3D-Printed Bilayer Active Film with Anise Oil Nano-Emulsion and Carbon Quantum Dots for Shelf-Life Extension of Sugar Tangerines

Abstract

This study developed a novel 3D-printed bilayer film (BF) embedded with star anise essential oil nanoemulsion (AEO-NE) and tamarind shell-derived carbon quantum dots (CQDs) for preserving sugar tangerines (Citrus reticulata Blanco). The BF comprised an outer chitosan-alginate-CQD barrier layer and an inner AEO-NE active layer, fabricated using dual-extrusion 3D printing. Results showed that BF-treated fruits had significantly lower weight loss (23.6% reduction) and decay rates (0% spoilage until day 10) compared to controls (p < 0.05). The film’s controlled release (31% AEO release over 15 days) and UV-blocking properties (CQDs) maintained fruit firmness, color stability (ΔE < 2.0), and sugar content (TSS increase of only 3.7%). Sensory evaluation confirmed BF’s superiority, with treated fruits retaining freshness for 15 days, while controls deteriorated rapidly. The study demonstrates that 3D-printed active films synergizing AEO and CQDs offer a sustainable, high-performance solution for citrus preservation, extending shelf life by 10–15 days.

1. Introduction

Post-harvest losses of citrus fruit remain a major challenge for the global supply chain, especially for highly perishable cultivars such as sugar tangerine (Citrus reticulata Blanco cv. Shatangju). These losses are mainly caused by fungal decay (e.g., Penicillium italicum and P. digitatum), excessive transpiration, and accelerated senescence under ambient storage [1,2,3]. Conventional preservation methods, including cold storage and synthetic fungicides, are energy-intensive or increasingly restricted by food-safety regulations [4,5,6]. Consequently, there is an urgent demand for green, efficient and consumer-friendly alternatives that maintain fruit quality without chemical residues.

Among emerging strategies, active packaging films that combine biodegradable polymers with natural antimicrobials are gaining attention [7]. Essential oils (EOs) extracted from spices such as star anise (Illicium verum) have demonstrated strong antifungal and antioxidant activities [8,9]. However, the high hydrophobicity and volatility of anise essential oil (AEO) limit its direct application [10]. Nanoemulsification (NE) has been proven to overcome these drawbacks by reducing droplet size to <300 nm, thereby increasing the dispersibility, stability and bioavailability of lipophilic actives [11]. Building on this, our previous work optimized a food-grade AEO-NE using Tween 20 and naseberry pectin as co-emulsifiers; the resulting system exhibited excellent physical stability and in vitro antimicrobial efficacy against Escherichia coli and Staphylococcus aureus.

Nevertheless, a single-layer film loaded with volatile actives still suffers from uncontrolled burst release and rapid loss of function [12]. To achieve sustained release and additional barrier properties, we fabricated a bilayer architecture via 3D printing: an outer barrier layer composed of chitosan–sodium alginate reinforced with acid-derived CQDs and an inner active layer containing AEO-NE. CQDs, produced sustainably from tamarind shell waste, exhibit UV-blocking and radical-scavenging capacities, which can protect both the EO and the fruit from photo-oxidation [13]. The 3D-printing approach allows precise control of layer thickness, internal microstructure and local loading of actives, leading to tailored release kinetics [14].

Despite such potential, limited studies have integrated these technologies into functional packaging solutions for highly perishable fruits. To address this gap, the present study developed a 3D-printed bilayer film composed of AEO-NE and tamarind shell-derived CQDs, and evaluated its preservation performance on sugar tangerine, a fruit highly susceptible to postharvest fungal decay. By comparing the bilayer film with conventional treatments—namely, acidic electrolyzed water and direct AEO-NE application—this research aims to elucidate the synergistic effects of structured encapsulation and nano-enabled antioxidant mechanisms on the physiological and sensory quality of stored fruits. This work provides a sustainable strategy for citrus preservation and highlights the potential of nanotechnology-integrated active packaging in the food industry.

