Multifunctional composite films by incorporating carboxylated cellulose nanocrystals stabilized p-coumaric acid pickering emulsion for cherry tomato preservation

Abstract

Bioactive films effectively prevent the quality deterioration of fruits during storage and transportation. In this study, multifunctional konjac glucomannan/methylcellulose (KGM/MC)-based films were developed by incorporating p-coumaric acid Pickering emulsion (p-CA PE) stabilized with carboxylated cellulose nanocrystals. The results indicated that the incorporation of PE promoted the uniform loading of hydrophobic p-CA within the KGM/MC-based network and exhibited good compatibility through hydrogen bonding interactions. p-CA PE films (PEF) exhibited excellent mechanical properties, hydrophobicity (WCA of 82.62°), UV/visible light barrier properties and antioxidant activity. The low hemolysis rate and high cell viability demonstrated the film biosafety. The application of PEF coating maintained quality of cherry tomatoes and activated key enzymes of the phenylpropanoid pathway. Importantly, fruits with PEF coatings defensed the growth of Botrytis cinerea during storage. Therefore, the multifunctional films coating successfully demonstrated the potential for maintaining fruit freshness and preventing spoilage during supply chain.

Graphical abstract

Highlights

• •p-CA pickering emulsion (PE) was stabilized by carboxylated cellulose nanocrystals.
• •Incorporation of PE improved mechanical and water resistance of the films.
• •Films maintained fruit quality and prevented spoilage of cherry tomatoes.
• •Key enzymes of phenylpropane metabolism in fruits with films were activated.

1. Introduction

With the increasing attention of consumers on nutrition and healthy diet, the demand for fruit and vegetable products rich in vitamins, minerals and dietary fiber continues to grow. However, the rapid physiological changes and fungal infections occur in fresh fruit and vegetables during transportation and storage, resulting in great food waste and economic losses (Kumar et al., 2023). Appropriate food packaging to address the preservation challenges of high-moisture and enzyme-activity fruit and vegetable products has become an urgent priority. Traditional food packaging lack resistance to atmospheric oxygen, moisture and fungal spores, and the prolonged degradation cycle raises concern of environmental pollution. In contrast, environmentally friendly bioactive films provide barrier properties, antioxidant and antifungal activities by incorporating the active components, representing a promising option within the food packaging (Dai et al., 2020). Especially, natural polyphenol compounds influence physiological metabolism of fruits and vegetables, and displayed vital roles to defense various environmental stress (Mao, Zhang, Wu, et al., 2025). Therefore, the development of bioactive films containing natural polyphenols represents a promising candidate strategy for the preservation of fruit and vegetables.

Phenolic compounds in fruits and vegetables are important secondary metabolites that play a key role in resisting adverse fruit stress (Luo et al., 2026). p-coumaric acid (p-CA) is a phenolic acid of the hydroxycinnamic acid family and synthesized in phenylpropanes metabolism, existing as free or ester form in fruits and vegetables (El-Seedi et al., 2012). It acts as a precursor and can be converted into phenolic acids (caffeic acid, ferulic acid, chlorogenic acid), flavonoids, lignin precursors and other secondary metabolites, thereby enhancing the stress tolerance of plant (Pei et al., 2016). Meanwhile, the unique phenolic acid structure confers antioxidant, antibacterial and anti-inflammatory properties (Li et al., 2023; Tehami et al., 2023). For practical applications on food preservation, p-CA exhibited broad-spectrum antibacterial activity against Gram-negative, Gram-positive bacteria and fungi (El-Seedi et al., 2012; Liu, Shen, et al., 2024). Additionally, p-CA inhibited the activity of chlorophyll degradation enzymes and preventing over-accumulation of ROS, thus significantly delayed yellowing and senescence of fruits and vegetables (Zhang, Yang, et al., 2023). However, the direct incorporation of p-CA in composite films applications existed limitations, due to low solubility of p-CA and susceptibility of environmental variables (Ma et al., 2025a). Therefore, exploring straightforward and feasible strategy to enhance phenolic acid utilization presents a challenge for food packaging.

Recently, nanoemulsions have garnered significant attention as delivery vehicles for protecting active substances in food preservation (Ma et al., 2025b). Compared to conventional nanoemulsions, Pickering emulsion (PE) exhibited superior stability and controlled-release potential as green encapsulation systems, which was achieved through solid particles derived from polysaccharides, proteins, and lipids acting as emulsion stabilizers that adsorb at the oil-water interface (Tian et al., 2024). Cellulose nanocrystals (CNCs) derived from plants and bacteria exhibited significant potential as food emulsifiers due to their high crystallinity, small size, large specific surface area and amphiphilic. However, the high hydrophilicity from the abundant hydroxyl groups on the CNC surface limited their practical application (Ji & Wang, 2025). Carboxylated cellulose nanocrystals (C-CNCs) were obtained by oxidizing hydroxyl groups on C6 in CNC to carboxyl groups via ammonium sulphate, and this modification resulted in numerous negative charges on the surface of C-CNC, which led to better dispersion through electrostatic repulsion (Rosalina et al., 2023). Therefore, the utilization of p-CA PE stabilized by C-CNCs holds particularly promising for enhancing film properties.

Natural polysaccharide represents promising alternatives to conventional petroleum-based materials due to their excellent film-forming properties, biodegradability, safety, wide availability and mild odor (Wu et al., 2026). Especially, konjac glucomannan (KGM), as a neutral nonionic polysaccharide extracted from Konjac tuberb (Wang et al., 2024), which has been used to prepare films due to good biocompatibility, renewability and safety. The KGM molecular chain consists with d-glucose and D-mannose linked via complex network of hydrogen bonds, helical structures and topological structures, which was available for encapsulating active substances (Sun et al., 2023). However, individual KGM films had low mechanical and barrier properties and was susceptible to dissolve in water (Chen et al., 2018; Wang et al., 2024). To overcome these limitations, mixing with other biopolymers or nanomaterials were benefit for improving physical properties (Du et al., 2013; Li et al., 2019; Zou et al., 2021). Methyl cellulose (MC) is a hydrophobic-modified cellulose produced by methylation with good film-forming properties, and improved the mechanical, barrier and hydrophobic properties of individual polysaccharide matrixes with adding MC (Pérez et al., 2013). Studies have shown that compared with carboxymethyl cellulose (CMC), microcrystalline cellulose (MCC) and hydroxypropyl methyl cellulose (HPMC), MC exhibited reinforcing effect on natural polysaccharide matrices (Li et al., 2026). Therefore, the combination of KGM and MC is alternative matrixes for preparing bioactive films.

This study aims to develop KGM/MC-based packaging films for fruits and vegetable preservation by incorporating C-CNC-stabilized p-CA PE. The p-CA PE composite films (PEF) were investigated the structure, barrier property, optical property, antioxidant activity, release kinetics and biosafety. Additionally, the PEF coating was applied in the preservation of cherry tomatoes, systematically analyzing the effects on physiological indicators of the fruits and the resistance to the pathogen B cinerea. These results provide potential application of active coatings incorporating phenolic acid-pickling emulsions in the field of fruit and vegetable preservation.

