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Abstract

The objective of this novel research was to examine the unique effects of Salix Aegyptiaca extract microcapsules (MEX; 0, 7.5, 15% v/v) and chitosan nanoparticles (NP_S; 0, 2, 4% w/v) within a central composite design on the physicochemical and structural properties of bionanocomposite films based on cottonseed meal protein isolate/pectin. The results showed that the thickness, moisture content, solubility, and antioxidant capacity, increased, while Hue angle decreased (p ≤ .05). Furthermore, the tensile strength and elongation at break of the samples increased significantly. Fourier-transform infrared spectroscopy and scanning electron microscopy results confirmed minor alterations in the chemical structure and strong molecular interactions between the biopolymer network and the factors under investigation. In addition, X-ray diffraction, differential scanning calorimetry, and antimicrobial activity analyses respectively indicated a decrease in crystallinity index, an increase in thermal stability, and an expansion of the inhibition zone diameter against both Gram-positive and Gram-negative bacteria with the addition of the extract and nanoparticles compared to the control sample. In conclusion, based on all results, it can be stated that the two unique factors examined in this study enhanced the structural and physicochemical properties of the produced film, thereby highlighting its potential application in food packaging to extend the shelf life of the product.

Keywords

Nanocomposite cottonseed protein isolate microencapsulation nano-chitosan

Introduction

Over the last few decades, researchers and industry professionals have examined and developed various cutting-edge packaging techniques to safeguard food and address the environmental risks associated with petroleum-based packaging materials.¹ Biodegradable and edible films are a contemporary solution that has a minimal impact on the environment. They provide several advantages, including the prevention of oxidation, acting as a barrier against the transfer of substances such as moisture, fat, and gas, controlling enzymatic activation, and incorporating bioactive agents like plant extracts and essential oils that possess potent antioxidant and antimicrobial properties. Additionally, they can also include halochromic compounds.^2–7

Cottonseed is a highly abundant oilseed and a significant source of plant protein. It is commonly utilized as animal feed because of its exceptional nutritional content.^8,9 Studies have shown that protein from cottonseed meal produces edible films that are biodegradable, transparent, but brittle, with low tensile strength; therefore, it is advantageous to blend it with other biopolymers, as done with pectin in this research.¹⁰

Pectin, a linear anionic polysaccharide composed of α-(1→4)-D-galacturonic acid units, is derived from citrus fruit peels and plant cell walls. The degree of esterification classifies pectin into three categories: high, medium, and low.¹¹ This degree of esterification influences pectin’s physical, chemical, and functional properties. A non-toxic, transparent, easily accessible, biodegradable, and environmentally friendly layer with good mechanical strength is produced when pectin is used to formulate edible films and coatings. On the other hand, it has high permeability to water vapor and lacks antimicrobial and antifungal properties.¹² Incorporating nanomaterials, antioxidants, and antimicrobial compounds into the formulation of edible films is preferred to improve the resulting films’ physicochemical and functional properties.¹³

Salix aegyptiaca (Salicaceae family) is recognized in traditional medicine for its anti-inflammatory benefits, its ability to relieve kidney stones and rheumatism, its heart-calming effects, fever-reducing and blood pressure–lowering effects, and its role in alleviating anemia, dizziness, and other disorders.¹⁴ The major constituents found in Salix aegyptiaca essence and extract are flavonoids, phenolic compounds, vanillin, myristicin, p-coumaric acid, gallic acid, caffeic acid, catechin, epigallocatechin gallate, salicin, luteolin, 1,4-dimethoxybenzene, carvone, tannins, citronellol, eugenol, and methyl eugenol.^15,16

Although bioactive compounds have many health benefits, they also have several disadvantages, including the high volatility of Salix aegyptiaca extract components (such as myristicin and eugenol), which easily evaporate and lose their effect over time; the poor stability of many phenolic compounds and flavonoids during processing and storage due to environmental factors such as oxygen, light, and heat, which lead to rapid degradation and loss of biological activity; the strong and bitter taste of some of these compounds, which reduces their effectiveness in food products; and limited controlled release, meaning that without protection, bioactive compounds may be released prematurely into the environment and fail to provide targeted effects. Therefore, various encapsulation methods are used to increase efficiency and protect valuable bioactive compounds.^17,18

In nanotechnology, one dimension ranges between 1 and 100 nm; such materials have a much greater surface-to-volume ratio compared to larger particles with the same composition, resulting in improved desirable properties. Nanoparticles can be made from various materials such as proteins, polysaccharides, synthetic polymers, and metals. Recently, the use of biodegradable and biocompatible nanoparticles has gained considerable attention.^19,20

In this study, chitosan nanoparticles were utilized to enhance the properties of the produced film. Chitosan is composed of N-acety-l-D-glucosamine and D-glucosamine units linked by β-(1→4) glycosidic bonds. It is derived from renewable sources, including the exoskeletons of insects, crustaceans such as shrimp and crabs, and fungal cell walls. Chitosan can be obtained through both chemical and microbiological methods.^21,22 Noteworthy characteristics of chitosan include non-toxicity, barrier properties, biodegradability, biocompatibility, and antibacterial and antifungal activities.²³

In this research, a bionanocomposite film based on cottonseed protein isolate/pectin containing microencapsulated Salix aegyptiaca extract and chitosan nanoparticles was prepared. This formulation introduces several innovations, such as the use of a new plant extract in an edible film, the application of cottonseed protein isolate as the film matrix, and the synergistic potential derived from combining an encapsulated natural extract (for stability and controlled release) with chitosan nanoparticles (for structural reinforcement and improved functionality). Therefore, the present work represents a significant innovation, and the aim of this study is to investigate these two factors at different concentrations on the physicochemical and functional properties of the produced bionanocomposite films.

