A self-adhesive glutenin-based coating cross-linked by genipin for suppressing microplastic shedding in harsh environments

Abstract

The large-scale application of plastic packaging has raised concerns on the generation of microplastics (MPs). The presence of MPs in food has been increasingly reported, and plastic packaging is one of the main sources. However, few studies have focused on strategies to inhibit shedding of MPs. Herein, a simple, green, and durable coating, which was inspired from soybean milk skin, was developed to suppress MP shedding from food-grade plastics in seven harsh simulation environments (considering the five factors of heat, acid, alkali, salt, and oil). This coating was formed via the phase transition of glutenin after treatment with tris(2-carboxyethyl)phosphine, and it spontaneously adhered to any plastic surfaces under mild conditions. To improve its physical properties, genipin was used as a cross-linking coating. The performance of the cross-linked coating improved in the following aspects: (1) increase in its contact angle from 84.7° to 96.1°; (2) improvement in its tightness; (3) reduction in its roughness from 0.59 μm to 0.31 μm; (4) improvement in its elastic modulus from 19.85 GPa to 30.87 GPa; (5) improvement in its gas barrier permeance by 24.46%; and (6) and decrease in the shedding abundance of MPs by 61.14%. Notably, the inhibition rate of the cross-linked coating on MP shedding under any harsh conditions ranged from 92% to 98%. After coating, MPs with sizes exceeding 100 μm were not observed through fluorescence and micro-Raman microscopies. Moreover, the covalent crosslinking mechanism of genipin on the coating was investigated using Fourier transform infrared and X-ray photoelectron spectroscopies. Furthermore, cell proliferation was used to demonstrate the safety of the coating. Overall, this work provides new insights into the control of MPs, inspiring researchers to focus more on the front-end source and design new materials to defend against the threat of MPs to human health.

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Green foundation

1. This study developed a glutenin-based coating for inhibiting the shedding of microplastics from plastic packaging materials, resulting in sustainable environmental benefits. The preparation method of this coating is carried out under mild conditions without the involvement of toxic reagents.

2. This study provides a novel method for simulating the shedding of microplastics in harsh environments. We quantitatively evaluated the effectiveness of the coating in suppressing the shedding of microplastics under seven harsh environments.

3. This strategy will be beneficial for further exploration works on microplastics control in the future. Moreover, the development of protein-based coatings is of great significance.

1. Introduction

Plastic products are indispensable in human life for the foreseeable future owing to their affordability, convenience, and ease of manufacturing. In recent years, the pervasive presence of microplastics (MPs), which are plastic particles ranging from 1 μm to 5 mm in size, have garnered significant attention from the research community. This emerging form of pollution poses a new and pressing environmental challenge in the modern era.^1,2 MPs have been detected in various environmental systems,³ and traces of them have also been found in numerous food items, including drinking water,⁴ milk,⁵ eggs,⁶ fruits and vegetables,⁷ ultimately reaching our dining tables. MPs can pose risks to human health through three primary routes, namely, ingestion, inhalation, and direct skin contact.⁸ More seriously, studies have revealed that MPs have detrimental effects on both the ecosystem and human health.^9–11

Plastic products are exposed to various environmental conditions, including wind, rain, ultraviolet light, and microbial activity, resulting in their break down into smaller particles of micro and even nano sizes.¹² The methods for controlling and regulating MPs can be divided into two as follows: 1. avoiding their generation and 2. their enrichment and removal. There have been numerous reports on the enrichment and removal of MPs after their production.^13,14 Previously, we have established a detection method for MPs in eggs¹⁵ and investigated the interactions between MPs and pesticides¹⁶ and their impact on toxicity.¹⁷ According to these studies, we realized that proactively controlling the emergence of MPs is of great significance, given that humans still require a large amount of plastic in the foreseeable future. In the textile and clothing industry, low-friction polymer brushes on fabrics could restrain MP shedding during laundering by over 93%.^18,19 Also, oxygen plasma treatment reduced the shedding of MPs from PET by 43% by mass (accumulative of prewash and 5 accelerated washes) and 73% by count compared to the untreated sample.²⁰ However, there is limited research on how to inhibit the generation of MPs through heating and other non-friction forces. Notably, plastic and paper cups released significant amounts of MPs when exposed to heat, such as during hot water soaking or microwave treatment.^21,22 According to statistics, steeping a plastic teabag would release about 11.6 billion MPs into a single cup of tea at the brewing temperature of 95 °C.²³ In addition, food plastic products are often exposed to other high-temperature environments such as hot filling, thermoforming, microwave heating, and pasteurization. This may cause the detached MPs to directly enter different food. Consequently, suppressing the shedding of MPs in hot or harsh environments remains an important topic.

