# Cricket protein-based composite film containing Andrographis paniculata leaf extract-based nanoparticles for meat emulsion

Aunzar Bashir Lone, Sunil Kumar, Rana Muhammad Aadil, Abdo Hassoun, Abderrahmane Aït-Kaddour, Zuhaib F Bhat

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Aunzar Bashir Lone, Sunil Kumar, Rana Muhammad Aadil, Abdo Hassoun, Abderrahmane Aït-Kaddour, et al.. Cricket protein-based composite film containing Andrographis paniculata leaf extract-based nanoparticles for meat emulsion. Applied Food Research, 2025, 5 (1), pp.100922. 10.1016/j.afres.2025.100922. hal-05201419

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# Cricket protein-based composite film containing *Andrographis paniculata* leaf extract-based nanoparticles for meat emulsion

Aunzar Bashir Lone<sup>a</sup>, Sunil Kumar<sup>a</sup>, Rana Muhammad Aadil<sup>b, c</sup>, Abdo Hassoun<sup>c</sup>, Abderrahmane Aït-Kaddour<sup>d, e, *</sup>, Zuhaib F. Bhat<sup>a, *</sup>

## ARTICLE INFO

### Keywords:

- Cricket protein

- Bioactive film

- *Andrographis paniculata*

- Nanoparticles

- Meat emulsion

- Storage quality

## ABSTRACT

A cricket (*Acheta domestica*) protein (Crik-Prot)-based composite film was developed to preserve the quality of the goat meat emulsion (Got-met-Emul). The *A. paniculata* leaf extract-based nanoparticles (And-Pan-NPs) were used as an active agent for Crik-Prot-based film. The addition of And-Pan-NPs [0.5 % (T₁), 1.0 % (T₂) and 1.5 % (T₃)] imparted antimicrobial and antioxidant properties to the film [indicated by the results of total phenolic and flavonoid contents, DPPH, ABTS and FRAP activities, antioxidant release (%), and inhibitory halos against *S. aureus* and *Escherichia coli*] and significantly (P < 0.05) altered its physicochemical [increased density (g/ml) whereas reduced the WVTR (mg/mt²), transmittance (%) and moisture content (%)] and colour [increased a* and b* and reduced L* values] properties. Crik-Prot-based films incorporated with And-Pan-NPs (0.5–1.5 %) significantly (P < 0.05) enhanced the oxidative stability [protein oxidation (total carbonyl content) and lipid oxidation (TBARS and free fatty acids)], antioxidant potential (ABTS, DPPH, and FRAP activities), and microbiological quality of Got-met-Emul during two weeks storage at 4 ± 1 °C. The Crik-Prot-based film (T₁, T₂ and T₃) enhanced the sensory quality of Got-met-Emul on day 7 of storage and gastrointestinal digestion increased (P < 0.05) the antioxidant potential of Got-met-Emul. The Crik-Prot-based film incorporated with And-Pan-NPs enhanced the storage quality and functional value of Got-met-Emul.

## 1. Introduction

Enhancing the storage quality of food products, especially highly perishable items, such as meat and meat emulsion, is one of the main aims of the food industry to provide consumers with these products with their best sensory attributes (Shankar Tumuluru, 2023). To meet this end, researchers and food processors evaluate the use of several emerging technologies and novel ideas along with traditional approaches such as food additives. Given their negative effects on human health and concerns about safety hazards, the use of synthetic food additives is generally discouraged by informed and educated consumers, which has led to increased research on green alternatives such as the use of plant-based phytochemicals (Munekata et al., 2021). While the use of plant-based bioactive molecules (e.g., polyphenols, terpenoids, and

alkaloids) and essential oils to enhance the shelf life and storage quality of foods has attracted global attention, their inclusion in food matrices at effective levels can sometimes have negative consequences on sensory profile and the acceptance of the food products due to their intense colour, volatile flavour compounds, and astringent taste (Rutkowska & Pasqualone, 2025). To fully exploit the bioactive and preservative properties of plant extract-based bioactive compounds without affecting the sensory quality of foods, researchers have recently used edible and biodegradable films as carriers which act as controlled delivery systems and release minimal amounts of these bioactive molecules on the surface of food products (Singh et al., 2023). Recent studies have revealed that the use of plant-based bioactive compounds in the form of nanoparticles as bioactive agents in the films is more effective in controlling microbial, lipid oxidative and sensory changes in food products during storage

<footnote><sup>a</sup> Division of Livestock Products Technology, SKUAST-J, Jammu, India</footnote>

<footnote><sup>b</sup> National Institute of Food Science and Technology, University of Agriculture, Faisalabad, Pakistan</footnote>

<footnote><sup>c</sup> Sustainable AgriFoodtech Innovation & Research (SAFIR), Arras, France</footnote>

<footnote><sup>d</sup> Université Clermont-Auvergne, INRAE, VetAgro Sup, Unité Mixte de Recherche sur le Fromage UMRF, 89 Avenue de l'Europe, France</footnote>

<footnote><sup>e</sup> Department of Food Technology, Faculty of Agroindustrial Technology, University of Padjadjaran, Sumedang 45363, Jawa Barat, Indonesia</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: muhammad.aadil@uaf.edu.pk (R.M. Aadil), a.hassoun@saf-ir.com (A. Hassoun), abderrahmane.aitkaddour@vetagro-sup.fr (A. Aït-Kaddour), zuhaibbhatvet@gmail.com (Z.F. Bhat).</footnote>
(Bhat et al., 2024). Therefore, the present study evaluated the use of *A. paniculata* leaf extract-based nanoparticles as a bioactive agent for the development of a novel bioactive film for enhancing the storage stability of meat emulsions.

Studies have widely evaluated the use of different biopolymers (proteins, carbohydrates, and lipids) for the development of edible and biodegradable films for food preservation utilizing main products, byproducts, food waste and side streams (Abdullah et al., 2022; Perera et al., 2023; Ahangari et al., 2025; Amjadi et al., 2024). However, only a limited number of studies have reported edible and biodegradable films developed using insect protein, a widely available natural resource, highlighting the need for research in this area. Insects can provide a novel and sustainable source of material for developing packaging systems for food preservation, especially in those countries where insects are not traditionally consumed as food and have low consumer acceptance. Therefore, the present study attempted to develop a cricket protein (Crik-Prot) based bioactive film to augment the storage quality of goat meat emulsion (Got-met-Emul) using silver nanoparticles as a bioactive agent. An ultrasonication-mediated green method of synthesis was used to develop the silver nanoparticles (And-Pan-NPs) using *Andrographis paniculata* leaf extract as a reducing and capping agent. The *A. paniculata* leaf extract has been reported to have a wide range of therapeutic applications due to its varied bioactive properties ranging from anticancer, anti-tuberculosis, anti-inflammatory, anti-diabetic, antibacterial, antifungal, antioxidant, antiparasitic, antiviral, immunostimulatory, and hepatoprotective due to the presence of a wide spectrum of secondary metabolites such as phenolic compounds, saponins, tannins, alkaloids, and terpenoids (Bhati et al., 2023; Chokthaweepanich et al., 2023). The leaves are a rich source of diterpenoid lactones especially andrographolide, which is a major bioactive compound and associated with antimicrobial and antioxidant properties, in addition to neoandrographolide and isoandrographolide (Chokthaweepanich et al., 2023). The strong antimicrobial and antioxidant properties of leaf extract make it an ideal candidate for the development of bioactive films for food preservation. The developed nanoparticles were incorporated in the Crik-Prot-based film at different levels and used for packaging Got-met-Emul samples stored for two weeks under refrigerated conditions. The Got-met-Emul samples were analysed for quality during storage and compared with control samples.

