Abstract

BACKGROUND During the lyophilization (i.e., freeze-drying) of probiotics, the addition of a protective agent is essential for the survival of the frozen bacterial cells. Although many studies have explored the effects of exogenous protective agents during freeze-drying, whether these protective agents play an intracellular role requires study. Sorbitol is one of the most commonly used protective agents. In Lactiplantibacillus plantarum , srlD1/srlD2 is a key gene for sorbitol synthesis, and the pts-srlD gene cluster controls sorbitol transport. The objective of this study was to analyze the effect of sorbitol as an endogenous protective agent during the lyophilization of L. plantarum . Intracellular sorbitol levels were successfully controlled by the overexpression and knockout of sorbitol synthesis ( srlD ) or transport ( pts-srlD ) genes. Meanwhile, knockout of ldh will shift the metabolism of Fru-6P to the synthesis of sorbitol. RESULTS In phosphate-buffered saline alone, the survival rate of the AR113Δ ldh-srlD2 strain, which had a large amount of intracellular sorbitol, reached 41.9% – a rate 3.2 times higher than that of AR113- srlD2 . The addition of sorbitol decreased the survival of transport system-knockout strains compared with the wild-type strain, and the transport system-restored strain had a survival rate 3.1 times higher than that of the wild-type strain. CONCLUSION These results show that sorbitol plays an intracellular role in L. plantarum , and exogenous sorbitol is transported into the cell, where it exerts a protective effect. This study provides a theoretical basis for the application of protective agents. © 2026 Society of Chemical Industry.