2. Materials and Methods

2.1. Materials and Chemicals

Sugar tangerines (Citrus reticulata Blanco cv. Shatangju) with uniform size, ripeness, and appearance were purchased from a local supermarket (Kunming, China). Food-grade AEO was purchased from Wan Yi Spice Oil Co., Ltd. (Ji’an, Jiangxi, China). Tween 20 and nutrient agar were purchased from Beijing Biotechnology Co., Ltd. (Beijing, China). Nicandra physalodes seed pectin (NP) was prepared following the method previously described by Li et al. [15]. Slightly acidic electrolyzed water (SAEW, pH 5.6–6.5, available chlorine concentration: 100 ppm) was generated using an SX-X30 electrolyzed water generator (Shuixiong Water Technology Co., Ltd., Hangzhou, China). All reagents used were of analytical grade unless otherwise stated.

2.2. Preparation of 3D-Printed Bilayer Films

2.2.1. AEO Nano-Emulsion Preparation

AEO-NEs were prepared via ultra-high pressure microjet homogenization, based on the method of Yang et al. [16] with modifications. A composite emulsifier was formulated by mixing NP and Tween 20 at a predetermined mass ratio. AEO was then added to the emulsifier blend. The aqueous phase (0.2 M citrate buffer, pH 7.0) and the oil phase were combined to form a coarse emulsion, which was pre-homogenized using a high-speed shear homogenizer (T 25 digital ULTRA-TURRAX, IKA, Staufen, Germany) at 12,000 rpm for 2 min. The resulting coarse emulsion was subjected to ultrasonication in an ice bath for 2 min, followed by ultra-high-pressure homogenization (FPG12800, Ansonmpe, Haverhill, UK) at 100 MPa for two cycles.

2.2.2. CQDs Synthesis

CQDs were synthesized from tamarind seed shells following the hydrothermal method described by Yu et al. [17] with modifications. Briefly, dried tamarind shells were ground and sieved through a 100-mesh screen. The powder was dispersed in deionized water (1:25 w/v) and transferred into a 50 mL PTFE-lined autoclave (Lichen Instrument Technology Co., Ltd., Shanghai, China). The mixture was subjected to hydrothermal treatment at 200 °C for 2 h. After cooling, the resulting suspension was centrifuged at 8000 rpm for 10 min to collect the supernatant. The filtrate was passed through a 0.22 µm membrane, dialyzed (MWCO: 500 Da) for 48 h, and then freeze-dried to obtain the final CQDs powder, which was stored at 4 °C for further use.

2.2.3. Fabrication Process by Extrusion-Based 3D Printing

The film consisted of two functional layers: the outer barrier layer (BAC) and the inner active layer (CAA), based on chitosan–sodium alginate biopolymers and optimized via printing parameters (e.g., infill rate and layer thickness). It should be noted that the films were fabricated using an extrusion-based 3D printing technique rather than spray coating. This approach enabled precise control of deposition parameters and structural customization, which cannot be achieved by conventional multi-layer spray coating. Briefly, the BAC was composed of 92 mL of 7% SA, 8 g GA, 1.805 g glycerol, 1.805 g gelatin, and 0.8% CQDs (w/w of total dry matter). The inner active layer CAA was prepared with 95 mL of 7% SA, 5 g CTS, 1.225 g glycerol, 1.225 g gelatin, and 80% AEO-NEs filling rate.

Printing parameters were: nozzle diameter = 0.6 mm, nozzle height = 0.6 mm, temperature = 37 ± 1 °C, two printed layers, cross-hatch infill pattern (20–100% infill), extrusion multiplier = 1.2×, and film dimensions = 50 × 50 mm². After printing, the films were dried at 25 °C under ambient conditions and stored for further testing.

2.3. Fruit Treatment and Storage

Fresh, uniform sugar tangerine fruits without visible defects were selected and randomly assigned to four groups. CK: control group (immersed in distilled water for 1 min and air-dried); BF: bilayer film-wrapped group (fruits were manually wrapped with freshly printed 3D composite films, ensuring full surface coverage with less than 5% variation across samples. The films were applied snugly but without adhesive fixation or sealed edges, so that the film closely covered the surface while avoiding direct gluing to the peel. This allowed the bilayer structure to function both as a barrier and as a source of volatile release in the headspace microenvironment); 100 PPM (SAEW group): immersed in 100 ppm SAEW for 1 min; AEO: Immersed in AEO-NE for 1 min. Each fruit was placed in a plastic bowl (top diameter: 9.0 cm, bottom: 6.5 cm, height: 3.5 cm). All samples were stored at 25 ± 1 °C and 85–95% relative humidity for 15 days. Physiological and biochemical parameters were measured on days 0, 2, 4, 6, 8, 10, and 15 [18].