# 2. Material and methods

## 2.1. Crik-Prot flour and *A. paniculata* extract

The Crik-Prot flour supplied by JR Unique Foods Ltd. (contained approximately 7 g protein, 2 g fat and 1 g carbohydrates per 10 g flour) was defatted using food-grade hexane (1:5 ratio, w/v) following the method elaborated by Lone, Bhat, Aït-Kaddour et al. (2023). The flour was developed using 100 % *Acheta domestica* crickets farmed in an HACCP and FDA-approved factory in Thailand (Udon Thani) and contained no additives (artificial colours, preservatives or flavours). The films were prepared using this defatted Crik-Prot flour, which was packaged under a vacuum and stored in a refrigerator (4 ± 1 °C) till used. The *A. paniculata* extract (containing 20 % w/w andrographolide) manufactured by Bixa Botanicals Pvt Ltd (Mumbai, India) from the leaves of the plant was used for developing the And-Pan-NPs (bioactive agent) for the Crik-Prot-based films. The extract dissolved in ethanol (85 % in water) was stirred for 2 h on a magnetic stirrer maintained at 50 °C and filtered through two filter papers (Whatman No.1 and 42) separately. The filtrate was reduced in volume using a rotary vacuum-evaporator (35 ± 2 °C) and finally freeze-dried and converted to a powder form (And-Pan-Ext) that was stored in a freezer (−20 °C) in the laminate pouches. This powder was used for the development of And-Pan-NPs, which were used as a bioactive agent for the development of Crik-Prot-based films to impart bioactive properties.

## 2.2. Ultrasonication-mediated And-Pan-NPs

And-Pan-NPs were developed following a green method using a Cole-Parmer ultrasonicator (WW-04,711-45, U.S.A), the And-Pan-Ext (5 %, w/v), and 1 mM aqueous solution of silver nitrate. The And-Pan-Ext (10 ml) and silver nitrate solution (90 ml) were processed at 500 w and 20 kHz (5/5 s on/off) for 10 min and observed for a colour change on a magnetic stirrer (1 h). The formation of And-Pan-NPs was indicated by a colour change initiated by the reduction of metal ions by the phytochemicals and proteins present in And-Pan-Ext turning the pale-yellow solution to a brown. This was followed by a centrifugation step (12,000 rpm x 30 min) and the And-Pan-NPs were obtained in a powder form after the freeze-drying process and were stored in a freezer (−20 °C) until used for the development of the Crik-Prot-based films as a bioactive agent. The particle characteristics [average particle size (nm), zeta potential (mV), and polydispersity index] and confirmation of the And-Pan-NPs synthesis were done using a Zetasizer Nano-ZS particle analyzer (Malvern, UK), a scanning electron microscope (SEM, JEOL, USA) and by evaluating the maximum absorption peak using a UV-Vis spectrophotometer which was found to be around 430 nm. The And-Pan-NPs were subjected to evaluation for their antimicrobial and antioxidant potential by assessing FRAP (ferric reducing antioxidant power, µM TE/100 g) and ABTS and DPPH radical scavenging activities (% inhibition) and determining total flavonoid and total phenolic contents in addition to inhibitory halos (mm) and minimum inhibitory concentration (MIC, mg/ml) against *Escherichia coli* and *S. aureus* following the details elaborated in our previous paper (Lone et al., 2023).

## 2.3. Characterization and preparation of the Crik-Prot-based films

A solvent casting method of film formation was employed to develop the Crik-Prot-based films containing four levels of And-Pan-NPs [0.0 % (control), 0.5 % ($T_1$), 1.0 % ($T_2$) and 1.5 % ($T_3$), w/v] (Singh et al., 2023). The final method (processing and ingredient levels) was chosen after a trial-and-error technique using different levels and conditions to obtain a film with desirable characteristics and optimum bioactive properties. An equal quantity of defatted Crik-Prot (0.75 g) and carrageenan (0.75 g) was dissolved in 100 ml distilled water using a glass rod and was heated for 15 min at 70 °C using a hot plate cum magnetic stirrer (@1500 RPM) to prepare a filmogenic solution. A plasticizer (14 ml glycerol) was added to the solution while it was agitated on the magnetic stirrer. This was followed by the addition of four levels of And-Pan-NPs to prepare four different types of Crik-Prot-based films [0.0 % (control), 0.5 % ($T_1$), 1.0 % ($T_2$) and 1.5 % ($T_3$), w/v]. The uniform layered films were developed by pouring uniform layers of the filmogenic solutions (60 °C) on glass plates which were dried in an air-drying oven (6 h at 55 °C). The Crik-Prot-based films were stored in a refrigerator for further analysis after being peeled from the glass surface and kept inside laminate pouches. The Crik-Prot-based films were subjected to characterization by evaluating their colour (L*, a*, b*), physicochemical characteristics (density, thickness, water vapour transmission rate, elongation at break, transmittance and moisture content) and antioxidant and antimicrobial potential (radical scavenging and ion-reducing activities, total phenolic and flavonoid contents and inhibitory halos against *E. coli* and *S. aureus*).

## 2.4. Goat meat emulsion (Got-met-Emul) and storage study

The Got-met-Emul was prepared following the method described by Lone, Bhat, Aït-Kaddour et al. (2023) using lean meat (67.40 %), ice flakes (10 %), vegetable oil (9 %), condiment mixture (5 %), refined wheat flour (4 %), spice mixture (2 %), monosodium glutamate (0.5 %), sodium tripolyphosphate (0.3 %), sodium nitrite (150 ppm) and sodium chloride (1.75 %) using a Sirman meat mincer (6 mm plate) and bowl chopper. The Crik-Prot-based films containing And-Pan-NPs [0.0 % (control), 0.5 % ($T_1$), 1.0 % ($T_2$) and 1.5 % ($T_3$), w/v] were used to wrap
the uniform samples of Got-met-Emul which were placed in a refrigerator at 4 ± 1 °C for 14 days and compared with control Got-met-Emul samples without any film. The Got-met-Emul was analysed on different storage time points (days 0, 7, and 14) for microbial and lipid stability, protein oxidation, physicochemical and digestion properties, and bioactivities (antimicrobial and antioxidant) to assess the impact of Crik-Prot-based films on quality and functional value of the Got-met-Emul.

## 2.5. Bioactive potential (antioxidant and antimicrobial)

The And-Pan-NPs, Crik-Prot-based films, and Got-met-Emul samples stored within the films were assessed for antioxidant potential by determining the total phenolic [expressed as µg Gallic acid equivalents (GAE)/mg and using the Folin-Ciocalteu method] and total flavonoid [expressed as µg Quercetin equivalents (QE)/mg and using the aluminium chloride colourimetric assay] contents and/or free radical and ion reducing capabilities by evaluating the ferric ion reduction (FRAP) capacity (µM TE/100 g) and DPPH and ABTS radical quenching activities (% inhibition) using calorimetric methods and following the details described by Lone, Bhat, Aït-Kaddour et al. (2023) and Singh et al. (2023). The Crik-Prot-based films were also assessed for antioxidant release % to express the release rate of phenolic compounds from Crik-Prot-based films using the method followed by Singh et al. (2023).

The And-Pan-NPs, Crik-Prot-based films, and Got-met-Emul samples were assessed for antimicrobial properties. The MIC (mg/ml) of And-Pan-NPs was determined using a protocol (microdilution technique using a 96-well plate) described by Lone, Bhat, Aït-Kaddour et al. (2023). The And-Pan-NPs (0.5–2.0 %) and Crik-Prot-based films (0.5, 1.0 and 1.5 %) were evaluated for inhibitory halos [disk agar diffusion tests using Muller Hinton agar (24 h incubation at 37 °C)] against both gram-positive and gram-negative bacteria (S. aureus and E. coli, 10⁶ cfu/ml). The Got-met-Emul samples stored for 14 days within Crik-Prot-based films were enumerated (log₁₀ cfu/g) for various microbial counts [yeast/moulds using potato dextrose agar, coliform using violet-red bile agar, psychrophilic using total plate agar and total plate using total plate agar] on regular storage time points (days 0, 7 and 14) (Lone, Bhat, Aït-Kaddour et al., 2023).

