Abstract: The present study aimed to optimize the medium formulation for high cell density culture of an invasive tumor-targeting engineered strain, EcNA. Using one-factor-at-a-time method and a Plackett-Burman design, yeast extract and dipotassium phosphate were found to be the most important factors affecting cell concentration. The optimal medium, as determined using central composite design and response surface methodology, was composed of soluble starch 2 g/L, yeast extract 50.3 g/L, K2HPO4 14.26 g/L, KH2PO4 3 g/L, MgSO4 0.3 g/L, and trace element stock solution 2 mL/L, and the inoculum amount was 3%. The optical density at 600 nm (OD600 nm) of the bacterial culture obtained using the optimized medium reached 7.602 and the biomass was 2.96 × 109 CFU/mL, which was 78.3% higher than that before optimization. Moreover, using orthogonal array design, a lyoprotectant formulation consisting of skim milk 14.25 g/100 mL, sucrose 3 g/100 mL, VC-Na 3 g/100 mL was found to be optimal for the freeze-drying of the strain. The survival rate of the engineered bacterium was up to 86.32% during freeze-drying using the lyoprotectant, being beneficial for of prolonged storage life of oral capsules.

Key words: tumor-targeting engineered strain, high cell density fermentation, response surface analysis, cryoprotectant, bacterial powder