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Article

Eugenol@Natural Zeolite Nanohybrid vs. Clove Powder as Active and Reinforcement Agents in Novel Brewer’s Spent Grain/Gelatin/Glycerol Edible, High Oxygen Barrier Active Packaging Films

by
Zoe Ntari
1,
Achilleas Kechagias
1,
Areti A. Leontiou
1,
Alexios Vardakas
1,2,
Margarita Dormousoglou
3,
Tarsizia Angelari
1,
Konstantinos Zaharioudakis
1,
Panagiota Stathopoulou
3,
Panagiota Karahaliou
4,
Grigorios Beligiannis
1,
Charalampos Proestos
5,
Constantinos E. Salmas
6,* and
Aris E. Giannakas
1,*
1
Department of Food Science and Technology, University of Patras, 30100 Agrinio, Greece
2
GAEA Products S.M. S.A., 1st km Agriniou—Karpenisiou National Rd., 30100 Agrinio, Greece
3
Department of Sustainable Agriculture, University of Patras, 30100 Agrinio, Greece
4
Department of Physics, University of Patras, 26504 Patras, Greece
5
Laboratory of Food Chemistry, Department of Chemistry, National and Kapodistrian University of Athens Zografou, 15771 Athens, Greece
6
Department of Materials Science Engineering, University of Ioannina, Dourouti, 45110 Ioannina, Greece
*
Authors to whom correspondence should be addressed.
Appl. Sci. 2025, 15(17), 9282; https://doi.org/10.3390/app15179282
Submission received: 10 July 2025 / Revised: 11 August 2025 / Accepted: 20 August 2025 / Published: 23 August 2025
(This article belongs to the Special Issue Unlocking the Potential of Agri-Food Waste for Innovative Applications and Bio-Based Materials, 2nd Edition)
Browse Figures
Versions Notes

Abstract

Following the trend of food waste valorization to produce innovative bio-based materials, this study proposes the conversion of brewer’s spent grain (BSG) into added value edible, high oxygen barrier, flexible, active packaging films via an extrusion molding compression method. Gelatin (Gel) was used as both a reinforcement and barrier agent and glycerol (Gl) as a plasticizer. Eugenol was nanoencapsulated on natural zeolite (EG@NZ), and pure clove powder (ClP) was used as an active agent to obtain BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP (x = 5, 10, and 15 %wt.) active films. Both BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP films show enhanced tensile, oxygen barrier, antioxidant, and antibacterial properties, and low toxicity and genotoxicity values. All BSG/Gel/Gl/xEG@NZ films presented a higher oxygen barrier, higher total phenolic content (TPC) values, higher antioxidant activity according to a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, higher inhibition zones against Staphylococcus aureus and Escherichia coli, and lower toxicity and genotoxicity than all BSG/Gel/Gl/xClP films. Thus, the superiority of the nanoencapsulated EG in NZ as compared to the physical encapsulated EG in ClP is proved. Briefly, BSG/Gel/Gl/15EG@NZ active film exhibited ~218% higher tensile strength, ~93% higher TPC value, and ~90% lower effective concentration for a 60% antioxidant activity value (EC60) as compared to the pure BSG/Gel/Gl film. The zones against S. aureus and E. coli were 45 and 30 mm, respectively, and the oxygen barrier was zero. The use of this film extended the shelf life of fresh minced meat by two days and exhibited the high potential to be used as active packaging material.

