Binding and corona formation of ovalbumin to polystyrene and polyethylene terephthalate microplastics under neutral and acidic conditions

Microplastic represents one of the major types of pollutants in modern era. Over several years of research in the field of microplastic, there are still many unknown gaps, including the effects and mechanisms of action of these particles on human health. Studies in this field conducted experiments on cells and human tissues or animals like rats and mice. While these studies suggest the toxic effects of microplastic, it is not clear if concentrations used for exposure are relevant for humans. Also, most of the studies used spherical polystyrene, which does not reflect well the diversity of microplastic particles found in nature. Another gap is lack of studies describing direct interactions of microplastics and proteins. While it is generally known that proteins form corona around microplastic particles, affinity studies and consequences on protein structure are usually missing. The aim of this work was to analyze interaction of a major egg white protein and allergen, ovalbumin to several to microplastic particles, including polystyrene (PS) of 120 and 500 μm in size and polyethylene terephthalate (PET) of 120 μm in size. Binding affinity was determined at both acidic, pH 3 and neutral, pH 7 conditions, at the room temperature, by measuring bulk ovalbumin concentration in supernatants at the equilibrium time. Several binding models, including Langmuir, Freundlich, Redlich–Peterson and Guggenheim-Anderson-de Boer (GAB), were used to determine binding parameters. The formation of soft and hard corona was analyzed according to the published protocol [1]. Structural analysis was performed using near and far-UV CD spectrometry. Obtained results showed that ovalbumin binds to both PS and PET. All binding models indicated that ovalbumin binds with higher affinity to tested microplastics on pH 3, compared to pH 7, with the highest affinity being calculated for PS 120 μm. Further analysis showed that ovalbumin forms both soft and hard corona onto the surface of all three microplastics. Structural alterations of ovalbumin as a consequence of its interaction with microplastic was shown to be both pH and microplastic type dependent. Also, more pronounced effect on its tertiary structure was observed, compared to secondary. At pH3, tertiary structure of bulk ovalbumin becomes destabilized, especially in the presence of PET 120 μm and PS 500 μm, while at pH 7, structural stabilization is observed, especially in the presence of PS 120 μm. Considering that the microplastic was discovered in eggs [2], obtained results suggest that direct interactions of native ovalbumin with microplastic particles could have influence on its structure and thus affect its techno-functional properties. Acknowledgments: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 96517. References: [1] D. Magrì, P. Sánchez-Moreno, G. Caputo, F. Gatto, M. Veronesi, G. Bardi, T. Catelani, D. Guarnieri, A. Athanassiou, P.P. Pompa, D. Fragouli, ACS Nano, 12 (2018) 7690-7700. [2] Q. Liu, Z. Chen, Y. Chen, F. Yang, W. Yao, Y. Xie, Food Chemistry, 397 (2022) 13377

Binding and corona formation of ovalbumin to polystyrene and polyethylene terephthalate microplastics under neutral and acidic conditions

Microplastic represents one of the major types of pollutants in modern era. Over several years of research in the field of microplastic, there are still many unknown gaps, including the effects and mechanisms of action of these particles on human health. Studies in this field conducted experiments on cells and human tissues or animals like rats and mice. While these studies suggest the toxic effects of microplastic, it is not clear if concentrations used for exposure are relevant for humans. Also, most of the studies used spherical polystyrene, which does not reflect well the diversity of microplastic particles found in nature. Another gap is lack of studies describing direct interactions of microplastics and proteins. While it is generally known that proteins form corona around microplastic particles, affinity studies and consequences on protein structure are usually missing. The aim of this work was to analyze interaction of a major egg white protein and allergen, ovalbumin to several to microplastic particles, including polystyrene (PS) of 120 and 500 μm in size and polyethylene terephthalate (PET) of 120 μm in size. Binding affinity was determined at both acidic, pH 3 and neutral, pH 7 conditions, at the room temperature, by measuring bulk ovalbumin concentration in supernatants at the equilibrium time. Several binding models, including Langmuir, Freundlich, Redlich–Peterson and Guggenheim-Anderson-de Boer (GAB), were used to determine binding parameters. The formation of soft and hard corona was analyzed according to the published protocol [1]. Structural analysis was performed using near and far-UV CD spectrometry. Obtained results showed that ovalbumin binds to both PS and PET. All binding models indicated that ovalbumin binds with higher affinity to tested microplastics on pH 3, compared to pH 7, with the highest affinity being calculated for PS 120 μm. Further analysis showed that ovalbumin forms both soft and hard corona onto the surface of all three microplastics. Structural alterations of ovalbumin as a consequence of its interaction with microplastic was shown to be both pH and microplastic type dependent. Also, more pronounced effect on its tertiary structure was observed, compared to secondary. At pH3, tertiary structure of bulk ovalbumin becomes destabilized, especially in the presence of PET 120 μm and PS 500 μm, while at pH 7, structural stabilization is observed, especially in the presence of PS 120 μm. Considering that the microplastic was discovered in eggs [2], obtained results suggest that direct interactions of native ovalbumin with microplastic particles could have influence on its structure and thus affect its techno-functional properties

