CS/N-CD composites enhance physical barriers, antioxidant activity and microbial modulation for improved chili pepper preservation

Abstract

Chili peppers (Capsicum annuum L.) have a high postharvest metabolism, causing moisture loss and microbial spoilage, which shortens their shelf life, thereby imposing environmental burdens through resource waste, greenhouse gas emissions, and secondary pollution. Carbon dots (CDs), zero-dimensional carbon-based nanomaterials with particle sizes below 10 nm, show promise in food packaging and postharvest preservation. In this study, a chitosan/N-CD (CS/N-CD) composite material was developed with superior barrier, antioxidant activity, and antibacterial properties. CS/N-CD films with different N-CD ratios showed good compatibility, enhanced UV absorption, improved barrier properties (0.5% film with 11.4% lower WVP), and higher antioxidant activity (2.5% film with 66.8% DPPH scavenging). The 0.5% films showed high antibacterial rates against Escherichia coli and Staphylococcus aureus (89.2–99.6% vs. 14.9–62.5% for pure CS). After being applied to chili pepper fruits via spraying, dipping, and film-coating, the material reduced weight loss and preserved fruit firmness (2.5-fold reduction by day 21 vs. 4.8-fold for the control). High-throughput 16S rRNA gene sequencing showed that CS/N-CDs altered the microbial structure; dipping increased Actinobacteria by 355.4% and suppressed Enterobacter by 98.2%, while spraying reduced Enterobacter by 82.9% and enriched Pseudomonas by 87.1%, thereby improving the microbial microenvironment during storage of the chili pepper fruit. These results show that the CS/N-CD composite exerts a synergistic preservation through a physical barrier and microbial modulation. Given the eco-friendly properties of CS/N-CDs, these findings offer insights into advancing sustainable nanocomposite-enabled postharvest preservation.

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Environmental significance

Eco-friendly CS/N-CD composites exert synergistic preservation effects via enhanced barrier, antioxidant and antibacterial properties, improving the microbial microenvironment of stored chili peppers.

1. Introduction

Chili peppers (Capsicum annuum L.) are one of the most widely consumed culinary spices and an important economic crop worldwide due to their distinctive pungent flavor.¹ Their fruits are rich in diverse bioactive substances, including vitamins, flavonoids, carotenoids, and capsaicin, which endow them with significant antioxidant, antibacterial, and metabolic regulatory properties.² However, despite their high nutritional and economic value, chili peppers exhibit a high respiration rate and metabolic activity, making them highly vulnerable to water loss, oxidative stress, and microbial infection.³ It has been reported that approximately 46% of pepper production is lost annually across multiple stages of the supply chain, including cultivation, postharvest handling, transportation, storage, distribution, and consumption.⁴ These factors usually result in wrinkling, flesh softening, and spoilage, significantly shortening the shelf life and reducing the commercial value.^5–7

Currently, cold storage, modified atmosphere packaging (MAP), and chemical preservatives are applied to reduce respiration and inhibit microbes. However, cold storage systems require high energy costs, MAP requires complex gas control, and chemical preservatives pose safety risks.^8–10 Given these limitations, developing greener, simpler, and safer fruit preservation technologies is critical for maintaining fruit quality and ensuring food safety.

Polysaccharide-based edible coatings have attracted increasing interest due to their excellent mechanical strength, transparency, and chemical stability.¹¹ Among them, chitosan (CS), the second most abundant natural polysaccharide after cellulose, presents numerous advantages, including abundant availability, low cost, biodegradability, and broad-spectrum antimicrobial activity.¹² It has been widely used in food packaging, gas adsorption, and wastewater treatment.¹³ Studies have shown that CS can form a compact protective film on the surface of fruits and vegetables via spraying, brushing, or immersion.¹⁴ This film effectively mitigates water loss, inhibits microbial growth, and retards aging and decay.¹⁵ Nevertheless, pure CS films still display deficiencies in mechanical strength, gas barrier properties, and functional activity.¹⁶ To address these limitations, researchers have explored the incorporation of functional additives to enhance the structural and functional performance of CS-based films.¹⁷ For example, plant-derived essential oils, such as thyme oil, cinnamon oil, and clove oil, were introduced for antimicrobial and antioxidant properties.¹⁸ However, their strong volatility and intense aroma limited their application by altering the flavor and membrane stability.¹⁹ Furthermore, metal and metal oxide nanoparticles (e.g., Ag, CuO, ZnO, and TiO₂) with broad-spectrum antibacterial and photocatalytic properties have been widely studied for use in food packaging and preservation.²⁰ However, their large-scale application remains limited by challenges such as biotoxicity, particle aggregation, poor dispersibility, and matrix instability. Although ZnO and TiO₂ are generally considered relatively safe at low concentrations, recent reviews have indicated that their long-term ingestion safety remains uncertain and that comprehensive toxicological evaluations are still lacking.^17,21 In contrast, Ag and CuO nanoparticles are more frequently associated with cytotoxic and oxidative stress responses.^22,23 Previous studies suggest that their toxicity mainly results from metal ion release, excessive reactive oxygen species (ROS) generation, membrane disruption, and interference with intracellular enzymatic and metabolic processes.²⁴ These adverse effects not only impair microbial viability but also present potential risks to food quality, human health, and environmental safety, thus constraining the sustainable application of metal-based nanomaterials in food preservation.^25,26

