Sustainable packaging using Aloe vera infused mango starch–wool keratin biocomposite films to extend the shelf life of mango

Abstract

The development of biodegradable packaging films from agro-waste is gaining prominence as a sustainable alternative to synthetic materials. This study explores the potential of Aloe vera (AV) gel extract as a bioactive agent to enhance the structural and functional properties of biocomposite films derived from mango starch (MS) and wool keratin (WK). The study aimed to investigate the impact of AV incorporation on film characteristics and its efficacy in extending mango shelf life at 33 ± 2 °C. Biocomposite films were formulated with 0, 20, and 40 mg of AV, designated as MS-WK-AV0, MS-WK-AV2, and MS-WK-AV4, respectively. Physico-mechanical, morphological, functional, and thermal properties were analyzed. AV incorporation reduced film thickness and water solubility by 23.8% and 17.6%, respectively, while increasing the contact angle by up to 67.6%. Structural analysis revealed enhanced intermolecular interactions, leading to improved smoothness and homogeneity. The MS-WK-AV4 film exhibited a complete loss of crystallinity. Notably, AV-enriched films prolonged mango shelf life by up to 12 days and preserved quality attributes during storage. This study demonstrates the potential of repurposing sheep wool and mango seed kernels for sustainable packaging, aligning with circular economy principles and offering an eco-friendly alternative for food preservation.

Introduction

Mango (Mangifera indica), often referred to as the “king of fruits,” is a widely cultivated tropical fruit with significant economic and nutritional importance^1,2. Major producers, including India, China, and Thailand, account for a substantial portion of global mango production, with India alone cultivating over 1500 varieties^3,4. Despite its rich nutritional profile of essential vitamins (A and C), antioxidants, polyphenols, and dietary fiber, mango is highly perishable due to its climacteric nature, undergoing rapid biochemical and microbial deterioration during storage and transportation⁵. Postharvest losses of mangoes are particularly severe in developing countries, where inadequate preservation techniques contribute to fruit wastage rates ranging between 20 and 50%⁶.

Traditional techniques such as refrigeration, chemical preservatives, and modified atmosphere packaging are effective; however, they present drawbacks including high costs, potential health risks, and environmental concerns⁷. Increasing consumer preference for minimally processed foods has catalyzed the adoption of alternative preservation methods that reduce thermal processing. Edible coatings and biodegradable films have emerged as sustainable alternatives, offering barriers to moisture loss, controlling respiration, and maintaining food quality^8–10. Among bio-based packaging materials, starch, a widely available polysaccharide, has demonstrated potential due to its biodegradability and renewability. However, starch films possess inherent limitations such as hydrophilicity, poor mechanical strength, and brittleness, rendering them unsuitable as standalone packaging solutions^11,12. To address these limitations, research has increasingly focused on blending starch with other biodegradable polymers and incorporating bioactive agents. These approaches aim to enhance the mechanical strength, water resistance, and antimicrobial properties of starch-based films, thereby meeting consumer demand for effective, safe, and eco-friendly food preservation strategies.

Mango seed kernels, a major byproduct of industrial mango processing, offer a sustainable alternative starch source with high starch content (~ 60%)¹³. Despite its potential as a functional ingredients in biopolymer development, research on mango seed starch (MS) based films remains limited. Similarly, wool keratin (WK), a protein derived from slaughterhouses and tannery waste, exhibits excellent hydrophobic and barrier properties, making it a suitable reinforcing agent in biopolymer matrices¹⁴. However, optimizing the compatibility and functionality of starch-keratin composites remains a challenge. Incorporating bioactive agents with antioxidant and antimicrobial properties into these biocomposite films presents a viable approach for enhancing their efficacy in food preservation^15,16.

Aloe vera (AV) gel, a plant-derived biopolymer rich in polysaccharides, organic acids, and bioactive compounds, has been widely recognized for its antimicrobial and preservative properties¹⁷. Recent studies have demonstrated the effectiveness of AV-based coatings in reducing weight loss, microbial spoilage, and ethylene production in various fruits, including peaches, plums, and tomatoes^18,19. Additionally, AV gel enhances film-forming properties, improves cross-linking within polymer matrices, and contributes to the overall functional performance of edible coatings^20,21.

This study aimed to develop an innovative, sustainable packaging solution by integrating AV gel into mango starch-wool keratin (MS-WK) biocomposite films. By leveraging renewable waste-derived polymers and natural bioactive agents, this study seeks to address the limitations of conventional starch-based films while providing an eco-friendly approach to extend the postharvest shelf life of mangoes.

Materials and methods

Materials

Sheep wool, a renewable waste-derived material, was sourced from a local tannery in Ranipet, Tamil Nadu, India. The wool material was transported to the laboratory in polyethylene bags. Mature green mango fruits of superior quality and free of defects (extra class as defined by Codex Standards)²² were harvested from the orchard of the CSIR-Central Leather Research Institute, Chennai (India) with due authorization from the competent authority. Freshly peeled Aloe vera leaves, a renewable plant-derived material, were collected from the local market in Chennai. Mango fruits were sorted and washed thoroughly with distilled water. Analytical grade bromothymol blue, dinitro salicylic acid, 2,6-dichlorophenol indophenol, D-galacturonic acid, metaphosphoric acid, 1,1-diphenyl-2- picrylhydrazyl (DPPH), pectin, polygalacturonic acid, sodium hydroxide, sodium phosphate, sodium trichloroacetic acid, and phosphoric acid were purchased from Sigma–Aldrich. All other chemicals and reagents used in this study were of analytical grade.

Extraction of starch from mango seed kernel

The wet grinding method described by Oluba et al.²³ was used to extract starch from the mango seed kernels, a renewable waste-derived material. First, the kernels were manually cut into small pieces and soaked in 0.1% sodium metabisulphite for 1 h. The cut pieces were pulverized into slurry using a food-grade grinder (Preethi^® Cheffpro, India). The slurry was screened through a 100 μm mesh, followed by sieving with a mesh size of 300 μm. The slurry was then submerged in distilled water and centrifuged (Thermo Scientific Sorvall MX120+) at 8,000 rpm for 5 min to purify the starch. This procedure was repeated five times to obtain a light-brown starch. The wet material was dried in a hot-air oven for 10 h at 50 °C to yield mango starch (MS) powder.

Keratin extraction from sheep wool

Keratin was extracted from sheep wool waste using the method described by Oluba et al.²⁴ with minor modifications. Briefly, 10 g of sheep wool fibers (SWF) were cleaned with detergent (sodium dodecyl benzene sulfonate), rinsed with distilled water, and dried in an oven at 60 °C for 8 h. The wool was soaked in a mixture of dichloroethane and n-hexane for 12 h and then oven-dried at 60 °C for 12 h. The defatted wool fibers were cut into 1 cm pieces and placed in a solution containing urea (8 M) and sodium hydroxide (0.8 M). The mixture was stirred continuously for 5 h at 55 °C using a magnetic stirrer. Subsequently, the solution was filtered through a nylon filter cloth with a mesh size of 800 μm. The pH of the dissolved wool solution was adjusted to 6.8 using a 2% acetic acid solution. The precipitated wool keratin was obtained using a nylon filter cloth and then placed in a dialysis tube with a molecular weight cut-off of 12,000–14,000 and dialyzed in distilled water at room temperature for 48 h. Water was changed every 4 h. Keratin obtained after dialysis was dried in a hot air oven at 50 °C for 6 h.