Graphical Abstract

1. Introduction

Climate change global issues impose the development of new technologies to reduce the carbon fingerprint, the use of fossil fuels, and to turn the global economy from linear to circular and sustainable [1,2,3,4,5,6,7]. In the food industry and food technology sector, these trends promote the development of new technologies/processes to reduce food waste and utilize food by-products [8,9,10,11]. Thus, food technology in the era of bioeconomy and sustainability tries to reduce food waste, recovers biopolymers and bioactive compounds from food and agricultural by-products, and reuses them in the development of bio-based/edible packaging, bio-based food additives, and the development of functional foods [1,2,3,4,5].
Valorization of industrial by-products is a key factor for building a circular economy and creating a sustainable production [12]. Recently, attention has been given to the generation of large quantities of spent grain from brewing and distilling processes. Researchers have given emphasis in the development of different valorization processes for the added-value use of brewer’s spent grain (BSG) beyond its traditional use in animal feed (70%) for biogas production (10%) and disposition in landfills (20%) [13]. Brewer’s spent grain (BSG) is a brewing industry by-product that makes up 85 % of brewing waste [14]. The annual production of wet BSG stands at approximately 8 million tons in Europe and 40 million tons worldwide [12,13]. BSG is a lignocellulosic biomass rich in proteins, lipids, minerals, and vitamins. The wet BSG contains up to 80% moisture, while in the dried BSG, moisture has decreased to 5–8%. Dried BSG also contains 14–30% crude protein, 3–10% lipids, 0.4–2.17% starch, and 50–70% total fiber, which has a nutrient-added value. Fiber, a crucial nutrient with significant health benefits, contains ~16% cellulose, ~28% hemicellulose, and ~7% lignin and various monosaccharides, oligosaccharides, and polysaccharides, as well as micronutrients such as vitamins, minerals, amino acids, and polyphenols [14]. Thus, BSG has a large volume of nutrient dense by-products that have great potential to be used in the context of sustainable food transition [14,15]. Although BSG has already been used for animal feed, biogas production, and disposition in landfills, its use in the development of biodegradable/edible food packaging is limited [16]. BSG has great potential as a raw material for the development of sustainable packaging because of the ability of its proteins to interact with the polypeptide chains, and because cellulose, which has been utilized in food packaging, is one of the major components of BSG. BSG exhibited enhanced antioxidant potential, which make it a byproduct for the development of antioxidant active packaging materials [17,18,19,20]. To use BSG in the development of packaging materials, plasticizers such as polyethylene glycol (PEG) or glycerol are added [14] to reduce its brittleness and increase its ductility. BSG has been used in the development of a six-pack beer carrier paperboard alternative [14]. BSG has also been added in polyurethane to enhance its thermal stability and has been blended with chitosan to enhance its antibacterial activity [10,21]. Nanocomposite films with UV-barrier, antioxidant, and antimicrobial properties and enhanced thermal and mechanical properties based on nanocellulose (NFC) and BSG arabinoxylans were prepared [22]. BSG protein-based active films have been developed by using PEG or glycerol as plasticizers [23]. The water barrier properties, solubility, optical properties, antioxidant (reducing power, ABTS•+, and lipidic radical scavenging) properties, and the antimicrobial properties of obtained films were studied [23]. Recently, BSG-based composite films have been developed by employing poly (vinyl alcohol) (PVA) and glycerol as binder materials, along with hexa-methoxy-methyl-melamine (HMMM) as a water-repelling agent [24]. The study results that films with a practical BSG content varying from 20 to 40 %wt. exhibited a balance between moisture absorption and mechanical strength [24]. The addition of glycerol enhanced the ductility and toughness of the films, while the addition of HMMM enhanced their water resistance [24].
In this paper, a nanohybrid made by natural zeolite (NZ) modified with eugenol (EG) essential oil (EO) and pure clove powder (ClP) is used as both the reinforcement and active agent for a BSG/gelatin, (Gel)/Glycerol (Gl) composite film. It is well known that EO derivatives such as EG are bio-based antioxidant/antibacterial compounds, which could potentially replace chemical additives used as antioxidant/antibacterial agents in food preservation [25,26,27,28,29]. It is also known that the adsorption of such EOs on natural nanocarriers such as nanoclays, natural zeolites, silicates and activated carbons ensures the slow loss of their activity and give rise to their controlled release in food [30,31,32,33,34,35]. EG, which can be extracted from clove powder, has been previously widely used in active food packaging and meat preservation [36,37,38,39]. Clove powder (ClP), which contains physically encapsulated EG, is well known for its antioxidant/antibacterial activity and has also been previously studied in meat preservation [40,41].
Specific innovative points of the current study are as follows: (i) The preparation via an extrusion molding compression method and the tensile properties, oxygen barrier properties, antioxidant activities, antibacterial activities, and toxicity/genotoxicity characterization of such BSG/Gel/Gl/xEG@NZ (where x = 5, 10 and 15 %wt.) and BSG/Gel/Gl/xClP (where x = 5, 10, and 15 %wt.) active films is, for first time, reported and (ii) to the best of our knowledge, a comparison study between EG@NZ nanohybrid and clove powder as both the reinforcement and antioxidant/antibacterial agents in BSG/Gel/Gl-based active packaging films is, for first time, reported, too.

2. Materials and Methods

2.1. Materials

Gelatin type A used (CAS 9000-70-8) was purchased from Thermo Scientific Chemicals (Thermo Fisher Scientific. 168 Third Avenue. Waltham, MA, USA). Eugenol, 2-Methoxy-4-(2-propenyl) phenol, 4-Allyl-2-methoxyphenol, 4-Allylguaiacol (CAS 97-53-0), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (CAS 1898-66-4, caffeic acid (CAS 331-39-5), 2-Thiobarbituric acid reagent for sorbic acid (CAS 504-17-6), Absolute ethanol (C2H6O) (CAS 64-17-5), sodium carbonate (Na2CO3) (CAS 497-19-8), and Folin Ciocalteü (47641) were purchased from Sigma-Aüldrich (Darmstadt, Germany). Ethanol ROTIPURAN® ≥ 99.8%, p.a. (CAS 64-17-5) was purchased from Carl Roth (Karlsruhe, Germany). Gallic acid (3,4,5-trihydrobenzoic acid) 99% is isolated from Rhus chinensis Mill (JNK Tech. Co., Ulsan, Republic of Korea). All the other reagents and solvents used were of analytical grade. Natural zeolite powder, 100 g, with Product Code: 102.057.004 and purity higher than 99% was purchased from Health Trade (Patras, Greece). Dried brewer’s spent grain was gifted from KYKAO handcrafted brew (Platani 26504, Patra, Greece https://kykao.gr/, accessed on 5 May 2025). Clove powder was purchased for the local Super Market.

2.2. Preparation of BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP Films

For the preparation of the EG@NZ nanohybrid used in development of BSG/Gel/Gl/xEG@NZ films, the recently reported vacuum-assisted adsorption process was followed [38]. Briefly, 2 g of as-received NZ was placed in a round-bottom glass flask and subjected to heating at 100 °C under a vacuum for 15 min to eliminate any adsorbed moisture. Following the drying process, EG was gradually added dropwise to the dried NZ under continuous stirring to facilitate uniform adsorption. The resulting EG@NZ nanohybrid was then collected and weighed. The calculated loading content of EG on the nanohybrid was approximately 58 %wt. For the preparation of BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP films, as well as BSG/Gel/Gl film via with the extrusion method, a twin-screw mini lab extruder (Haake Mini Lab II, Thermo Scientific, ANTISEL, S.A., Athens, Greece) was operated. For all the prepared films, twin-extruder conditions were as follows: 110 °C, 250 rounds per minute (rpm), and 5 min operation time. The amounts of BSG, Gel, Gl, H2O, EG@NZ nanohybrid, and ClP used, as well as the twin extruder operating conditions with sample code names, are listed in Table 1. After the extrusion process, the collected pellets were transformed into films via the compression molding method. Films with an average diameter of 10 cm and an average thickness of 0.2 mm were obtained by heat pressing approximately 2 g of pellets at 110 °C with 1 tone (tn) pressure for 2 min by using a thermostatic hydraulic press (Specac Atlas™ Series Heated Platens, Specac, Orpinghton, UK). This temperature, i.e., 110 °C, was chosen because the used polymeric matrices melting stage started after 90 °C. After repetitions and visual observations, we empirically chose the temperature of 110 °C as the preparation temperature, because we observed homogeneity in the final film samples in both cases with or without the zeolite nanohybride. The temperature and pressure conditions were the same for all mixtures used for the film preparation.

