Beyond viability: comparative functional evaluation of live and inactivated industrial biomasses of probiotic lactobacilli

Introduction: Probiotic microorganisms are well known for their ability to modulate host immune responses and support intestinal barrier function1. However, concerns related to the use of live probiotics (such as safety in immunocompromised individuals, and stability during storage) have led to growing interest in non-viable microbial preparations, often referred to as inactivated, paraprobiotic or postbiotic preparations2,3. This study aimed to investigate the mechanisms by which different inactivated probiotic strains may influence intestinal functions, with a focus on adhesion to epithelial cells and modulation of inflammation-related pathways4. Moreover, we addressed a practical and often overlooked aspect in functional characterization studies: the impact of using small-scale laboratory-prepared bacterial suspensions versus industrially produced microbial biomass. This comparison is particularly relevant for non-viable probiotics, where industrial preparations are generally more standardized, reproducible, and ultimately preferable for translational applications. Methods: The industrial biomass of four commercially available probiotic strains (Lacticaseibacillus paracasei LPC1114, Lacticaseibacillus rhamnosus LRH020, Lactiplantibacillus plantarum PBS067, and Lactobacillus acidophilus PBS066) was inactivated by thermal and mechanical methods. Cell inactivation was confirmed through colony-forming unit (CFU) enumeration and flow cytometry. Membrane integrity and potential were assessed using SYTO24, Propidium Iodide, and DiOC2 staining; metabolic activity was evaluated with carboxyfluorescein diacetate. Protein structure alterations were analyzed using SDS-PAGE. Adhesion to intestinal epithelial cells was assessed using Caco-2 monolayers. NF-κB signaling and epithelial barrier integrity were evaluated using NF-κB-reporter Caco-2 cells, HEK-TLR2 cells, and transepithelial electrical resistance (TEER). Immunomodulatory capacity was studied by measuring TNF-α and IL-2 secretion in murine peritoneal macrophages and dendritic cells. Results: Industrial inactivation protocols were highly effective and yielded standardized, non-viable biomass. The probiotic features (adhesive and immunomodulatory activities) were strain-specific and, in several cases, preserved even after inactivation. Furthermore, the method of inactivation significantly affected biological properties, underscoring the importance of optimizing inactivation protocols for functional applications. Notably, a comparison with laboratory-prepared cells revealed substantial variability in functional outcomes, suggesting that the use of industrial biomass is preferable for reliable and translatable data, particularly for inactivated probiotics. Discussion: These findings confirm that non-viable probiotics retain strain-specific bioactivities relevant for gut health and immune modulation. The study reinforces the concept that inactivated microbial cells can be functionally active and highlights the importance of using industrially standardized preparations in functional studies. These insights are critical for guiding future development of next-generation interventions with non-viable microbial ‘biotics. References: (1) OHLAND, Christina L.; MACNAUGHTON, Wallace K. Probiotic bacteria and intestinal epithelial barrier function. American journal of physiology-gastrointestinal and liver physiology, 2010, 298.6: G807-G819. (2) GUGLIELMETTI, Simone, et al. Commercial and regulatory frameworks for postbiotics: non-viable microbial agents conferring a beneficial physiological effect. Zenodo, 2025. (3) BRUNELLI, Laura, et al. In vitro assessment of the probiotic properties of an industrial preparation containing. Lacticaseibacillus paracasei in the context of athlete health. Frontiers in Pharmacology, 2022, 13: 857987. (4) TAVERNITI, Valentina; GUGLIELMETTI, Simone. The immunomodulatory properties of probiotic microorganisms beyond their viability (ghost probiotics: proposal of paraprobiotic concept). Genes & nutrition, 2011, 6.3: 261-274.