Probing sustainable cross-linked quince seed mucilage–sodium alginate biocomposite hydrogels through the development of biodegradable films

Abstract

Biocomposite hydrogels based on quince seed mucilage (QSM) and sodium alginate (SA) incorporated with crosslinkers (ZnO, CaCl₂ and NC) were developed and characterized. Biocomposite hydrogels were characterized for their rheological, structural and thermal properties. Hydrogels with a QSM-to-SA ratio of 80 : 20 and CaCl₂ as a crosslinker agent exhibited more elastic character and stronger gelation, as revealed by rheological studies, and improved structural and thermal properties, as revealed by Fourier transform infrared spectroscopy and differential scanning calorimetry. Hydrogels were further used for the development of films. The prepared biocomposite films were characterised for thickness, tensile strength (TS), water vapour transmission rate (WVTR) and oxygen transmission rate (OTR), biodegradability, instrumental color and antimicrobial activity. Significantly higher TS and lower WVTR and OTR (P ≤ 0.05) were observed in the biocomposite films with CaCl₂ as a crosslinker agent. Biocomposite films with NC as a crosslinker agent exhibited significantly higher instrumental color parameters. Films incorporated with NC showed lower biodegradation by the soil burial test. Films with ZnO as a crosslinker agent exhibited greater inhibitory effect against Aspergillus niger and Rhizopus stolonifer. Overall, biocomposite films with an 80 : 20 ratio of QSM to SA and CaCl₂ as a crosslinker agent as food packaging can provide better protection to extend the food shelf life.

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Sustainability spotlight

Sustainability provides robust solutions towards everyday challenges in society without having any detrimental effects on the surrounding environment; hence, it is important for achieving SDGs. Presently, food-packaging materials are creating an extra burden on Earth due to their inherent toxicity and non-degradability, viz., the average lifespan of plastic on Earth is several years. Therefore, to address this issue, in this study, bioinspired polymers such as quince seed mucilage and sodium alginate were used for the development of composite hydrogels with CaCl₂, nanoclay and ZnO as crosslinkers. Furthermore, the as-prepared biodegradable films were fully characterized with various parameters and studied with the help of rheology, thermal and spectroscopic investigations. This work highlights the importance of the UN sustainable development goals, such as good health and well-being (SDG 3) and industry, innovation and infrastructure (SDG 9).

1. Introduction

Quince seed mucilage (QSM) is generally recognised as safe and is inexpensive, non-toxic, biocompatible, renewable and biodegradable. These properties make it suitable for the preparation of biopolymer films.¹ The swelling and deswelling properties of QSM are primarily due to the polysaccharide glucuronoxylan.² Moreover, QSM has a greater molecular weight (9.61 × 10⁶ g mol⁻¹) than some commercially available and most applicable gums like xanthan gum, guar gum and gellan gum.³ The poor strength and barrier properties of films and coatings prepared using QSM limit its use in food packaging despite being an economic polymer with great water absorption capacity.⁴ These drawbacks of QSM are overcome by blending it with other biopolymers like sodium alginate to prepare biocomposites.⁵ Sodium alginate has been used as a blending of diverse biopolymers to tailor the properties of coatings/films.⁶ It is a polysaccharide consisting of (1,4)-linked β-D -mannuronic acid units and α-L-guluronic acid units. Alginate is obtained commercially from seaweed and has gel forming properties, which increase its scope in the packaging of food.^5,7 Moreover, it has applications in various pharmaceutical as well as cosmetic industries.⁸ However, the disadvantages like high water vapor and gas permeability, low mechanical strength and solubility limit its applications in biodegradable films.⁵ These drawbacks are overcome by the addition of a number of components like other biopolymers,⁹ plant extracts,¹⁰ pure active ingredients¹¹ and crosslinker additives.¹²

Hydrogels are three-dimensional water-absorbable polymeric networks commonly employed in the medical and pharmaceutical field due to their resemblance to the extracellular matrix constituents.^13,14 Moreover, its non-toxic, inert and biocompatible nature ensures that it does not influence the metabolic processes of living organisms. Formulation of hydrogels mostly requires the addition of crosslinkers to prevent the solubilization of polymeric chains before use. More recently, several promising crosslinkers with improved biocompatibility, biodegradability, toxicity and aqueous solubility have been investigated.^15–18 Moreover, crosslinking agents have been incorporated in hydrogel formulations to enhance the tensile strength and attain desired drug release characteristics.^19,20 Hydrogels exhibit viscoelastic to elastic behavior due to the incorporation of crosslinkers between the polymeric chains.^21,22

Films from synthetic polymers are common applied materials in food packaging. Nevertheless, the mass use of plastics results in waste that is stored for many years, leading to environmental pollution.²³ Moreover, various studies have found the apparent ill effects of plastics in various tissues and organs,²⁴ human cells²⁵ and marine organisms.²⁶ Scientists have also reported the occurrence of microplastics in human milk.²⁷ Therefore, researchers nowadays are focused more on smart biomaterials for packaging that are non-toxic, edible, and biodegradable and can serve as potential substitutes to the existing polymer plastics.²⁸ In this study, QSM and sodium alginate (SA)-based biocomposite polymers were enriched with crosslinkers like nanoclay, ZnO and CaCl₂ to form hydrogels. The hydrogels based on QSM and sodium–alginate incorporated with crosslinkers were then used for the development of a biocompatible film.

2. Materials and methods

2.1. Materials

Sodium alginate (91% purity), nanoclay and ZnO were purchased from Sigma-Aldrich Co. Ltd, (St. Louis, MO, USA), while glycerol and CaCl₂ were purchased from Hi-Media Pvt. Ltd (India). All other chemicals and reagents used in this study were of analytical grade, and were procured from standard commercial suppliers (Sigma-Aldrich and HiMedia).