Extraction of Aloe vera gel

The pulp (500 g) of freshly peeled Aloe vera leaves was collected and homogenized using a kitchen blender (Preethi^® Cheffpro, India). It was then combined with 500 mL of distilled water containing 1.2 g of tannic acid²⁵. The solution was heated at 30 °C for 2 h, after which the resulting suspension was separated by centrifugation at 1500 rpm. The supernatant was carefully removed and filtered using Whatman™ filter paper (No. 4) and the filtrate was evaporated at 50–55 °C. The residue was dehydrated, crushed into a fine powder, and dissolved in anhydrous ethanol (25 mL). The turbid solution obtained was filtered to obtain a clear extract solution, which was concentrated and freeze-dried to yield Aloe vera gel extract (AV).

Preparation of Aloe vera gel extract-loaded Mango starch-wool keratin (MS-WK-AV) biocomposite films

The method described by Gutiérrez et al.²⁶ was used to prepare the MS-WK-AV films. The process flow diagram for the fabrication of the MS-WK-AV biocomposite is shown in Scheme 1. A series of films were prepared using film-forming solutions comprising of 5% (w/v) mango kernel starch (MS; 5 g of mango starch powder was added to 100 mL distilled water, stirred thoroughly and heated at 70 °C until the starch solution thickened to form starch gel), 5% (w/v) wool keratin (WK) prepared in 0.1 M NaOH solution (5 g of wool keratin powder was added to 100 mL 0.1 M NaOH solution), and 30% (v/v of total MS-WK solution) glycerol in a 250 mL beaker. Various concentrations containing 0, 20, and 40 mg of AV, designated MS-WK-AV₀, MS-WK-AV₂, and MS-WK-AV₄, respectively, were added to this solution. The choice of 20 and 40 mg AV in this study was based on the results of our preliminary studies. The solutions were heated in a water bath with constant shaking at 95 °C for 10 min to ensure starch gelatinization and inactivation of the enzymes present in the AV²⁷. The resulting gel was poured into stainless steel trays measuring 40 × 30 cm² and dried in a hot air oven for 48 h at 50 °C. Before further analysis, we carefully removed the films from the casting molds and conditioned them at approximately 57% relative humidity for seven days at room temperature in a dark room to prevent the photooxidation of polyphenolic compounds.

Scheme 1.

Schematic representation of the process flow diagram for the fabrication of Aloe vera-functionalized mango starch-wool keratin (MS-WK-AV) biocomposite films.

Application of MS-WK-AV biocomposite film for preservation of mango fruit

For this experiment, a sample of twenty-four (24) matured, unripe mango fruits were chosen. The mango fruits exhibited consistent shapes and sizes, and were meticulously selected to guarantee the absence of any apparent lesions or postharvest illnesses. Prior to experimentation, fruits were washed with distilled water and air-dried for 6 h. The fruits were randomly allocated into four groups of six fruits each, designated as control, MS-WK-AV₀, MS-WK-AV₂, and MS-WK-AV₄, respectively. The mango fruits in the MS-WK-AV₀, MS-WK-AV₂, and MS-WK-AV₄ groups were wrapped with their corresponding films to guarantee complete coverage of the entire surface. The fruits in the control group were exposed without wrapping. Subsequently, the fruits were subjected to controlled laboratory conditions, precisely maintained at 33 ± 2 °C, relative humidity of 50%, and a light/dark cycle of 12 h for a duration of 12 days. Fruit weight was periodically measured on days 0, 4, 8, and 12 to determine any changes. Other fruit quality metrics were also evaluated at the end of the treatment period.

Characterization analyses

Measurement of amylose content

The amylose content in MS was determined using the ISO 6647-1²⁸ standard technique.

Measurement of particle size

The particle sizes of MS and WK were determined using a Zetasizer Nano ZS (ZEN 3600; Malvern Instruments). Both samples were prepared before analysis by dispersing the 0.01% sample in Milli-Q water and sonicated for 30 min at 40 kHz.

Physicochemical characteristics

The thickness of the film was determined using a digital micrometer (Mitutoyo, Japan) with measurements performed at 10 random locations around each sample, and the average was reported. The water solubility of the films was calculated according to the method described by Pavin et al.²⁹. After drying to a constant mass in a hot-air oven at 100 °C, the moisture content of the film was calculated using Eq. (1).

1

Where correspond to the initial and final weights of the biocomposite film before and after drying, respectively.

Measurement of water vapor permeability

The water vapor permeability (WVP) of each film was determined using a modified version of ASTM procedure E96-90, as documented by Wu et al.³⁰. A 68 mm diameter film sample was shaped into a sphere and positioned on top of the opening of a cylindrical cup. The cup contained a specific quantity of anhydrous CaCl₂, which had a relative humidity of 0%. This was performed to ensure a consistent 75% disparity in the relative humidity between the samples. The airtight cylindrical container was then placed in a desiccator and exposed to a saturated sodium chloride solution at 25 °C and relative humidity of 75%. Weight measurements were taken at eight-hour intervals following a two-hour period to enable the equipment to stabilize. The rate of change in cup weight, measured to the nearest 0.0001 g, was determined as a function of time using linear regression analysis, with an r² value greater than 0.99. The water vapor transmission rate (WVTR) was estimated by calculating the ratio of the slope to the exposed area of the sample. Conversely, WVP was determined using Eq. (2).

2

S represents the saturated vapor pressure of water at 25 °C, R1 represents the relative humidity in the desiccator, R2 represents the relative humidity in the permeation cell, and D refers to the thickness of the sample. The analysis was replicated thrice.

Measurement of tensile strength and elongation at break

The tensile strength of the sample was measured by applying a force of 100 N at a rate of 10 cm/min at room temperature, using a Universal Testing Machine (Hounsfield Series S, UK) according to the ASTM standard D882, as described by Rahman and Jamalulail³¹. The data from the tensile strength analysis were used to calculate the elongation at break of the sample. The tensile stress was calculated using Eq. (3).

3

Measurement of contact angle

The contact angle of the films was determined by analyzing the images captured when a 2-µL drop of distilled water was placed onto the film’s surface to measure the angles produced at the point where the liquid met the solid surface and the point where the liquid met the air (tangent to the edge of the droplet). Images were captured using a Holmarc Digital Microscope (model HO-IAD-CAM-01) equipped with an Image Analysis Software. Droplets were deposited onto the films using a syringe, and the contact angles were measured before the onset of swelling. The contact angle was calculated as the mean of five measurements for each film.

NMR spectroscopy

¹³C-NMR spectrum of the extracted mango seed kernel starch was obtained using a 600 MHz Bruker Biospin spectrometer at 25 °C. Chemical shifts were reported in ppm.