Considering factors such as raw material sources, environmental friendliness, sustainability, biocompatibility, and durability, it is easy to choose biomass materials as the first choice for the preparation of materials, especially plant protein.²⁴ Due to their unique physical and chemical properties such as modifiability, precise construction, and biocompatibility, protein-based materials have been widely developed.^25,26 Interestingly, we observed that soybean milk skin (yuba) can easily adhere to substrates such as chopsticks, bowls, and metal. Milk skin also exhibits a similar phenomenon.²⁷ Inspired by this, the development of simple, green, and widely sourced protein self-adhesion coatings may be valuable in inhibiting MP shedding.

In this study, we developed a self-adhesive coating designed to reduce the shedding of MPs in harsh environments. The coating was fabricated through the rapid phase-transition of glutenin (PTG) facilitated by tris(2-carboxyethyl)phosphine (TCEP) reduction. To enhance its mechanical properties and tightness, genipin was employed as a crosslinking agent for the PTG coating (genipin@PTG). The crosslinking mechanism of genipin on PTG was analyzed by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). Scanning electron microscopy (SEM) was conducted to observe the changes in the surface and cross-sectional morphology of the coatings before and after cross-linking. The hydrophobicity and stability of the coatings were evaluated using the water contact angle (CA). Furthermore, the elastic modulus of the coatings with and without genipin crosslinking was compared. This research focused on assessing the performance of the final coating under various conditions specified in China's national standards for food contact materials and product migration, including water-based or oil-based, acidic or alkaline, and high salt content systems. Two key factors were considered to improve the ability of the coating to suppress MP shedding, i.e., the concentration of genipin and the number of coating layers. Additionally, cell proliferation tests were conducted to demonstrate the biocompatibility of both PTG and genipin@PTG coatings. In summary, this work offers a new perspective on MP control and explores the potential applications of protein-based materials (Scheme 1).

Scheme 1 Schematic of the coatings developed in this work to suppress the shedding of MPs.

2. Experimental

2.1. Materials

Glutenin (from wheat, PN: S25995, CAS Reg. no. 8002-80-0) and tris(2-carboxyethyl)phosphine hydrochloride (≥98%, PN: S16054, CAS Reg. no. 51805-45-9) were provided from Yuanye Biotechnology Co., Ltd (Shanghai, China). Genipin (>98%, PN: G810337, CAS Reg. no. 6902-77-8) was purchased from Macklin Biochemical (Shanghai, China). Polypropylene (PP, food grade, CAS Reg. no. 9003-07-0) was procured from Binzhou Samsung Packaging Technology Co., Ltd (Shandong, China). Polyethylene terephthalate (PET, food grade, CAS Reg. no. 25038-59-9) was purchased from Binzhou Longcheng Plastic Industry Co., Ltd (Shandong, China). Nile Red (97%, CAS Reg. no. 7385-67-3) was acquired from Meryer Biochemical Technologies Co., Ltd (Shanghai, China). Ethanol (chromatographic grade), acetone, acetic acid (CH₃COOH) (chromatographic grade), sodium chloride (NaCl) and sodium carbonate (Na₂CO₃) were procured from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Ultrapure water was prepared using a Milli-Q system (EMD Millipore Company, Billerica, MA, USA).

2.2. Preparation of PTG coating and genipin@PTG coating

The method for the preparation of the PTG coating was based on our previous research.²⁸ Briefly, soluble glutenin was extracted with alkali and the supernatant was collected by centrifugation to obtain the glutenin stock solution. The PTG solution was prepared by mixing glutenin solution (25 mg mL⁻¹) and TCEP solution (50 mM, pH 8.2) in the same volume. When TCEP solution was mixed with gluten solution, the mixed solution immediately turned to a milky white liquid due to the scattering of glutenin aggregates into particles, as shown in Fig. S1.† Plastic sheets (6 dm²) were soaked in the PTG solution (30 mL) and incubated at 28 °C for 24 h. After that, the plastic sheets were washed clean with 67 mL dm⁻² ultrapure water using a water cannon and dried, finally obtaining a one-layer PTG coating. The above-mentioned operation was repeated to ensure that both sides of the plastic sheets were coated with the PTG coating.

Genipin was first dissolved in acetone solution, and then diluted with ultrapure water. The final volume ratio of acetone to water was 1 : 4. The plastic sheets with PTG coating were soaked in genipin solution (0.01–1 mg mL⁻¹) and incubated for 8 h at 150 rpm and 55 °C in a sealed glass container. Next, the plastic sheets were fully washed with ultrapure water and dried. Then, the genipin@PTG coating was obtained on the surface of the plastics.

