Development of Bioactive Carboxymethyl Cellulose-Based Films via Dual Crosslinking with Citric Acid and X-Ray Irradiation

Abstract

This study developed biodegradable carboxymethyl cellulose (CMC) films crosslinked with citric acid (CA) and X-ray irradiation as sustainable packaging alternatives to reduce plastic use. CMC/CA films were subjected to three doses of X-ray irradiation at two energy levels. CMC/CA films exposed to 10 kGy at 350 kV exhibited a significant three-fold reduction in water solubility compared to non-irradiated films, while also lowering water vapor and oxygen permeability without affecting mechanical strength (p ≤ 0.05). FTIR analysis confirmed the esterification between CMC and CA, which reduced the film hydrophilicity. Onion peel extract (OPE) was added as a bioactive compound to provide antifungal properties. Release studies showed reduced OPE diffusion in irradiated films, with lower release rate constant (k_kp) values. The in situ test on cheese inoculated with Penicillium commune showed that the irradiated bioactive films prolonged shelf life, reducing fungal counts to log 2.3 CFU/g after 18 days compared to log 5.7 CFU/g in control samples. Cheese wrapped with irradiated bioactive films had weight loss from 1.05 to 9.37%, whereas uncovered samples exhibited the highest weight loss (2.07 to 15.07%). Overall, irradiation-assisted crosslinking and OPE incorporation improved film functionality, offering a sustainable and effective packaging solution for cheese preservation within a circular economy framework.

1. Introduction

Fossil fuel-based single-use plastics, derived from petrochemicals, are discarded after a single use. Only 9% of used plastics are recycled, whilst the rest is incinerated (19%) or ends up in landfills or the environment (72%) [1]. The widespread use of plastic in packaging, bags, and bottles results in millions of tons of non-degradable waste daily. For instance, global plastic manufacturing reached 458 million metric tons in 2022 [2]. This problem, combined with global waste production, presents a significant challenge.

The conversion from the plastic-linear economy to renewable circular approaches includes exploring biodegradable polymers to replace conventional plastics in the food packaging sector. Several biopolymers, including cellulose, chitosan, pectin, starch, alginate, and agar, are commonly used to create bio-based packaging and films [3]. Carboxymethyl-cellulose (CMC) is an attractive cellulose derivative in sustainable food packaging due to its biodegradability, cost-effectiveness, non-toxicity, and film-forming capabilities. CMC has a high degree of hydroxyl group (-OH) substitution and cross-linking potential for film packaging production [4,5]. CMC is also widely utilized as a thickener and stabilizer in food, pharmaceutical, cosmetic, and dye formulations [6]. However, its inherent high water solubility presents a limitation for certain applications [7]. To overcome this, researchers have employed strategies to enhance its water resistance, among which nanofiller addition and chemical or physical crosslinking are particularly effective [8].

The incorporation of nanofillers is a promising strategy to enhance the mechanical and barrier properties of biopolymer-based films due to their high surface area and interfacial interactions [9]. Cellulose nanocrystals (CNCs), rod-like nanoparticles derived from acid hydrolysis of lignocellulosic biomass, exhibit high crystallinity, aspect ratio, and surface functionality [10]. Their integration into polymer matrices facilitates strong hydrogen bonding, reinforcing the polymer network, while their nanoscale dispersion introduces a tortuous diffusion path that restricts water vapor permeability [11]. Citric acid (CA), a non-toxic tricarboxylic acid, serves as a green crosslinker by esterifying hydroxyl groups in polymers like CMC, forming a three-dimensional network that enhances structural integrity and water resistance [12,13]. CA and its salts are allowed as food additives by the EU, US, and WHO; they are considered Generally Recognized As Safe (GRAS) by the FDA (21 CFR 184) and are classified as Group I food additives by the EU (Commission Regulation 1129/2011) for use in food preservation [14].

Irradiation is considered a physical treatment that could also induce crosslinking. This treatment can activate reactive sites within the polymer structure, thereby enhancing the functional performance of biopolymers [15,16]. X-ray irradiation can enhance the crosslinking reactions between CA and CMC, leading to the improved structural and functional properties of the resultant films [17]. In this method, irradiation must be performed under oxygen-free conditions, as the irradiation of biopolymers generates free radicals that can react with oxygen to form peroxide radicals, promoting chain scission and hindering the crosslinking process [16].

Valorizing food waste to develop active packaging systems provides a sustainable approach by transforming food by-products into bioactive ingredients. These compounds, when integrated into packaging materials, can actively regulate the internal environment of the package, contributing to extended shelf life and improved food quality [18]. Agricultural waste valorization converts food waste into high-value products through the extraction of bioactive compounds, including antioxidants, polyphenols, and dietary fibers [19]. These bioactive substances offer numerous advantages, such as their natural origin, biodegradability, and broad applicability across the food or pharmaceutical industries due to their antimicrobial and antioxidant properties [20]. By transforming food waste into functional ingredients, this approach minimizes the environmental impact, which will contribute to achieving the zero-waste concept and meet the growing consumers’ interests for eco-friendly ingredients and safer food products. Among various food wastes, onion peels contain a rich source of polyphenolic compounds with significant antioxidant and antimicrobial effects [21]. Peels and skins make up the majority of the 37% waste produced during onion processing [22]. In Europe, countries like Spain, the Netherlands, and the United Kingdom generate up to 500,000 tons of onion waste annually [23]. To exploit the bioactivity of onion peel (OP), they have been incorporated into various forms, including extracts, powders, and microencapsulated products, showing preservative functions, reducing food spoilage, and enhancing food quality in packaging [24].

The global dairy industry is expected to continue expanding over the coming decade, with volumes projected to reach approximately 14.2 million tons by 2031, representing a 15% increase compared with the base period. Within this sector, the cheese industry is anticipated to grow steadily at an average annual rate of about 1.6% [25]. Europe remains the dominant cheese market, accounting for more than 60% of global consumption, while other major consuming regions include North America, the Asia-Pacific region, and Latin America. The global cheese market is characterized by remarkable diversity, with over 1000 varieties currently available. These cheeses differ widely in sensory and physical attributes, such as flavor, aroma, texture, color, appearance, yield, and nutritional composition [26]. Owing to this diversity and broad consumer acceptance, cheese ranks among the most widely consumed dairy products and contributes substantially to human nutrition. However, it is highly prone to mold contamination, leading to quality deterioration, food safety concerns, and economic losses [27]. Although various mold genera can colonize cheese, only a few species typically predominate, with Penicillium being the most common, followed by Aspergillus. Moreover, some cheese-associated fungi can produce mycotoxins, posing additional risks to consumer health [28].

This study aimed to develop a dual-crosslinked bioactive CMC film and to evaluate the influence of crosslinking on its physicochemical and antimicrobial properties, as well as its effectiveness in extending the shelf life of cheese. Based on a comprehensive review of the literature, no prior study has reported the combined use of CA and X-ray irradiation for crosslinking CMC film containing onion peel extract (OPE), nor their application as active packaging for cheese preservation.

