Aims: Our aim was to evaluate calcium alginate beads as a physical cultivation platform for probiotic bacteria and investigate their effects on bacterial growth localization, release dynamics, and viability under different environmental conditions. Methods and Results: Calcium alginate beads were fabricated using sodium alginate at different concentrations (1.0–2.0%, w/v), and their structural stability, permeability, and pH resistance were assessed. Lacticaseibacillus paracasei was used as the primary model organism. Among the tested formulations, 2.0% alginate exhibited the lowest permeability (~40%) compared with 1.0% and 1.5% alginate (~60%) and maintained structural integrity under alkaline conditions (pH 11–12). Encapsulated L. paracasei proliferated predominantly within the bead matrix, while less than 1% of total bacteria were released into the surrounding medium during the first 12 h and approximately 8% released after 24 h. Encapsulation also maintained higher bacterial viability over 24–48 h compared with conventional liquid culture conditions. Encapsulated cells could be efficiently recovered using sodium citrate without marked loss of viability. In addition, encapsulation enabled the growth of Bifidobacterium animalis subsp. lactis under aerobic incubation conditions, suggesting the formation of locally protective microenvironments within the gel matrix. Conclusions: Calcium alginate beads function as controllable microenvironmental regulators that influence bacterial growth, release behavior, and long-term viability. These findings suggest that alginate-based encapsulation systems may provide a simple and scalable platform for controlled microbial cultivation and probiotic-related applications.

The vaginal microbiota contributes to mucosal immune homeostasis, yet the immunomodulatory properties of individual vaginal lactic acid bacteria (LAB) strains remain poorly characterized. In this study, eight LAB strains previously isolated from healthy Algerian women and selected based on their probiotic-related properties were evaluated for their capacity to modulate macrophage immune responses. Seven Lactiplantibacillus plantarum strains (N1, N2, N7, N11, N17, 87JG8, and 87BG2) and one Limosilactobacillus fermentum strain (70BJ2) were initially screened using porcine alveolar macrophages (PAM cells) under basal conditions and following lipopolysaccharide (LPS) challenge. Distinct strain-dependent cytokine profiles were observed. Hierarchical clustering separated the strains into two major groups: N1, N2, N7, and N11 were characterized mainly by stronger inflammatory responses, whereas N17, 87BG2, 87JG8, and 70BJ2 displayed comparatively lower inflammatory responses together with enhanced IL-6 and IL-27 expression at later time points. Based on these profiles, N17, 87JG8, and 70BJ2 were selected for further characterization in porcine intestinal macrophages (IPIM cells) and RAW 264.7 murine macrophages (RAW cells). In IPIM cells, 70BJ2 reduced LPS-induced TNF-α and IL-6 expression, while N17 enhanced IL-10 and IL-27 expression following LPS challenge. In RAW cells, N17 and 87JG8 enhanced several inflammatory and chemokine responses, whereas all three strains increased IFN-γ, IL-12, CSF3, IL-10, and IL-27, with 70BJ2 inducing the strongest IL-10 and IL-27 responses and uniquely increasing CSF2. Overall, vaginal LAB displayed distinct strain- and macrophage-dependent immunomodulatory profiles rather than uniformly suppressing or enhancing LPS-induced inflammation. Among the selected strains, Lmb. fermentum 70BJ2 exhibited particularly prominent regulatory properties across macrophage models. These findings extend the functional characterization of vaginal LAB and support their strain-level evaluation for the identification of candidate immunobiotics capable of modulating mucosal immune responses.

Lacticaseibacillus rhamnosus CRL1505 enhances antiviral immunity at mucosal sites, but its capacity to modulate liver immune responses remains unclear. Therefore, this study evaluated whether this immunomodulatory bacterium protects against Toll-like receptor 3 (TLR3)-mediated acute hepatitis induced by poly(I:C), and whether this effect depends on mucosal adhesion. BALB/c mice received the wild-type CRL1505 strain or the Δmbf CRL1505 mutant lacking the mucus-binding factor gene prior to poly(I:C) challenge. Liver injury, serum transaminases, and hepatic expression of interferons (IFNs), antiviral factors, inflammatory mediators, and regulatory cytokines were evaluated 48 h later. Poly(I:C) challenge induced acute hepatitis characterized by increased ALT/AST levels, leukocyte infiltration, and elevated hepatic IFNs and proinflammatory cytokines. The CRL1505 strain administration significantly reduced TNF-α, IL-1β, and IL-6 while enhancing IFNs, antiviral factors, and the regulatory cytokines IL-10 and IL-27, resulting in improved transaminase levels and attenuated liver damage. Notably, the Δmbf CRL1505 mutant conferred protection comparable to the wild-type strain. These findings demonstrate that L. rhamnosus CRL1505 exerts immunomodulatory and hepatoprotective effects during TLR3-driven hepatitis and that mbf-mediated adhesion is not required for this protection. Overall, CRL1505 emerges as a promising preventive strategy to enhance antiviral defenses and limit inflammation-associated liver injury.

Immunomodulatory lactic acid bacteria (immunobiotics) have been proposed to prevent bovine mastitis through the enhancement of host immune responses. Furthermore, accumulating evidence suggests that non-viable immunobiotics (postimmunobiotics) can be as effective as viable bacteria in modulating immunity in the bovine host. However, the molecular mechanisms underlying the immunomodulatory effects of heat-inactivated lactic acid bacteria in bovine mammary epithelial cells remain incompletely understood. In the present study, we evaluated the effects of heat-killed Ligilactobacillus salivarius FFIG56 (hk-FFIG56) on the Toll-like receptor (TLR)-4-mediated innate immune response in bovine mammary epithelial cells (BMECs) in vitro. BMECs were pre-stimulated with hk-FFIG56 and subsequently challenged with Escherichia coli-derived lipopolysaccharide (LPS). Transcriptomic analyses revealed that, compared with cells stimulated with LPS alone, hk-FFIG56-pretreated BMECs exhibited 743 upregulated and 881 downregulated genes, indicating substantial changes in the LPS-induced transcriptional response. Among the inflammatory mediators evaluated, hk-FFIG56 pretreatment significantly reduced LPS-induced interleukin-6 (IL-6) and interleukin-8 (IL-8) expression at 12 h, as well as CCL2 expression at 6 and 12 h, compared with cells challenged with LPS alone at the corresponding time points. Differential regulation of the negative regulators of TLR signaling, IRAK-M, Tollip, and Mitogen-activated protein kinase phosphatase 1 (MKP-1), was also detected in BMECs pretreated with hk-FFIG56. In addition, hk-FFIG56 pretreatment prevented LPS-induced reduction in IκBα protein levels, whereas no significant changes were detected in the p-ERK/ERK ratio at the evaluated time points. Furthermore, hk-FFIG56 pretreatment attenuated the LPS-induced reduction in CSN3 (κ-casein) mRNA expression in BMECs. Overall, these results suggest that hk-FFIG56 modulates LPS-induced inflammatory responses and CSN3 mRNA expression in BMECs, supporting its potential as a postimmunobiotic candidate for further investigation in the context of bovine mastitis prevention.