2.3. Physicochemical Characterization of BSG/Gel/Gl, BSG/Gel/Gl/xEG@NZ, and BSG/Gel/Gl/xClP Films

All obtained BSG/Gel/Gl, BSG/Gel/Gl/xEG@NZ, and BSG/Gel/Gl/xClP films were characterized with X-Ray Diffraction (XRD), Fourier Transform Infrared (FTIR) spectroscopy with Attenuated Total Reflectance (ATR), and Scanning Electron Microscopy (SEM) measurements by using the instrumentation and following the methodology and experimental settings given in detail in the Supplementary Materials file.

2.4. Charactrization of Packaging Properties of BSG/Gel/Gl, BSG/Gel/Gl/xEG@NZ, and BSG/Gel/Gl/xClP Films

Tensile and oxygen barrier properties of all obtained films were determined according to ASTM D638 and ASTM D 3985 methods correspondingly, by using the instrumentation and following the methodology and experimental settings described in the Supplementary Materials. Total phenic content (TPC) and in Vitro Antioxidant Activity according to a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay of all obtained films were determined according to the methodology described in detail in the Supplementary Materials file.

2.5. Antimicrobial Activity of BSG/Gel/Gl/10EG@NZ, BSG/Gel/Gl/10ClP Active Films

Standard and isolated strains of the following two bacteria, including one Gram-positive (Staphylococcus aureus (ATCC25923)) and one Gram-negative (Escherichia coli (RM03) bacteria, were used in screening the antimicrobial activity. The antimicrobial activity of the food film samples was determined using the disk diffusion method according to EUCAST guidelines. Bacteria from frozen glycerol stock cultures were grown aerobically at 37 °C in Mueller–Hinton broth (MHB) according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2018). Before each experimental procedure, cultures were transferred to a liquid medium and incubated for 24 h. Broth cultures were then adjusted to a specific optical density (OD) at 600 nm (OD600) using a pre-established calibration curve unique to each microorganism to achieve a cell density of 108 colony forming units per milliliter (CFU/mL). OD measurements were performed using a Q5000 micro-volume UV-Vis spectrophotometer (Quawell Technology, San Jose, CA, USA).

Disk Diffusion Susceptibility Test

Aseptically, a 100 mg sample of each food film sample was weighed and directly applied as a defined circular deposit onto the surface of Mueller–Hinton agar (MHA) plates, which had been previously inoculated with the respective test microorganism (108 CFU mL−1). The inoculated MHA plates were then incubated at 37 °C for 24 h. After the incubation, the diameter of the Zone of Inhibition was calculated using a calibrated ruler. The test was performed in triplicate for each test microorganism and each food film concentration.

2.6. Cytotoxic and Genotoxic Effects of Films in Human Lymphocytes

2.6.1. Ethics Statement and Approval

The experimental use of human lymphocytes was conducted in accordance with international bioethical criteria, following approval by the Research Ethics Committee of the University of Patras (Ref. No 11584/6 March 2018).

2.6.2. Whole Blood Collection and Cell Culture Preparation

Whole blood samples were collected in heparinized vectors from three healthy and non-smoking male donors (one 20 and two 25 years old) who previously declared that they were not exposed to radiation, drug treatment, or any viral infection in the recent past.

2.6.3. CBMN Assay

The cytotoxic and genotoxic potential of films (at concentrations ranged from 50 to 500 μg mL−1) in human lymphocytes was assessed using the Cytokinesis-Block Micronucleus (CBMN) assay with cytochalasin-B [42].
For each of the two donors, a 0.5 mL whole blood aliquot was introduced into 6.5 mL of Ham’s F-10 medium, supplemented with 1.5 mL fetal bovine serum (FBS), and 0.3 mL phytohemagglutinin (PHA) to stimulate lymphocyte proliferation. Twenty-four hours after initiating the cultures, the films were added to the 8.8 mL culture volume to achieve final concentrations of 50, 100, and 500 μg mL−1. Concurrently, separate cultures were treated solely with mitomycin C (MMC, 0.5 μg mL−1) to serve as a positive control, following established methods (OECD Test Guideline 487, 2023). At 44 h of incubation, cytochalasin-B (Cyt-B) was added to a final concentration of 6 μg mL− 1 to arrest cytokinesis in dividing cells. The cultures were maintained for a total of 72 h from initiation at 37 °C in a humidified atmosphere containing 5% CO2. Subsequently, cells were harvested by centrifugation at 1500 rpm for 10 min at room temperature. The resulting cell pellets were subjected to a 3 min mild hypotonic treatment at room temperature using a 3:1 solution of Ham’s medium and Milli-Q H2O. Fixation was then performed four times with a freshly prepared 5:1 methanol/acetic acid mixture, each for 10 min.
Cell monolayers, prepared on microscope slides, were stained with 10% Giemsa solution and subsequently mounted using DPX. Slides were then scored for the presence of one or more micronuclei within binucleated cells. For each experimental condition, a minimum of 2000 binucleated (BN) cells exhibiting intact cytoplasm were evaluated (1000 cells per donor culture, N = 2). Micronucleus (MN) frequency was subsequently expressed in parts per thousand (‰) [43]. Cytotoxicity was evaluated by determining the Cytokinesis-Block Proliferation Index (CBPI), Replicative Index (RI), and percentage of cytostasis (%Cyt). These indices were derived from scoring a minimum of 1000 cells per treatment condition (500 cells from each donor culture), applying established formulas as outlined in [41]. CBPI is given by the following equation:
CBPI = [M1 + 2 × M2 + 3 × (M3 + M4)]/N,
where M1, M2, M3, and M4 correspond to the numbers of cells with one, two, three, and four nuclei, and N is the total number of cells [44].