2.2. Extraction of quince seed mucilage

Quince seeds were purchased from a local market at Shalimar Srinagar, Jammu and Kashmir (J&K), India. After manual cleaning of the seeds, the extraction of aqueous QSM from whole seeds was carried out in distilled water for 30 min at 40 °C to induce mucilage exudation. The ratio of water to seed was 25 : 1 (v/w). The water-seed slurry was agitated with an electric mixing paddle throughout the extraction period to remove the mucilage layer from the surface of the seed. The solutions were then filtered using a cotton cloth, and the collected mucilage was oven-dried at 45 °C ± 1 °C to a constant weight. The dried mucilage was then bottled and kept under a cool and dry atmosphere. The QSM yield was approximately (7%) on dry weight basis.

2.3. Formulation of QSM/sodium alginate hydrogels

QSM and SA powders were dissolved in distilled water with continuous stirring at 80 °C for 1 h with the addition of glycerol (2 mL) as a plasticizer. The suspension was ultra-sonicated to remove air bubbles. Finally, various additives like nanoclay, ZnO and CaCl₂ were added to the hydrogels of QSM and sodium alginate mixtures at constant concentrations (Table 1). The hydrogels were homogenized at 8000 rpm for 15 min using a high-speed IKA T18 digital Ultra-Turrax.

Table 1 QSM, sodium alginate and NC/ZnO/CaCl₂ film-forming solution compositions

Treatments	Quince seed mucilage (%)	Sodium alginate (%)	Crosslinker concentration (mg/50 mL of hydrogel)
QAH1-NC (QSM + SA + NC)	100	0	0
QAH1-ZnO (QSM + SA + ZnO)	100	0	0
QAH1-CaCl2 (QSM + SA + CaCl2)	100	0	0
QAH2-NC (QSM + SA + NC)	80	20	25
QAH2-ZnO (QSM + SA + ZnO)	80	20	25
QAH2-CaCl2 (QSM + SA + CaCl2)	80	20	25
QAH3-NC (QSM + SA + NC)	60	40	25
QAH3-ZnO (QSM + SA + ZnO)	60	40	25
QAH3-CaCl2 (QSM + SA + CaCl2)	60	40	25
QAH4-NC (QSM + SA + NC)	40	60	25
QAH4-ZnO (QSM + SA + ZnO)	40	60	25
QAH4-CaCl2 (QSM + SA + CaCl2)	40	60	25
QAH5-NC (QSM + SA + NC)	20	80	25
QAH5-ZnO (QSM + SA + ZnO)	20	80	25
QAH5-CaCl2 (QSM + SA + CaCl2)	20	80	25

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2.3.1. Characterization of hydrogels.
2.3.1.1. Rheological studies. Rheological characterization of the composite hydrogels was performed using a rheometer (Anton Paar MCR-101) fitted with a cone and plate geometry (diameter of 25 cm with a cone angle of 1.998 °C) having a Peltier temperature control system with an accuracy of ±0.01 °C in accordance with the procedure of Afzal et al.²⁹ The tests were performed at room temperature. In amplitude sweep tests, determination of the linear viscoelastic (LVE) region of the composite hydrogels was done at a frequency of 1 Hz to perform all the measurements within this range. For the determination of the flow properties and viscoelastic behavior of the composite hydrogels, steady shear tests (r = 0.1–100 s⁻¹) and frequency sweep tests (r = 0.1–100 s⁻¹) were performed at a constant strain (1).
2.3.1.2. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR). Hydrogels were characterized using ATR-FTIR (CARY 630, Agilent Technologies, USA) at room temperature to determine the polymer interactions. An IR spectrum of the samples was obtained in the frequency range of 4000–400 cm⁻¹. The spectral resolution was 4 cm⁻¹ and 32 scans were used. The IR spectrum of air was first determined under the same conditions, which was then automatically deducted from the IR spectra of the samples.
2.3.1.3. Thermal properties (differential scanning calorimeter). Determination of the thermal properties of QSM/sodium alginate hydrogels with crosslinkers was done by using a differential scanning calorimeter (DSC) (DSC-1 STAR System, Mettler-Toledo). A hydrogel sample of 3.5 mg was taken in an aluminum pan, followed by the addition of about 8 μl of deionized water. The aluminum pans carrying the sample were kept at room temperature overnight after sealing them hermetically. The samples were then heated at the rate of 10 °C min⁻¹ in the range of 30 °C to 300 °C. The reference for the determination of the thermal properties was an empty aluminum pan.

2.4. Preparation of the biocomposite films

Preparation of the biocomposite films was done according to the method of Zhang et al.¹⁸ with some modifications. The biocomposite films from different combinations of hydrogels were developed by solution casting method, followed by evaporation of the solvent. A known amount of hydrogel solution was poured into the Petri plates (made of polypropylene) and dried in an oven at 60 °C for 12 h, and then peeled from the plate and conditioned at 50% ± 1% relative humidity (RH) and 25 °C before analysis.

