Probiotics exert antimicrobial activity through both direct inhibition of pathogens and ecological competition. Besides producing antimicrobial metabolites such as organic acids, bacteriocins, and hydrogen peroxide, probiotics can disrupt adhesion and invasion mechanisms in pathogens. Lactobacillus spp. were shown to significantly reduce Campylobacter jejuni adhesion and translocation in epithelial models, suggesting interference with pathogen entry and dissemination (Šikić Pogačar et al., 2020). Similarly, Bifidobacterium strains utilize host-derived glycans and express mucus-binding proteins that facilitate colonization and inhibit pathogens such as Clostridium difficile (O’Callaghan and Van Sinderen, 2016). L. plantarum ATCC 14917 showed strong anti-biofilm and antibacterial activity against multiple Listeria monocytogenes isolates, inhibiting planktonic growth and disrupting mature biofilms (Abou Elez et al., 2023).

Significantly, probiotics can interfere with quorum-sensing (QS) networks and biofilm formation, which are critical for multidrug resistance. Bacillus subtilis-derived lipopeptides suppress the S. aureus Agr system, reducing toxin production and enhancing antibiotic susceptibility (Piewngam et al., 2018; Leistikow et al., 2024). Similarly, metabolites from L. plantarum and L. rhamnosus downregulate QS-regulated virulence factors such as pyocyanin and elastase in Pseudomonas aeruginosa, weakening biofilm structure and facilitating host clearance (Zhang et al., 2025; Chappell and Nair, 2020),. Probiotic metabolites can also downregulate MRSA virulence and enhance β-lactam efficacy by disrupting QS, highlighting their potential as adjuncts to conventional antibiotics for biofilm-associated infections (Cella et al., 2023). These combined mechanisms illustrate the multifaceted ecological strategies through which probiotics counteract pathogen persistence and resistance.

Bacteriocins are ribosomally synthesized antimicrobial peptides that disrupt the membranes of competing bacteria, leading to ion and ATP leakage and eventual cell death (Demirgül et al., 2025). Reuterin, an antimicrobial aldehyde produced by L. reuteri from glycerol metabolism, inhibits pathogens such as E. coli, Salmonella, and Shigella by inducing oxidative stress and inhibiting DNA synthesis (Yang et al., 2024). Pediococcus acidilactici produces pediocin PA-1, a well-characterized bacteriocin with strong antilisterial and anti-Salmonella activity (Todorov et al., 2023). Enterocins from Enterococcus spp. display potent inhibitory effects against MRSA, particularly when combined with conventional antibiotics (Hanchi et al., 2017). Certain strains of Bifidobacterium longum, particularly the subsp. infantis has been found to contain multiple genetic clusters encoding bacteriocins, indicating the potential of this species to enhance probiotic antimicrobial activity (Yu et al., 2023). Newer analyses corroborate these modes of action, demonstrating that Lactobacillus bacteriocins compromise cell-envelope integrity and macromolecular synthesis, and identifying candidates with activity against resistant Staphylococcus strains (Kranjec et al., 2025; Chen et al., 2025). In parallel, mechanistic studies of reuterin confirm thiol-reactivity, membrane damage, and oxidative stress responses in target bacteria (Yang et al., 2024; Sun M-C. et al., 2023).

Probiotics and pathogens compete for essential nutrients such as iron, amino acids, and vitamins. By rapidly utilizing available substrates, probiotics limit the resources available to pathogen growth. L. rhamnosus GG effectively competes with E. coli and Salmonella Typhimurium for epithelial adhesion sites, reducing colonization (Lee and Puong, 2002). Bifidobacterium species possess specialized enzymatic systems that enable the utilization of host-derived mucin glycans, providing a competitive advantage for intestinal colonization and limiting pathogen persistence (Turroni et al., 2016). Certain Lactobacillus strains also sequester iron, further hindering pathogen proliferation (Sassone-Corsi and Raffatellu, 2015). Competition-mediated exclusion is especially relevant in dysbiotic or antibiotic-perturbed ecosystems. By restoring resource landscapes and adhesion-site occupancy, probiotics reduce the probability that MDR Enterobacteriaceae and non-fermenters establish durable niches.