2.7. Fresh Minced Pork Packaging Preservation Test with BSG/Gel/Gl/15EG@NZ, BSG/Gel/Gl/15ClP Active Films

Fresh minced pork was given by the Ayfantis local meat processing company. Twelve BSG/Gel/Gl/15EG@NZ and twelve BSG/Gel/Gl/15ClP active films with a ~11 cm diameter and ~0.15 mm thickness were prepared and used for wrapping fresh minced pork. Two films were used to aseptically wrap ~40–50 g of minced pork. The pork wrapped with BSG/Gel/Gl/15EG@NZ and BSG/Gel/Gl/15ClP active films was then placed inside the Ayfantis company’s commercial wrapping paper without the inner film by Ayfantis. A total of 40–50 g of minced pork was aseptically wrapped in the commercial packaging paper of the Ayfantis company and labeled as the control sample. For all tested packaging systems, samples for the 2nd, 4th, and 6th day of storage were prepared and stored at 4 ± 1 °C.
During the 6 days of storage, the determination of the Thiobarbituric acid reactive substances (TBARS) values, the heme iron content, and the total viable count (TVC) of minced pork were carried out by following the methodology described in detail in the Supplementary Materials file.

2.8. Statistical Analysis

All obtained data from tensile properties measurements, along with antioxidant activity, antibacterial activity, toxicity, total viable count, TBARS, and heme iron content, were subjected to statistical analysis. For the statistical analysis, the Krüskal–Wallis non-parametric method was applied to indicate the significance of difference between the properties’ mean values. Assuming a significance level of p < 0.05, all measurements were conducted using three separate samples. Although this non-parametric method is the appropriate statistical method for 3 repetitions, in the case of OTR measurements, the goodness of convergence was very low, and the ANOVA method was adopted only for these measurements. Statistical analysis was performed using SPSS software (v. 28.0, IBM, Armonk, NY, USA). Detailed statistical analysis results are also included in Supplementary Materials file.

3. Results

3.1. XRD Analysis

In Figure 1, the XRD plots of all the BSG/Gel/Gl/xEG@NZ (Figure 1a) and the BSG/Gel/Gl/xClP (Figure 1b) films are presented.
As is observed in Figure 1, the XRD plot of the pure BSG/Gel/Gl film (see plot line (1) in both Figure 1a,b) shows a broad peak at around 20° 2theta corresponding to an amorphous crystal phase. The addition of both the EG@NZ nanohybrid and ClP do not affect the amorphous crystal phase of pure BSG/Gel/Gl, suggesting a good dispersion of both the EG@NZ nanohybrid and ClP in the BSG/Gel/Gl matrix. In the case of BSG/Gel/Gl/xEG@NZ films, small inflection peaks between 20 and 30° 2theta were assigned to the NZ crystal phase, suggesting the presence of NZ in the BSG/Gel/Gl matrix [45,46].

