Application of novel egg albumin-based biodegradable films fortified with antioxidant pomegranate peel extract to enhance the storage stability of chicken cookies

Introduction

Food packaging is an integral part for the product safety, quality preservation and shelf-life extension across the food supply chain (Hillier et al.2017) [20] . Conventional plastic packaging dominates the market due to its low cost and favourable mechanical properties however, increasing global plastic production and waste accumulation intensified environmental pollution and greenhouse gas emissions (Dirpan et al.2023) [12] . The persistence of plastics and the release of toxic additives pose substantial risks to ecosystems and human health, necessitating the development of sustainable packaging alternatives (Mangaraj et al. 2019) [34] . Biopolymers loaded with bioactive compound are considered one of the most promising and safest alternatives to other antimicrobial compounds. Biodegradable polymers sourced from renewable materials have attracted significant interest as environmentally friendly alternatives to conventional petroleum-derived plastics. Within this group, protein-based films are valued for their excellent film-forming properties, satisfactory mechanical strength and efficient oxygen barrier characteristics. Egg albumin is considered as a particularly promising biopolymer due to its globular protein nature and the coiled structure of ovalbumin (Lopez Mata et al., 2016) [31] , which encourages disulfide cross-linking and supports the formation of transparent and flexible films. Although egg albumin films offer several benefits, their high nutrient content renders them vulnerable to oxidative degradation and microbial contamination, which limits their practical use in food packaging. To mitigate these drawbacks, the incorporation of plant-based bioactive compounds has been explored as a promising approach to improve the functional characteristics of biodegradable films.

Pomegranate peel (Punica granatum L.), an underutilized by-product of agro-processing industries, is a rich source of phenolic compounds, flavonoids, and ellagitannins that exhibit strong antioxidant and antimicrobial properties (Kumar et al., 2022b) [29] . Ethanolic extracts of pomegranate peel demonstrate notable radical-scavenging activity and have been earlier reported to enhance the mechanical integrity and protective performance of biopolymer films (Malviya et al., 2014) [33] . Recently, consumer demand for ready-to-eat, proteinenriched snack products has increased distinctly, especially for poultry-based items due to their high nutritional value and economic affordability (Berwal et al., 2013) [7] . We are passing through a crucial juncture of global insecurity against the right of everyone to have access to safe and nutritious food (Rindhe et al., 2017) [46] . However, lipid oxidation and microbial proliferation remain major constraints affecting the shelf life and quality of meat-based snacks such as chicken cookies. A number of foods in India have been reported to have high incidence of bacteria, however, there is little/scanty information about the extent of microbial contamination of chicken products sold in Indian supermarkets (Rindhe et al., 2008) [47] . Therefore, the present study was designed to develop an egg albuminbased biodegradable film incorporated with varying levels of pomegranate peel extract and to evaluate its potential in enhancing oxidative stability, microbial safety, and overall storage quality of chicken cookies.

Materials and methods

Peel preparation and extraction

Pomegranate (Punica granatum L.) fruits were obtained from the local market of Parbhani city (M.S.). Peels of pomegranate fruit were peeled, washed, oven-dried at 40±5 °C, ground into powder. Ethanolic extracts were obtained by macerating 1 kg of pomegranate peel powder in ethanol:water (3500:1500 mL) for 72 h at room temperature. The extract was filtered by muslin cloth and Whatman's filter paper no.1. which was concentrated by evaporation on hot plate and stored at refrigeration temperature until use.

Preparation of egg albumin film incorporated with pomegranate peel extract

Films were prepared following Rindhe et al. (2020) [44] with modifications. Egg albumin film-forming solution was prepared by dissolving 6 g of egg albumin powder in 100 mL distilled water under continuous stirring at 150 rpm using a magnetic stirrer. Pomegranate peel extract was incorporated at concentrations of 0.03, 0.06, and 0.09%, followed by the addition of 2 mL glycerol as a plasticizer. The pH of the solution was adjusted to 9.0 using 1.0 N sodium hydroxide, after which the mixture was incubated at 60 °C for 40 min. The solution was then cooled, filtered through muslin cloth, poured onto Teflon sheets and dried in hot air oven at (32±2 °C) to obtain the films.