## 2.6. Characteristics of Crik-Prot-based films

The Crik-Prot-based films containing And-Pan-NPs (0.0, 0.5, 1.0 and 1.5 %) were evaluated for colour [brightness (L*), yellowness (b*) and redness (a*)] employing a Hunterlab colourimeter (U.S.A)] and various physicochemical and mechanical characteristics such as thickness (mm, digital micrometre), transmittance (%, UV–Vis spectrophotometer), moisture content (%, hot air drier), water vapour transmission rate (mg/m²t, test cell with water), and density (g/ml, floatation method using heptane and CCl₄) (Singh et al., 2023).

## 2.7. Lipid stability, protein oxidation, digestion simulation and physicochemical parameters

The methodologies elaborated by Lone, Bhat, Aït-Kaddour et al. (2023) for Got-met-Emul were applied to determine the two most commonly used lipid stability parameters [free fatty acids (% oleic acid) and TBARS (mg MDA/kg)] and total carbonyl content (protein oxidation was measured as nmol/mg protein) of Got-met-Emul samples during 14 days storage. The Got-met-Emul samples from days 0 and 7 were subjected to digestion simulation (gastrointestinal phases) following details elaborated by Lone, Bhat, Aït-Kaddour et al. (2023) for meat emulsion. Got-met-Emul samples (~437.50 mg protein) were subjected to two phases of digestion [gastric using pepsin at pH 1.9 for 1 h followed by intestinal using pancreatin for 2 h (substrate: enzyme = 100: 1 w/w for both pepsin and pancreatin)] in plastic (polyvinyl) containers placed on magnetic multi-stirrer to maintain temperature (37 °C) and stirring

<table><caption>**Table 1**

Characteristics of And-Pan-NPs and Crik-Prot-based film.</caption><thead><tr><th colspan="2">And-Pan-NPs</th></tr></thead><tbody><tr><td>Parameters</td><td>Mean ± S.E</td></tr><tr><td>Total phenolic content (µg GAE/mg)</td><td>22.55 ± 0.59</td></tr><tr><td>Total flavonoid content (µg QE/mg)</td><td>39.29 ± 0.75</td></tr><tr><td>DPPH (% inhibition)</td><td>74.4 ± 0.58</td></tr><tr><td>ABTS (% inhibition)</td><td>81.6 ± 0.32</td></tr><tr><td>FRAP (µM TE/100 g)</td><td>40.2 ± 0.65</td></tr><tr><td>Average size (nm)</td><td>22.83 ± 3.24</td></tr><tr><td>Zeta potential (mV)</td><td>-15.12 ± 0.84</td></tr><tr><td>Polydispersity index (PDI)</td><td>0.346</td></tr><tr><td>Maximum absorption peak (nm)</td><td>430</td></tr><tr><td>MIC (mg/ml)</td><td>S. aureus 1.0<br>E. coli 3.0</td></tr><tr><td>Inhibitory halos (mm)</td><td>S. aureus 0.5 % 13.50 ± 0.08<sup>C</sup><br>1.0 % 15.00 ± 0.05<sup>B</sup><br>2.0 % 16.00 ± 0.05<sup>A</sup><br>E. coli 0.5 % 10.00 ± 0.06<sup>C</sup><br>1.0 % 12.50 ± 0.08<sup>B</sup><br>2.0 % 13.50 ± 0.04<sup>A</sup></td></tr><tr><td>Crik-Prot-based film</td><td></td></tr><tr><td>Parameters</td><td>T<sub>0</sub> (0.0 %) T<sub>1</sub> (0.5 %) T<sub>2</sub> (1.0 %) T<sub>3</sub> (1.5 %)</td></tr><tr><td>Thickness (mm)</td><td>0.143 ± 0.01<sup>a</sup> 0.144 ± 0.02<sup>a</sup> 0.144 ± 0.03<sup>a</sup> 0.145 ± 0.03<sup>a</sup></td></tr><tr><td>Moisture content (%)</td><td>34.94 ± 0.21<sup>a</sup> 34.61 ± 0.11<sup>b</sup> 33.87 ± 0.22<sup>c</sup> 33.19 ± 0.21<sup>d</sup></td></tr><tr><td>Density (g/ml)</td><td>1.01 ± 0.008<sup>c</sup> 1.02 ± 0.004<sup>bc</sup> 1.04 ± 0.009<sup>ab</sup> 1.06 ± 0.007<sup>a</sup></td></tr><tr><td>WVTR (mg/mt<sup>2</sup>)</td><td>2.09 ± 0.007<sup>a</sup> 2.06 ± 0.006<sup>b</sup> 2.03 ± 0.008<sup>c</sup> 2.01 ± 0.009<sup>d</sup></td></tr><tr><td>Transmittance (%)</td><td>83.05 ± 0.21<sup>a</sup> 81.19 ± 0.11<sup>b</sup> 79.33 ± 0.22<sup>c</sup> 77.24 ± 0.12<sup>d</sup></td></tr><tr><td>Elongation at break (%)</td><td>54.45 ± 1.25<sup>a</sup> 54.24 ± 1.40<sup>a</sup> 53.30 ± 0.95<sup>a</sup> 52.80 ± 1.40<sup>a</sup></td></tr><tr><td>L* (brightness)</td><td>78.22 ± 0.11<sup>a</sup> 75.20 ± 0.21<sup>b</sup> 73.78 ± 0.10<sup>c</sup> 71.29 ± 0.20<sup>d</sup></td></tr><tr><td>a* (redness/greenness)</td><td>0.93 ± 0.008<sup>b</sup> 0.95 ± 0.008<sup>a</sup> 0.97 ± 0.006<sup>a</sup> 0.97 ± 0.004<sup>a</sup></td></tr><tr><td>b* (yellowness/blueness)</td><td>3.19 ± 0.01<sup>d</sup> 3.40 ± 0.01<sup>c</sup> 3.54 ± 0.009<sup>b</sup> 3.60 ± 0.01<sup>a</sup></td></tr><tr><td>Total phenolic content (µg GAE/mg)</td><td>0.86 ± 0.03<sup>d</sup> 0.38 ± 0.28<sup>c</sup> 0.68 ± 0.32<sup>b</sup> 10.14 ± 0.26<sup>a</sup></td></tr><tr><td>Total flavonoid content (µg QE/mg)</td><td>1.12 ± 0.04<sup>d</sup> 0.42<sup>c</sup> 0.28<sup>b</sup> 12.64 ± 0.28<sup>b</sup> 17.25 ± 0.52<sup>a</sup></td></tr><tr><td>DPPH (% inhibition)</td><td>1.42 ± 0.03<sup>d</sup> 31.42 ± 0.62<sup>c</sup> 34.64 ± 0.22<sup>b</sup> 38.39 ± 0.34<sup>a</sup></td></tr><tr><td>ABTS (% inhibition)</td><td>2.32 ± 0.04<sup>d</sup> 35.65 ± 0.23<sup>c</sup> 39.43 ± 0.34<sup>b</sup> 44.22 ± 0.72<sup>a</sup></td></tr><tr><td>FRAP (µM TE/100 g)</td><td>0.98 ± 0.03<sup>d</sup> 14.10 ± 0.46<sup>c</sup> 16.23 ± 0.52<sup>b</sup> 19.03 ± 0.75<sup>a</sup></td></tr><tr><td>Antioxidant release (%)</td><td>– 82.45 ± 1.10<sup>c</sup> 85.32 ± 1.25<sup>b</sup> 88.24 ± 0.95<sup>a</sup></td></tr><tr><td>Inhibitory halos (mm)</td><td>S. aureus 0 11.00 ± 0.04<sup>c</sup> 13.00 ± 0.02<sup>b</sup> 14.50 ± 0.05<sup>a</sup><br>E. coli 0 9.50 ± 0.03<sup>c</sup> 10.00 ± 0.04<sup>b</sup> 12.00 ± 0.04<sup>a</sup></td></tr></tbody></table>

Mean ± SE with different superscripts in a row (lower case alphabet) or column (upper case alphabet) differ significantly (P < 0.05), n = 6 for each treatment. T₀ (0.0 %) = film without any And-Pan-NPs, T₁ (0.5 %) = film with 0.5 % And-Pan-NPs, T₂ (1.0 %) = film with 1.0 % And-Pan-NPs, T₃ (1.5 %) = film with 1.5 % And-Pan-NPs, WVTR = water vapour transmission rate.
(using stir bars or fleas) during digestion. Sodium phosphate buffer (0.1 M) was used to change the pH of the digesta after gastric digestion. The samples for analysis were taken after completion of the digestive simulation and were subjected to centrifugation for 15 min (4000 x g) and clear supernatants were collected and analyzed (DPPH, ABTS and FRAP activities). The stored Got-met-Emul samples were analysed for pH and moisture content employing the methods described by Lone, Bhat, Aït-Kaddour et al. (2023) for meat emulsion.