Method and Materials

Materials

Salix aegyptiaca was collected from orchards located in Urmia, West Azerbaijan Province, Iran. Chitosan (average molecular weight: 50 kDa; degree of deacetylation: 70%; purity: >99%) was procured from Merck (Germany). Cottonseed meal was obtained from Tenco Dasht-e Shomal Company (Mazandaran, Iran), and Whey Protein Isolate (WPI) was purchased from the Arla brand (Denmark). All other chemical reagents used in this study were also supplied by Merck (Germany).

Methods

Isolation of Protein from Cottonseed Meal

The protein isolate from cottonseed meal was prepared following the method outlined by Tan et al. (2022), with minor adjustments. Initially, the purchased cottonseed meal was pulverized using a household grinder (Model SC-223FS, China) and subsequently filtered through a 0.5 mm mesh. The resulting powder was then mixed with sodium hydroxide at a volumetric ratio of 1:81 (powder to NaOH solution). The pH of this mixture was adjusted to 10.5–10.9, and the suspension was agitated for 1 hour at ambient temperature using a magnetic stirrer. Following agitation, the suspension was centrifuged at 5000 rpm for 15 min. The resulting supernatant (the liquid portion above the sediment) was carefully decanted into a new tube. To precipitate the protein, the pH of the supernatant was adjusted to 4.5 by the addition of either 0.1 or 1 M HCl. The suspension was again centrifuged at 5000 rpm for 15 min. After discarding the supernatant, the remaining precipitate was neutralized using 0.1 or 1 M NaOH and subsequently freeze-dried. Finally, the protein content was determined using the Kjeldahl method and reported to be 82%.²⁴

Extraction of Salix Aegyptiaca Extract

Fresh Salix Aegyptiaca were sun-dried, and powdered. The extraction was then performed using the percolation method. For this purpose, 250 g of the prepared Salix Aegyptiaca powder were combined with 96% ethanol (3-4 cm above the powder level) in a percolator. After 72 hours, the extraction was initiated by opening the percolator’s spigot. The remaining powder from the first stage was then subjected to a second round of ethanol addition and extraction after 48 hours. A third extraction was conducted after 24 hours in the same manner. Ultimately, the extracts from the first to the third stages were collected in a dark glass bottle and concentrated using a rotary evaporator (Hei-VAP Core, Germany) under vacuum conditions.²⁵

Encapsulation of Salix Aegyptiaca Extract

The Salix aegyptiaca extract was encapsulated using Whey Protein Isolate (WPI) as the wall material. Initially, 1 g of WPI was dispersed in 50 mL of deionized water. The Salix aegyptiaca extract was then added to this polymeric solution at a WPI-to-extract ratio of 40:60 (% w/w).

To ensure complete dissolution and initial homogenization, the mixture was subjected to high-speed homogenization (IKA T25 digital Ultra-Turrax, Germany) at 11,000 rpm for 10 min.

Then, in the microcapsules preparation stage, the final solution was prepared using ultrasound (model UP400S, Schreck Hoelscher, Germany), with 150 W and 20 kHz and a H7 type titanium probe with a diameter of 7 mm and a length of 100 mm for 5 min.²⁶

Particle Size Analysis and Zeta Potential of Microcapsules

The particle size of the emulsion was determined using dynamic light scattering (DLS) and PolyDispersity Index (PDI) with a Zetasizer device (Malvern Instruments, Worcestershire, UK).

A Zetasizer (Model Nano ZS, Malvern Instruments, UK) was used at 25°C to measure the average particle size (Z-Average), the particle size distribution index (PDI), and the surface electrical charge of particles against pH. The material was diluted to a 1:100 ratio with deionized water before analysis.²⁷

Stability Analysis of Microcapsules

A predetermined portion of the sample was sealed to avoid evaporation and kept at room temperature for 3 weeks to evaluate the stability of samples against phase separation and coalescence.²⁸

Preparation of Nanocomposite Films

The bio-nanocomposite films were prepared through a sequential casting and drying process. First, 1.25 g of Cottonseed Protein Isolate (CPI) powder was dispersed in 50 mL of distilled water and mixed at 80°C for 30 min. The pH of this resulting solution was then adjusted to 8.0 using 0.1 N NaOH. Concurrently, 1.25 g of apple pectin was dissolved in 50 mL of deionized water over 12 hours at 30°C. Once the individual polymer solutions were prepared, they were combined at a 50:50 ratio. To investigate the effects of the factors under study, the microencapsulated Salix aegyptiaca Extract was added to the combined polymer solution according to Table 1and homogenized for 2 min at 13,000 rpm. Subsequently, chitosan nanoparticles were incorporated according to Table 1 and homogenized for an additional 10 min. Glycerol was added as a plasticizer at a level 40% of the total dry weight of the film components. This final mixture was stirred for 5 min followed by 10 min of degassing.

Table 1.

List of experiments in the CCD

Run	NPS (W/V)	MEX (V/V)
1	2	15
2*	4	0
3	2	7.5
4	2	0
5*	0	15
6	2	7.5
7	4	7.5
8	2	7.5
9	0	7.5
10*	0	0
11	2	7.5
12*	4	15
13	2	7.5

*Optimum samples.

