Mild ultrasound-assisted alkali de-esterification modified pectins: Characterization and structure–activity relationships in immunomodulatory effects

Apple pectin (AP), a well-established dietary fiber, offers significant health benefits, particularly in immunomodulation. However, the structure-activity relationship (SAR) in this context remains poorly understood. This study aimed to elucidate the impact of varying degrees of esterification (DE) on AP's SAR in immunomodulatory activity. AP-Es (AP-E1, AP-E2, AP-E3) with different DE were prepared using mild ultrasound-assisted alkali de-esterification, followed by SAR analysis. Results revealed that AP-E3, with the lowest DE (5.08 ± 0.22 %), demonstrated a significant reduction in homogalacturonan (HG) domains and a corresponding increase in rhamnogalacturonan-I (RG-I) domains, which coincided with enhanced immunomodulatory effects. The molecular weights of AP-E1, AP-E2, and AP-E3 were determined to be 30.94 ± 0.83 kDa, 27.61 ± 0.65 kDa, and 22.17 ± 0.57 kDa, respectively. To further explore the underlying mechanism, transgenic zebrafish with fluorescent macrophages were utilized. A positive correlation was observed between AP-E3 concentration and the number of fluorescent microspheres engulfed by macrophages. Additionally, AP-E3 significantly upregulated the expression of key immune response genes (tnf-α, il-1β, il-6, cox-2, inos, and nf-κb) and restored the gut microbiota composition and abundance in chloramphenicol-induced immunocompromised zebrafish. Metabolomics analysis revealed that AP-E3 effectively restored metabolic homeostasis by activating multiple signaling pathways associated with signal transduction, immune regulation, and metabolism. These findings highlight the potential of low-esterified AP enriched with RG-I domains as a promising candidate for applications in immune modulation and gut health management.