Protecting DNA and improving freeze-drying survival rate of Lactiplantibacillus plantarum LIP-1 through metabolism of hypoxanthine.

Abstract

Freeze drying is widely employed in the industrial production of probiotic powders. However, it often results in a marked reduction in strain viability due to exposure to low temperatures and dehydration, with DNA damage being a key contributor to cell death. Given the established link between purine metabolism and DNA repair, this study explored the effects of exogenous added hypoxanthine supplementation on freeze-drying survival and its underlying mechanisms, as well as its impact on the room-temperature storage stability of Lactiplantibacillus plantarum LIP-1. Our results showed that supplementation with 0.03 g/L hypoxanthine in MRS culture medium significantly improved both the freeze-drying survival rate and room-temperature stability of L. plantarum LIP-1 compared with the control group (P < 0.05). Mechanistically, the strain metabolized hypoxanthine to enhance the synthesis of inosine monophosphate (IMP) which in exerted feedback inhibition on the conversion of glutamine to IMP, leading to intracellular accumulation of its precursor substance, glutamate. Concurrently, hypoxanthine addition facilitated the establishment of a low-pH environment during bacterial growth, promoting the conversion of glutamate to γ-aminobutyric acid (GABA). This conversion irreversibly consumed intracellular H⁺, and given the alkaline nature of GABA, further elevated the intracellular pH, thereby mitigating DNA damage. This study presents the first evidence of hypoxanthine's beneficial role in improving both freeze-drying resistance and storage stability in Lactobacillus. These findings provide a novel strategy to optimize freeze-drying processes for producing highly viable probiotic powders.