2. Materials and methods

2.1. Materials

Konjac glucomannan (a molecular weight of ∼1000 kDa, purity ≥95%, viscosity ≥15,000 mpa·s), glycerol, rutin, gallic acid and methanol were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Methyl cellulose (viscosity 4000 cP) was purchased from Shanghai Biyuntian Biotechnology Co., Ltd. (Shanghai, China). p-CA, ethyl acetate, sodium carbonate and other inorganic reagents were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Carboxycellulose nanocrystals were purchased from Guilin Qihong Science and Technology Co. Ltd. (Guilin, Guangxi, China). The cherry tomatoes were harvested in local orchard and delivered to the laboratory (Changchun, China).

2.2. Preparation and characterization of p-CA Pickering emulsion

The p-CA Pickering emulsion (PE) was prepared with C-CNC as emulsifier according to the method of Mikulcová et al. (2018). Ethyl acetate (10%, v/v) was used as oil phase and was mixed thoroughly with p-CA powder (15 mg/mL, maximum solubility). Subsequently, 0.54 g of C-CNC was dissolved in 54 mL distilled water and was sonicated for 20 min (400 W). The C-CNC suspension obtained as emulsion with 90% aqueous phase. Finally, PE was treated with ultrasonic cell crusher for 15 min (Sonics Vibra-Cell, U.S.), with 3 s of ultrasound and 2 s of stopping. The entire procedure was performed on ice bath and PE was stored at 4 °C. The particle size distribution, zeta potential and polydispersity index of PE were measured using Zetasizer Nano ZS 90 (Malvern Instruments ZEN3600, U.K.). The microstructure of PE was observed by scanning electron microscopy (SEM) (S-4800, Japan) at an accelerating voltage of 3 kV. The PE were heated in water bath at 40 °C, 50 °C, 60 °C and 70 °C for 30 min, then stability and particle size of emulsion were measured. Storage stability was assessed by storing the PE at 4 °C and 25 °C, with samples taken at 0, 2, 4, 6 and 8 d to measure particle size and PDI.

2.3. Preparation of composite films

KGM was dissolved in distilled water (2%, w/v) and glycerol (30% w/w of KGM) was added. The solution was heated and stirred at 70 °C for 30 min. The MC (1%, w/w) solution was dispersed in water at 80 °C and then stirred at room temperature. The MC (10%, w/w, based on KGM) was added into KGM solution and stirred for 1 h. Subsequently, PE was added at 5%, 10%, 15% and 20% of film matrixes. The film-forming solution (15 g) was poured over disposable petri dishes (diameter 90 mm) and dried at 40 °C for 12 °C. The composite films were named PEF5, PEF10, PEF15 and PEF20, and PEF0 was the control sample without PE.

2.4. Characterization of composite films

2.4.1. SEM and TEM-energy dispersive spectrometer (EDS)

The micro-morphological structure of film surface was observed using SEM (S-4800, Japan) with reference to the method of Liu, Li, et al. (2022). The films were cut using a sharp blade into 6 mm squares with neat edges, free from stretching or scratches. Then the samples were glued to a cross-section of aluminium with double-sided tape and sprayed a thin layer of gold on the surface. Subsequently, the morphology of film surface was observed at accelerating voltage of 3 kV under 2000 × magnification. EDS was used to analyze the elemental composition of film surface and the distribution of PE in film matrixes.

2.4.2. Attenuated total reflection-Fourier transform infrared (ATR-FTIR)

The ATR-FTIR spectra of the films were collected in the range of 4000–400 cm⁻¹ by scanning the film samples with a resolution of 4 cm⁻¹ using an ATR-FTIR spectrometer (INVENIO-R, China) (Chen, You, et al., 2023).

2.4.3. X-ray diffraction (XRD)

Diffraction angles (2θ) in the range of 3–60° of the films were detected at a rate of 0.5°/s using an X-ray diffractometer (D8 Advance, Germany) at 25 °C. The X-rays produced Cu Kα radiation at 40 kV and 40 mA (Bu, Huang, et al., 2022).

2.4.4. Thermal stability

Thermogravimetric analysis (TGA) was performed by a thermogravimetric analyzer (Pyris Diamond, USA) in a nitrogen atmosphere, and analyzed the mass change of composite films in the temperature range from 30 to 600 °C (Roy et al., 2022).

2.5. Mechanical properties of composite films

The samples were cut into rectangular strips with size of 80 mm × 10 mm, and the thickness was measured at eight positions using a micrometer (JDE01, Japan). The tensile strength (TS) and elongation at break (EAB) of the films were measured using a tensile testing machine (INSTRON-1121, UK). The rectangular specimens were fixed in the tensile gripper with initial gauge length of 20 mm and stretched at speed of 2 mm/min (Xu et al., 2026).

2.6. Barrier properties of composite films

2.6.1. Water vapor permeability (WVP)

The glass bottles (12 mm inner diameter) containing 5 mL of distilled water (100% RH) were sealed with films and placed in a container containing dry anhydrous calcium chloride (0% RH) for 8 h and weighed every 2 h (Priyadarshi et al. (2021). The WVP of the films was calculated by following equation:

$$\mathrm{WVP}\left(\mathrm{g}\bullet \mathrm{mm}\bullet {\mathrm{h}}^{-1}\bullet {\mathrm{m}}^{-2}\bullet {\mathrm{Pa}}^{-1}\right)=\frac{S\times d}{A\times P\times △\mathrm{R}}$$ (1)

In the equation S was the slope of the weight loss curve fit (g/h), d was the thickness of the film (mm), A was the area of the film at the top of bottle (m²), P was the saturated water vapor pressure (Pa), and ΔR was the difference in relative humidity (%) between two sides of the films, with a value of 100%.

2.6.2. Oxygen permeability (OP)

A composite deoxidiser was prepared by mixing sodium chloride, reduced iron powder and activated carbon in a ratio of 1:3:2, and oxygen was continuously consumed via electrochemical oxygen-absorption corrosion. The 3 g of deoxidant was put into glass bottles, then sealed with the films and placed in a dry dish with 55% humidity. The oxygen permeability was determined by measuring the weight change after 48 h (Rao et al., 2023). The OP was calculated as follows:

$$\mathrm{OP}\left(\mathrm{g}·\mathrm{mm}·{\mathrm{m}}^{-2}·{\mathrm{s}}^{-1}\right)=\frac{\left(m_{f-}{m}_0\right)\times d}{A\times t}$$ (2)

where m_f was the final weight of glass bottle after 48 h equilibration and m₀ was the initial weight of glass bottle. The d was the thickness of the films, t was the equilibration time and s was the area of top bottle.

2.6.3. Moisture content (MC)

The standardized samples (diameter 19 mm) were placed in an oven at 105 °C for 12 h, according to the method of Bu, Sun, et al. (2022). The MC of samples was obtained by the weight change during dying.

2.6.4. Water contact angle (WCA)

The film samples were fixed on a holder and 2 μL water was added to film surface for 30 s (Liu, Li, et al., 2022). The WCA of the films was determined by obtaining digital images of the droplets by a contact angle meter (DSAHT17C, Germany).

2.6.5. Water solubility (WS)

The films (diameter 19 mm) were placed in 15 mL of distilled water with shaking. The integrity and dissolution process of the film were recorded at 0, 2.5 and 7.5 h by taking photos (Han et al., 2024).

2.7. Optical properties of composite films

2.7.1. Color parameter

The a*, b*, L* and total color difference (ΔE) of the films were measured using a colorimeter (Konica Minolta CM-2300d, Japan). ΔE was calculated using the following formula:

$$\mathrm{\Delta E}=\sqrt{{\left(L^{\ast }-{L_0}^{\ast }\right)}^2+{\left(a^{\ast }-{a_0}^{\ast }\right)}^2+{\left(b^{\ast }-{b_0}^{\ast }\right)}^2}$$ (3)

L*, a*, b* were film color parameter values and L₀*, a₀*, b₀* were color parameter values of whiteboard.