Scanning Electron microscopy (SEM)

The samples were affixed to stubs using double-sided sticky tape and then coated with a 20-nm thick layer of gold using a sputtering process. Examination and observation were conducted using a scanning electron microscope (SEM; TESCAN, CLARA).

Atomic force microscopy (AFM)

The surface properties of the MS-WK-AV composite films were assessed using a Multimode 8 AFM instrument (Bruker, Ettlingen, Germany). The tapping mode was employed, which allows for the direct control of the tip-sample interaction and ensures that the maximum repulsive force does not exceed 500 pN. Each image covered a 1 × 1 µm² scan area and was composed of 256 × 256 steps, resulting in a lateral resolution slightly below 4 nm. AFM measurements were conducted using a silicon scanning probe (TAP150GD-G, BudgetSensors, Sofia, Bulgaria) with a nominal spring constant of 5 N/m, a resonance frequency of 150 kHz, and a tip radius of 10 nm. Prior to analysis, the height images were plane-fitted to remove surface tilt and then flattened row-by-row to align the neighboring lines in the image.

Fourier-Transformed infrared spectroscopy (FTIR)

FTIR spectra of the samples were recorded using the attenuated total reflection (ATR) mode, with 32 scans. Spectral scanning was conducted using an FTIR spectrometer (Jasco, FT-IR 4200) operating in the frequency range of 400–4000 cm^–1 and a resolution of 2 cm^–1.

X-ray diffraction (XRD) analysis

X-ray diffraction measurements were performed using an X-ray diffractometer (Rigaku, Germany) equipped with a Cu-Kα radiation source (40KV, 30 mA) at a wavelength of 1.54 Aº. The XRD scan was performed between 2θ = 2.0º and 80º. The scan speed was set at 2.0º/min. The integral intensity of the selected speed was generated using Rigaku profile-fitting analysis (Rigaku XRD-6000 software version 4.1). Gaussian peak fitting was performed using OriginPro 2016 software. The degree of crystallinity of the samples was computed as the ratio of the intensity of the diffraction peak in the crystal area to the total intensity of all the peaks using Eq. (4).

4

Where A_t is the total area under the curve of the diffracted intensity at 2θ = 5º–45º, and A_a is the corresponding area under the baseline³².

Thermogravimetric analysis (TGA)

Thermogravimetric analysis was conducted using a Perkin Elmer Pyris 1 TGA analyzer (Waltham, Massachusetts, USA). Briefly, a 5 mg sample was subjected to heating in a temperature range of 30 to 800 °C at a heating rate of 10 °C /min under a nitrogen atmosphere.

Differential scanning calorimetry (DSC)

Approximately 5 mg of each sample was placed in sealed aluminium pans. DSC analysis was carried out using a 2920 Modulated DSC (TA Instruments (USA) in a nitrogen environment. The temperature range for the analysis was 30–250 °C for MS, WK, SWF samples and 30–350 °C for hybrid MS-WK-AV biocomposite films with a heating rate of 10 °C/min.

Evaluation of post-treatment quality attributes of the Mango fruits

At the end of the treatment period, mango fruits in each treatment group were homogenized separately, filtered using Whatman™ No. 1, and kept in plain, sterile bottles at 4 °C until further analysis.

Determination of weight loss

Mango fruits per treatment group were weighed individually using an electronic weighing balance (Sartorius BSA224S-CW, Germany, 0.01 accuracy) on day 0 (W₀) and on days 4, 8, and 12 and recorded as W_f for each day. The percentage of weight loss was calculated using Eq. (5).

5

Measurement of pH

Ten millilitres (10 mL) of the filtrate obtained after the homogenization and filtration of the mango fruit were diluted with 50 mL of distilled water, and the pH of the resulting solution was determined using an electronic pH meter (H12210 pH meter).

Measurement of titratable acidity

The titratable acidity of mango fruits was determined by titrating 10 mL of each sample with 0.05 M sodium hydroxide, resulting in a consistently pink-coloured solution, as described by Dagnew et al.³³. Using Eq. (6), the titratable acidity value was computed and expressed in grams of lactic acid per 100 g of sample.

6

Measurement of total soluble solid (ºBrix value)

The total soluble solid (TSS) or brix content was determined using a handheld digital refractometer (DR 201-95). TSS (%) was recorded by directly reading the refractometer in ºBrix and integrating the required temperature correction parameters. Three measurements were obtained for each treatment, and the resulting average values were used for the analysis.

Measurement of pectin Methyl esterase (PME) activity

The activity of pectin methyl esterase was determined using the method developed by Hagerman and Austin³⁴ and modified by Ali et al.³⁵. A homogenized mixture of 1 g mango fruit sample and 2 mL NaCl (8.8% w/v) solution was centrifuged at 1000 × g for 20 min at 4 °C, and the enzyme mixture was labeled. The enzyme mixture was then mixed with 100 L of 1% bromothymol blue and 1 mL of 0.5% pectin and allowed to stand for 10 min before measuring absorbance at 620 nm. Enzyme activity was measured in units per gram of protein.

Measurement of polygalacturonase (PG) activity

The polygalacturonase activity was estimated by measuring the formation of reducing sugars using the dinitrosalicylic acid method described by Pathak and Sanwal³⁶ and modified by Chen et al.³⁷. The assay mixture contained 200 µL of 0.2 M sodium acetate buffer (pH 4.5), 100 µL of 0.2 M NaCl, 300 µL of 1% polygalacturonic acid (pH 4.5), and 0.1 mL of homogenized mango fruit extract. The reaction was initiated by adding the fruit extract and allowing it to stand for 30 min at 37 °C before adding the dinitrosalicylic acid reagent. The reaction was stopped by immersion in boiling water for 5 min. The production of reducing sugars was measured at 520 nm using d-galacturonic acid as the standard. Polygalacturonase enzyme activity was measured in units per gram of protein.

Statistical analysis

The analyses were repeated three times, and the results are presented as the means ± SD. Tukey’s test with a 95% confidence interval was used to compare means using analysis of variance (ANOVA) in GraphPad Prism 8.4.3. OriginPro 2016 was used to plot these needs.

Results and discussion

¹³C NMR spectroscopy

The ¹³C solid-state nuclear magnetic resonance spectrum of mango seed kernel starch (Fig. 1) showed well-defined chemical shifts consistent with typical polysaccharide structures. The observed resonances at 101.06, 81.71, 72.03, 61.63, and 33.75 ppm correspond to the C1 to C6 carbon environments of the α-D-glucopyranose units that constitute the starch polymer backbone. The anomeric carbon (C1) resonated at ~ 101 ppm, confirming the α-configuration of the glycosidic linkages. The signal at ~ 81.7 ppm corresponds to C4, often associated with crystalline domains in starch, while overlapping signals around ~ 72 ppm reflects the C2, C3, and C5 carbons. The resonance at 61.6 ppm is assigned to the primary alcohol group at C6. Notably, a smaller signal at 33.75 ppm may suggest the presence of residual modifying groups or minor impurities, such as CH₂ groups, indicating potential partial modification or plasticizer inclusion. These assignments align with Zhu³⁸ and El Nokab et al.³⁹ on native and modified starch systems, particularly in relation to the semi-crystalline organization of amylopectin and the amorphous nature of the amylose-rich regions. These chemical shifts confirm the α-configuration of the starch’s glycosidic linkages, important for ensuring the biocomposite film’s structural integrity and performance.