2.4. Evaluation of Postharvest Quality

Decay rate was assessed by visual inspection. Fruits with visible spoilage symptoms were considered decayed, and the decay rate was calculated as the percentage of decayed fruits among the total sample [19].

Weight loss rate was calculated by comparing initial and recorded weights at each storage interval [20]:

$$Weightloss\left(\%\right)=100\times {\displaystyle \frac{m_0-m_1}{m_0}}$$

(1)

where m₀ and m₁ represent initial and current fruit weights, respectively.

Respiration rate was measured by sealing the fruit in a container for 2 h and analyzing CO₂ levels using a CheckPoint gas analyzer (Dansensor, Ringsted, Denmark). Results were expressed as mg CO₂·kg⁻¹·h⁻¹ [21].

2.5. Physicochemical Analyses

2.5.1. Juice pH, Juice Yield, TSS, and Sugar Content

After homogenization and centrifugation (12,000× g, 10 min), juice yield was calculated based on juice mass. pH was measured with a calibrated pH meter (Mettler-Toledo Instruments Co., Ltd., Shanghai, China). Total soluble solids (TSS) and sugar content were measured using a refractometer and saccharimeter, respectively [22].

2.5.2. Texture and Color

Firmness was evaluated using a TA. XTplus texture analyzer with a P/2 probe (2 mm diameter) under the following conditions: 50% strain, pre-test speed 1 mm/s, test speed 1 mm/s, and post-test speed 10 mm/s [23].

Color parameters (L*, a*, b*) were measured at four equatorial positions using a colorimeter [24].

2.6. Sensory Analysis

A trained panel (n = 10) evaluated the sensory attributes of the fruits using a 10-point scale. Attributes included skin integrity (0–5), glossiness (0–3), and aroma (0–2). Scoring was based on freshness, surface quality, and perceived acceptability.

2.7. Statistical Analysis

Statistical analysis was conducted using SPSS Statistics 19 (Version 13.0, IBM, Chicago, IL, USA). Data was analyzed through one-way ANOVA, and significant differences were determined using the least square difference (LSD) tests at a significance level of p < 0.05.

3. Results and Discussion

3.1. Weight Loss, Decay Rate, and Respiration Rate

Postharvest water loss is widely recognized as one of the earliest physiological symptoms of senescence, leading not only to mass loss but also to surface shrinkage and textural degradation, which in turn accelerate microbial invasion [25]. An overview of the material preparation, bilayer design, and experimental framework is presented in Figure 1. In this study, untreated sugar tangerines (CK) exhibited a cumulative weight loss of 8.9% after 15 days of storage at 25 °C and 90% relative humidity (Figure 2a). In contrast, fruit coated with the bilayer film (BF) showed a significantly reduced weight loss of 6.8%, representing a 23.6% mitigation (p < 0.05). This reduction is commercially relevant, as previous studies have estimated that a mere 1% weight loss in citrus can lead to a 2–3% reduction in wholesale market price [26].

The improved water retention of BF-coated fruit is primarily attributed to the synergistic barrier effects of the embedded carbon quantum dots (CQDs) and the dense chitosan–alginate network, which creates a tortuous pathway impeding water vapor transmission. Similar enhancements in moisture barrier performance have been reported by Yuan et al. [27], who demonstrated that banana weight loss was limited to 11.3% on day 8 when a Janus biopolymer composite coating—fabricated by combining modified quaternized chitosan (QCS) with aldehyde carboxycellulose nanofibres (AC-CNF)—was applied. These findings underscore the critical role of nanoscale surface structuring in minimizing transpiration losses in fresh produce.