2. Materials and Methods

2.1. Materials

Carboxymethyl cellulose sodium salt (CMC, high viscosity) was obtained from Sigma-Aldrich (Oakville, ON, Canada). Glycerol (≥99%), citric acid (CA) (≥99%), sodium carbonate (Na₂CO₃), ethyl acetate HPLC (≥99%), and methanol HPLC (≥99%) were purchased from Fisher Scientific (Ottawa, ON, Canada). Folin–Ciocalteu reagent and gallic acid standard were supplied by Sigma-Aldrich (Oakville, ON, Canada). Cellulose nanocrystals (CNCs) were provided by CelluForce (Montreal, QC, Canada). Microbiological media: Potato Dextrose Broth (PDB) and Potato Dextrose Agar (PDA) were purchased from Alpha Biosciences Inc. (Baltimore, MD, USA).

2.2. Extraction of Active Compounds

Onion peels (OPs) (yellow variety, Allium cepa L.) were collected from a local restaurant in Laval, Canada, and air-dried at room temperature. The active compounds were extracted from the dried ground OP by maceration using ethyl acetate–methanol (85:15 v/v) solvent at a ratio of 1:12.5 (peels:solvent). The solution was left in dark conditions at 20 °C for 3 days. The mixture was then sonicated in an ice bath for 10 min at 400 W, 80% amplitude, and 20 kHz (UP400S, Hielscher, Teltow, Germany) (pulse mode: 5 s on/5 s off), then filtered under vacuum. The solvent was evaporated by reduced pressure rotary evaporator, and the remaining crude extract was weighed to quantify the yield. Then, the extract was freeze-dried in a FreeZone Benchtop Freeze Dryer (Labconco, Kansas City, MO, USA), and the dried powder was kept at 4 °C until analysis.

2.3. Characterization of Onion Peel Extract

2.3.1. Total Phenol Content

The total phenol content (TPC) of onion peel extract (OPE) was measured using the Folin–Ciocalteu method [29]. The OPE solution (1 mL) was mixed with 6 mL of distilled water and 1 mL of Folin–Ciocalteu reagent. The mixture was vortexed for 30 s and left to stand for 6 min, after which 2 mL of 20% Na₂CO₃ solution was introduced. Then, samples were incubated at 40 °C for 20 min, and absorbance was measured using a UV–Vis spectrophotometer (S-3100, Scinco, Betatek Inc., North York, ON, Canada) at 765 nm. The TPC was calculated from a calibration curve of gallic acid (20–100 µg GAE/mL) and expressed as mg gallic acid equivalents (GAEs) per gram of dried extract.

2.3.2. Determination of Total Flavonoid Content

The total flavonoid content (TFC) of the OPE was assessed using the colorimetric procedure outlined by Chang et al. [30]. A volume of 0.5 mL of the extract was combined with 1.5 mL of 95% ethanol, followed by the addition of 0.1 mL of 10% aluminum chloride, 0.1 mL of 1 M potassium acetate, and 2.8 mL of distilled water. The reaction mixture was left to stand at room temperature for 30 min. Absorbance was then recorded at 370 nm using a UV–Vis spectrophotometer (S-3100, Scinco, Betatek Inc., North York, ON, Canada). TFC was quantified using a quercetin standard calibration curve and reported as micrograms of quercetin equivalents per milliliter of extract (µg QE/mL).

2.3.3. Minimal Inhibitory Concentration of the OPE

The minimal inhibitory concentration (MIC) of the OPE was evaluated according to the method of Hossain et al. [31] with modifications against mold strains of Penicillium commune, Aspergillus niger, and Botrytis cinerea. The OPE was diluted in PDB medium to obtain serial two-fold dilutions ranging from 18,350 to 18 ppm. Then, 50 µL of the extract solution was added directly to each well of a 96-well microplate. Each well was then inoculated with 100 µL of each mold to achieve a final concentration of 1 × 10⁴ conidia/mL. The microplate was then incubated aerobically at 28 °C for 48 to 72 h. The negative control consisted of PDB medium with the OPE, while the positive control contained the mold inoculum without the OPE. After that, the absorbance was measured at 595 nm to quantify fungal growth using a microplate reader (BioTek Instruments Inc., ELx800^® Winooski, VT, USA).

2.4. Film Preparation

CMC-based films were prepared via the solvent-casting method with the incorporation of crosslinkers CA (50% w/w of CMC) and CNCs (5% w/w of CMC). These concentrations were selected based on preliminary optimization experiments aimed at reducing the water solubility of the films. Briefly, CNCs were dispersed in distilled water and then shear-mixed with an Ultra-Turrax at 10,000 rpm for 2 min. Subsequently, CMC (2% w/v) was gradually incorporated and stirred at 60 °C for 2 h until fully dissolved. Glycerol (40% w/w of CMC) was then added and mixed at 350 rpm for 30 min, followed by the addition of CA (50% w/w of CMC) and stirring for 1 h. For the bioactive film preparation, OPE was incorporated at concentrations of 0.095, 0.19, or 2.85% (w/w of CMC), determined according to the MIC value. The final film-forming solutions were degassed under vacuum and cast onto Teflon plates to dry at room temperature for 48 h. Dried films were then conditioned in a desiccator at 55% relative humidity (RH) until further analysis. Dried films were exposed to low-energy X-ray irradiation under inert conditions (nitrous oxide) using a Precision 350 irradiator at two settings: 350 kV, 10.8 mA (1.5 kGy/h) or 55 kV, 29.8 mA (1.5 kGy/h), at doses of 5, 10, or 15 kGy. Non-irradiated films were used as controls. After irradiation, films were stored in the desiccator (55% RH) until further analysis.

2.5. Characterization of Films

2.5.1. Water Solubility

The water solubility (WS) of the films was determined following the method described by Sarmast et al. [32]. Briefly, film samples (2 × 2 cm²) were dried at 105 °C for 24 h and weighted. The pre-weighed dried films were then placed into 100 mL of distilled water in a beaker. Then, the undissolved films were filtered and dried at 110 °C to a constant weight. The WS was determined using the following Equation (1):

$$\mathit{W}\mathit{S} (\%)= \frac{{\mathit{W}}_0-{\mathit{W}}_1}{{\mathit{W}}_0}\times 100$$

(1)

where W₀ = initial weight and W₁ = weight of the undissolved film after drying.

2.5.2. Barrier Properties

The water vapor permeability (WVP) of the films was determined according to the ASTM E96/E96M—16 cup method, which tests the moisture transfer ability through the film [33]. The films with an area of ≈ 32 cm² were placed at the top of a cup (model 68–3000; Twhing-Albert Instrument Co., West Berlin, NJ, USA) containing anhydrous CaCl₂ (30 g) as a desiccant. The initial weight of the cups was taken before placing them into a controlled humidity chamber (25 °C and 50 ± 2% RH). The final weight was measured after 24 h, representing the amount of water vapor that passed through the film and was absorbed by the desiccant. WVP was calculated by Equation (2):

$$\mathit{W}\mathit{V}\mathit{P}(\mathit{g}·\mathit{m}\mathit{m}\mathit{/}{\mathit{m}}^2·\mathit{d}\mathit{a}\mathit{y}·\mathit{k}\mathit{P}\mathit{a}) = \frac{\mathit{∆}\mathit{w}·\mathit{x}}{\mathit{A}·\mathit{t}·\mathit{∆}\mathit{P}}$$

(2)

where: ∆w = Final weight of the cell − Initial weight of the cell, g; x = Average thickness, mm; A = Area of the films, m²; t = time, days; ∆P = 3.282 KPa.