Fig. 1.

¹³C-NMR spectrum of mango seed kernel starch.

Particle size and zeta potential analysis

The particle size distribution of MS granules exhibited a bimodal distribution, as shown in Fig. 2. A primary peak with an area of 78.5% was observed at approximately 1.12 μm, whereas a secondary peak was noticed with an average particle size of 0.2 μm. The particle size of the mango starch used in this study was smaller than that reported by other authors. Espinosa-Solis et al.⁴⁰ reported that mango starch extracted from the Tommy Atkins cultivar exhibited granules with diameters ranging from 10 to 15 μm. According to another study, starches from the Manila and Criollo types showed reduced granular sizes ranging from 5 to 10 μm⁴¹. Lagunes-Delgado et al.⁴² found that the degree of ripening of mangoes may affect the shape and dimensions of their starch granules. The study conducted by Kaushal et al.⁴³ found that mango starch granules varied in size from 7 to 28 μm. Ramírez-Brewer et al.² showed that after being treated with microwave, the Sugar and Tommy mango cultivars had particle sizes ranging from 8.05 to 24.14 μm and 7.37 to 24.3 μm, respectively. WK exhibited an average particle diameter of approximately 502.7 nm, as depicted in Fig. 2. This value is smaller in comparison to the measurement of 750 nm reported by Shavandi et al.⁴⁴ and the measurement of 1000 nm reported by Fujii and Li⁴⁵. Nevertheless, the particle size of the WK produced in this study exceeded the range of 100–150 nm, as described by Ntumba et al.⁴⁶ for keratin derived from wool through enzymatic degradation. The reduced particle sizes of MS and WK achieved in this study are expected to facilitate the creation of a homogeneous and compact MS-WK film owing to their increased surface area. Conversely, the presence of larger particles can result in the formation of uneven, coarse films^46,47. Furthermore, the characteristics of film formation, such as its ability to adhere to the surface of an object, are controlled by the size of the particles in the film⁴⁸. The reduced particle sizes of MS and WK, as observed, contributed to the formation of a more homogeneous and compact film.

Fig. 2.

Particle size distribution of mango seed kernel starch (MS) and wool keratin (WK).

Amylose content

The MS exhibited an amylose concentration of 24.7%. According to the study by Colussi et al.⁴⁹, this concentration categorizes MS as intermediate or medium. Our findings closely align with those reported by Silva et al.⁵⁰. Starches with moderate to high amylose content are more conducive to film formation due to the predominantly linear amylose chains, which facilitate the formation of more hydrogen bonds compared to amylopectin⁴⁹. The linear configuration of amylose enhances re-association more effectively than amylopectin, occurring through retrogradation, which involves the formation of a double-helical structure⁵¹. Starches with elevated amylose levels yield films with enhanced tensile strength and barrier properties, although they tend to be more rigid and susceptible to fracture⁵².

Physico-mechanical properties of the film

The incorporation of Aloe vera (AV) into mango starch–wool keratin (MS-WK) composite films significantly altered their visual characteristics, producing a distinct dark-brownish coloration (Fig. 10). The resulting color is crucial for consumer acceptability, influencing consumer decisions directly. Films without AV displayed a lighter brown hue, likely attributable to the browning of mango kernel-derived starch. The deeper coloration of AV-enriched films is due to anthraquinones or aloins in AV, which may chemically convert into Aloe emodin during drying⁵³. Similar color modifications were reported in AV-chitosan composite films²¹.

Fig. 10.

Images of the (a) mango fruits wrapped with Aloe vera-enriched-mango starch-sheep wool keratin composite film on day 0, (b) unwrapped mango fruits after storage for 12 days at 33 ± 2 °C. Note: MS-WK-AV₀, MS-WK-AV₂ and MS-WK-AV₄, are mango fruits wrapped with mango seed kernel starch-sheep wool keratin composite films enriched with 0, 20, and 40 mg Aloe vera gel extract, respectively. Control mango fruits were not wrapped with any material.

Films enriched with AV demonstrated significantly (p < 0.05) reduced moisture content compared to those lacking AV (Table  1). Correspondingly, AV incorporation also resulted in thinner films. These findings align with those reported by Cyras et al.⁵⁴ and Gutiérrez and González⁵⁵, suggesting a plasticizing effect due to the organic acids in AV, reducing starch hygroscopicity through strong starch–AV interactions. Unlike the findings of Gutiérrez and González⁵⁵, increasing AV content by 50% did not significantly alter moisture or film thickness, a behavior similarly observed in cross-linked cassava and yam starch films by Gutiérrez et al.²⁶. Additionally, the integration of keratin into starch-based films, as previously reported ^56–58, consistently decreases moisture content and thickness. The notable interaction among mango starch, wool keratin, and AV in this study likely explains these reduced properties, which is further supported by the observed decrease in film solubility (Table 1).

Table 1.

Physico-mechanical characteristics of Aloe vera-enriched Mango starch-wool keratin biocomposite films.

Film characteristics	MS-WK-AV0	MS-WK-AV2	MS-WK-AV4
Moisture (%)	6.93 ± 0.8b	5.01 ± 0.5a	4.98 ± 0.4a
Thickness (mm)	0.21 ± 0.001b	0.16 ± 0.001a	0.15 ± 0.001a
Solubility (%)	45.5 ± 1.9b	39.7 ± 1.3a	37.5 ± 1.5a
Water vapour permeability (g.cm–2.s–1.mmHg–1)	0.0042 ± 0.00b	0.0023 ± 0.00a	0.0021 ± 0.00a
Tensile strength (MPa)	12.86 ± 1.3a	15.83 ± 1.6b	14.93 ± 1.3b
Elongation at break (%)	7.11 ± 0.8a	8.53 ± 0.3b	8.76 ± 0.5b
Contact angle (º)	40.91 ± 2.2a	51.57 ± 1.7b	68.58 ± 2.3c
Roughness (Ra / RMS)	101.51 / 106.61	102.45 / 108.48	106.21 / 110.37

Values are mean ± SD of three measurements. Note: Values in the same row carrying different superscript are statistically significant (p < 0.05). Note: MS-WK-AV₀, MS-WK-AV₂ and MS-WK-AV₄ are mango seed kernel starch-sheep wool keratin composite films enriched with 0, 20, and 40 mg Aloe vera gel extract, respectively. Ra and RMS are average roughness and root mean square roughness values, respectively.

The inclusion of AV significantly (p < 0.05) enhanced the water barrier properties of MS-WK composite films, as evidenced by the reduced water vapor permeability (WVP) (Table 1). This finding concurs with earlier studies by Tambe et al.⁵⁹ and Kaur et al.²¹, where AV incorporation markedly decreased WVP in starch-PVA and chitosan films, respectively. Additionally, AV significantly (p < 0.05) improved the mechanical properties, specifically increasing the tensile strength and elongation at break of MS-WK films. These improvements correlate positively with the reduced solubility and suggest decreased hydrophilicity driven by overlapping interactions between AV constituents and hydrophilic groups in starch. Organic acids present in AV may enhance film compactness through hydrogen bonding and ionic cross-linking, resulting in improved mechanical properties.