Carbon dots (CDs), zero-dimensional carbon-based nanomaterials with particle sizes below 10 nm, have emerged as promising candidates for food packaging and postharvest preservation due to their low toxicity, excellent water solubility, high biocompatibility, and tunable surface functionalities.²⁷ CDs can enhance the mechanical properties, UV shielding capacity, and antimicrobial efficacy of CS films and facilitate stable CS cross-linking.²⁸ Notably, nitrogen-doped (N-doped) CS films introduce surface amide and amine groups, increasing microbial membrane penetration via increased positive charge, which results in stronger electrostatic interactions and membrane-disruptive effects and improved antibacterial performance.²⁹ Therefore, the combination of CS and N-CDs synergizes the physical barrier and biological antimicrobial properties for high-performance preservation systems.

In recent years, CS/CD composite materials have attracted increasing attention in fruit and vegetable preservation due to their multifunctional properties. For example, Ananda et al. incorporated coffee-ground-derived CDs into chitosan to produce composite films with enhanced UV-shielding capacity, mechanical stability, and photoresponsive properties, and demonstrated their preservation efficacy in shrimp and dairy product models.³⁰ Similarly, Liu et al. developed multifunctional films by co-incorporating CDs and cellulose nanocrystals (CNCs) into CS, which improved gas selectivity, photodynamic antibacterial activity, and preservation performance.³¹ However, most current studies still focus on conventional microbiological assessments such as inhibition zone, total bacterial count, and MIC/MBC. Although previous studies have demonstrated the antibacterial and preservative properties of chitosan-based coatings, insight into how CS/CDs-induced microenvironments modulate the surface microbiota of fresh produce remains limited.¹³ Elucidating microbial microenvironments is of critical importance for fruit and vegetable preservation, as this microscale habitat directly modulates the structure and metabolic activity of surface microorganisms, which can greatly affect fruit respiration, tissue softening, and decay progression.³² In this study, CS/N-CD composite suspensions and corresponding films were prepared, characterized, and applied to the postharvest preservation of chili peppers via spraying, immersion, and direct coating methods. Their effects on weight loss, visual quality, texture maintenance, and surface microbial communities were systematically evaluated.

These findings shed light on the microenvironmental mechanisms underlying the preservation process, providing theoretical and practical insights into green, efficient, and sustainable preservation technologies for fruits and vegetables.

2. Materials and methods

2.1 Materials

Citric acid monohydrate, urea, glycerol, chitosan, acetic acid, anhydrous calcium chloride, deoxidizer, potassium hydroxide, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sinopharm Group Co. Ltd. The above chemicals were all of analytical grade purity.

2.2 Synthesis of N-CDs and CS/N-CD films

N-CDs were synthesized according to the method reported by He et al.³³ with slight modifications. 4.0 g of citric acid monohydrate and 6.0 g of urea were weighed and added to 40 mL of ultrapure water. Subsequently, the mixed solution was transferred into a 100 mL Teflon-lined stainless-steel autoclave. The reaction was carried out at 180 °C for 6 h. After cooling to room temperature, the obtained suspension was filtered through a 0.22 μm polyethersulfone (PES) membrane to remove large particles. After washing with ultrapure water, the N-CDs were obtained by freeze-drying. According to Lin et al.,³⁴ CS/N-CD films were prepared by the solvent casting method. Film-forming solutions (FFS) were prepared by mixing CS solution with N-CDs at different incorporation ratios of 0%, 0.1%, 0.5% and 2.5% (N-CDs: CS w/w). The obtained FFS was evenly spread over culture dishes and then dried in an oven at 60 °C for 8 h. The resulting films were designated as 0% CS/N-CD, 0.1% CS/N-CD, 0.5% CS/N-CD and 2.5% CS/N-CD films, respectively.

2.3 Characterization and performance

2.3.1 Morphology analysis. The morphology, hydrodynamic diameter, and zeta potential of the N-CDs were characterized by transmission electron microscopy (TEM, JEM-2100, Japan) and dynamic light scattering (DLS, Nano-ZS90, UK).

The surface and cross-section of the CS/N-CD films were examined by scanning electron microscopy (SEM, Hitachi, Japan) at 10 kV. Atomic force microscopy (AFM, multimode, Germany) was used to characterize the surface structure and roughness, with R_a and R_q values calculated with Nanoscope software.

2.3.2 Fourier transform infrared spectroscopy. The functional groups of the N-CDs and composite films with varying N-CD contents were analyzed using a Fourier transform infrared spectrometer (FTIR, PerkinElmer Inc., USA) over the wavenumber range of 500–4000 cm⁻¹.

