Effect of pectinase addition in juice processing on the structural characteristics, immunological activity and <i>in vitro</i> and <i>in vivo</i> prebiotic properties of apple pomace pectic polysaccharides

Apple pomace is a waste in fruit juice processing and is an important raw material for pectin extraction. The addition of pectinase will not only change the juice characteristics but also affect the apple pomace. However, the differences in the structure and function of pectin obtained from the pectinase-treated (TAPP) and untreated apple pomace (NTAPP) are unclear. In this paper, TAPP and NTAPP (APPs) were prepared using the subcritical-water method. Structural analysis showed that the APPs were acidic-pectin polysaccharides with low molecular weight (<i>M</i>_w) and a high esterification degree (DE), but TAPP had a lower <i>M</i>_w, DE, and galacturonic acid content and smoother surface. Immune activity detection demonstrated that NTAPP can stimulate macrophage proliferation, phagocytosis, and cytokine release by activating the TLR4/p-ERK/p-NFκB pathway, while TAPP activates the TLR4/p-NFκB to stimulate macrophage phagocytosis and the cytokine release. <i>In vitro</i> fermentation characteristics indicate that anaerobic fermentation using APPs as the sole carbon source can significantly promote the production of lactic acid and the short-chain fatty acids (SCFAs). Microbial diversity analysis revealed that the APPs exhibit prebiotic properties, but their effects on the gut microbiota composition differ: TAPP mainly promotes the enrichment of <i>Akkermansia</i>, while NTAPP primarily enhances the abundance of <i>Faecalibaculum</i> and <i>Dubosiella</i>. Finally, structure-function correlation analysis suggests that monosaccharide composition (particularly mannose) and molecular weight (<i>M</i>_w) are key factors influencing the gut microbiota composition, providing a research direction for future studies on their structure-activity relationships.