AV significantly (p < 0.05) increased the hydrophobicity of MS-WK biocomposite films, indicated by a marked increase in water contact angle (Table 1). The highest contact angle (68.58°) was observed in the MS-WK-AV₄ film, compared to the lowest (40.9°) in films without AV. This finding contrasts earlier observations by Jouki et al.⁶⁰, who reported a reduced contact angle following glycerol incorporation into sesame gum films. In the current study, enhanced hydrophobicity is attributable to robust hydrogen bonding and ionic interactions among AV, starch, keratin, and glycerol, promoted by organic acids present in AV. Higher contact angles reflect increased surface hydrophobicity⁶¹, consistent with Karbowiak et al.⁶², who linked enhanced biopolymer film hydrophobicity to extensive subsurface intermolecular hydrogen bonding. Consequently, the films exhibit reduced surface polarity, rendering starch-based films less prone to surface moisture retention.

Scanning electron microscopic analysis

SEM analysis revealed that sheep wool fibres (SWF) exhibited concentric layers composed of outer cuticles (thin scale layers) and an inner cortex (thicker central region) (Fig. 3a). The scales displayed regular length variations along the fibre axis. Previous studies have similarly reported variations in scale thickness among different sheep breeds and fibres⁶³. The overlapping scale structure significantly influences wool fibre properties such as felting, tangling, and curl formation. Additionally, the protective function of the cuticle shields fibres from chemical and physical damage⁶³. Keratin extracted from sheep wool demonstrated a globular and compact morphological structure (Fig. 3b). Mango starch (MS) granules appeared oval-shaped with smooth surfaces under SEM analysis (Fig. 3c), consistent with the previously reported morphology for mango starch granules from various Indian cultivars by Kaur et al.⁶⁴.

Fig. 3.

Scanning electron microscopic images showing sheep wool fibres (a), wool keratin (b), mango kernel starch (c), SEM images showing surface (d, e, and f) and cross-sectional (g, h, and i) micrographs of MS-WK-AV₀, MS-WK-AV₂, and MS-WK-AV₄ films, respectively. Arrows indicate spongy pores in the cross-sectional morphology of the films. Note: MS-WK-AV₀, MS-WK-AV₂ and MS-WK-AV₄ are mango seed kernel starch-sheep wool keratin composite films enriched with 0, 20, and 40 mg Aloe vera gel extract, respectively (Scale: 50 μm). Arrows indicate spongy pores present in the MS-WK-AV₂ and MS-WK-AV₄ films.

Surface microstructure analysis of MS-WK composite films indicated significant morphological differences resulting from Aloe vera (AV) incorporation. Films without AV (MS-WK-AV₀) showed rough surface textures (Fig. 3d). However, the smoothness of film surfaces notably improved with increased AV concentration, as evident in the SEM images of MS-WK-AV₂ (Fig. 3e) and MS-WK-AV₄ (Fig. 3f) biocomposite films. Contrastingly, cross-sectional SEM images revealed a porous, spongy internal structure in AV-enriched films (Fig. 3h and i), as opposed to the compact structure in MS-WK-AV₀ films (Fig. 3g). Increased porosity correlated positively with higher AV concentration. The spongy structure induced by AV incorporation potentially enhances the biocomposite films’ suitability for food packaging applications by providing cushioning and protection against mechanical damage.

Atomic force microscopic analysis

Atomic force microscopy (AFM) revealed detailed insights into the surface roughness of MS-WK-AV biocomposite films. Figure 4 displays the 2D and 3D AFM images, and the calculated roughness average (Ra) and root mean square (RMS) values are summarized in Table 1. Enhanced inter- and intra-molecular cross-linking between mango starch and wool keratin through hydrogen bonding and ionic interactions could significantly lower surface roughness and contact angle in films without Aloe vera (AV).

Fig. 4.

Atomic force micrographs showing 2D images (a, b, and c) and 3D images (d, e, and f) for the MS-WK-AV₀, MS-WK-AV₂, and MS-WK-AV₄ films, respectively. Note: MS-WK-AV₀, MS-WK-AV₂ and MS-WK-AV₄ are mango seed kernel starch-sheep wool keratin composite films enriched with 0, 20, and 40 mg Aloe vera gel extract, respectively.

However, incorporating increasing amounts of AV into the MS-WK biocomposite films significantly altered their nanostructure, resulting in homogeneous and compact microstructures with a corresponding increase in surface roughness. The most prominent change in nanoscale roughness, indicated by significant increases in Ra and RMS values, occurred between AV₂ and AV₄ samples. This substantial roughness enhancement at higher AV concentrations correlates positively with increased hydrophobicity, as evidenced by corresponding increases in contact angle measurements. These findings align closely with previously reported observations by Tesfaye et al.⁶⁵.

Correlation between material properties and surface roughness

The physico-mechanical properties of the biocomposite films were analyzed in relation to surface roughness values (Ra and RMS) using a heat map and scatter plots (Fig. 5). The physicochemical properties of MS-WK-AV biocomposite films exhibited a significant dependence on surface roughness characteristics. Specifically, moisture content, solubility, and water vapor permeability (WVP) demonstrated a negative correlation with surface roughness parameters (Ra and RMS), indicating that MS-WK-AV films with higher nanoscale roughness showed reduced hydrophilic interactions and water diffusion capacity. This trend suggests that the progressive incorporation of Aloe vera extract enhances the barrier functionality of the MS-WK composite matrix, likely through densification or restructuring of the surface morphology, thereby impeding moisture transmission, a phenomenon consistent with findings in similar biopolymer systems^66,67. The strong positive correlation between surface roughness and water contact angle further supports the interpretation that the inclusion of Aloe vera improves surface hydrophobicity. Increased contact angles with increasing roughness are indicative of enhanced liquid repellency in alignment with surface energy models such as the Wenzel or Cassie-Baxter theory^68,69. Conversely, the tensile strength displayed a nonlinear relationship with roughness. Peak mechanical strength was observed in the MS-WK-AV₂ formulation, corresponding to an intermediate roughness level, suggesting an optimal Aloe vera loading threshold that balances structural reinforcement without compromising film integrity. Excessive Aloe vera content may disrupt matrix cohesion, thereby reducing strength, a phenomenon previously reported in other polysaccharide-protein blends⁷⁰. Elongation at break showed a positive association with roughness, indicating that surface roughening may also contribute to enhanced elasticity or ductility. This may stem from morphological irregularities that facilitate stress dispersion or elongation under loads. Collectively, these findings suggest that modifying the surface roughness through Aloe vera enrichment serves as a dual-functional strategy, enhancing both barrier and mechanical performance. While MS-WK-AV₂ offers an optimal balance between tensile strength and permeability resistance, MS-WK-AV₄ maximizes hydrophobicity and barrier integrity, making it potentially more suitable for applications that demand higher resistance to moisture ingress.

