Prebiotic-matrix protective stabilization enables long-term probiotic storage for vaginal intervention.

Probiotic therapy is an emerging non-antibiotic strategy for bacterial vaginosis (BV). However, the susceptibility of probiotics to environmental factors leads to poor storage stability, and the complex, multi-step processes involved in current protection strategies increase production costs, thereby limiting their further clinical application. Here, we report a single-pot co-lyophilization formulation (termed prebiotic-matrix protective stabilization, PMPS), in which probiotics are uniformly dispersed within a protective prebiotic matrix. The formulation was characterized for microstructure, storage stability, and in vitro antibacterial activity. Furthermore, it was incorporated into a thermosensitive gel for in vivo efficacy evaluation. The probiotic formulation via PMPS method exhibited uniform probiotic distribution by scanning electron microscopy and confocal laser scanning microscopy. It retained >90% viability after 30 weeks at 4 °C under air (vs. ∼20% within 3 weeks for bare probiotic powders). The formulation achieved >99% inhibition of Gardnerella vaginalis, with probiotic viable counts remaining above with probiotic viable counts remaining above 109 CFU/mL after co-culture. Furthermore, the thermosensitive gel system showed superior in vivo efficacy to commercial products. The PMPS method integrates physical protection with nutritional support, enabling long-term storage stability through a simple and cost-effective preparation process, and offers a promising platform for vaginal microecological intervention.