Process Optimization Of Reuterin Produced By Lactobacillus Reuteri

Reuterin,a broad spectrum antibacterial substance,was reported to inhibit the growth of gram negative bacteria and positive bacteria,yeasts,molds and protozoa.Reuterin produced by biotechnological could be used in health care,food industry and chemical industry.No commercial reuterin has been produced until now,mainly due to low microbial productivity via glycerol dehydratase(GDHt).Based on this background,the fermentation conditions of glycerol dehydratase by Lactobacillus reuteri and whole-cell-biotransformation process were firstly developed to ferment reuterin practically feasible for commercial production in the present study.Fermentation medium composition and culture conditions were optimized to enhance the enzyme activity of glycerol dehydratase.The results showed that the enzyme activity was significantly influenced by the cell age and the highest enzyme activity was obtained at initial stationary phase of cell grown.A certain amount of dissolved oxygen in fermentation medium was beneficial to the growth of bacteria.Lactose was the best carbon source for improving glycerol dehydratase activity,and the yeast extract made contribution to enhancing the glycerol dehydratase activity.Using yeast extract and corn steep liquor as the compound nitrogen source could maintain the high enzyme activity and control the production cost.A certain concentration of K+ and Mn2+ in the culture medium could promote the growth of Lactobacillus reuteri and the production of GDHt enzyme.The optimized fermentation conditions were as follows: lactose 20 g/L,corn steep liquor 30 g/L,yeast extract 4 g/L,K2HPO4 3 g/L,MnSO4 0.15 g/L and incubated at 37℃ for 26 h.The effects of production parameters on the biotransformation were investigated to enhance the yield of reuterin by Lactobacillus reuteri.The biotransformation conditions were optimized by orthogonal tests as follows: glycerol concentration 400 mmol/L,cell concentration 110 g(wet weight)/L,conversion time 2 h,temperature 30℃ and pH 6.2.The optimized fermentation conditions of GDHt and whole-cell-biotransformation process were used to enlarge the production in 7.5 L fermentor.The maximum GDHt enzyme activity and reuterin concentration finally reached to 2.81±0.12 U/mg and 309.9±7.0 mmol/L respectively.So after optimization,the cell production efficiency(0.313 g(wet weight)/L/h)and the yield of reuterin were enhanced by 58% and 82% respectively and the commercial cost of fermentation medium was reduced by 43%.The free cells were immobilized to enhance the recycling ability,and the effects of immobilization condition on reuterin production were investigated.The tolerance ability of L.reuteri for reuterin was increased under low temperature,and it would conducive to cells recycling.The optimum condition were: enzyme conversion temperature of immobilized cells is 15℃,sodium alginate concentration 2%,CaCl2 concentration 3%.Under the optimized condition,half of its original activity could be maintained after five runs.Storage stability of raw reuterin could be influenced by temperature and pH.The retention ratios decreased with the increasing of temperature(25~50℃)and pH(4.0~7.0).In the early storage period(20 days),pH showed a more significant effect than temperature,but after 25 days,the effect of temperature rather than pH on the stability was the dominating factor.