2.3.3 Optical properties. The optical barrier properties of the composite films and pure chitosan films were evaluated using a UV-visible spectrophotometer (Shimadzu UV-1800, Japan). Light transmittance was recorded in the wavelength range of 200–800 nm.

2.3.4 Determination of water contact angle. Composite films and pure chitosan films were cut into 2 × 2 cm rectangular pieces and mounted onto glass slides. The water contact angle (WCA) was measured using a contact angle instrument (DSA100, KRÜSS GmbH, Germany) to evaluate the surface hydrophilicity of the films.

2.3.5 Barrier properties. The barrier properties of the film samples were assessed according to a reported method.¹² Water vapor permeability (WVP), oxygen permeability (OP), and carbon dioxide permeability (CDP) were measured to assess the film's resistance to water vapor, oxygen, and carbon dioxide, respectively. Briefly, 5 g of anhydrous calcium chloride, deoxidizer, or potassium hydroxide was placed into separate centrifuge tubes, which were then sealed with the film samples. The initial weights were recorded, and the tubes were incubated in a controlled environment chamber at 25 °C and 75% relative humidity. After 48 h, the final weights of the tubes were recorded.³⁵ The WVP, OP, and CDP were calculated using eqn (1)–(3), respectively:

WVP = (W × d)/(A × t × ΔP) (1)

OP = (W × d)/(A × t) (2)

CDP = (W × d)/(A × t) (3)

where W denotes the weight increase of the centrifuge tube (g), d is the thickness of the film (m), A is the effective permeation area (m²), t is the storage duration (s), and ΔP is the water vapor pressure difference across the film (Pa).

2.3.6 Antioxidant property (DPPH scavenging activity). The antioxidant activity of the films was evaluated based on their free radical scavenging ability using the DPPH assay. Briefly, 50 mg of the CS/N-CD composite films with different N-CD doping levels (0%, 0.1%, 0.5%, and 2.5%) were dissolved in 1 mL of 1% (v/v) acetic acid. The solution was then transferred into a 10 mL centrifuge tube, followed by the addition of 3 mL of DPPH solution. The mixture was incubated in the dark at room temperature for 30 minutes. Absolute ethanol was used as the blank control. The absorbance was measured at 517 nm.³⁶ The DPPH radical scavenging activity was calculated according to the following equation:

DPPH Scavenging Activity (%) = [1 − (A₁ − A₂/A₀)] × 100% (4)

where A₀ represents the absorbance of the mixture of distilled water and DPPH solution, A₁ represents the absorbance of the mixture of the sample solution and DPPH solution, and A₂ represents the absorbance of the mixture of the sample solution and absolute ethanol.

2.3.7 Antibacterial properties. The antibacterial performance of the film samples was evaluated by measuring their antibacterial activity against gram-negative bacteria (E. coli) and Gram-positive bacteria (S. aureus).³⁷ Specifically, 0.5 mL of bacterial suspension (∼10⁷ CFU mL⁻¹) was mixed with CS/N-CD film samples (20 mm × 20 mm) containing different N-CD doping levels (0%, 0.1%, 0.5%, and 2.5%) in sterile 10 mL centrifuge tubes. The mixtures were then incubated on a shaker at 37 °C and 190 rpm under two different conditions: one under UV irradiation and the other in the dark. After 1 h, 150 μL of the treated bacterial suspension was evenly spread onto LB agar plates and incubated at 37 °C for 16 h, followed by colony counting. A blank control group was treated in the same way, except no film sample was added to the bacterial suspension. The relative antibacterial rate was calculated using the following equation:

Relative Antibacterial Rate (%) = (N₀ − N₁)/N₀ × 100% (5)

where N₀ is the number of bacterial colonies in the control group, and N₁ is the number of bacterial colonies in the experimental group.

2.3.8 Biosafety evaluations. The biosafety of the N-CDs was assessed using hemolysis and cytotoxicity assays, following the procedure reported by Li et al.¹² with minor modifications. The tested N-CD concentrations (20, 100, and 500 μg mL⁻¹) corresponded to their incorporation ratios in the CS/N-CD composite films (0.1%, 0.5%, and 2.5%, w/w), ensuring consistency between the free N-CD solutions and the film matrix.
Hemolysis test. A 0.5 mL erythrocyte suspension (2%, v/v) was prepared from fresh rat blood and mixed with equal volumes of N-CD solutions at concentrations of 20, 100, and 500 μg mL⁻¹. Fresh rat blood used to prepare the erythrocyte suspension was obtained as a commercial product from Guangzhou Hongquan Biological Technology Co., Ltd. (Guangzhou, China). A 0.9% NaCl solution was used as the negative control, while distilled water served as the positive control. The mixtures were incubated at 37 °C for 4 h and then centrifuged at 2000 rpm for 5 min. The absorbance of the resulting supernatant was recorded at 492 nm using a UV-vis spectrophotometer. The hemolysis ratio was calculated using the following equation:where A₁, A₂, and A₀ represent the absorbance of the sample and the positive and negative control groups, respectively.
Cytotoxicity assay. NIH3T3 cells were divided into groups (control with complete medium and samples treated with varying concentrations of N-CDs) and incubated for 24 h. NIH/3T3 cells were purchased from Wuhan Procell Life Science & Technology Co., Ltd. (Wuhan, China). The medium was removed, and the wells were rinsed three times with PBS. Fresh medium containing 0.5 mg mL⁻¹ MTT was added to each well and incubated at 37 °C with 5% CO₂ for 4 h. After removing the supernatant, 100 μL of DMSO was added to each well, and the absorbance was recorded at 570 nm after gentle shaking for 10 min.where A₁ and A₂ represent the absorbance values of the sample and control wells, respectively.