MEX: Salix Aegyptiaca extract microcapsules, NPS: chitosan nanoparticles.

Exactly 25 mL of the final film solution was poured onto identical glass plates (diameter: 25 cm) and dried in a forced-air convection oven at 40°C for 24 hours.²⁹

Nanocomposite Film Testing

Moisture Content

The moisture content (MC) of the prepared films was determined gravimetrically. Film samples, precisely cut to 3 × 3 cm² pieces, were initially weighed to determine the initial mass (M₁). These samples were then dried in a forced-air convection oven at 130 ± 2°C until a constant weight (M₂) was achieved. The moisture content was subsequently calculated using equation (1)³⁰:

MC % = [(M_{1} - M_{2}) / M_{1}] \times 100

(1)

Solubility

After immersing the film samples in water for a day, solubility tests were performed to determine the percentage of dissolved dry matter. To achieve this, films were divided into 2 × 2 cm² pieces, weighed (W_i), and left in 30 mL of distilled water for 3 hours while stirring. After removing extra water with filter paper, the samples were baked for a day at 105 ± 2°C to dry them out (W_f). The solubility was calculated using the equation (2).³¹

Solubility % = [(W_{i} - W_{f}) / W_{i}] \times 100

(2)

Colorimetry

A CIE colorimeter (Minolta CR300 Series, Minolta Camera Co. Ltd., Osaka, Japan) was used to measure film color parameters such as L*, a*, b*. Subsequently, Hue angle, Chroma, yellowness index, whiteness index, and total color difference (ΔE) were calculated using the equations (3)–(7) respectively.

Hue angle = \tan^{- 1} (b / a)

(3)

Chroma = \sqrt{{(a)}^{2} + {(b)}^{2}}

(4)

Yellow Index = {(Δ E)}^{2} - {(Δ L)}^{2} - {(Δ C)}^{2}

(5)

Whiteness Index = 100 - \sqrt{{(100 - L^{*})}^{2} + (a^{* 2} + b^{* 2})}

(6)

Δ E = \sqrt{{(Δ L)}^{2} + {(Δ a)}^{2} + {(Δ b)}^{2}}

(7)

Where: the color parameters (ΔL, Δa, and Δb) were compared to a standard white plate (L = 97.39, a = −5.11, b = 7.16).

Thickness

The thickness of the films was randomly measured at several points (10 locations) using a digital micrometer (Mitutoyo-Co, Japan) with a precision of 0.001 mm.³²

Antioxidant Activity

To evaluate the antioxidant activity of the film samples, 25 mg of the samples were dissolved in 4 mL of distilled water. Then, 2 mL of the extract was mixed with 0.2 mL of a 1 mM methanolic DPPH solution and stirred well for 5 min, followed by incubation in the dark at room temperature for 1 hour. The absorbance of the samples was read at 517 nm using a spectrophotometer (Model T60 UV, USA). The percentage of DPPH free radical scavenging activity was calculated using the equation (8)^33–35:

DPPH scaveging activity (%) = \frac{{A b s}_{c} - {A b s}_{s}}{{A b s}_{c}} \times 100

(8)

Where: Abs_c: Absorption of the control, Abs_s: Absorption of the film sample

Mechanical Properties

Using a texture analyzer (Model TA.XT plus C, UK) the mechanical characteristics of the film samples were determined. Film samples were made in 1 × 5 cm dimensions and conditioned at room temperature with 55% relative humidity (calcium nitrate) for 72 hours. The grips were initially spaced 30 mm apart, and their speed was 0.83 mm/s.³² The starting length of the film sample (L₀), the maximum force at the breaking point (F_max), the cross-sectional area of the film (A), and the maximum elongation of the film at the breaking point (L_max) were all noted. Then the tensile strength and elongation at break was calculated using equations (9) and (10) respectively.

Tensile strength (TS) = \frac{F_{\max}}{A}

(9)

Strain to break (STB) = \frac{L_{\max}}{L_{0}} \times 100

(10)

FTIR

FTIR spectroscopy (Spectrum Two, Perkin-Elemer, USA) investigated the structure and interactions within the optimized film samples. Tablets of the film (thickness ≤1 mm) were prepared by mixing with water, grinding the samples, coating them with potassium bromide at a 20:1 ratio, and applying a pressure of about 60 kPa for 10 min. The prepared samples were then analyzed with a resolution of 0.5 cm⁻¹ in the wavenumber range of 4000-400 cm⁻¹.³⁶

SEM

The microstructure of the produced bionanocomposite films was analyzed using a scanning electron microscope (Philip, Netherlands). For this purpose, samples fractured in liquid nitrogen were mounted on a metal base with carbon adhesive. During preparation for analysis, the samples were coated with gold particles, and imaging was performed at various magnifications.³⁶

XRD

X-ray diffraction (PHILIPS_PW1730, Netherlands) characterized the crystalline structure of the optimized bionanocomposite films. During this examination, the X-ray generator operated at an energy of 40 kV and a current of 30 mA. The diffracted rays were measured throughout a 2°θ angle range of 2 to 70°θ, using a scan rate of one degree and a step size of 0.02°θ.³⁷

DSC

The thermal behavior of the optimized bionanocomposite films was investigated using differential scanning calorimetry (DSC- Model DSC-R, Japan). A specific amount of the samples (ranging from 0.02 to 0.03 g) was analyzed in an aluminum pan at a heating rate of approximately 10°C/min, over a temperature range of −20 to 180°C, under a constant flow of nitrogen atmosphere. The melting temperature (T_M), crystallization temperature (T_c), and glass transition temperature (T_g) were determined based on the obtained thermograms.³⁸