3.2. FTIR-ATR Spectroscopy

In Figure 2, the recorded FTIR-ATR plots of the EG@NZ nanohybrid (plot line (1)) and pure ClP are presented for comparison.
The FTIR-ATR plot line (1) corresponds to the EG@NZ nanohybrid, and it is a mix of EG and NZ bands [47]. The bands corresponding to NZ are as follows: The bands at 3619 and 3465 cm−1 are assigned to the O–H stretching vibrations, the band at 1650 cm−1 corresponds to the O–H bending vibration, the band at 1090 cm−1 is attributed to the Si–O stretching vibration, and the band at 468 cm−1 corresponds to the Si–O bending vibration of NZ [48].
The bands corresponding to EG molecules adsorbed in NZ are as follows: the broad band in the region of 3300–3550 cm−1 is attributed to the stretching vibrations of O–H groups of EG [49]. The peaks at 3000 and 3040 cm−1 correspond to the stretching vibrations of the CH=CH–H groups of EG, while the absorption bands in the 650–1000 cm−1 region are assigned to the bending vibrations of the same functional groups of EG [47,49,50]. The peaks at 2870 and 2960 cm−1 are attributed to the symmetric and asymmetric stretching vibrations of the methyl (CH3) groups of EG, respectively. The peaks at 1370 and 1450 cm−1 are assigned to the symmetric and asymmetric bending vibrations of CH3 groups of EG [49]. Finally, the peaks at 1514, 1608, and 1637 cm−1 are assigned to the aromatic C=C stretching vibrations of the EG [47,49,50].
In the FTIR-ATR plot line (2) of pure ClP, many of the characteristic peaks assigned to EG and denoted also in the FTIR plot line (2) of the EG@NZ nanohybrid are recorded, indicating the presence of EG molecules physically encapsulated in the pure ClP. More specifically, the broad peak around 3400 cm−1 indicates the presence of hydroxyl (-OH) groups, likely from EG and other phenolic compounds [51]. The peaks at around 2920 cm−1 and 2852 cm−1 represent the stretching vibrations of C-H bonds in methyl (CH3) and methylene (CH2) groups, commonly found in aromatic and aliphatic compounds likely from EG [52]. The peaks at around 1600 cm−1 and 1500 cm−1 are attributed to the aromatic C=C bonds, characteristic of EG and other phenylpropanoids [51]. The peaks around 1260 cm−1 and 1030 cm−1 are assigned to the C-O stretching vibrations, likely from alcohols, ethers, and esters [52]. Finally, the plot region below 1000 cm−1 provides a unique “fingerprint” for the ClP, allowing for the differentiation from other substances. The specific peak positions and intensities in this region are highly dependent on the overall chemical composition and structure of the sample [51,52,53].
In Figure 3, the FTIR-ATR plot of the pure BSG/Gel/Gl film (line (1)), as well as the FTIR plots of all BSG/Gel/Gl/xClP active films (lines (2), (3), and (4)) and all BSG/Gel/Gl/xEG@NZ active films (lines (5), (6), and (7)), are presented for comparison.
In all obtained films’ FTIR-ATR plots, the following peaks are observed: the peak between 800 and 600 cm−1 represented the out-of-plane N-H bonding (amide V) of BSG’s protein [54]. The characteristic peak at 1032 cm−1 observed in all spectra corresponds to the O–H groups of Gl [55]. The peaks at 1535 cm−1 and 1632 cm−1 are assigned to the N–H bending vibrations of amide II and the C=O stretching vibrations of the amide I group of Gel, respectively. The peak at 1535 cm−1 is also assigned to the C–N stretching vibrations of Gel [56,57,58]. The peaks in the range of 1350–1200 cm−1 were due to a combination of N-H bending and C-H stretching vibrations of the amide III group of BSG’s protein. The peaks at 2926 cm−1 and 2852 cm−1 are assigned to saturated C–H stretching vibrations, attributable to both Gel and Gl. The broad peak between 3300 and 3600 cm−1 corresponds to O–H stretching vibrations [54,56,57,58]. Thus, in all the obtained films’ FTIR plots, the presence of BSG, Gel, and Gl is suggested. No additional peaks corresponding to pure ClP and the EG@NZ nanohybrid are obtained, implying a good dispersion of both the EG@NZ nanohybrid and ClP in the BSG/Gel/Gl matrix, in line with the XRD results.

3.3. SEM Analysis

The cross-section morphology, as well as the surface topography of all the prepared samples, was tested via Scanning Electron Microscopy (SEM), and the results are illustrated in Figure 4.
Both the cross-section and surface images confirm the production of rather uniform films with a homogeneous dispersion of both the EG@NZ nanohybrid and ClP in the BSG/Gel/Gl matrix, as expected by the twin-extrusion procedure that followed. In the case of the BSG/Gel/Gl/xEG@NZ films, a uniform dispersion of the EG@NZ nanohybrid is observed in the cross-section images, with only a few agglomerations. A smooth surface topography is observed in all cases, suggesting no deterioration of the quality of the composite films in comparison to the BSG/Gel/Gl matrix.

3.4. Tensile Properties of BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP Films

In Table 2, the calculated mean values of the Elastic Modulus (E), ultimate strength (σuts), and % elongation at break (%ε) are listed for comparison.
As is obtained from the listed mean values in Table 2 of the Elastic Modulus (E), ultimate strength (σuts), and % elongation at break (%ε) for all tested films, both the EG@NZ nanohybrid and ClP react as reinforcement agents in BSG/Gel/Gl-based films. As the content of both the EG@NZ nanohybrid and ClP increases, the obtained E and σuts values increase and %ε values decrease. As detailed for the BSG/Gel/Gl/5EG@NZ, BSG/Gel/Gl/10EG@NZ, and BSG/Gel/Gl/15EG@NZ films, the ultimate strength increases up to 88.5%, 173.3%, and 207.7% in comparison to the pure BSG/Gel/Gl film, while for BSG/Gel/Gl/5ClP, BSG/Gel/Gl/10ClP, and BSG/Gel/Gl/15ClP films, it increases up to 213.6%, 210.5%, and 211.5% in comparison to the pure BSG/Gel/Gl film. This means that in the case of BSG/Gel/Gl/xEG@NZ films, the increase in e is affected by the EG@NZ nanohybrid content, and in the case of BSG/Gel/Gl/xClP films, it is not affected by the ClP content. Simultaneously, for both BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP, the % elongation at break values decrease up to 40–50% in comparison to the pure BSG/Gel/Gl film. This means that the addition of both the EG@NZ nanohybrid and ClP do not dramatically decrease ductility, which is crucial for the packaging application of such films [59,60]. Overall, the tensile properties results presented here imply that both the EG@NZ nanohybrid and pure ClP act as reinforcement agents in BSG/Gel/Gl by keeping the ductility of obtained films. Tensile properties results are in agreement with the SEM image morphology results, presented hereabove, and show the uniform dispersion of both the EG@NZ nanohybrid and pure ClP in the BSG/Gel/Gl matrix.

