Study on the Freezing Protection Effect of Melatonin on Lactobacillus plantarum FQR

This study aimed to investigate the regulatory effect and cryoprotective mechanism of melatonin (MT) on the physiological functions of Lactobacillus plantarum FQR during freezing and freeze-drying. Results indicated that the addition of 5 mg/mL MT as a cryoprotectant maximized the freeze-drying survival rate to 32.04 ± 2.14%. MT effectively alleviated low-temperature and freeze-drying stress by reducing extracellular alkaline phosphatase activity, enhancing intracellular lactate dehydrogenase activity, and decreasing extracellular β-galactosidase activity without significant differences. Higher survival rates in defining medium further suggested that MT reduced damage to cell wall and membrane structures during lyophilisation, decreased membrane permeability, and preserved cellular physiological functions. In addition, MT supported cellular energy metabolism and protein synthesis, enhanced transmembrane potential to facilitate ATP transport, and helped maintain intracellular and extracellular pH balance. The prepared freeze-drying protectant containing 69.80 mg/mL exopolysaccharides (EPS) and 4.25 mg/mL MT showed better protective effects than the control group. MT also increased bound water content, lowered the freezing point of the solution, and inhibited ice crystal formation. Transcriptomic analysis revealed that amino acid biosynthesis, amino acid metabolism, and ABC transport systems were the primary pathways affected by MT treatment. These findings demonstrate that MT improves freeze-drying tolerance by maintaining membrane integrity, regulating cellular metabolism, and enhancing oxidative stress resistance. Given its natural biosynthetic origin, generally recognized as safe (GRAS) status, and absence of residual solvents or allergenic proteins, MT can be safely considered for incorporation into food and nutraceutical products. This study underscores the practical relevance of MT as a functional component in compound cryoprotectants, providing a feasible strategy to enhance the viability, stability, and industrial applicability of Lactobacillus plantarum during freeze-drying and storage.