Study On The Optimum Fermentation Time Of Recombinant Lactobacillus In Scale-up Production

It is of great significance to determine the optimum fermentation time for the production of recombinant Lactobacillus casei(L.casei).On the one hand,the quality of the product can be guaranteed,and on the other hand,the cost can be reduced.Therefore,in this study,a traditional L.casei scale-up MRS medium was used for production of recombinant ETEC K99/L.casei and PEDV-S1/L.casei in shake flask and 200 L fermenter.Fluorescent quantitative PCR was employed to detect the plasmid copy numbers(PCNs).The cell number of the expressed target protein was detected by Flow cytometry.The correlation between the PCNs and the number of target protein-expressing bacteria was explored to determine the optimum fermentation time for the industrial production.Firstly,The growth curves were drawn according to the fermentation of the recombinant L.casei,and the optimal growth period had been founded.After that,p LA-K99/L.casei and p LA-PEDV-S1/L.casei were cultured on MRS medium with and without antibiotic respectively for the stability of the recombinant L.casei.The PCNs and foreign protein expression in recombinant L.casei were measured by Fluorescent quantitative PCR and Flow cytometry,respectively.The results showed that both two strains of recombinant L.casei reach the growth peak at 8 h and the PCNs did not lost in the 120 th generation.Both PCNs of K99 and S1 reached their respective peak value of 31.12 and 29.97 at 8 h.What's more,the bacteria expressed target protein K99 and S1 also reached their respective peak value of 98.64% and92.76% at 8 h.During scale up cultures in 200 L fermentor,it was found that the number of bacteria increased compared to that in flask while the PSNs and the expression level of the target protein had no different.Above experimental results indicated that the PCNs and the expression level of the target protein are the highest at the end of the logarithmic growth phase.In the decline phase of the recombinant L.casei growth,the PCNs and the the expression level of the target protein gradually decrease with the fermentation time gradually increasing.Therefore,there is a positive correlation between PCNs and the expression level of the target protein in the logarithmic growth phase of the recombinant L.casei,suggesting that the optimal harvest time is6-10 h after fermentation.The recombinant L.casei can be applied to industrial fermentation.