2.4 Preservation of pepper fruits

Fresh chili peppers (Capsicum annuum L.) were purchased from the Fangmiao Neighborhood Center in Binhu District, Wuxi City, Jiangsu Province, China. Peppers of uniform size, vivid color, moderate weight, and free from mechanical damage were carefully selected. Three treatment methods were established: immersion, spraying, and film packaging, with untreated fruits serving as the control. For the immersion treatment, fruits were immersed in a 0.5% CS/N-CD suspension for 2 min. 2% CaCl₂ solution and distilled water were used as control treatments. After immersion, excess liquid was gently removed, and the fruits were air-dried at room temperature (25 ± 1 °C) before storage. For the spraying treatment, a handheld sprayer was used to uniformly apply a 0.5% CS/N-CD suspension onto the fruit surface. Each fruit received approximately 1.25 mL of coating solution, as estimated from the spray volume. Similarly, 2% CaCl₂ solution and distilled water served as controls. The sprayed fruits were air-dried at room temperature for 30 min before storage. For the film-packaging treatment, fruits were wrapped with either 0% CS/N-CD film (pure CS film) or 0.5% CS/N-CD composite film. Commercial polyethylene (PE) film was used as the control, and unwrapped fruits served as the blank control. All samples were stored at 25 ± 1 °C under ambient humidity conditions. The pepper fruits were taken out and photographed on days 7, 14, 21, and 28, respectively, and subsequent measurements of weight loss and firmness were conducted. The fruit weight loss rate was determined by the gravimetric method. The initial weight of the fruit was recorded as M₀, and the weight at the sampling time was recorded as M₁. The weight loss rate was calculated using the following equation:

Weight Loss Rate (%) = [(M₀ − M₁)/M₀] × 100% (8)

The hardness of the pepper fruits during storage was measured using a texture analyzer. The test parameters were set as follows: puncture speed of 0.5 mm s⁻¹ and puncture depth of 7 mm. The fruit hardness is expressed in Newtons (N).

2.5 Microbial communities on the fruit surface

On the 21st day of storage after harvesting, the pepper fruits were frozen in liquid nitrogen and subsequently sent to Shenzhen WeGene Biotech Co., Ltd. for sequencing analysis. Total DNA was first extracted and quality-checked. The V5–V7 hypervariable regions of the bacterial 16S rRNA gene were then amplified using the forward primer 799F (AACMGGATTAGATACCCKG) and the reverse primer 1193R (ACGTCATCCCCACCTTCC). After PCR amplification, the products were purified and used for library construction and quality assessment, followed by sequencing on the Illumina platform.

2.6 Statistical analysis

The statistical analysis was conducted using IBM SPSS Statistics 26.0. All experimental results were from at least triplicate biological replicates and presented as mean value and standard deviation. One-way analysis of variance (ANOVA) followed by the Tukey HSD posthoc test (p < 0.05) was used to evaluate differences between all treatments.

3. Results and discussions

3.1 Characterization of N-CDs and CS/N-CD films

The morphology of the prepared N-CDs was characterized by TEM. The results showed that the N-CDs were well dispersed, spherical in shape, and uniform in size (Fig. 1A). The synthesized N-CDs were light yellow under visible light and exhibited strong blue fluorescence when exposed to ultraviolet light at 365 nm, with a maximum excitation wavelength of 440 nm (Fig. 1B). The FTIR spectrum of the N-CDs (Fig. 1C) showed an absorption peak at 3200 cm⁻¹ corresponding to the stretching vibrations of O–H and N–H,³⁸ and the peak at 1670 cm⁻¹ was attributed to the stretching vibration of C=O.³⁹ In addition, the peak at 1568 cm⁻¹ represented the stretching vibration of N–H. These results confirmed the presence of amino groups and carboxyl groups on the prepared N-CDs. These functional groups endow N-CDs with high hydrophilicity in aqueous solution, which can enhance their dispersibility in the chitosan matrix.

Fig. 1 Characterizations of the synthesized N-CDs. (A) TEM image. (B) The fluorescence spectra of N-CDs (inset: N-CD photographs under indoor daylight and 365 nm illumination). (C) The FTIR spectrum of N-CDs.