The film’s Oxygen Transmission Rate (OTR) was measured according to Sharmin et al. [34], using an OX-TRANS^® 1/50 permeation analyzer (MOCON^®, Brooklyn Park, MN, USA). The temperature and RH were set at 23 °C and 0% RH, respectively, with a film area of 5 cm². After the system was purged with nitrogen for 30 min, the films were exposed to 100% oxygen for 6 to 8 h. The tests were performed in triplicate for each film, and readings were recorded as cm³/m²·day.

2.5.3. Mechanical Properties

Film thickness, tensile strength (TS, MPa), tensile modulus (TM, MPa), and elongation at break (Eb, %) were measured following the ASTM D638-99 test method [35], using a Universal Testing Machine (UTM) H5KT from Tinius Olsen Testing Machine Co., Inc. (Horsham, PA, USA) and Test Navigator program (ver. 7.02.11). The film samples were stretched under tensile conditions with a 30 mm initial gauge length, a crosshead speed of 50 mm/min, and a 100 N load cell.

2.5.4. Appearance and Optical Properties

Film opacity (OP) was assessed by measuring the absorbance at 660 nm (A660), using a UV–vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) according to Jaiswal et al. [16]. The OP was calculated by Equation (3):

$$\mathit{O}\mathit{P}=\frac{\mathit{A}660}{\mathit{d}}$$

(3)

where A₆₆₀ = absorbance of the film at 660 nm; d = film thickness (mm).

2.5.5. Gel Fraction

The gravimetric method was utilized to analyze the insoluble gel fraction (GF) of the CMC film, following the method of Tran Vo et al. [36]. Prior to the analysis, film samples measuring 3.0 × 3.0 cm² in the area were dried at 60 °C for 48 h and weighed (W₀). Subsequently, the dried films were immersed in distilled water for 24 h to remove the soluble portion. The remaining solid films were then dried in a vacuum oven at 60 °C to a constant weight (W₁). The GF was calculated according to the following Equation (4):

$$\mathit{G}\mathit{F} \left(\%\right)= \frac{{\mathit{W}}_1}{{\mathit{W}}_0}\times 100$$

(4)

2.5.6. Fourier Transform Infrared Spectroscopy

The fourier transform infrared spectroscopy (FTIR) spectra of the films were analyzed using a Spectrum One spectrophotometer (PerkinElmer, Woodbridge, ON, Canada) according to the method of Mahmud et al. [37]. This instrument was equipped with an attenuated total reflectance (ATR) accessory for solid analysis and a high-linearity lithium tantalate (HLLT) detector. Film specimens were pre-conditioned at 25 °C/57.6% RH for 48 h, in a desiccator containing a saturated solution of sodium bromide. Spectra were analyzed by using Spectrum^® 10.7.2.1630 software. Spectra were recorded from 650 to 4000 cm⁻¹, using 64 scans at a resolution of 4 cm⁻¹. After attenuation of total reflectance and baseline correction, spectra were normalized within the whole infrared region with a minimum transmission peak set at 75%. Tests were performed at 23 ± 2 °C and 50 ± 5% RH in triplicate (n = 3).

2.5.7. Antimicrobial Activity of Films Against P. commune

The antimicrobial activity of the films was assessed at 3 different values of MIC according to Mohammadi et al. [38]. The antimicrobial activity of the bioactive films irradiated at 10 kGy (energy level of 350 kV; optimal dose) and non- irradiated (control) was performed using the disk diffusion test on PDA. A 100 µL suspension of mold spores (1 × 10⁴ conidia/mL) was cultured on the PDA culture media. Sterile film disks (15 mm in diameter) were placed at the center of Petri dishes (Sarstedt Inc., Saint-Leonard, QC, Canada) containing the medium. The dishes were then incubated at 28 °C for 72 h. After incubation, the zone of inhibition was measured, and the inhibitory capacity (IC %) was calculated as an indication of the film’s activity. To ensure consistent mold growth on the surface of the Petri dish, a positive control Petri dish without film was used. Additionally, a Petri dish without mold but with film was included as a negative control to confirm that no mold contamination occurred. The IC % was calculated according to Equation (5):

$$\mathit{I}\mathit{C} (\%)=\frac{\mathit{D}\mathit{i}\mathit{a}\mathit{m}\mathit{e}\mathit{t}\mathit{e}\mathit{r} \mathit{i}\mathit{n}\mathit{h}\mathit{i}\mathit{b}\mathit{i}\mathit{t}\mathit{i}\mathit{o}\mathit{n} \mathit{z}\mathit{o}\mathit{n}\mathit{e}\left(\mathit{m}\mathit{m}\right)-\mathit{D}\mathit{i}\mathit{a}\mathit{m}\mathit{e}\mathit{t}\mathit{e}\mathit{r} \mathit{o}\mathit{f} \mathit{t}\mathit{h}\mathit{e} \mathit{f}\mathit{i}\mathit{l}\mathit{m} (\mathit{m}\mathit{m})}{\mathit{D}\mathit{i}\mathit{a}\mathit{m}\mathit{e}\mathit{t}\mathit{e}\mathit{r} \mathit{o}\mathit{f} \mathit{P}\mathit{e}\mathit{t}\mathit{r}\mathit{i} \mathit{d}\mathit{i}\mathit{s}\mathit{h}\mathit{,} (\mathit{m}\mathit{m})}\times 100$$

(5)

The diameter of all Petri dishes was 83 mm.

2.5.8. In Vitro Release Test

The in vitro release test was performed following Mahmud et al. [37] with modifications. The experiment was conducted under standardized conditions, using 50% ethanol (v/v) as a food simulant for cheese with a 48 h contact period at 20 °C. The simulant was selected for foods with lipophilic properties, covering both dairy and non-dairy fatty foods according to the European commission [39,40]. Briefly, 0.5 g of the bioactive film (irradiated at 10 kGy and energy level of 350 kV) containing 0.19% of OPE was immersed in 100 mL of simulant in glass flasks. The flasks were sealed and stored in a refrigerator (at 4 °C). The release of bioactive compounds was measured using a UV–Vis spectrophotometer (Scinco S-3100, Betatek Inc., North York, ON, Canada) at 300 nm (λ_max). A standard calibration curve was prepared using OPE concentrations ranging from 12 to 96 ppm. Absorbance was then recorded for each sampling interval (2–4 h), with measurements continued until a constant value was obtained. The cumulative release (%) was determined using the following Equation (6):

$$\mathit{C}\mathit{u}\mathit{m}\mathit{u}\mathit{l}\mathit{a}\mathit{t}\mathit{i}\mathit{v}\mathit{e} \mathit{r}\mathit{e}\mathit{l}\mathit{e}\mathit{a}\mathit{s}\mathit{e} (\%)=\left(0.05{\mathit{P}}_{\left(\mathit{t}-1\right)}\mathit{+}{\mathit{P}}_{\mathit{t}}\right)\times 100$$

(6)

where: Pt: Percentage release (w/v) at time t; P_{(t − 1)}: Percentage release (w/v) at a time before ‘t’; 0.05 factor corresponds to the withdrawn sample volume (5 mL) divided by the total simulant volume (100 mL).