Organic acids and SCFAs produced by probiotics are critical mediators of pathogen inhibition. Lactic acid penetrates bacterial membranes, lowering intracellular pH and disrupting metabolism. L. acidophilus and L. plantarum reduce the growth of E. coli and Salmonella enterica through lactic acid production (Alakomi et al., 2000). Acetate from Bifidobacterium longum protects epithelial cells from E. coli O157:H7 by strengthening tight junctions and reducing translocation (Fukuda et al., 2011). Butyrate and propionate, generated by probiotic consortia, induce intracellular acidification and inhibit DNA replication in pathogens (Louis and Flint, 2017). SCFAs also act as signaling molecules, modulating immune responses and promoting the differentiation of regulatory T cells (Tregs). Recent reviews and translational studies reinforce the link between probiotic-boosted SCFAs, barrier repair, and inflammation control, effects that are clinically relevant to infection susceptibility and to resilience during antibiotic treatment (Li et al., 2025; Zhu and Lee, 2025).

Hydrogen peroxide (H₂O₂) production by Lactobacillus species provides an oxygen-dependent antimicrobial mechanism, particularly important in mucosal niches. H₂O₂-producing L. crispatus and L. jensenii inhibit Gardnerella vaginalis and Candida albicans, reducing risks of bacterial vaginosis and yeast infections (O’Hanlon et al., 2011). Although the intestinal lumen is largely anaerobic, transient oxygen-rich microenvironments may still permit probiotic H₂O₂ generation, contributing to mucosal defense (Aldunate et al., 2015). Updated microbiome analyses continue to highlight H₂O₂ production as a competitive trait among vaginal lactobacilli, which contributes to pathogen suppression and community stability (Nori et al., 2025).

Beyond direct antimicrobial actions, probiotics reinforce mucosal defenses and regulate immune homeostasis. Several Lactobacillus and Bifidobacterium strains enhance epithelial integrity by upregulating tight-junction (TJ) proteins (occludin, claudins) and stimulating mucin secretion, thereby preventing pathogen adhesion and translocation (Anderson et al., 2010; Segers and Lebeer, 2014). Soluble proteins p40 and p75 from L. rhamnosus GG activate the epidermal growth-factor receptor (EGFR) pathway, promoting epithelial survival and protection against cytokine-induced apoptosis (Yan et al., 2007; Christensen et al., 2002).

At the immune interface, structural components such as peptidoglycan and lipoteichoic acid are recognized by Toll-like receptor 2 (TLR2) and NOD-like receptors, leading to NF-κB and MAPK activation and the release of defensins and anti-inflammatory cytokines (De LeBlanc et al., 2007; Lebeer et al., 2010). Metabolites, including short-chain fatty acids and tryptophan-derived indoles, act as signaling molecules activating the aryl hydrocarbon receptor (AhR) and pregnane X receptor (PXR), which attenuate inflammation and promote regulatory T-cell differentiation (Claes et al., 2012; Tanoue et al., 2016; Fanning et al., 2012). Together, these mechanisms highlight how probiotics contribute to epithelial protection, mucosal tolerance, and balanced immune responses.

The combined actions of these antimicrobial metabolites and structural components are summarized in Figure 1, which integrates the principal pathways through which probiotics exert both antimicrobial and immunoregulatory effects.

The vulnerabilities of specific probiotic strains, including acid sensitivity, oxygen intolerance, and other biological constraints, highlight the need for protective delivery systems. These systems not only preserve probiotic viability but also maintain their functional activity at the target site. Consequently, it is crucial to select an appropriate encapsulation strategy that accounts for each strain’s unique characteristics.

Because many probiotic antimicrobial mechanisms, including bacteriocin secretion and SCFA production, are highly sensitive to gastric acidity and bile exposure, natural biopolymer matrices such as alginate, pectin, and chitosan are primarily selected for their ability to buffer pH, preserve membrane integrity, and maintain metabolic activity during gastrointestinal transit.

Biopolymers serve as biocompatible, biodegradable matrices that protect probiotic cells during processing, storage, and gastrointestinal (GI) transit. Among these, sodium alginate is particularly prominent owing to its mild gelation and ability to form stable hydrogels via ionic cross-linking. In alginate, the guluronic acid blocks preferentially bind divalent calcium ions, yielding a three-dimensional “egg-box” structure that traps viable cells in a semipermeable, water-rich network. It permits metabolite diffusion while shielding the entrapped microorganisms from exposure to acids or bile (Colin et al., 2024; Tordi et al., 2025).