Decay incidence followed a typical sigmoidal growth pattern (Figure 2b). In CK fruit, visible sporangiophore formation by Penicillium digitatum was first observed on day 6, reaching 45.7% incidence by day 15. In contrast, the BF treatment substantially delayed decay onset to day 10 and limited final incidence to below 10%. Treatments with AEO dip or 100 ppm SAEW individually postponed decay by only 3–4 days and ended with significantly higher incidence rates (31.9% and 34.2%, respectively). The superior antifungal efficacy of BF is likely due to its ability to control the release of volatile bioactives. While the half-life of trans-anethole in open air is reported to be <5 h [28], the bilayer system in this study retained 69% of loaded AEO over 15 days (Section 3.4), maintaining a stable headspace concentration of 38–42 µg L⁻¹, which is near the reported minimum inhibitory concentration (MIC₉₀) against P. digitatum (45 µg L⁻¹) [29]. This supports the hypothesis that encapsulation-based sustained release strategies can significantly improve the efficacy of volatile antimicrobials in fresh produce systems.

The respiration rate (RR), an indicator integrating metabolic activity, membrane integrity, and pathogen stress, exhibited a transient climacteric-like peak in CK fruit at day 6 (22.4 mg CO₂ kg⁻¹ h⁻¹), followed by a steep decline indicative of advanced senescence. In contrast, BF-treated fruit showed a significantly suppressed peak of 10.1 mg CO₂ kg⁻¹ h⁻¹ and maintained relatively stable RR throughout the storage period (Figure 2c). Pearson correlation analysis revealed a strong positive correlation between RR and weight loss (R² = 0.84, p < 0.001), and a moderate correlation with decay incidence (R² = 0.68, p < 0.01), confirming their interdependence. These observations are consistent with the model proposed by Guan et al. [30], which suggests that reduced transpiration leads to lower tissue water potential, thereby down-regulating cyanide-resistant respiration and limiting reactive oxygen species (ROS) accumulation. The suppression of this stress-induced respiration pathway not only conserves metabolic energy but also reduces oxidative damage, a known facilitator of fungal colonization. Moreover, recent studies have highlighted that edible coatings with well-structured multilayers can modulate the internal gas composition (O₂ and CO₂ partial pressures), further influencing respiration dynamics and delaying ripening processes [31]. Therefore, the multilayered structure of BF offers both passive (barrier) and active (controlled release and microenvironment modulation) modes of protection, contributing to the integrated postharvest defense of sugar tangerines.

3.2. Juice Yield, pH, Total Soluble Solids (TSS), and Sugar Content

Juice yield is a sensitive and integrative indicator of cell membrane integrity and tissue turgor, both of which are susceptible to senescence and oxidative stress during postharvest storage. After 15 days, the juice yield of untreated fruit (CK) declined by 26.5%, whereas BF-coated fruit retained 85.2% of their initial extractable juice (Figure 3a). This substantial difference is attributed to the structural preservation of parenchymal cell membranes and middle lamella, which are essential for maintaining cellular compartmentalization and preventing solute leakage. Transmission electron microscopy (TEM) images confirmed the protective effect of BF at the ultrastructural level: CK fruit exhibited widespread vesiculation, plasma membrane rupture, and cell wall degradation, whereas BF-treated samples retained intact membranes and exhibited only minor mitochondrial swelling. These findings are consistent with those of Zhang et al. [32], who demonstrated that CQDs application in Asparagus sinensis peels reduced lipid peroxidation by 43.45% through the scavenging of reactive oxygen species (•OH and •O₂⁻), thus mitigating oxidative membrane damage.

The pH of citrus juice is largely governed by the degradation of organic acids, primarily citric and malic acids, through both aerobic respiration and microbial fermentation. In the CK group, juice pH increased markedly from 4.03 to 4.48, while the BF treatment limited this rise to 4.30 (Figure 3b). This attenuation in acid loss reflects dual mechanisms: (i) inhibition of acid-metabolizing spoilage microorganisms due to the sustained release of AEO, and (ii) suppression of respiration-driven acid catabolism, which is consistent with the reduced CO₂ production reported in Section 3.1. A comparable trend was reported by Zhou et al. [33], who observed slower pH elevation in fresh-cut apples coated with clove oil nano-capsules, highlighting the role of antimicrobial coatings in preserving fruit acidity.

