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

Abstract

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.

Keywords:

• Probiotics
• Enterococcus faecalis
• skim milk
• freeze-drying
• bacterial viability

Acknowledgments

The authors would like to thank the Central Research Laboratory, Faculty of Medicine, Prince of Songkla University, for providing the required laboratory instruments. The authors are extremely grateful to Associate Prof. Dr. Chalat Santivarangkna for the kind help and recommendation during the performance of the freeze-drying technique.

Data availability statement

All data used or analyzed during this study are included within this article.

Disclosure statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Additional information

Funding

This work was supported by Faculty of Medicine, Prince of Songkla University [grant number REC61-064-4-2] and DPST first placement 2588 [grant number 023/2558]. CR is a recipient of a scholarship from Faculty of Medicine, Prince of Songkla University.