Fig. 5.

Correlation between material properties and surface roughness of Aloe vera-infused mango starch-wool keratin biocomposite film.

Fourier-Transform infrared (FTIR) spectroscopic analysis

FTIR spectroscopy analysis exhibited signature functional groups of sheep wool fiber (SWF), wool keratin (WK), and mango starch (MS), and revealed significant molecular interactions among the MS, WK and AV constituents in the biocomposite films (MS-WK-AV₀, MS-WK-AV₂, and MS-WK-AV₄) (Fig. 6). SWF exhibited characteristic amide I and II peaks at approximately 1634 and 1528 cm^–1, reflecting protein secondary structures. Similarly, WK showed distinct amide bands (~ 1627 and 1522 cm^–1) along with peaks between 1235 and 1166 cm^–1, indicating C–N and C–O stretching vibrations. MS displayed typical polysaccharide absorption features at 3285 cm^–1 (O–H), 2915–2849 cm^–1 (C–H), and 1151–925 cm^–1 (C–O–C).

Fig. 6.

FTIR spectra of SWF, WK, and MS powders; and MS-WK-AV₀, MS-WK-AV₂, and MS-WK-AV₄ composite films. Note: SWF, sheep wool fiber; WK, wool keratin; MS, mango starch; MS-WK-AV₀, MS-WK-AV₂ and MS-WK-AV₄ are mango starch-sheep wool keratin composite films enriched with 0, 20, and 40 mg Aloe vera gel extract, respectively.

Integrating WK into the MS matrix caused a shift of the O–H absorption band to 3278 cm^–1 and introduced amide I and II peaks at 1638 and 1542 cm^–1. These changes imply significant hydrogen bonding and integration of keratin within the polysaccharide-protein composite matrix. Incorporating Aloe vera further broadened the O–H absorption band and introduced new peaks at 1742 cm^–1, attributed to carbonyl stretching vibrations of Aloe polysaccharides. This indicates enhanced protein–polyphenol interactions and intensified hydrogen bonding within the films^59,71,72. These molecular-level interactions confirm improved compatibility and structural networking within the composite, underpinning the observed enhancement in the films’ functional properties.

X-ray diffraction analysis

X-ray diffraction (XRD) analysis of sheep wool fiber (SWF), wool keratin (WK), mango starch (MS), and the composite films (MS-WK-AV₀, MS-WK-AV₂, MS-WK-AV₄) revealed significant structural transformations indicative of molecular interactions and structural rearrangements, essential for their application in food packaging (Fig. 7). SWF and WK displayed broad peaks around 21.6º and 24.9º, characteristic of partially crystalline α-helix keratin structures. The broad peaks illustrate the semi-crystalline nature of keratin, contributing flexibility and barrier functionality to biocomposite materials⁷³. MS exhibited distinct crystalline peaks at 15.1º, 17.7º, and 23.2º, representative of an A-type crystalline pattern associated with amylopectin double helices in native starch granules. These well-defined peaks indicate moderate crystallinity, typically linked to limited solubility and reduced film-forming capabilities of starch-based materials⁷⁴.

Fig. 7.

X-ray diffractograms of SWF, WK, MS powders and MS-WK-AV₀, MS-WK-AV₂, and MS-WK-AV₄ composite films. Note: SWF, sheep wool fiber; WK, wool keratin; MS, mango starch; MS-WK-AV₀, MS-WK-AV₂ and MS-WK-AV₄ are mango starch-sheep wool keratin composite films enriched with 0, 20, and 40 mg Aloe vera gel extract, respectively.

Blending WK with MS resulted in significant crystallinity reduction, evidenced by a broad single peak centered at 19.9º in the MS-WK composite. This indicates disruption of the native starch crystalline structure and the formation of an amorphous composite matrix, driven by ionic interactions and hydrogen bonding between starch hydroxyl and keratin amide groups⁷⁵. Such molecular interactions improve film homogeneity and functionality. Further incorporating Aloe vera gel extract into the composite films (MS-WK-AV₂ and MS-WK-AV₄) led to more pronounced crystallinity disruption, reflected by broader and less intense XRD patterns. Specifically, MS-WK-AV₂ showed a weak broad peak at approximately 21.3º (with an area of 232.84), and MS-WK-AV₄ displayed near-complete amorphization. MS-WK-AV₂ exhibited a crystallinity index (CI) of 29.18%. MS-WK-AV₄ exhibited a complete absence of crystallinity (0% CI), displaying a typical amorphous character. Aloe vera’s polyphenols and polysaccharides effectively disrupted residual crystallinity, enhancing film flexibility and structural homogeneity⁷⁵.

Collectively, the progressive reduction in crystallinity across modified samples aligns with the goal of enhancing biocomposite film functionality. Reduced crystallinity contributes to improved flexibility, solubility, and homogeneity, crucial for optimal food packaging performance. These structural modifications, promoted by keratin blending and Aloe vera incorporation, highlight the importance of ionic and hydrogen bonding interactions in enhancing the physicochemical and barrier properties of the biocomposite films.

The incorporation of AV into MS-WK composite induces a structurally amorphous yet functionally reinforced matrix that enhances mechanical flexibility without significantly compromising strength. Simultaneously, water vapor permeability remains controlled due to AV’s crosslink-induced densification and hydrogen bonding network. This dual functionality, as both an amorphizing agent and a natural cross-linker, makes AV particularly valuable in developing biodegradable, water-resistant, and mechanically compliant films suitable for food packaging applications. The synergistic incorporation of Aloe vera (AV)⁷⁶ and calcium ions (Ca²⁺)⁷⁷ in biopolymer films has been shown to significantly enhance both mechanical strength and water vapor barrier properties. In the study by Abd Karim et al.⁷⁶, the addition of AV gel to thermoplastic starch (TPS) films resulted in a marked improvement in tensile strength, increasing from 1.0 to 9.1 MPa, and a substantial rise in Young’s modulus from 51.9 to 769.0 MPa. Our results are in agreement with the previous reports.

Thermal stability analysis

Thermogravimetric analysis (TGA) and derivative thermogravimetry (DTG) revealed multi-step degradation patterns for mango starch-wool keratin (MS-WK) biocomposite films and their Aloe vera-enriched variants (MS-WK-AV₂ and MS-WK-AV₄), indicating complex decomposition involving moisture loss, polymer degradation, and residue oxidation (Fig. 8). The initial weight loss (~ 4.37%) in the unmodified MS-WK film occurred around 88.3 °C due to bound moisture evaporation. The primary decomposition, representing starch, Aloe vera, and keratin polymer degradation, peaked around 231 to 233 °C across all films. A significant secondary decomposition at approximately 292.7 °C involved substantial mass loss (~ 78.5%) due to polymer backbone degradation, followed by gradual third-stage decomposition around 397.2 °C from residual carbonaceous char oxidation.

Fig. 8.

TGA thermograms of (a) MS-WK-AV₀, (b) MS-WK-AV₂, and (c) MS-WK-AV₄ composite films. Note: MS-WK-AV₀, MS-WK-AV₂ and MS-WK-AV₄ are mango seed kernel starch-sheep wool keratin composite films enriched with 0, 20, and 40 mg Aloe vera gel extract, respectively.