2.3. Characterization of PTG coating and genipin@PTG coating

The surface and cross-sectional morphology of the PTG and genipin@PTG coatings were observed using SU8020 (HITACHI, Tokyo, Japan) and Apreo (Thermo Fisher Scientific, MA, USA) SEM. ATR-FTIR was performed to identify the functional groups on the surface of the coatings using a NEXUS (Nicolet, USA). The XPS spectra of the plastics with the coatings were analyzed using a Thermo Escalab 250XI (MA, USA), and the binding energies were calibrated by setting the C 1s peak at 284.8 eV. Water contact angles (CA) were tested on an OCA115EC (Deerfield, Stuttgart, Germany) video-optical measuring instrument, equipped with a CCD camera and image processing software under laboratory conditions (temperature of 25 °C and relative humidity of 50%–60%). The roughness of the PTG coating and genipin@PTG coating was measured using an LSM880 (Carl Zeiss AG, Oberkochen, Germany) confocal laser scanning microscope. Nanoindentation was measured using a TI-PREMIER (Hysitron, Minnesota, USA) nano mechanics testing system. Moreover, the PET sheets with PTG coating and genipin@PTG coating were used for testing the water vapor permeance using the ASTM E96/E 96 M-05 standards, utilizing a gas permeability analyzer (C130H model from Languang Electromechanical Technology, Jinan, China).

2.4. Experiments on MP shedding

2.4.1. Quality control. In the course of our research, ultrapure water served as a crucial blank control, undergoing the identical experimental procedure as the experimental group. This was to eliminate any potential interference from airborne particles that may settle and affect the results. Furthermore, to prevent the introduction of new contaminants, all experimenters wore cotton lab coats and gloves devoid of polymeric materials throughout the experiment. Additionally, we ensured that the samples were kept at a safe distance from the plastic fittings of all the instruments employed. Lastly, all containers used in the experiment were crafted from glass, meticulously cleaned by soaking them in aqua regia, rinsed thoroughly three times with ultrapure water, and dried thoroughly prior to use. Each of these measures was implemented to maintain the integrity and accuracy of our findings.

2.4.2. Experimental conditions for MP shedding. When selecting solvents to serve as food simulants for plastic food contact materials, it is imperative to consider the type of food the material will come into contact with. In our study, we specifically investigated the impact of food system acidity, alkalinity, and oil content on plastic packaging. Consequently, food products were classified into six categories including neutral aqueous, acidic aqueous, alkaline aqueous, acidic low-oil, acidic high-oil, and alkaline high-oil food systems. To prepare the food simulants used in this study, we referenced and enhanced the guidelines provided in “(EU) No 10/2011”, “GB 5009.156-2016” and “GB 31604.1-2023”. Additionally, recognizing the complexity of ingredients and high salt content in many food products, we also prepared simulants for high-salt food products, drawing inspiration from “GB/T 32095.3-2015 Performance and Test Methods of Non-Stick Surface of Domestic Metal Utensils for Household Food – Part 3: Test Specification of Corrosion Resistance” to assess the corrosion performance of the plastic packaging.

Determining the most extreme temperatures and durations that lead to the highest shedding of MPs is essential for setting the parameters. Our aim was to expose plastic packaging products to the harshest possible conditions. The specific experimental conditions that were ultimately selected are detailed in Table S1.† It is important to note that during the MP shedding experiments, the plastic products must adhere to the relevant requirements stipulated by several national standards, as previously mentioned. During the experiment, there should be no deformation, melting, or swelling of the plastic products, given that they can compromise the accuracy of the results. If any such physical changes occur, the experimental conditions must be reviewed and adjusted to ensure they are more reasonable and appropriate.

Regarding the experimental procedures for assessing MP shedding, our approach adhered to the guidelines outlined in “(EU) No 10/2011”, “GB 5009.156-2016” and “GB 31604.1-2023”. Given that the plastics used in our study had a thickness of 0.5 mm or less, we opted for the total immersion method. According to the requirements of national standards, when the ratio (S/V) of the contact area (S) of food contact materials and products to the mass or volume (V) of food is unknown, it is necessary to conduct experiments using 6 dm² of food contact materials and products in contact with 1 kg or 1 L of food. Then, the plastic pieces and simulants were placed together in a glass container and heated in an oil bath, with reflux condensation to minimize evaporation.

2.4.3. Detection method for MPs. Following the experiment designed to assess the release of MPs from plastic packaging, we vacuum-filtered the solution in a glass container through glass fiber filter paper featuring a pore size of 0.45 μm. This step aimed to effectively isolate the MPs. Once all the released MPs were concentrated on the filter paper, they were stained and examined under a microscope, adhering to our previously established research method.¹⁵ Several drops of Nile Red-acetone aqueous solution (20 μg mL⁻¹) were added dropwise to the glass fiber filter paper enriched with MPs. Then, the glass Petri dish containing the filter paper was placed in a water bath and incubated at 60 °C for 15 min. Subsequently, we conducted preliminary visual inspections and measurements of the MPs on the filter paper using a fluorescence microscope (Axio Vert A1, Zeiss, Germany). These observations were carried out under both bright field and fluorescent conditions, allowing us to evaluate the number, size, and shape of the shed MPs. Initial identification of the MPs was achieved using blue excitation light (with an excitation wavelength of 450–490 nm and an emission wavelength of 515–565 nm). During the observation process, we photographed the identified particles and measured their size (the longest dimension) digitally, utilizing the MShot Image Analysis System 1.1.4 software. This comprehensive approach ensured the accurate and detailed analysis of the MPs released from the plastic packaging.