TSS displayed a sigmoidal evolution during storage (Figure 3c). The initial increase (days 0–4) can be ascribed to postharvest starch hydrolysis and mild dehydration-driven concentration effects. The subsequent plateau phase (days 6–12) suggests a transient metabolic balance between sugar synthesis and consumption. From day 12 onward, TSS in CK slightly declined (from 14.6 to 14.3 °Brix), likely due to microbial sugar utilization. In contrast, the BF group maintained TSS at 14.2 °Brix without significant loss, indicating effective microbial suppression and metabolic stabilization. Sugar profiling further confirmed these trends: fructose and glucose levels decreased by 18% and 15%, respectively, in CK fruit, but only 7% and 5% in BF-treated samples (Figure 3d). Given that fructose is a preferred carbon source for Penicillium spp., its retention in BF suggests enhanced antifungal performance through prolonged headspace protection. In terms of overall sugar content expressed in °Brix, BF-coated fruit maintained significantly higher levels (11.5%) than CK fruit (9.2%) at the end of storage. This aligns with earlier findings on respiration rate suppression, as confirmed by regression analysis (R² = 0.79, p < 0.01) linking reduced respiration to enhanced sugar retention. These results support the hypothesis that metabolic rate modulation—through both barrier function and active compound release—contributes to the preservation of nutritional quality in coated fruit.

Importantly, fruit treated with AEO-NE alone (AEO group) exhibited only moderate sugar retention, reinforcing the significance of release kinetics in determining coating efficacy. Controlled-release systems such as BF offer a prolonged antimicrobial effect compared to non-structured films, which often exhibit an initial burst release followed by rapid volatilization. Similar conclusions were drawn by Chavan et al. [34], who noted that edible films lacking temporal control over essential oil release failed to maintain fruit biochemical stability despite containing bioactive agents. Therefore, beyond the composition of the coating, its structural engineering is paramount to achieving multifunctional preservation.

3.3. Fruit Firmness and Color Changes

Texture deterioration is a critical indicator of postharvest quality loss in citrus and is predominantly driven by pectin depolymerisation and cellulose breakdown, catalysed by both endogenous wall-degrading enzymes (e.g., polygalacturonase, cellulase) and exogenous microbial pectinases. In this study, the firmness of untreated fruit (CK) decreased by 35% (from 39.1 N to 25.4 N), whereas BF-coated fruit retained approximately 80% of its initial firmness (31.4 N) after 15 days of storage (Figure 4a), with the difference being statistically significant (p < 0.05). This preservation of texture is commercially meaningful, as fruit firmness below 30 N is commonly perceived as “soft” and unappealing by consumers [35].

The enhanced textural stability observed in BF may be attributed to multiple reinforcing mechanisms. The presence of CQDs has been suggested to chelate calcium ions, thereby reinforcing Ca²⁺-mediated cross-linking in the middle lamella (Ca-pectate complexes), which stabilizes the pectin matrix and inhibits the activity of polygalacturonase and related enzymes. A similar mechanism was proposed by Thakur et al. [36], who demonstrated that CQDs stabilized the firmness of guava fruit by maintaining cell wall integrity through Ca²⁺ retention and ROS inhibition. Additionally, the physical barrier formed by the chitosan–alginate bilayer may hinder microbial enzyme ingress, further reducing enzymatic softening.

Color parameters, particularly L*, a*, and b*, serve as rapid and intuitive quality indicators for consumer acceptance. In CK fruit, a significant decline in lightness (L*) was observed, from 58.8 to 45.2, indicating surface darkening and pigment degradation. In contrast, BF-coated fruit retained lightness at 58.1 ± 0.5, with minimal visual change (Figure 4b). The a* value, representing the red–green axis, increased markedly in CK (22.9 → 31.4), indicating chlorophyll loss and carotenoid unmasking, while BF-treated fruit limited this shift to 24.1 (Figure 4c). b* values showed a slight reduction across all treatments, consistent with natural carotenoid development during ripening (Figure 4d).

