Feasibility of using cellulose nanofibers as a cryoprotectant for freeze-drying Bifidobacterium animalis subsp. lactis IDCC 4301

Cryoprotectants are essential in probiotic product manufacturing, ensuring osmotic pressure balance and minimizing mechanical damage caused by ice crystals formation during freeze-drying. However, conventional cryoprotectants, such as sugar-based compounds, are typically required at concentrations exceeding 5 %, which may introduce formulation challenges. In this study, cellulose nanofibers (CNFs), one of the most abundant and sustainable biomaterials, were investigated for their potential as a low-concentration cryoprotectant. To assess their effectiveness, freeze-dried Bifidobacterium animalis subsp. lactis IDCC 4301 was subjected to different concentrations of CNFs with subsequent evaluation of bacterial growth and survival. Based on the comparison with trehalose, skim milk, and glycerol as a control, 0.05 % CNF was selected as the optimal cryoprotectant. Notably, B. animalis IDCC 4301 preserved with 0.05 % CNF exhibited comparable or superior survival rates under acid and bile conditions, as well as enhanced adhesion to gut epithelial cells relative to 0.05 % trehalose. In addition, during freeze-thaw cycles, CNF-treated B. animalis IDCC 4301 demonstrated higher viability with comparable metabolic activity, suggesting that CNFs effectively mitigate cellular damage by forming a viscous, flexible protective layer. These findings highlight the potential of CNFs as an innovative and efficient cryoprotectant, providing effective bacterial preservation at lower concentrations than traditional usage. Given their abundance, biocompatibility, and functionality, CNFs represent a promising alternative for improving probiotic stability in food applications.