Differential mechanism of casein phosphopeptide-quercetin covalent/non-covalent complex on gastrointestinal response behavior

Casein phosphopeptide-quercetin (CPP-QR) covalent and non-covalent complex were constructed, and the activity changes and difference mechanisms of these two complexes were explored after gastrointestinal digestion using dynamic simulation digestion and molecular dynamic (MD) simulation. Compared with CPP, the soluble calcium binding capacity of CPP-QR covalent and non-covalent complex increased by 44.35% and 34.57% respectively, and the DPPH· scavenging rate increased by 77.97% and 25.46% respectively after static simulation digestion. Thereinto, the activity of CPP-QR covalent complex was significantly better than that of CPP-QR non-covalent complex (P < 0.05). Under acidic conditions, the DPPH· scavenging rate of CPP-QR covalent and non-covalent complexes was higher than that of CPP (P < 0.05). While the antioxidant activities of two complexes decreased significantly at pH=8, they still exhibited markedly superior performance relative to CPP alone (P < 0.05). The CPP-QR covalent and non-covalent complex exhibited 66.72% and 70.72% higher inhibition rates than CPP against pepsin, and 83.85% and 46.60% higher inhibition against trypsin, respectively. MD simulation had revealed that the binding free energies were -60.71 kcal/mol for the non-covalent complex in the pepsin system versus -84.21 kcal/mol for the covalent complex in the trypsin system. The CPP-QR non-covalent complex has exhibited minimal radius of gyration (Rg) and root-mean-square deviation (RMSD) in pepsin systems, while the CPP-QR covalent complex had demonstrated superior stability with correspondingly minimal Rg and RMSD in trypsin systems. In conclusion, the soluble calcium binding capacity and DPPH· scavenging rate of the CPP-QR covalent complex were superior to those of the non-covalent complex and CPP. CPP-QR covalent complexes had higher stability in the intestinal phase due to their resistance to trypsin, while non-covalent complexes maintain higher activity during gastric digestion due to their resistance to pepsin and acid adaptation.