Preparation, Structural Elucidation, and In Silico Prebiotic Evaluation of Pectin Oligosaccharides from Apple Pectin

Pectin oligosaccharides (POS) recovered from agro-industrial by-products are attracting attention as candidate prebiotics, yet obtaining structurally defined neutral POS and screening their activity remains challenging. This study prepared neutral POS from commercial apple pectin by controlled acid hydrolysis (0.05 M TFA, 80 °C, 1 h), ethanol precipitation, ultrafiltration (<3 kDa), and DEAE-Sepharose CL-6B chromatography, yielding a neutral P-1 fraction (3.6% w/w). The fraction was characterized by PMP-HPLC, HPSEC-RID, and UPLC-HILIC-MS/MS, revealing a bulk composition dominated by galactose, glucose, and arabinose, with five selected neutral oligosaccharides (DP 2–5). The prebiotic potential of these five selected, tentatively assigned oligosaccharides was evaluated in silico by simulation against 13 microbial glycosidases and by simulating colonic fermentation with CompuGUT using kinetic parameters derived from binding energies. The docking matrix showed favorable and enzyme-selective binding (−10.3 to −4.6 kcal/mol), with the strongest complexes involving component D–A7LXS9 (−8.9 kcal/mol) and component E–Q93GI5 (−10.3 kcal/mol). CompuGUT predicted complete hydrolysis of all five components within 48 h and increased mucosal biomass, indicating that these five selected compounds are predicted to be fermentable and to support microbial growth at the mucosal surface. These findings provide an integrated strategy for the preparation, structural characterization, and rapid functional screening of neutral POS, facilitating the identification of structurally defined pectin-derived prebiotic candidates.