All particles suspected to be MPs were characterized at 532 nm using a micro-Raman spectrometer (LabRAM HR Evolution, HORIBA, France) to analyze the functional groups. The resulting spectra were matched against a library of reference spectra (https://academic.knowitall.com), and the particles with less than 70% match to the reference spectrum were excluded from the MPs and not counted.²⁹

2.5. Cell proliferation evaluation

The cell viability of NCM460 cells was measured using the CCK-8 assay for the PTG coating and genipin@PTG coating. Briefly, the plastic sheets coated with PTG and genipin@PTG were immersed in physiological saline at 37 °C for 24 h and at 95 °C for 2 h to obtain the extract. Then, the extract was added to RPMI-1640 (90%) and FBS (10%) to prepare the medium. 100 μL of cell suspension with a cell density of 30 000 cell per cm² was transferred to a 96-well plate and inoculated for 24 h at 37 °C in an incubator with 5% CO₂. Then, medium containing different concentrations of extract (3.2, 1.6, and 0.8 cm² mL⁻¹) was added. After culturing for 24, 48, and 72 h, 5 μL of CCK-8 was added to each well. After incubation for 2 h, a shaker was employed to mix the solutions. 5-Fu (5 μg mL⁻¹) was set as the positive control and the medium without extract was set as the negative control. Finally, a microplate reader was used to test the absorbance value at 450 nm. The cell inhibition ratio was calculated using the following formula:
where A_nc is the absorbance value of the negative control and A_s is the absorbance value of the sample.

3. Results and discussion

3.1. Self-adhesive PTG coating inspired by soybean milk skin

After being ground and boiled, the disulfide bonds in the protein of soybean milk were broken, as illustrated in Fig. 1B. The proteins in soybean underwent further denaturation during continuous heating. As a result, their hydrophobic residues became exposed, causing the proteins to become unstable and prone to aggregating. Thus, to minimize their interfacial free energy, these proteins and aggregates migrated to the air–water interface, ultimately forming a two-dimensional nanofilm (Fig. 1A).³⁰ Alternatively, protein nanofilms are rich in functional groups such as hydroxyl, amino, and carboxyl, which are helpful to spontaneously adhere to various substrates.³¹ The surface morphology of soybean milk skin before and after washing clean was characterized by SEM. As shown in Fig. 1C, irregular protein aggregates were found in the nanofilm. Interestingly, after thorough washing, almost no trace of the nanofilm structure remained, as shown in Fig. 1D, indicating that the soybean milk skin had been washed away. This special phenomenon prompted us to consider its reasons more deeply. Soybean milk skin consists of a protein–lipid film composed primarily of approximately 57.6% protein and 24.1% lipid.^32,33 Its formation involves protein denaturation, an endothermic polymerization process, and interactions between proteins and lipids. This multi-faceted and intricate composition leads to decreased adhesion to the underlying surface, making it unable to resist the impact of water. In the food processing industry, this soybean milk skin is further processed into yuba through drying.³⁴ Given these insights, it became intriguing to explore the possibility of creating self-adhesive nanofilms under gentle conditions by utilizing a single protein through assembly techniques.

Fig. 1 PTG coating inspired from soybean milk skin. Soybean milk skin (A); schematic of the mechanism for the formation of soybean milk skin (B); SEM image of soybean milk skin using silicon wafer as the substrate (C); SEM image of soybean milk skin after thorough washing using silicon wafer as the substrate (D); and photo of tweezer adhered to silicon wafer coated with PTG (E).

We used TCEP to reduce the disulfide bonds in glutenin, forming PTG that spontaneously adhere to the substrate surface.²⁸ As shown in Fig. 1E, the PTG coating before washing, similar to soybean milk skin, could also adhere to tweezers and other substrates. More importantly, the ultra-thin PTG coating after thorough washing could resist high-strength water impact (force >0.25 N) and had excellent physical and chemical properties. Hydrophobic interactions and intermolecular disulfide bonds may be the main forces that form strong coatings.^28,35

3.2. Morphology of PTG coating and genipin@PTG coating

The concentration of glutenin was one of the important factors affecting the formation of the PTG coating. As shown in Fig. 2A–C, the morphology of the PTG coating was observed at three different concentrations (5 mg mL⁻¹, 15 mg mL⁻¹, and 25 mg mL⁻¹). PTG was composed of 400–500 nm spherical particles, which was similar to that of soybean milk skin.³² As the concentration of glutenin increased, more and more aggregates particles were formed on the surface. When the glutenin concentration was 25 mg mL⁻¹, a dense coating was formed by PTG adhering to the substrate (Fig. 2C). However, the pores on its surface may not be able to suppress the detachment of MPs. Furthermore, a superior mechanical performance is necessary to meet the needs of industrialization. Therefore, surface modification and cross-linking are the most commonly used methods to improve the performance of coatings.^36–38