2.7.2. Transparency property

The films were cut into rectangular strips and placed into the sample slot, and optical properties of the films were measured using a UV–Vis spectrophotometer (Shimadzu UV-2550, Japan) with wavelength scans from 200 to 800 nm, obtaining transmittance and opacity of the films (Liu, Lin, et al., 2020).

$$\text{Opacity}\left({\mathrm{mm}}^{-1}\right)=\frac{A_{600}}{d}$$ (4)

where A₆₀₀ was the absorbance of the film at a wavelength of 600 nm, and d was the film thickness (mm).

2.8. The antioxidant activities and sustained-release effect of composite films

2.8.1. The antioxidant activities of composite films

ABTS and DPPH radical scavenging activity were determined by the method of Ding et al. (2022). 200 mg of the films was weighed in a centrifuge tube containing 5 mL of 80% methanol and extracted for 12 h at 25 °C. ABTS radicals were generated by mixing 7 mM ABTS solution with 2.45 mM potassium persulfate and reacted in dark for 12 h. The absorbance at 734 nm of ABTS reaction solution was adjusted to 0.7 ± 0.02 with distilled water and reacted with sample extract at a volume ratio of 1:3, using 80% ethanol as a blank, and the absorbance at 734 nm was measured after 5 min reaction. For the DPPH reaction, the sample extract was reacted with 0.2 mmol DPPH reaction solution at a volume ratio of 1:3, and the absorbance at 517 nm was measured after 30 min. The free radical scavenging rate was calculated as follows:

$$\text{Free radical scavenging rate}\left(\%\right)=\frac{A_C-A_S}{A_C}\times 100\%$$ (5)

where A_C and A_S corresponded to the absorbance of blank and samples.

2.8.2. The sustained-release of composite films

Distilled water, 20% ethanol, 50% ethanol, 90% ethanol and 3% acetic acid were used as food simulation systems. The cumulative release of p-CA was determined according to the method of Liu, Lin, et al. (2022). 200 mg of PEF15 and PEF20 samples were weighed into centrifuge tubes containing 30 mL of simulation solution, and 100 μL solution was taken at regular intervals. The absorbance of p-CA was measured at 308 nm and the content of released p-CA was calculated.

2.9. Biosafety assessment of composite films

2.9.1. Hemolysis test and cytotoxicity assays

The hemolysis test and cytotoxicity assays were performed to evaluate the safety of PEF20 films. The hemolysis assay was estimated according to the method of Ni et al. (2023). The 1 mL anticoagulated lamb blood was centrifuged at 5000 rpm for 12 min. The lamb blood was washed three times and resuspended in PBS solution. Subsequently, the different concentrations of PEF20 film extracts (0.25, 0.50, 0.75, 1.00 mg/mL) were incubated with diluted blood cell at 37 °C for 1 h. The sterile water and PBS were used as positive and negative controls, respectively. After centrifugation at 5000 rpm for 12 min, the absorbance of supernatant was measured at 540 nm and relative cell viability was calculated as follows.

$$\text{Hemolysis rate}\left(\%\right)=\frac{A-A_{\mathbf{0}}}{A_{\mathbf{1}}-A_{\mathbf{0}}}\times 100\%$$ (6)

where A₀, A₁ and A represent the absorbance at 540 nm of positive control, negative control and experimental group, respectively.

The biocompatibility of film samples was evaluated by MTT cytotoxicity assays, and human hepatocytes (L−02) were selected as a model (Wu et al., 2026). L-02 cells with a density of 5 × 10³ cells/well were incubated for 24 h in CO₂ incubator. Different concentrations of film extracts (0.25, 0.50, 0.75, 1.00 mg/mL) were added and incubated for 24 h. Then, the solution was removed and incubated with 100 mL of MTT solution (0.5 mg/mL) for 4 h. The supernatant was discarded and 100 uL DMSO was added and the absorbance was measured at 490 nm. The cells without film extract as control group, and the cells with separate film extract and cell culture medium were as blank groups. Cell viability was calculated according to following equation.

$$\text{Cell viability}\left(\%\right)=\frac{A_2-A_{\mathbf{0}}}{A_{\mathbf{1}}-A_{\mathbf{0}}}\times 100\%$$ (7)

where A₀, A₁, and A₂ represent the absorbance of blank group, control group and experimental group at 490 nm, respectively.

2.9.2. Determination of ethyl acetate residues

Residues of ethyl acetate were analyzed using a gas chromatograph-mass spectrometer (Thermo Fisher TSQ 9000) via headspace solid-phase microextraction coupled with gas chromatography–mass spectrometry (HS-SPME-GC–MS). The 0.1 g of film sample and 1 μL of 2,4,6-trimethylpyridine (1000 ppm) were placed in a 20 mL headspace vial. Added 2.0 g of sodium chloride to promote the release of volatile components, sealed the vial and equilibrated in 40 °C water bath for 15 min. The fiber tip was aged for 45 min at 250 °C, inserted into the headspace vial and exposed to the sample headspace, and then extracted for 40 min at 60 °C. Upon completion of extraction, the samples were desorbed at 250 °C for 5 min. An HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm) was used, with high-purity helium (purity ≥99.999%) as the carrier gas at a flow rate of 1.0 mL/min, and the splitless injection was employed. The injector temperature was set at 250 °C. The temperature procedure was as follows: the initial temperature was held at 40 °C for 3 min, then increased at a rate of 5 °C/min to 150 °C, followed by increase at 10 °C/min to 250 °C and was held for 5 min. The mass spectrometer detector utilized electron impact (EI) ionisation with an ionisation energy of 70 eV, an ion source temperature of 230 °C and transfer line temperature of 280 °C. The scan range was m/z 35–550, and data were acquired in full-scan mode. Compounds were identified by comparison with standard ethyl acetate samples (purity >99.9% by GC) under same conditions, and searching the NIST 21 and Wiley 275 mass spectrometry databases. Identification based on forward and reverse matches exceeded 80%, and retention indices (RI) was used for auxiliary confirmation. The retention index was calculated from the retention times, applying the linear retention index formula. Quantification of the compounds was performed using the internal standard method with 2,4,6-trimethylpyridine as the internal standard.

2.10. Application composite films for cherry tomatoes preservation

2.10.1. Tomato fruits with PEF coating

The tomato fruits of similar size and maturity were selected and immersed in sodium hypochlorite solution (0.3%, w/v) for 3 min, and then washed with distilled water. The fruits were soaked in PEF0, PEF15 and PEF20 film-forming solutions for 30 s and dried at room temperature. The control group was treated with distilled water under the identical conditions.

2.10.2. Determination of color change, weight loss and firmness

For analysis of color change, four points at equator of tomato fruits were measured randomly and repeated eight times using a colorimeter (Konica Minolta CM-2300d, Japan) to record the values of L*, a*, b* parameters. The formula in 2.8.1 was used to calculate ΔE. Weight loss was determined by measuring the weight of fruits and calculating the percentage reduction based on the initial weight. A texture analyzer (Brookfield, CT3, USA) and a flat-tipped probe with a diameter of 10 mm were used to determine fruit firmness. The test traversed 3 mm distance at speed of 1 mm/s and maximum force was recorded as fruit firmness (Arabpoor et al., 2021; Wang et al., 2019).