Incorporating 20 mg Aloe vera extract (MS-WK-AV₂) shifted the thermal degradation pattern, slightly reducing the onset degradation temperature to 79.1 °C but raising the secondary decomposition peak temperature to 296.5 °C, indicating improved thermal stability. Enhanced stability likely results from antioxidant and cross-linking interactions of Aloe vera bioactive components with the biopolymer matrix. The MS-WK-AV₄ film, enriched with 40 mg Aloe vera extract, exhibited the highest thermal stability, with onset degradation at 101.3 °C and maximum secondary decomposition at 303.0 °C. The increased stability suggests stronger cross-linked polymer networks facilitated by higher Aloe vera phytochemical concentrations. These findings align with previous studies highlighting natural polyphenolic compounds’ role in delaying polymer degradation, increasing char yield, and improving thermal resistance in starch-based composites^78,79. Overall, Aloe vera enrichment significantly enhanced the thermal stability of MS-WK films, confirming its potential as a natural stabilizing agent in biodegradable packaging applications.

Differential scanning calorimetric (DSC) analysis

Differential scanning calorimetry (DSC) thermograms revealed distinct thermal transitions in the native components (SWF, WK, MS, Fig. 9a) and the fabricated biocomposite films (MS-WK-AV₀, MS-WK-AV₂, MS-WK-AV₄) (Fig. 9b). Mango starch (MS) exhibited a distinct endothermic gelatinization peak at 110.3 °C. Wool keratin (WK) showed transitions at 107.8 and 204.3 °C, indicating polypeptide chain unfolding and protein denaturation. Sheep wool fiber (SWF) displayed a lower onset temperature at 87.5 °C, suggesting comparatively lower thermal resistance.

Fig. 9.

DSC thermograms of (a) SWF, WK, and MS; (b) MS-WK-AV₀, MS-WK-AV₂, and MS-WK-AV₄. Note: SWF, sheep wool fiber; WK, wool keratin; MS, mango starch; MS-WK-AV₀, MS-WK-AV₂ and MS-WK-AV₄ are mango seed kernel starch-sheep wool keratin composite films enriched with 0, 20, and 40 mg Aloe vera gel extract, respectively.

The fabricated MS-WK-AV₀ biocomposite film exhibited enhanced thermal stability relative to individual components, with an onset temperature of 58.7 °C and prominent peaks at 110.4 and 260.7 °C. These transitions demonstrate strong intermolecular interactions between starch and keratin. However, the incorporation of Aloe vera extract led to slightly reduced thermal transition temperatures. The MS-WK-AV₂ film presented endothermic peaks at 77.4 and 239.9 °C, whereas MS-WK-AV₄ showed transitions at 87.1 and 250.5 °C, indicating a plasticizing effect. This effect is attributed to the disruption of hydrogen bonding and reduced crystallinity by Aloe vera in agreement with previous research on the effects of incorporating bioactive agent in starch-based films^75,79,80.

Overall, the DSC results indicate that keratin incorporation into the starch matrix significantly enhanced thermal resistance, while Aloe vera integration modulated film flexibility and thermal properties by decreasing melting enthalpy and transition temperatures. The TGA data corroborate enhanced thermal stability, particularly at higher temperature degradation stages. These thermal characteristics affirm successful matrix integration and demonstrate the ability to adjust film properties by varying Aloe vera concentrations.

The extraction method employed in this study using urea/NaOH for wool keratin may raise environmental concerns due to chemical use, wastewater generation, eutrophication potential from urea, and alkalinity issues from NaOH. These impacts can be effectively mitigated through careful waste management, solvent recycling, and adoption of greener extraction alternatives such as enzyme-assisted processes. Despite these concerns, converting wool waste into keratin-based biocomposite films supports sustainable, circular economy principles, significantly reducing reliance on petrochemical-based packaging. Future research should prioritize more eco-friendly extraction approaches to further improve sustainability.

Application study

Post-storage appearance

The images of the wrapped fruits before storage are shown in Fig. 10a. After storage for 12 days at 33.0 ± 2.0 °C, images of the mango fruits in each treatment group are displayed in Fig. 10b. In the control (unwrapped) group, only two fruits were left at the end of the storage period, as 67% of the fruits were lost owing to deterioration. Most of the fruits enclosed in the MS-WK-AV biocomposite films exhibited a green hue resembling that of unripe mangoes, whereas the control group displayed a yellow coloration resulting from the breakdown of the green chlorophyll pigment and the buildup of carotenoids throughout the ripening process. Nevertheless, we have not tested the mangoes for taste, aroma, or other textural properties.

Weight loss and pH

The MS-WK-AV₂ biocomposite film significantly reduced weight loss in mangoes during storage, outperforming MS-WK-AV₄, MS-WK-AV₀, and the control (Fig. 11a). A gradual increase in weight loss was observed across all treatment groups as storage time increased; however, MS-WK-AV₂-treated fruits consistently exhibited the lowest weight reduction. The difference in weight loss between MS-WK-AV₂ and MS-WK-AV₄ groups was statistically significant (p < 0.05). Given the high water content in fruits, moisture retention is a crucial indicator of freshness. Weight loss in stored fruits is often triggered by temperature and humidity variations, which increase respiration and transpiration rates. The MS-WK-AV films reduced these effects, likely due to enhanced crosslinking between starch and keratin as facilitated by Aloe vera incorporation. SEM analysis confirmed that Aloe vera contributed to a denser film microstructure, reducing water infiltration. These findings align with those of Nasrin et al.⁸¹ and Eshetu et al.⁸², who observed similar moisture retention effects using beeswax and coconut oil-based coatings.

Fig. 11.

Weight loss (a) and pH values (b) of mango fruits wrapped with mango seed kernel starch-sheep wool keratin composite film enriched with Aloe vera gel extract at room temperature for 12 days. Note: MS-WK-AV₀, MS-WK-AV₂ and MS-WK-AV₄, are mango fruits wrapped with mango seed kernel starch-sheep wool keratin composite films enriched with 0, 20, and 40 mg Aloe vera gel extract, respectively. Control mango fruits were not wrapped with any material. Each variable that shares a mean that is statistically different from another one will not share the same letter. The variable with the lowest mean was named “a” (if it is statistically different from all the others, otherwise it may be called “b”, etc.) and the variable with the highest mean was named with the highest letter among the tested variables.

The MS-WK-AV biocomposite films also effectively stabilized the pH of mangoes during storage. After 12 days at 33.0 ± 2.0 °C, treated mangoes exhibited significantly lower (p < 0.05) pH values than the untreated control (Fig. 11b). Fruits wrapped in MS-WK films containing Aloe vera had slightly lower pH values compared to those without Aloe vera, although the difference between MS-WK-AV₂ and MS-WK-AV₄ was not statistically significant. The Aloe vera component of the film likely influenced the preservation of organic acids by moderating the respiration-driven conversion of acids into sugars. In contrast, control fruits experienced a sharp increase in pH, indicating accelerated ripening and degradation. These results are consistent with the findings of Topno⁸³, who reported that chitosan-beeswax coatings helped preserve the pH and quality of strawberries during storage.

