In this study, the effects of heat shock-repair combined with addition of trehalose in lyoprotectant on the freeze-drying resistance and room temperature storage stability of Lactiplantibacillus plantarum LIP-1 were investigated. The results showed that the freeze-drying survival rate of the combined treatment group with heat shock-repair and 4 g/100 mL trehalose addition was 81.65%, which was significantly higher than those of the control group without heat shock-repair treatment (59.92%) and the heat shock-repair treatment group (70.23%) (P < 0.05). The damage degree of cell wall and cell membrane in the combined treatment group was significantly reduced compared with that in the control group. The addition of trehalose maintained a high proportion of unsaturated fatty acids in the cell membrane, reduced cell membrane damage, and significantly enhanced the freeze-drying resistance of the strain while improving its room temperature storage stability. After eight weeks of storage, the survival rate of the combined treatment group was 51.66%, which was significantly higher than those of the control (12.09%) and heat shock-repair (43.79%) groups (P < 0.05), and the intracellular fluorescence intensity was 1969.23 ± 37.22, which was significantly lower than those in the control (3475.21 ± 106.56) and heat shock-repair (2843.95 ± 52.12) groups (P < 0.05). To sum up, the combined treatment could improve the freeze-drying resistance and room temperature storage stability of the strain.

Adenine acts as a growth promoter to promote the growth of the lactic acid bacteria (LAB), but the effect on the viability of freeze-dried strains has rarely been studied. In this study, adding 0.01 g/L of adenine to medium increased the growth and freeze-dried viability of Lactiplantibacillus plantarum LIP-1. Further research has found that L. plantarum LIP-1 synthesized large amounts of adenosine triphosphate (ATP) by metabolizing adenine. Elevated intracellular ATP content caused feedback inhibition on the conversion pathway of pyruvate to lactic acid, while promoting the conversion of pyruvate to acetyl coenzyme A (acetyl-CoA). After a large accumulation of acetyl-CoA in the cells, there was sufficient substrate for the synthesis of cell membrane fatty acids. Elevated intracellular ATP content also activated the acyl-CoA thioesterase activity to catalyse the conversion of saturated fatty acids to unsaturated fatty acids, thereby improving the integrity of the cell membrane and reducing damage to the cell membrane during the freeze-drying process. Additionally, a reduction in the amount of pyruvate converted into lactate prevented the decrease in intracellular pH (pH_in), which alleviated the degree of acid stress on the strain, resulting in less DNA damage and improved DNA stability. It is concluded that L. plantarum LIP-1 reduced the degree of cell membrane and DNA damage by metabolizing adenine and improved the freeze-dried viability of the strain.

Amino acids are often used as probiotic growth factors. Their addition to the growth medium is found to effectively enhance the resistance of the strain to adverse environments. In this research, we found that adding 0.05 g/L L-cysteine to culture medium improved the freeze-drying survival rate of the strain. We investigated the internal mechanism behind this phenomenon and found that the addition of L-cysteine can reduce DNA damage to bacterial cells during the freeze-drying process. In comparison to the control group without L-cysteine, the treatment group with the addition of 0.05 g/L of L-cysteine exhibited an up-regulation of the metC gene, leading to the metabolism of L-cysteine into pyruvate and NH₃, which raised the intracellular pH, reduced DNA damage, and consequently enhanced the resistance of Lactiplantibacillus plantarum LIP-1 to freeze-drying.