Preparation of chicken cookies

Chicken cookies were prepared with slight modifications in the method followed by Nemade et al. (2021) [39] . Chicken meat powder required for chicken cookies preparation was obtained by grinding of dried clean minced boneless chicken meat in a hot air oven at 60-65 °C for 20-22 hrs. As per table 1. the chicken cookies ingredient mixture was prepared, uniformly mixed, and adjusted to the desired dough consistency. The dough was rolled, shaped into cookies and baked in a microwave oven at 150-160 °C for 20 minutes then cooled.

Storage of chicken cookies

Chicken cookies were wrapped in bioactive egg albumin film incorporated with (T 2 ) or without 0.09% pomegranate peel extract (T 1 ) along with control (C) packed in a lowdensity polyethylene (LDPE) pouch and subsequently stored at room temperature. All packaged samples were stored under aerobic conditions at a temperature of 32±2 °C. Sampling of stored cookies was conducted at regular intervals of 10 days to evaluate the storage stability. Throughout the storage period, the shelf life and overall quality of the chicken cookies were assessed to determine the effectiveness of the packaging materials.

Analytical Procedures for bioactive protein films

Film thickness was measured using a digital electronic vernier caliper with an accuracy 0.0001 mm (Kumari et al. 2017) [30] . Film opacity was measured at 660 nm using a UV-VIS spectrophotometer following method of Domene et al. (2019) [13] with 3 × 1 cm film strips placed in water-filled cuvettes and opacity calculated from transmittance values. Film density was determined at 25 °C using the flotation method described by Quarashi et al. (1992) [42] . The moisture content of egg albumin films was determined using the gravimetric method demarcated by Rhim et al. (1998) [43] . Total soluble mass of egg albumin films was determined according to method outlined by Jiang et al. (2007) [22] . Water Vapour Transmission Rate of the coatings was determined according to ISO 2528:2017(E). Film penetrability was measured as per the method followed by Mahajan et al. (2017) [30] , while tensile strength (MPa) and elongation at break (%) were measured according to ASTM standards (ASTM, 1986; 2001) using texture analyzer (TMS-PRO, Food Technology Corporation, USA). The oxygen transmission rate (OTR) of film coatings was determined using the deoxidizer absorption method (Zhang et al. 2019) [57] .

Physicochemical Analysis of Cookies

The pH of chicken cookies was measured according to AOAC (1995). Ten gm of sample was homogenized with 50 mL distilled water, and the pH of the resulting suspension was recorded using a digital pH meter. The peroxide value of chicken cookies was determined based on the method described by Koniecko (1979) [28] , with slight modifications.

The Thiobarbituric Acid Reactive Substances value was estimated using the method described by Witte et al. (1970) [54] .

Microbial Analysis

Total plate count, psychrophilic count, yeast and mould count, staphylococcal count was determined according to method outline by APHA (1992; 2015) [2,3] .

Sensory Evaluation

Ready-to-eat chicken cookies were evaluated by a semitrained panel (n = 21) from COVAS, Parbhani, using an 8point descriptive scale (Keeton, 1983) [27] . Attributes assessed included appearance and color, texture, flavor, crispiness, and overall acceptability. Potable water was provided between samples for palate cleansing.

Statistical Analysis

The data obtained in the study on various parameters were statistically analysed on "SPSS-16.0" software package for one-way ANOVA as per standard methods of Snedecor and Cochran (1994) [51] .

Result and Discussion

Physicochemical Characteristics of Film

The impact of pomegranate peel extract on the physicochemical, mechanical and permeability properties of biodegradable films is presented in Table 4.1.

Thickness

Film thickness, an indicator of structural integrity, increased significantly (p<0.05) with increasing PPE concentration in chicken cookies, ranging from 0.18±0.00 mm in the control (C) to 0.20±0.00 mm in T 3 . This enhancement may be attributed to stronger intermolecular interactions facilitated by polyphenols and other bioactive compounds present in PPE. Comparable increase in film thickness with higher incorporation of PPE has also been reported in chitosanbased films (Min et al., 2021) and gluten-based films (Kumari et al., 2017) [36,30] . Similarly, Kavas and Kavas (2016) [26] reported that the incorporation of plant extracts increased the thickness of egg albumin films, supporting the present findings that PPE enhances film structure.