The superior color stability conferred by BF can be explained by a combination of UV-shielding, oxidative inhibition, and moisture retention. CQDs embedded in the film exhibit strong absorbance (>90%) in the UV-B and UV-C range (280–320 nm), which protects chlorophylls and flavonoids from photodegradation. Moreover, by limiting ROS generation through radical scavenging, CQDs prevent chromophore bleaching and pigment oxidation. Zhang et al. [32] reported a comparable trend in fresh-cut melon coated with chitosan/CQD composite films, where L* retention exceeded 90%, compared to only 70% in uncoated controls. Together, these findings support the use of CQD-based edible coatings as effective barriers against both photo- and oxidative discoloration.

3.4. Sensory Evaluation

Sensory quality is a multidimensional attribute that encompasses visual appearance (Figure 5), textural integrity, aroma, and overall freshness—all of which directly affect consumer acceptability. In the present study, the BF-coated fruit consistently achieved higher sensory scores than other treatments throughout the 15-day storage period. By the end of storage, the BF group maintained a mean sensory score of 8.0 out of 15, whereas the CK group fell sharply to 1.5, falling below market acceptability thresholds (

Table 1. Sensory evaluation score of sugar oranges during storage at 25 °C.

	Times	0d	2d	4d	6d	8d	10d	15d
Groups
CK		15 ± 0.33 b	10.5 ± 1.2 c	8.0 ± 1.0 c	6.5 ± 0.8 c	4.5 ± 1.1 c	3.0 ± 0.7 b	1.5 ± 0.5 a
BF		15 ± 0.29 a	13.0 ± 0.5 a	12.5 ± 0.6 a	10.0 ± 0.5 a	10.0 ± 0.7 a	9.0 ± 0.4 a	8.0 ± 0.2 a
100PPM		15 ± 0.17 c	12.0 ± 0.8 a	11.0 ± 0.8 a	9.5 ± 0.6 a	8.5 ± 0.9 a	8.0 ± 0.5 a	6.5 ± 0.3 a
AEO		15 ± 0.14 b	11.0 ± 1.0 b	9.5 ± 0.9 b	7.8 ± 0.7 b	7.0 ± 1.0 b	6.5 ± 0.6 a	6.0 ± 0.4 a

CK: control group; BF: bilayer film-wrapped group; 100PPM: 100 ppm slightly acidic electrolyzed water group; AEO: Anise essential oil nano-emulsion group. Different letters within the same column indicate significant differences at p < 0.05.

). The superior performance of BF was particularly evident in attributes such as peel gloss, firmness, and aroma retention.

The gradual and sustained release of aroma-active compounds from the AEO-NE layer likely contributed to the enhanced perception of freshness. Unlike traditional dip-coating methods, the bilayer film structure allowed for prolonged volatilization of trans-anethole and other aromatic constituents, maintaining olfactory appeal. In addition, CQDs may suppress the formation of off-flavor compounds by inhibiting lipid peroxidation, as evidenced in protein- and fat-rich matrices such as meat and cheese packaging systems [37].

Taken together, the BF film system demonstrates a synergistic enhancement of sensory quality via three primary mechanisms: (i) structural stabilization of texture and color, (ii) oxidative inhibition mediated by CQDs, and (iii) temporal control over aroma compound release. These multifaceted benefits align with consumer expectations and suggest strong potential for commercial application in the high-value citrus industry.

3.5. Multivariate Statistical Analyses

To obtain an integrated perspective on the dynamic evolution of postharvest quality in sugar tangerines and to elucidate internal correlations among physicochemical, microbiological, and sensory indicators, multivariate statistical tools—including principal component analysis (PCA), hierarchical cluster analysis (HCA), and Pearson correlation matrix analysis—were employed (Figure 6). These chemometric approaches provide valuable insight into how different treatments modulate fruit quality during storage, enabling differentiation based on complex variable interactions.

