Biosynthesis Of Phenyllactic Acid By Lactobacillus Crustorum

Phenyllactic acid（PLA）is is a new type of food preservative discovered in recent years.PLA has strong inhibitory effect on bacteria,yeast and mold bacteria.It can also be used to produce poly（phenyllactic acid）s,which is environmentally friendly,high mechanical mild,chemically resistant,strong ultraviolet absorbed.In this paper,high-yield PLA strains were screened from fermented vegetables,and then the fermentation conditions for producing PLA by growing cells and conditions of batch fermentation and continuous fermentation by resting cells to produce PLA were optimized.Finally,as the key enzyme of PLA synthesis,lactate dehydrogenase（LDH）properties were analyzed to provide a theoretical basis for the efficient production of PLA.The traditional fermented vegetables were used as the separation source.The lactic acid bacteria（LAB）were isolated from the fermented vegetable juice and then were identified by16 s RNA.The results showed that the PLA yield of L.crustorum was significantly higher than that of other lactic acid bacteria.The top three strains were NWAFU1019,NWAFU1060 and NWAFU1078,respectively,and their yields were more than 1 mmol/L,which was significantly higher than other strains.Therefore,the three strains were selected for further research.The optimum conditions for PLA production of by growing cells were as follows:the fermentation time was 48 h,the temperature was 30°C,the substrate was phenylpyruvate,the concentration was 40 mmol/L and 0.5%（g/L）Ca CO₃was added into MRS.Under the optimum condition,the PLA yields of NWAFU1019,NWAFU1060,NWAFU1078 were 27.2mmol/L,29.2 mmol/L,30.8 mmol/L with conversion rate of 65.3%,69.6%,73.3%,respectively.Compared with the pre-optimization,the yield increased by 23.7,23.3,19.5times.The optimum conditions for PLA production by resting cells were as follows:without shaking the reaction time was 2 h,the temperature was 30°C,the p H was 6.5,and the substrate was phenylpyruvate,the concentration was 20 mmol/L,the glucose concentration was 1.0%,under the optimum conditions,the PLA yields of NWAFU1019,NWAFU1060.NWAFU1078 were 15.1 mmol/L,14.6 mmol/L,15.2 mmol/L,with conversion rate of 75.5%,73%,76%,respectively.At the same time,the recyclability test was carried out under the optimal conditions.The results showed that NWAFU1078 had the best recyclability.Even reused for 10 times,the conversion rate did not decrease significantly.Therefore,it was used to produce PLA continuously,according designing a continuous production device to control the feed discharge speed is equal to achieved dynamic equilibrium.Through the optimization of the continuous conversion dilution rate,it was determined that the optimum dilution rate is 0.6,and the average conversion rate is 54.5%,the space-time production is 6.5 mmol/L/h.In addition,it was found that both D-PLA and L-PLA can be produced by L.crustorum after analysis with hand column,so it is presumed that at least two kinds of lactate dehydrogenase（LDH）are contained in L.crustorum.6 LDHs containing 3 D-LDH and 3L-LDH were obtained by whole-genome sequencing from the fermented vegetable source NWAFU1078.Then the physical and chemical properties,functional sites,spatial structure and evolution relationship were predicted through bioinformatics software.The results showed that all of them were heat-resistant and stable hydrophilic proteins,and had active sites.The bioinformatics method predicts can provide a theoretical basis for further research on LDH in L.crustorum in this laboratory,and help us to clone and express active LDH protein in the future,thus avoiding the blindness of the experiment.