Functional Equivalence of Heat-Inactivated (HI) and Live Probiotic RSB11 in Suppressing Inflammation: Expanding Formulation and Application Potential

Abstract

The clinical potential of probiotics has been widely recognized, but their translation into reliable therapeutic products has been hindered by major limitations such as undesirable immunogenic responses, the need to maintain viability, instability during storage and transport, and concerns regarding safety in vulnerable populations. Postbiotics, defined as inanimate microbial cells or their components with pro-health activities, overcome many of these limitations by offering enhanced stability, reproducibility, and safety. However, it is very vital to understand if the heat inactivation (conversion of a probiotic to its postbiotic inert form) compromise its functional efficacy. Here, we systematically compared a novel probiotic-derived candidate, Lactiplantibacillus plantarum RSB11 strain, in its live (RSB11 Life, probiotic) and heat-inactivated (RSB11-HI, postbiotic) forms across multiple human epithelial and non-epithelial models relevant to inflammation driven pathologies. To investigate the gut-tissue(s)-axis concept we used gut (Caco-2), lung (HBE), ovary (BG1), bone (osteoblasts, MG-63), kidney (A-498) and liver (HepG2) cells exposed to E-coli or lipopolysaccharide (LPS), and quantified matrix metalloproteinase-9 (MMP-9), an inflammatory mediator, by qPCR and pro-inflammatory cytokines such as tumor necrosis factor- (TNF-), IL-6, and IL-1{beta} by ELISA. In addition, we assessed {beta}-glucuronidase activity and estrogen modulation to explore gut-ovarian axis signaling. Across all models, both RSB11 Life and RSB11-HI robustly suppressed MMP-9, TNF-, IL-6 and IL-1{beta} induction, with equivalent magnitude of effect. The inactivated form retained full cytokine-suppressive capacity and, notably, enhanced {beta}-glucuronidase activity, suggesting additional benefits in microbiome hormone cross-talk. Our findings demonstrate that heat inactivation does not compromise, and may even expand, the functional range of RSB11. By maintaining bioactivity while eliminating the drawbacks of live biotics, heat inactivated RSB11 emerges as a robust, scalable, and versatile postbiotic with potential applications in systemic inflammatory disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/705228v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@7bb1e6org.highwire.dtl.DTLVardef@dc86c7org.highwire.dtl.DTLVardef@147448org.highwire.dtl.DTLVardef@de5232_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstract of RSB11-HI activityHeat-inactivated postbiotic RSB11-HI retains the anti-inflammatory efficacy of its live counterpart (RSB11Life) across diverse organ-relevant cell models. Upon LPS or E-coli stimulation, epithelial, immune, and tissue-specific cells (gut, lung, ovary, bone, kidney, liver) upregulate pro-inflammatory mediators including MMP-9, TNF-, IL-6, and IL-1{beta}. Both RSB11Life and RSB11-HI effectively suppress these inflammatory responses, with RSB11-HI exhibiting more consistent and robust reductions of inflammatory markers across models. Additionally, RSB11-HI uniquely enhances {beta}-glucuronidase activity, facilitating estrogen metabolism and signaling through the gut-ovary axis. Together, these findings highlight RSB11-HI as a stable, safe, and multifunctional postbiotic candidate suitable for therapeutic formulation. Image was designed using ChatGPT.