Investigating the impact of common migration substances found in milk packaging on proteases: A multispectral and molecular docking approach

• Published in: Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy Vol. 318; p. 124517
• Main Authors: Xiong, Zijian, He, Yue, Guan, Weiyan, Lv, Xinguang, Chen, Jing, Ma, Da
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 05-10-2024
• Subjects: Animals; Circular Dichroism; Digestive protease; Food Packaging; Inhibitory kinetics; Migrations; Milk - chemistry; Milk packaging; Molecular docking; Molecular Docking Simulation; Pepsin A - chemistry; Pepsin A - metabolism; Peptide Hydrolases - chemistry; Peptide Hydrolases - metabolism; Spectrometry, Fluorescence; Stearic Acids - chemistry; Stearic Acids - metabolism; Thermodynamics; Trypsin - chemistry; Trypsin - metabolism; Inhibitory kinetics; Digestive protease; Milk packaging; Molecular docking; Migrations
• ISSN: 1386-1425; 1873-3557
• DOI: 10.1016/j.saa.2024.124517

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•Migration substances from milk packaging (stearic acid and stearamide) can reduce the activity of proteases.•500 μg/mL stearic acid and stearamide decreases the activity of gastric protease by 18 %.•The inhibitory effect of stearic acid and stearamide on gastric protease is non-competitive.•The binding of stearic acid and stearamide to proteases induces conformational changes in the enzyme.
The effects of common migration substances in milk packaging on digestive protease were studied. We choose the common migrants found in eight types of multi-layer composite milk packaging. Enzyme activity experiments revealed that pepsin activity decreased by approximately 18 % at 500 μg/mL of stearic acid and stearamide treatment, while trypsin activity decreased by approximately 18 % only by stearic acid treatment (500 μg/mL). Subsequently, fluorescence spectroscopy, circular dichroism spectroscopy, and molecular docking technology were employed to investigate the inhibition mechanism of protease activity by migrating substances in three systems: stearic acid-trypsin, stearic acid-pepsin, and stearamide-pepsin. Results showed that the inhibitory effect of stearic acid on trypsin is a reversible mixed inhibition, whereas the inhibitory effects of stearic acid and stearamide on pepsin are non-competitive. In all three systems, ΔH < 0, ΔS < 0, and ΔG < 0, indicating the binding process between the migrant and the protease is a spontaneous exothermic process primarily driven by hydrogen bonding and van der Waals forces. In addition, their binding constants are all around 104 L/moL, indicating that there are moderate binding affinities exist between migrants and proteases. The binding process results in the quenching of the protease's endogenous fluorescence and induces alterations in the enzyme's secondary structure. Synchronized fluorescence spectroscopy showed that stearic acid enhanced the hydrophobicity near the Tyr residue of trypsin. The molecular docking results indicated that the binding affinity of stearic acid-trypsin, stearic acid-pepsin, and stearamide-pepsin was –22.51 kJ/mol, −12.35 kJ/mol, −19.28 kJ/mol respectively, which consistent with the trend in the enzyme activity results. This study can provide references for the selection of milk packaging materials and the use of processing additives, ensuring food health and safety.