Frost-induced structural modification of mulberry leaf polysaccharides enhances hypoglycemic activity and Maillard reaction inhibition in biscuits

This study established an efficient protocol involving ultrasound-assisted deep eutectic solvent extraction and chromatographic purification to obtain two high-purity polysaccharide fractions-MLP-3-1 from standard mulberry leaves and FMLP-3-1 from post-frost leaves. Frost treatment significantly altered the structure of FMLP-3-1, increasing its molecular weight, total sugar content, uronic acids (notably mannuronic acid), and structural order. Correspondingly, FMLP-3-1 demonstrated superior in vitro hypoglycemic activity, including stronger inhibition of α-amylase/α-glucosidase and improved glucose metabolism in insulin-resistant HepG2 cells. Molecular docking supported the role for mannuronic acid in enzyme inhibition. Furthermore, FMLP effectively suppressed the formation of Maillard reaction products (5-HMF and AGEs) and modulated volatile flavor profiles in biscuit baking. These findings indicate that frost treatment beneficially modifies mulberry leaf polysaccharides, boosting their hypoglycemic potential and suitability as functional food ingredients for reducing harmful compounds during thermal processing.