The CS/N-CD composite films displayed a smooth, uniform surface with high transparency and a faint yellow tint, suggesting good dispersion of N-CDs within the chitosan matrix. The immersion and spraying solutions exhibited homogeneous and stable colloidal properties, with no visible precipitation or aggregation, confirming their suitability for direct food-contact applications (Fig. S1). The visual features of these materials indicate favorable consumer acceptability and strong potential for application in practical food preservation systems. As reported by Bucher et al., visual attributes such as color uniformity, clarity, and perceived naturalness play a crucial role in shaping consumers' acceptance of edible packaging materials.⁴⁰ The visually appealing appearance of CS/N-CD films may therefore enhance consumer trust and acceptance in practical applications.

The AFM images depicted the 3D (Fig. 2A) and 2D (Fig. 2B) morphologies of the CS/N-CD films with different N-CD incorporation ratios (0%, 0.1%, 0.5% and 2.5%). The results show that film roughness gradually increases with the increase in N-CD concentration. The wettability of the composite films was evaluated by WCA. In comparison to the pure CS film, the inclusion of N-CDs significantly reduces the contact angle of the CS/N-CD films (Fig. 2C). This phenomenon could be attributed to the hydrophilicity of N-CDs.⁴¹ The microstructure of the surface and cross-section of the composite films was characterized by SEM (Fig. 2D). Overall, no pores or cracks were observed in the composite films, indicating good compatibility. However, morphological differences were evident for CS films with and without the incorporation of N-CDs. As compared with the pure CS film, the surface and cross-section of the films incorporated with N-CDs exhibited more texture, especially for the 0.5% and 2.5% CS/N-CD films, whose surface and cross-section became rougher. In addition, the arithmetic mean roughness (Ra) and root mean square roughness (R_q) parameters of the film surface were quantitatively analyzed. With the increase in the N-CD concentration, both R_a and R_q gradually increase, and the roughness of the film increases (Fig. S2). This is consistent with previous studies, where the increase in NMs led to agglomeration and affected the roughness of the film.⁴²

Fig. 2 Characterizations of CS/N-CD films with different incorporation ratios (0%, 0.1%, 0.5%, and 2.5%) of N-CDs. (A) Atomic force microscopy (AFM) 3D surface topography (scan area = 5 μm × 5 μm and height scale = −20 nm to 20 nm). (B) AFM 2D surface topography of CS/N-CD films (scan area = 5 μm × 5 μm; height scale = −20 nm to 20 nm; and scale bar = 1.0 μm). (C) Water contact angle images of the films. (D) Scanning electron microscopy (SEM) images of CS/N-CD films showing surface morphology and cross-sectional structure. Surface image: accelerating voltage = 5.0 kV; magnification = 10 000×; and scale bar = 5 μm. Cross-sectional image: accelerating voltage = 5.0 kV; magnification = 500×; and scale bar = 100 μm.

3.2 FTIR spectra, barrier properties and antioxidant capacities of the prepared composite films

The functional groups of the CS/N-CD films with different N-CD incorporation ratios (0%, 0.1%, 0.5%, and 2.5%) were characterized by FTIR (Fig. 3A). The broad peak at 3300 cm⁻¹ was mainly due to the stretching vibration of O–H in both CS films and CS/N-CD films.⁴³ The absorption peak at 1550 cm⁻¹ was attributed to the stretching vibration of N–H.⁴⁴ Additionally, the peaks at 1650 cm⁻¹ and 1070 cm⁻¹ corresponded to the characteristic bands of C=C and C–O–C.⁴⁵ Therefore, after incorporating different proportions of N-CDs, no characteristic changes occurred in the functional groups of the chitosan-based films, indicating good compatibility between the N-CDs and CS. As shown in Fig. 3B, the absorbances of the CS/N-CD films with different N-CD incorporation ratios (0%, 0.1%, 0.5%, and 2.5%) were recorded when exposed to ultraviolet radiation from 200 nm to 800 nm. The results indicated that as the N-CD content increased, the ultraviolet absorption capacity of the composite films increased, enabling the films to effectively block ultraviolet light from reaching the food. Ultraviolet light is a strong oxidant that can have adverse effects on food. Therefore, the CS/N-CD films can not only delay the oxidation of lipids in photosensitive foods, but also significantly enhance the heat resistance of the composite films by increasing the absorbance.

Fig. 3 The FTIR spectra, barrier properties and antioxidant capacities of the prepared composite films. (A) The FTIR spectra of the films. (B) The UV-vis absorption spectra of the films. (C) Water vapor permeability. (D) Oxygen permeability. (E) Water vapor permeability. (F) DPPH-radical scavenging activity; the letters indicate significant differences among the groups (p < 0.05).