Opacity

Opacity, a key parameter determining light-barrier efficiency, increased significantly (p<0.05) from 1.35±0.02 (C) to 2.04±0.03 (T 3 ) with increasing PPE concentrations. The higher opacity indicates reduced light transmission, due to availability of phenolic compounds and natural pigments in PPE which absorb and scatter light. Comparable results have been reported in plant-extract-incorporated protein films by Moghadam et al., (2020) [37] . Increased opacity is beneficial in protecting photosensitive foods from UV induced oxidation (Galus, 2018) [16] , demonstrating the effectiveness of PPE in enhancing light-barrier properties.

Density

Film density increased significantly (p<0.05) as PPE concentration rose, ranging from 1.20±0.01 g mL⁻¹ in the control (C) to 1.31±0.01 g mL⁻¹ in T 3 . This could be attributed to the development of a denser and more compact film structure, possibly resulting from stronger interactions between the polyphenolic compounds of PPE and the protein matrix (Elsebaie et al., 2023) [15] . Similar consequence of polymer-plasticizer interactions on density have been documented in protein-based films by Atik et al., (2024) [6] .

Moisture Content

Moisture content decreased significantly (p<0.05) with increasing PPE concentration, from 0.36±0.01 (C) to 0.12±0.03 (T 3 ). The decrease may be attributed to enhanced intermolecular interactions and reduced water-binding capacity after PPE incorporation, which restricts moisture retention within the matrix (Min et al., 2021; Cao et al., 2021) [36,9] . Hamarashid et al. (2025) [19] also reported a similar reduction in moisture content in protein-based films on addition of plant extract addition have been reported by), indicating improved film stability.

Penetrability

Penetrability (puncture resistance) increased significantly (p<0.05) from 4.85±0.01 N (C) to 7.15±0.04 N (T 3 ) as the level of PPE increased. This improvement reflects enhanced mechanical strength and suitability for food packaging applications. Singh et al. (2018) [50] also reported similar effects of film composition and plasticizer content on the puncture resistance of protein-and polysaccharide-based films. In contrast, Kanatt et al. (2012) [24] reported no significant effect of plant extract incorporation on the penetrability of chitosan-PVA composite films.

Total Soluble Mass (TSM)

Total soluble mass decreased significantly (p<0.05) from 40.0±0.58% (C) to 26.0±0.10% (T 3 ) with the progressive increase in PPE concentration. Lower TSM suggests improved water resistance and greater structural stability, likely resulting from stronger interactions between PPE constituents and the protein matrix. Cao et al. (2021) [9] also observed comparable results in carvacrol-enriched egg albumin films, where decreased solubility was attributed to reduced water-binding capacity. Similar decreases in film solubility with incorporation of plant extracts have been reported by Hu et al. (2019) and Moghadam et al. (2020) [21,37] .

Tensile Strength

Tensile strength increased significantly (p<0.05) from 0.98±0.01 MPa (C) to 4.75±0.00 MPa (T 3 ) as the concentration of PPE increased. This enhancement reflects improved mechanical resistance and durability, may be attributed to stabilizing interactions between PPE polyphenols and the egg albumin matrix. Similar improvements in tensile properties of polyphenol-enriched biopolymer films were reported by Mushtaq et al. (2018) [38] .

Elongation at Break

Elongation at break decreased significantly (p<0.05) from 35.00±0.15% (C) to 32.80±0.15% (T 3 ) with increasing PPE concentration. The reduced flexibility may result from enhanced hydrogen bonding between phenolic compounds and the protein matrix, producing a more rigid and compact structure. Similar trends were reported in PPE-incorporated biopolymer films by Min et al., (2021) [36] .

Water Vapour Transmission Rate (WVTR)

Water vapour transmission rate decreased significantly (p<0.05) with increasing PPE concentration, declining from 13.52±0.03 g/m²•day (C) to 11.92±0.05 g/m²•day (T 3 ). This reduction may be attributed to improved moisture barrier properties, possibly due to hydrophobic phenolic compounds enhancing polymer cross-linking and limiting water diffusion (Deseta et al., 2023) [11] . A similar declining trend was reported by Mushtaq et al. (2018) [38] in phenolicenriched protein films, confirming the role of PPE in enhancing film compactness.

