Identification of exopolysaccharides as the principal immunomodulatory components of <scp> <i>Lactobacillus helveticus</i> </scp> postbiotics and potential targets for quality control

Abstract BACKGROUND As a novel immunomodulatory strategy alternative to live bacterial agents, postbiotics remain incompletely understood with respect to their principal functional components and mechanisms of action. This study isolated and identified the active ingredients – namely, exopolysaccharides (EPS) and bacteriocins – from the crude postbiotics (CP) of Lactobacillus helveticus KLDS 1.8701, and investigated their immunomodulatory and antioxidant properties and potential underlying mechanisms of action. RESULTS Characterization showed that the purified exopolysaccharide (PEPS) had a weight‐average molecular weight of 134.6 kDa and an ordered triple‐helical conformation. Functional comparison revealed that PEPS played a dominant role in bidirectional immunomodulation, significantly outperforming CP in promoting the proliferation of normal macrophages (viability reaching 230.61% at 5 mg mL −1 ) and inhibiting the abnormal proliferation of inflammatory cells ( P &lt; 0.05). Moreover, PEPS exhibited superior antioxidant capacity and significantly increased antioxidant enzyme activity in inflammatory cells ( P &lt; 0.05). In contrast, high concentrations of purified bacteriocin (PB) (&gt; 1.25 mg mL −1 ) were cytotoxic, and its combination with PEPS did not yield a synergistic immunomodulatory effect. Intervention with the reactive oxygen species (ROS) inhibitor N ‐acetylcysteine (NAC) further confirmed that PEPS inhibited the activation of the TLR4/NF‐ κ B pathway by efficiently scavenging ROS. This anti‐inflammatory effect was equivalent to that of the NAC group and significantly superior to that of the CP group ( P &lt; 0.05), whereas CP showed no compensatory effect via alternative pathways after ROS blockade. CONCLUSION This study identified and verified PEPS as the principal immunomodulatory component of the postbiotics of L. helveticus , providing a theoretical foundation for the development of standardized preparations using PEPS as a quality control target. © 2026 Society of Chemical Industry.