2.10.3. Determination of total soluble solids (TSS) and titratable acid (TA)

Fruits were crushed in a mortar and centrifuged with 8000 rpm at 25 °C for 10 min, which was used for determination of TSS and TA. A drop of supernatant was placed in panel on the refractometer and observed TSS value (Gang et al., 2024). The acid-base titration method was used to assess the TA of cherry tomatoes. The supernatant was diluted and 20 uL phenolphthalein was added, and the solution was titrated using 0.1 mol/L NaOH solution (Long et al., 2022).

2.10.4. Determination of total phenols and total flavonoids content

Total phenols and total flavonoids were determined according to the method of (Tabaszewska et al., 2023).

2.10.5. Determination of antioxidant capacities

Determination of DPPH and ABTS free radical scavenging capacities of tomato fruits referred to 2.9.1.

2.10.6. Measurement of enzymatic activities

To determine polyphenol oxidase (PPO) and catalase (CAT) activities, 0.8 g of tomato fruits were ground with liquid nitrogen and mixed with 4 mL of pre-cooled phosphate buffer (20 mmol/L, pH 7.2) containing 2% (w/v) polyvinyl pyrrolidone. Subsequently, the mixture was centrifuged at 12,000 rpm (10 min, 4 °C) and the supernatant was used for further analysis. PPO and CAT were determined with reference to Zeng et al. (2024). The key enzymes of phenylpropane metabolism phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL) were determined (Liu, Ji, et al., 2020). The unit of enzyme activity was defined as an increased absorbance of 0.01 per minute per gram of samples.

2.10.7. Disease resistance of tomato fruits with composite films

To determine disease resistance of Botrytis cinerea, the tomato fruits were wound (3 mm × 1 mm) on the surface using sterilized tip and then immersed in different concentrations of PE solution. The incubated spore suspension (1 × 10⁶ CFU/mL) 20 uL was inoculated into the fruit wounds. The fruits were stored at room temperature and 95% RH, and the lesion diameter of fruits was measured (Liu et al., 2019).

2.11. Statistical analysis

Each parameter was repeated at least three times and the data were expressed as mean ± standard deviation. Homogeneity of variances was assessed using the homogeneity of variance test, and the normality of the data was verified using nonparametric test (Kolmogorov–Smirnov test). Statistical analysis was performed using SPSS 26.0 software, employing analysis of one-way ANOVA and Duncan's test, and p < 0.05 was considered a significant difference. For postharvest indicators evaluated over storage time, a two-way repeated measures ANOVA was employed. Where the interaction effect was significant (p < 0.05), simple effect analysis was conducted. Where the interaction effect was not significant (p > 0.05) and the main effect was significant, multiple comparisons of main effects were performed. Graphs were plotted using Origin 2025 software, with different letters indicating significant differences (p < 0.05).

3. Results and discussion

3.1. Characterization of PE

To improve the solubility and bioavailability of p-CA, p-CA PE was prepared using carboxylated cellulose nanocrystals as a stabilizer. As shown in Table 1, p-CA PE exhibited excellent dispersion, with a polydispersity index (PDI) of 0.26, suggesting that the preparation of the emulsion effectively improved the solubility of p-CA. Furthermore, the emulsion composite exhibited a negative potential of −24.05 mV, which was primarily attributable to the negative charge carried by the C-CNC surfaces stabilizing the interface (Rosalina et al., 2023). The particle size distribution, SEM analysis and appearance of PE are shown in Fig. 1. The PE was transparent yellow liquid with spherical oil droplets and concentrated particle size distributed with average particle size of 199.93 nm (Fig. 1A), which was consistent with the droplet size observed by SEM. The stability of PE was evaluated with particle size and state changes at different temperatures. As shown in Fig. 1B, the particle size achieved maximum value at 40 °C, which the particle size increased by 22.67% compared with that of control samples. When the temperature constantly increased, the oil droplets might aggregate and form stable state, leading to the increase of particle size (Xu et al., 2023). However, as the temperature continued to increase, the particle size gradually decreased. Studies have shown that C-CNC formed stable two-dimensional structure on the oil droplets through irreversible adsorption, which was proportional to the temperature changes (Rahmi et al., 2025; Wen et al., 2014). When the temperature rose to 70 °C, the temperature close to the boiling point of the oil phase caused the vaporization of ethyl acetate, resulting in a reduction in the volume of the core oil and significant reduction in particle size at high temperatures. As can be observed in Fig. 1C, no demulsification phenomenon occurred during the heating process, indicating that PE was stable in the process of film development. In addition, the storage stability of PE over an 8-day period was evaluated, with the results shown in Fig. S1. Under conditions of 4 °C and 25 °C, the particle size exhibited an overall increasing trend as the emulsion underwent aggregation and flocculation due to factors such as Brownian motion and van der Waals forces. In particular, C-CNC may undergo processes of desorption and re-adsorption, leading to a temporary decrease in particle size (Cai et al., 2024). Meanwhile, the PDI continued to increase during storage, which was attribute to the aggregation or sedimentation resulting from the effects of gravity, interparticle interactions or rearrangement at the oil-water interface, leading to an uneven particle size distribution. After 8 d of storage, the particle sizes of the emulsion at 4 °C and 25 °C were 361.83 ± 9.61 nm and 365.00 ± 7.05 nm respectively, remaining at the nanometer level, and the PDI were both less than 0.4. These results indicated that the emulsion was stable at common temperature conditions.

Table 1.

Particle size, polydispersity index and zeta-potential of the p-CA PE.

Sample	Particle size (nm)	Polydispersity index	Zeta-potential (mV)
p-CA PE	199.933 ± 7.086	0.263 ± 0.017	−24.050 ± 2.333

Values are expressed as mean ± standard deviation.

Fig. 1.

(A) The visual appearance, SEM image and particle size distribution of p-CA Pickering emulsions. (B) Average particle size and (C) visual appearance of p-CA Pickering emulsion at temperatures ranging from 40 to 70 °C, different letters represent significant differences (p < 0.05). (D) SEM image of composite film surface. (E) EDS analysis of elements C, O and Na distribution in PEF20. (F) ATR-FTIR analysis of composite films. (G) Schematic representation of potential mechanism and (H) XRD analysis of composite films.

3.2. Characterization of composite films

3.2.1. SEM analysis of the films

As shown in Fig. 1D, the surface of PEF0 films was flat, smooth and no cracks, indicating that MC and KGM combined tightly and had good compatibility. The addition of PE slightly increased the surface roughness of the films, with a minor free p-CA crystals appearing on the surface, which were not aggregated seriously and changed the micromorphology of KGM/MC matrixes. Specially, no crystals were observed on the surface of PEF20, which might be attributed to the uniform distribution of droplets and less flocculation and agglomeration in the films. The results indicated that the PE was evenly dispersed in film matrixes, which was related to the size of emulsion droplets and film compatibility (Dai et al., 2023; Wei et al., 2024). Therefore, incorporating PE in the films did not adversely affect the surface structure, which due to hydrogen bonding interactions existed between film matrixes and PE. Meanwhile, few spherical particles precipitated on the film surface, indicating that emulsion was successfully encapsulated in film matrixes. The uniformity of PE distribution and elemental surface composition in PEF20 were analyzed by EDS. The results showed that C, O and Na elements existed in PEF20, and the Na element was related with the distribution of PE in matrixes (Fig. 1E), indicating the PE was uniformly distributed in film matrixes.

