Evolutionary Screening of Lacticaseibacillus rhamnosus MP108 for Freeze-Thaw Tolerance

Freeze-drying is the most commonly used method for preserving probiotics. The freeze tolerance of probiotics has a significant impact on both their survival rate and the expression of their functional properties. To enhance the freeze tolerance of probiotics, this study established an adaptive evolution protocol combining cold stress with repeated freeze-thaw cycles to screen for freeze-thaw-tolerant evolved strains of Lacticaseibacillus rhamnosus MP108. The safety, metabolic, and functional characteristics of these strains were then evaluated. The results showed that the combination of the 8 h cold stress treatment at 4 °C and nine cycles of freezing and thawing at -20 °C effectively enhanced the strain's freeze tolerance, and the evolved strain L134 was successfully screened through adaptive evolution. Its freeze-dried survival rate and storage survival rate after 6 months of storage were both significantly higher than those of the parental strain (p < 0.05). Furthermore, it exhibited good passage stability. At the same time, the safety and acid-producing characteristics of L134 did not show significant changes compared to the parental strain. Furthermore, its tolerance to simulated gastric fluid, antibacterial activity, and antioxidant capacity were significantly enhanced (p < 0.05). In particular, compared to MP108, L134 exhibited significantly increased hydroxyl radical scavenging capacity as well as higher activities of the antioxidant enzymes SOD and CAT (p < 0.05); the improvement in its freeze tolerance may be related to this enhanced antioxidant capacity.