Fig. 2 Surface and cross-sectional morphology of PTG coatings and genipin coatings. PTG coating prepared with 5 mg mL⁻¹ glutenin (A); PTG coating prepared with 15 mg mL⁻¹ glutenin (B); PTG coating prepared with 25 mg mL⁻¹ glutenin (C); genipin@PTG coating prepared with 15 mg mL⁻¹ glutenin and 0.1 mg mL⁻¹ genipin (D); genipin@PTG coating prepared with 25 mg mL⁻¹ glutenin and 0.1 mg mL⁻¹ genipin (E); genipin@PTG coating prepared with 25 mg mL⁻¹ glutenin and 1 mg mL⁻¹ genipin (F); cross-sectional morphology of single-layer genipin@PTG coating (G and H); and cross-sectional morphology of three-layer genipin@PTG coating (I). Schematic of the preparation process of genipin@PTG coating (J).

Genipin, a natural substance extracted from gardenia fruit, has been widely used for the cross-linking of coatings.^39–41 Moreover, it has been demonstrated to be 10 000 times less cytotoxic than the commonly used cross-linking agent glutaraldehyde.^42,43 Consequently, the PTG coating exhibited a higher degree of crosslinking when treated with genipin due to the exposure of numerous modifiable side chains. Fig. 2D–F present a comparison of the surface structure of the PTG coating crosslinked with genipin. In the case of the non-compact PTG coating (Fig. 2D), it was evident that genipin was wrapped around the aggregates and interconnected with other particles (highlighted by the white arrow), which is consistent with previous reports.^43,44 Additionally, in the case of the more compact coatings, an increase in genipin concentration significantly enhanced their crosslinking degree, as shown in Fig. 2E and F. Notably, the cross-linked genipin@PTG coatings lacked obvious pores on their surface. As illustrated in the schematic diagram in Fig. 2J, genipin compacted and densified the aggregates, preventing gas permeation, which was further validated later in this work. Examining the cross-sectional SEM image, it was observed that the thickness of the single-layer coating was not uniform (Fig. 2G), potentially due to the variations between the oligomers and aggregates.²⁸ The average thickness of the one-layer coating was about 142 ± 18 nm, as shown in Fig. 2H. To enhance the thickness and uniformity of the coating, a three-layer coating was developed through a straightforward and repeatable preparation process. It was found that the thickness of the three-layer coating was roughly three times that of a single-layer coating (Fig. 2I), indicating that manually controllable coating thickness can meet practical requirements.

3.3. Comparison of physical properties between the PTG coating and genipin@PTG coating

The physical properties of both the PTG coating and genipin@PTG coating were compared in detail. The CA was used to assess the hydrophobicity of the coating surface. To demonstrate the strong and non-specific adhesion, PTG coatings were prepared on different plastic surfaces (PET, PS, PI, PE, PP, PC and PTFE). Despite the differences in the surface structure, hydrophobicity and functional groups among these plastics, the PTG coatings adhered equally well to all the surfaces, as evidenced by their consistent CA values (Fig. 3A). Moreover, as shown in Fig. 3B and C, the PTG coating was slightly hydrophilic (CA ∼ 84.7°),²⁸ while the genipin@PTG coating was hydrophobic (CA ∼ 96.1°), which was attributed to the exposure of the hydrophobic side chains of genipin. Surface roughness is another crucial factor influencing the properties of coatings.^44–46 After crosslinking with genipin, the roughness of the PTG coating (prepared with 25 mg mL⁻¹ glutenin) decreased by 47%, as shown in Fig. 3D and E. This reduction can be due to genipin filling the pores and the height differences between aggregates, as also observed in the SEM images (Fig. 2E and F). Moreover, nanoindentation was performed to evaluate the mechanism properties of the coatings. The elastic modulus, a measure of the resistance of a coating to elastic deformation, reflects the bonding strength between atoms, ions, or molecules. Notably, the substrate-particle adhesion strength does not significantly affect the elastic modulus when measured under low denaturation conditions.⁴⁷ The results, as shown in Fig. 3F, indicate that the elastic modulus of the genipin@PTG coating (30.87 ± 1.85 GPa) was approximately 55% higher than that of the PTG coating (19.85 ± 1.32 GPa). This improvement is likely due to genipin crosslinking, which enhanced the packing density of aggregates and their resistance to external pressure.⁴⁸ Furthermore, the tolerance of the genipin@PTG coating to complex environments is crucial for practical applications. Organic solvents, pH, common food processing techniques, and repeated tape application were the main factors we considered, as shown in Fig. 3G. The results show that the genipin@PTG coating exhibited resistance to erosion in harsh environments and maintained excellent adhesion (Fig. 3G and Fig. S2, S3†). These favorable physical and chemical properties position the genipin@PTG coating as an effective solution for inhibiting microplastic (MP) detachment in plastic food packaging. Additionally, the gas barrier performance of the genipin@PTG coating was further investigated. Water vapor permeance was used as an indicator to evaluate the gas barrier efficacy, with bare PET plastic serving as the coating substrate and control group. As shown in Fig. 3H, increasing the number of coating layers significantly reduced the water vapor permeance of the PTG coatings. However, when the coating density was low (due to a low glutenin concentration), the crosslinking effect of genipin was limited. Conversely, a dense PTG coating (prepared with a high glutenin concentration) exhibited a significantly improved gas barrier performance after crosslinking with genipin, which is attributed to its smaller pores and higher degree of crosslinking (Fig. 3I). The water vapor permeance after crosslinking of the three-layer coating decreased by 24.46% compared to that before crosslinking. Given that gas molecules such as water, oxygen, and carbon dioxide are essential for the physiological metabolic activities of fresh food,^49,50 the PTG and genipin@PTG coatings can enhance the application value of plastic packaging.