3.2.2. ATR-FTIR analysis of the films

As shown in Fig. 1F, the films show broad peaks at 3600–3000 cm⁻¹, corresponding to the presence of -OH groups in MC and KGM, and the peak around 1640 cm⁻¹ is associated with intermolecular hydrogen bonding. Compared with PEF0, the absorption peaks of the films were red-shifted from 1645 cm⁻¹ to 1635 cm⁻¹, which might be attributed to hydrogen bonding interactions between PE and film matrixes, resulting in dense structures of PE films (Yang et al., 2024). The absorption peak at 2924 cm⁻¹ was the symmetrical vibration peak of -CH group in KGM and MC. In addition, the peaks at 1016 cm⁻¹ and 1074 cm⁻¹ were mainly attributed to the stretching vibration of C-O-C in polysaccharides (Chen, You, et al., 2023). Incorporation of PE in the films, the asymmetric vibration of C O group of the carboxyl anion in C-CNC and p-CA showed peaks at 1728 cm⁻¹. In addition, new peaks appeared at 1605 cm⁻¹ and 1514 cm⁻¹ correspond to the C—C stretching vibrations of a small number of p-CA aromatic rings that have been freed from emulsion aggregation on the film surface (Liu, Shen, et al., 2024). Meanwhile, no new covalent bonds were formed between emulsion and film matrixes (Fig. 1G), and displayed physical crosslinking with hydrophobic interaction and hydrogen bonding.

3.2.3. XRD analysis of the films

As shown in Fig. 1H, all composite films showed obvious diffraction peaks at 2θ = 7.7°. The diffraction peak became broadened and diffused at 2θ = 10° - 28°, which was mainly due to the dislocations of incomplete crystals in KGM and MC. The diffraction of fine crystals led to peak broadening, indicating the semi-crystalline properties of the films (Wang et al., 2024). Although the addition of PE decreased peak intensity, the position and width of peak was no changes, indicating that PE did not destroy the semi-crystalline structure of the films. Interestingly, the intensity of diffraction peak at 2θ = 22.2° increased with adding PE, which was probably due to the hydrogen bonding between PE and film matrixes according to the FTIR results (Panda et al., 2019). The films with incorporating PE showed no other peaks in diffraction angles, indicating that PE had good compatibility with film matrixes, which was consistent with the SEM results. Additionally, Fig. 1H showed that the intensity of 2θ = 7.7° peak was slightly increased in the composite films. This might be due to the intermolecular interaction between MC, KGM and PE, which stimulated the formation of CH⋯O, CH⋯N, H⋯ON and OH⋯O, thereby forming a semi-crystalline domain (Shen et al., 2021; Zhang et al., 2024), which was verified by ATR-FTIR analysis.

3.2.4. TGA analysis of the films

Three weight loss stages of the films were indicated in Fig. 2A-B. The first phase occurred between 40 and 130 °C, which might be caused by the evaporation of free water and volatile components in the films. Meanwhile, the degradation rate in this stage slowed down with the increase of PE content, indicating that PE has barrier effect against the evaporation of moisture and volatile components (Yang et al., 2024). The second phase was at 140–270 °C and two rate acceleration in this stage, which might be related to the degradation of glycerol and bound water. Furthermore, the peak of the films shifted to low temperature with the increase of PE, which might be related to the degradation of partially encapsulated p-CA by high temperatures. The main phase of weight loss between 270 °C and 360 °C, which was corresponded to the thermal decomposition of polysaccharide backbone degradation. The PEF0 had the fastest degradation rate and the mass retention rate of the films loaded PE was higher than that of PEF0 (Bu, Huang, et al., 2022). The above results showed that addition of PE improved the thermal stability of composite films, which was attributed to the interaction between PE and film matrixes through hydrogen bonds, and formed dense network structure.

Fig. 2.

Thermogravimetric analysis of (A) TGA curves and (B) DTG curves for composite films. (C) Thickness, (D) density, (E) tensile strength and (F) elongation at break of composite films. (G) Water vapor permeability and oxygen permeability, (H) water contact angle, (I) moisture content, (J) water swelling of composite films. Different letters indicate significant differences (p < 0.05).

3.3. Mechanical properties of composite films

The film thickness and density were shown in Fig. 2C-D and addition of PE displayed significantly differences. The thickness of PEF15 and PEF20 was significantly lower than that of PEF0, whilst their density increased significantly. This might be attributed to the uniform filling of PE within the network voids of KGM/MC. Meanwhile, addition of C-CNC in the films reduced the spatial resistance of polysaccharide molecules, while the hydrogen bonding improved dense network structure of the films, reduced film thickness and enhanced film density (Roy et al., 2022; Zhang, Chen, et al., 2023). As shown in Fig. 2E and F, incorporation of PE significantly enhanced TS values of the films, and TS increased from 5.72 MPa (PEF0) to 8.73 MPa (PEF15). The TS of PEF20 decreased slightly and no significant difference compared with PEF15. The improvement of TS could be attributed to the uniform dispersion of PE in the film matrixes and high film continuity, avoiding stress concentration under external loading. Furthermore, the solid crystals stabilized PE formed hydrogen bonding interactions with the films, thereby increasing the degree of cross-linking and forming dense network structure (Li et al., 2025). The EBA of PE-added films was significantly higher than that of PEF0, and the EBA of PEF15 films was 31.85% higher than that of PEF0. This might be attributed to that PE was available to deform in the form of tiny oil droplets, thereby improving the ductility of composite films. The decrease in EBA of PEF15 and PEF20 might be due to the increase of PE content, which cross-linked with the films matrixes and enhanced the rigid structure of the films, acting as a hindrance to film ductility compared with PEF5 (Dai et al., 2023; Zhou et al., 2024). However, the highest loading level of PEF20 did not show any significant adverse effects on EAB compared with PEF0. Therefore, PE stabilized with C-CNC improved mechanical properties of composite films.

3.4. Barrier properties of composite films

The WVP and OP of the films are important indicators for evaluating barrier properties of the films, and the values are inversely proportional to the barrier properties. As shown in Fig. 2G, the WVP and OP of PEF0, PEF5, PEF10, PEF15 and PEF20 exhibited significant differences. Compared to PEF0, the WVP of films with high PE content were significantly decreased, indicating that the addition of high content PE can improve the moisture barrier properties of the films. This might be due to that addition of PE enhanced the network structural density of the films, resulting in an increased tortuosity of water molecules through the films (Zhang et al., 2024). In addition, the addition of PE increased the film hydrophobicity by reduced the number of free hydroxyl groups in the films, which inhibited the binding and adsorption of water vapor. The results of OP were essentially similar with those of WVP. The reason might be that emulsion droplets create intermolecular forces with film matrixes by filling gaps in the film-forming matrixes, reducing the mobility of polymer molecular chains and oxygen passage (Yang et al., 2024). Therefore, the incorporation of PE was effective for improving barrier properties of composite films.

