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Article

Development of Antifungal Packaging Based on Pectin/Gelatin Containing Azadirachta indica Bioactive Extracts for Carica papaya L. Fruit Coating

by
Thais Trindade de Brito Ribeiro
1,2,
Andriele Mendonça Barbosa
1,
Tatiana Pacheco Nunes
3,
Anabela Silvia Guedes da Costa
4,
Maria Beatriz Prior Pinto Oliveira
4,
Gustavo Rodrigues Borges
1,
Francine Ferreira Padilha
1,2,
Claudio Dariva
1,2 and
Klebson Silva Santos
1,*
1
Center for Study on Colloidal Systems (NUESC), Institute of Technology and Research (ITP), Av. Murilo Dantas, 300, Aracaju 49032-490, Sergipe, Brazil
2
Postgraduate Program in Process Engineering (PEP), Tiradentes University (UNIT), Av. Murilo Dantas, 300, Aracaju 49032-490, Sergipe, Brazil
3
Departamento de Tecnologia de Alimentos—DTA/UFS, Universidade Federal de Sergipe (UFS), Av. Marechal Rondon, São Cristóvão 49100-000, Sergipe, Brazil
4
REQUIMTE/LAQV, Laboratory of Bromatolgy, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, R. Jorge de Viterbo Ferreira, 228, 4050-313 Porto, Portugal
*
Author to whom correspondence should be addressed.
Appl. Sci. 2025, 15(8), 4423; https://doi.org/10.3390/app15084423
Submission received: 9 March 2025 / Revised: 31 March 2025 / Accepted: 8 April 2025 / Published: 17 April 2025
(This article belongs to the Special Issue Advances in Food Safety and Microbial Control)
Browse Figures
Versions Notes

Abstract

:
The deterioration of the Carica papaya L. fruit caused by Colletotrichum gloeosporioides highlights the importance of postharvest packaging for extending papaya shelf life. To this end, in this present study, pectin/gelatin-based food packaging (FPC) was enriched with Azadirachta indica hydroethanolic extract (HNE), obtained by pressurized liquid extraction (PLE) and microwave-assisted extraction (MAE). The HNE showed a high concentration of phenolic compounds, with values of 2893 mg GAE/100 g extract (PLE) and 3136 mg GAE/100 g extract (MAE). The packaging thickness incorporated with HNE (FPC + HNE) did not significantly differ (FPC + HNE-PLE: 0.10 ± 0.01, and FPC + HNE-MAE: 0.16 ± 0.04) from the packaging control (FPC: 0.11 ± 0.00). Nevertheless, the FPC + HNE exhibited enhanced elongation (FPC + HNE-PLE: 10.33 ± 0.2%, and FPC + HNE-MAE: 9.50 ± 0.2%) compared to FPC (8.00 ± 0.0%). Variations in water vapor permeability (FPC: 5.2 g·mm/d·m2·kPa, FPC + HNE-PLE: 2.0 g·mm/d·m2·kPa, and FPC + HNE-MAE: 6.9 g·mm/d·m2·kPa) and tensile strength (FPC: 13.76 ± 0.79 MPa, FPC + HNE-PLE: 16.45 ± 2.25 MPa, and FPC + HNE-MAE: 9.24 ± 2.01 MPa) values were observed among all samples. FPC + HNE-PLE resulted in 0% deterioration by Colletotrichum gloeosporioides over 15 days. The antifungal FPC + HNE-PLE provides a promising way to reduce postharvest losses and extend the shelf life of papaya fruit.

Graphical Abstract

1. Introduction

Nowadays, polymeric packaging is widely used for food protection and preservation. However, plastic production has exceeded hundreds of millions of tons annually. These packages are often discarded as waste within a year or less after purchase, contributing to increasing environmental pollution due to massive trash accumulation [1,2,3]. Biopolymer-based edible packaging films have emerged as a relevant alternative to mitigate the impacts of conventional polymers [1,3,4].
Edible packaging can be produced from gelatin [5,6] and/or pectin [6,7,8]. These biopolymers have garnered significant interest from the scientific community due to their biocompatibility and edibility, making them suitable for use in food coating production [8,9]. Moreover, pectin and gelatin films can be enriched with bioactive extracts, providing biological properties such as antioxidant and antimicrobial activity and favorable food-coating characteristics [6,10,11].
Azadirachta indica A. Juss (neem) is a medicinal plant widely studied by the scientific community for its bioactive compounds, such as alkaloids, terpenoids, and phenolic compounds [12,13,14,15], which exhibit antimicrobial activities, including antifungal effects [16]. Antifungal polyphenols with activity against Colletotrichum can be obtained using ethanol [17,18]. However, the efficiency of bioactive compound extraction is influenced by the process used, with pressurized liquid extraction (PLE) and microwave-assisted extraction (MAE) emerging as promising techniques for isolating active biomolecules [19,20,21].
PLE has been widely utilized for extracting polar and non-polar compounds, as the use of solvents under high pressure enhances extraction efficiency. This process allows the solvent to penetrate more effectively into the plant matrix compared to extractions conducted at room pressure, thereby increasing the yield of target compounds from medicinal plants [14,19,20,21,22]. The MAE process involves the extraction of bioactive compounds from biomass under elevated temperature and pressure, which enhances solvent diffusion into the biomass, resulting in the release of target molecules into the solvent. Both PLE and MAE provide more rapid and efficient biomolecule extractions compared to conventional methods [21].
Papaya fruit (Carica papaya L.) is a relevant crop cultivated in tropical and subtropical conditions [23]. It represents one main share of global fruit production, with the five leading producers being India (5.34 million tons/Mt), the Dominican Republic (1.28 Mt), Mexico (1.14 Mt), Brazil (1.11 Mt), and Indonesia (1.09 Mt) [24]. Nevertheless, postharvest diseases, mainly caused by Colletotrichum gloeosporioides, have significantly reduced the overall quality of papayas [25,26,27,28]. To this end, the present study aimed to develop antifungal food packaging based on pectin and gelatin enriched with Azadirachta indica bioactive extracts to coat papayas (Carica papaya L.) and improve their postharvest shelf life.

