Characterization of a co-fermented citrus pectin preparation: structural and compositional changes, in vitro bioactivities, and antioxidant-related responses in Caenorhabditis elegans

Co-fermentation yields assay-dependent improvements in in vitro bioactivities. • Apparent peak molecular weight shifts from 113.6 to 31.9 kDa. • CRP-LrLl elevates basal antioxidant enzymes in C. elegans . • Multi-omics suggests coordinated proteostasis, lipid, and redox responses. A co-fermented citrus pectin preparation (CRP-LrLl) was characterized for structural, functional, and organism-level properties across multiple experimental levels. CRP systems were incubated for 48 h without inoculation or with single strains or a mixed culture; in the mixed system, Lacticaseibacillus rhamnosus and Lactococcus lactis reached 8.26 and 8.79 log CFU/mL, respectively, and pH decreased from 6.53 to 3.95. The co-fermented preparation (CRP-LrLl) exhibited a balanced in vitro functional profile, characterized by improved ABTS and hydroxyl radical scavenging, strong inhibition of carbohydrate-hydrolyzing enzymes, substantial pancreatic lipase inhibition, and the highest apparent bile salt binding, although the magnitude of improvement was assay-dependent. Following co-fermentation, the apparent peak molecular weight (Mp) of the predominant RI peak shifted from 113.6 to 31.9 kDa, the relative galacturonic acid (GalA) proportion increased from 38.98% to 44.40%, and the degree of esterification and methoxyl content decreased. In Caenorhabditis elegans under non-stressed conditions, CRP-LrLl elevated glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD), and catalase (CAT) activities relative to CRP administered at the same nominal preparation concentration. Exploratory integrated transcriptomic and metabolomic analyses characterized the molecular response of CRP-LrLl-treated worms relative to untreated controls, suggesting coordinated changes in proteostasis, lipid metabolism, and redox regulation rather than activation of a single canonical pathway. These findings describe associations among co-fermentation, structural remodeling of the pectin-containing matrix, and host responses to a cell-free fermentation-derived preparation. Because the preparation also contains soluble fermentation-derived constituents, causal attribution to individual components requires further fractionation and validation.