The release kinetics study was conducted following the Korsmeyer–Peppas model presented in Equation (7).

Mt/M∞ = k_kp tⁿ

(7)

where Mt is the amount of bioactive compounds released at time t; M∞ is the total release at infinite time; k_kp is the release constant; n is the release exponent; and t is the sampling time.

2.6. In Situ Study on Cheese

The optimum irradiated bioactive films containing 0.19% OPE were selected for assessing their potential as cheese packaging material. Gouda cheese commercial product (moisture 41.0%, fat 26%, protein 26%, and salt 9.0%) was purchased from a local shop (Laval, QC, Canada). Three pieces of cheese (4.0–5.0 g) were placed in sterile Petri dishes and covered with the films, ensuring full coverage of the surface. The samples were divided into four groups: a control group with only Gouda cheese; a second group covered with irradiated film without OPE; a third group covered with irradiated film containing 0.19% OPE; and a fourth group kept in its original packaging (Nylon-EVOH-LLDPE/MPE). To test antifungal activity, the target fungus P. commune (1 × 10⁴ conidia/mL) was inoculated onto the surface of cheese. Then, every set in a Petri dish was packed in a sealed container and stored at 8 ± 2 °C. Samples were microbially analyzed every three days. At each sampling time, the sample was mixed with 90 mL of sterile peptone water (0.1%) and homogenized for 1 min at 230 rpm using a Lab-blender 400 stomacher (Seward Medical, London, UK). Serial dilutions were prepared, and 100 µL was plated onto PDA plates. Then. The dilutions are incubated at 28 °C for 48 to 72 h, and the results were presented as CFU/g. While conducting microbial analysis, mold growth on the sample surface was visually observed throughout the storage period.

Weight Loss and pH Analysis of Cheese

Weight loss and pH analyses were carried out according to Aleksandrovas et al. [41] following Equation (8):

$$\mathit{W}\mathit{e}\mathit{i}\mathit{g}\mathit{h}\mathit{t} \mathit{l}\mathit{o}\mathit{s}\mathit{s} (\%)=\frac{{\mathit{m}}_1-{\mathit{m}}_2}{{\mathit{m}}_1}\times 100$$

(8)

where m₁ = initial weight of the cheese, g; m₂ = final weight at each sampling, g.

The pH of the cheese was determined using a calibrated Accumet Basic AB15 pH meter (Fisher Scientific, Pittsburgh, PA, USA).

2.7. Statistical Analysis

Each experiment was performed in triplicate (n = 3), and results were presented as mean ± standard deviation. Data analysis was conducted using the ANOVA test (SPSS Statistics 26, IBM Corp., Armonk, NY, USA), followed by Tukey’s test for multiple pairwise comparisons. A p-value ≤ 0.05 was considered statistically significant.

3. Results and Discussion

3.1. Characterization of OPE

3.1.1. Total Phenol and Flavonoid Content

The extraction of OPE yielded an average of 1.47%. When diluted in water (3.67% w/v), the extract had a TPC of 122 ± 2.25 mg GAE/g (Table 1). This concentration was higher than the values reported for onion peel and skin, which ranged from 27.8 to 68.2 mg GAE/g [42]. However, the result was consistent with other studies, for instance, Lee et al. [43] reported 120.6 mg GAE/g in orange-colored onion peel extracts, while Benito-Román et al. [44] found up to 102 mg GAE/g in onion skin waste extracts. Additionally, TFC of the extract was 2.07 ± 0.51 mg QE/g (Table 1), which aligns closely with values reported by Ren et al. [45], who found a TFC ranging from 2 to 4 mg QE/g in OPE collected between 2009 and 2014.

Table 1. Characterization of onion peel extract in terms of total phenolic content (TPC), total flavonoid content (TFC), and antimicrobial activity (MIC).

Properties of OPE
TPC	122 ± 2.25 mg GAE/g
TFC	2.07 ± 0.51 mg QE/g
Minimal Inhibitory Concentration (MIC) (ppm)
Penicillium commune	950 ± 11.0
Botrytis cinerea	3860 ± 12.4
Aspergillus niger	3860 ± 12.4

3.1.2. Minimal Inhibitory Concentration of the OPE

The minimal inhibitory concentration (MIC) of OPE against P. commune, B. cinerea, and A. niger is presented in Table 1.

The extract exhibited the strongest antifungal activity against P. commune, with an MIC of 950 ppm, whereas higher concentrations (3860 ppm) were required to inhibit the growth of B. cinerea and A. niger. OP is recognized for its antimicrobial properties, largely attributed to polyphenols, including flavonoids, which exert antifungal effects through multiple mechanisms [46,47,48]. Flavonoids can disrupt fungal plasma membranes, induce mitochondrial dysfunction, interfere with cell wall synthesis, inhibit cell division, block RNA and protein synthesis, and impair efflux pump activity [49]. Da et al. [50] reported that flavonoids caused the degeneration of cytoplasmic organelles and leakage of cytosolic contents in pathogenic fungi such as A. fumigatus, suggesting the disruption of plasma and intracytoplasmic membranes. Morphological changes, including partial swelling and shrinkage or cracking of fungal cell walls, indicate the inhibition of cell wall biosynthesis. Additionally, flavonoids may induce apoptosis-like programmed cell death via excessive production of reactive oxygen species. Škerget et al. [48] compared the antifungal activity between onion bulb and peel extracts against A. niger and found that peel extracts inhibited fungal growth more effectively (35.6%) than bulb extracts. This was attributed to the higher concentration of phenolic compounds and quercetin in the peel, which were 3–5 times greater than in the edible bulb. Conversely, Fredotović et al. [51] reported that A. niger was largely unaffected by the methanolic extracts of red or yellow onion varieties, although A. cornutum exhibited slight susceptibility (MIC = 500 µg/mL). These findings, together with previous studies, indicate that the antifungal efficacy of OPE is influenced by cultivar-specific chemical composition, extraction methods, and the analytical techniques used to assess antimicrobial activity [52].

3.2. Characterization of the Films

3.2.1. Water Solubility

Various strategies have been proposed to enhance the physicochemical properties of biopolymer films, including chemical and physical crosslinking [53]. In the present study, dual cross-linking using CA and X-ray irradiation was applied to CMC-based films to evaluate the effect on water solubility (WS) (Table 2).

Table 2. Water solubility (WS, %) of the CMC/CA-based films.

Films	WS, %
Control (CMC/CA)	48.9 ± 2.8 c
CMC/CA- X-ray (55 kV, 5 kGy)	34.2 ± 5.6 b
CMC/CA-X-ray (55 kV, 10 kGy)	38.1 ± 2.2 b
CMC/CA-X-ray (55 kV, 15 kGy)	33.1 ± 3.6 b
CMC/CA-X-ray (350 kV, 5 kGy)	37.8 ± 5.0 b
CMC/CA-X-ray (350 kV, 10 kGy)	12.9 ± 3.0 a
CMC/CA-X-ray (350 kV, 15 kGy)	15.8 ± 1.2 a
Bioactive film	50.3 ± 1.3 c
Bioactive film-X-ray (350 kV, 10 kGy)	11.6 ± 1.0 a

Within column, groups having the same letter are not significantly different (p > 0.05). Values are expressed as mean ± standard deviation for each measurement.