Fig. 3 Physical properties of PTG coating and genipin@PTG coating. CA of coatings on different plastic surfaces (A); CA of PTG coating (B); CA of genipin@PTG coating (C); roughness of PTG coating (D); roughness of genipin@PTG coating (E); and elastic modulus of PTG coating and genipin@PTG coating. The three colors represent the results of three independent experiments (F); effects of different environments (pH 2, pH 12, ethanol and acetone for 1 h, liquid nitrogen for 5 min, ultrasonic, ultraviolet and plasma activated water for 1 h, pasteurization at 85 °C for 30 min, UHT for 10 min, repeat the tape application 10 times) on CA of genipin@PTG coating (G); water vapor permeance of PET plastic sheet, PTG coating and genipin@PTG coating (H); and schematic of the superior gas barrier performance of genipin@PTG coating compared to PTG coating (I).

3.4. Mechanism of genipin-crosslinked PTG coating

ATR-FTIR was conducted to analyze the functional groups in the PTG coating and genipin@PTG coating. As the concentration of genipin increased, the stretching vibration of the C–H bond gradually increased at 2920 cm⁻¹ (Fig. 4A and Fig. S4†). After acetone treatment of the PTG coating, a large amount of the carbonyl groups in the amide bonds in glutenin aggregates was buried. However, the newly formed carbonyl peak after crosslinking with genipin was blue-shifted from 1680 cm⁻¹ to 1650 cm⁻¹, which may be due to the formation of new amide bonds on the surface. Moreover, the amino group on the glutenin attacked the alkene carbon atom at the C-3 position of genipin, causing the opening of the dihydropyran ring and the formation of heterocyclic amines.^43,48,51 In an alkaline environment, genipin has the capability to undergo a self-polymerization process, resulting in the creation of long-chain cross-linking bridges. Subsequently, these bridges interact with proteins through crosslinking. The nucleophilic attack of OH– ions in aqueous solution leads to the ring opening of genipin and the formation of an aldehyde intermediate.^43,48 Next, the open-loop genipin molecule undergoes aldol condensation reaction internally to form a macromolecular polymer. These terminal aldehyde groups of polymerized genipin can undergo Schiff base reactions with amino groups in proteins, further forming cross-linked structures. Genipin may also undergo self-polymerization reaction to form long-chain genipin cross-linking bridges, and then crosslink with proteins. Therefore, the formation of a C–N bond at 1470 cm⁻¹ was an important structural basis for cross-linking (Fig. 4A).

Fig. 4 Mechanism for the crosslinking PTG with genipin. ATR-FTIR spectra of glutenin, genipin, PTG coating and genipin@PTG coating prepared with 0.01 mg mL⁻¹ genipin (A); wide-scan XPS spectra of PTG coating and genipin@PTG coating prepared with 0.01 mg mL⁻¹ genipin (B); proportion of C, N, and O elements from arrow-scan XPS spectra in the PTG coating and genipin@PTG coating prepared with 0.01 mg mL⁻¹ genipin (C); binding energy of C, N, and O elements from arrow-scan XPS spectra in the PTG coating and genipin@PTG coating prepared with 0.01 mg mL⁻¹ genipin (D); Gaussian peak fitting deconvolution of XPS N_1s spectra (E); Gaussian peak fitting deconvolution of XPS C_1s spectra (F); and schematic of genipin cross-linked PTG (G).