Investigation of structural changes in ovalbumin induced by two types of MPs and its impact on protein digestibility

Ovalbumin (OVA) is the most abundant protein in chicken egg white. It is one of the major allergens in eggs. Micro- and nanoplatic particles (MNPs) are a widespread contaminant and have been found in food and water. It is still unclear how MNPs might affect human health. However, due to their large surface area they have been found to bind various biopolymers, including proteins. These biopolymers can be bound more strongly or loosely, and are referred to as hard and soft corona, respectfully [1]. MPs have been found in eggs, in the size range of 50-100 μm [2]. It is shown that these particles can interact with proteins and induce structural changes, but there is still not enough information on this topic [3]. These structural changes could lead to a decreased digestibility in the gastrointestinal tract, which could increase the immune response to known allergens. The aim of this study was to determine whether there are structural changes present in the OVA after incubation with two types of MPs – 120 μm polyethylene terephthalate (PET) and 120 μm polystyrene (PS) and whether they could influence digestion of OVA with gastrointestinal enzymes. 20 mg of MPs were incubated with 1.3 mg/mL ovalbumin for 4 h at room temperature in a 20 mM phosphate buffer at pH 7. Bulk ovalbumin was separated from the MPs by centrifugation and by filtration through a 0.22 μm PVDF filter. Soft corona was obtained by washing the MPs with water, and the MPs were later removed as described with bulk ovalbumin. Formation of amyloids was monitored with a Thioflavin T (ThT) assay at room temperature and after thermal treatment, and additional structural analysis was performed by circular dichroism (CD) spectrometry in the far-UV region. Thermal stability was also determined by spectrofluorimetry. Digestion with two proteases (pepsin and trypsin) was performed to determine whether there is a change in the gastrointestinal digestibility of OVA. Results from the ThT assay show that at room temperature there is no significant difference between the fluorescence emission obtained for all samples, with bulk OVA from both MPs showing a slight decrease. However, there is an increase of fluorescence after thermal treatment in all OVA samples, where OVA from the soft corona emits significantly less fluorescence than control and bulk samples for both types of MPs. Additionally, soft coronas have been shown to have more β-sheet content than other samples, which is more pronounced for OVA incubated with PET. For the heated samples there is a sharp change from α-helix to β-sheets in all the samples, but it is the most dramatic in the soft coronas. This could impose rigidity to the tertiary structure, which would explain why the ThT molecule does not bind as strongly. Despite differences in both the secondary and tertiary structure, the thermal stability is almost the same in all samples. Digestion of the samples shows that the soft corona incubated with PS tends to be more resistant to trypsin than other samples after 2 min, but it is not significant. For digestion with pepsin there is no difference between the samples. In conjunction with the previous results, which indicates a structural stabilisation of the soft corona at pH 7, it is not surprising that there is an increased resistance to trypsin, compared to pepsin which is a gastric enzyme and for which digestion is performed at an acidic pH. In conclusion, there is a structural change present in samples upon contact with MPs, particularly in the soft corona, of which the most pronounced is a decrease of α-helix content and increase in β-sheet content as determined by far-UV CD. This leads to a structural stabilization which could further impact the digestibility of the OVA protein and impact its allergenicity. However, this must be confirmed with further experiments. Acknowledgments: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 965173. References: [1] M.P. Monopoli, C. Åberg, A. Salvati, K.A.Dawson, Nat. Nanotechnol., 7 (2012) 779-786. [2] Q. Liu, Z. Chen, Y. Chen, F. Yang, W. Yao, Y. Xie. Food Chem., 397 (2022) 133771. [3] P. Ju, Y. Zhang. Y. Zheng, F. Gao, F. Jiang, J. Li, C. Sun, Sci. Total Environ., 734 (2020) 139219