2. Materials and Methods

2.1. List of Materials

The experimental procedures in this research employed a range of high-quality materials and reagents to ensure proper experimental control and reproducibility. Detailed specifications and sources for these components are outlined as follows: high-purity nitrogen gas (N₂), grade 5.0 (99.999% purity), supplied by White Martins- Laranjeiras, Brazil, was used to create inert atmospheres as necessary. Glacial acetic acid (99.8% purity, Synth, São Paulo, Brazil) was used throughout this study. The pectin (GastronomyLab, Brasilia, Brazil) was derived from citrus fruits and classified as high-methoxyl, with a degree of esterification between 65% and 75% and a degree of amidation below 10%. Other key materials included food-grade gelatin, propylene glycol (≥99% purity, Sigma-Aldrich-Merck KGaA, Darmstadt, Germany), and analytical-grade borax (sodium tetraborate decahydrate, ≥99.5% purity, BC Labs-João Pessoa, Brazil). A 5% active chlorine solution of sodium hypochlorite (Casa dos Químicos, São Paulo, Brazil) was employed during the experiments.
The phenolic content was quantified using the commercially available Folin–Ciocalteu reagent (Dinâmica Química Contemporânea Ltda, Campinas, Brazil). Anhydrous sodium carbonate (ACS grade, ≥99.5% purity, Química Moderna Indústria e Comércio Ltda, Barueri, Brazil) and anhydrous aluminum chloride (ACS grade, ≥99% purity, Dinâmica Química Contemporânea Ltda, São Paulo, Brazil) were also crucial for the analytical procedure. Quantitative analyses were executed through the employment of quercetin (≥98% purity, Merck/Sigma-Aldrich, Barueri, Brazil) and gallic acid (≥98% purity, Vetec, Darmstadt, Germany) as standards. The critical solvents for this investigation were Milli-Q purified distilled water and anhydrous ethanol (99.8% purity, Synth, São Paulo, Brazil). Additionally, microbiological investigations were conducted utilizing potato dextrose agar (PDA, Oxoid, Basingstoke, UK), prepared from a medium powder from a dehydrated culture.

2.2. Obtaining and Preparation of Material Containing Antifungal Biocompounds

Neem (Azadirachta indica A. Juss) leaves and seeds were collected from February to March 2015 at the Brazilian Agricultural Research Center—Embrapa Coastal Tablelands, located in Aracaju, Sergipe, Brazil [29]. The leaves and seeds were cleaned using water and then washed with a 150 ppm sodium hypochlorite solution for 10 min, as described by Martínez-Castro et al. (2024) [30] with some modifications. The samples were dried in a tray dryer with hot-air circulation at 60 °C until reaching a water activity of approximately 0.4. All dried neem samples were milled to a particle size ranging from 8 to 16 mesh using a series of Tyler sieves (Model 1868 Bertel, Caieiras, Brazil).

2.3. Extraction Processes

2.3.1. Pressurized Liquid Extraction (PLE)

PLE was performed using 10 g of dried neem leaves and seeds with an ethanol/water (80:20 v/v) mixture at 150 bar at 50 °C, with a flow rate of 1 mL/min for 60 min. The extraction temperature was controlled via a thermostatic bath connected to a jacketed extractor. A positive displacement pump was used to pressurize and monitor the hydroethanolic solvent system. Pressure control during extraction was controlled using a pressure indicator, transducer, and needle valve. For further details about the experimental apparatus, see Barbosa et al. (2019) [22]. The hydroethanolic neem extracts obtained through PLE (HNE-PLE) were dried at 40 °C in a hot-air circulation oven for 24 h to evaporate the extraction solvent. All PLEs were carried out in triplicate as described by Santos et al. (2023) [29].

2.3.2. Microwave-Assisted Extraction Process (MAE)

MAE was conducted in quartz vessels containing 5 g of dried neem leaves and seeds with 30 mL of an ethanol/water (80:20 v/v) mixture. The quartz vessels were sealed and pressurized with nitrogen for 2 min. The extractions were performed at 50 °C for 60 min using a multimode batch reactor (Anton Paar GmbH, Graz, Austria). The hydroethanolic neem extracts obtained through MAE (HNE-MAE) were filtered and then dried in a hot-air circulation oven at 40 °C for 24 h to evaporate the extraction solvent. All extractions were carried out in triplicate, with modifications as described by Nonglait et al. [31] and Martínez-Castro et al. (2024) [30].

2.4. Determination of Polyphenols by Spectrophotometric Methods

2.4.1. Total Phenolic Compounds (TPC)

The total phenolic content (TPC) was determined using the Folin–Ciocalteau method, with light modification from the literature [32]. A 150 µL sample was mixed with 2400 µL of distilled water and 150 µL of Folin–Ciocalteau reagent (0.25 N), followed by homogenization using a vortex homogenizer. Subsequently, 300 µL of 1 N sodium carbonate was added, and the mixture was homogenized again using the vortex homogenizer. The resulting mixture was incubated in the dark at room temperature for 120 min. The absorbance was measured at 725 nm using a UV-2601 spectrophotometer (RayLeigh, Beijing, China). The calibration curve was generated using gallic acid as the standard (y = 0.0052x + 0.0579; R2 = 0.9986). Total phenolic content values were expressed as milligrams of gallic acid equivalents per 100 g of extract (mg GAE/100 g).