## 2.8. Sensory analysis

The panels used to evaluate the cooked Got-met-Emul [3 replications for each time point (days 0, 7, and 14)] comprised ten members (30–50 years old five males and five females) with years of experience with routine sensory testing of meat products. The participants provided written consent, followed all ethical guidelines and regulations and used a 9-point scale to evaluate the cooked Got-met-Emul samples (marked by 3-digit codes and randomized) for five significant attributes (juiciness, flavour, texture, overall acceptability, and colour and appearance) (Lone et al., 2023). The Got-met-Emul samples (30 g) were cooked in a hot air oven (180 °C for 20 min) and served warm (40 °C) using a palate cleanser (potable water) between different samples.

## 2.9. Statistical analyses

The results presented in the Tables and Figures (as means±standard errors) were obtained after analysing data from various experiments (replicated six times, n = 6) using ANOVA [one-way ANOVA (film and particle characteristics), two-way ANOVA (storage characteristics) using a General linear model at 0.05 significance level or repeated measurements ANOVA (sensory data, 0.05 significance level)] using SPSS-21. The individual pair of means were subjected to DMRT to determine the significance.

# 3. Results and discussion

## 3.1. Physico-mechanical characteristics of the Crik-Prot-based film

Table 1 presents the data relating to the physico-mechanical properties of Crik-Prot-based composite film. The moisture content (%) of the Crik-Prot-based film decreased significantly with increasing levels of And-Pan-NPs (0.0–1.5 %). Due to their hydrophilic nature, proteins and polysaccharides generally allow the interaction of water molecules within the film matrix. However, the addition of bioactive agents can disrupt the interactions and impede hydrogen bond formation with water molecules, thus reducing the moisture content of the film (Rashid et al., 2023). A polymeric film with a low moisture content is generally preferred for food packaging (Yavari Maroufi et al., 2021). Adame et al. (2024) found a significant ($P < 0.05$) decrease in the moisture content of pullulan and starch-based composite film with increasing concentrations of *Litsea cubeba* essential oil (1.0–7.0 %). The addition of silica nanoparticles loaded with caffeic acid has also been reported to decrease the moisture content of the starch and CMC-based composite films (Lin et al., 2023).

While the thickness (mm) and elongation at break (%) of the Crik-Prot-based film showed no significant ($P > 0.05$) change with increasing levels of And-Pan-NPs (0.0–1.5 %), WVTR (mg/mt²) and transmittance (%) showed a significant decreasing trend with increasing concentration of And-Pan-NPs (0.0–1.5 %). In general, the thickness of a polymeric film should not exceed 0.25 mm for optimal performance. The thickness of the Crik-Prot-based film produced in our study falls within the recommended thickness range for biodegradable films meant for food packaging (Gniewosz et al., 2022). While polysaccharides and proteins generally produce films with poor water vapour barrier properties (Thangavelu & Kulandhaivelu, 2022), the addition of bioactive agents affects the density and film thickness and reduces the WVTR. A

significant ($P < 0.05$) increasing trend was observed in the density of the Crik-Prot-based film with increasing concentration of And-Pan-NPs (0.0–1.5 %) which contains silver metal. The bioactive agents can disrupt the interaction between the binding sites and water molecules within the film matrix and can also reduce the availability of water-binding sites. The addition of silica nanoparticles loaded with caffeic acid has been reported to increase the density and decrease the WVTR and oxygen permeability of the starch and CMC-based composite films (Lin et al., 2023). Studies such as Elsherif et al. (2024) have reported a significant decrease in the WVTR of chitosan films incorporated with rosemary and carvacrol nano-emulsions. A film with a low WVTR is preferred for food packaging as it reflects its moisture barrier properties and can reduce water availability for microbial growth and limit some adverse food reactions (Silva et al., 2023).

A significant decreasing trend was observed in the transmittance (%) of the Crik-Prot-based film with increasing concentrations of And-Pan-NPs (0.0–1.5 %). A clear film with a high transmittance is generally preferred for food packaging due to its clear food visibility and high consumer acceptance (Simmonds et al., 2018). However, low transmittance and high density can help preserve the sensory quality of food by reducing lipid oxidation by reducing exposure to UV light. A significant decrease in the transmittance of chitosan-based edible film has been reported on the incorporation of rosemary and carvacrol nano-emulsions (Elsherif et al., 2024).

## 3.2. Colour and bioactive properties of Crik-Prot-based film

Table 1 presents the results relating to the colour and bioactive properties of Crik-Prot-based film And-Pan-NPs. The Crik-Prot-based film showed a significant ($P < 0.05$) decreasing trend in L* (lightness/brightness) values and a significant ($P < 0.05$) increasing trend in a* [redness (positive values)/greenness (negative values)] and b* [yellowness (positive values)/blueness (negative values)] values with increasing concentration of And-Pan-NPs (0.0–1.5 %). The reduction in the transmission of light and increased scattering of light by And-Pan-NPs distributed uniformly in the film matrix might be responsible for this reduction in the brightness of the Crik-Prot-based film (Rashid et al., 2023). Lin et al. (2023) reported a significant decrease in the L* and an increase in a* and b* values of the starch and CMC-based composite film with the addition of silica nanoparticles loaded with caffeic acid, indicating that the brightness of the film decreased and its colour was close to red and yellow. These results indicate that the Crik-Prot-based film's redness and yellowness values increased with increasing concentrations of And-Pan-NPs, possibly due to the colourful compounds and pigments present in And-Pan extract. Adame et al. (2024) found a significant decrease in the L* and an increase in a* values of pullulan and starch composite films with increasing concentrations of L. cubeba essential oil (1–7 %). Elsherif et al. (2024) reported an increase in the b* values of chitosan edible films on the incorporation of rosemary and carvacrol nano-emulsions.