Antimicrobial Properties

The antimicrobial properties of the optimized bionanocomposite film samples were assessed using the agar diffusion method. For this purpose, the bacteria used in this method were sub cultured twice consecutively in Mueller-Hinton broth. This was followed by injection with 0.1 mL of broth culture containing bacteria (10⁵ - 10⁶ CFU/mL) prepared the night before. Cut to 15 mm film samples were placed on plates containing Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (E. coli). The plates were incubated for 24 hours at 37°C, after which the inhibition zones were measured using calipers.³⁹

Statistical Analysis

In this study, a central composite design was employed at three levels for two variables: microencapsulation of Salix Aegyptiaca extract (0, 7.5 and 15% v/v) and chitosan nanoparticles (0, 2 and 4% w/v) across 13 treatments as per Table 1, using Design Expert software version 11.0.0. A significance level of 95% was established.

Results and Discussion

Particle Size, Particle Size Distribution, Zeta Potential, and Microcapsule Stability

Zeta potential and particle size of microcapsules act as pivotal indexes of their physical properties, including electrical charge and dimensions properties, and provide insights into their potential to interact with surrounding environments and biological systems. The zeta potential, particle size, and particle size distribution of the produced Salix Aegyptiaca extract microcapsules are depicted in Figure 1. The zeta potential indicates the net surface charge of materials or the electric potential difference at the solid-liquid interface, particularly in colloidal systems.⁴⁰ The acceptable range for the zeta potential of electrostatically stabilized compounds is ± 30 mV; thus, samples within this range are more stable, and their particles settle more slowly. Using surfactants with varying charges in encapsulation leads to different surface charges on the particles. In this study, whey protein isolate was used as the encapsulation wall material, and based on the obtained zeta potential (−9.32), the negative potential can be attributed to the anionic structure of the used material.^41,42 Considering that smaller particle sizes contribute to higher stability and resistance to gravitational forces, the particle size of the sample under investigation was reported as 218.1 nm, which indicates increased stability; furthermore, the Polydispersity Index (PDI) of the samples was found to be 0.250, indicating a uniform and stable distribution of the produced samples. The stability of the produced microcapsules over 3 weeks at room temperature was confirmed, as no phase separation or creaming was observed on the surface of the samples, which could be due to the whey protein isolate used in the microencapsulation process.

Figure 1.

Particle size, particle size distribution and zeta potential of Salix Aegyptiaca extract, microcapsules (MEX).

Particle size has a significant impact on numerous characters of microcapsules, particularly including permeability, specific surface area and solubility.^43,44

Physicochemical and Instrumental Tests

Moisture Content

Table 2 and Figure 2(a) demonstrate that the moisture content of the bionanocomposite films generated was significantly affected (p ≤ .05) by the individual and combined effectts of the components under investigation and the quadratic effect of chitosan nanoparticles. The moisture content of films increased with increasing the amount of chitosan nanoparticles and Salix Aegyptiaca extract microcapsules; the samples with the highest concentrations of both components showed the highest moisture content (∼from 14% to 20%). The microcapsules of Salix Aegyptiaca extract cause the produced bionanocomposite films to become moister due to their high concentration of hydrophilic functional groups like flavonoids and phenolic compounds, as well as their presence of hydroxyl groups, molecular interactions, and changes like the biopolymer matrix^15,43; moreover, research has shown that the addition of chitosan nanoparticles reduces hydrogen and covalent bonds with the biopolymer chain, increasing the amount of free hydroxyl groups available for forming hydrophilic bonds and increasing moisture.⁴⁴ Comparable outcomes were noted by Nurdiani et al. (2023) with the inclusion of silver nanoparticles in gelatin-based edible films⁴⁵ and by Kumar et al. (2021) with the addition of pomegranate peel extract in chitosan-based edible films.²³

Table 2.

Regression coefficients of physicochemical properties of nanocomposite films.

Factor	Moisture	Solubility	Thickness	DPPH
Model	10.64^**	57.62^**	0.024^**	558.83^**
Intercept	12.37	30.98	0.33	16.22
A	2.26^**	−1.41^**	0.07^**	20.14^**
B	0.44^**	−1.06^**	0.02^**	2.79^**
AB	−0.03^*	0.14^*	−0.0008	−0.03
A2	−0.23^*	0.57^*	−0.003	−3.05^**
B2	−0.009	−0.04^*	−0.0005	−0.08^*
R2	0.98	0.96	0.96	0.98
Adj-R²	0.96	0.93	0.94	0.97
CV%	2.23	3.10	2.47	2.13

*: p ≤ .0.

**: p < .01, A: Chitosan nanoparticles (NPS), B: Salix Aegyptiaca extract microcapsules (MEX).

Figure 2.

Effect of Chitosan nanoparticles (NPX) and Salix Aegyptiaca extract microcapsules (MEX) on Moisture (a) Solubility (b) Thickness (c) and DPPH (d) of Cottonseed Protein Isolate/Pectin Nanocomposite Film.

Solubility

According to the results of the analysis of variance (Table 2) and Figure 2(b), the individual, interactive, and quadratic effects of the studied factors were significant (p ≤ .05) on the solubility of the bionanocomposite films.