2.4.2. Total Flavonoid Content (TFC)

TFC was determined using the aluminum chloride colorimetric method, with modifications adapted from the method of Barbosa et al. (2019) [22]. A 500 µL sample was mixed with 250 µL of aluminum chloride solution (5.0% prepared in ethanol) and 4.25 mL of ethanol. The mixture was homogenized and incubated at room temperature for 30 min. Subsequently, the absorbance of the reaction was measured at 425 nm using a UV-2601 spectrophotometer (RayLeigh, Beijing, China). The calibration curve was generated using quercetin as the standard (y = 0.0047x + 0.00671; R2 = 0.9942). The results were expressed as milligrams of quercetin equivalents per 100 g of extract (mg QE/100 g).

2.5. Development of the Antifungal Food Packaging

Antifungal food packaging, based on edible pectin/gelatin containing Azadirachta indica bioactive extracts, was produced using the solvent casting technique [33]. The filmogenic formulation was prepared as outlined in Table 1.
The filmogenic solution was prepared using acetic acid, pectin, gelatin, and propylene glycol, as detailed in Table 1, and homogenized under moderate agitation at 40 °C for 30 min. To achieve antifungal effects, neem extracts (1 g) were added to the filmogenic solution and homogenized for 24 h. Subsequently, 1% borax was added to the filmogenic solution, followed by homogenization for 15 min. A volume of 30 mL of the filmogenic solution was then poured onto a polystyrene plate and subjected to solvent evaporation in a hot-air circulation oven at 40 °C for 24 h, resulting in the production of the antifungal food packaging. The food packaging control (FPC) was prepared using the same procedure but without the addition of neem extracts.

2.6. Characterization of the Food Packaging

2.6.1. Fourier Transform Infrared (FTIR) Spectroscopy

The spectra were obtained using a diffuse reflectance technique with a FTIR spectrometer (Perkin Elmer, Shelton, CT, USA). Measurements were performed in the range of 4000–400 cm−1 with a resolution of 4 cm−1 at room temperature [32].

2.6.2. Mechanical Properties

The mechanical properties of food packaging were determined following the ASTM standard method D 882-97 [34], with slight modifications. Briefly, three random locations of each food packaging sample were measured for thickness determination using a digital micrometer caliper (Mitutoyo, Suzano, São Paulo, Brazil). The average thickness of each food packaging strip was used to evaluate mechanical properties. The tensile strength (TS) and percentage elongation at break (EAB) of four rectangular strips (10 × 25 mm) from each food packaging sample were tested using a texture analyzer (TA-XT plus, Stable Micro System Ltd., Surrey, UK), with the initial grip separation set to 20 mm and the mechanical crosshead speed at 1 mm/s.

2.6.3. Water Vapor Permeability (WVP)

The WVP of food packaging was determined gravimetrically, following the procedure described by Narasagoudr et al. (2020) [35] with modifications. Briefly, 10 mL of a saturated sodium bromide solution (58% relative humidity) was added to the test cups, and the food packaging was sealed onto the cup with parafilm. Controls were conducted using one cup wrapped and sealed with parafilm and another left uncovered, without food packaging or parafilm. Subsequently, all cups were weighed and placed in a desiccator (closed system) containing silica gel (0% theoretical RH) at room temperature. The cups were weighed for 48 h, with intervals of 2 h between each weighing. The WVP was calculated using Equation (1):
WVP = (wx)/(tAΔP)
where w: the weight change of the cup (g), x: the packaging thickness (mm), t: time (s), A: the area of exposed packaging (m2), and ΔP: vapor pressure difference between both sides of the packaging.

2.7. In Vitro Evaluation of Antifungal Food Packaging

The mycelial growth inhibition percentage (MGI%) was established using 10 µL of the inoculum of Colletotrichum gloeosporioides (0.97 × 105 conidia/mL) seeded on PDA plates containing a filter paper disc of 10 mm. Food packaging was cut into 1 × 1 cm and exposed on both sides to a 110 V UV lamp with an emission wavelength of 254 nm (Coospider, Jinyun, China) for 15 min; then, the food packaging was placed on the PDA plates reported above. The plates were kept at 25 ± 1 °C in a BOD incubator (7Lab, Rio de Janeiro, Brazil) with alternation of the light–dark cycle of 12 h. Radial mycelial growth was evaluated in 6 days using a ruler to measure the two orthogonal axes on Colletotrichum gloeosporioides colony. The MGI% was calculated as Equation (2):
MGI% = (control diameter − treatment diameter/control diameter) × 100.

2.8. Evaluation of Antifungal Packaging on Postharvest Quality of Papaya Fruit

Papaya fruits (Carica papaya) were obtained at the local market and then washed with a 200 ppm sodium hypochlorite solution. After this, the fruits were separated according to their maturity stage and subdivided into four groups: papaya without packaging (control), papaya packaged without neem extracts, papaya packaged with HNE-PLE, and papaya packaged with HNE-MAE. Papaya fruit was packed by immersion in each filmogenic solution previously described in Table 1 using the solvent casting method. After this, the packed papaya was kept at 18 °C ± 2 °C under a relative humidity of 90 ± 5% and evaluated for 0, 2, 4, 7, 9, 11 and 15 days using the following parameters:
Fresh weight loss was calculated from the difference between the initial weighing of fruits and the final weighing at each evaluation period, and the values were expressed as a percentage.
External appearance regarding fungal incidence was evaluated through visual inspection and quantified as the percentage of the fruit’s deteriorated area, assessed using a scale from 0 to 5, where 1—no deterioration; 2—up to 5% of the deteriorated area; 3—up to 10% of the deteriorated area; 4—up to 15% of the deteriorated area; and 5—more than 15% of the deteriorated area. Fruits rated three or higher were considered unfit for consumption. External appearance included observation of damage caused by temperature, depressions, spots, wilting, and manifestations of diseases. Fruits with fungal infections on the surface were classified as deteriorated.
A visual evaluation of fruit skin color was performed according to the subjective rating scale, where 0—fruit grown and developed (100% green); 1—up to 15% of the yellow surface; 2—up to 25% of the yellow surface; 3—up to 50% of the yellow surface; 4—50 to 75% of the yellow surface; and 5—75 to 100% of the yellow surface.
Instrumental evaluation of fruit skin color: The fruit skin color was determined using a Minolta CR 400 colorimeter (Konica Minolta, Osaka, Japan). The L*, a*, and b* values were used to determine a three-dimensional color space, where L* indicates color lightness, with values ranging from 0 (completely black) to 100 (completely white). The a* dimension correlates variation from green (−60) to red (+60) and b* from blue (−60) to yellow (+60).
Determination of fungus growth on papaya fruit: The mold and yeast count was performed on the last day of storage. For this, the microorganisms were seeded on PDA plates and then homogenized by using a drigalski spatula. The plates were kept at 25 ± 1 °C in a BOD incubator (7Lab, Rio de Janeiro, Brazil) for 3 to 5 days, and the results were expressed as colony-forming units per gram (g−1).