A significant ($P < 0.05$) increasing trend was observed in the antioxidant properties [ABTS and DPPH radical scavenging (% inhibition) and ferric ion reduction (FRAP)] of Crik-Prot-based film with increasing concentrations of And-Pan-NPs (0.0–1.5 %). This increase in the antioxidant potential of Crik-Prot-based film might be due to the increase in total phenolic and flavonoid contents which also showed an increasing trend with increasing concentrations of And-Pan-NPs. Further, the antioxidant release rate (%) of Crik-Prot-based film also showed an increasing trend with increasing concentrations of And-Pan-NPs. The And-Pan-NPs contain a high level of total phenolic and flavonoid contents (22.55 ± 0.59 µg GAE/mg and 39.29 ± 0.75 µg QE/mg, respectively) and also exhibited high DPPH, ABTS and FRAP activities [74.4 ± 0.58 (%), 81.6 ± 0.32 (%), and 40.2 ± 0.65 µM TE/100 g]. The And-Pan-leaf extract has been reported to have a wide spectrum of secondary metabolites with antioxidant properties such as phenolic compounds, saponins, tannins, alkaloids, and terpenoids (Bhati et al., 2023;
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

Mean ± SE with different superscripts differ significantly [alphabets (A, B, C, D) for each time point (days 0, 7 or 14) and numerals (1, 2, 3) for each treatment (T₀, T₁, T₂, T₃ or control)].

n = 6 (for each treatment)

TE = Trolox equivalents

Two-way ANOVA was used at a 0.05 level of significance

Control = samples without any film

T0 = samples with a film without And-Pan-NPs

T1 = samples with a film containing 0.5% And-Pan-NPs

T2 = samples with a film containing 1.0% And-Pan-NPs

T3 = samples with a film containing 1.5% And-Pan-NPs

<caption>**Fig. 1.** Effect of Crik-Prot-based film incorporated with And-Pan-NPs on the antioxidant potential of the goat meat emulsion</caption>

Mean ± SE with different superscripts differ significantly

[alphabets (A, B, C, D) for each time point (days 0, 7 or 14) and numerals (1, 2, 3) for each treatment (T₀, T₁, T₂, T₃ or control)].

n = 6 (for each treatment)

TE = Trolox equivalents

Two-way ANOVA was used at a 0.05 level of significance

Control = samples without any film

T0 = samples with a film without And-Pan-NPs

T1 = samples with a film containing 0.5% And-Pan-NPs

T2 = samples with a film containing 1.0% And-Pan-NPs

T3 = samples with a film containing 1.5% And-Pan-NPs

Chokthaweepanch et al., 2023). The And-Pan leaves are a rich source of diterpenoid lactones especially andrographolide, which is a major bioactive compound and associated with antioxidant and antimicrobial properties, in addition to neoandrographolide and isoandrographolide (Chokthaweepanch et al., 2023). The silver nanoparticles developed using And-Pan leaf extract have been reported to have strong antioxidant (DPPH and nitric oxide scavenging activities) and metal chelating properties (FRAP and CERAC) (Anantharaman et al., 2020). Yong et al. (2023) reported a significant increase in the DPPH radical scavenging activity of the soy-protein isolate-based film incorporated with curcumin. The addition of curcumin also increased the total phenolic content of the film. Similarly, the addition of silica nanoparticles loaded with caffeic acid has been reported to significantly increase the antioxidant properties (ABTS and DPPH radical scavenging activity) of the starch and CMC-based composite films compared to the control films (Lin et al., 2023). Adame et al. (2024) found a significant (P < 0.05) effect of concentrations of L. cubeba essential oil (1.0–7.0%) on the antioxidant release rate from the pullulan and starch-based composite films.

In general, the addition of plant extracts in the form of nanoparticles is considered a more effective way of preserving food products due to their small size and greater surface area that can accommodate more phytochemicals and due to better release rates. The And-Pan-NPs produced in our study showed an average particle size of 22.83 ± 3.24, zeta potential of -15.12 ± 0.84, a polydispersity index of 0.346 and plasmon resonance (UV-Vis spectroscopy) at 430 nm (indicated by a brown colour). Gajbhiye et al. (2024) reported an average particle size of 18–52 nm for silver nanoparticles developed using A. paniculata leaf extract. Gopalakrishnan et al. (2019) reported an average particle size of 20–50

nm and a peak absorbance at 430 nm for the silver nanoparticles prepared using A. paniculata leaf extract. Kotakadi et al. (2014) found a zeta potential of -50.7 mV for the silver nanoparticles prepared using A. paniculata plant extract.

A significant (P < 0.05) effect of the addition of And-Pan-NPs (0.5–2.0%) was observed on the antimicrobial properties (inhibitory halos) of Crik-Prot-based film against S. aureus and E. coli. Adame et al. (2024) found a significant (P < 0.05) increase in the size of inhibitory halos with increasing concentrations of L. cubeba essential oil (1.0–7.0%) in composite films against E. coli and S. aureus. Similarly, the addition of silica nanoparticles loaded with caffeic acid has been reported to significantly increase the antimicrobial properties of the composite films against E. coli and S. aureus (Lin et al., 2023). The And-Pan-NPs (0.5–2.0%) inhibited the growth of S. aureus and E. coli and showed inhibitory halos of size ranging from 13.50–16.0 mm and 10.0–13.50, respectively. A MIC value of 1.0 and 3.0 mg/ml were found against S. aureus and E. coli, respectively. In general, the zone of inhibitions greater than 14 mm represents strong antimicrobial activity and the size between 14.0–9.0 mm reflects intermediate activity and those of <9 mm indicate a weak activity. The And-Pan leaves are a rich source of diterpenoid lactones, such as andrographolide, neoandrographolide, and isoandrographolide, with antimicrobial properties (Chokthaweepanch et al., 2023). Sinha and Paul (2015) reported strong antimicrobial properties for the silver nanoparticles prepared using A. paniculata leaf extract. The presence of phytochemicals, such as alkaloids, flavonoids, amino acids, saponins, tannins, and terpenoids, imparted antimicrobial properties to the silver nanoparticles against seven examined bacteria including Pseudomonas aeruginosa (Sinha & Paul, 2015). The
<caption>**Table 2**</caption>

Effect of Crik-Prot-based film incorporated with And-Pan-NPs on lipid stability, protein oxidation and physicochemical parameters of goat meat emulsion.