The findings demonstrated that when the amounts of Salix Aegyptiaca extract microcapsules and chitosan nanoparticles increased, the solubility of the samples also increased. The samples with the highest levels of both factors exhibited excellent solubility (∼from 35% to 45%). The increase in solubility with higher levels of materials used in the bionanocomposite film formulation can be attributed to the presence of phenolic compounds and a high number of hydroxyl groups in the Salix Aegyptiaca extract microcapsules and the reduction of biopolymer chain interactions due to the addition of chitosan nanoparticles.^43,44 Similar findings were reported with the addition of zinc oxide nanoparticles in pectin/alginate-based edible films⁴⁶ and ficus extract in chitosan/sodium alginate-based edible films.⁴⁷

Thickness

Based on the regression coefficients shown in Table 2 and Figure 2(c), the individual effects of Salix Aegyptiaca extract microcapsules and chitosan nanoparticles were significant (P ≤ .05) on the thickness of the bionanocomposite films. The results indicated that with an increase in the levels of the factors studied, the thickness of the films increased, with the greatest thickness observed in samples with the maximum levels of both factors (∼from 0.4 to 0.7 mm). The increased thickness of the produced biocomposite films can be attributed to the high-water absorption by the Salix Aegyptiaca extracts microcapsules and chitosan nanoparticles. As the levels of the factors under investigation increased, water molecules became trapped in the biopolymer matrix, leading to swelling and increased thickness; additionally, adding the factors used in the film formulation increased the solid content and created electrostatic bonds between the biopolymer network and the Salix Aegyptiaca extract microcapsules and chitosan nanoparticles, resulting in increased thickness.^48,49 Similar results were reported by Ngo et al. (2020) with increased levels of nanochitosan in pectin-based films⁵⁰ and by Silva et al. (2020) with the addition of Eriobotrya japonica leaf extract in ripe banana peel and starch-based films.⁵¹

Antioxidant Activity

The antioxidant capacity of the created bionanocomposite film samples was significantly affected (p ≤ .05) by the individual and quadratic impacts of the parameters under investigation, as indicated by the data displayed in Table 2. As shown in Figure 2(d), the films’ antioxidant capacity increased as the amounts of chitosan nanoparticles and Salix Aegyptiaca extract microcapsules increased. The samples with the highest concentrations of these components also showed the highest antioxidant capacity values (∼from 20% to 60%). The high concentration of antioxidant components inside the extract’s structure, such as phenolic compounds and flavonoids, is responsible for increased antioxidant capacity with increased Salix Aegyptiaca extract.^15,52 These results are in line with those of Hamann et al. (2022), who observed comparable outcomes when green tea extract was added to gelatin-based films⁵³; moreover, chitosan is known to have antioxidant qualities, which include the ability to chelate metal ions by electron pairing or hydrogen donation, as well as to inhibit free radical reactions.⁵⁴ Comparable results were reported by Dong et al. (2022) in chitosan-based films containing silica nanoparticles.⁵⁵

Colorimetriy

Based on the Analysis of Variance (Table 3) and Figure 3, the concentrations of chitosan nanoparticles and Salix Aegyptiaca extract microcapsules significantly affected the color parameters (p ≤ .05) of the bionanocomposite films, with the exception of the Yellowness Index (YI).Specifically, Chroma (C∗), the Whiteness Index (WI), and the Total Color Difference (ΔE) all showed a significant increase as the levels of both the extract microcapsules and chitosan nanoparticles increased, reaching their maximum values at the highest incorporation rates. In contrast, the Hue index (H) exhibited an inverse relationship, decreasing proportionally with increasing component concentrations, reaching its minimum value at the highest tested levels. The increase in Chroma (color saturation/purity) and ΔE (shift from the pure polymer baseline) is likely attributable to the inherent pigmentation introduced by the Salix Aegyptiaca extract, which becomes more express at higher level. The simultaneous decrease in Hue further confirms that the characteristic color imparted by the extract becomes increasingly dominant in the film matrix.

Table 3.

Regression coefficients of colorimetric parameters of nanocomposite film.

Factor	Chroma	Hue	Yellow index	White index	ΔE
Model	25.97^**	21.42^**	48.02^ns	13.4^**	41.05^**
Intercept	19.40	86.26	55.40	45.31	52.73
A	0.35^**	−2.08^**	−1.43	3.85^**	3.26^**
B	0.12^**	−0.30^*	0.02^*	0.35	0.58^**
AB	0.10^*	0.08^*	0.24	−0.05	0.05
A2	0	0	0	−0.53	−0.30
B2	0	0	0	−0.01	−0.02
R2	0.88	0.88	0.56	0.88	0.97
Adj-R²	0.84	0.84	0.41	0.80	0.95
CV%	1.64	1.21	4.61	2.17	1.48

*p ≤ .05, **p < .01, ns (non-significant). A: Chitosan nanoparticles (NPS), B: Salix Aegyptiaca extract microcapsules (MEX).

Figure 3.

Effect of Chitosan nanoparticles(NPS) and Salix Aegyptiaca extract microcapsules(MEX) on Colorimetric Parameters of Cottonseed Protein Isolate/Pectin Nanocomposite Film.

