Abstract
Probiotics hold considerable promise for treating and preventing inflammatory disease; however, their application is often limited by unclear anti-inflammatory mechanisms and reduced viability following lyophilization. In this study, I thoroughly evaluated the anti-inflammatory potential of Lactiplantibacillus plantarum FS4722 (L. plantarum FS4722) and substantially enhanced strain viability through optimization of the lyoprotectant formulation. Functional assays demonstrated that the fermented supernatant, heat-inactivated bacterial suspension, and cell lysate derived from L. plantarum FS4722 effectively suppressed transcription and expression of inflammatory cytokines in LPS-stimulated RAW 264.7 macrophages. The fermented supernatant exhibited the strongest inhibitory effects, surpassing the reference probiotic Lacticaseibacillus rhamnosus GG (LGG). Mechanistic investigations revealed that anti-inflammatory activity is primarily mediated via inhibition of the MAPK and NF-κB signaling pathways. Furthermore, using component screening combined with response surface methodology, the lyoprotectant formulation (10.00% trehalose, 1.00% sodium carboxymethyl cellulose, and 5.00% skim milk) was optimized, resulting in a lyophilization survival rate of 82.32% while maintaining cellular integrity; in this accelerated stability assessment, the strain retained 78.89% of its activity after 28 days of storage at 4 °C. Collectively, this study provides a robust and efficient approach for probiotic formulation while systematically elucidating the underlying anti-inflammatory mechanisms, thereby offering practical guidance for the development and clinical application of high-performance probiotic products.