Heat-Inactivated Lacticaseibacillus paracasei subsp. paracasei 431 Modulates Gut Microbiota Composition and Enhances Short-Chain Fatty Acid Production During In Vitro Colonic Fermentation

Paraprobiotics offer safer alternatives to live probiotics without requiring cell viability. Here, heat-inactivated bacterial preparations (paraprobiotics) were produced from Lacticaseibacillus paracasei subsp. paracasei 431 (L. casei 431) using thermal treatments (65–121 °C). No viable colonies were detected by conventional plating in any treatment, and flow cytometry indicated that more than 96% of cells were membrane-compromised or dead. The most favorable condition (65 °C, 60 min) was identified using a TOPSIS multi-criteria ranking approach integrating loss of culturability with antimicrobial activity against Enterococcus faecalis and Escherichia coli with inactivation efficiency. This paraprobiotic and the corresponding probiotic were subjected to in vitro gastrointestinal digestion and colonic fermentation, followed by short-chain fatty acid (SCFA) and 16S rRNA-based gut microbiota analysis. The fecal inoculum was pooled from five healthy donors and used to run three replicate fermentation vessels per group. Both probiotic and paraprobiotic supplementation significantly increased acetate, butyrate, and total SCFA concentrations compared with the control, and propionate and valerate concentrations were also significantly higher in the paraprobiotic group than in the control. Both interventions increased Chao1 richness and Simpson diversity (p < 0.05), although Shannon diversity did not differ significantly, and induced significant shifts in beta diversity, alongside marked enrichment of butyrate- and SCFA-producing taxa such as Roseburia, Anaerostipes, and Akkermansia muciniphila. However, not all observed microbial changes were unequivocally beneficial; increases in Bilophila wadsworthia and Collinsella aerofaciens warrant cautious interpretation given their context-dependent associations with host health. Spearman correlation analysis identified positive associations between enriched taxa and SCFA concentrations, which should be interpreted as exploratory rather than evidence of causal metabolic cross-feeding. Under the conditions of this in vitro fermentation model, no statistically significant differences were detected between the viable and heat-inactivated preparations for most measured outcomes, indicating that thermal inactivation did not markedly diminish the capacity of L. casei 431 to influence gut microbial composition and short-chain fatty acid production. The multi-criteria selection strategy offers a practical approach for optimizing paraprobiotic production for functional food applications.