For strains whose antimicrobial activity depends on sustained bacteriocin release, oxygen sensitivity, or site-specific intestinal delivery, semi-synthetic and synthetic polymers such as PLGA, Eudragit^®, and PVA-based systems offer tunable release kinetics, oxygen-barrier properties, and enteric protection that directly align encapsulation design with probiotic functional mechanisms.

Semi-synthetic and synthetic polymers extend the scope of probiotic encapsulation beyond the structural and chemical limitations of natural biopolymers by providing tunable mechanical strength, precise release control, and enhanced resistance to acidic and enzymatic degradation. Hybrid formulations that combine natural and synthetic components bridge biocompatibility with improved physicochemical stability (Sun Q. et al., 2023; Vijayaram et al., 2024). Polyvinyl alcohol (PVA)–alginate hybrids and other PVA-based fibers enhance the robustness of probiotics under acidic/GI) conditions, while encapsulation can preserve antimicrobial (bacteriocin) activity. Together, these findings support semi-synthetic reinforcement for probiotic protection (Ilomuanya et al., 2024; Yilmaz et al., 2020).

Poly(lactic-co-glycolic acid) (PLGA) is among the most widely used synthetic polymers for enteric and colon-targeted probiotic delivery owing to its biodegradability and regulatory approval. PLGA micro- and nanoparticles effectively protect encapsulated cells from gastric acidity and enable pH-responsive release in the intestine (Wang et al., 2024). Multilayer PLGA–pullulan–PLGA electrospun fibers carrying L. rhamnosus GG preserved viability during gastrointestinal simulation and supported intestinal colonization in vivo (Ajalloueian et al., 2022). PLGA carriers improve acid tolerance and enable controlled intestinal release of L. plantarum and L. rhamnosus, though solvent-based fabrication can reduce viability (Sun Q. et al., 2023).

Beyond PLGA and PVA, fully synthetic polymers have also been used for controlled probiotic release. Methacrylate copolymers such as Eudragit^® provide reliable enteric protection by remaining intact under gastric acidity and dissolving at intestinal pH, enabling site-specific delivery of Lactobacilli and Bifidobacteria. For example, enteric-coated capsules with Eudragit L100–55 did not disintegrate in simulated gastric fluid and were designed for release in the intestine, confirming the suitability of methacrylate coatings for oral probiotic delivery (Fredua-Agyeman, 2024). Microencapsulation with Eudragit S100 has likewise maintained viable probiotic counts above 6 log CFU mL⁻¹ after simulated gastric exposure, further evidencing gastric protection and downstream release (Akanny et al., 2020). In contrast, specific PEG–PVP multilayer carriers with quantified probiotic benefits are sparsely documented. Recent work instead supports PEG-containing synthetic matrices, such as PCL-PEG-PCL coatings in dairy systems, which improved the stability of formulated probiotics during storage and processing (Bayat et al., 2024). Contemporary reviews concur that PEG and PVP are explored as moisture-stabilizing, fully synthetic excipients within probiotic encapsulation toolkits, but emphasize that detailed strain-level performance still depends on formulation and process variables (Agriopoulou et al., 2024; Sun Q. et al., 2023). Probiotics microencapsulated in a polyacrylate resin matrix exhibited superior gut-protective effects in C57BL/6 mice compared with non-encapsulated controls. In the study by Gyawali et al., mice were fed encapsulated Lactobacillus paracasei for four weeks, which significantly increased mucin production and secretory immunoglobulin A, thereby enhancing intestinal health and favorably modulating the microbiota composition (Gyawali et al., 2023). In a related approach, L. rhamnosus was encapsulated in Pullulan nanofibers, with two electrospun PLGA layers covering it. The study showed that LGG, delivered by a construct, was able to survive and recover from all segments of the simulated gastrointestinal (Ajalloueian et al., 2022). Together, these studies highlight that synthetic and hybrid polymeric matrices can complement natural biopolymers by offering controlled release profiles, enhanced stability, and synergistic prebiotic–probiotic functionality for intestinal health applications.