2.4.1. Characterization of the biocomposite films.
2.4.1.1. Film thickness. The biocomposite films were first cut into small strips of size 1 × 6 cm. The thickness was then measured at different points by a handheld micrometre (YAMAYO Instruments).
2.4.1.2. Instrumental color (L*, a* and b*). A Hunter Lab colorimeter (Model SN3001476, Accuracy Micro-sensors, New York) was used to determine the L*, a* and b values of the biocomposite films. After placing the samples on the plate, their exposure at different places was recorded according to the CIE LAB system of color measurement. CIE Lab is a color space where L* indicates lightness, a* represents the red-green axis, and b* represents the yellow–blue axis. This color system allows for quantitative evaluation of color differences in materials.
2.4.1.3. Water vapor transmission rate (WVTR) and WVP (water vapour permeability). The WVTR of the film samples was determined by using a Labthink W3/031 automated water vapor transmission tester and the ASTM (American Society for Testing and Materials) standard method E96-80 procedure. The RH and temperature were set to 90% and 38 °C, respectively. For the experiments, the continuous mode was chosen. Three specimens of each film were measured and the average value was reported. WVP was calculated by the following given equation:
where WVTR is the water vapor transmission rate (g h⁻¹ m⁻²), thickness is the film thickness (mm), and ΔP is the water vapor partial pressure difference across the film (kPa).
2.4.1.4. Oxygen transmission rate (OTR). The OTR of the film samples was determined in accordance with ISO 15105 2/ASTM D3985 standard using an oxygen transmission rate test apparatus (Model, OX2/230; Lab-think Instruments Co., Ltd). Prior to measurements, the samples were conditioned for 24 hours. The diffusion cell was clamped with flat film samples. Then, pure oxygen (99.99%) was supplied into the diffusion cell chamber, and the carrier gas carried oxygen molecules that had permeated through the film to the interior chamber to the color-metric sensor (N₂).
2.4.1.5. Tensile strength (TS). The tensile strength (MPa) and elongation at break film samples was determined at 25 °C with a texture analyser (TA. XT Plus Texture stable microsystem). Specimens were taken in dumbbell shapes for the analysis.
2.4.1.6. Biodegradability test of the biocomposite films. The biodegradability of the films was ascertained by burying the samples under soil and then monitoring the weight loss after regular intervals of time. The films were cut in pieces with a size of 5 × 2 cm. The pieces were then tied with a thread at one corner, followed by burying them about 2–3 cm under the soil surface. The soil was kept humid by sprinkling it with water once daily. The pre-weighed pieces of film were then removed from the soil at intervals of 20, 40, 60 and 90 days. The adhered soil particles were cleaned off by washing the films with distilled water. The samples were then dried at room temperature to constant weight. The equation given below was used for the calculation of weight loss:³⁰
where W_i = initial weight of the specimen and W_d = dry weight of the specimen after degradation in soil.
2.4.1.7. Antimicrobial activity. To investigate the antimicrobial activity of the biocomposite films, two fungal strains Rhizopus stolonifer and Aspergillus niger were used in this study. The antimicrobial activity was examined by disc diffusion assay. The potato dextrose agar medium was prepared and sterilized by autoclaving for 30 min at 121 °C. The media was allowed to solidify after being transferred into the Petri plates. Fungal inoculation was carried out using a spore suspension prepared in sterile distilled water, adjusted to a concentration of approximately 1 × 10⁵ CFU mL⁻¹. The test microorganisms were then swabbed on the surface of the solidified media plates. The biocomposite film samples were punched to make discs (7 mm in diameter). The discs were inoculated on the Petri plates, which were then incubated at 37 °C for 48 h. After incubation, the diameter of microbial inhibition was measured in mm scale.³¹

2.5. Statistical analysis

One-way and two-way analysis of variance (ANOVA) was employed using SPSS software at 5% level of significance. The difference between means was determined using Duncan's multiple range test (DMRT). The data are presented as an average of triplicates.

3. Results and discussion

3.1. Characterization of hydrogels

3.1.1. Rheological studies. Frequency sweep tests of the hydrogels were performed between the angular frequency of 0.1 to 100 sec⁻¹ (Fig. 1). The hydrogels exhibited significant variation in viscoelastic behaviour by changing the proportion of QSM and sodium alginate with constant concentrations of crosslinker agents (ZnO, CaCl₂ and NC). The storage modulus (G′) was higher than the loss modulus (G′′) (G′ > G′′) in hydrogels with CaCl₂ as the crosslinking agent, denoting a more elastic nature, followed by hydrogels with NC and ZnO as crosslinkers when a load was applied. Thus, the results indicated a more elastic response and solid-like behaviour in hydrogels with CaCl₂. This could be attributed to the stronger ionic interaction of the Ca²⁺ ions with polymer reactive groups or backbone, and consequently a stronger hydrogel.³² A higher storage modulus (G′) indicates a solid-like or elastic behaviour of hydrogels, which in turn indicates higher strength and mechanical rigidity.

Fig. 1 (a–e) Frequency sweep test of QSM/sodium alginate hydrogels with different QSM : SA ratios (a) QAH1 (100 : 0), (b) QAH2 (80 : 20), (c) QAH3 (60 : 40), (d) QAH4 (40 : 60), (e) QAH5 (20 : 80), each crosslinked with CaCl₂, NC and ZnO.

As can be observed from the results, hydrogels with an 80% concentration of sodium alginate and 20% QSM with ZnO, CaCl₂ and NC as crosslinkers exhibited a lower storage modulus (G′). However, increasing the concentration of QSM up to 80% and decreasing alginate resulted in a higher storage modulus (G′), indicating the dominance of the elastic behaviour on application of the load. Thus, the storage modulus (G′) improves more quickly than the loss modulus (G′′) when the concentration of QSM is increased and alginate is decreased, indicating that the hydrogel phase has converted into a solid-like gel with supreme elastic behaviour. This might be due to a greater amount of three-dimensional cross-linked networks of hydrogels.³³ QSM at 80% concentration exhibited a higher cross-linking level. This could be attributed to the availability of more carboxylate groups for crosslinking with Ca²⁺, resulting in strong hydrogen-bond interactions and greater stabilization of the calcium-induced gelation than at higher alginate concentration.⁷ This improves the mechanical and viscoelastic gelation behaviour. Similar results were reported by Cuomo et al.,³⁴ who found that the storage modulus (G′) showed a certain dependence in hydrogels with higher value at low alginate content in the hydrogels.