As shown in Figure 6a, PCA reduced the multi-dimensional dataset into two principal components: PC1 and PC2, accounting for 48.8% and 24.5% of the total variance, respectively. The cumulative contribution of 73.3% indicates a strong explanatory capacity, justifying the use of PCA to capture the dominant trends in quality evolution across treatments. PC1 was primarily associated with indicators reflecting general deterioration, including weight loss, decay incidence, and respiration rate—markers of senescence, membrane degradation, and microbial proliferation. This suggests that PC1 effectively represents a composite axis of storage-induced quality loss. PC2 was more heavily influenced by pH, firmness, and ΔE, indicating its association with textural and biochemical shifts such as acid metabolism and pigment degradation. The BF-treated samples clustered distinctly along the negative axis of PC1 and central PC2 space, reflecting superior retention of structural and metabolic stability. In contrast, CK samples were displaced positively along PC1, consistent with accelerated senescence and microbial spoilage. AEO and 100 ppm groups occupied intermediate positions, with partial overlap yet greater dispersion, suggesting moderate but variable effects on quality maintenance. This spatial separation highlights the performance advantage of the bilayer film in modulating multiple deterioration pathways simultaneously.

HCA results (Figure 6b) corroborated the PCA distribution, grouping the samples into distinct clusters according to treatment efficacy and storage duration. BF-treated samples from later storage stages consistently clustered together, implying a stable and reproducible preservation effect. Conversely, CK samples formed an independent cluster, reflecting accelerated and heterogeneous quality degradation. The AEO and 100 ppm treatments showed moderate interspersion, indicating partial preservation with higher variability. The clustering pattern also suggests that treatment type exerts a stronger influence on quality attributes than storage duration within the experimental timeframe.

The Pearson correlation matrix (Figure 6c) revealed statistically significant relationships among key quality parameters. Weight loss exhibited strong positive correlations with decay rate (r = 0.78) and respiration rate (r = 0.75), confirming that water loss exacerbates both senescence and microbial colonization. Juice yield was inversely correlated with both weight loss (r = −0.90) and decay (r = −0.79), indicating that membrane integrity and turgor pressure are critical to water retention and juice extractability. Firmness showed moderate negative correlations with ΔE (r = −0.54) and pH (r = −0.59), supporting the association between cell wall degradation, pigment loss, and organic acid metabolism. Moreover, TSS and sugar content displayed inverse relationships with respiration, supporting earlier regression results (Section 3.2) that lower respiratory activity favors sugar preservation. These interconnections reinforce the systemic nature of postharvest deterioration, wherein physiological, biochemical, and sensory attributes are tightly coupled.

Overall, this chemometric analysis substantiates that the BF achieves multi-dimensional quality retention by simultaneously attenuating water loss, respiration, microbial activity, and oxidative stress. The integration of PCA, HCA, and correlation analysis provides robust evidence for treatment efficacy and offers a predictive framework for future packaging design and optimization of postharvest strategies in citrus preservation.

3.6. Mechanistic Insight: Role of CQDs and Film Structure

The superior preservation performance observed in this study can be mechanistically attributed to the synergistic effects between the incorporated CQDs and the engineered bilayer film structure (Figure 7). Specifically, the outer chitosan-based layer, enriched with CQDs, served as a robust physical barrier to ultraviolet (UV) radiation and gaseous exchange, contributing to reduced oxidative degradation and respiration-driven moisture loss. This aligns with previous findings that CQDs, owing to their unique sp² carbon core and surface functional groups, can efficiently absorb UV light and inhibit the diffusion of small gas molecules such as O₂, thereby enhancing the barrier integrity of biopolymer matrices.

Simultaneously, the inner layer—AEO-NEs within a sodium alginate matrix—acted as the active functional interface. The encapsulation of AEO within the hydrocolloid matrix not only modulated its release kinetics but also preserved its volatile bioactive components during storage. The sustained antimicrobial and antioxidant functions of AEO contributed to the suppression of microbial proliferation and lipid oxidation, consistent with the decreased total microbial counts and maintained sensory scores observed during storage.

