optimize the fermentation medium and high cell density fermentation process.The microcapsules were prepared by spray drying and their effects on growth performance of broilers were studied.The main results were described as follows:1.Single factor experiment and Plackett-Burman design were separately applied to screen the medium compositions and significant influencing factors.The components affecting the biomass of S.boulardii were determined as:glucos,peptone,corn steep liquor,KNO₃,yeast nutrients,KH₂PO₄ and MgSO₄.Then the central composite design was designed to the significant influencing factors including glucose,peptone and corn steep liquor.Response surface methodology?RSM?and artificial neural network?ANN?were used to model the relationship between the biomass and the medium composition.The results showed that the ANN model had better fitness and forecasting accuracy than the RSM model.2.The optimal compositions of medium were obtained via genetic algorithm:glucose 40.52 g/L,peptone 36.8 g/L,corn steep liquor 17.32 g/L,KNO₃ 14 g/L,yeast nutrients 1.5 g/L,KH₂PO₄ 0.6 g/L and MgSO₄ 0.8 g/L.Under the optimal conditions,the biomass of S.boulardii reached 8.21 g/L by shake-flask fermentation,which was1.3 times higher than before optimization.On this basis,the optimal high cell density culture conditions in a one-liter fermentation tank were determined as follows:30?,10%inoculum,pH 5.0 and 40%DO.Furthermore,we expanded the culture in a fifty-liter fermentation tank.Glucose and peptone were added to maintain the glucose concentration at 3 g/L and the ammonia nitrogen concentration at 0.06 g/L,the biomass of S.boulardii reached 51.21 g/L.3.To improve the tolerance to the digestive juices,S.boulardii microcapsules were made by spray drying.Composite wall materials were prepared by modified starches,?-cyclodextrin,gelatin,and the proportion of three wall materials showed significant influence on S.boulardii viable counts.Then several spray drying parameters were performed.The results showed that the optimum ratio of modified starches,?-cyclodextrin and gelatin was 6:3:5,composite wall material concentration of 16%,inlet temperature of 80°C,air flow of 550 L/h and feeding speed of 15mL/min.Besides,the most leading influence factor on the production of microcapsules was inlet temperature,followed by compound wall material concentration and feeding speed,and finally air flow.4.Microcapsules were prepared under optimal conditions.The resault showed that they were spherical without holes and cracks on the surface by using scanning electron microscopy,and the particle sizes of the most microcapsules were between100 and 180?m,with an average of 138.65?m.The viable counts of S.boulardii microcapsules were 1.48×10¹⁰ CFU/g.Moreover,the microcapsules showed good tolerance to artificial digestive juices.The survival rate of cell still had 90.57%in artificial gastric juice for 2 h.Meanwhile,the release of the microcapsules was continuously observed,and they were completely released in artificial intestinal juices for 1.5 h.5.To study the effect of S.boulardii microcapsules on the growth performance of broilers.120 1-day-old healthy broilers were randomly divided into 6 groups,including 3 experimental groups and 3 control groups.6‰of microcapsules were added to the feed of experimental groups for a six-week experiment.The results showed that the average weight of the broilers in the experimental groups was 791.04g,803.58 g and 816.72 g,and that of the control groups was 637.28 g,631.96 g and643.49 g,the difference was significant?P<0.01?.Moreover,the average intake of the experimental groups was 12.62%higher than that of the control groups,and the average feed to gain ratio of the experimental groups was decreased by 10.63%.Therefore,S.boulardii microcapsules increased the average gain weight and feed conversion rate,which had a significant effect on growth of broilers.