Investigation of structural changes in ovalbumin induced by two types of MPs and its impact on protein digestibility

Ovalbumin (OVA) is the most abundant protein in chicken egg white. It is one of the major allergens in eggs. Micro- and nanoplatic particles (MNPs) are a widespread contaminant and have been found in food and water. It is still unclear how MNPs might affect human health. However, due to their large surface area they have been found to bind various biopolymers, including proteins. These biopolymers can be bound more strongly or loosely, and are referred to as hard and soft corona, respectfully [1]. MPs have been found in eggs, in the size range of 50-100 μm [2]. It is shown that these particles can interact with proteins and induce structural changes, but there is still not enough information on this topic [3]. These structural changes could lead to a decreased digestibility in the gastrointestinal tract, which could increase the immune response to known allergens. The aim of this study was to determine whether there are structural changes present in the OVA after incubation with two types of MPs – 120 μm polyethylene terephthalate (PET) and 120 μm polystyrene (PS) and whether they could influence digestion of OVA with gastrointestinal enzymes. 20 mg of MPs were incubated with 1.3 mg/mL ovalbumin for 4 h at room temperature in a 20 mM phosphate buffer at pH 7. Bulk ovalbumin was separated from the MPs by centrifugation and by filtration through a 0.22 μm PVDF filter. Soft corona was obtained by washing the MPs with water, and the MPs were later removed as described with bulk ovalbumin. Formation of amyloids was monitored with a Thioflavin T (ThT) assay at room temperature and after thermal treatment, and additional structural analysis was performed by circular dichroism (CD) spectrometry in the far-UV region. Thermal stability was also determined by spectrofluorimetry. Digestion with two proteases (pepsin and trypsin) was performed to determine whether there is a change in the gastrointestinal digestibility of OVA. Results from the ThT assay show that at room temperature there is no significant difference between the fluorescence emission obtained for all samples, with bulk OVA from both MPs showing a slight decrease. However, there is an increase of fluorescence after thermal treatment in all OVA samples, where OVA from the soft corona emits significantly less fluorescence than control and bulk samples for both types of MPs. Additionally, soft coronas have been shown to have more β-sheet content than other samples, which is more pronounced for OVA incubated with PET. For the heated samples there is a sharp change from α-helix to β-sheets in all the samples, but it is the most dramatic in the soft coronas. This could impose rigidity to the tertiary structure, which would explain why the ThT molecule does not bind as strongly. Despite differences in both the secondary and tertiary structure, the thermal stability is almost the same in all samples. Digestion of the samples shows that the soft corona incubated with PS tends to be more resistant to trypsin than other samples after 2 min, but it is not significant. For digestion with pepsin there is no difference between the samples. In conjunction with the previous results, which indicates a structural stabilisation of the soft corona at pH 7, it is not surprising that there is an increased resistance to trypsin, compared to pepsin which is a gastric enzyme and for which digestion is performed at an acidic pH. In conclusion, there is a structural change present in samples upon contact with MPs, particularly in the soft corona, of which the most pronounced is a decrease of α-helix content and increase in β-sheet content as determined by far-UV CD. This leads to a structural stabilization which could further impact the digestibility of the OVA protein and impact its allergenicity. However, this must be confirmed with further experiments