<table><thead><tr><th rowspan="2">Treatments</th><th colspan="3">Storage days (4 ± 1 °C)</th></tr><tr><th>0</th><th>7</th><th>14</th></tr></thead><tbody><tr><td>TBARS (mg malondialdehyde/kg)</td><td></td><td></td><td></td></tr><tr><td>Control</td><td>0.38 ± 0.03<sup>Ac</sup></td><td>0.64 ± 0.03<sup>Ab</sup></td><td>0.86 ± 0.02<sup>AA</sup></td></tr><tr><td>T<sub>0</sub> (0.0 %)</td><td>0.37 ± 0.04<sup>Ac</sup></td><td>0.60 ± 0.02<sup>Ab</sup></td><td>0.83 ± 0.02<sup>AA</sup></td></tr><tr><td>T<sub>1</sub> (0.5 %)</td><td>0.36 ± 0.02<sup>Ac</sup></td><td>0.51 ± 0.02<sup>Bb</sup></td><td>0.59 ± 0.03<sup>Ba</sup></td></tr><tr><td>T<sub>2</sub> (1.0 %)</td><td>0.35 ± 0.01<sup>Ac</sup></td><td>0.46 ± 0.01<sup>Cb</sup></td><td>0.53 ± 0.02<sup>Ca</sup></td></tr><tr><td>T<sub>3</sub> (1.5 %)</td><td>0.35 ± 0.02<sup>Ac</sup></td><td>0.39 ± 0.02<sup>Db</sup></td><td>0.46 ± 0.02<sup>Da</sup></td></tr><tr><td>Free fatty acids (% oleic acid)</td><td></td><td></td><td></td></tr><tr><td>Control</td><td>0.093 ± 0.003<sup>Ac</sup></td><td>0.149 ± 0.003<sup>Ab</sup></td><td>0.214 ± 0.003<sup>AA</sup></td></tr><tr><td>T<sub>0</sub> (0.0 %)</td><td>0.093 ± 0.002<sup>Ac</sup></td><td>0.142 ± 0.002<sup>Ab</sup></td><td>0.209 ± 0.006<sup>AA</sup></td></tr><tr><td>T<sub>1</sub> (0.5 %)</td><td>0.089 ± 0.004<sup>Ac</sup></td><td>0.125 ± 0.003<sup>Bb</sup></td><td>0.153 ± 0.002<sup>Ba</sup></td></tr><tr><td>T<sub>2</sub> (1.0 %)</td><td>0.089 ± 0.003<sup>Ac</sup></td><td>0.110 ± 0.004<sup>Cb</sup></td><td>0.135 ± 0.004<sup>Ca</sup></td></tr><tr><td>T<sub>3</sub> (1.5 %)</td><td>0.089 ± 0.003<sup>Ac</sup></td><td>0.097 ± 0.003<sup>Db</sup></td><td>0.123 ± 0.003<sup>Da</sup></td></tr><tr><td>Total carbonyl content (nmol/mg protein)</td><td></td><td></td><td></td></tr><tr><td>Control</td><td>1.05 ± 0.04<sup>Ac</sup></td><td>1.79 ± 0.03<sup>Ab</sup></td><td>2.03 ± 0.02<sup>AA</sup></td></tr><tr><td>T<sub>0</sub> (0.0 %)</td><td>1.04 ± 0.03<sup>Ac</sup></td><td>1.76 ± 0.03<sup>Ab</sup></td><td>1.98 ± 0.01<sup>AA</sup></td></tr><tr><td>T<sub>1</sub> (0.5 %)</td><td>1.04 ± 0.04<sup>Ac</sup></td><td>1.65 ± 0.02<sup>Bb</sup></td><td>1.90 ± 0.02<sup>Ba</sup></td></tr><tr><td>T<sub>2</sub> (1.0 %)</td><td>1.03 ± 0.05<sup>Ac</sup></td><td>1.58 ± 0.01<sup>Cb</sup></td><td>1.84 ± 0.01<sup>Ca</sup></td></tr><tr><td>T<sub>3</sub> (1.5 %)</td><td>1.02 ± 0.03<sup>Ac</sup></td><td>1.50 ± 0.02<sup>Db</sup></td><td>1.73 ± 0.02<sup>Da</sup></td></tr><tr><td>pH</td><td></td><td></td><td></td></tr><tr><td>Control</td><td>6.25 ± 0.02<sup>Ac</sup></td><td>6.41 ± 0.02<sup>Ab</sup></td><td>6.62 ± 0.02<sup>AA</sup></td></tr><tr><td>T<sub>0</sub> (0.0 %)</td><td>6.24 ± 0.03<sup>Ac</sup></td><td>6.40 ± 0.01<sup>Ab</sup></td><td>6.60 ± 0.01<sup>AA</sup></td></tr><tr><td>T<sub>1</sub> (0.5 %)</td><td>6.22 ± 0.03<sup>Ac</sup></td><td>6.36 ± 0.01<sup>Bb</sup></td><td>6.50 ± 0.01<sup>Ba</sup></td></tr><tr><td>T<sub>2</sub> (1.0 %)</td><td>6.22 ± 0.01<sup>Ac</sup></td><td>6.31 ± 0.01<sup>Cb</sup></td><td>6.45 ± 0.02<sup>Ca</sup></td></tr><tr><td>T<sub>3</sub> (1.5 %)</td><td>6.21 ± 0.02<sup>Ac</sup></td><td>6.27 ± 0.01<sup>Db</sup></td><td>6.39 ± 0.01<sup>Da</sup></td></tr><tr><td>Moisture (%)</td><td></td><td></td><td></td></tr><tr><td>Control</td><td>64.48 ± 0.25<sup>Aa</sup></td><td>63.13 ± 0.29<sup>Bb</sup></td><td>62.12 ± 0.24<sup>Bc</sup></td></tr><tr><td>T<sub>0</sub> (0.0 %)</td><td>64.53 ± 0.42<sup>Aa</sup></td><td>64.03 ± 0.38<sup>Aa</sup></td><td>63.57 ± 0.08<sup>Aa</sup></td></tr><tr><td>T<sub>1</sub> (0.5 %)</td><td>64.54 ± 0.27<sup>Aa</sup></td><td>64.16 ± 0.16<sup>Aa</sup></td><td>63.73 ± 0.32<sup>Aa</sup></td></tr><tr><td>T<sub>2</sub> (1.0 %)</td><td>64.59 ± 0.53<sup>Aa</sup></td><td>64.28 ± 0.17<sup>Aa</sup></td><td>63.87 ± 0.34<sup>Aa</sup></td></tr><tr><td>T<sub>3</sub> (1.5 %)</td><td>64.60 ± 0.44<sup>Aa</sup></td><td>64.33 ± 0.25<sup>Aa</sup></td><td>64.17 ± 0.43<sup>Aa</sup></td></tr></tbody></table>

Mean ± SE with different superscripts in a row (lower case alphabet) and column (upper case alphabet) differ significantly (P < 0.05).

n = 6 for each treatment.

Control = samples without any film.

T<sub>0</sub> (0.0 %) = samples with a film without any And-Pan-NPs.

T<sub>1</sub> (0.5 %) = samples with a film with 0.5 % of the And-Pan-NPs.

T<sub>2</sub> (1.0 %) = samples with a film with 1.0 % of the And-Pan-NPs.

T<sub>3</sub> (1.5 %) = samples with a film with 1.5 % of the And-Pan-NPs.

A. paniculata leaf extract-based silver nanoparticles have been reported to exhibit antifungal properties against Aspergillus niger and Penicillium sp. (Inhibition zone: 12 mm and 14 mm, respectively) (Kotakadi et al., 2014). Eka Idorenyin Idorenyin et al. (2020) used A. paniculata leaf extract of 2.50 mg/ml strength and reported a zone of inhibition of 15 mm size against E. coli. Generally, the phytochemicals in the plant extracts affect the microbes by damaging their cell membranes, thereby slowing their growth or causing death.

### 3.3. Antioxidant potential of Got-met-Emul packed within Crik-Prot-based film

Fig. 1 (A–C) presents the data related to the antioxidant potential of Got-met-Emul packed within the Crik-Prot-based film. The Got-met-Emul samples packed within the Crik-Prot-based films containing And-Pan-NPs [0.5 % (T<sub>1</sub>), 1.0 % (T<sub>2</sub>), and 1.5 % (T<sub>3</sub>)] showed significant (P < 0.05) higher antioxidant potential in terms of DPPH, ABTS and FRAP compared to the Got-met-Emul samples packed within T<sub>0</sub> films containing no And-Pan-NPs and samples without any film (control) during entire storage of two weeks. The concentration of And-Pan-NPs also showed a significant (P < 0.05) positive impact on days 7 and 14 and the Got-met-Emul packed within T<sub>3</sub> films showed higher antioxidant potential than T<sub>2</sub> films which in turn showed higher antioxidant potential than the Got-met-Emul packed within T<sub>1</sub> films. The A. paniculata leaf extract contains a wide spectrum of secondary metabolites with strong antioxidant properties such as phenolic compounds, saponins,

tannins, alkaloids, and terpenoids (Bhati et al., 2023; Chokthaweepanch et al., 2023). These phytochemicals leach out from the film matrix and enhance the antioxidant potential of Got-met-Emul. The higher release rate (%) of the films containing higher concentrations of And-Pan-NPs might be the reason for the higher antioxidant activities of Got-met-Emul samples packed within. The antioxidant activities (DPPH, ABTS, and FRAP) of Got-met-Emul packed within the films showed a significant (P < 0.05) increase on day 7 of storage and thereafter a significant decline was recorded on day 14 indicating a higher rate of release of antioxidant molecules from the films during the first week followed by a decreased release rate during the second week of storage. Studies have reported a significant increase in the antioxidant activities (DPPH and ABTS activities) of food products, such as cheese, packed within the bioactive films during storage (Flórez et al., 2024). A similar increase in the antioxidant activity of the stored products during the initial phase of storage and a decrease during the last phase of storage has been reported for food products packed within the edible bioactive films (Noor et al., 2024). This biphasic release rate of active molecules (initial high release rate followed by lower sustained rate) from film matrices has been reported by previous studies. The decrease in the antioxidant activity of Got-met-Emul after day 7 may also be attributed to the higher rate of lipid and protein oxidation resulting in quenching of more and more bioactive molecules.