2.9. Statistical Analysis

All experimental data are presented as the mean ± standard deviation, based on a minimum of triplicate measurements, and analyzed using the free software Assistant 7.7 Beta. Statistical significance was determined at a value of p < 0.05 through one-way ANOVA, followed by Tukey’s post hoc test [30].

3. Results

3.1. Obtaining and Determination of Polyphenols from Neem

The overall extraction yield of molecules from neem leaves and seeds using a hydroethanolic solvent was significantly higher with PLE compared to MAE. Additionally, neem leaves were found to contain more extractable global molecules than neem seeds when using the hydroethanolic solvent, leading to the highest overall extraction yield being obtained from neem leaves via PLE. However, extracts obtained through MAE were richer in TPC and TFC compared to those from PLE (Table 2). As shown in Table 2, neem leaf extracts obtained through both PLE and MAE demonstrated a significantly higher polyphenol content compared to neem seed extracts.

3.2. Fourier Transform Infrared (FTIR) Spectroscopy

Fourier transform infrared (FTIR) absorption spectroscopy is a technique that provides spectral data on the vibrational frequencies of molecular structures in both inorganic and organic compounds. FTIR spectra act as unique fingerprints for each material studied, as the intrinsic physicochemical properties of materials produce specific wavenumbers corresponding to functional groups (e.g., C–H, O–H, C=O) [36].
In Figure 1, the FTIR spectra of the food packaging materials—FPC + HNE-PLE (a), FPC (b), and FPC + HNE-MAE (c)—are presented. Bands at wavelengths 3457 cm−1 (a and c) and 3445 cm−1 (b) are attributed to O–H stretching, while bands at 1641 cm−1 (a) and 1653 cm−1 (b and c) correspond to C=O stretching.

3.3. Mechanical Properties and Water Vapor Permeability (WVP)

The incorporation of neem extracts into pectin-/gelatin-based packaging (FPC) significantly influenced the mechanical and barrier properties of the material (Table 3). The addition of neem extracts increased the elongation (E) of the packaging, likely due to the interaction between the hydroxyl groups of the phenolic compounds and the polymeric side chains. As a result, FPC + HNE-PLE and FPC + HNE-MAE exhibited greater flexibility compared to FPC.
Among the analyzed samples, FPC + HNE-PLE demonstrated higher tensile strength (TS), greater flexibility, and lower water vapor permeability (WVP) (Table 3). FPC + HNE-PLE showed a lower WVP value (2.0 g·mm/d·m2·kPa) than the control (FPC, 5.2 g·mm/d·m2·kPa), suggesting reduced permeability. Conversely, FPC + HNE-MAE demonstrated a higher WVP value (6.9 g·mm/d·m2·kPa) than the control, indicating increased permeability. HNE-PLE significantly reduced (p ≥ 0.05) the WVP of FPC, while HNE-MAE did not result in a statistically significant difference (p ≥ 0.05).
These findings suggest that PLE, due to its higher extraction capacity compared to MAE (Table 2), was able to isolate biomolecules from neem leaves with both hydrophilic and hydrophobic characteristics, leading to the observed reduction in WVP for FPC + HNE-PLE.

3.4. Antifungal Activity of Food Packaging

The mycelial growth inhibition percentages of Colletotrichum gloeosporioides by FPC, FPC + HNE-PLE, and FPC + HNE-MAE are presented in Figure 2. As shown in Figure 2, all food packaging materials studied in the present research effectively reduced the mycelial growth of Colletotrichum gloeosporioides, with inhibition values of 41%, 64.5%, and 71.66% for FPC, FPC + HNE-MAE, and FPC + HNE-PLE, respectively.

