Preparative high-performance liquid chromatography purification and structural characterization of xylobiose isomers...

This research focused on synthesizing xylobiose isomers via β-xylosidase reaction and developing a preparative high-performance liquid chromatography (HPLC) separation method for their purification. The reverse reaction of β-xylosidase was conducted using 6.68 M xylose substrate at 50 °C for 24 h, resulting in 18.5 % (w/w) total xylobiose yield. Three distinct product peaks were identified and purified using preparative HPLC. Each xylobiose isomer was refined to >95 % purity through two different single-recycling techniques. Final purities ranged from 85.6 % to 97.3 %, with recovery rates between 36.2 % and 75.2 %. Molecular weight and linkage structures were examined using liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. All peaks exhibited 281.09 Da, corresponding to disaccharides linked by β-1,3-, β-1,2-, and β-1,4-glycosidic bonds. In simulated digestion, these xylobiose isomers remained intact and stimulated growth of Bifidobacterium strains. Production via single enzyme followed by preparative HPLC purification significantly enhances research on linkage-specific prebiotic effects.