Impact of MPs on trypsin activity in simulated intestinal fluid

Mircoplastics (MPs) are an abundant contaminant in the environment with ingestion being the most common way of exposure for humans. Binding of protein to MPs is proposed to be multilayered with the formation of a soft and hard corona. It has been proven that MPs interact with enzymes present in the digestive system and impact their activity. The aim of this study is to investigate the impact of MPs on the activity of trypsin in simulated intestinal fluid (SIF). For this purpose, two sizes of polypropylene (large – 180-500 μm, small – 63-180 μm) and one size of polyethylene terephthalate (<80 μm) have been studied. Activity in bulk and soft corona was determined in SIF at 405 nm with N-α- Benzoyl-DL-arginine 4-nitroanilide hydrochloride after different times of incubation. Activity in hard corona was determined after 1 h of incubation with the MPs. Although specific activity in the control decreases through time, there is a tendency for all MPs to preserve activity in bulk and soft corona trypsin after 4 h of incubation. Trypsin remains active in the hard corona, with the activity being an order of magnitude lower than in the control, possibly due to significant changes in structure

Ovalbumin interaction with polystyrene and polyethylene terephthalate microplastics alters its structural properties

Related to research data no. 1: [https://cherry.chem.bg.ac.rs/handle/123456789/6465]Related to research data no. 2: [https://cherry.chem.bg.ac.rs/handle/123456789/6469

Biocorona formation of hen egg white proteins onto the surface of polystyrene and polyethylene terephthalate

Ovalbumin (OVA), a main protein of egg white, has characteristic structural fold of a serpin-family of proteins, propensity to fibril formation and stability to digestion. Microplastics (MPs) contaminating our food can interact with food proteins in the food matrix and during digestion. In this study adsorption of OVA to polystyrene (PS) (110 μm and 260 μm), polyethylene terephthalate (PET) (140 μm) MPs were investigated in acidic (pH 3) and neutral (pH 7) conditions. Formations of corona on MPs were investigated using isolated OVA and egg white protein extract comparatively. OVA adsorption depends on MPs size, polymer chemistry and pH, being highest in acidic pH and higher for PS. Adsorption of OVA to PS and PET reaches dynamic equilibrium after 4h resulting in disruption of tertiary structure and formation of hard and soft corona around MPs. Shorter fragments of OVA populate hard corona, while soft corona exclusively consist of full length OVA, albeit in its non-native conformation. The conformational changes resemble those induced by heat treatment with re-arrangement of α-β secondary structures. Structural changes are striking for the OVA in corona around MPs. Soft corona OVA preserves thermal and proteolytic stability, but loses ability to form fibrils upon heating. OVA is abundantly present in corona around MPs also in the presence of other egg white proteins. MPs contaminating food may bind and change structure and functional properties of main egg white protein

Impact of MPs on trypsin activity in simulated intestinal fluid

Mircoplastics (MPs) are an abundant contaminant in the environment with ingestion being the most common way of exposure for humans. Binding of protein to MPs is proposed to be multilayered with the formation of a soft and hard corona1. It has been proven that MPs interact with enzymes present in the digestive system and impact their activity2. The aim of this study is to investigate the impact of MPs on the activity of trypsin in simulated intestinal fluid (SIF). For this purpose, two sizes of polypropylene (large – 180-500 μm, small – 63-180 μm) and one size of polyethylene terephthalate (<80 μm) have been studied. Activity in bulk and soft corona was determined in SIF at 405 nm with Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride after different times of incubation. Activity in hard corona was determined after 1 h of incubation with the MPs. Although specific activity in the control decreases through time, there is a tendency for all MPs to preserve activity in bulk and soft corona trypsin after 4 h of incubation. Trypsin remains active in the hard corona, with the activity being an order of magnitude lower than in the control, possibly due to significant changes in structure. Acknowledgements This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 965173. References 1. Monopoli MP, Åberg C, Salvati A, Dawson, KA. Biomolecular coronas provide the biological identity of nanosized materials. Nat Nanotechnol 2012;7:779-86. 2. de Guzman MK, et al. Small polystyrene microplastics interfere with the breakdown of milk proteins during static in vitro simulated human gastric digestion. Environ Pollut 2023;335:122282

Proteomic insight into allergenic food corona on polyethylene terephthalate microplastics