3.5. Evaluation of Antifungal Packaging on Postharvest Quality of Papaya Fruit

As shown in Table 4, the postharvest coating of papaya fruits with FPC, FPC + HNE-PLE, and FPC + HNE-MAE significantly reduced the mass loss of papaya fruits after 10 days of storage compared to the uncoated control fruits. The results presented in Table 4 also indicate that FPC + HNE-PLE and FPC + HNE-MAE were particularly effective as barriers around the papaya fruits, minimizing water loss and helping to maintain postharvest fruit quality.
According to Table 5, the use of FPC exerted a protective effect on papaya fruits, as fruits without FPC showed signs of deterioration by the fourth day of storage. In contrast, fruits coated with FPC exhibited fungal growth only on the 10th day of storage. Furthermore, the results in Table 5 indicate that the incorporation of HNE into FPC enhanced its ability to control the growth of Colletotrichum gloeosporioides on the surface of papaya fruits during storage.
The peel coloration of the papaya fruit transitioned from green to yellow during the 15-day storage period. However, the yellow color became more pronounced in the control fruits as early as the second day of storage, as shown in Figure 3. By the fourth day, the control fruits exhibited greater maturity compared to the other samples (both coated and uncoated with neem extract), as also depicted in Figure 3. These samples were categorized as stage 5 on the peel color scale, representing a 75–100% yellow surface. This change likely resulted from the degradation of chlorophyll and the synthesis of carotenoids during fruit ripening.
On the sixth day of storage, a noticeable difference in coloration was observed only in the fruits coated with FPC + HNE-PLE and FPC + HNE-MAE, which displayed a less intense yellow hue compared to the other samples. From the eighth day onward, all papaya fruits in this study were classified as stage 5 according to the peel color scale. This trend persisted until the end of the 15-day storage period.
Based on the results obtained, it was evident that food packaging containing neem extracts effectively delayed ripening up to the sixth day, as papaya fruits exhibited a less-yellow color compared to the control samples and those coated with FPC. The color parameters (L*, a*, and b*) measured using the Minolta colorimeter are presented in Figure 4.
The luminosity values (L *), ranging from 0 (black) to 100 (white), were observed to increase during storage. In general, control fruits (uncoated) and those coated with FPC showed higher L* values (50.53 and 51.05, respectively) compared to fruits coated with FPC + HNE-PLE and FPC + HNE-MAE (38.07 and 40.67, respectively). This decrease in L* values could be attributed to the dark-green coloration of the neem-based extracts, which reduced the brightness of the coated samples.
Regarding the a * coordinate, which ranges from green (−a*) to red (+a*), control fruits and those coated with FPC displayed higher a* values (8.27 and 8.75, respectively) at the end of the storage period compared to fruits coated with FPC + HNE-PLE and FPC + HNE-MAE (4.02 and 5.38, respectively). Similarly, the b* parameter, which ranges from blue (−b*) to yellow (+b*), showed that control fruits and those coated with FPC had higher b* values (58.43 and 52.60, respectively) when compared to FPC + HNE-PLE and FPC + HNE-MAE coatings (44.53 and 50.13, respectively).
This trend was also observed in the visual evaluation of papaya peel coloration. Uncoated fruits turned more yellow over time, whereas fruits coated with FPC + HNE-PLE and FPC + HNE-MAE retained a greener appearance. This could be due to the green coloration of the neem-based coatings interfering with the a* and b* values, indicating that ripening occurred more slowly in these samples.