3.5. Hydrophilicity of composite films

The WCA reflects the hydrophilicity of surface, and when the WCA of the films was higher than 65°, it could be considered hydrophobic. It has been shown in Fig. 2H that the WCA increased from 56.58° to 82.62°, indicating that the films transformed from hydrophilic surface to hydrophobic surface. This might be due to hydrogen bond interaction between the films and hydrophilic groups of PE, which led to the exposure of PE hydrophobic groups and reduced hydroxyl groups on film surface (Zhao et al., 2022). As shown in Fig. 2I, the PEF0 exhibited the highest MC, which was attributed to the abundance of hydrophilic groups of KGM/MC matrixes (Chen et al., 2018; Wang et al., 2024). The MC gradually decreased from 12.93% to 11.32% with increasing PE content in the films. The matrix-water interaction is weakened due to hydrogen bonding between hydrophilic group of PE and the films, which was consistent with the first decomposition step of TGA curves (Zhao et al., 2023). The WS of films are shown in Fig. 2J, and the PEF0 films ruptured after 2.5 h and completely dissolved in distilled water by 7.5 h. In contrast, the PE-loaded films retained relatively intact shape after 7.5 h. These phenomena demonstrated that the incorporation of PE effectively enhanced resistance to water of composite films by forming robust network structure with the polysaccharide matrix.

3.6. Optical properties of composite films

The color parameters of the films are represented by the values of L*, b*, a* and ΔE in Table S1. The addition of PE showed decrease of b*, increase of a*, and significant decrease in L* of PEF15 and PEF20 compared with PEF0, indicating that addition of PE increased the darkness and yellow color of the films. Based on the SEM results, addition of PE led to the appearance of crystals on film surface, resulting in increased diffuse reflection and light absorption, thereby a decrease in L* and an increase in ΔE (Yao et al., 2022). The increase of b* values was related with the light yellow of PE, and the film color changed to yellow with adding high content of PE.

It can be seen from Fig. 3A that the film containing PE at 335–370 nm had good ultraviolet shielding effects, which was mainly due to the high ultraviolet absorption ability of aromatic group in PE (Bu, Sun, et al., 2022). This has a positive effect on preventing oxidation, discoloration and lipid spoilage of food. It is worth noting that addition of PE increased transparency of the films in visible region, which corresponded to a decrease in opacity at 600 nm. This phenomenon correlated with the uniform distribution of PE within the film substrate, which improved polymer compatibility. (Zhang et al., 2024). The results indicated that the films could shield ultraviolet light and maintain good transparency in visible light region, which can improve consumer acceptance and provide a new strategy for application in food packaging.

Fig. 3.

(A) Light transmittance, (B) DPPH and ABTS radical scavenging activity of composite films, different letters represent significant differences (p < 0.05). p-CA release from (C) PEF15 and (D) PEF20 in water, 20% ethanol, 50% ethanol and 3% acetic acid systems. p-CA release from (E) PEF15 and (F) PEF20 in 90% ethanol system. (G) Hemolysis test and (H) cytotoxicity evaluation of PEF20 films (****p < 0.0001).

3.7. The antioxidant activity of composite films

As shown in Fig. 3B, the radical scavenging capacities of different composite films towards DPPH and ABTS radicals reflect antioxidant activity. The addition of PE effectively enhanced the antioxidant activity of films, and increased gradient with PE content. With the PE content in the films reached 20%, the DPPH scavenging rate increased significantly from 32.08% to 63.42%, and the ABTS scavenging rate increased from 2.50% to 74.69%, indicated that PE had significant effect on improving the antioxidant activity of composite films. This was mainly due to the role of hydroxyl group on the aromatic ring of the released p-CA. The hydroxyl group on the bicyclic ring acted as a hydrogen atom or electron donor, cooperating with the lone electron pairs of nitrogen in ABTS and DPPH to break free radicals and inhibiting the oxidative chain reaction (Liu, Sun, et al., 2024). Oxidation is the primary reason of browning and quality deterioration of fruit and vegetables. Therefore, incorporating p-CA PE into the film provided antioxidant protection for fresh produce, reducing oxidation-related losses and thereby extending shelf life.

3.8. Sustained-release effect of composite films

Based on the excellent mechanical, barrier and optical properties of PEF15 and PEF20, these were selected for simulated release analysis. Cumulative release of p-CA from PEF15 and PEF20 films in five food simulation systems (distilled water, 20% ethanol, 50% ethanol, 90% ethanol, and 3% acetic acid) was determined. First, the p-CA was released rapidly in first hour in the systems of distilled water, 20% ethanol, 50% ethanol and 3% acetic acid (Fig. 3C and D), and then slowly released in the following 2–4 h and achieved equilibrium. It was possible that the entry of water molecules caused the rapid expansion of PEF15 and PEF20, enhancing the contact of PE and simulated solution, while the polarizing properties of ethanol and acetic acid molecules led to the rapid release of p-CA. Meanwhile, acetic acid had a degradation effect on the film substrate and accelerated the release of p-CA. However, as shown in Fig. 3D and E, PEF15 and PEF20 achieved slow release in a 90% ethanol system, which might be due to the low water of system inhibiting film swelling and the PE remained firmly embedded within the film matrixes (Mao, Ren, et al., 2023). The results indicated that PE had sustained release effect and can migrate to the film surface effectively. Interestingly, composite films had potential applications in lipid-containing foods (Mao, Li, et al., 2023; Zhao et al., 2024). It is worth noting that in vitro release using liquid simulation systems merely reflected Fickian/non-Fickian matrix diffusion processes of active substance in coating matrix. In practical applications, compared with pre-formed films that physically attached to the fruit peel surface, the polymer network cross-linked with the lipophilic cuticular wax during the impregnation and coating process, further the partition equilibrium of the coating and the cuticular wax changed the retention and permeation behavior of active substance. Consequently, the release profile was regulated by the partition coefficient (Kwax/coating) at the interface between the lipophilic cuticular wax and the coating, except diffusion in the coating matrix. Future study on mass transport patterns at the interface of fruits will be available for elucidating the retention and penetration mechanisms of active substance in the films.

3.9. The biosafety of composite films

The biosafety of composite films was performed by hemolysis, cell cytotoxicity and ethyl acetate residue. The hemolysis rates are shown in Fig. 3G, the positive group showed distinct red due to complete dissolution, while the negative (PBS) and PEF20 samples showed transparent and colorless supernatant, indicating that the PEF20 had good hemocompatibility. Specifically, even after incubating with PEF20 samples at a concentration of 1.00 mg/mL, the hemolysis rate was only 0.3%, which was lower than the acceptable limit of 5% (Ni et al., 2023). These results suggested that PEF20 did not damage the permeability and integrity of erythrocyte membranes. Meanwhile, studies have reported that phenolic acids can improve erythrocyte stability based on their antioxidant properties, which protected erythrocytes by inhibiting intracellular ROS production and counteracting the pro-oxidant effects of cell membrane (Cruz et al., 2024; Tang & Liu, 2008). The cell viability decreased slightly with increasing concentration of PE in the films compared with control (Fig. 3H), but the difference was not significant. It is worth noting that cell viability of PEF20 films was higher than 93.2%, indicating low toxicity of the films. Generally, antioxidants protect cells at low concentrations and induce cell death at high concentrations, while the sustained release of PEF films controlled the concentration of phenolic acids and was non-toxic to cells (Xu et al., 2021). The volatile components of the PEF20 film were analyzed by HS-SPME-GC–MS to assess the residual ethyl acetate content. The total ion chromatogram (Fig. S2) showed that the ethyl acetate standard sample produced a single main peak at a retention time of approximately 3.46 min. In contrast, the PEF20 sample exhibited baseline fluctuations at retention time (RT ≈ 3.46 min, RI = 612), with no detectable peaks exceeding the signal-to-noise ratio threshold. Furthermore, no peaks corresponding to ethyl acetate were detected in the library search, indicating that ethyl acetate residues were essentially absent from the samples. Overall, the low hemolysis rate, high cell survival rate and undetectable levels of ethyl acetate residues indicated that the composite films provided high level of safety for food preservation.