Microplastics is abundant in the environment, food and beverages and get ingested by humans. Its complex interplay with proteins lead to formation of corona. Tightly bound proteins represent hard corona, while weaker binding partners are found in soft corona. Separation of hard and soft corona of allergenic proteins of shrimps, eggs and cow’s milk, tropomyosin (TPM), ovalbumin (OVA) and beta-lactoglobulin (BLG) and identification of binding partners by proteomics was aim of our study. Allergenic proteins were purified from egg white, shrimps and cow’s milk. Binding to polyethylene terephthalate microplastics (PET) (70-100 m) was probed at pH 7 for purified allergens and egg white proteins. After establishment of binding equilibrium, soft and hard corona were separated and analyzed by SDS PAGE, followed by identification of bound proteins by nanoLC-HRMS. Binding of all allergenic proteins was observed in both soft and hard corona. Soft corona contains exclusively intact, full length OVA, TPM and BLG. Hard corona is enriched for truncated OVA and oligomers of TPM. OVA fragments are partially or fully enfolded and have higher level of exposed hydrophobic patches resulting in higher affinity for PET microplastics. In comparison to OVA and TPM, hard corona of BLG is less abundant under similar conditions. BLG is compact globular protein with lower level of exposed hydrophobic patches in comparison to ovalbumin and tropomyosin. In hard corona, trace amounts of contaminating alfa-lactalbumin become enriched. In the presence of egg white protein extract OVA forms both SC and HC on microplastics, being the dominant protein of hard corona (with ovotransferrin). Lysozyme and ovomucin are present only in hard corona. Both proteins are known for their strong bioactivity and represent a small fraction of total egg white proteins. Our results show that allergenic proteins form hard corona on PET microplastics. Among egg white proteins, minor proteins such as lysozyme and ovomucin become enriched. Denaturing effect of strong binding to microplastics may change functional characteristics of allergens and bioactive proteins of foods and should be further investigated in functional assays. Acknowledgment: This study was supported by IMPTOX European Union's Horizon 2020 research and innovation program (grant number 965173).Book of Abstract

Proteomic insight into allergenic food corona on polyethylene terephthalate microplastics

Microplastics is abundant in the environment, food and beverages and get ingested by humans. Its complex interplay with proteins lead to formation of corona. Tightly bound proteins represent hard corona, while weaker binding partners are found in soft corona. Separation of hard and soft corona of allergenic proteins of shrimps, eggs and cow’s milk, tropomyosin (TPM), ovalbumin (OVA) and beta-lactoglobulin (BLG) and identification of binding partners by proteomics was aim of our study. Allergenic proteins were purified from egg white, shrimps and cow’s milk. Binding to polyethylene terephthalate microplastics (PET) (70-100 m) was probed at pH 7 for purified allergens and egg white proteins. After establishment of binding equilibrium, soft and hard corona were separated and analyzed by SDS PAGE, followed by identification of bound proteins by nanoLC-HRMS. Binding of all allergenic proteins was observed in both soft and hard corona. Soft corona contains exclusively intact, full length OVA, TPM and BLG. Hard corona is enriched for truncated OVA and oligomers of TPM. OVA fragments are partially or fully enfolded and have higher level of exposed hydrophobic patches resulting in higher affinity for PET microplastics. In comparison to OVA and TPM, hard corona of BLG is less abundant under similar conditions. BLG is compact globular protein with lower level of exposed hydrophobic patches in comparison to ovalbumin and tropomyosin. In hard corona, trace amounts of contaminating alfa-lactalbumin become enriched. In the presence of egg white protein extract OVA forms both SC and HC on microplastics, being the dominant protein of hard corona (with ovotransferrin). Lysozyme and ovomucin are present only in hard corona. Both proteins are known for their strong bioactivity and represent a small fraction of total egg white proteins. Our results show that allergenic proteins form hard corona on PET microplastics. Among egg white proteins, minor proteins such as lysozyme and ovomucin become enriched. Denaturing effect of strong binding to microplastics may change functional characteristics of allergens and bioactive proteins of foods and should be further investigated in functional assays.[https://itpa.it/index.php/past-events/xvii-itpa-hps-and-sepa-international-congress-rome-italy-november-29-december-1-2023/