Regarding the yellowness index, its lack of statistical significance suggests that while the overall color profile shifted markedly (ΔE increased), the yellow component itself was either present at a very low concentration or its variation across factor levels was too consistent to yield a significant statistical result. These results underscore that the concentration of active components dictates the final color quality of the film. This dependence of film color on formulation type and concentration aligns with established literature.²³

SEM

Scanning electron microscopy images of the optimized bionanocomposite films are shown in Figure 4. As seen in Figures 4(a), the cottonseed protein isolate-pectin bionanocomposite film exhibited a uniform microstructure with no pores, surface cracks, or irregularities, contributing to mechanical properties and low water vapor permeability.⁵⁶ The addition of 15% Salix Aegyptiaca extract microcapsules and 4% chitosan nanoparticles (Figures 4(b) and 4(c)) resulted in a uniform and dense structure on the film surface, indicating strong molecular interactions within the biopolymer network, matrix space closure, and increased thickness, thereby reducing water permeability.⁵⁷ In other words, the addition of extract microcapsules and nanoparticles acted as fillers in the biopolymer matrix and led to strong intermolecular interactions, mainly hydrogen bonding, between the functional groups of the nanoparticles, extract microcapsules, and the biopolymer network; the results obtained are consistent with the results obtained from FTIR (Figure 5), which confirm the slight change in the intensity and position of the resulting peaks due to the increase in hydrogen bonding. As depicted in Figure 4(d), the film surface appears cohesive and rough, with no visible pores or cracks, and the extract and nanoparticles are well dispersed within the polymeric matrix, indicating good integration of pectin, cottonseed protein isolate, extract, and nanoparticles. On the other hand, the observed roughness can be attributed to the presence of nanoparticles, which create a more tortuous path for the diffusion of molecules and further improve the barrier properties. Similar results were presented by Shan et al. (2023) for multilayer composite films containing green tea extract based on gelatin/sodium alginate.⁵⁸ Shapi’i et al. (2022) reported strong intermolecular interactions with starch chains in starch-based edible films containing chitosan nanoparticle.⁵⁹

Figure 4.

SEM of active films: (a) (pure film), (b) (film containing 15% Salix Aegyptiaca extract microcapsules), (c) (film containing 4% Chitosan nanoparticles) and (d) (film containing 15% Salix Aegyptiaca extract microcapsules and 4% Chitosan nanoparticles).

Figure 5.

FTIR of active films: (a) (pure film), (b) (film containing 15% Salix Aegyptiaca extract microcapsules), (c) (film containing 4%Chitosan nanoparticles) and (d) (film containing 15% Salix Aegyptiaca extract microcapsules and 4% Chitosan nanoparticles.

FTIR

FTIR spectra of the optimized bionanocomposite film samples are presented in Figure 5. The absorption pattern of the samples is reported in the range of 4000 to 500 cm⁻¹. The FTIR spectrum of the control film sample shown in Figure 5(a) indicates the presence of asymmetric stretching vibrations of the NH2 group in the protein structure at approximately 3226.85 cm⁻¹.⁶⁰ The peak at approximately 2861.96 cm⁻¹ corresponds to CH2 stretching vibrations in the pectin chain,⁶¹ and the peaks at approximately 1547.44 cm⁻¹ and 1397.10 cm⁻¹ represent the bending vibration of N-H and the stretching vibration of C-N in the amide groups of the cottonseed protein isolate structure.⁶² The measured peak in the range of 1014.46 cm⁻¹⁶³ is attributed to the stretching vibration of C-O, whereas the peak at 614.77 cm⁻¹⁶⁴ is attributed to the stretching vibrations of the polysaccharides C-O-C. Spectrum 5-b shows that adding 15% Salix Aegyptiaca extract microcapsules caused slight peak intensities and position changes. This implies a planned interaction between the extract and the biopolymer matrix occurred, So that the peak at 3226.85 cm⁻¹ changed to 3231.33 cm⁻¹, the peak at 2861.96 cm⁻¹ changed to 2856.94 cm⁻¹, the peak at 1547.44 cm⁻¹ and 1397.10 cm⁻¹ changed to 1567.47 cm⁻¹ and 1364.70 cm⁻¹, respectively, the peak at 1014.46 cm⁻¹ changed to 1012.80 cm⁻¹, and the peak at 614.77 cm⁻¹ changed to 623.49 cm⁻¹; these changes, along with the increase in peak intensity, are a clear indicator of the strengthening of the hydrogen bond network between the polyphenols in the extract and the functional groups of the biopolymer matrix. Strong molecular interactions between the nanoparticles and the biopolymer matrix are suggested by Spectrum 5-c, which demonstrates that the addition of 4% chitosan nanoparticles did not create new peaks but rather slightly intensified the ones that already existed and the peak changes were almost similar to Spectrum 5-b; finally, spectrum 5-d shows that the utilization of 4% nanoparticles and 15% extract together decreased peak intensities, which indicates a weak electrostatic interaction of the factors used together.

XRD Analysis

The XRD spectra of the optimized bionanocomposite film samples are presented in Figure 6. According to the results, Figure 6(a) shows a broad peak at a 2θ angle of approximately 20.10° in the control sample, indicative of the semi-crystalline structure of the film based on cottonseed protein isolate and pectin; And also the crystallinity index for the pure film sample was equal to 15.2%. Upon the addition of 15% Salix Aegyptiaca extract microcapsules (Figure 6(b)), there was no significant shift in the peak positions compared to the control sample, but a slight increase in peak intensity was observed. In other words, the peak position remained unchanged at around 20.10°, but the crystallinity index increased to 18.5%. The incorporation of 4% chitosan nanoparticles (Figure 6(c)) did not alter the peak positions, yet the peak heights and crystallinity index increased to 22.3%, suggesting the crystalline structure of the nanoparticles; finally, the simultaneous addition of the extract and nanoparticles (Figure 6(d)) resulted in a reduction in peak intensities and crystallinity index to 8.9% compared to the control sample and those containing the individual factors under study, indicating a decrease in the crystalline structure of the biopolymer being examined. These findings are consistent with those reported by Amjadi et al. (2020), who observed similar effects with the addition of ZnO nanoparticles in a gelatin/chitosan-based edible film⁶⁵; in addition, Moghadam et al. (2020) reported comparable results by incorporating pomegranate peel extract in a mung bean protein-based edible film.⁶⁶

Figure 6.