As shown in Table 2, the WS of CMC/CA films decreased from 48.9% to 34.2%, corresponding to a 30% reduction when exposed to 5 kGy at 55 kV. No significant differences (p > 0.05) were observed among the CMC/CA films irradiated at 55 kV (34.2% at 5 kGy; 38.1% at 10 kGy; 33.1% at 15 kGy). In contrast, a pronounced decrease in WS was observed under 350 kV irradiation, where WS declined from 37.8% (5 kGy at 350 kV) to 12.9% (10 kGy at 350 kV; 65.9% reduction). However, WS values between 10 and 15 kGy did not differ significantly (p > 0.05), indicating that 10 kGy is the optimal dose. The greater reduction in WS observed at 350 kV compared with 55 kV can be attributed to the variation in voltage, as higher voltage X-ray photons with higher energies can penetrate deeper into polymers than a low-voltage X-ray [54]. Furthermore, the formation of free radicals is directly related to the energy and absorbed doses applied during irradiation [55]. The high numbers of generated free radicals throughout the film matrix at the 350 kV treatment enhances crosslink formation, leading to a denser polymer network with fewer hydrophilic sites available for water interaction [14,56]. The incorporation of OPE into the film irradiated at 350 kV and 10 kGy did not significantly influence WS. These findings are consistent with the work of Shahbazi et al. [57] who reported a 27% reduction in WS following photo and chemical crosslinking of CMC films using UV-C and sodium benzoate. CA acts as a chemical crosslinker by promoting the esterification of CMC hydroxyl groups via a cyclic anhydride intermediate [58]. The formation of ester bonds decreases solubility by reducing available hydroxyl groups, thereby limiting interactions with water molecules and improving resistance to dissolution [59]. Simultaneously, X-ray irradiation contributes to physical crosslinking by activating reactive sites within the CMC matrix [15]. Although irradiation can induce polysaccharide chain scission through radical formation in the presence of oxygen. Previous studies have reported that the presence of air (oxygen) affects polymer crosslinking and may induce degradative reactions [60,61]. The use of nitrous oxide regulates radical behavior and prevents excessive degradation. This gas reduces the concentration of free radicals, enabling a controlled interaction between the crosslinker and the polysaccharide [62,63]. In the absence of oxygen, the most significant products of radiolysis of water are hydroxyl radicals (^•OH), hydrogen atoms (H^•), and hydrated electrons (e_aq⁻), which can be transformed into ^•OH by reacting with nitrous oxide [32,64].

3.2.2. Mechanical and Barrier Properties

Table 3 presents the key characteristics of the CMC/CA films, listed in order of thickness, tensile strength (TS), tensile modulus (TM), elongation at break (EB), water vapor permeability (WVP), oxygen transmission rate (OTR), opacity (OP) and gel fraction (GF).

Table 3. Physicochemical properties of the CMC/CA-based films.

Films	Thickness (mm)	TS (MPa)	TM (MPa)	EB (%)	WVP (g·mm/m2·day·kPa)	OTR (cm3/m2·day)	OP	GF (%)
Control (CMC/CA)	0.08 ± 0.01 a	19.3 ± 1.02 a	89.6 ± 1.3 a	26 ± 1.12 a	8.22 ± 0.7 c	29.6 ± 6.80 b	10.8 ± 0.57 a	47.01 ± 1.62 a
CMC/CA-X-ray (350 kV, 10 kGy)	0.07 ± 0.01 a	34.2 ± 3.30 b	202.6 ± 6.4 b	24.1 ± 1.05 a	3.98 ± 0.8 a	9.62 ± 0.86 a	9.71 ± 0.59 a	76.90 ± 8.02 b
Bioactive film	0.08 ± 0.01 a	18.2 ± 0.60 a	90 ± 3.78 a	26.03 ± 0.35 a	6.23 ± 0.3 b	29.43 ± 1.04 b	12 ± 0.54 b	47.55 ± 2.14 a
Bioactive film-X-ray (350 kV, 10 kGy)	0.08 ± 0.01 a	34.1 ± 2.93 b	204 ± 5.29 b	25.93 ± 0.40 a	5.25 ± 0.9 ab	10.28 ± 0.47 a	15.8 ± 0.33 c	77.61 ± 6.58 b

Within the same column, groups having the same letter are not significantly different from each other (p > 0.05). Values are expressed as mean ± standard deviation for each measurement.

The thickness of the films showed no significant differences among treatments (p > 0.05), with a mean value of 0.08 mm. The tensile strength (TS) of the control film was 19.3 MPa, which increased significantly to 34.2 MPa following irradiation (~77% increase). The incorporation of OPE into the film did not significantly affect the TS (18.2 MPa; p > 0.05). However, when the bioactive film was irradiated, the TS increased to 34.1 MPa, mirroring the improvement observed in the irradiated CMC/CA films. Similarly, non-irradiated CMC/CA and bioactive films exhibited comparable TM values (~90 MPa; p > 0.05), whereas irradiation significantly increased the TM to 204 MPa (p ≤ 0.05). This increase reflects a significant enhancement in film rigidity, associated with restricted polymer chain mobility due to radiation-induced crosslinking. These results indicate that X-ray irradiation effectively strengthened the polymer network, likely through enhanced crosslinking, while the bioactive addition did not hinder the formation of this reinforced structure. There were no statistically significant differences in EB among the four groups (p > 0.05), values ranged from 24.1% to 26%, indicating that irradiation and bioactive extract incorporation did not impact the flexibility of the films.

The control film exhibited a WVP of 8.22 g·mm/(m²·day·kPa). Irradiation at 350 kV and 10 kGy significantly reduced WVP to 3.98 g·mm/(m²·day·kPa), representing a ~52% decrease (p ≤ 0.05). Incorporating the OPE into the film increased WVP to 6.23 g·mm/m²·day·kPa, which was significantly higher than the irradiated film (p ≤ 0.05). Similar results have been reported by Thivya et al. [65], who observed that the addition of onion extract increased WVP and the surface hydrophilicity of composite films, likely due to polyphenols disrupting the ordered polymer structure and creating voids and channels. In addition, when the bioactive film was X-ray irradiated, the WVP decreased to 5.25 g·mm/(m²·day·kPa). However, no significant difference was observed between the irradiated CMC/CA film and the irradiated bioactive film (p > 0.05). These results indicate that while X-ray irradiation alone effectively improves the moisture barrier of CMC/CA films, the addition of OPE does not further enhance WVP significantly. Similar trends have been reported by Sarmast et al. [32], where gamma irradiation at 5 kGy reduced the WVP of gelatin films by nearly 9.5-fold (from 53.20 to 5.4 g·mm/(m²·day·kPa)), whereas the incorporation of plant-based essential oils did not produce a further reduction (7.09 g·mm/(m²·day·kPa)).

