Research On Breeding And Fermentation Optimiztion Of EPS Highly Produced Latobacilli

Bacteria exopolysaccharides (EPS) is widely used in food and non-food industry because of its unparalleled advantages compared with plant and algae sourced polymers. However, subculture for too many times will lead to katagenesis and reduction of EPS amount. Protoplast regeneration is an advanced method to improve strains by gene recombination and exchange. It provides a way to obtain the EPS highly produced lactobacilli. This research used two kinds of lactobacilli, Lb. plantarum and Lb. reuteri as research material to study protoplast dissociation, regeneration and fusion.1. Inoculation amount, lysozyme concentration, dissociation temperature, treatment time, pH as well as osmotic pressure were studied byvariance analysis and orthogonal design.In summary, optimal dissociation condition of Lb. plantarum was3-6%inoculation amount,5-7h cell age,15-20mg/L lysozyme concentration and37-42℃treated temperature, while a5h aged6%inoculated cell which treated with20mg/ml lysozyme at37℃was the best condition for Lb. reuteri.2. The regeneration of protoplast was greatly influenced by the previous dissociation factor and latter cultured condition. The best enzyme concentration for regenerating Lb. plantarum (26.5%) and Lb. reuteri (42.98%) was15mg/mL. The optimal osmotic pressure of Lb. plantarum and Lb. reuteri was0.5-0.65M and0.5M separately.4ml/L and6ml/L BSA enhanced Lb. plantarum and Lb. reuteri regeneration frequency to30.58%and52.85%. These two lactobacilligrew betteron0.02mol/L Mg2+、0.02mol/L Ca2+and0.02mol/L Mg2+、0.01mol/L Ca2+ionic contained medium.3. For the protoplast fusion of Lb. plantarum×Lb. reuteri and Lb. reuteri×Lb. reuteri, heated at53℃for5min and UV for2min were the best inactivation condition. Fusion rate was greatly influenced by PEG-6000(fusogen), fusion temperature and fusion time. The optimal fusion rate of53.65-54.5×10-5was achieved by Lb. plantarum×Lb. reuteri with the follow conditions:24-28℃,40%PEG-6000,5min. And for Lb. reuteri×Lb. reuteri, the fusion frequency of42.10×10-5was achieved with the condition of using40%PEG-6000for5min at28℃. We obtained one EPS highly produced fusant (Lb. reuteri-YR) from all14hybridization fusant. Fermentation condition optimization experiment was designed using Planckett-burman to screen the key factors (sucrose concentration, fermentation temperature and time) which most related to EPS production. The highest EPS production of 7344.8mg/L was obtained at the optimal fermentation condition:ferment the fusant in100g/L sucrose MRS broth for21h under34℃. The prediction was done by using Center combination experiment and response surface analysis to5levels of sucrose concentration, fermentation temperature and time respectively-Anactual EPS production of7214.4mg/L was obtained by ferment under the theoretical optimum condition.