4. Discussion

Air bubbles in the vegetal matrix hinder the solvent from accessing the biomolecules. However, the application of high pressure during PLE helps to manage these air bubbles, which, combined with enhanced solvent penetration into the vegetable matrix, facilitates the transfer of biomolecules from the solid phase to the liquid phase, thereby increasing the overall extraction yield [37]. Consequently, the results of the present study suggest that the physical effects of PLE were more effective in extracting overall biomolecules from neem leaves and seeds than the physical effects of MAE.
On the other hand, the heat generated by MAE leads to the evaporation of moisture within the cell matrix, creating intense pressure on the cell walls, which causes ruptures and the subsequent release of biomolecules from the vegetal matrix [38,39,40]. Therefore, we propose that the physical properties of MAE were more effective in releasing TPC and TFC from neem leaves and seeds compared to the physical effects of PLE.
Previous studies have reported varying levels of TPC and TFC in NEEM extracts [15,41]. TPC in neem leaf extracts has ranged from 20.80 to 107.29 mg/100 g [41]. TFC is influenced by the plant organ and solvent, with leaf concentrations between 65.06 and 72.96 mg mL−1 and seed concentrations between 18.99 and 93.03 mg mL−1 [15]. Furthermore, different solvents (ethyl acetate and hydroethanolic) yielded a TFC of 74.17 mg g−1 QE and 118.57 mg g−1 QE in leaf extracts [15,42].
Phenolic compounds are essential biomolecules that act as natural chemical agents in plants, protecting against insects, bacteria, viruses, and fungi by inhibiting the invasion of resistant pathogens [15,43]. These compounds, along with their flavonoid content, are recognized for their antifungal properties [15,44]. Therefore, HNE-PLE and HNE-MAE extracts obtained from neem leaves were incorporated into edible pectin/gelatin to create antifungal food packaging (Illustrative images of the food packaging are provided in Figure A1), as they exhibited higher amounts of TPC and TFC compared to seed extracts in this study (Table 2).
Based on the FTIR spectra presented in Figure 1, only the food packaging materials containing neem extracts (FPC + HNE-PLE (a) and FPC + HNE-MAE (c)) exhibited bands in the regions of 1043 and 1056 cm−1, attributed to C-O-C stretching, and at 934 and 946 cm−1, corresponding to C-H stretching. According to Elumalai and Velmurugan (2015) [45], the C-H bands are indicative of phenolic compounds and flavonoid content. The C-O-C stretching bands, on the other hand, have been associated with terpenoids from neem extracts [46].
The differences observed in the FTIR spectra of food packaging with and without neem extracts confirm the presence of phenolic compounds from neem leaf extracts in FPC + HNE-PLE and FPC + HNE-MAE. Therefore, it can be suggested that FPC + HNE-PLE and FPC + HNE-MAE contain antimicrobial biomolecules.
The results of this study show that the addition of neem extracts did not significantly alter the thickness of the food packaging. This consistent thickness can be attributed to the controlled standardization of the film-forming solution volume and the plate area during production. This result aligns with Tongnuanchan et al. (2014) [47], who observed a homogeneous packaging structure due to the ordered alignment of gelatin chains, unaffected by the presence of extract compounds.
Neem extracts were also observed to enhance the elongation (E) of pectin-/gelatin-based packaging (FPC). This improvement likely resulted from interactions between the hydroxyl groups of phenolic compounds in the neem extracts and the polymeric side chains [48], which increased the flexibility of FPC + HNE-PLE and FPC + HNE-MAE compared to FPC. These mechanical properties, particularly tensile strength (TS) and flexibility, are essential for packaging to withstand external stress while maintaining its structural integrity [49,50].
Among the tested formulations, FPC + HNE-PLE showed superior mechanical and barrier properties, including higher TS, greater flexibility, and significantly lower water vapor permeability (WVP). These enhancements can be linked to the higher extraction efficiency of PLE (Table 2), which enabled the extraction of more plasticizing biocompounds from neem leaves compared to MAE. In contrast, FPC + HNE-MAE exhibited the lowest TS and highest WVP values, indicating reduced performance.
The barrier properties of food packaging, especially WVP, play a crucial role in controlling moisture transfer and extending the shelf life of food products [35,51,52]. Lower WVP values are particularly desirable as they minimize water transfer between the food product and its surrounding environment [35,53,54,55]. In this study, FPC + HNE-PLE significantly reduced the WVP of FPC (p ≥ 0.05), while FPC + HNE-MAE did not cause any statistically significant changes (p ≥ 0.05). This improvement with HNE-PLE can be attributed to PLE’s ability to extract biomolecules with both hydrophilic and hydrophobic properties, resulting in an enhanced barrier effect. Consequently, FPC + HNE-PLE provided the lowest WVP value and emerged as the most effective material for food packaging applications requiring moisture control.
Several studies have explored the mechanical and barrier properties of packages incorporated with phenolic compounds to application as intelligent films. To this end, Jati et al. (2024) [56] demonstrated that roselle-based films had a tensile strength of 13.42 MPa and an elongation at break of 25.53%. In contrast, litchi shell extract-based films exhibited enhanced mechanical properties, including a higher tensile strength of 18.60 MPa and a significantly greater elongation at break of 116%, along with improved barrier properties such as water vapor transmission rates of 1.62–1.65 × 10−12 g·cm/cm2·Pa·s [57]. Xue Mei et al. (2020) [58] reported that torch ginger-based films had the highest elongation at break (85.14%). Purple sweet potato anthocyanin films provided a tensile strength of 9.28 MPa and an elongation at break of 9.87% [59]. Anthocyanin-based films, Wahyuningtiyas and Suryanto (2018) [60] highlighted cassava starch-based bioplastics reinforced with nanoclay, which showed an increase in tensile strength from 5.2 MPa to 6.3 MPa.
When correlating the color results with the water vapor permeability (WVP) data (Table 3), it was observed that the incorporation of HNE-PLE into FPC significantly reduced its permeability (2.0 g·mm/d·m2·kPa) compared to FPC alone (5.2 g·mm/d·m2·kPa). The different treatments applied to the papayas are shown in Figure A2. Based on the results of the present study, the observed reduction in WVP likely influenced the respiratory metabolism of the papaya fruits, consequently contributing to the delayed ripening. A lower WVP could restrict both moisture loss and gaseous exchange across the fruit’s surface, potentially limiting the availability of oxygen. This limitation in oxygen supply would be expected to result in a decreased respiratory rate.
As respiration is a key metabolic process providing energy for various physiological changes during ripening, a slower rate could directly impact the biosynthesis and action of ethylene—a primary plant hormone that triggers and regulates fruit maturation. Therefore, the reduced gas exchange due to lower WVP likely suppressed respiration, leading to diminished ethylene production and a subsequent delay in the ripening process [61].
As suggested by Basiak et al. (2019) [62] and L. Zhang et al. (2020) [63], bioactive food packaging gradually releases active molecules into the surrounding environment or absorbs compounds that contribute to food spoilage. This mechanism helps delay the ripening process and minimizes water loss, thereby preserving the quality of the fruits during storage. The antifungal activity of FPC can primarily be attributed to the presence of borax in its composition. Although no previous studies have explored the antifungal effects of food packaging based on a pectin/gelatin blend containing borax against Colletotrichum gloeosporioides, Li et al. (2012) [64] reported that borax exhibits antifungal effects, possibly through its ability to disrupt fungal cell membranes and induce pathogen death.
However, FPC containing neem leaf extracts significantly reduced the growth of the food contaminant Colletotrichum gloeosporioides compared to FPC alone, regardless of the biocompound extraction method (PLE or MAE) used. This suggests that neem extracts enhanced the antifungal activity of FPC against Colletotrichum gloeosporioides. A plausible explanation for this effect is that neem-derived compounds, including polyphenols (Table 2), present in HNE-MAE and HNE-PLE, were released onto the food packaging surfaces, inhibiting the growth of Colletotrichum gloeosporioides. According to Gilles, Zhao, An, and Agboola (2010) [65], polyphenols from plant-based matrices exhibit antimicrobial properties by causing the leakage of essential intracellular molecules or by disrupting microbial enzyme systems.
Mahmoud et al. (2011) [66] also reported that ethanolic extracts of neem leaves have antifungal effects against Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Candida albicans, and Microsporum gypseum. Similarly, neem leaf extracts have been shown to reduce the mycelial growth of Aspergillus niger, Aspergillus flavus, Botryodiplodia theobromae, and Fusarium moniliforme in African yam bean seeds [67]. These findings indicate that neem leaf extract holds promise as an antifungal agent against several major fungal pathogens.
The FPC enriched with neem extracts has demonstrated significant potential in preserving papaya fruits during storage. These coatings not only inhibit pathogen growth and reduce weight loss but also delay the ripening process, contributing to the extended commercial shelf life of the fruits. The efficacy of these materials stems from the synergistic interaction between polysaccharides, which act as a physical barrier against moisture loss, and neem extracts, which contain bioactive compounds with antifungal and antimicrobial properties.
Furthermore, the application of these coatings offers both aesthetic and functional advantages. By forming a protective layer around the fruits, they help maintain visual and structural quality while limiting microbial activity, presenting a sustainable and efficient approach to tropical fruit preservation. The use of such coatings serves as a practical solution to reduce waste in the production chain, particularly in tropical regions.
However, further research is needed to optimize the formulations of these coatings, considering factors such as sensory properties, large-scale economic viability, and durability during transportation and storage. Future studies can provide valuable insights to further enhance the impact of these coatings on food control.

