Development of a new freeze-drying protection system for potential probiotic Enterococcus faecalis L118 and...

The purpose of this study was to investigate the freeze-drying protective effect of cold stress and arginine on Enterococcus faecalis L118 and to develop a new freeze-drying protection system in combination with glycerol and skim milk. The freeze-drying protection effect of cold stress and arginine on E. faecalis L118 was evaluated using one-way ANOVA and response surface methodology (RSM), and the freeze-drying protection system was optimized. Subsequently, the protective mechanism and efficacy of the optimized system were further assessed via cell membrane integrity analysis, storage stability tests, probiotic property assays, and RT-qPCR. In this study, cold stress on E. faecalis L118 before freeze-drying and arginine as a cryoprotectant significantly enhanced its survival rate. The optimal protection system was optimized via RSM to consist of cold stress at 8 °C for 8 h, 1.5 % arginine, 10.4 % skim milk, and 9.8 % glycerol, and the survival rate of E. faecalis L118 achieved under this system was 87.90 ± 1.28 %. The E. faecalis L118 treated with the optimal protection system had relatively high storage stability at -20 °C and could maintain the cell membrane integrity and probiotic properties of E. faecalis L118. RT-qPCR results indicated that cold stress significantly upregulated (P < 0.05) the expression of atpA, plsX, luxS, and SOD genes, while arginine upregulated (P < 0.05) atpA, cspC, LDH1, FabH, FabF, plsX, and mprF2 genes. All genes in the optimal protection system were upregulated (P < 0.05). Our study demonstrated the protective effects of cold stress and arginine on E. faecalis L118 during freeze-drying and established a novel freeze-drying protective system, providing a theoretical basis for the preservation and industrial-scale production of E. faecalis L118.