3.5. Oxygen Barrier Properties of BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP Films

In Table 3, the observed oxygen transmission rate (OTR) values, as well as the calculated oxygen permeability PeO2 mean values of all tested BSG/Gel/Gl, BSG/Gel/Gl/xEG@NZ, and BSG/Gel/Gl/xClP films, are listed for comparison [61].
Edible films can provide excellent oxygen barrier properties, significantly impacting food preservation and shelf life [62]. As is obtained from Table 3, pure BSG/Gel/Gl has a high OTR value of 8301.2 mL·m−2·day−1, which corresponds to a 2.59 × 10−7 cm2·s−1 of oxygen permeability. It is known that when BSG is added in polyhydroxybutyrate–valerate-based blowing films, the O2 and CO2 permeability increased [63]. In the case of all BSG/Gel/Gl/xEG@NZ films, zero O.T.R. and PeO2 values are observed. This means that BSG/Gel/Gl/xEG@NZ films are impermeable to oxygen. In the case of BSG/Gel/Gl/xClP films, the film with a 5wt. ClP content is impermeable to oxygen, while 10 and 15 %wt. ClP content films exhibited a 5.87 × 10−10 and 5.17 × 10−10 mean PeO2, correspondingly. In other words, all BSG/Gel/Gl/xEG@NZ films and BSG/Gel/Gl/5ClP films are oxygen impermeable, while BSG/Gel/Gl/10ClP and BSG/Gel/Gl/15ClP films are high oxygen barrier films. According to our knowledge from the literature, such high and/or zero oxygen barrier results for BSG-based films are received for the first time. The oxygen barrier results presented here are in line with previous reports claiming that protein-based films such as gelatin-based exhibit a high oxygen barrier [64,65]. The zero and/or high oxygen results presented here are also in line with recent reports validating the use of extrusion molding and compression process methods for the preparation of such films [55,66,67].

3.6. Total Phenolic Content (TPC) of BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP Films

Table 4 presents the calculated EC60 and TPC mean values of all tested films.
Calculated mean TPC values for all tested films are listed in Table 4 for comparison. As is obtained, all BSG/Gel/Gl/xEG@NZ films exhibited much higher TPC values than the BSG/Gel/Gl/xClP ones. Moreover, for both BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP active films, the recorded TPC values affected by the content of the EG@NZ nanohybrid and ClP dispersed in the BSG/Gel/Gl matrix, correspondingly. Thus, the higher the EG@NZ and ClP content, the higher the recorded TPC values. The highest TPC value of 291.77± 12.81 mgGAE/L is observed for the BSG/Gel/Gl/15EG@NZ active film, and it is equal to those reported recently by Vieira et al., who prepared films with BSG and cassava starch and poly (vinyl alcohol) and obtained a TPC value of 263.23 ± 10.97 mg GAE/L [20].

3.7. Antioxidant Activity of BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP Films

Edible films and coatings can be designed to have antioxidant properties, enhancing their ability to preserve food by preventing oxidation and extending shelf life [68]. EC60 values of all BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP films, as well as the pure BSG/Gel/Gl film, are listed in Table 4. All experimental data and the obtained linear plots used for the calculation of EC60 values of all tested films are shown in the Supplementary file. EC60 values are recommended for films with high antioxidant activity [69]. As is obtained from Table 4, the pure BSG/Gel/Gl film exhibited significant antioxidant activity up to 69.6 mg/L. The antioxidant capacity of BSG is well known due to its content of various phenolic compounds [18,19,20,23]. The addition of both the EG@NZ nanohybrid and ClP enhances even more the antioxidant capacity of the obtained BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP films. Thus, all obtained films could be characterized as films with a high antioxidant capacity. Overall, BSG/Gel/Gl/xEG@NZ films exhibited a higher antioxidant activity than BSG/Gel/Gl/xClP films, and films with a 10%wt. content of EG@NZ and ClP are the most active ones. The higher antioxidant activity values recorded for BSG/Gel/Gl/xEG@NZ films against BSG/Gel/Gl/xClP ones are in accordance with the higher TPC values recorded for BSG/Gel/Gl/xEG@NZ films against BSG/Gel/Gl/xClP ones.

3.8. Antibacterial Activity of BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP Films

The antibacterial activity results of all obtained films against the Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) are presented in Table 5.
S. aureus showed sensitivity to BSG/Gel/Gl/10EG@NZ, BSG/Gel/Gl/15EG@NZ, and BSG/Gel/Gl/15ClP films, presenting halos of 25, 45, and 25 mm, respectively. In the case of E. coli, antibacterial activity was observed only for the BSG/Gel/Gl/10EG@NZ and BSG/Gel/Gl/15EG@NZ films, which produced inhibition zones of 20 mm and 30 mm, respectively. No antibacterial effect was observed for the unmodified BSG/Gel/Gl film against either bacterial strain under the test conditions, indicating that the incorporation of EG@NZ and ClP components was essential for imparting antibacterial activity. A comparative analysis indicates that films containing EG@NZ exhibited superior antibacterial activity, particularly at higher concentrations (15%), against both bacterial strains. This result is in agreement with previous reports suggesting there is antibacterial activity of EG when it is incorporated in packaging films [70,71,72]. Herein, it is reported for the first time how the nanoencapsulated EG of EG@NZ-based films excel over the physically encapsulated EG in ClP-based films. It seems that NZs encapsulate higher amounts of EG than ClP due to their unique pore structure [73]. In particular, BSG/Gel/Gl/15EG@NZ demonstrated the most potent effect, with the largest inhibition zones against both S. aureus and E. coli. This suggests a concentration-dependent enhancement of antimicrobial properties due to the increased availability of EG. The stronger activity against S. aureus compared to E. coli may be likely due to structural differences between the Gram-positive and Gram-negative bacteria. The outer membrane in E. coli can act as a barrier, making it more resistant.