The apparent viscosity curves of the QSM-alginate hydrogels incorporated with ZnO, CaCl₂ and NC as crosslinking agents are shown in Fig. 2. The steady-state flow of the QSM-alginate hydrogels demonstrate that all of the hydrogels showed shear rate ([small gamma, Greek, dot above]) dependence and shear thinning behaviour. Thus, all hydrogels exhibited low viscosity as the shear rate increased from 0 to 100 1 S⁻¹. It was observed that the hydrogels immediately recovered into solid-like materials as the shear rate approached 0. Depending on the type of crosslinker agent, results showed that hydrogels with added CaCl₂ exhibited higher viscosity due to strong gelation. The shear rate of these hydrogels approached zero, indicating that the gel does not flow at rest. Moreover, hydrogels with NC as the crosslinker agent exhibited stronger gelation than hydrogels with ZnO, as revealed from the steady-state flow behaviour. Thus, the data indicate that hydrogels with CaCl₂ as the crosslinker constitutes the strong gel network.⁷ Hydrogels with 80% concentration of QSM and 20% sodium alginate added with ZnO, CaCl₂ and NC as crosslinking agents showed higher viscosity values as the shear rate increases; thus, exhibiting stronger gelation followed by hydrogels formulated from pure QSM, which was independent of the specific shear rate and duration. Furthermore, the increase in the concentration of alginate from 20% to 80% showed lower viscosity values, resulting in weaker gelation. Thus, the results concluded that hydrogels with a higher concentration of sodium alginate have more shear thinning properties compared to hydrogels with QSM. While comparing the crosslinking agents used, ZnO exhibited more shear thinning properties, followed by NC and CaCl₂. The notable shear-thinning behavior of the hydrogels suggests excellent processability for film production, as viscosity decreases under applied stress during casting or extrusion, enabling smoother, more uniform film formation while minimizing energy input. Additionally, a higher elastic modulus within the linear viscoelastic region supports structural integrity during drying and handling, which is critical for the scalable manufacturing of biodegradable films.

Fig. 2 Apparent viscosity curves of the QSM/alginate hydrogels with different QSM : SA ratios (a) QAH1 (100 : 0), (b) QAH2 (80 : 20), (c) QAH3 (60 : 40), (d) QAH4 (40 : 60), (e) QAH5 (20 : 80), with constant concentration of CaCl₂, NC and ZnO as crosslinker agents.

3.1.2. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR). ATR-FTIR spectroscopy was used to characterize hydrogels with varying concentrations of QSM and sodium alginate and with ZnO, CaCl₂ and NC as crosslinking agents, and Fig. 3 shows the spectral differences acquired from 4000 to 400 cm⁻¹. The FTIR spectra of hydrogels crosslinked with ZnO revealed spectroscopic bands at 1554–1630 cm⁻¹, hydrogels crosslinked with CaCl₂ at 1559–1655 cm⁻¹ and hydrogels crosslinked with NC at 1555–1651 cm⁻¹, which were related to the asymmetric and symmetric axial deformations of the –COO⁻ groups.³⁵ The strong bands of hydrogels with CaCl₂ as crosslinker agents could be beneficial to improve the physical performance of biopolymer films. The shift in peaks to higher wavenumbers upon the addition of crosslinking agents might be attributed to the bonding between –COO⁻ groups and crosslinkers ZnO, CaCl₂ and NC, which forms a three-dimensional crosslinked network of hydrogels. Loria et al.³⁶ also found a shift of peaks (1621 and 1414 cm⁻¹) to higher wavenumbers on interaction of ions (Ca²⁺) with anionic groups (COO⁻). A similar shift was observed in the present study, where the majority of –COO⁻ anionic groups were crosslinked at lower concentrations of sodium alginate with the constant concentration of crosslinker agents (25 mg/50 mL of hydrogel). The FTIR spectra revealed a wide band between 3288 and 3345 cm⁻¹ in QSM hydrogels crosslinked with ZnO, 3308 and 3680 cm⁻¹ in those crosslinked with CaCl₂, and 3306 and 3500 cm⁻¹ in those crosslinked with NC, corresponding to the stretching of the –OH groups present in the QSM and sodium alginate polymer chains.³⁷

Fig. 3 FTIR spectra of the QSM/alginate hydrogels with constant concentration of ZnO, CaCl₂ and NC as crosslinker agents, with different QSM : SA ratios (a) QAH1-5 cross-linked with ZnO (b) QAH1-5 cross-linked with CaCl₂ (c) QAH1-5 cross-linked with NC.

QSM hydrogels containing NC as a crosslinker showed a shift of peaks to higher wavenumbers, which might be due to the elongation of the –OH groups. The change was confirmed by the slight upward shift of the band.³⁸ The strong absorption bands were observed at 1106–1400 cm⁻¹ for hydrogels with ZnO as the crosslinker agent, 1124–1401 cm⁻¹ with NC as the crosslinker agent, and 1138 to 1407 cm⁻¹ for hydrogels crosslinked with CaCl₂, corresponding to the C–O–C and C–O stretching vibrations.³⁹ Thus, the ATR-FTIR spectra confirmed that the CaCl₂ crosslinker agent, followed by NC, had good biocompatibility in QSM and sodium alginate films.

