Human ingestion of microplastics (MPs) is common and inevitable due to the widespread contamination of food items, but implications on the gastric digestion of food proteins are still unknown. In this study, the interactions between pepsin and polystyrene (PS) MPs were evaluated by investigating enzyme activity and conformation in a simulated human gastric environment in the presence or absence of PS MPs. The impact on food digestion was also assessed by monitoring the kinetics of protein hydrolysis through static in vitro gastric digestion of cow's milk contaminated with PS. The binding of pepsin to PS showed that the surface chemistry of MPs dictates binding affinity. The key contributor to pepsin adsorption seems to be π-π interactions between the aromatic residues and the PS phenyl rings. During quick exposure (10 min) of pepsin to increasing concentrations (222, 2219, 22188 particles/mL) of 10 μm PS (PS10) and 100 μm PS (PS100), total enzymatic activities were not affected remarkably. However, upon prolonged exposure at 1 and 2 h, preferential binding of pepsin to the small, low zeta-potential PS caused structural changes in the protein which led to a significant reduction of its activity. Digestion of cow's milk mixed with PS10 resulted in transient accumulation of larger peptides (10-35 kDa) and reduced bioavailability of short peptides (2-9 kDa) in the gastric phase. This, however, was only observed at extremely high PS10 concentration (0.3 mg/mL or 5.46E+05 particles/mL). The digestion of milk peptides, bound preferentially over pepsin within the hard corona on the PS10 surface, was delayed up to 15 min in comparison to bulk protein digestion. Intact caseins, otherwise rapidly digested, remained bound to PS10 in the hard corona for up to 15 min. This work presents valuable insights regarding the interaction of MPs, food proteins, and pepsin, and their dynamics during gastric digestion.

Oxidative modifications of proteins are key to many applications in biotechnology. Metal-catalyzed oxidation reactions efficiently oxidize proteins but with low selectivity, and are highly dependent on the protein surface residues to direct the reaction. Herein, we demonstrate that discrete inorganic ligands such as polyoxometalates enable an efficient and selective protein oxidative cleavage. In the presence of ascorbate (1 mM), the Cu-substituted polyoxometalate K₈[Cu²⁺(H₂O)(α₂-P₂W₁₇O₆₁)], (Cu^IIWD, 0.05 mM) selectively cleave hen egg white lysozyme under physiological conditions (pH =7.5, 37 °C) producing only four bands in the gel electropherogram (12.7, 11, 10, and 5 kDa). Liquid chromatography/mass spectrometry analysis reveals a regioselective cleavage in the vicinity of crystallographic Cu^IIWD/lysozyme interaction sites. Mechanistically, polyoxometalate is critical to position the Cu at the protein surface and limit the generation of oxidative species to the proximity of binding sites. Ultimately, this study outlines the potential of discrete, designable metal oxo clusters as catalysts for the selective modification of proteins through radical mechanisms under non-denaturing conditions.

In this study, we investigated structural aspects of covalent binding of food derived blue pigment phycocyanobilin (PCB) to bovine β-lactoglobulin (BLG), major whey protein, by spectroscopic, electrophoretic, mass spectrometry and computational methods. At physiological pH (7.2), we found that covalent pigment binding via free cysteine residue is slow (k_a = 0.065 min^-1), of moderate affinity (K_a = 4 × 10⁴ M^-1), and stereo-selective. Binding also occurs at a broad pH range and under simulated gastrointestinal conditions. Adduct formation rises with pH, and in concentrated urea (k_a = 0.101 min^-1). The BLG-PCB adduct has slightly altered secondary and tertiary protein structure, and bound PCB has higher fluorescence and more stretched conformation than free chromophore. Combination of steered molecular dynamic for disulfide exchange, non-covalent and covalent docking, favours Cys119 residue in protein calyx as target for covalent BLG-PCB adduct formation. Our results suggest that this adduct can serve as delivery system of bioactive PCB.

Infants may ingest large quantities of micro- and nanoplastics (MNP) due to the heating and sterilization of the feeding bottles, which promotes the generation and migration of MNP into the milk or milk formula. The effect of MNP on the digestion of proteins, which are crucial for the growth and development of infants remain unknown. The current study investigated the in vitro digestion of cow's milk proteins (1 mg/mL) with or without polypropylene (PP) microplastics (MP, pristine or oxidized) (20 mg/mL and 63-180 μm) in simulated gastric fluids (SGF) using an infant (pH 5.0; pepsin activity; 268 U/mL) or adult (pH 3.0; pepsin activity; 2000 U/mL) digestion model at 37 °C for 5, 30, and 120 min. Secondly, the effect of the presence of agglomerated PP nanoplastics (NP, 10, 50, and 100 μg/mL) in milk on the in vitro digestion process using the infant model of gastric digestion was investigated. The profiles of protein digestion products, soft corona and hard corona, were analyzed using SDS-PAGE, and nLC-MS/MS, respectively. Cow's milk protein digestion with or without PP-MP was significantly slower in the infant compared to the adult model, and oxidation of the PP-MP enriched some proteins in the soft and hard corona. In the presence of agglomerated PP-NP, an additional decrease in the rate of milk protein digestion was observed, especially proteins of Mw between 18 and 20 kDa, presumably allergenic β-lactoglobulin. Irrespective of the type of PP, six different types of proteins, including casein α-S1, α-S2, β and κ, β-lactoglobulin, and α-lactalbumin, were present both in the soft and hard corona. Our results indicate a direct impact of PP-MNP on the rate of milk protein digestion in the infant model of gastric digestion. Aging of PP MP through oxidation and smaller size nanoplastics exert more pronounced effects on the digestion of cow's milk proteins in vitro. This suggests that PP-MNP could affect the biological functions of milk proteins and promote chronic health problems.