Thermal treatment of alkali lignin to eliminate its inhibition of pancreatic proteases in vitro

This study aims to investigate how alkali lignin inhibits protein digestion and explore thermal treatment as a potential solution. Solid alkali lignin species pre-heated at different temperatures (150, 200, and 250 °C) and soluble acid-differentiated fractions are subjected to in vitro protein digestion. A range of techniques, including Thermogravimetric Analysis (TGA), Size-Exclusion Chromatography (SEC), Zeta Potential Analyzer, 1H NMR, Isothermal Titration Calorimetry (ITC), and Molecular Docking, were used to investigate the inhibitory mechanism of alkali lignin on pancreatic proteases hydrolysis. Our results suggest that soluble alkali lignin inhibits pancreatic trypsin and chymotrypsin, with the acid-differentiated soluble fraction (LgpH<1) displaying the strongest inhibition and proteases' binding affinity due to the abundance of polar groups (e.g., -OH, -CHO), which facilitate hydrogen-bond formation. Furthermore, pre-heating lignin (200 °C) was confirmed effective for removing LgpH<1 and its negative nutritional influence, providing a feasible strategy for overcoming the negative impact of alkali lignin on protein digestion.