### 3.4. Lipid stability and protein oxidation of Got-met-Emul during storage

Table 2 presents the results related to lipid stability [TBARS (mg MDA/kg) and free fatty acids (FFA, % oleic acid)] and protein oxidation [total carbonyl content (TCC, nmol/mg protein)] of Got-met-Emul packed within Crik-Prot-based film during storage. The Got-met-Emul packed within Crik-Prot-based film containing And-Pan-NPs [0.5 % (T<sub>1</sub>), 1.0 % (T<sub>2</sub>), and 1.5 % (T<sub>3</sub>)] showed significantly (P < 0.05) lower values for TBARS and FFA on days 7 and 14. An effect of the concentration of And-Pan-NPs in the film was observed and Got-met-Emul packed within T<sub>3</sub> films showed the lowest values for both TBARS and FFA followed by T<sub>2</sub> and T<sub>1</sub> in that sequence. The Got-met-Emul packed within the control film (T<sub>0</sub>) and without any film showed comparable values for both TBARS and FFA during the entire storage time. Similar results were found for TCC and Got-met-Emul packed within Crik-Prot-based film containing And-Pan-NPs showed significantly (P < 0.05) lower values than control samples. The lowest TCC values were observed for the Got-met-Emul samples packed within T<sub>3</sub> films followed by T<sub>2</sub> and T<sub>1</sub> in that sequence. This effect of concentration of And-Pan-NPs in film on lipid and protein oxidation of Got-met-Emul might be attributed to the antioxidant release rate (%) which is higher for T<sub>3</sub> films followed by T<sub>2</sub> and T<sub>1</sub>. The And-Pan-leaf extract has been reported to have a wide spectrum of secondary metabolites with antioxidant properties such as phenolic compounds, saponins, tannins, alkaloids, and terpenoids (Bhati et al., 2023; Chokthaweepanch et al., 2023). The hydroxyl groups present in antioxidant molecules such as polyphenolic compounds can donate hydrogen atoms which can scavenge the free radicals and form stable macromolecules, thereby reducing lipid and protein oxidation (Bhat et al., 2024). Further, the metal chelating properties of And-Pan-NPs can reduce the oxidation rate as metal ions are known to catalyse the oxidation process of biological macromolecules. The antimicrobial properties of And-Pan-NPs can further help in reducing the microbial load and thereby the lipolysis of lipids by microbial lipases and the production of fatty acids. Farahani et al. (2024) developed an active edible coating using seed mucilage as a base material and *Satureja hortensis* essential oil as an active agent for extending the shelf life of lamb meat. The meat samples with the edible coating containing essential oil showed significantly lower values for TBARS and peroxide values compared to the uncoated samples during 18 days of refrigerated storage (4 °C). A significant impact of the concentration of the essential oil (0.5–1.5 %) was reported and the samples with coatings containing the highest concentration of essential oil showed the lowest values for
<figure><img src="image_5.png" /><figcaption>Fig. 2. Effect of Crik-Prot-based film incorporated with And-Pan-NPs on the microbial quality of goat meat emulsion</figcaption></figure>

Mean ± SE with different superscripts differ significantly [alphabets (A, B, C, D) for treatments (T0, T1, T2, T3, and control) and numerals (1, 2) for digestion (before or after)]

n = 6 (for each treatment), TE = Trolox equivalents

Two-way ANOVA was used at a 0.05 level of significance

Control = samples without any film.

T0 = samples with a film without And-Pan-NPs

T1 = samples with a film containing 0.5 % And-Pan-NPs

T2 = samples with a film containing 1.0 % And-Pan-NPs

T3 = samples with a film containing 1.5 % And-Pan-NPs

Mean ± SE with different superscripts differ significantly [alphabets (A, B, C, D) for each time point (days 0, 7 or 14) and numerals (1, 2, 3) for each treatment (T₀, T₁, T₂, T₃ or control)].

n = 6 (for each treatment)

Detection limit <10 cfu/g

Two-way ANOVA was used at a 0.05 level of significance.

Control = samples without any film

T0 = samples with a film without And-Pan-NPs

T1 = samples with a film containing 0.5% And-Pan-NPs

T2 = samples with a film containing 1.0% And-Pan-NPs

T3 = samples with a film containing 1.5% And-Pan-NPs

TBARS and peroxide values during the entire storage time. Similarly, Yan et al. (2024) reported a significant decrease in lipid oxidation (TBARS) and protein oxidation (total carbonyl content and total sulfhydryl groups) of fresh beef samples coated with gelatin-chitosan supplemented with *Cyclocarya paliurus* flavonoid (0.1–0.3 %) during 14 days of refrigerated storage in comparison to the control beef samples (without film and with control film). Further, Lin et al. (2023) reported a significant decrease in the lipid oxidation (TBARS values) of fresh beef and chicken samples packed within the composite films containing silica nanoparticles loaded with caffeic acid during the entire storage time.

### 3.5. Effect of Crik-Prot-based film on the pH and moisture content of Got-met-Emul

The Got-met-Emul packed within Crik-Prot-based film containing And-Pan-NPs [0.5 % (T₁), 1.0 % (T₂), and 1.5 % (T₃)] showed significantly (*P* < 0.05) lower values for pH on days 7 and 14 (Table 2). An effect of the concentration of And-Pan-NPs in the film was observed and Got-met-Emul packed within T₃ films showed the lowest values followed by T₂ and T₁ in that sequence. The Got-met-Emul packed within the control film (T₀) and without any film showed comparable (*P* > 0.05) values during the storage. This positive effect on the pH might be attributed to the antimicrobial properties of Crik-Prot-based film which can reduce the microbial production of alkaline metabolites in the Got-met-Emul during storage. Adame et al. (2024) found significantly (*P* < 0.05) lower pH for fresh beef packed within pullulan and starch-based composite film containing *L. cubeba* essential oil (3.0 %) during refrigerated storage for one week compared to the control samples packed

within plastic packaging and without any packaging. A similar effect of a mucilage-based active film has been reported on the pH of fresh lamb by Farahani et al. (2024) who reported significantly higher pH values for uncoated samples whereas coated samples showed significantly lower values during entire storage. An effect of concentration was observed and meat samples with coatings with higher levels of *S. hortensis* essential oil (0.5–1.5 %) showed more decline in pH compared to the uncoated samples. Significantly lower pH values have been reported for fresh beef and chicken samples packed within the starch and CMC-based composite films containing caffeic acid-loaded silica nanoparticles during the entire storage time compared to the control samples without any film (Lin et al., 2023).

The Got-met-Emul packed within Crik-Prot-based film (T₀, T₁, T₂, and T₃) showed significantly (*P* < 0.05) higher moisture content on days 7 and 14 compared to the control samples without any film (Table 2). Farahani et al. (2024) has reported a positive impact of a mucilage-based active coating on the moisture content of fresh lamb meat. An effect of concentration was observed (0.0–1.5 %) and coatings with higher levels of *S. hortensis* essential oil were more effective in retaining the moisture content of the meat during refrigerated storage. While an increasing trend was observed in the moisture content of Got-met-Emul with the increasing concentration of And-Pan-NPs in the film, however, no significant (*P* > 0.05) difference was observed between the films in our study.
<figure><img src="image_6.png" /></figure>

Mean ± SE with different superscripts differ significantly [alphabets (A, B, C, D) for treatments (T₀, T₁, T₂, T₃, and control) and numerals (1, 2) for digestion (before or after)]
n = 6 (for each treatment), TE = Trolox equivalents
Two-way ANOVA was used at a 0.05 level of significance
Control = samples without any film.
T0 = samples with a film without And-Pan-NPs
T1 = samples with a film containing 0.5% And-Pan-NPs
T2 = samples with a film containing 1.0% And-Pan-NPs
T3 = samples with a film containing 1.5% And-Pan-NPs

<caption>Fig. 3. Effect of in vitro gastrointestinal digestion on the antioxidant potential of goat meat emulsion</caption>

Mean ± SE with different superscripts differ significantly
[alphabets (A, B, C) for each time point (days 0, 7 and 14)
and numerals (1, 2, 3) for each treatment (T0, T1, T2, T3 or
control)].
10 panellists performed the sensory evaluation thrice for each
treatment at each time point (days 0, 7, and 14) using a 9-point
descriptive scale (1 denoted ‘disliked extremely’ and 9 denoted
‘liked extremely’).

