Ethanol-graded fractionation reveals coordinated changes in composition, chain conformation, and rheology of hawthorn...

Hawthorn pectin was separated into four fractions (HHP20-HHP80) using graded ethanol precipitation to examine how compositional changes shape molecular organization and solution behavior. As the ethanol concentration increased from 20% to 80%, the yield decreased from 2.14% to 0.11%, the weight-average molecular weight dropped from 1.09 × 10⁵ g/mol to 1.13 × 10⁴ g/mol, and the degree of esterification declined sharply from 62.16% to 0.96%, accompanied by a marked shift from GalA-dominant fractions (GalA 65.06-71.77% in HHP20-HHP40) to a neutral sugar-enriched fraction (Ara 31.06% in HHP80) and increased branching indices ((Gal+Ara)/Rha: 2.60 → 27.55). HPSEC-MALS-RI and AFM showed that all fractions adopted compact, coil-like to near-globular conformations in 0.1 M NaNO₃, with β = 0.14-0.16 (Rg-Mw scaling exponent reflecting chain conformation) and ρ = 0.04-0.10 (Rg/Rh ratio indicating molecular compactness), consistent with attenuated viscoelasticity. Rheological measurements confirmed shear-thinning, liquid-like behavior with reduced apparent viscosity, storage modulus (G'), and loss modulus (G″) at higher ethanol grades. Congo red assays revealed the strongest red-shift in HHP60, suggesting the presence of more ordered chain associations under alkaline conditions. Correlation analysis further showed that Mw, DE, GalA, and HG varied in parallel, whereas branching sugars and RG-I indices displayed opposite trends. These results elucidate how ethanol fractionation modulates structure and conformation, providing guidance for targeted functional use of hawthorn pectin.