Oxygen Gas Transmission Rate (O₂ GTR)

Oxygen gas transmission rate (O₂ GTR) decreased significantly (p<0.05) with increasing pomegranate peel extract (PPE) concentration, from 7.81±0.03 to 7.09in C±0.03 cm³/m²•day•atm in T 3 . This enhanced oxygen barrier property may be attributed to the formation of a denser microstructure through polyphenolic interactions, thereby restricting oxygen diffusion. Similar reductions in oxygen permeability of plant-extract-enriched egg albumin films were reported by El-Sayed et al. (2018), demonstrating the barrier-enhancing potential of PPE.

Quality evaluation of chicken cookies stored at room

Chicken cookies were packaged using three different materials: control (C) in LDPE bags, T 1 in egg albumin films, and T 2 in egg albumin films incorporated with 0.09% PPE. The samples were stored at room temperature and periodically analyzed for various quality parameters.

Sensory evaluation

The average sensory scores for chicken cookies either wrapped or unwrapped in egg albumin films during room storage are presented in Table 3. Sensory scores declined significantly (p<0.05) with increasing storage duration across all treatments. However, T 2 consistently retained higher scores for appearance, flavour, texture, crispiness, and overall acceptability. The deterioration in scores of sensory attributes viz, appearance, flavour, texture, crispiness, and overall acceptability of chicken cookies during storage may be attributed to lipid and protein oxidation, moisture migration, formation of free fatty acids, and microbial growth. Oxidative lipid degradation negatively affects colour, flavour, and texture, as previously reported for meat-based products by Wu et al., (2022) [55] . Comparable reductions in sensory scores associated with lipid oxidation during storage have also been documented by Jung et al. (2025) [23] after 270 days in chicken stored at-20 °C. However, T 2 showed better retention of sensory qualities, which may be attributed to the presence of polyphenols and flavonoids in pomegranate peel extract. These bioactive compounds delayed oxidative deterioration, thereby extending shelf life. The improved flavour stability in T 2 may be attributed to antioxidant phenolics that minimize oxidative damage (Giri et al., 2023) [18] . Similar storage-related flavour deterioration in meatbased snacks has been reported previously (Pavan Kumar et al. 2016). Throughout storage, T 2 maintained higher texture scores, likely due to improved barrier properties and the stabilizing effect of bioactive compounds in pomegranate peel extract, which limited moisture uptake and structural degradation (Giannakourou et al.2025) [17] . Similar effects of edible packaging on minimizing textural deterioration in snack foods have been reported previously by Karnwal et al. (2025) [25] . The reduction in crispiness may be may be linked to moisture uptake and lipid oxidation, which compromise structural integrity and freshness perception. Similar decline in the crispiness of stored meat-based snack products previously reported by Sonia (2024), who also reported that the sensory quality of products declines as the storage period increases. Overall acceptability of chicken cookies declined significantly (p<0.05) with progression of ambient storage period. However, C was not subjected for sensory evaluation after 40 th day and T 1 after 60 th day of storage due to existence of signs of incipient spoilage. According to Park et al. (2025) [40] , the quality of meat products during storage is largely influenced by chemical reactions involving lipids and proteins. The deterioration in sensory attributes may be due to lipid and protein oxidation, release of free fatty acids, and progressive microbial growth during storage (Domínguez et al., 2019) [14] . However, T 2 recorded relatively higher acceptability scores, suggesting the protective role of pomegranate peel extract, as its polyphenolic antioxidants helped to retard oxidative deterioration and spoilage (Giri et al. 2023) [18] . Similar shelf-life extension using pomegranate peel extract has been reported previously in meat products (Turgut et al. 2016; Mehdizadeh et al. 2020) [53,35] . Chicken cookies packaged in egg albumin film incorporated with 0.09% pomegranate peel extract (T 2 ) exhibited higher sensory scores than the control. Pomegranate peel extract acts as a natural additive due to its strong antioxidant properties which helped delay spoilage and improve the retention of flavour, texture, and overall acceptability, thereby demonstrating its effectiveness in extending shelf life (Giri et al., 2023) [18] .

