Structure and dual enzymatic resistance mechanisms of ethanol-precipitated A-type crystalline resistant starch

Resistant starch is recognized for its capacity to modulate postprandial glycemia and promote gut health, yet the molecular basis of its enzymatic resistance remains poorly understood. In this study, highly ordered A-type crystalline RS3 was prepared from debranched waxy corn starch using an ethanol-assisted recrystallization approach, and its structure-digestibility relationship was systematically investigated. The obtained RS3 exhibited a high resistant starch content (65.62%), significantly higher than the control (25.68%), confirming the formation of a compact, enzyme-resistant crystalline framework. Multi-scale characterization, including scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC), revealed that the A-type crystals consisted of dense crystalline structures with well-aligned double helices that remained intact during enzymatic hydrolysis. XPS results suggested that digestive enzymes were non-catalytically adsorbed on the crystal surface, forming an enzyme adsorption layer that hindered substrate recognition and catalytic access. Collectively, these findings provide supporting evidence that the A-type crystalline structure resists enzymatic attack through a dual mechanism involving physical shielding and molecular recognition barriers, providing mechanistic insight into RS3 digestion resistance and guidance for designing starches with controlled digestibility and low glycemic response.