3.10. Effect of composite coatings on cherry tomatoes preservation

3.10.1. Effect of PEF coatings on color change, weight loss and firmness of fruits

The appearance changes of cherry tomatoes with PEF0, PEF15 and PEF20 are shown in Fig. 4A. The appearance changes reflected the intuitive indicator of fruit quality, and mature tomato fruits were rich in carotenoids and lycopene (Song et al., 2023). The color changes of cherry tomatoes during 8 d storage were shown in Fig. 4B. The appearance of tomato fruits changed significantly at 2 d, and the control group and the fruit treated with PEF0 turned red gradually. The results might be due to that the effect of p-CA on lycopene synthesis in tomato fruits. As shown in Table 2S, the a* values of all fruits increased while the b* values decreased during storage, and the L* values of the coated fruits were higher than those of the control group. Compared with the control group, the PEF15 and PEF20 coatings significantly reduced changes in color parameters, effectively delaying the process by which the skin color changed from yellow to red. In addition, ΔE value of tomato fruits continued to increase during storage. Compared with the PEF15 and PEF20-coated samples, significant increase of ΔE was observed in the control sample on the day 8 (Fig. 4B).

Fig. 4.

(A) Appearance changes of cherry tomatoes with the coatings during storage. (B) Changes of ΔE, (C) weight loss, (D) firmness and (E) titratable acid of cherry tomatoes with the coatings during storage. The interaction effect between treatment and time was significant, and simple effect analysis was conducted. Different letters represent significant differences in treatment at the same time point (p < 0.05).

As shown in Fig. 4C, the fruits with different treatment group exhibited upward trend in weight loss during storage due to the loss of tissue moisture and the nutrient depletion. The fruit with PEF0 coatings exhibited the greatest weight loss, which might be due to the high water content of KGM/MC matrix. Hernández-Carrillo et al. (2021) reported that the coatings of strawberry surfaces with polysaccharides showed similar trend. However, as the increased p-CA PE content, the weight loss of the fruits gradually decreased, and fruit treated with the PEF20 coatings maintained the low level during storage. Moreover, the addition of PE enhanced the barrier properties of the coatings, thereby inhibiting the respiration and transpiration of fruits. The decreased water content of the coating minimized the direct migration of water into the air.

The Fig. 4D depicted the firmness of tomato fruits with coated and uncoated treatments. The firmness of all samples decreased during storage, which was related to the respiration and enzyme activities of fruits during storage (Wang et al., 2025). The firmness of control fruits was significantly lower than that of coated samples. This was attribute to the barrier properties of the coating, which effectively reduced the respiration rate, thereby inhibiting pectinase activity associated with cellular structure degradation (Mao et al., 2024). The firmness of fruits in the control group was 44.34% and 47.35% lower than those of PEF15 and PEF20 group at 8 d, and was significantly different from the fruits coated with PEF0. This could be that the release of p-CA stimulated the synthesis of lignin, which was the major component involved in cell wall composition, and had a positive effect on resistance to cell wall degradation (Liu, Ji, et al., 2020).

3.10.2. Effect of PEF coatings on TSS and TA of fruits

TSS and TA are key indicators of the soluble sugar and organic acid content in the fruit, which directly influence the flavor and ripeness of cherry tomatoes. As shown in Table. S2, the total TSS content of tomato fruits increased continuously during ripening due to the conversion of organic acids and the hydrolysis of insoluble carbohydrates (Li et al., 2024). The TSS of fruits with PEF20 coating was lower than that of control at 8 d. cherry tomatoes are rich in organic acids, which is mainly regulated by respiration effect. Fig. 4E showed that TA of all fruits decreased due to the involvement of organic acids in ATP production, which was constantly consumed as substrate. At late period of storage, the TA of fruits with coatings was higher than that of control, which was due to barrier property of the coatings, reducing oxygen penetration and forming high carbon dioxide environment. While the coatings decreased the intensity of fruit respiration and reduced TA consumption by respiration, thereby delaying the TA decrease of tomato fruits (Chen, Tian, et al., 2023).

3.10.3. Effect of PEF on total phenols and total flavonoids of fruits

The content of phenolic compounds is closely related to the shelf life of cherry tomatoes. As shown in Fig. 5A-B, the PEF15 and PEF20 coating significantly affected the total phenolic and total flavonoid contents of tomato fruits compared with the control group. It was worth noting that total phenol and total flavonoid contents of all fruits increased slowly during 4 d storage and then increased rapidly. In particular, the total phenol and total flavonoid contents of PEF15 and PEF20 coated tomato fruits were significantly higher than those of PEF0 samples at initial. This might be due to that free p-CA stimulated the phenylpropane metabolic pathway of the fruits and increased content of total phenol and total flavonoid (Yuan et al., 2019; Zhang et al., 2025). After storage for 4 d, the p-CA further released from the coating effectively stimulated an increased in the total phenolic content of cherry tomatoes. The total phenolic content of fruits with PEF15 and PEF20 achieved maximum values on day 6 and 8 d, and increased by 18.98 μg/g and 16.31 μg/g compared with that of control. Furthermore, there was no significant difference in total flavonoid content between fruits coated with PEF0 and control group throughout the storage period. In conclusion, the p-CA released from composite coatings accelerated synthesis of total phenols and total flavonoids in fruits.

Fig. 5.

(A) Changes of total phenols and (B) total flavonoid contents, (C) ABTS and (D) DPPH radical scavenging activities of cherry tomatoes with composite coatings. The interaction effect between treatment and time was not significant, but the main effect of treatment was significant, and multiple comparisons were performed for the main effect of treatment. *** indicate p < 0.001, ** indicate p < 0.01, * indicate p < 0.05, and ns indicate no significant difference.

3.10.4. Effect of PEF on antioxidant capacity of fruits

As shown in the Fig. 5C and D, the antioxidant capacities showed decreasing changes at 4 d storage, which might be related to the slow increase of phenolic content at this stage, (Du & He, 2024). The rapid increase of phenolic content in fruits during 4–8 d storage, which was consist with the increase of antioxidant capacity in fruit at later storage. The DPPH radical scavenging capacity of fruits with PEF15 and PEF20 coatings increased to 73.36% and 74.14% on 8 d, while the control group was only 52.87%. Meanwhile, the ABTS free radical scavenging of fruits with PEF15 and PEF20 was also higher than that of control during storage and achieved maximum value (80.60% and 81.65%) on 8 d. This result might be due to the limitations of DPPH analysis, and study reported that the ABTS analysis was more effective in detecting antioxidant capacity due to the high water content of fruit and vegetables (Floegel et al., 2011). Throughout the storage period, PEF15 and PEF20 exhibited more significant free radical scavenging effect compared with the control group, which was consistent with the accumulation trends of total phenolics and flavonoids. Therefore, PEF coatings treatment can improve antioxidant capacity by accumulating phenolic content of fruits.