3.1.3. Thermal properties (DSC). The thermal properties of QSM-sodium alginate hydrogels incorporated with ZnO, CaCl₂ and NC were determined to find out the thermal energy required to break the chemical bonds present in the hydrogels (Table 3). The hydrogels containing ZnO as a crosslinking agent exhibited exothermic peaks at 244.01 °C (QAH1-ZnO), 245.65 °C (QAH2-ZnO), 241.55 °C (QAH3-ZnO), 240.66 °C (QAH4-ZnO), and 240.33 °C (QAH5-ZnO). Before these temperatures are attained, an increase in temperature results in an absorption of heat by the hydrogels. After these temperatures are attained, the temperature increase breaks the bonds present in the gel, resulting in disintegration of the gel structure.^40,41 From the heat flow curves of hydrogels containing ZnO as the crosslinking agent, it was observed that a higher exothermic temperature was exhibited by QAH2-ZnO and the lowest temperature was seen with QAH5-ZnO treatment. When QSM-sodium alginate hydrogels with a constant concentration of CaCl₂ as the crosslinker absorbed the heat, exothermic peaks were observed at 246.32 °C (QAH1-CaCl₂), 248.22 °C (QAH2-CaCl₂), 245.22 °C (QAH3-CaCl₂), 244.21 °C (QAH4-CaCl₂), and 243.17 °C (QAH5-CaCl₂). From the heat flow curves, it was observed that a higher exothermic temperature was exhibited by QAH2-CaCl₂ and the lowest temperature was seen with QAH5-CaCl₂ treatment. When QSM-sodium alginate hydrogels with a constant concentration of NC as the crosslinker agent absorbed the heat, exothermic peaks were observed at 243.12 °C (QAH1-NC), 244.33 °C (QAH2-NC), 243.07 °C (QAH3-NC), 242.78 °C (QAH4-NC), and 241.03 °C (QAH5-NC). From the heat flow curves, it was observed that a higher exothermic temperature was exhibited by QAH2-NC and the lowest temperature was seen with QAH5-NC treatment. Overall, the results showed that higher exothermic peaks were observed in hydrogels with CaCl₂ as the crosslinker agent. This could be attributed to the strong interactions between the Ca²⁺ ions of CaCl₂ and the polymer matrices in the hydrogels. However, in comparing the different biopolymer combinations, hydrogels with an 80% : 20% concentration of QSM and sodium alginate exhibited higher exothermic peaks, while hydrogels with a 20% : 80% concentration of QSM and sodium alginate showed lower peaks. This might be attributed to the increased entanglement of polymer chains and crosslinking, leading to increased hydrophobic interactions and thus resulting in higher exothermic peaks.³⁸ The thermal transitions observed by DSC are critical for defining the processing conditions and final film performance. Higher degradation onset temperatures ensure thermal stability during film casting, drying, and application in food packaging. These thermal properties therefore support scalable manufacturing, and ensure that the developed biodegradable films maintain their mechanical and barrier functions under typical handling and storage conditions.

3.2. Characterization of the biocomposite films

3.2.1. Film thickness. The thickness of QSM and sodium alginate-based biocomposite films with added ZnO, CaCl₂ and NC as crosslinking agents is presented in Table 2. The thickness of the biocomposite films varied from 110.38 to 114.32 μm. It was observed that an increase in the sodium alginate concentration increased the thickness of the film. Significantly, the highest thickness was observed in biocomposite films with 20% : 80% concentration of QSM and sodium alginate (P ≤ 0.05). The biocomposite films with higher QSM and lower alginate concentration exhibited lower value of thickness. This lower value of thickness might be due to the greater amount of three-dimensional cross-linked networks in the biocomposite films associated with the compact structure of the films.⁶ Similar variations in film thickness based on biopolymer difference were observed by earlier research studies.^42,43 The type of crosslinker agent used showed a non-significant variation in the thickness of the biocomposite films among various treatments (P ≥ 0.05).

Table 2 Thickness, TS, WVTR, WVP, EB and OTR of QSM/sodium alginate biocomposite films with NC/ZnO/CaCl₂ as crosslinker agents^a,b

Treatments	Thickness (μm)	Tensile strength (MPa)	Elongation at break (%)	WVTR (g per m^2 per day)	WVP g m per m^2 per day per Pa	OTR (cm^3 per m^2 per day)
a All values are mean ± SD of three replicates. b Different letters in the same column indicate significant differences (p < 0.05).
QAH1-NC	110.85 ± 0.23^a	33.37 ± 0.43^e	37.35 ± 0.32^d	306.17 ± 1.09^c	6.04 × 10^−4 ± 0.19^a	1017.10 ± 2.23^c
QAH1-ZnO	110.33 ± 0.19^a	32.80 ± 0.27^d	35.26 ± 0.23^cd	335.11 ± 1.23^f	6.58 × 10^−4 ± 0.23^c	1028.66 ± 1.88^d
QAH1-CaCl2	110.22 ± 0.41^a	34.55 ± 0.33^f	40.12 ± 0.36^f	301.48 ± 1.33^b	5.92 × 10^−4 ± 0.37^a	1009.11 ± 1.65^b
QAH2-NC	110.56 ± 0.33^a	34.83 ± 0.27^f	42.23 ± 0.41^f	306.14 ± 0.98^c	6.03 × 10^−4 ± 0.16^a	1016.25 ± 2.34^c
QAH2-ZnO	110.54 ± 0.28^a	32.95 ± 0.21^d	36.12 ± 0.29^d	335.55 ± 1.22^f	6.60 × 10^−4 ± 0.28^c	1029.70 ± 2.45^d
QAH2-CaCl2	110.60 ± 0.44^a	36.12 ± 0.39^g	44.05 ± 0.13^g	299.72 ± 1.11^a	5.90 × 10^−4 ± 0.29^a	1003.22 ± 2.01^a
QAH3-NC	110.38 ± 0.31^a	30.22 ± 0.58^b	34.26 ± 0.54^c	306.67 ± 1.14^c	6.02 × 10^−4 ± 0.41^a	1042.26 ± 1.76^ef
QAH3-ZnO	110.41 ± 0.26^a	28.45 ± 0.36^a	38.02 ± 0.28^e	336.47 ± f0.86^g	6.61 × 10^−4 ± 0.35^c	1049.77 ± 2.09^h
QAH3-CaCl2	111.44 ± 0.18^ab	32.12 ± 0.41^c	41.11 ± 0.62	303.50 ± 1.29^b	6.02 × 10^−4 ± 0.32^a	1031.33 ± 2.20^d
QAH4-NC	111.15 ± 0.45^ab	29.40 ± 0.85^b	30.07 ± 0.36^b	308.45 ± 0.96^cd	6.11 × 10^−4 ± 0.15^ab	1045.23 ± 2.04^g
QAH4-ZnO	112.22 ± 0.52^ab	27.44 ± 0.33^a	28.62 ± 0.38^a	337.12 ± 1.22^g	6.74 × 10^−4 ± 0.07^d	1049.88 ± 1.67^h
QAH4-CaCl2	113.05 ± 0.33^ab	31.39 ± 0.57^bc	33.25 ± 0.45^c	303.41 ± 0.83^b	6.12 × 10^−4 ± 0.11^ab	1038.33 ± 2.04^e
QAH5-NC	113.35 ± 0.22^c	30.31 ± 0.76^b	28.03 ± 0.11^a	314.55 ± 1.11^e	6.34 × 10^−4 ± 0.34^b	1058.25 ± 2.22^i
QAH5-ZnO	113.33 ± 0.46^c	27.02 ± 0.29^a	29.12 ± 0.26^a	338.49 ± 1.09^g	6.82 × 10^−4 ± 0.18^e	1069.33 ± 2.41^j
QAH5-CaCl2	114.32 ± 0.42^c	32.04 ± 0.24^c	34.17 ± 0.39^c	309.45 ± 0.93^d	6.30 × 10^−4 ± 0.06^b	1044.22 ± 1.87^g

