Although rare, the ability to produce surface S-layer proteins is beneficially associated with particular Lactobacillus strains being investigated as probiotics. Therefore, this work aimed to study specific probiotic functionalities of selected Levilactobacillus brevis strains MB1, MB2, MB13 and MB20, isolated from human milk microbiota, and to assess the contribution of S-proteins. Firstly, Rapid Annotation using Subsystem Technology revealed that cell wall-related genes were abundant in analysed L. brevis genomes. Furthermore, the results demonstrated that S-proteins mediate aggregation capacity and competitive exclusion of selected pathogens by L. brevis strains. The improvement of Caco-2 epithelial monolayer barrier function was demonstrated by the increase in JAM-A and occludin expressions when L. brevis strains or S-proteins were added, with the effect being most pronounced after treatment with MB2 and S-proteins of MB1. L. brevis strains, especially MB20, exerted the potential to adhere to recombinant human ZG16. Strain MB2 and MB20-S-proteins improved the barrier function of HT29 epithelial monolayer, as evidenced by increased ZG16 expression. Analysed L. brevis strains and S-proteins differentially affected the protein expression of IL-1β, IL-6 and IL-8, and IL-10 cytokines. The most prominent effect was observed by S-proteins of MB20, since IL-1β production was decreased while IL-10 production was significantly increased.

The presence of surface (S)-layer proteins as a rare property of lactic acid bacteria plays an important role in the expression of the probiotic properties of the producer strain and protects the cells from unfavourable environmental conditions. Therefore, the aim of this work was to evaluate the role of S-layer proteins of four Levilactobacillus brevis MB1, MB2, MB13, and MB20 strains, previously isolated from human milk. The results showed better survival under simulated gastrointestinal tract (GIT) conditions and better adhesion to Caco-2 cells, achieved when S-proteins were present on the surface of the examined probiotic Levilactobacillus brevis strains. Free cells and cells microencapsulated in alginate, with or without galactooligosaccharides (GOS) and fructooligosaccharides (FOS), survived in high numbers during freeze-drying and one-month storage at 4 °C, as well as during exposure to simulated GIT conditions (>108 CFU/g), thus fulfilling the primary technological criterion for the selection of probiotic strains. Higher survival of freeze-drying of encapsulated cells than free cells, can be attributed to the protective effect of alginate, GOS and FOS as prebiotic agents. However, GOS was more protective than FOS in maintaining the viability of microencapsulated Levilactobacillus brevis MB1, MB2, MB13 and MB20 cells in alginate during storage and under the harsh conditions of GIT and freeze-drying.

Probiotic lactic acid bacteria are widely recognized for their health-promoting effects. However, the use of live microorganisms may pose safety concerns and stability limitations. Consequently, postbiotics, defined as inactivated microbial cells and/or their components, have emerged as a promising alternative. This study integrates genome-guided evaluation of probiotic potential, experimental validation of in silico predictions and process optimization for the production of inactivated Lactiplantibacillus plantarum DM1 and KK1 cells as postbiotics. Genome mining identified genes and gene clusters associated with metabolic versatility, antimicrobial activity, gastrointestinal stress tolerance, adhesion and prebiotic substrate utilization. Building on these findings, to generate postbiotics, the efficiency of thermal, enzymatic, mechanical and radiation-based inactivation methods was evaluated in bacterial suspensions prepared in three dairy by-product matrices: milk permeate, sweet whey and sour whey. Complete inactivation of both strain cells was achieved by thermal treatment (3 min pasteurization), γ-irradiation (3 kGy), and combined lysozyme–pasteurization treatment, whereas other treatments showed partial and matrix-dependent effects. Matrix composition significantly influenced treatment efficacy, suggesting a protective role of food components used. These findings highlight the importance of combining genome mining for potential probiotic strain characterization with robust, matrix-adapted inactivation strategies for the development of stable postbiotic formulations.

Limosilactobacillus fermentum MC1 is an exopolysaccharide (EPS)-producing strain with previously determined probiotic potential in vitro. This study aimed to investigate the in vivo capacity of the MC1 strain or its EPSs to modulate intestinal microbiota and assess its anti-inflammatory effects in both healthy and dysbiotic conditions. Therefore, Lb. fermentum MC1 and its EPSs were administered to a mouse model of dextran sulfate sodium (DSS)-induced colitis (DIC) and to a healthy group, and the effects were observed. Microbiome analysis was used to detect taxonomic differences between treatments. According to the results, administration of the MC1 strain and MC1-EPSs significantly altered gut microbiome composition at different taxonomic levels. The most notable effect was an increased relative abundance of Firmicutes and decreased levels of Candidatus saccharibacteria. Llb. fermentum MC1, and its EPS administration positively affected several disease parameters: reduced disease activity index (DAI), reduced mouse colitis histology index (MCHI), reduced expression of inflammation-related genes and levels of bleeding, and induced polarization of M1 macrophages to the M2-like macrophage phenotype in the DIC mice. These results, along with those related to the induction of antioxidant enzymes and changes in NF-κB-related gene expression, suggest that strain MC1 and MC1-EPSs could be further investigated for their capacity to alleviate DSS-induced histopathological changes and modulate pro-inflammatory cytokine gene expression in colon tissue, which positively correlates with the secretion of inflammatory cytokines, the delay of intestinal inflammation and the maintenance of intestinal barrier function. The obtained data provide a basis for further research into the potential application of intact or microencapsulated Llb. fermentum MC1 cells and its EPSs in colitis therapy.