Food packaging films with low water vapor permeability (WVP) effectively limit moisture transfer through the film, thereby maintaining product humidity and extending the shelf life.⁴⁶ As shown in Fig. 3C, the pure CS film exhibited the highest WVP value. The 0.1% CS/N-CD film, with a low N-CD content, showed no significant difference in WVP compared with the pure CS film. However, increasing the N-CD content to 0.5% and 2.5% significantly reduced the WVP, with the 0.5% CS/N-CD film showing an 11.4% decrease relative to the pure CS film. This improvement is attributed to the compact polymer network formed by strong interfacial interactions between the N-CDs and the chitosan matrix, which increased the tortuosity of the moisture diffusion pathways.⁴⁷ Controlling oxygen permeability (OP) and carbon dioxide permeability (CDP) is essential to regulate fruit respiration and delay senescence.⁴⁸ As shown in Fig. 3D and E, both OP and CDP exhibit trends similar to WVP, decreasing markedly with increasing N-CD content. Specifically, the OP and CDP of the 0.5% CS/N-CD film decreased by 46.6% and 40.2%, respectively, compared with the CS film, indicating enhanced gas-barrier performance. These findings are consistent with previous studies, which similarly reported that incorporating nanoscale fillers into chitosan-based films enhances barrier properties by reducing molecular mobility and increasing the tortuosity of the diffusion pathways.^48–50 This consistent trend confirms that N-CD incorporation improves the structural compactness and barrier integrity of chitosan films, reinforcing their potential for practical food preservation. The antioxidant capacity of the CS/N-CD films was reflected by measuring the DPPH radical scavenging activity. Among all the films, the CS film had the lowest DPPH radical scavenging activity. After incorporating N-CDs, the DPPH radical scavenging activity of the films increased with the elevated N-CD content (Fig. 3F). The DPPH radical scavenging activity of the CS film was 25.4%, while that of the 0.1%, 0.5%, and 2.5% CS/N-CD films was 44.7%, 65.8%, and 66.8%, respectively. This may be attributed to the synergistic antioxidant effect of N-CDs and CS. Thus, the incorporation of N-CDs endows CS/N-CD films with good barrier capabilities against water vapor, carbon dioxide, and oxygen, while enhancing their antioxidant activity, and holds potential for extending the shelf-life of food upon application.

3.3 Inhibitory effects on pathogenic microorganisms of the CS/N-CD films

Antibacterial performance is a key characteristic for evaluating the inhibitory effect of packaging films on microbial infections of postharvest fruits. To assess the antibacterial ability of the CS/N-CD films, their relative antibacterial rates were measured under ultraviolet (UV) and dark conditions. For Escherichia coli, under dark conditions, the relative antibacterial rate of the pure CS film was 14.9%, whereas that of the 0.5% CS/N-CD film reached 89.2%. When exposed to UV light, the relative antibacterial rates of the CS and 0.5% CS/N-CD films increased to 42.4% and 97.4%, respectively. Similar trends were observed against Staphylococcus aureus. Under dark conditions, the relative antibacterial rates of the CS and 0.5% CS/N-CD films were 27.8% and 91.2%, respectively; under UV exposure, these values rose to 62.5% and 99.6%, respectively (Fig. 4).

Fig. 4 The antibacterial properties of the prepared composite films. (A) Effect of films on the growth of E. coli in the dark and under UV. (B) The relative inhibitory rates of films against E. coli in the dark and under UV. (C) Effect of films on the growth of S. aureus in the dark and under UV. (D) The relative inhibitory rate of films against S. aureus in the dark and under UV. The letters indicate significant differences among groups.

The antibacterial capacity of the composite films was enhanced with increasing N-CD content. This enhancement can be attributed to two mechanisms: (1) as zero-dimensional NMs with a high specific surface area, N-CDs increase the contact area between the film and bacterial cell walls, causing mechanical damage to the bacteria.⁵¹ (2) The amide and amino groups in N-CDs endow them with a positive charge, enabling binding to the negatively charged bacterial surfaces.^52,53 This interaction results in severe damage to the bacterial cell membranes, leakage of intracellular components, and ultimately, bacterial death.^13,54 The results indicated that incorporating N-CDs endowed the composite films with prominent photocatalytic antibacterial properties under UV conditions, effectively promoting bacterial inactivation.

3.4 Effects of dipping and spraying of CS/N-CD suspensions on the physical appearance and microbial community of chili fruits

The dipping and spraying of CS/N-CD suspensions showed similar trends in overall preservation performance. As shown in Fig. 5A, the pepper fruits treated with the control (CK) and CaCl₂ solution exhibited significant microbial decay over the 21-day storage period. In contrast, the CS/N-CD-dipped fruits maintained a visually intact and fresh appearance throughout storage, suggesting superior resistance to microbial spoilage.⁵⁵ Furthermore, after 21 days of storage, the pepper fruits treated with CS/N-CDs exhibited the lowest weight loss, at 8.5%, which was significantly lower than that of the control group (Fig. 5B).

Fig. 5 Effects of dipping with the CS/N-CDs on pepper fruits' appearance (A) and weight loss rate (B). Microbial changes in the pepper fruits after dipping with CS/N-CDs during storage. Alpha diversity index (C); principal components analysis (D); relative abundance of dominant bacterial communities at the phylum level (E); and relative abundance of dominant bacterial communities in major genera (F). The letters indicate significant differences among groups (p < 0.05).

