The cell membrane of Lactiplantibacillus plantarum is a key structure for cell survival. In this study, we aimed to improve the lyophilization resistance of L. plantarum by regulating the cell membrane structure. Unsaturated fatty acids or cell membrane-regulating substances were added during culturing to determine their effect on the composition of cell membrane fatty acids and the survival rate of the cells after freeze-drying. The results showed that Tween 80, β-carotene and melatonin increased the lyophilization survival rate of L. plantarum by 9.44, 14.53, and 18.34%, respectively. After adding a lyophilization protective agent at a concentration of 21.49% at a 1:1 ratio, a combination of Tween 80, melatonin, and β-carotene was added to regulate the cell membrane, which increased the lyophilization survival rate by 32.08–86.05%. This study proposes new research directions and ideas for improving the survival rate of probiotics for industrial production.
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