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3.2.2. Tensile strength (TS) and elongation break of biocomposite films. The tensile strength (TS) and elongation at break (EB) of QSM and sodium alginate-based biocomposite films are presented in Table 2. Significant difference (P ≤ 0.05) was observed in the tensile strength and elongation at break, depending on the type of crosslinking agent and the QSM : sodium alginate ratio. Films crosslinked with CaCl₂ exhibited the highest TS and elongation at break values. The interfacial interaction among Ca²⁺ ions and polymer matrices is the main reason for the higher tensile strength of the biocomposite films. Moreover, the Ca²⁺ ions of CaCl₂ are positively charged, and are involved in electrostatic interaction with the negatively charged groups of the polymer matrix. This interaction could also contribute to the increased tensile strength.⁴⁴ NC-crosslinked films showed moderate TS and EB. This is likely due to interfacial reinforcement and the layered dispersion of nanoclay within the matrix, which promotes stress distribution while maintaining structural cohesion.⁴⁵ In contrast, ZnO-crosslinked films exhibited lower TS and the least elongation. This is possibly due to their rigid, aggregated structures restricting polymer chain mobility. Among the different biopolymer combinations, films prepared with a QSM : sodium alginate ratio of 80% : 20% demonstrated superior mechanical properties, achieving the highest TS and EB, followed by 100% QSM, 60% : 40%, 40% : 60% and 20% : 80%. The mechanical properties of the film depend on the microstructure and intermolecular forces of the polymer matrix. The variation in TS and EB with increasing concentration of sodium alginate in the biopolymer combination might be due to the weak intermolecular interactions, leading to structural discontinuity.⁴⁶ The higher TS and EB of pure QSM films than those of QSM films with a higher concentration of sodium alginate could be due to the compact structure and development of strong hydrogen bonds within the polymer matrix of pure QSM.

3.2.3. Barrier properties (WVTR and OTR) of biocomposite films. The barrier properties are considered an important parameter of films for application in food products. Table 2 shows the effect of crosslinking agents on WVTR of biocomposite films prepared with varying concentration of QSM and sodium alginate. Biocomposite films with CaCl₂ as the crosslinker exhibited significantly lower WVTR, while films with ZnO as the crosslinker exhibited higher WVTR (P ≤ 0.05). Similarly, the OTR was significantly lowest in films with CaCl₂ as the crosslinker and highest in films with ZnO as the crosslinker (P ≤ 0.05). This might be attributed to the strong interactions among Ca²⁺ ions of CaCl₂ and polymer matrices in the films. After adding CaCl₂ to the biocomposite suspension, a dense microstructure might be formed, which enhanced the barrier properties of the developed biocomposite films. Furthermore, the addition of CaCl₂ to the biopolymer leads to the formation of egg-box like structures, which results in a zigzag path for water vapour and oxygen movement through the biocomposite film, thus reducing their diffusion.⁴⁷ Similarly, films crosslinked with CaCl₂ exhibited significantly lower water vapour permeability (WVP) values (e.g., 5.92 × 10⁻⁴ to 6.12 × 10⁻⁴ g mm m⁻² h⁻¹ Pa⁻¹), suggesting improved moisture barrier properties. Conversely, ZnO-crosslinked films demonstrated higher WVP (e.g., 6.58 × 10⁻⁴ to 6.82 × 10⁻⁴ g mm m⁻² h⁻¹ Pa⁻¹), consistent with the WVTR and OTR trends. These results suggest that stronger crosslinking leads to a tighter polymer matrix, restricting moisture transmission. However, the different biopolymer combinations films with 80% : 20% concentrations of QSM and sodium alginate exhibited lower WVTR and OTR, followed by 100% QSM, 60% : 40%, 40% : 60% and 20% : 80%. The reason might be the hydrogen bonding and electrostatic interactions taking place due to the dense network arrangement within the polymer matrix, which enhances the water vapour and oxygen barrier capacity of the films. The increased concentration of sodium alginate caused discontinuity in the network structure, thus weakening the intermolecular interactions, which further results in an increase in the pore size, leading to an opportunity for water vapour and oxygen to diffuse though the films.⁴⁶ Similar trends in water vapor permeability (WVP) were also reported by Karambasti and Shavisi,⁵⁸ who developed guar gum–pectin nanofiber mats. Likewise, Heydarian and Shavisi⁵⁹ observed comparable WVP behavior in electrospun gelatin–xanthan gum mats containing chitin nanofibers and anthocyanins.