Additionally, the bacterial communities of the chili fruits were analyzed. No significant differences were observed in the alpha diversity indices (Chao1, Shannon, and Simpson) between the CS/N-CD-dipped group and the control, indicating comparable overall richness and evenness (Fig. 5C and S2C). However, β-diversity analysis revealed a significant shift in bacterial community structure (Fig. 5D), suggesting a stable diversity but altered microbial composition. Dominant phyla among the treatments were Proteobacteria (81.4%), Bacteroidetes (5.4%), Firmicutes (5.2%), and Actinobacteria (6.9%). Notably, CS/N-CDs increased the relative abundance of Actinobacteria by 355.4% versus a 5.1% decline in the control (Fig. 5E). At the genus level, the CS/N-CD suspensions significantly suppressed spoilage-associated taxa: Enterobacter and Aureobacter, both implicated in fruit rot, decreased by 98.2% and 65.0%, while beneficial genera such as Pseudomonas, Arthrobacter, and Sphingomonas were enriched by 61.4%, 96.9%, and 92.8%, respectively (Fig. 5F). Thus, CS/N-CD dipping effectively modulated the microbial community on the surface of pepper fruits by suppressing decay-associated bacteria and promoting the growth of potentially beneficial taxa, enhancing postharvest preservation.

Meanwhile, throughout the 21-day storage period, no visible mold growth or brown spots were observed on the CS/N-CD-sprayed pepper fruits, which retained better texture compared to other treatments. In contrast, fruits in the control and CaCl₂ treatments exhibited obvious microbial damage, including significant mold growth (Fig. S3A). The trend in weight loss of the peppers upon CS/N-CD spraying was consistent with that of the dipping treatment. On day 21, the CS/N-CD-sprayed fruits showed a significantly lower weight loss rate (7.3%) compared to the CK group (16.0%) (Fig. S3B). Furthermore, β-diversity analysis showed a clear separation between the CS/N-CDs and the control (Fig. S3D), suggesting that the CS/N-CD suspensions altered the bacterial community structure on the fruit surface. Among all the sprayed treatments, the dominant bacterial phyla included Proteobacteria (74.9%), Bacteroidetes (3.4%), Firmicutes (9.2%), and Actinobacteria (9.6%). Compared with the control, the relative abundance of Bacteroidetes increased by 10.5% in the CS/N-CD group, whereas Proteobacteria decreased by 12.1% (Fig. S3E). At the genus level, spoilage-related taxa such as Enterobacter, Aureobacterium, and Ralstonia decreased by 82.9%, 91.8%, and 72.6%, respectively, in the CS/N-CD-treated fruits compared to the control. Several beneficial genera were also enriched. The abundance of Pseudomonas and Arthrobacter, known biocontrol agents, increased by 87.1% and 88.7%, respectively. The abundance of Sphingobacterium and Paenibacillus, which can degrade fungal cell walls, rose by 95.3% and 99.9%, respectively (Fig. S3F). Notably, Ochrobactrum, a genus known to suppress bacterial scab disease in chili and tomato fruits, increased by 95.0% compared to CK. In summary, similar to the dipping method, the spraying of CS/N-CD suspensions effectively reduced the abundance of decay-related bacterial genera (Enterobacter, Aureobacterium, and Ralstonia) and promoted the proliferation of beneficial taxa (Pseudomonas, Arthrobacter, Sphingobacterium, Paenibacillus, and Ochrobactrum), thereby improving the microbial microenvironment during storage of chili pepper fruit.⁵⁶

3.5 Preservation performance and biosafety evaluation of CS/N-CD films on chili fruits

Based on the previously discussed structural, barrier, antioxidant, and antibacterial properties, the prepared CS/N-CD composite film was confirmed to be a multifunctional active packaging material. Subsequently, its effectiveness in preserving the postharvest quality of chili fruits was evaluated. After 21 days of storage, fruits without any film treatment and those wrapped with PE film exhibited significant browning, decay, and mold growth. In contrast, the fruits packaged with CS/N-CD film maintained a lower ripening level and showed no visible signs of mold contamination (Fig. 6A). Notably, the CK and PE groups began to show decay as early as day 7 and were completely spoiled by day 21. Meanwhile, the CS film group only suffered microbial deterioration by the end of the storage period. However, fruits packaged with the CS/N-CD film displayed no obvious signs of decay throughout the whole 21-day storage duration. Additionally, on day 21, the CS/N-CD film-wrapped fruits exhibited a weight loss of 67.2%, which was lower than that of the CK group (84.5%) and the CS film group (77.2%). The lowest weight loss was observed in the PE film treatment (18.1%) (Fig. 6B). This can be attributed to the ultralow permeability of PE film, which effectively reduced moisture loss during storage.⁵⁷ However, such high barrier properties also created a humid microenvironment, making the fruits more susceptible to microbial infection and decay, as clearly evidenced in their visual appearance (Fig. 6A).