Titratable acidity and total soluble solid

Effect on titratable acidity and total soluble solids

Mango fruits wrapped in MS-WK-AV₂ biocomposite films exhibited significantly higher (p < 0.05) titratable acidity compared to the control, with an approximate 650% increase observed in MS-WK-AV₂ and a 600% increase in MS-WK-AV₄ (Fig. 12a). This elevated acidity is attributed to the reduced oxygen environment created by the film, which slows respiration and the conversion of organic acids into sugars⁸⁴. The titratable acidity trend closely mirrored the pH pattern observed earlier, reinforcing the role of Aloe vera in moderating respiratory metabolism. Given the climacteric nature and high respiration rate of mango fruits, maintaining acid levels is critical for prolonging shelf life. Conversely, mangoes wrapped in MS-WK films without Aloe vera experienced reduced acidity and increased pH, indicating elevated respiratory activity and the loss of organic acids such as citric and malic acids⁷.

Fig. 12.

(a) Titratable acidity, (b) total soluble solids (TSS), (c) pectin methyl esterase (PME) activity, and (d) polygalacturonase (PG) activity of mango fruits wrapped with mango seed kernel starch-sheep wool keratin composite film enriched with Aloe vera gel extract at room temperature for 12 days. Note: MS-WK-AV₀, MS-WK-AV₂ and MS-WK-AV₄, are mango fruits wrapped with mango seed kernel starch-sheep wool keratin composite films enriched with 0, 20, and 40 mg Aloe vera gel extract, respectively. Control mango fruits were not wrapped with any material. Each variable that shares a mean that is statistically different from another one will not share the same letter. The variable with the lowest mean was named “a” (if it is statistically different from all the others, otherwise it may be called “b, c”, etc.) and the variable with the highest mean was named with the highest letter among the tested variables.

MS-WK-AV films also significantly reduced the total soluble solids (TSS) content in mangoes compared to the control group (Fig. 12b). TSS values were lowered by 31.3, 43.8 and 34.4% in MS-WK-AV₀, MS-WK-AV₂, and MS-WK-AV₄ wrapped fruits, respectively. This reduction is likely due to the decreased conversion of organic acids to sugars over the 12-day storage period. Jordan et al.⁸⁵ noted that an increase in TSS typically reflects sugar accumulation and weight loss due to carbohydrate breakdown. The ability of the MS-WK-AV biocomposite film to reduce TSS levels highlights its effectiveness in slowing down metabolic processes that would otherwise accelerate ripening and sugar production. This suggests that Aloe vera integration into the composite film provides added functionality in maintaining fruit quality during postharvest storage.

Effects on pectin methylesterase (PME) and polygalacturonase (PG) activities

Mango fruits wrapped in MS-WK-AV composite films exhibited significant inhibition of pectin methylesterase (PME) and polygalacturonase (PG) enzyme activities after 12 days of storage compared to the control (Fig. 12c and d). These findings underscore the film’s role in mitigating fruit softening during ripening. Fruit softening is largely driven by the enzymatic hydrolysis of cell wall components⁸⁶. PME initiates this process by catalyzing the de-esterification of polygalacturonans, converting protopectin into water-soluble pectin and making the fruit susceptible to softening. PG then depolymerizes the resulting pectin into galacturonic acid⁸⁷. The reduced PME and PG activity observed in this study suggests that the MS-WK-AV composite film effectively delays the enzymatic degradation of the fruit cell wall. This enzymatic suppression contributes to better maintenance of mango firmness during storage and further supports the efficacy of Aloe vera-enriched biocomposite films in extending the postharvest shelf life of climacteric fruits. The combined action of PME and PG significantly influences the extent of cell wall expansion. It also contributes to the decrease in turgor pressure, both of which are key features of softened and ripened fruits. The modified atmosphere created by the AV-active MS-WK biocomposite film may have contributed to the reduced activities of PME and PG, thereby delaying the ripening process. Previous studies suggest that starch-based coatings can inhibit PME and PG activities by interacting with pectin and restricting the enzymes’ accessibility to the fruit cell wall substrate^88,89.

It is important to note that the efficient application of MS-WK-AV (like other starch-based composites), as reported in this study, is restricted by certain environmental conditions. The mango starch and wool keratin components confer significant hydrophilicity. Consequently, high relative humidity (> 70%) may lead to excessive moisture uptake, potentially compromising film stability, mechanical strength, and barrier efficacy. To ensure optimal performance, the MS-WK-AV biocomposite films are recommended for use in moderately humid conditions, ideally within a relative humidity range of approximately 50–70%, which aligns well with standard conditions during the storage and retail distribution of fresh produce⁹⁰. In addition, the MS-WK-AV biocomposite films were primarily designed and evaluated at ambient temperatures, specifically within the range of 25–40 °C. Temperatures exceeding this range may induce undesirable thermal transitions in starch and keratin structures, resulting in decreased mechanical integrity, reduced barrier function, and overall compromised stability of the film^58,91. Therefore, it is advisable that the films be utilized within ambient temperature and humidity conditions typically encountered during post-harvest storage and transport operations to achieve maximum effectiveness and ensure practical feasibility. In summary, Aloe vera-infused mango starch–wool keratin biocomposite films are best utilized under controlled or moderate environmental conditions, specifically within the relative humidity range of 50–70% and temperatures between 25 and 40 °C for effective and sustainable packaging applications. Adhering to these practical guidelines ensures optimal preservation performance, thereby enhancing the shelf life of mangoes sustainably.

Conclusion

This study demonstrated the successful development and application of a biodegradable biocomposite film composed of mango starch, wool keratin, and Aloe vera for the sustainable packaging of mango fruits. The incorporation of Aloe vera significantly enhanced the film’s physicochemical, mechanical, and thermal properties, as evidenced by improved moisture resistance, tensile strength, surface morphology, and thermal stability. Molecular interactions confirmed by FTIR and reduced crystallinity observed via XRD further substantiated the structural synergy among the biopolymers. Application of the Aloe vera-infused biocomposite films effectively extended the postharvest shelf life of mangoes up to 12 days, reduced the enzymatic degradation, and preserved key nutritional parameters such as titratable acidity, pH, and weight loss. These outcomes highlight the dual functionality of the film, acting both as a passive moisture and gas barrier and as an active preservation matrix, owing to the bioactivity of Aloe vera constituents. Overall, the findings present a promising, eco-friendly alternative to synthetic packaging materials, aligning with global efforts toward sustainable food preservation. Future work should explore industrial scalability and biodegradability in real-world environments, and the biocomposite film’s applicability to other climacteric fruits.

Author contributions

O.M. O. conceptualize the study, carried out the bench work, took part in data acquisition, data analysis, wrote the original draft. S. M. and N. S carried out the bench work, took part in data acquisition and data analysis, T. P. assisted in study design, methodology, supervised the study, reviewed and edited the original draft.

Data availability

All data generated or analyzed during this study are included in the manuscript.

Competing interests

The authors declare no competing interests.