Anti-Inflammatory Effect of Excretion-Secretion Products of Clinostomum marginatum (Digenea: Clinostomidae) and Its Effect over the Viability and Antioxidative Activity of a Mix of Lactobacillus and/or Bifidobacterium

The trematode <i>Clinostomum marginatum</i>, secretes excretory-secretory products (ESPs) which have the potential to increase the viability and antioxidant activity of probiotic strains. The aim of this study was to identify the ESP profile of <i>C. marginatum</i> and to evaluate its anti-inflammatory activity in RAW 264.7 macrophages, as well as its effect on the viability and antioxidant activity of a consortium of bacteria comprising <i>Lactobacillus</i> and/or <i>Bifidobacterium</i>. <i>C. marginatum</i> was maintained in RPMI-1640 medium for ESP collection. Anti-inflammatory activity was assessed in LPS-stimulated RAW264.7 cells treated with 800 µg/mL of ESPs, measuring cell viability, nitric oxide production, and the relative expression of pro-inflammatory cytokines (IL-6, TNF-α, INF-γ) and the COX-2 gene by qPCR. The influence of ESPs (800-1600 µg/mL) on probiotic viability and antioxidant activity was determined using MTT, DPPH, hydroxyl, and superoxide radical scavenging assays. <i>C. marginatum</i> showed 74% survival in vitro, and SDS-PAGE analysis revealed three major protein bands in the ESPs (47, 54, and 58 kDa). ESP treatment significantly reduced nitric oxide and the mRNA expression of pro-inflammatory markers in LPS-activated macrophages. ESPs supplemented at 1200 µg/mL optimized the growth kinetics of <i>Lactobacillus</i> (specific growth rate <i>μ_L</i> = 1.12 h^-1, doubling time <i>t_d</i> = 0.62 h) and <i>Bifidobacterium</i> (<i>μ_B</i> = 1.09 h^-1, <i>t_d</i> = 0.63 h) compared to control conditions. In conclusion, ESPs from <i>C. marginatum</i> exhibited significant anti-inflammatory and antioxidant effects while enhancing bacterial viability, which positions them as promising candidates for biotherapeutics agents in the management of inflammatory control and gut microbiota modulation.