5. Conclusions

In this study, we present innovative food packaging with the potential to reduce Colletotrichum gloeosporioides growth isolated from papaya fruits, marking a novel application of neem leaf extracts in food safety. Therefore, we suggest that FPC + HNE could serve as a valuable alternative for the food industry and rural producers, acting as a packaging material for papaya fruits to extend their shelf life. However, further research is needed to optimize the formulations of these coatings, considering factors such as sensory properties, large-scale economic viability, and durability during transportation and storage. Moreover, incorporating scanning electron microscopy analysis would deepen our understanding of these food control coatings by revealing their microstructural properties, such as surface morphology and interactions with food matrices. This detailed information would then enable the optimization of formulations and application processes, leading to improved barrier and mechanical properties for better food preservation.

6. Patents

The work reported in this manuscript resulted in the granting of patent number BR 102018000947-8.

Author Contributions

Conceptualization C.D. and F.F.P.; methodology, T.T.d.B.R. and A.M.B.; validation, T.T.d.B.R. and A.M.B.; formal analysis, T.T.d.B.R., T.P.N. and G.R.B.; investigation, T.T.d.B.R. and A.M.B., data curation, T.T.d.B.R., T.P.N. and G.R.B., writing—original draft preparation, C.D., K.S.S., A.S.G.d.C. and M.B.P.P.O.; writing—review and editing, C.D., K.S.S., A.S.G.d.C. and M.B.P.P.O.; supervision C.D. and F.F.P. All authors have read and agreed to the published version of the manuscript.

Funding

The Article Processing Charge was funded by the National Council for Scientific and Technological Development (CNPq), under the CNPq/MCTI Call No. 10/2023 (Process: No. 407015/2023).

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors acknowledge the support received by the CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior)—Finance Code 001, CNPq (National Council for Scientific and Technological Development), FAPITEC/SE (Foundation for Research and Technological Innovation Support of the State of Sergipe).

Conflicts of Interest

All authors declare that they have no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
PLEPressurized liquid extraction
MAEMicrowave-assisted extraction process
TPCTotal phenolic compounds
mg GAE/100 gMilligrams of gallic acid equivalents per 100 g of extract
TFCTotal flavonoid contents
mg QE/100 gMilligrams of quercetin equivalents per 100 g of extract
HNE-PLEHydroethanolic neem extracts obtained through PLE
HNE-MAEHydroethanolic neem extracts obtained through MAE
PCPackaging control
HNE Hydroethanolic neem extract
MAEMicrowave-assisted extraction
PECPectin
GELGelatin
PROPPropylene glycol
VEVegetable extract
AA-0.5Acetic acid (0.5 mol/L)
TSTensile strength
EABElongation at break
WVPWater vapor permeability
MGI%Mycelial growth inhibition percentage
OEYOverall extraction yield
TThickness

Appendix A

Applsci 15 04423 g0a1
Figure A1. The image illustrates (from left to right) representations of FPC, FPC + HNE-PLE, and FPC + HNE-MAE.
Figure A1. The image illustrates (from left to right) representations of FPC, FPC + HNE-PLE, and FPC + HNE-MAE.
Applsci 15 04423 g0a1
Applsci 15 04423 g0a2
Figure A2. Illustrative representation of uncoated papayas, coated papayas with FPC, papayas coated with FPC + HNE-PLE, and papayas coated with FPC + HNE-MAE.
Figure A2. Illustrative representation of uncoated papayas, coated papayas with FPC, papayas coated with FPC + HNE-PLE, and papayas coated with FPC + HNE-MAE.
Applsci 15 04423 g0a2