The oxygen barrier properties followed a similar trend to WVP. The control film showed an OTR of 29.6 cm³/m²·day, which was significantly reduced by nearly 3.1 times (9.62 cm³/m²·day; p ≤ 0.05) upon irradiation. The incorporation of OPE alone to the control film did not significantly affect OTR (29.43 cm³/m²·day, p > 0.05); however, the combination of irradiation and bioactive film lowered OTR to 10.28 cm³/m²·day, with no significant difference compared to the irradiated CMC/CA film (p > 0.05), indicating that irradiation predominantly enhances oxygen barrier properties. Jaiswal et al. [16] reported that the OTR value of the trans-cinnamaldehyde-gelatin film decreased drastically after X-ray irradiation at 10 kGy, with a value of 12.5 ± 2.1 cm³/m²·day. In this study, irradiation significantly reduced the permeability of water vapor and oxygen through the films, likely due to the formation of effective intramolecular bridges between CMC and the crosslinker during irradiation, which increased the tortuosity of diffusion pathways and thereby hindered the diffusion pathways for both water and oxygen [66,67].

3.2.3. Appearance and Optical Properties

Since the color of the films can influence consumer perception and purchasing decisions regarding packaged food, the appearance of the CMC/CA films was visually evaluated. The films were uniform, flexible, and self-supporting, lacking any visible cracks or irregularities, and could be easily handled and peeled off. No noticeable changes were observed between the control and irradiated films, all of which remained transparent (Supplementary File). In contrast, the bioactive films appeared translucent and showed a light-yellow color with increasing OPE concentration, likely due to the presence of phenolic compounds and flavonoids present in the OPE.

In this study, no significant changes in opacity were observed between the control film (10.8) and the irradiated CMC/CA film (9.71) (p > 0.05) (Table 3). In contrast, bioactive films showed a significantly increased opacity of the films to 12 (p ≤ 0.05). Similar results were reported when OPE was incorporated into methylcellulose films, supporting the present study [68]. The increased opacity is primarily attributed to higher light absorption and reduced light transmission through the film, enabling it to function as a light barrier and thus helping to protect light-sensitive foods from spoilage. Furthermore, the opacity values significantly increased (p ≤ 0.05) from 11.9 to 15.8 when the bioactive film was exposed to irradiation. This effect may be attributed to the action of ionizing irradiation, which induces oxidation of some sensitive phenolic compounds in the incorporated extract, resulting in color changes, higher opacity, and reduced light transmittance [16,69,70].

3.2.4. Gel Fraction

Gel fraction (GF) was used to determine the degree of crosslinking via quantifying the insoluble portion of CMC films. The resulting percentage of GF is presented in Table 3. The control and bioactive films (non-irradiated films) showed the GF of ~47%, with no significant difference between them (p > 0.05), indicating that the incorporation of the bioactive compound did not affect the crosslinking efficiency of the polymer matrix. The observed GF is primarily attributed to CA-mediated crosslinking within the CMC matrix. During thermal treatment, CA forms cyclic anhydrides that react with the hydroxyl groups of CMC, resulting in ester bond formation. These ester linkages facilitate the development of a three-dimensional polymer network, thereby increasing the insoluble fraction of the films [7]. In contrast, X-ray irradiation resulted in a pronounced increase in GF for both film formulations. The GF of the irradiated CMC/CA and irradiated bioactive films increased significantly to ~77% compared with their respective non-irradiated films (p ≤ 0.05). This enhancement is attributed to radiation-induced free radical formation along the polymer chains, which promotes additional crosslinking reactions and further stabilizes the polymer network [71]. Ibrahim and El Salmawi [72] observed that the GF of CMC-based blends increased from approximately 55% to 65% as the irradiation dose increased from 10 to 20 kGy, confirming that irradiation enhances crosslinking efficiency in CMC/sodium alginate blends films.

3.2.5. Fourier Transform Infrared Spectroscopy

The fourier transform infrared spectroscopy (FTIR) spectra of all CMC/CA films are shown in Figure 1.

Figure 1. FTIR spectra of CMC/CA, bioactive CMC/CA, cross-linked CMC/CA, and bioactive cross-linked CMC/CA films.

All films showed a broad -OH stretching band between 3100 and 3600 cm⁻¹. For the CMC/CA film, the band at 3310 cm⁻¹ shifted to higher wavenumbers in the bioactive film and bioactive-X-ray film, implying that the possible formation of intramolecular and intermolecular hydrogen bonding between the CMC and OPE. The bands observed at 2925–2930 and 2865–2885 cm⁻¹ corresponded to C-H stretching vibrations, while peaks around the band at 1580–1585 cm⁻¹ represented conjugated carbonyl (C-O) groups of CMC. Additionally, the peaks in the range of 1000–1200 cm⁻¹ were related to the stretching vibrations of the C-O-C groups within the polysaccharide structure [73]. Notably, a distinct peak is present at 1718–1724 cm⁻¹, which was attributed to the stretching vibration of ester carbonyl C=O groups due to CA possibly combined with the crosslinking process [59,74]. According to the possible mechanism of esterification between CA and CMC proposed by Mali et al. [58], the cyclic anhydride intermediate formed from the CA reacts with the -OH functional groups of the CMC. In addition, the intensity of this ester carbonyl peak (C=O) increased in the irradiated films compared to the unirradiated films, possibly due to irradiation-enhanced crosslinking reactions. Previous studies have reported similar effects of irradiation. Bekhit et al. [75] found that the carbonyl peak at 1745 cm⁻¹ increased after irradiation due to the formation of new carbonyl groups. Likewise, Ferrante et al. [76] observed that the C=O peak at 1725 cm⁻¹ intensified with increasing irradiation dose and explained that γ-irradiation can cause both chain scission and crosslinking through free radical formation. Overall, these results indicate that irradiation induces structural modifications in the polymer, including carbonyl formation and increased crosslinking through free radical generation, consistent with the enhanced ester C=O peak and higher gel fraction observed in irradiated samples.

3.2.6. Antifungal Activity of Bioactive Films Against P. commune

Since P. commune is the most common spoilage mold in cheeses, and was the most sensitive to the OPE compared to B. cinerea and A. niger, it was selected for further analysis. Accordingly, the antifungal activity of the films against P. commune was evaluated using three concentrations of the incorporated OPE, 1 × MIC, 2 × MIC, and 3 × MIC by measuring the inhibitory capacity (IC, %) (Table 4 and Figure 2).

Table 4. Inhibitory capacity (IC, %) of bioactive films at varying MIC values against P. commune.

Films	IC (%)
Bioactive film (1 × MIC)	48 ± 5 ab
Bioactive film (2 × MIC)	52 ± 2 a
Bioactive film (3 × MIC)	53 ± 6 a
Bioactive film-X-Ray (1 × MIC)	30 ± 5 c
Bioactive film-X-Ray (2 × MIC)	41 ± 6 b
Bioactive film-X-Ray (3 × MIC)	42 ± 5 b

Within the same column, groups having the same letter are not significantly different from each other (p > 0.05). Values are expressed as mean ± standard deviation for each measurement.

Figure 2. Antimicrobial activity of bioactive CMC/CA films at varying MIC values, (a) control film, (b) non-irradiated film × 1 MIC, (c) non-irradiated film × 2 MIC, (d) non-irradiated film × 3 MIC, (e) irradiated film × 1 MIC, (f) irradiated film × 2 MIC, and (g) irradiated film × 3 MIC.