XRD of active films: (a) (pure film), (b) (film containing 15% Salix Aegyptiaca extract microcapsules), (c) (film containing 4% Chitosan nanoparticles) and (d) (film containing 15% Burdock extract microcapsules and 4% Chitosan nanoparticles).

DSC Thermograms

Figure 7 shows the DSC thermograms of the optimized bionanocomposite film samples. The control sample (Figure 7(a)) showed two endothermic peaks: the glass transition temperature (Tg) is represented by the peak at 42.14°C, and the melting temperature (Tm) of the cottonseed protein isolate/pectin is defined by the minor peak at 73.74°C. The spectrums of the pectin, Salix Aegyptiaca extract, and cottonseed protein isolate (Figure 7(b)) demonstrate that the microcapsule addition had no discernible impact on the film sample’s glass transition and melting temperatures. The film sample’s glass transition and melting temperatures were elevated by the addition of nanoparticles, as demonstrated by the cottonseed protein isolate/pectin/chitosan nanoparticles spectrum (Figure 7(c)). This implies that the nanoparticle inclusion enhanced the films’ thermal stability. In the bionanocomposite films containing both factors under study, it was observed that the addition of both factors increased the glass transition and melting temperatures more than when the factors were used individually, demonstrating excellent interaction between the cottonseed protein isolate/pectin/chitosan nanoparticles/Salix Aegyptiaca extract microcapsules and the high thermal stability of the produced sample (Figure 7(d)).

Figure 7.

DSC of active films: (a) (pure film), (b) (film containing 15% Salix Aegyptiaca extract microcapsules), (c) (film containing 4%Chitosan nanoparticles) and (d) (film containing 15% Salix Aegyptiaca extract microcapsules and 4% Chitosan nanoparticles).

Similar results were reported by Jridi et al. (2019) with the addition of blood orange peel extract in a gelatin-based edible film.⁶⁷ Additionally, Oleyaei et al. (2016) also noted that the addition of TiO₂ nanoparticles in a starch-based edible film increased the glass transition and melting temperatures.⁶⁸

Mechanical Properties

Table 4 shows the mechanical property analysis results, including tensile strength (TS) and elongation at break (ETB), of the optimized bionanocomposite film based on cottonseed protein isolate/pectin, including Salix Aegyptiaca extract microcapsules and chitosan nanoparticles. The study found that adding Salix Aegyptiaca extract microcapsules did not significantly affect tensile strength compared to the control sample; however, adding chitosan nanoparticles alone and combining the two factors resulted in a significant increase in tensile strength (p < .05). On the other hand, in examining elongation at break, no significant difference was observed in the control samples and those containing chitosan nanoparticles; however, the addition of Salix Aegyptiaca extract microcapsules, both individually and in combination with chitosan nanoparticles, resulted in a significant increase in the elongation at break factor (p < .05). According to the studies conducted, the addition of plant extracts can improve the mechanical properties of edible films; in this research, the addition of Salix Aegyptiaca extract microcapsules increased the ETB of the film samples compared to the control due to uniform dispersion and appropriate interaction between the Salix Aegyptiaca extract microcapsules and the polymeric matrix.⁶⁹ The increase in tensile strength with the addition of chitosan nanoparticles can be attributed to the excellent and uniform dispersion of nanoparticles in the biopolymer network, strong molecular interaction between the nanoparticles and the biopolymer network, and the high surface-to-volume ratio of the nanoparticles.⁴⁸ The optimized bionanocomposite film containing Salix Aegyptiaca extract microcapsules and chitosan nanoparticles, due to the synergistic effect of both factors used, had the highest tensile strength (TS) and elongation at break (ETB) compared to the control sample. The results agree with the findings of Grabska-Zielińska et al. (2021), who reported that with an increase in the percentage of olive leaf extract, the elongation at the break of poly-lactic acid-based films increased⁷⁰; in addition, Dos Santos et al. (2023) reported comparable results for an increase in tensile strength with the addition of chitosan nanoparticles to a pectin-based film containing garlic essence.¹²

Table 4.

Regression coefficients of colorimetric parameters of nanocomposite film.

Films	TS (MPa)	ETB (%)	Diameter of inhibition zone (mm)
Films	TS (MPa)	ETB (%)	S. aureus	E. coli
Run 10	6.51 ± 0.01^a	6.12 ± 0.04^a	6.23 ± 0.1^a	7.17 ± 0.01^a
Run 5	6.56 ± 0.24^a	7.54 ± 0.04^b	10.43 ± 0.3^c	8.66 ± 0.01^c
Run 2	7.72 ± 0.14^b	6.77 ± 0.01^a	8.23 ± 0.01^b	7.5 ± 0.08^b
Run 12	9.64 ± 0.02^c	9.45 ± 0.02^c	21.3 ± 1.32^d	17.32 ± 1.13^d

Different superscript letters (a-d) in each column show significant differences (p < 0.05).

