Response surface analysis and anti-inflammatory activity of a potential postbiotic exopolysaccharide produced by probiotic Lacticaseibacillus paracasei K56

Exopolysaccharides (EPSs) produced by lactic acid bacteria (LAB) have been extensively studied, but the potential postbiotic activities of the EPSs are not well characterized. A probiotic strain of Lacticaseibacillus paracasei K56 previously characterized by strong postbiotic activities was investigated by response surface analysis for production of an EPS, designated K56-EPS, and by testing EPS for anti-inflammatory effects in a LPS-induced RAW264.7 macrophage inflammation model. The optimal protocol for EPS production (76.27 mg/L) in skim milk employed 8.7 % ( w /w) inoculum for 34.2 h fermentation at 33.9 °C, followed by extraction with 6 % ( w / v ) TCA and treatment of the culture supernatant with 4 volumes of ethanol at 4 °C for 24 h. The chromatographically purified K56-EPS had a monosaccharide composition of rhamnose, glucosamine hydrochloride, galactose, and glucose in a molar ratio of 0.387:0.053:0.188:0.371, with a molecular weight of 3.39 × 10 5 Da. Furthermore, K56-EPS (200 μg/mL) effectively suppressed expression of key genes within the TLR4/ NF-κB signaling pathway and inhibited the nuclear translocation of the NF-κB p65 protein to interrupt transcriptional activation of inflammatory mediators. Higher dose of K56-EPS (400 μg/mL) was shown to significantly attenuate phagocytosis, reduce acid phosphatase (ACP) activity, decrease nitric oxide (NO) release, and downregulate TNF-α, IL-6, IL-1β, and IL-10 cytokine levels ( p < 0.05). Therefore, K56-EPS demonstrated prominent in vitro anti-inflammatory activity with significance as a potential novel postbiotic of LAB for applications in functional dairy foods. • Optimal EPS production by strain K56 with 8.7 % inoculum at 33.9 °C for 34.2 h. • The EPS was extracted with 6 % TCA and 4:1 ethanol/supernatant treated for 24 h. • K56-EPS consisted of rhamnose, glucosamine, galactose, and glucose. • K56-EPS exhibited potent macrophage anti-inflammatory activity. • K56-EPS suppressed TLR4/NF-κB pathway and NF-κB p65 nuclear translocation.