XPS was used to analyze the content and binding energy of the surface elements in the PTG coating and genipin@PTG coating, as shown in Fig. 4B. Fig. 4C indicates that the proportion of carbon elements on the surface of the coating increased from 65.02% to 89.38%, which meant a higher crosslinking degree of genipin. This result is consistent with the conclusion from the CA measurement, which is the transition from hydrophilicity to hydrophobicity (Fig. 3B and C). According to Fig. 4D, the binding energy of N and O elements increased with an increase in the concentration of genipin. The formation of more amide bonds and hydrogen bonds may be the main reason for this. By fitting the peaks of N and O elements, we found that the genipin@PTG coating contained more abundant amide bonds on its surface than the PTG coating (Fig. 4E and F). Finally, we present a schematic of the cross-linking of the PTG coating with genipin in Fig. 4G, which is of great significance for understanding the crosslinking of PTG by genipin.

3.5. Inhibition of MP shedding by the coating

PP is a versatile material commonly used in the production of disposable tableware, plastic bags, and beverage bottles, while PET is highly valued for its transparency, excellent barrier properties, and strong recyclability, making it a popular choice for packaging such as beverage bottles and food containers. Both PP and PET occupy a substantial market share in the plastics industry. Thus, to gain insight into the MP shedding from these packaging materials, microscopy provided an intuitive and clear visualization method. By capturing both bright-field and fluorescence images using a microscope, we could initially identify, count, and analyze the MPs trapped on glass fiber filter paper. Fig. 5 presents the microscopy images of the MPs, showing their characteristics. Furthermore, Fig. 7 displays the counting statistics of the released and migrated MPs, which were processed in conjunction with the subsequent micro-Raman analysis for comprehensive evaluation. These findings are crucial for understanding the effectiveness of our newly developed coating in inhibiting the shedding of MPs from plastic packaging.

Fig. 5 Representative photos of MPs under bright field and fluorescence conditions. Bare PP plastic (A); PP plastic coated with PTG coating (B); PP plastic coated with genipin@PTG coating (C); bare PET plastic (D); PET plastic coated with PTG coating (E); and PET plastic coated with genipin@PTG coating (F).

As shown in Fig. 5, the location of the MPs could be very easily found on the glass fiber filter paper by combining the images taken in the bright field condition and the fluorescence condition. This is due to the fact that the chosen stain, Nile Red, is a very good lipophilic stain, which has been used in many studies to stain MPs, thus making it very easy to distinguish MPs from other substances.^6,52Fig. 6 shows the Raman peaks of the representative PP and PET MPs detected under a 532 nm wavelength laser. Firstly, it could be observed that in ultrapure water without plastic samples, the number of MPs detected was extremely low, less than 10 particles per 100 mL, which is negligible. This finding aligns with the results reported in previous studies concerning blank control samples.⁵³ Further analysis using micro-Raman spectroscopy revealed that the particles present in these control samples were primarily composed of polyester, polypropylene (PP), and polyethylene terephthalate (PET). This qualitative analysis confirmed the composition of the detected MPs.⁵⁴ Even under tightly controlled conditions, air pollution is difficult to eliminate.⁵³

Fig. 6 Representative micro-Raman spectra of PP and PET MPs collected from glass fiber filter papers.

Based on the findings presented in Fig. 7D and F, it is evident that the shedding of PP and PET MPs was the most significant under acidic conditions with a high oil content when no coating was applied. This observation is attributed to the inherent sensitivity of plastics to acidic environments. Consequently, many current extraction methods prefer alkaline digestion over acid digestion when isolating MPs.⁸ Furthermore, the release of MPs is also facilitated in high oil environments. This can be rationalized by the hydrophobic nature of MPs and the principle of like dissolves like.⁵⁵ We elaborated on the mechanism of MPs shedding under various conditions in another study, which is not within the scope of consideration herein. Therefore, we conducted a single factor experiment using acidic high oil parameters to compare the crosslinking effect of genipin. As shown as Fig. 7A, when the concentration of genipin reached 1 mg mL⁻¹, the shedding amount of PP and PET MPs decreased by 57.81% and 64.46%, respectively. This indicated that the cross-linked PTG had a better physical and chemical properties than before, which is consistent with previous results. More interestingly, the particle sizes of MPs were further compared under acidic high oil conditions (Fig. 7B and C). Through the resistance of PTG coating and genipin@PTG coating, MPs with particle size exceeding 500 μm were not detected. Moreover, the genipin@PTG coating could further inhibit the shedding of MPs with size in the range of 100–500 μm. We believe that the cross-linked network structure of genipin@PTG coating could restrict the shedding of large-size MPs. According to Fig. 7D–G, MP shedding in various harsh environments could be effectively controlled by the genipin@PTG coating, and the shedding rates decreased by 90%–98%. Furthermore, the effect of multi-layer genipin@PTG coatings in suppressing MP shedding was further compared. Under the three-layer genipin@PTG coating, the shedding rate of PP MPs decreased by 97.28%, which is significantly higher than that by the one-layer genipin@PTG coating of 91.60%. Also, the inhibition rate of PET MP shedding increased from 92.76% to 97.09%. A small amount of MP shedding may be due to the lack of a dense coating on the plastic side. However, overall, the genipin@PTG coating was a highly effective method for inhibiting the shedding of MPs.

