Study On High-density Culture And Microencapsulation Of Selenium-enriched Lactobacillus Reuteri GG001

Lactobacillus is a kind of probiotics widely used in food.As a class of lactobacillus that has been approved by FDA and Ministry of Health for food applications,Lactobacillus reuteri have good probiotic effects.But there are some problems such as high desity fermentation,encapsulation which limited the application of of L.reuteri as probiotic.In this study,L.reuteri was used as the research object,We studied its metabolic properties,high density culture,selenium enrichment,preparation technology of microencapsulation.The main findings are as follows:First,sucrose was used as a carbon source instead of glucose to optimize the culture conditions of L.reuteri CG001.The aim is to improve the concentration of biomass in the fermentation broth and reduce the fermentation cost.The final sucrose-MRS medium was determined by RSM analysis as follows: 93.0 g / L sucrose,93.0 g/L yeast extract,3.5 g / L ammonium citrate,7.0 g / L sodium acetate,4.0 g/L K2HPO4,0.4 g/L MnSO4·H2O,0.6 g/L MgSO4,1.5 g/L Tween 80.The CDW(g/L,Cell dry weight)of L.reuteri CG001 reached 10.9 g/L with optimized medium in an anaerobic bottle.The survival cell count of the fermentation broth reached 4.4×1010 CFU/ml after 18 h.When L.reuteri CG001 was fermented in a5 L fermentor,CDW reached 13.7 g/L.Compared to 1.7 g/L before optimization,there was a significant increase.The purpose of high-density culture has been achieved.Secondly,the effects of sodium selenite concentration,selenium absorption characteristics and sodium selenite supplementation on selenium enrichment of L.reuteri CG001 were studied,based on the modified SucMRS medium.The administration of sodium selenite in dosage of 1-8 mg/L was suitable for selenium enrichment in L.reuteri CG001.Cultures were found to turn red due to the occurrence of Se(0)during the stationary phase under higher selenite stress(>4 mg/L Na2SeO3)and the color rose with selenite concentration increase,suggesting that L.reuteri was able to tolerate higher selenite by a detoxification process of reducing Se(IV)to nonsoluble Se(0).The results showed that selenium enrichment was mainlyin the logarithmic growth phase of L.reuteri CG001.The conversion rate of selenium was 75-83%.The selenium content in L.reuteri CG001 reached 173 ?g/g in anaerobic bottle,however,5L fermenter was 211 ?g/g.When the residual selenium concentration in the fermentation broth was less than 320 ?g/L,the cells were no longer biotransformed to sodium selenite.Finally,the L.reuteri CG001 was encapsulated in core-shell alginate@chitosan microcapsule using high pressure air spray method.The hydrodynamic parameters of the microencapsulation process which influence the size of microspheres,encapsulation yied(EY)and the protective effect of microencapsulation on L.reuteri CG001 was investigated.It was found that the EY of microcapsulation was approximately 60~90 % and the highest was 93 %.The mean diameter of capsules ranged from 40 to 100 ?m.After coating with chitosan,microcapsules could be cultured in salt-free SucMRS fermentation broth for more than 18 hours without any impact.The survival rate of L.reuteri embedded in microcapsules was also checked under varying cryoprotectant conditions.The survival rate of L.reuteri in microcapsules was 76.6 % by adding 20% skimmed milk as cryoprotective agent,which was superior to sucrose and glycerol.The theoretical value of viable cells was8.7×109 CFU/g microcapsules.Chitosan coated alginate microcapsules had better protective effect and sustained release effect.The viability of L.reuteri CG001 was4×107 CFU/g in simulated gastric juice in two hours.Microcapsules remained spherical shape in simulated gastric juice for more than 4 hours,while L.reuteri CG001 embeded in microcapsule was completely released in less than 1 hour if microcapsule was treated with simulated intestinal fluid.The survival rate of L.reuteri was 10.4 % storage at 4 ? after four weeks and the viability was maintained at 9.2 ×108 CFU/g.Thus,the study demonstrated that high pressure air spray method associated with the freeze drying process could be considered as feasible technology for the protection,application and controlled release of probiotics.