Analysis of microplastic (MP) occurrence in commercially relevant species is a prerequisite for food risk assessment. Using a standardized methodology, we aimed to investigate MP contamination in point-of-sale clams, mussels, and Crustacea shellfish collected from various markets (Belgium, Croatia, Serbia, and South Korea). An improved digestion protocol yielded ≥ 99.8% digestion efficiency for all species analyzed. In a total of 190 samples analyzed individually by microFTIR, MPs were identified in 43.68% of the samples with less than 1 MP/individual average (0–4 MP/individual, 0–1.35 MPs/g tissue). Significant differences between species were observed when considering samples contaminated with MPs, with Crustacea shellfish having the lowest MPs/g of edible tissue. Polystyrene, polypropylene, and polyethylene were dominant MPs found in clams and mussels, while polyamide, polyethylene terephthalate, and polyvinyl chloride were the most abundant in Crustacea shellfish. Our data do not support the bioaccumulation or biomagnification of MPs with the size of the animal in the shellfish group. MP contamination is more strongly associated with the type of shellfish than with the geographical origin of the market.

Ingestion is one of the main exposure routes of humans and animals to microplastics (MPs). During digestion, MPs can interact with both gastrointestinal enzymes and food proteins. This study investigated the adsorption of trypsin onto polypropylene (PP) and polyethylene terephthalate (PET) MPs, the influence of MPs on trypsin structure and activity, and the in vitro trypsin digestibility of bovine meat extract (BME) sarcoplasmic proteins and BME α-Gal-carrying allergens (α-GalA) in the presence of PP and PET MPs. Trypsin, BME and α-GalA proteins interact with MPs, resulting in the formation of a soft (SC) and hard (HC) corona. This interaction is dynamic, leading to the adsorption and desorption of protein through time. Trypsin adsorption onto MPs results in slight structural changes in the SC and bulk solution, while a trypsin fraction residing in the HC loses most of its specific activity. The presence of MPs slightly slows down the digestibility of proteins with a mass of 38 kDa, while it does not affect the digestion of α-GalA. According to our results, it is unlikely that realistic concentrations of MPs in the intestine would have significant effects on meat extract proteins’ and allergens’ digestibility by trypsin. We confirmed that during trypsin digestion, the corona on PP and PET MP is composed of BME sarcoplasmic proteins and allergenic α-Gal-carrying proteins.

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.

Microplastics entering the gastrointestinal environment rapidly acquire protein coronas that alter their surface chemistry and analytical detectability. We investigated the physicochemical interactions between fluorescently labeled bovine serum albumin (BSA) and polyethylene terephthalate (PET) microplastics during simulated intestinal exposure and evaluated the stability of the resulting hard corona. Using fluorescence tracking, SDS-PAGE, and FTIR spectroscopy, we showed that BSA forms a persistent corona that resists oxidative-only treatments. Only a combination of oxidation with an alkaline (KOH) or surfactant step (SDS) effectively removed the corona. None of the protocols applied affected polymer integrity. Residual protein in less effective protocols did not show changes on PET spectra in ATR FTIR. To validate the protocol under physiologically relevant complexity, we extended it to PET incubated with single digestive enzymes. FTIR spectra confirmed the removal of protein-specific signals in both systems, with no degradation of PET ester or aromatic functional groups nor signals of protein–polymer interactions. Our results highlight the robustness of protein–PET interactions in biological conditions and provide a variety of protocols for protein corona removal, suitable for diverse applications of microplastic analysis and toxicological studies.

Ingestion is one of the main exposure routes of humans to microplastics (MPs). During digestion, MPs can interact with both gastrointestinal enzymes and food proteins. This study investigated adsorption of trypsin to polypropylene (PP) and polyethylene terephthalate (PET) MPs, the influence of MPs on trypsin structure and activity, and in vitro trypsin digestibility of bovine meat extract (BME) sarcoplasmic proteins and BME α-Gal-carrying allergens (α-GalA) in the presence of PP and PET MPs. Trypsin, BME and α-GalA proteins interact with MPs resulting in the formation of a soft (SC) and hard (HC) corona. This interaction is dynamic, leading to adsorption and desorption of protein through time. Trypsin adsorption to MPs results in slight structural changes in SC and bulk solution, while a trypsin fraction residing in the HC lost most of its specific activity. The presence of MPs slightly slows down the digestibility of proteins with a mass of 38 kDa, while it does not affect digestion of α-GalA. According to our results, it is unlikely that realistic concentrations of MPs in the intestine would have significant effects on meat extract proteins and allergens digestibility by trypsin. We confirmed that during trypsin digestion, corona on PP and PET MP is composed of BME sarcoplasmic proteins and allergenic α-Gal-carrying proteins.

Microplastics entering the gastrointestinal environment rapidly acquire protein coronas that alter their surface chemistry and analytical detectability. We investigated the physicochemical interactions between fluorescently labelled bovine serum albumin (BSA) and polyethylene terephthalate (PET) microplastics during simulated intestinal exposure and evaluated the stability of the resulting hard corona. Using fluorescence tracking, SDS-PAGE, and FTIR spectroscopy, we show that BSA forms a persistent corona that resists oxidative and surfactant-only treatments. Only a combined alkaline-surfactant protocol (SDS/KOH) effectively removed the corona. None of the protocols applied affected polymer integrity. Residual protein in less effective protocols did not show changes on PET spectra in ATR FTIR. To validate the protocol under physiologically relevant complexity, we extended it to PET incubated with a digestive enzyme mixture. FTIR spectra confirmed the removal of protein-specific signals in both systems, with no degradation of PET ester or aromatic functional groups, nor signals of protein-polymer interactions. Our results highlight the robustness of protein–PET interactions in biological conditions and provide a variety of protocols for protein corona removal, suitable for diverse applications of microplastic analysis and toxicological studies.