Two-way ANOVA was used at a 0.05 level of significance.

Control = samples without any film.

T0 = samples with a film without And-Pan-NPs

T1 = samples with a film containing 0.5 % And-Pan-NPs

T2 = samples with a film containing 1.0 % And-Pan-NPs

T3 = samples with a film containing 1.5 % And-Pan-NPs

3.6. Effect of Crik-Prot-based film on the microbiological quality of Got-
met-Emul

The Got-met-Emul packed within Crik-Prot-based film containing And-Pan-NPs [0.5 % (T₁), 1.0 % (T₂), and 1.5 % (T₃)] showed significantly (*P* < 0.05) lower total plate counts (log₁₀ CFU/g) on days 7 and 14 (Fig. 2). An effect of the concentration of And-Pan-NPs in the film was observed and Got-met-Emul packed within T₃ films showed the lowest total plate counts followed by T₂ and T₁ in that sequence. The other enumerated counts (psychrophilic, coliforms, and yeast and moulds) were detected on day 14 and exhibited similar trends i.e., the lowest counts were detected for Got-met-Emul packed within T₃ films followed by T₂ and T₁ in that sequence. The Got-met-Emul packed within the control film (T₀) and without any film showed comparable (*P* > 0.05) counts during the storage. The presence of Crik-Prot-based film containing And-Pan-NPs improved the microbial quality of Got-met-Emul during storage. The silver nanoparticles developed using And-Pan leaf extract have been reported to have strong antimicrobial activities against both Gram-positive (such as Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus) and Gram-negative (such as Klebsiella pneumonia, Salmonella typhimurium) bacteria and fungi (Candida albicans, Aspergillus niger, Penicillium sp. and Botrytis cinerea) (Anantharaman et al., 2020; Kotakadi et al., 2014). A similar effect of a mucilage-based active film has been reported on the microbiological quality (log CFU/g) of fresh lamb by Farahani et al. (2024) who reported

significantly lower counts for total viable counts, psychrophilic counts,
*E. coli*, *S. aureus* and yeast/moulds for the coated samples compared to
the uncoated samples during the entire storage time. An effect of con-
centration was observed and meat samples with coatings with higher
levels of the essential oil (0.5–1.5 %) showed significantly lower counts
for all the enumerated microbes compared to the coatings with lower
levels. Ali Ghoflogar Ghasemi et al. (2024) reported significantly (*P* <
0.05) lower total plate and *S. aureus* counts and TVB-N values for fresh
beef samples with CMC coating containing ZnO nanoparticles and
nanostructured lipid carriers loaded with savory essential oil compared
with the uncoated samples during 12 days of refrigerated storage. The
total aerobic bacteria and total coliforms were significantly lower in the
beef burgers packed within chitosan film containing chitosan nano-
particles incorporated with cinnamaldehyde during refrigerated storage
compared to the control samples without film and with chitosan film
(Mondéjar-López et al., 2024).

3.7. Digestion simulation of Got-met-Emul

The Got-met-Emul packed within Crik-Prot-based film containing And-Pan-NPs (T₁, T₂, and T₃) showed significantly (*P* < 0.05) higher values for antioxidant potential (DPPH, ABTS, and FRAP activities) before and after digestion simulation compared to the control samples (Fig. 3). The same trends were observed on days 0 and 7. No effect of the concentration of And-Pan-NPs was observed on day 0 whereas a
<figure><img src="image_7.png" /><figcaption>Fig. 4. Effect of Crik-Prot-based film incorporated with And-Pan-NPs on sensory quality of goat meat emulsion.</figcaption></figure>

significant effect was found on day 7 and Got-met-Emul packed within T₃ films showed the highest values followed by T₂ and T₁ in that sequence. The Got-met-Emul packed within the control film (T₀) and without any film showed comparable values before and after digestion. This positive effect of the film containing And-Pan-NPs on the antioxidant potential of Got-met-Emul is beneficial for the health of consumers. Previous studies have reported a positive impact of bioactive films on the antioxidant properties of foods during gastrointestinal digestion indicating enhanced functional value (Jatav et al., 2023; Noor et al., 2024).

### 3.8. Effect of Crik-Prot-based film on the sensory quality of Got-met-Emul

The Got-met-Emul packed within Crik-Prot-based film containing And-Pan-NPs (T₁, T₂, and T₃) showed significantly (P < 0.05) higher scores for colour and appearance on days 7 and 14 (Fig. 4). An effect of the concentration of And-Pan-NPs in the film was observed and Got-met-Emul packed within T₃ films obtained the highest scores followed by T₂ and T₁ in that sequence. The Got-met-Emul packed within the control film (T₀) and without any film showed comparable values during the storage. Similar patterns were found for other sensory attributes and significantly (P < 0.05) higher scores were observed for Got-met-Emul packed within Crik-Prot-based film containing And-Pan-NPs for flavour, texture and overall acceptability on day 7. The samples were not evaluated for these parameters on day 14. For juiciness, Got-met-Emul packed within the Crik-Prot-based film showed significantly (P < 0.05) higher scores compared to the control samples without any film. The higher sensory scores of Got-met-Emul packed within Crik-Prot-based film containing And-Pan-NPs might be attributed to lower oxidation of lipids and proteins and lower microbial spoilage. These deteriorative processes affect the sensory quality of meat and meat products. A similar effect of a mucilage-based active film has been reported on the sensory quality of fresh lamb by Farahani et al. (2024) who reported significantly higher scores for colour, odour, and overall

acceptability for the coated samples compared to the uncoated samples during the entire refrigerated storage. An effect of concentration was observed and meat samples with coatings with higher levels of the essential oil showed significantly higher scores for all the attributes compared to the coatings with lower levels. Similarly, Ali Ghoflgar Ghasemi et al. (2024) reported significantly (P < 0.05) higher scores for odour, colour and overall acceptability during 12 days of refrigerated storage for fresh beef with CMC coating containing ZnO nanoparticles and nanostructured lipid carriers loaded with savory essential oil compared with the uncoated control.

### 4. Conclusions

A Crik-Prot-based composite film was developed to enhance the quality of Got-met-Emul during refrigerated storage. The And-Pan-NPs (0.5–1.5 %) were added to impart antimicrobial and antioxidant properties to the film, which also affected its barrier and colour properties. Applying Crik-Prot-based film for preserving Got-met-Emul enhanced its lipid and protein oxidative stability, antioxidant potential, and microbiological quality during two weeks of storage (4 ± 1 °C). Both Crik-Prot-based film and gastrointestinal digestion exhibited a positive impact on the antioxidant potential of Got-met-Emul. Future studies should examine the efficacy of the film using other food products and evaluate the durability of the film under factory and field conditions.

#### Ethical statement

The study was not performed in animals or humans.

#### CRediT authorship contribution statement

**Aunzar Bashir Lone:** Methodology, Investigation, Formal analysis.
**Sunil Kumar:** Supervision, Methodology, Data curation.
**Rana Muhammad Aadil:** Writing – review & editing, Software, Methodology,
Data curation. Abdo Hassoun: Writing – review & editing, Validation, Methodology. Data curation. Abderrahmane Aït-Kaddour: Writing – review & editing, Visualization, Software, Resources, Methodology, Data curation. Zuhaib F. Bhat: Writing – original draft, Supervision, Resources, Methodology, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

None.

Data availability

Data will be made available on request.

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