For the non-irradiated bioactive films, the IC % increased from 48% (1 × MIC) to 52% (2 × MIC) and 53% (3 × MIC). However, no significant differences were observed between these concentrations (p > 0.05). For the X-ray-treated bioactive films, IC % values were consistently lower compared to non-irradiated bioactive films. The IC % significantly increased (p ≤ 0.05) from 30% (1 × MIC) to 42% (3 × MIC). There was no significant difference between 2 × MIC and 3 × MIC (p > 0.05), yet significantly higher than the 1 × MIC treatment (p ≤ 0.05). The reduced antifungal activity observed in the irradiated bioactive films may be associated with the effects of crosslinking on the release behavior of the active constituents within the extract. Several studies have shown that a highly crosslinked and compact polymer network can slow or limit the diffusion of antimicrobial agents. For example, Khan et al. [77] reported that the gamma-irradiated genipin crosslinked chitosan film initially showed lower antimicrobial activity compared to the uncrosslinked film; however, the activity increased during 35 days of storage as the network gradually relaxed.

Likewise, Mousavi et al. [78] reported that a crosslinked gelatin–chitosan film limited the release of ε-poly-lysine and Liang et al. [79] found that incorporating crosslinkers into chitosan–bacterial cellulose films resulted in noticeably lower antimicrobial performance compared with the uncrosslinked films. The crosslinked structure likely restricted the initial desorption of active compounds from the film surface, consistent with the release study outlined in the following Section 3.2.6, where the crosslinked film showed a slower release profile. With extended storage, the relaxation of the network allowed more active molecules to reach the surface. In this study, the films prepared using both X-ray irradiation and citric acid crosslinking exhibited the highest antimicrobial activity during storage, as further confirmed in the in situ cheese experiment.

3.2.7. In Vitro Release Test

The release profile of OPE from the bioactive film, with and without X-ray irradiation, is presented in Figure 3.

Figure 3. Release profile of the bioactive compounds from CMC/CA bioactive films.

Both films exhibited a gradual increase in cumulative release over time, indicating a diffusion-controlled mechanism. However, significant differences (p ≤ 0.05) were observed after 1 h in the cumulative release rates between the two films. The non-irradiated bioactive film showed a faster initial release, reaching approximately 60% within the first 10 h, followed by a steady increase until reaching a sustained release at 96% after 22 h. In contrast, the bioactive film exposed to X-ray irradiation exhibited a slower release rate during the first 10 h, reaching only 36% of the cumulative release (Mt/M∞ × 100) (p ≤ 0.05). Despite this slower early release, the irradiated film continued to release bioactive compounds over time and ultimately reached a maximum of about 75% at 28 h, after which the release remained stable and reached a plateau. This plateau indicates that a portion of the OPE was strongly retained within the CMC/CA film matrix, suggesting a potential irradiation-induced modification in the release profile, due to crosslinking or structural changes within the CMC/CA film matrix. Consequently, the plateau represents the equilibrium state where the readily diffusible fraction of OPE has been released, while the remaining extract remains entrapped within the crosslinked network. Irradiation may have enhanced interactions between the biopolymers and the OPE, leading to the entrapment of a substantial portion of the extract. This is further supported by the apparent enhancement of mechanical properties, including increased tensile strength and lower film permeability (Table 3). Likewise, Benbettaïeb et al. [80] observed that electron-beam irradiation delayed the release and increased the quercetin retention in the chitosan–gelatin film. After release, the quercetin content (%) in the film was higher in irradiated samples than in the non-irradiated one. It increased from 6.6 mg/g in the non-irradiated film to 9.5 mg/g after 40 kGy and 8.3 mg/g after 60 kGy of irradiation. Moreover, Vasile et al. [81] observed that PLA-based blend/rosemary extract exposed to γ-irradiation (30 kGy) exhibited slower migration and released smaller amounts of bioactive compounds than non-irradiated samples.

The release behavior of bioactive compounds from the films was fitted using the Korsmeyer–Peppas model (Figure 3). High R² values (>0.9) for both film samples confirmed a good fit of the model to the experimental data. The calculated release exponent (n) values were 0.77 for the bioactive non-irradiated film and 0.66 for the bioactive irradiated film, indicating anomalous (non-Fickian) transport, in which both diffusion and polymer chain relaxation contribute to the release [82,83]. According to the Korsmeyer-Peppas model, n ≤ 0.45 corresponds to Fickian diffusion, 0.45 < n < 0.89 indicates anomalous transport (combining diffusion and polymer relaxation), n = 0.89 reflects zero-order release predominantly driven by swelling, and n > 0.89 denotes non-Fickian transport dominated by polymer relaxation or erosion [37]. However, the release rate constant (k_kp) was lower for the irradiated films compared to the non-irradiated film, indicating a slower release in the irradiated films.

3.3. In Situ Study on Cheese

Based on the previously demonstrated MIC test and in vitro release test, irradiated films showed better efficiency than non-irradiated films; therefore, irradiated films were considered for the in situ cheese preservation study. Figure 4 shows the mold counts (log CFU/g) during 18 days of incubation at 8 ± 2 °C, for cheese samples covered with CMC/CA-X-ray (Treatment 2) and bioactive film-X-ray (Treatment 3) compared to uncovered cheese (Treatment 1) and conventional packaging (Treatment 4).

Figure 4. P. commune counts for an uncovered cheese (Treatment 1), cheese covered with CMC/CA-X-ray (Treatment 2), bioactive film-X-ray (Treatment 3), and conventional package (Treatment 4) storing at 8 ± 2 °C for 18 days. Means with the same uppercase letter are not significantly different (p > 0.05).

The initial growth of mold for Treatment 1 was log 2.3 at day 6, which significantly increased to log 4.9 at day 12 and eventually reached log 5.7 at day 18 (p ≤ 0.05). Similar results were observed for Treatment 4, there were no significant differences between Treatments 1 and 4 during 18 days of storage (p > 0.05). In contrast, Treatment 2 showed mold growth after day 6 and reached log 4.7 by day 18, which corresponds to approximately 1 log reduction compared to Treatments 1 and 4. However, Treatment 3 exhibited the strongest inhibition; no detectable growth was observed until day 12, after which mold counts increased slowly, reaching 2.3 log by day 18. This corresponds to a 3.2–3.4 log reduction compared to Treatments 4 and 1, and a 2.4 log reduction compared to Treatment 2 at day 18. In all cases, the growth of inoculated mold was significantly higher compared to the bioactive-X-ray irradiated film, due to the inhibitory effect of the released OPE on the cheese (p ≤ 0.05). This behavior aligns with the in vitro observations (Table 4). The smaller inhibition zone observed for the irradiated bioactive film at early time points corresponds with its slower, sustained-release profile (Figure 3), suggesting that the denser dual-crosslinked network delays the initial burst of bioactive compounds. As a result, antimicrobial activity is maintained over an extended period on the cheese surface, which explains the prolonged suppression of mold observed in Treatment 3.