Physico-chemical characteristics

The changes in the physicochemical characteristics of chicken cookies stored at room conditions (32±2 °C) for a period of 70 days, and summarized in Table 4. The pH of chicken cookies increased significantly (p<0.05) during ambient storage across all treatments. The control showed the greatest rise (6.31-6.98), followed by T 1 (6.12-6.87), while T 2 recorded the lowest increase (6.09-6.52) over 70 days at room temperature. The comparatively lower pH in T 2 suggests better product stability, likely due to the antimicrobial effect of pomegranate peel extract. Similar pH stabilization effects have been reported by Mehdizadeh et al. (2020) and Caglak et al. (2024) [35,8] in pomegranate peel extract treated products. The gradual increase in pH during storage may be attributed to microbial growth and protein degradation producing alkaline compounds (Rushitha et al., 2024) [48] . Peroxide value (PV) increased significantly in all stored cookies, indicating progressive lipid oxidation. The control packed in LDPE showed the highest increase (1.32-4.25 meq/kg), while T 1 exhibited a moderate rise (up to 3.82 meq/kg). The lowest PV was observed in T 2 (1.03-2.10 meq/kg), packaged in egg albumin film containing 0.09% pomegranate peel extract. The enhanced oxidative stability of T 2 is attributed to phenolic compounds that scavenge free radicals and inhibit lipid peroxidation, as reported by Cava and Ladero (2023) [10] . Similar reductions in peroxide formation for pomegranate peel extract-based film wrapped Kalari cheese during storage have been reported previously by Mushtaq et al. (2018) [38] . Similarly, thiobarbituric acid (TBA) values increased significantly (p<0.05) during storage. The control showed the highest rise (0.57-2.50 mg MDA/kg), followed by T 1 (0.51-2.01 mg MDA/kg), whereas T 2 recorded the lowest increase (0.48-1.62 mg MDA/kg), indicating superior oxidative stability due to the presence of PPE. This reduction in lipid oxidation can be linked to the strong radical-scavenging activity of phenolic compounds in pomegranate peel extract (Xiang et al. 2022) [56] . Present findings are consistent with findings obtained by Mehdizadeh et al. (2020) [35] during storage of beef wrapped in pomegranate peel extract added film.

Microbial quality

The changes in the microbiological quality of chicken cookies stored at room temperature (32±2 °C) for 70-days period enumerated in the table 5. Total plate count (TPC) increased significantly (p<0.05) in all cookies during storage. The control packaged in LDPE exhibited the greatest increase (3.38-4.10 log₁₀ cfu/g), followed by T 1 (3.37-3.78 log₁₀ cfu/g), whereas T 2 (egg albumin film incorporated with 0.09% pomegranate peel extract) exhibited the lowest microbial growth (3.33-3.55 log₁₀ cfu/g), indicating the antimicrobial efficacy of pomegranate peel extract, attributed to its bioactive phenolic compounds such as ellagic and gallic acids, as reported by Chen et al. (2020). Similar observations regarding the antimicrobial effects of pomegranate peel extract in meat products have also been reported by Shahamirian et al. (2019) [49] , with emphasizing its potential for extending the shelf life of meat-based snack products. Psychrophilic bacteria were not detected in any cookie samples during storage at 32±2 °C, as such temperatures are unfavourable for psychrophilic microorganisms, that thrive at lower temperatures (around 13 °C). The absence of psychrophilic bacteria in this study might be linked to unfavourable thermal conditions and the protective effect of packaging, which limited the activity of cold-adapted microbes (Son et al., 2023) [52] . Yeast and mould counts remained undetectable in treated cookies up to day 60 and in the control up to day 50. By day 70, the control exhibited the highest fungal count (3.50 log₁₀ cfu/g), followed by T 1 (2.98 log₁₀ cfu/g), while T 2 recorded the lowest count (2.60 log₁₀ cfu/g). The suppressed fungal growth in T 2 can be ascribed to the antifungal properties of phenolic compounds present in pomegranate peel extract. As per Turgut et al. (2016) [53] pomegranate peel extract inhibits yeast and mould growth by disrupting microbial cell membranes. Similar findings on the antifungal efficacy of pomegranate peel-based active packaging have been reported by Deseta et al. (2023) [11] , with confirming its role in improving the microbial stability of meat-based bakery products during storage. From the study it is concluded that the inclusion of 0.09% pomegranate peel extract was found to be optimal for enhancing the physicochemical, mechanical, and barrier properties of the egg albumin film and storage stability of chicken cookies for up to 70 days at ambient temperature without significant deterioration in quality attributes.

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