Fig. 7 Abundance of MPs based on particle size percentage. MP abundance in simulated environment containing acidic with high oil content (A); PP MP size percentage in simulated acidic environment with high oil content (B); PET MP size percentage in simulated acidic environment with high oil content (C); PP MP abundance in different simulation environments (D and E); PET MP abundance in different simulation environments (F and G); MP abundance under different coating layers (*p < 0.05) (H); and MPs abundance under different proteins and preparation methods (I).

Additionally, glutenin is commonly used as a raw material for protein packaging coatings in the past.⁵⁶ Thus, it is easy to speculate whether other protein packaging materials can be used to suppress the shedding of MPs. Casein, collagen, and zein have been used as packaging materials and coatings.^57–60 However, the coating formed by these proteins after TCEP and genipin treatment did not have the ability to inhibit the MP shedding, as shown in Fig. 7I. The observed failure of certain protein-based coatings to effectively adhere may be attributed to the absence of disulfide bonds, which are crucial for self-assembly. Despite forming a coating with inherent adhesion properties, these coatings underwent significant peeling when exposed to an acidic environment with a high oil content at 95 °C for 2 h. To further investigate this, we compared the impact of various operational steps on the performance of the coating. Notably, when glutenin was first crosslinked in solution, and subsequently treated with TCEP, the resulting coating did not significantly inhibit the shedding of PP and PET MPs. These findings underscore the critical role of the genipin@PTG coating preparation processes in achieving the desired adhesive and inhibitory properties. Therefore, it is essential to meticulously control the coating preparation steps to ensure the effectiveness of the coating in preventing MP shedding.

3.6. Safety assessment of PTG coating and genipin@PTG coating

Cell proliferation experiments were conducted to evaluate the effect of coatings on the growth of intestinal epithelial NCM460 cells. As shown in Fig. 8, the inhibition rates in the extracts obtained after 24 h of treatment at 37 °C and 2 h of treatment at 95 °C were both less than 15%. This indicates that both the PTG coating and genipin@PTG coating exhibited low toxicity or non-toxicity to cells. Coatings formulated with proteins as fundamental building blocks are known to exhibit low cytotoxicity and elicit minimal immune responses.^26,61 Overall, the PTG coating and genipin@PTG coating had excellent safety and can meet the requirements of food packaging.

Fig. 8 Inhibition rate of NCM460 cell proliferation by PTG and genipin@PTG coating. Note, “37” and “95” refer to the incubation temperature at which the extraction solution was obtained from the coating, respectively.

4. Conclusion

This study introduced a method for the preparation of a green and biocompatible coating for suppressing the shedding of MPs from plastic packaging. This coating was produced by the phase transition of glutenin after TCEP reduction, and could spontaneously adhere to any plastic substrate surface under mild conditions. To further enhance its mechanical properties and tightness, genipin was used for cross-linking the coating. After crosslinking, the hydrophobicity of the coating increased (from 84.7° and 96.1°), the roughness decreased (from 0.59 to 0.31 μm), the elastic modulus increased (from 19.85 GPa to 30.87 GPa), and the water vapor permeability decreased (from 21.45 g per m² per 24 h to 16.31 g per m² per 24 h). More importantly, the shedding numbers and sizes of MPs were significantly reduced. Overall, the suppression rate of MP shedding by the coating under the harsh simulation environments ranged from 92% to 98%. Compared to animal protein, we believe that the glutenin used in this work is more economically acceptable to manufacturers.^62,63 However, based on our experience, the friction resistance of the coatings is limited. Thus, this study only considered the release of microplastics in liquid environments and ignored the impact of mechanical damage. To further enhance the practical application value of coatings, it may be feasible to incorporate polysaccharides or modify polymer chains in them in the future.^63,64 In summary, it is necessary to explore more advanced packaging materials in the future to resist the shedding of MPs.

Author contributions

Yulun Chen: conceptualization, methodology, writing – original draft, review & editing; Qingrun Liu: investigation, data curation, formal analysis; Jianjun Ding: visualization, data curation; Shaofeng Yuan: supervision, visualization. Hang Yu: formal analysis, writing – review & editing, supervision. Yahui Guo: supervision, review & editing. Yuliang Cheng: review & editing, supervision. He Qian: review & editing, supervision. Weirong Yao: formal analysis, supervision, funding acquisition, writing – review & editing, project administration.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

The work described in this article was supported by National Natural Science Foundations of China (32302247 and 32172326) and Jiangxi Province High-Level Innovation and Entrepreneurship Team Project (jxsq2023105005). Yulun Chen sincerely thanks the China Scholarship Council (CSC, 202406790044) for its support for this work.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4gc06154c

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