3.2.4. Biodegradability (%) of biocomposite films. The ability of biodegradable polymers to decompose by the action of microorganisms is their most significant advantage over petroleum-based plastics. According to Kochkina, and Lukin,⁴⁸ a bioplastic is considered to be biodegradable if it is degraded by 90% in 90 days. Table 4 depicts the influence of crosslinker agents and various combinations of QSM and sodium alginate on the biodegradability of biocomposite films using the soil burial test for 90 days. All treatments were almost destroyed at the 90th day of testing. However, the biocomposite films incorporated with NC as the crosslinker agent showed significantly less biodegradation compared to the other treatments after 90 days (P ≤ 0.05). The water solubility of the biofilms is an important criterion for biodegradability because the penetration of soil moisture content in the polymer chains of biofilm weakens them and renders them susceptible to the action of microorganisms. The soil microorganisms hydrolyse the glycosidic linkages of the polymer, leading to its degradation.^30,49 Thus, on the basis of NC as an inorganic material that enhances the mechanical strength, the films incorporated with NC were stronger than the films with CaCl₂ and ZnO as crosslinking agents, which might have delayed the degradation rate. Shekarabi et al.⁵⁰ demonstrated that the incorporation of NC decreased the water solubility of QSM films. Slavutsky et al.⁵¹ reported that films prepared by using 5% clay and starch exhibited a decrease in water solubility compared to the control without clay. The authors illustrated that this decrease could be due the interaction of clay with starch chains in the nanocomposite films. Ojagh et al.⁴³ observed similar results for methylcellulose films. Comparing the biofilms with different biopolymer combinations, films with 80% : 20% concentrations of QSM and sodium alginate exhibited lower biodegradability, followed by 100% QSM, 60 : 40%, 40 : 60% and 20 : 80% QSM and sodium alginate. The reason might be due to the strong intermolecular interactions and the enhanced mechanical properties of the biocomposite films.⁵²

3.2.5. Instrumental color (L*, a*, and b*) of biocomposite films. The results of the color parameters (L*, a* and b*) of the biocomposite films are presented in Table 3. The films were homogeneous, uniform and transparent with a slight whitish tint. Biocomposite films with NC as the crosslinking agent exhibited significantly higher L* and b* values and lower a* value (P ≤ 0.05). However, the color parameters among films prepared with CaCl₂ and ZnO as the crosslinking agents exhibited non-significant variation (P ≥ 0.05). The variation in L* and b* values of biofilms with NC as the crosslinker might be due to the increase in the reflection of light and subsequent decrease in the passage of light through films.⁵³ Abdollahi et al.⁵⁴ reported similar results of bionanocomposite films containing NC. Comparing the biocomposite films with different biopolymer combinations, a non-significant variation was observed among all of the color parameters (P ≥ 0.05).

Table 3 DSC data, instrumental color and antimicrobial activity of QSM/sodium alginate biocomposite films with NC/ZnO/CaCl₂ as crosslinker agents^a,b

Treatments	DSC data		Instrumental color			Antimicrobial activity
	Endothermic peak °C	Exothermic peak °C	L*	a*	b*	Inhibition zone-diameter (mm) (Aspergillus niger)	Inhibition zone-diameter (mm) (Rhizopus stolonifer)
a All values are mean ± SD of three replicates. b Means with different superscripts (lowercase) in the same row differ significantly (p < 0.05).
QAH1-NC	127.22 ± 2.30^b	243.12 ± 1.54^c	88.51 ± 1.23^c	−0.59 ± 0.07^a	5.99 ± 0.10^b	13.16 ± 0.03^a	10.04 ± 0.04^a
QAH1-ZnO	126.05 ± 1.09^ab	244.01 ± 1.37^cd	86.44 ± 0.98^a	−0.53 ± 0.06^b	4.32 ± 0.14^a	15.29 ± 0.04^b	12.32 ± 0.03^b
QAH1-CaCl2	137.17 ± 1.13^c	246.32 ± 2.01^d	86.32 ± 1.11^a	−0.52 ± 0.03^b	4.98 ± 0.11^a	13.18 ± 0.04^a	10.34 ± 0.07^a
QAH2-NC	127.55 ± 2.02^b	244.33 ± 1.69^cd	88.89 ± 0.87^c	−0.58 ± 0.03^a	6.03 ± 0.17^b	13.21 ± 0.07^a	10.56 ± 0.03^a
QAH2-ZnO	126.32 ± 1.87^ab	245.65 ± 1.11^d	87.81 ± 1.14^ab	−0.51 ± 0.07^b	4.33 ± 0.15^a	15.83 ± 0.05^b	12.61 ± 0.04^b
QAH2-CaCl2	137.33 ± 1.19^c	248.22 ± 2.20^e	87.80 ± 1.10^ab	−0.55 ± 0.08^b	4.12 ± 0.14^a	13.44 ± 0.03^a	10.65 ± 0.07^a
QAH3-NC	126.22 ± 1.54^ab	243.07 ± 2.03^c	88.22 ± 1.23^c	−0.58 ± 0.03^a	5.93 ± 0.09^b	13.55 ± 0.07^a	10.57 ± 0.07^a
QAH3-ZnO	126.07 ± 1.33^ab	241.55 ± 1.47^a	87.25 ± 1.09^ab	−0.53 ± 0.07^b	4.21 ± 0.11^a	15.82 ± 0.08^b	12.67 ± 0.04^b
QAH3-CaCl2	137.22 ± 1.07^c	245.22 ± 1.44^d	87.13 ± 0.97^ab	−0.52 ± 0.10^b	4.22 ± 0.17^a	13.24 ± 0.05^a	10.55 ± 0.07^a
QAH4-NC	127.65 ± 2.01^b	242.78 ± 1.86^ab	88.42 ± 0.87^c	−0.57 ± 0.09^a	6.20 ± 0.13^b	15.83 ± 0.07^a	10.60 ± 0.06^a
QAH4-ZnO	124.33 ± 1.25^a	240.66 ± 2.01^a	87.11 ± 1.11^ab	−0.50 ± 0.07^b	4.77 ± 0.07^a	15.88 ± 0.08^b	12.62 ± 0.02^b
QAH4-CaCl2	137.01 ± 1.44^c	244.21 ± 2.12^cd	87.33 ± 1.11^ab	−0.51 ± 0.04^b	4.66 ± 0.15^a	13.03 ± 0.04^a	10.44 ± 0.07^a
QAH5-NC	125.33 ± 1.83^a	241.03 ± 1.34^a	88.33 ± 0.86^c	−0.60 ± 0.02^a	5.97 ± 0.09^b	13.87 ± 0.07^a	10.63 ± 0.04^a
QAH5-ZnO	124.03 ± 1.79^a	240.33 ± 1.88^a	87.42 ± 1.14^ab	−0.51 ± 0.06^b	4.32 ± 0.16^a	15.84 ± 0.08^b	12.65 ± 0.03^b
QAH5-CaCl2	137.11 ± 1.46^c	243.17 ± 1.62^c	87.32 ± 1.10^ab	−0.52 ± 0.09^b	4.33 ± 0.11^a	13.85 ± 0.02^a	10.66 ± 0.04^a