Visual observations supported the microbial results. Cheeses in Treatments 1 and 4 were fully covered with mold between days 12 and 18, whereas Treatment 2 showed visible mold from day 12 onward. In contrast, Treatment 3 displayed visible growth only at the end of the experiment. According to Jurado and Vicente [84], who inoculated hard and extra-hard cheeses with P. commune at log 6 and ripened them at 8 ± 2 °C and 85% RH for either 2 or 5 weeks, the inoculated cheeses showed fungal growth after 6 days and were completely covered by mycelium after 10 days. Kontogianni et al. [85] reported yeast/mold levels between log 2.6 ± 0.5 and log 3.2 ± 1.4 after 20 days using whey-protein films enriched with 2% spirulina, an extract level nearly five times higher than that applied in the present work.

Traditional chemical preservatives, such as potassium sorbate and sodium benzoate, are commonly used to extend the shelf life of cheese due to their antimicrobial properties. However, evidence suggests that, when applied at typical concentrations ranging from 1000 ppm to 10,000 ppm, these additives may have limited or even negative effects on shelf stability [86]. In recent years, researchers have investigated the use of natural extracts obtained from food waste and by-products as alternatives to conventional chemical preservatives for extending the shelf life of cheese. This strategy not only improves the product’s quality and safety but also plays a significant role in mitigating food waste. Though bioactive films for cheese preservation have been previously explored, the present study represents the first to focus on CMC/CA bioactive/X-ray films containing a waste extract to extend the shelf life of cheese by analyzing P. commune growth after artificial inoculation.

3.4. Weight Loss and pH Analyses

Figure 5 illustrates the weight loss of cheese samples for 18 days, sampling every 3 days.

Figure 5. Weight loss for uncovered cheese samples (Treatment 1), cheese samples covered with CMC/CA-X-ray (Treatment 2), bioactive film-X-ray (Treatment 3), and conventional package (Treatment 4) stored at 8 ± 2 °C for 18 days. Means with the same uppercase letter are not significantly different (p > 0.05).

The weight loss of Treatment 1 from day 6 to day 9 was drastically augmented (from 3.89% to 10.33%), indicating rapid moisture loss once surface dehydration began, but afterward, the loss rate remained stable (from 13.77% to 15.07%). As expected, the uncovered cheese sample (Treatment 1) showed the highest weight loss compared to other treatments (Treatment 2–4) from day 9 to day 18 (p ≤ 0.05). Among the covered cheese treatments (Treatment 2–4), weight loss progressed more slowly. From day 9 to day 15, Treatment 2 showed an increase from 4.61% to 8.66%, Treatment 3 increased from 4.18% to 7.64%, and Treatment 4 showed an increase from 3.68 to 7.39%. Despite these increases, no significant differences were observed among these three treatments during this period (p > 0.05), suggesting that both the CMC/CA-X-ray film and the bioactive-X-ray film provided a moisture-retention performance comparable to that of conventional packaging. However, at day 18, Treatment 4 (conventional package) showed the lowest weight loss (7.52%), which was significantly different from the other treatments (p ≤ 0.05). Across all samples, a gradual mass loss was observed in all treatments, possibly due to water evaporation, contributing to the change in weight. Even so, the weight loss values obtained in this study were generally lower than those reported in the literature. For example, Ramos et al. [87] reported higher losses (>15%) for cheese coated with antimicrobial edible coatings after 20 days of ripening. In the case of commercially coated cheese, the losses were around 15%, double that reported in the present study (7.52%). Similarly, they reported the highest weight loss values for the uncoated treatment (≈22%), consistent with the findings of the present study, with a loss of 15.1%. Similarly, Jurado and Vicente [84] reported pronounced weight reductions, noting a loss of around 25% in cheeses inoculated with P. commune after 21 days. All these previous results suggest that covered cheese generally had decreased weight losses and a higher moisture content, also contributing to a reduction in fungal growth [88,89].

The pH of all cheese samples is shown in Table 5. At day 0, cheese samples had an initial pH of 5.31. Throughout the 18 days, Treatment 1 and Treatment 4 consistently maintained the highest pH values, with no significant differences between them at any time point (p > 0.05). In contrast, Treatment 2 and Treatment 3 displayed significantly lower pH values from day 9 onward (p ≤ 0.05). In addition, from day 3 to day 18, Treatment 2 exhibited pH values ranging from 4.78 to 4.52, while Treatment 3 ranged from 4.88 to 4.51, without significant differences from each other at all time points. Jurado and Vicente [84] obtained final pH values ranging from 5.3 to 5.4 in P. commune-inoculated cheeses. In contrast, Ramos et al. [87] observed a pH reduction in all cheeses wrapped with edible coatings plus natamycin and lactic acid, with values ranging from 4.7 to 4.8 on day 20. In this study, a lower pH was observed in the cheese samples covered with CMC/CA-X-ray (Treatment 2) and bioactive film-X-ray (Treatment 3), which can be attributed primarily to the presence of residual CA that remains unbound and can gradually diffuse into the cheese surface.

Table 5. pH values of uncovered and covered cheese samples with CMC/CA-based films.

	Treatment 1	Treatment 2	Treatment 3	Treatment 4
Day 0	5.31 ± 0.02 a
Day 3	5.16 ± 0.41 a	4.78 ± 0.20 b	4.88 ± 0.27 ab	5.21 ± 0.31 a
Day 6	5.12 ± 0.25 a	4.75 ± 0.14 b	4.91 ± 0.16 ab	5.17 ± 0.24 a
Day 9	5.19 ± 0.44 a	4.58 ± 0.25 b	4.66 ± 0.16 b	5.25 ± 0.43 a
Day 12	5.45 ± 0.41 a	4.66 ± 0.22 b	4.76 ± 0.25 b	5.58 ± 0.36 a
Day 15	5.32 ± 0.31 a	4.59 ± 0.07 b	4.52 ± 0.13 b	5.27 ± 0.36 a
Day 18	5.43 ± 0.34 a	4.52 ± 0.14 b	4.51 ± 0.18 b	5.49 ± 0.42 a

Within the column, groups having the same letter are not significantly different (p > 0.05). Values are expressed as mean ± standard deviation for each measurement.

4. Conclusions

The study successfully developed crosslinked bioactive CMC-CA films from natural, eco-friendly materials for potential application in cheese packaging. The dual crosslinking process, which combines chemical (CA) and physical (X-ray) methods, significantly enhanced the film’s physicochemical properties. The incorporation of phenolic-rich OPE provided strong antifungal activity and enabled the sustained release of bioactive compounds. The bioactive crosslinked film also proved effective in a real food system, preserving cheese quality, minimizing weight loss, and providing a moisture-retention performance comparable to the conventional packaging under in situ conditions. Thus, combining CA with X-ray irradiation acts as an effective dual-crosslinking strategy for reinforcing the structural integrity of CMC films. This approach not only improves their functionality for packaging applications but also enables the valorization of agricultural by-products by incorporating phenolic-rich extracts into food systems. Together, these advances support waste-reduction efforts, enhance food safety, and contribute to extending product shelf life. It should be noted that, while CA and the extract of an onion peel are food-grade and generally recognized as safe (GRAS), the films were not tested for the migration of these compounds into cheese. Future studies should include migration testing to fully assess the safety and compliance of the films for food-contact applications.