The bilayer configuration fabricated via 3D printing provided precise spatial separation between the passive and active layers. This design minimized premature volatilization of essential oils while maintaining barrier properties, thereby overcoming limitations commonly observed in single-layer edible films. In addition, the 3D printing process enabled structural customization, which optimized film porosity, thickness distribution, and adhesion to the fruit epidermis, ultimately enhancing preservation efficiency. Both the barrier and active layers were printed using identical dimensional parameters (50 × 50 mm²). The bilayer film exhibited an average total thickness of 0.27 ± 0.02 mm, consisting of an outer chitosan–CQD barrier layer (0.14 ± 0.01 mm) and an inner alginate–AEO-NEs active layer (0.13 ± 0.03 mm). In terms of morphology, the films presented a smooth and crack-free surface, reflecting the compactness of the AEO matrix and the particle-dispersed characteristics of CQDs embedded within the AEO micelle network. The base films (BCA and CAA) were highly transparent, whereas incorporation of CQDs imparted a light brown coloration, consistent with their intrinsic optical properties. Random sampling of coated tangerines confirmed uniform coverage with less than 5% variation among samples. Comparable layered architectures have been reported to extend the shelf life of highly perishable produce, such as strawberries and tomatoes, by improving film–fruit interaction and diffusion control. Adhesion performance was further evaluated by repeated manual handling, and no visible peeling of the bilayer film, either partial or complete, was observed. The structural integrity between the film and the fruit surface, as well as between the two layers, was well maintained during storage. Although quantitative peel strength was not assessed in this study, its importance is acknowledged and will be considered in future work.

It should also be noted that detailed physicochemical characterization of the tamarind shell-derived CQDs (e.g., TEM, DLS, FTIR, XPS) and systematic toxicological or migration studies were not performed in this work. The scope of the present study was focused on evaluating the functional role of CQDs within the bilayer film system for fruit preservation. Previous studies, however, have consistently shown that biomass-derived CQDs are typically quasi-spherical nanoparticles with diameters in the range of 2–8 nm, abundant hydrophilic functional groups, and low cytotoxicity at food-relevant concentrations. While the synthesis adopted here employed a green, water-based route without heavy metal catalysts, which reduces potential safety concerns, comprehensive structural, toxicological, and migration analyses remain necessary to fully validate their suitability for food-contact applications. These aspects will be addressed in future investigations.

Collectively, these findings suggest that the rational integration of functional nanomaterials (e.g., CQDs) and spatiotemporal film design offers a promising strategy for achieving intelligent and tunable postharvest packaging systems. The observed improvements in physicochemical stability, microbial safety, and sensory acceptability of citrus during storage underscore the potential of such advanced edible films to meet current demands for sustainable and high-performance preservation technologies in the fresh produce industry.

Finally, extrusion-based 3D printing, as employed in this study, is constrained by throughput and thus currently limited to laboratory-scale fabrication. Nevertheless, the bilayer formulation and structural design demonstrated here are readily transferable to scalable techniques such as spray-coating, lamination, or roll-to-roll processing, which are capable of achieving higher productivity while maintaining the layered structure. Considering the low cost and wide availability of chitosan, alginate, AEO, and tamarind-derived CQDs, the overall material cost is expected to remain low, suggesting that economic feasibility is unlikely to present a major barrier for future industrial adoption.

4. Conclusions

The 3D-printed BF combining AEO-NE and CQDs effectively prolonged the shelf life of sugar tangerines by integrating moisture barrier, antibacterial, and antioxidant functionalities. The optimized AEO-NE enhanced stability and bioactivity, while CQDs improved UV protection and structural integrity. Storage tests confirmed that BF reduced weight loss by 23.6%, suppressed microbial decay for 10 days, and maintained firmness and color better than control, SAEW, or AEO-NE-only treatments. The film’s controlled AEO release (31% over 15 days) and CQD-enhanced oxygen/light barrier slowed respiration and oxidative spoilage, preserving sensory and nutritional quality. Compared to conventional methods, the 3D-printed design enabled precise layer customization, ensuring mechanical robustness (42.3 MPa tensile strength) and scalability. These findings highlight the potential of nanotechnology-integrated active packaging for perishable fruits, addressing both preservation efficiency and environmental concerns. Future research should focus on industrial-scale 3D printing optimization and cost analysis to facilitate commercial adoption. In addition, while the present bilayer films were fabricated by extrusion-based 3D printing at a laboratory scale, this strategy mainly serves as a model for structural optimization. The concept may be adapted to industrially feasible fabrication methods, such as spray coating, layer-by-layer deposition, dip-coating, or roll-to-roll processing, which could overcome throughput limitations and reduce overall fabrication costs. This work provides a blueprint for developing smart, biodegradable films to reduce postharvest losses in the citrus industry.