Fig. 6 Effects of CS/N-CD packaging on pepper fruits' appearance (A), weight loss rate (B), and firmness (C). Hemocompatibility evaluation of the N-CDs based on red blood cell (RBC) hemolysis assay (D) and cytotoxicity assessment of the N-CDs on NIH3T3 fibroblasts after 24 h incubation (E). The letters indicate significant differences among groups (p < 0.05).

Firmness is another critical parameter for evaluating the quality deterioration of chili fruit during storage.⁵⁸ All treatments showed a progressive decline in firmness with extended storage. By day 21, the firmness of the chili fruits in the control, CS film, and CS/N-CD film decreased by 4.8-fold, 4.1-fold, and 2.5-fold compared to the initial value, respectively. Notably, CS/N-CD film-wrapped fruits retained significantly higher firmness at the end of storage, with 100% and 45.5% improvements compared to the control and CS film groups, respectively (Fig. 6C). In summary, the CS/N-CD composite film exhibited a strong preservative effect on chili fruits during storage. Its antimicrobial activity effectively inhibited microbial damage, while its excellent barrier properties against UV radiation, water vapor, carbon dioxide, and oxygen contributed to the overall delay in quality degradation.

Postharvest weight loss and firmness degradation are closely associated with changes in fruit juiciness, texture, and flavor quality. Excessive moisture loss accelerates dehydration and volatile loss, leading to reduced freshness and diminished flavor perception, while softening weakens cell wall integrity and adversely affects mouthfeel.^59,60 The CS/N-CD-treated chili peppers exhibited significantly reduced weight loss and slower firmness decline compared with the control, indicating improved water retention and delayed tissue degradation. These results suggest that the CS/N-CD coatings help maintain the characteristic flavor, texture, and sensory quality of chili peppers throughout storage, thereby enhancing their overall postharvest acceptability.

Biosafety evaluation is an essential prerequisite for packaging films used in fruit and vegetable preservation. Herein, hemolysis and cytotoxicity assays were conducted to evaluate the potential toxicity of N-CDs. The hemolysis assay used red blood cells to assess potential blood compatibility risks during food contact, while the NIH/3T3 fibroblast assay evaluated general cytotoxicity toward mammalian cells.^61,62 As shown in Fig. 6D, the supernatants of both the saline control and N-CD-treated groups remained pale red and transparent, whereas that of the distilled water control exhibited complete hemolysis and appeared bright red and turbid. These results indicate that the N-CDs exert minimal hemolytic effects on red blood cells. Notably, even at the highest tested concentration (500 μg mL⁻¹), the hemolysis rate remained far below the permissible threshold of 5%, confirming the excellent hemocompatibility and non-toxic characteristics of the N-CDs.⁶³ The cytotoxicity results showed that NIH/3T3 cell viability remained above 90% after exposure to varying concentrations of N-CDs, indicating no significant inhibition of cell proliferation (Fig. 6E). These consistently high cell survival rates confirm the excellent cytocompatibility of the N-CDs, and their safety and suitability for direct food-contact applications.

4. Conclusions

CS/N-CD composite films were successfully prepared by incorporating N-CDs into a CS matrix. The resulting films exhibited excellent multifunctional properties, including strong antibacterial and antioxidant activity, effective UV shielding, and enhanced barrier performance against water vapor, oxygen, and carbon dioxide. These characteristics make them promising candidates for active food packaging applications. The results of the chili pepper preservation experiment showed that both dipping and spraying treatments with CS/N-CD suspensions effectively maintained the postharvest quality, significantly reducing weight loss and preserving firmness during 21 days of storage. The film exhibited better efficacy than the suspension. Compared to the control and conventional film treatments, CS/N-CD films delayed visible decay and suppressed microbial spoilage. Microbial community analysis showed a clear shift in bacterial composition, with a decrease in spoilage-associated genera (Enterobacter, Chryseobacterium, and Ralstonia) and an increase in beneficial taxa (Pseudomonas, Arthrobacter, Sphingobacterium, Paenibacillus, and Ochrobactrum), contributing to a more favorable storage microenvironment. Among all treatments, the 0.5% CS/N-CD composite film exhibited optimal preservation efficacy, as evidenced by the lowest weight loss and highest firmness retention after storage. This superior performance arises from the synergistic enhancement of its barrier, antioxidant, and antibacterial functions, which together contribute to the stabilization of the fruit-surface microenvironment and effective delay of quality deterioration. This study provides insights that the CS/N-CD films not only enhance the physical preservation performance but also modulate microbial communities on the surface of the pepper fruits, thereby offering a sustainable and effective strategy for extending the shelf life of perishable fruits.

Conflicts of 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.

Data availability

The authors confirm that the data supporting the findings of this study are available within the article and its SI.

Supplementary information (SI): the SI file contains additional data and figures, including the appearance and surface properties of CS/N-CD solutions and films, as well as storage quality and microbial community analyses of chili peppers treated with CS/N-CDs. See DOI: https://doi.org/10.1039/d5en00743g.

Acknowledgements

Financial support from the National Natural Science Foundation of China (42421005, 42277225, 41807378) is gratefully acknowledged.

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