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Probiotic application remains a challenge since decades, as microorganisms lose their viability and functionality during the manufacture and storage of freeze-dried powders. Appropriate cryoprotectants are important in achieving particular probiotic preparation. Recently,  Enterococcus  spp. such as  Enterococcus faecalis  and  E. faecium  have been used as probiotics for human or animal health promotion. We aimed to determine a suitable protectant to protect the viability and pathogen inhibition potential of  E. faecalis  probiotic strains during freeze-drying and subsequent storage.  E. faecalis  probiotic strains were lyophilized in the presence of glucose, fructose, sucrose, skim milk, and distilled water. The lyophilized vials were stored at 4 °C for up to 30 days. Then, the cells from these vials were evaluated for their probiotic functionality. Among several protectants, skim milk exhibited highly protective capability to protect  E. faecalis  cell viability during freeze-drying and 30-day storage, with survival rates of  E. faecalis  strains ranging from 95.96 ± 0.20% to 103.42 ± 1.96% and 95.68 ± 0.72% to 102.95 ± 2.12%, respectively. The presence of skim milk could also effectively enhance the antimicrobial activity of all freeze-dried  E. faecalis  strains against toxigenic  Clostridium difficile  strains and maintain their activity after 30-day storage. Furthermore, hemolytic analysis demonstrated the safety of these freeze-dried  E. faecalis  strains. Overall, the results indicate that skim milk could be used as a suitable cryoprotective substance for freeze-dried  E. faecalis  probiotics, with potential applications in functional food, medicine, and feed industries.

Optimization of cryoprotectants for freeze-dried potential probiotic Enterococcus faecalis and evaluation of its storage stability