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Figure 1. FTIR spectrums of food packaging containing neem extracts (a,c) and without neem extracts (b).
Figure 1. FTIR spectrums of food packaging containing neem extracts (a,c) and without neem extracts (b).
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Figure 2. Mean of mycelial growth inhibition percentage (MGI%) of the fungus Colletotrichum gloeosporioides during six days in culture media containing FPC, FPC + HNE-PLE, and FPC + HNE-MAE. The same capital letter (A or B) above the bars indicates that they are not significantly different from each other (p < 0.05).
Figure 2. Mean of mycelial growth inhibition percentage (MGI%) of the fungus Colletotrichum gloeosporioides during six days in culture media containing FPC, FPC + HNE-PLE, and FPC + HNE-MAE. The same capital letter (A or B) above the bars indicates that they are not significantly different from each other (p < 0.05).
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Figure 3. The coloration of papaya peel with or without covering, during the storage of 15 days. 0—100% of the green surface; 1—up to 15% of the yellow surface; 2—up to 25% of the yellow surface; 3—up to 50% of the yellow surface; 4—50 to 75% of the yellow surface; 5—75 to 100% of the yellow surface.
Figure 3. The coloration of papaya peel with or without covering, during the storage of 15 days. 0—100% of the green surface; 1—up to 15% of the yellow surface; 2—up to 25% of the yellow surface; 3—up to 50% of the yellow surface; 4—50 to 75% of the yellow surface; 5—75 to 100% of the yellow surface.
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Figure 4. Determination of the color parameters (L*, a*, and b*) of papaya peel during a 15-day storage period.
Figure 4. Determination of the color parameters (L*, a*, and b*) of papaya peel during a 15-day storage period.
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Table 1. Filmogenic formulation composition for the development of antifungal food packaging based on edible pectin/gelatin containing Azadirachta indica bioactive extract (HNE) obtained via PLE and MAE.
Table 1. Filmogenic formulation composition for the development of antifungal food packaging based on edible pectin/gelatin containing Azadirachta indica bioactive extract (HNE) obtained via PLE and MAE.
PackagingPEC (g)GEL (g)PROP (g)VE (g)AA-0.5 (mL)B (%)
FPC2.00.20.3-50.01
FPC + HNE-PLE2.00.20.31.050.01
FPC + HNE-MAE2.00.20.31.050.01
FPC = food packaging control; HNE = hydroethanolic neem extract; PLE = pressurized liquid ex-traction; MAE = microwave-assisted extraction; PEC = pectin; GEL = gelatin; PROP = propylene glycol; VE = vegetable extract; AA-0.5 = acetic acid (0.5 mol/L); B = borax.
Table 2. Overall extraction yield (OEY), TPC, and TFC of neem extract obtained by PLE and MAE using a hydroethanolic (ethanol/water, 80:20 v/v) solvent.
Table 2. Overall extraction yield (OEY), TPC, and TFC of neem extract obtained by PLE and MAE using a hydroethanolic (ethanol/water, 80:20 v/v) solvent.
SampleOEY (%)TPC (mg AGE/100 g)TFC (mg QE/100 g)
PLEMAEPLEMAEPLEMAE
Leaves20 Aa 6 Bb2893 Ee3136 Ee542 Hh797 Ii
Seeds5 Bb 4 Dd 417 Fe641 Gg365 Jj348 Kk
Means followed by the equal capital letter on the same lines indicate that there is no significant difference (p ≥ 0.05) between the extraction methods for OEY, TPC, and TFC in the same sample (Leaves or seeds). Means followed by the equal lowercase on the same column indicate that there is no significant difference (p ≥ 0.05) between neem leaves and seeds for OEY, TPC, and TFC in the same extraction method (PLE or MAE).
Table 3. Mean values and standard deviation of mechanical properties (T: thickness, E: elongation, TS: tensile strength) and water vapor permeability (WVP) of FPC + HNE-PLE, FPC, and FPC + HNE-MAE.
Table 3. Mean values and standard deviation of mechanical properties (T: thickness, E: elongation, TS: tensile strength) and water vapor permeability (WVP) of FPC + HNE-PLE, FPC, and FPC + HNE-MAE.
PackagingT [mm]E [%]TS [Mpa]WVP (g·mm/d·m2·kPa)
FPC0.11 ± 0.00 a8.00 a ± 0.00 a13.76 ± 0.79 a5.2 ± 0.4 a
FPC + HNE-PLE0.10 ± 0.01 a10.33 ± 0.21 b16.45 ± 2.25 b2.0 ± 0.2 b
FPC + HNE-MAE0.16 ± 0.04 a9.50 ± 0.21 c9.24 ± 2.01 c6.9 ± 1.7 a
Different lowercase within the same column indicates significant differences among food packaging (p < 0.05).
Table 4. Assessment of fresh weight loss (%) of the coated papaya fruit with FPC, and FPC + HNE-PLE, FPC + HNE-MAE, and control (without covering) during storage at 18 ± 2 °C for 15 days.
Table 4. Assessment of fresh weight loss (%) of the coated papaya fruit with FPC, and FPC + HNE-PLE, FPC + HNE-MAE, and control (without covering) during storage at 18 ± 2 °C for 15 days.
PackagingMass Loss (%) over Days of Storage
2468101215
FPC2.56 a8.06 a10.77 a12.33 a14.26 b17.14 b20.73 b
FPC + HNE-PLE2.55 a7.15 a9.37 a10.91 b12.83 b15.94 b19.67 b
FPC + HNE-MAE2.89 a6.89 a9.22 a10.62 b12.50 b15.16 b18.56 b
CONTROL2.80 a9.81 a13.31 a16.75 a21.47 a28.16 a34.31 a
Values followed by the same lowercase letter within the same column indicate no significant differences (p < 0.05) among the papaya fruit coating.
Table 5. Evaluation of the external appearance of papayas coated or not with food packaging regarding the incidence of fungi.
Table 5. Evaluation of the external appearance of papayas coated or not with food packaging regarding the incidence of fungi.
PackagingExternal Appearance for Fungus Incidence in Papaya Fruit
Days of Storage
02468101215
FPC11112233
FPC + HNE-PLE11111111
FPC + HNE-MAE11111112
CONTROL11244455
1—no deterioration, 2—up to 5% deterioration, 3—up to 10% deterioration, 4—up to 15% deterioration and 5—greater than 15% deterioration.
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MDPI and ACS Style

Ribeiro, T.T.d.B.; Barbosa, A.M.; Nunes, T.P.; Costa, A.S.G.d.; Oliveira, M.B.P.P.; Borges, G.R.; Padilha, F.F.; Dariva, C.; Santos, K.S. Development of Antifungal Packaging Based on Pectin/Gelatin Containing Azadirachta indica Bioactive Extracts for Carica papaya L. Fruit Coating. Appl. Sci. 2025, 15, 4423. https://doi.org/10.3390/app15084423

AMA Style

Ribeiro TTdB, Barbosa AM, Nunes TP, Costa ASGd, Oliveira MBPP, Borges GR, Padilha FF, Dariva C, Santos KS. Development of Antifungal Packaging Based on Pectin/Gelatin Containing Azadirachta indica Bioactive Extracts for Carica papaya L. Fruit Coating. Applied Sciences. 2025; 15(8):4423. https://doi.org/10.3390/app15084423

Chicago/Turabian Style

Ribeiro, Thais Trindade de Brito, Andriele Mendonça Barbosa, Tatiana Pacheco Nunes, Anabela Silvia Guedes da Costa, Maria Beatriz Prior Pinto Oliveira, Gustavo Rodrigues Borges, Francine Ferreira Padilha, Claudio Dariva, and Klebson Silva Santos. 2025. "Development of Antifungal Packaging Based on Pectin/Gelatin Containing Azadirachta indica Bioactive Extracts for Carica papaya L. Fruit Coating" Applied Sciences 15, no. 8: 4423. https://doi.org/10.3390/app15084423

APA Style

Ribeiro, T. T. d. B., Barbosa, A. M., Nunes, T. P., Costa, A. S. G. d., Oliveira, M. B. P. P., Borges, G. R., Padilha, F. F., Dariva, C., & Santos, K. S. (2025). Development of Antifungal Packaging Based on Pectin/Gelatin Containing Azadirachta indica Bioactive Extracts for Carica papaya L. Fruit Coating. Applied Sciences, 15(8), 4423. https://doi.org/10.3390/app15084423

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