3.10.5. Effect of PEF on PPO activity and CAT activity of fruits

The PPO activities of fruits with coatings are shown in Fig. 6A. The PPO activity of control and PEF0 samples increased throughout whole storage, while PEF15 and PEF20 coatings showed an increase tendency and then decrease. And there was no significant difference of PPO activity among different samples during 4 d storage. Whereas the PPO activities of fruits with PEF coating were significantly lower than that of control at 8 d. The results might be related with acidic nature of p-CA, which reduced pH values and PPO activity of fruits (Allahveran et al., 2018). The CAT activity of fruits with coating was significantly higher than that of control throughout the storage (Fig. 6B). The highest CAT activity of PEF20 coated fruits achieved 3.56 U/g FW at 2 d storage, which increased by 44.55% compared to that of control. During ripening and senescence of tomato fruits, ROS was produced rapidly and activated antioxidant defense system, while p-CA released from the coatings induced CAT activity of fruits during storage (Kaur et al., 2017). This study illustrated that p-CA released from coatings inhibited PPO activity, improved CAT activity and reduced phenols degradation of fruits.

Fig. 6.

Changes of (A) PPO, (B) CAT, (C) PAL, (D) C4H and (E) 4CL of cherry tomatoes with composite coatings during storage.

3.10.6. Effect of PEF on key enzymes of phenylpropane metabolism

The phenylpropane pathway is key pathways of secondary metabolites, and PAL, C4H, and 4CL are key enzymes in the phenylpropanes metabolism (Yuan et al., 2019). Fig. 6C reflects PAL activity changes of tomatoes with coatings and all samples were consistently increased during storage. It was possible that the ROS increase with response to oxidative damage and simulated PAL activity of fruits. As shown in Fig. 6, the PAL, C4H and 4CL activities of fruits coated with PEF0 were lower than those of control. The results might be attributed to good barrier property of coatings, reducing gas exchange and forming favorable preservation atmosphere for fruits, thereby reduced oxidative damage and enzyme activities of phenylpropane metabolism. With the extension of storage time to 8 d, the PAL, C4H and 4CL activities of PEF20-coated fruits achieved the high value at 3.53 U/g FW, 3.36 U/g FW and 3.58 U/g FW. Therefore, release P-CA from composite coatings increased key enzyme activities of phenylapropane metabolism in tomato fruits.

3.10.7. Effect of PEF on resisting Botrytis cinerea of cherry tomatoes

The necrotic lesions of fruits with different treatment are significant differences at 6 d as shown in Fig. 8B. The visible mycelial growth can be seen in control samples at 3 d and the colony diameter expanded 1.82 times at 6 d. In contrast, the mycelial growth of fruits with PEF0 was delayed two days, indicating that the barrier properties of KGM/MC-based coating might inhibit fungal invasion into fruits. As expected, with the increased concentrations of PE incorporated into the coatings, the spore growth of Botrytis cinerea on fruits was completely inhibited by PEF15 and PEF20 treatment. The accumulation of flavonoids and phenol substances played a vital role in inhibiting spore germination and mycelia growth (Prusky & Romanazzi, 2023; Wei et al., 2017). Furthermore, the obvious antifungal activities of fruits coated with PEF were exhibited on the 6 d, suggesting that the encapsulation of p-CA provided continuous antifungal activities due to sustained release of the coatings. The results indicated that PEF coatings were able to defense fungal infection on cherry tomatoes. In general, PEF coatings improved the barrier properties against the water, O₂, ROS and UV light, showing excellent antioxidant activities and disease resistance by regulating metabolic pathways of fruits (Fig. 7B), which have a potential application for maintaining fruit quality.

Fig. 7.

(A) Resistance of cherry tomatoes with composite coatings against Botrytis cinerea. (B) Schematic illustration of structural properties and functional activities of composite coatings.

3.10.8. Correlation analysis and principal component analysis (PCA)

This study analyzed the correlations between various quality parameters of cherry tomatoes during storage. The Pearson correlation coefficient (r) in the heatmap indicated positive correlation with the respective parameters. As shown in the Fig. S3a, TSS exhibited significant positive correlation with ΔE and weight loss rate, while significant negative correlation with firmness and TA. During storage, postharvest fruits continued active physiological metabolism, characterized by red pericarps and accumulation of soluble solids in ripening process, which was accompanied by significant weight loss, firmness reduction and organic acids depletion. The significant correlation was observed between the phenolic compounds of cherry tomatoes and the free radical scavenging rate, which was related to the antioxidant capacity of postharvest fruits. Furthermore, the key enzymes (4CL and PAL) of phenylpropanoid metabolism were highly correlated with the accumulation of phenolic compounds in fruits. These results indicated that activation of key enzymes in the phenylpropanoid metabolism can impact the synthesis of phenolic compounds in the fruits, thereby enhancing the antioxidant defense capacity of the fruits. Additionally, PCA was employed to investigate the relationship between different treatments and fruit quality. As shown in Fig. 3Sb-c, the first three principal components and their contributions were PC1 (51.7%), PC2 (17.6%) and PC3 (9.9%), and the selected variables were sufficient to capture the trends in fruit quality parameters under different treatments (Mao, Zhang, Xu, et al., 2025). It can be seen that the PEF15 and PEF20 groups exhibited significant differences along the PC1 axis compared with the control and PEF0 groups. The control and PEF groups were closer to ripening-related variables including ΔE, TSS and PPO. In contrast, the PEF15 and PEF20 treatments were more closely aligned with variables such as high polyphenol content, enzyme activity and disease resistance, and PEF20 treatment exhibited more significant overlap. These results indicated that the PE-loaded coating effectively released the encapsulated p-CA, regulated fruit physiological metabolism and maintained fruit quality.

4. Conclusion

The KGM/MC based bioactive films were successfully prepared by incorporating p-CA emulsions (PE) stabilized by C-CNCs. FTIR and XRD results showed that PE exhibited good compatibility with KGM/MC through hydrogen bonding interactions. The addition of PE reinforced the network structure of the composite films and effectively enhanced the mechanical properties, hydrophobicity, and barrier performance against ultraviolet/visible light, oxygen and water vapor. Moreover, the composite films exhibited good antioxidant activities and p-CA achieved release from the films under water-based food simulant system. The low hemolysis rate and high cell survival rate confirmed the biosafety of films, which could be applied as biosafe packaging materials. The application of PEF coatings effectively delayed fruit ripening, increased total phenol and total flavonoid content, and enhanced antioxidant capacity of tomato fruits. The key enzymes PAL, C4H and 4CL of phenylpropane metabolism were increased in fruits with PEF coatings, and defensed the fungal infection of fruits. Therefore, the bioactive coatings incorporating PE have potential application for maintaining fruit quality during the supply chain.

In this study, the bioavailability of p-CA was enhanced by using ethyl acetate as the oil phase to formulate Pickering emulsion for the preparation of active packaging films. However, the high volatility of organic solvents and the risk of residues impose limitations on their application. It will be necessary to identify green and environmentally friendly solvents for comparison and substitution. Furthermore, future research needs to elucidate the molecular mechanisms of composite films incorporating PE on the regulation of postharvest fruits. Meanwhile, systematic comparisons with existing commercial packaging materials on the market are required to enhance the feasibility of their application.

CRediT authorship contribution statement

Linli Zhang: Writing – original draft, Methodology, Investigation, Data curation. Qiao Wu: Writing – original draft, Validation, Methodology. Shuo Mao: Methodology, Investigation. Yingjie Xu: Formal analysis. Tiehua Zhang: Supervision, Conceptualization. Chengwen Lu: Writing – review & editing, Supervision, Project administration, Methodology, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary material.

Data availability

Data will be made available on request.