Optimization of Cryoprotectant for Attenuated Salmonella Cholerae C500

The purpose of this study was to optimize the antifreeze protectant of attenuated Salmonella Cholerae C500 and improve the survival rate of C500.The effect of four types of cryoprotectant (permeating,semi-permeating,non-permeating cryoprotectants and antioxidants) on the survival rate of C500 before and after cryopreservation was investigated by single factor test.Each cryoprotectant was set at different concentrations and mixed in equal volume of C500.After two weeks of cryopreservation in liquid nitrogen,becteria plate count was carried out to calculate the survival rate,and the best one of the permeating cryoprotectant,semi-permeating cryoprotectant,non-permeating cryoprotectant and antioxidants and their optimal concentrations were beneficial to the preservation of bacteria was initially selected.Finally,the four protectors selected were tested by orthogonal test of four factors and three levels to get the optimal combination formula.The results of single factor test showed that the best permeating,semi-permeating,non-permeating cryoprotectants and antioxidants were glycol,sucrose,PVP and glycine,and the corresponding preservation concentrations were 15%,6%,6% and 0.25%,respectively.The orthogonal test showed that each factor had a significant impact on the survival rate of C500.The optimal combination formula was 17% glycol,9.0% sucrose,6.0% PVP and 0.35% glycine.The optimal combination had the optimal effect at -80 ℃,the survival rate was 88.34% in 6 weeks,followed by liquid nitrogen group,-20 ℃ group and 4 ℃ group.The results showed that the formulation of the protective agent obtained in this experiment greatly improved the antifreeze ability of C500 and its survival rate.