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Table 4 Biodegradability (%) of QSM/sodium alginate biocomposite films with NC/ZnO/CaCl₂ as crosslinker agents^a,b

Treatments	Biodegradability (%)
	20th day	40th day	60th day	90th day
a All values are mean ± SD of three replicates. b Means in the same column with different superscripts (uppercase) differ significantly, and the means with different superscripts (lowercase) in the same row differ significantly (p < 0.05).
QAH1-NC	29.85 ± 0.21^aA	60.12 ± 0.54^aB	80.22 ± 0.83^aC	91.23 ± 1.12^aD
QAH1-ZnO	33.30 ± 0.37^cA	64.66 ± 0.66^bBc	84.32 ± 0.67^cC	94.42 ± 1.10^bcD
QAH1-CaCl2	33.44 ± 0.19^cA	63.93 ± 0.51^bB	83.22 ± 0.92^bC	93.22 ± 0.96^bD
QAH2-NC	31.12 ± 0.27^bA	60.23 ± 0.46^aB	80.12 ± 0.76^aC	91.34 ± 0.87^aD
QAH2-ZnO	32.16 ± 0.31^cA	64.33 ± 0.39^bcB	84.54 ± 0.88^cC	94.82 ± 1.24^bcD
QAH2-CaCl2	33.30 ± 0.41^cA	63.54 ± 0.47^bB	83.22 ± 0.93^bC	93.08 ± 1.11^bD
QAH3-NC	31.29 ± 0.21^bA	60.46 ± 0.56^aB	80.37 ± 0.57^aC	91.55 ± 0.98^aD
QAH3-ZnO	34.47 ± 0.40^dA	66.97 ± 0.44^eB	86.11 ± 1.01^dC	95.33 ± 0.75^dD
QAH3-CaCl2	33.54 ± 0.18^cA	65.22 ± 0.37^dB	84.10 ± 0.67^cC	93.78 ± 0.10^bcD
QAH4-NC	31.62 ± 0.33^bA	61.14 ± 0.52^bB	80.53 ± 0.49^aC	92.10 ± 1.16^abD
QAH4-ZnO	32.64 ± 0.22^cA	67.45 ± 0.66^eB	87.22 ± 0.71^eC	96.11 ± 0.76^eD
QAH4-CaCl2	33.22 ± 0.40^cA	66.30 ± 0.71^eB	84.33 ± 0.83^cC	95.22 ± 0.77^dD
QAH5-NC	32.10 ± 0.16^bcA	61.22 ± 0.53^bB	80.88 ± 0.77^aC	92.62 ± 0.86^bD
QAH5-ZnO	35.22 ± 0.22^eA	71.12 ± 0.47^gB	89.07 ± 0.93^fC	97.32 ± 1.04^fD
QAH5-CaCl2	34.77 ± 0.31^dA	68.77 ± 0.63^fB	86.98 ± 0.87^eC	96.23 ± 1.01^eD

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3.3. Antimicrobial activity of biocomposite films

Evaluation of the antimicrobial efficacy of the developed QSM and sodium alginate films with added ZnO, CaCl₂ and NC as crosslinking agents was assessed against Aspergillus niger and Rhizopus stolonifer (Table 3). The diameter of the inhibition zone indicated the inhibitory activity of the films. Biocomposite films incorporated with ZnO clearly showed a greater inhibitory effect against Aspergillus niger and Rhizopus stolonifer in comparison to films containing CaCl₂ and NC (P ≤ 0.05). The exact mechanism of antimicrobial properties of ZnO is still not known. However, several authors have proposed that the release of Zn²⁺ ions, generation of reactive oxygen species (ROS) and physical interactions with the microorganisms are possible mechanisms in the inhibition of bacterial and fungal growth.⁵⁵ Earlier research studies also reported the antimicrobial properties of ZnO in packaging films.^56,57

4. Conclusion

A novel biocomposite film was successfully developed based on QSM and sodium alginate, crosslinked with CaCl₂, ZnO, and NC. The films were characterized in terms of the thickness, tensile strength, WVTR, OTR, biodegradability, color (L*, a*, b*), and antimicrobial activity, while the hydrogels were examined with rheology, ATR-FTIR, and DSC. Hydrogels with 80 : 20 QSM : sodium alginate crosslinked using CaCl₂ possessed better rheological properties, resulting in greater elastic behavior and higher gel network strength, followed by ZnO and NC. This was due to the three-dimensional crosslinked network resulting from hydrogen bonding and electrostatic interactions in the polymer matrices. Biocomposite films with a 20% : 80% ratio of QSM : sodium alginate exhibited the maximum film thickness, reflecting the enhanced film-forming ability. Meanwhile, the CaCl₂-crosslinked films exhibited much greater tensile strength, reflecting greater mechanical stability for future application in food protection. CaCl₂-crosslinked films also exhibited the minimum water vapor transmission rate (WVTR) and oxygen transmission rate (OTR), enhancing the barrier properties. NC-crosslinked films exhibited lower biodegradation in the 90-day soil burial testing compared to the other samples. ZnO-incorporated films had the maximum antimicrobial activity, making them suitable for active packaging. Overall, our findings determine that an 80% : 20% ratio of QSM to sodium alginate in biocomposite films exhibits strong potential to be applied as a food packaging material to extend the shelf life of perishable food products.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data used are provided in the manuscript.

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