3.9. Cytotoxic and Genotoxic Effects of BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP Films in Human Lymphocytes

Table 6 presents the cytotoxicity and genotoxicity results for the BSG/Gel/Gl/15EG@NZ and BSG/Gel/Gl/15ClP films, as well as for the unmodified BSG/Gel/Gl control film.
Cytotoxic and genotoxic tests are crucial for ensuring the safety of edible packaging, particularly for food contact applications [74]. The micronucleus frequency (‰MN), a well-established biomarker for assessing genotoxic effects in human lymphocytes, was evaluated for selected film formulations. These two modified formulations were specifically chosen for testing, as they represent the highest loadings of EG@NZ and ClP, respectively, and thus provide the most stringent conditions for evaluating potential toxicological effects.
The analysis aimed to determine whether the incorporation of bioactive components at maximum concentrations could induce cytotoxic or genotoxic responses in human lymphocytes. The results serve to assess the safety profile of the developed active films for potential applications in food contact materials or biomedical packaging.
As shown in Table 6, the BSG/Gel/Gl/15EG@NZ and BSG/Gel/Gl/15ClP films, along with the pure BSG/Gel/Gl film, exhibited lower micronucleus frequencies (‰MN, genotoxicity index) and cytostasis percentages (% cytostasis, cytotoxicity index) compared to the mitomycin C (MMC) positive control at all tested concentrations (50, 100, and 500 μg/mL). These results indicate that none of the film formulations induced significant genotoxic or cytotoxic effects relative to the established positive control.
In more detail, the ‰MN values of all films were equal to or lower than those observed in the control group, indicating no significant increase in micronuclei frequency at any of the tested concentrations. This suggests that none of the formulations exerted genotoxic effects under the conditions examined. These findings support the genetic safety of the developed materials, consistent with international guidelines for genotoxicity testing [42,75]. In terms of cytotoxicity, both BSG/Gel/Gl/15ClP and BSG/Gel/Gl/15EG@NZ films exhibited slightly elevated % cytostasis values compared to the pure BSG/Gel/Gl film. However, these values remained well within the acceptable range, as defined by OECD 487 guidelines [43,76]. The observed mild cytotoxic effects were only present at the highest concentrations tested and did not exceed regulatory thresholds. Importantly, no associated genotoxicity was observed, indicating that the inclusion of ClP or EG@NZ does not compromise genomic integrity. Notably, the lowest ‰MN and % cytostasis values were recorded for the BSG/Gel/Gl/15EG@NZ film. This result suggests that the nanoencapsulation of EG (the principal bioactive compound in ClP) in nanozeolite effectively reduces both genotoxicity and cytotoxicity compared to its direct incorporation via clove powder. These findings align with recent studies reporting that the nanoencapsulation of EG in carriers such as montmorillonite nanoclay and nanozeolite reduces the cytotoxic profile of active packaging films based on pork gelatin matrices [47,55].

3.10. Packaging Preservation Test—Minced Pork Wrapped with BSG/Gel/Gl/10EG@NZ and BSG/Gel/Gl/10ClP Active Films

3.10.1. TVC

The calculated TVC mean values of minced pork wrapped with BSG/Gel/Gl/15EG@NZ and BSG/Gel/Gl/15ClP active films, as well as with commercial paper (control), as a function of the storage time, are listed in Table 7 for comparison.
As is obtained from Table 7, both BSG/Gel/Gl/15EG@NZ and BSG/Gel/Gl/15ClP active films succeed in maintaining lower TVC growth rates of fresh minced pork than the control sample during the six days of storage. In addition minced pork wrapped with the BSG/Gel/Gl/15EG@NZ active film, a lower TVC growth rate than that of the minced pork wrapped with the BSG/Gel/Gl/15ClP active film was exhibited. This result is in line with the highest antibacterial activity recorded hereabove against Gram-negative E. coli and Gram-positive S. aureus for the BSG/Gel/Gl/15EG@NZ active film. As is listed in Table 7, minced pork wrapped with the commercial paper (control sample) exceeds the TVC limit of acceptance (7 logCFU/g) after the 4th day of storage [77]. On the contrary, minced pork wrapped with the BSG/Gel/Gl/15EG@NZ active film exhibited a TVC value lower than 7 logCFU/g after the 6th day of storage, while minced pork wrapped with the BSG/Gel/Gl/15ClP active film exhibited a TVC value a little bit higher from 7 logCFU/g on the 6th day of storage. In other words, it could be stated that the BSG/Gel/Gl/15EG@NZ active film succeeded at extending the shelf life of minced pork from a microbiological point of view for approximately two days, and the BSG/Gel/Gl/15ClP active film succeeded at extending the shelf life of minced pork from a microbiological point of view for approximately one day.

3.10.2. Lipid Oxidation

The calculated TBA mean values of minced pork wrapped with BSG/Gel/Gl/15EG@NZ and BSG/Gel/Gl/15ClP active films, as well as with commercial paper (control), as a function of the storage time, are listed in Table 8 for comparison.
As is obtained from the list in Table 8, the mean TBARS values for both the BSG/Gel/Gl/15EG@NZ and BSG/Gel/Gl/15ClP active films prevent minced pork lipid oxidation during the six days of storage, in comparison to minced pork wrapped in a commercial paper film. Additionally, minced pork wrapped in the BSG/Gel/Gl/15EG@NZ film recorded the lowest TBARS increment rate, implying the highest lipid oxidation protection of wrapped minced pork. Thus, on the 6th day of storage, minced pork wrapped in the BSG/Gel/Gl/15EG@NZ film had a TBARS value ~20% lower than that of minced pork meat wrapped in commercial film. This result is in accordance with the zero oxygen barrier of such an active film and its enhanced antioxidant activity.

