Structure-Dependent Triglyceride Protection of Freeze-Dried Lactiplantibacillus plantarum: Storage Protection Window, Lipid Oxidation, and Stress Adaptation

Lipid-rich matrices can protect probiotics by limiting direct exposure to moisture, oxygen, and gastrointestinal stressors. However, triglycerides are chemically dynamic during storage, and differences in fatty acid structure and oxidation susceptibility may determine whether they exert protective or detrimental effects. In this study, tricaprylin, triolein, and trilinolein were evaluated as post-drying lipid storage matrices for freeze-dried Lactiplantibacillus plantarum 124 during storage at 25 °C, and 37 °C was used to accelerate lipid oxidation. Survival during storage, fatty acid chain retention, oxidation-derived products, stress tolerance, qualitative confocal imaging of membrane-integrity patterns, and transcriptomic responses were analyzed. After 30 d at 25 °C, log reductions were 0.30, 0.24, and 0.24 log10 units in the tricaprylin, triolein, and trilinolein groups, respectively, compared with 0.60 log10 units in the oil-free control, defining a common protective window. Tricaprylin showed the highest fatty acid chain retention, whereas trilinolein exhibited the greatest loss and strongest oxidative change. GC–MS detected no representative oxidation-derived products in tricaprylin, while unsaturated triglycerides generated dimethyl azelate and methyl 9-oxononanoate. Cells recovered from the protective window showed improved acid and bile tolerance, with survival increasing from 30.48% to 53.43–55.65% and from 14.85% to 22.49–27.79%, respectively. Confocal imaging further showed better-preserved membrane-integrity patterns in the triglyceride-treated groups after acid exposure. Transcriptomics indicated enhanced transport, redox, DNA-related, and cell-envelope responses, accompanied by reduced translation and growth-associated metabolism. Within the experimental conditions tested, these results suggest that triglyceride-associated attenuation of viability loss varies among triglyceride systems and across storage stages, while excessive oxidation of unsaturated triglycerides may weaken probiotic stability.