Melanoidins (MLDs), the final products of the Maillard reaction, are high molecular-weight polymers formed during the processing of black garlic. These compounds contribute to the distinctive color, flavor, and bioactive properties of black garlic. However, the impact of storage conditions on the structural integrity and bioactivities of black garlic melanoidins (BG-MLDs) remains largely unexplored. By using various in vitro simulations, this study investigated the effects of different storage conditions (at 4 and 35 ℃ for 0, 3, and 6 months) on the digestion, absorption, metabolism, and intestinal microbial profile of BG-MLDs during in vitro gastrointestinal digestion. Our results demonstrated that BG-MLDs resisted digestion regardless of storage conditions. BG-MLDs stored at 4 ℃ for 3 months significantly enriched the abundance of beneficial bacteria like Bifidobacterium longum and decreased harmful bacteria like Escherichia. Additionally, MLD treatment significantly increased the production of short-chain fatty acids (SCFAs), particularly propionate, butyrate, and isovalerate, with the 35 ℃ for 3 months of storage showing the most significant effect (increased by 3.74-fold). However, a longer storage at 35 ℃ (6 months) showed a decline in SCFA production. These findings highlight the critical role of a certain storage time (3 months) in preserving and potentially enhancing the prebiotic activity of BG-MLDs. By optimizing storage parameters, the bioactive potential of BG-MLDs can be used for their potential application as functional food ingredients or nutraceuticals, contributing to the prevention and management of chronic diseases associated with gut dysbiosis.

In this study, we systematically investigated the regulatory effects of two hempseed peptides (APAM and RLPA) on pancreatic lipase (PL) and cholesterol esterase (CE) activities and the molecular binding mechanism using enzyme kinetics, synergistic inhibition, fluorescence spectroscopy, isothermal titration calorimetry and molecular docking. The results showed that RLPA had stronger inhibitory activity on PL and CE with half maximal inhibitory concentration (IC₅₀) values of (79.62 ± 3.20) and (301.27 ± 14.40) μmol/L, respectively. Kinetic analysis indicated that the inhibition mechanism of APAM on the two enzymes was competitive inhibition, whereas RLPA was a mixed-type inhibitor. Both APAM and RLPA synergistically inhibited PL and CE with orlistat at low concentrations, but the synergistic effect was weakened or even antagonized at high concentrations. Fluorescence spectroscopy results showed that both peptides could produce static quenching by binding to PL and CE, which changed the hydrophobic environment of aliphatic amino acids in the enzymes. Isothermal titration calorimetry demonstrated that the binding process between the peptides and the enzymes was spontaneous and exothermic, which was achieved mainly through hydrogen bonding and electrostatic interaction. Further analysis using molecular docking simulations showed that hydrogen bonding, salt bridges, and cation-π interactions played crucial roles in peptide-enzyme binding. This study enriches our understanding of the interaction mechanism of peptides with PL and CE, and provides a scientific basis for the development of functional foods based on hempseed protein.