Antimicrobial Properties

Table 4 shows the antibacterial efficiency of the optimized bionanocomposite film samples against E. coli and B. aureus. Because of the inherent antibacterial activity of the polymers utilized in the edible film formulation, the control film showed the least antimicrobial impact against these dangerous microorganisms. The antibacterial effectiveness of both chitosan nanoparticles and Salix Aegyptiaca extract microcapsules was demonstrated by their respective considerable enhancements of the antimicrobial properties against Gram-positive and Gram-negative bacteria compared to the control (p < .05). Due to a synergistic impact, films containing both components simultaneously showed the maximum antibacterial activity. Because Gram-positive bacteria have a thinner cell wall than Gram-negative bacteria, they are less resistant to antimicrobial drugs. This explains why the results showed a stronger impact on Gram-positive than Gram-negative bacteria.⁷¹ Compounds including phenolics, caffeic acid, rutin, hydrobenzoic acid, chlorogenic acid, and p-coumaric acid are responsible for the antibacterial action of Salix Aegyptiaca extract.⁷² The antimicrobial activity of chitosan nanoparticles can be attributed to the electrostatic interactions between the positively charged glucosamine amine groups of chitosan and the negatively charged bacterial cell membrane, which alter membrane permeability, bind to bacterial DNA, and ultimately cause cell death.⁷³ Chitosan itself has antibacterial and antifungal properties. These results align with those of Xu et al. (2021) in chitosan/gelatin-based films containing hop plant extract⁷⁴ and comparable outcomes documented by Ngo et al. (2020) in pectin-based edible films containing chitosan nanoparticles.⁵⁰

Conclusion

Improving food products’ quality and shelf life while being stored has become a significant concern in recent years. Researchers and business experts use various techniques to increase the quality and durability of food products. Using edible films and coatings for packaging that include bioactive substances like antioxidants and antimicrobials is a creative method. In light of this, this work investigated the physicochemical and structural characteristics of bionanocomposite films based on pectin and cottonseed protein isolate combined with chitosan nanoparticles (0, 2, 4% w/v) and Salix Aegyptiaca extract microcapsules (0, 7.5, 15% mL). The results indicated that the addition of Salix Aegyptiaca extract and chitosan nanoparticles improved the physicochemical properties (thickness, moisture content, solubility, and antioxidant capacity), color parameters (Chroma, white index, ΔE), and mechanical properties (elongation at break and tensile strength), with a decrease in Hue angle (P ≤ .05). FT-IR and SEM analyses confirmed minor changes in chemical structure and strong molecular interactions between the biopolymer network and the factors under study. XRD, DSC, and antimicrobial activity analyses, respectively, indicated a reduction in crystallinity index, increased thermal stability, and enhanced inhibition zone diameter against Gram-positive and Gram-negative bacteria with the addition of extract and nanoparticles compared to the control; ultimately, based on all the results obtained, the sample containing 15% Salix Aegyptiaca extract microcapsules and 4% chitosan nanoparticles exhibited superior properties among the produced samples and can be recommended as a suitable candidate for food packaging applications.

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

CRediT Authorship Contribution Statement

Aysan Mosaviyan: Conceptualization, Methodology, Investigation, Writing-Original Draft, Project administration. Shahin Zomorodi and Afshin Javadi: Supervision, Funding acquisition, Writing-Review & Editing. Farnaz Yahyavi: Writing-Review & Editing.

ORCID iD

Shahin Zomorodi

Data Availability Statement

Data will be made available on request.*

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Run	NPS (W/V)	MEX (V/V)
1	2	15
2*	4	0
3	2	7.5
4	2	0
5*	0	15
6	2	7.5
7	4	7.5
8	2	7.5
9	0	7.5
10*	0	0
11	2	7.5
12*	4	15
13	2	7.5

Run	NPS (W/V)	MEX (V/V)
1	2	15
2*	4	0
3	2	7.5
4	2	0
5*	0	15
6	2	7.5
7	4	7.5
8	2	7.5
9	0	7.5
10*	0	0
11	2	7.5
12*	4	15
13	2	7.5

Biodegradable film based on cottonseed protein isolated and pectin enriched with Salix aegyptiaca extract microcapsules and chitosan nanoparticles

Abstract

Keywords

Introduction

Method and Materials

Materials

Methods

Isolation of Protein from Cottonseed Meal

Extraction of Salix Aegyptiaca Extract

Encapsulation of Salix Aegyptiaca Extract

Particle Size Analysis and Zeta Potential of Microcapsules

Stability Analysis of Microcapsules

Preparation of Nanocomposite Films

Nanocomposite Film Testing

Moisture Content

Solubility

Colorimetry

Thickness

Antioxidant Activity

Mechanical Properties

FTIR

SEM

XRD

DSC

Antimicrobial Properties

Statistical Analysis

Results and Discussion

Particle Size, Particle Size Distribution, Zeta Potential, and Microcapsule Stability

Physicochemical and Instrumental Tests

Moisture Content

Solubility

Thickness

Antioxidant Activity

Colorimetriy

SEM

FTIR

XRD Analysis

DSC Thermograms

Mechanical Properties

Antimicrobial Properties

Conclusion

Footnotes

Declaration of conflicting interests

Funding

CRediT Authorship Contribution Statement

ORCID iD

Data Availability Statement

References

Run	NPS (W/V)	MEX (V/V)
1	2	15
2*	4	0
3	2	7.5
4	2	0
5*	0	15
6	2	7.5
7	4	7.5
8	2	7.5
9	0	7.5
10*	0	0
11	2	7.5
12*	4	15
13	2	7.5