3.10.3. Heme Iron Content

The calculated heme iron content mean values of minced pork wrapped with BSG/Gel/Gl/15EG@NZ and BSG/Gel/Gl/15ClP active films, as well as with commercial paper (control), as a function of the storage time, are listed in Table 8 for comparison.
As is obtained from the list in Table 8, the mean heme iron content values for both the BSG/Gel/Gl/15EG@NZ and BSG/Gel/Gl/15ClP active films succeeded in keeping wrapped minced pork with higher heme iron contents during the six days of storage in comparison to the minced pork wrapped in commercial paper (control sample). In other words, both BSG/Gel/Gl/15EG@NZ and BSG/Gel/Gl/15ClP active films succeeded at preserving fresh minced pork with a higher nutritional value during the six days of storage. Overall, the highest heme iron content values during the six days of storage were obtained for minced pork wrapped in the BSG/Gel/Gl/15EG@NZ active film. This result supports the lowest TBARS values obtained for the minced pork wrapped with the same BSG/Gel/Gl/15EG@NZ active film. So, BSG/Gel/Gl/15EG@NZ prevents minced pork from lipid oxidation and keeps it with the highest nutritional value.
TVC, TBARS, and heme iron content results presented hereabove for the preservation of fresh minced pork wrapped with BSG/Gel/Gl/15EG@NZ and BSG/Gel/Gl/15ClP are supported by several previous reports, where EG and ClP have been used for the preservation of fresh meat [41,78,79,80,81]. By inhibiting microbial growth and lipid oxidation, EG can help to maintain the color, texture, and overall quality of fresh meat during storage [78,79,82]. In addition, previous reports indicate that ClP can be effective in extending the shelf life of various meat products, including chicken and beef [41,80,81].

4. Discussion

In the current study, BSG was valorized to edible active packaging films via the extrusion–compression molding method. Gel and Gl were used as the reinforcement and plasticizer, correspondingly, to obtain an edible BSG/Gel/Gl matrix. This pure BSG/Gel/Gl matrix was farther reinforced and activated by adding the EG@NZ nanohybrid and pure ClP at 5, 10, and 15 %wt. To the best of our knowledge, the obtained BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP active films were, for the first time, developed and characterized here. The physicochemical characterization of the obtained BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP active films took place via XRD analysis, FTIR-ATR spectroscopy, and SEM images analysis (surface and cross-section), in line with XRD and FTIR-ATR spectroscopy, showing that both the EG@NZ nanohybrid and pure ClP were homogeneously dispersed in the BSG/Gel/Gl matrix. This effective dispersion of both the EG@NZ nanohybrid and pure ClP in the BSG/Gel/Gl matrix depicted the enhancement of the ultimate strength of all obtained BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP active films. Simultaneously, the % elongation at break of all the obtained films did not dramatically reduce, which means that all films kept their ductility and are suitable for flexible packaging applications. In addition, both BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP active films show high oxygen barrier properties. For all BSG/Gel/Gl/xEG@NZ active films, zero OTR values were recorded, while a zero OTR value was recorded for the BSG/Gel/Gl/5ClP active film, and low OTR values were recorded for both BSG/Gel/Gl/10ClP and BSG/Gel/Gl/15ClP active films. This means that EG@NZ-based active films excel over ClP-based active films in terms of oxygen barrier properties. Such zero and/or high oxygen barrier results presented here are initially reported here. To the best of our knowledge, there is no previous report on BSG-based films studying the oxygen barrier of such films. Cunha et al. reported the preparation, characterization, and film blowing of polyhydroxybutyrate–valerate (PHBV)/beer spent grain fiber (BSGF) composites and found that the addition of BSGF increases the permeability of PHBV films to O2, CO2, and water vapor. Permeability to CO2 and O2 increases tenfold when incorporating 5 %wt. BSGF [63]. Zero and/or high oxygen barrier results are presented, validating the use of a Gel protein as reinforcement and high barrier agent, and the use of the extrusion–compression molding process used for the preparation of films [47,55].
Both BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP active films exhibited high TPC contents, high antioxidant activity according to calculated EC60 values with THE DPPH assay method, and significant antibacterial activity against S. aureus and E. coli food pathogens. In addition, BSG/Gel/Gl/xEG@NZ active films exhibited a much higher TPC, EC60 values, and antibacterial activity against S. aureus and E. coli food pathogens than BSG/Gel/Gl/xClP active films. More specifically, the BSG/Gel/Gl/15EG@NZ active film exhibited a ~93% higher TPC value and ~90% lower EC60 value than the pure BSG/Gel/Gl film, while the BSG/Gel/Gl/15ClP active film exhibited a ~90% higher TPC value and ~88% lower EC60 value than the pure BSG/Gel/Gl film. In addition, the BSG/Gel/Gl/15EG@NZ active film exhibited a 45 mm and 30 mm inhibition zone against S. aureus and E. coli, respectively, while the BSG/Gel/Gl/15ClP has a 25 mm inhibition zone against S. aureus and antibacterial activity only under the contact area of the film against E. coli. At the same time, for both BSG/Gel/Gl/15EG@NZ and BSG/Gel/Gl/15ClP active films, the % cytostasis values remained well within the acceptable range, as defined by OECD 487 guidelines, and no associated genotoxicity was observed. The lowest ‰MN and % cytostasis values were recorded for the BSG/Gel/Gl/15EG@NZ film. Overall, the results of the TPC content, EC60 values, antibacterial activity tests against S. aureus and E. coli, and ‰MN and % cytostasis values suggest that the nanoencapsulation of EG (the principal bioactive compound in ClP) in NZ increases the antioxidant and antibacterial activity and effectively reduces both genotoxicity and cytotoxicity, compared to its direct incorporation via clove powder. This is the first report we have received that the nanoencapsulation of bioactive molecules, such as EG in NZ, excels over the physical encapsulation of such bioactive molecules. It is known that EG interacts with other compounds in the clove plant; it is not chemically bonded to them in the same way that it would be in a synthesized molecule [83]. Instead, it is present as a component of the EOs within the plant cells, which are then released when the powder is processed or used [84]. The amount of EG released from ClP can vary depending on several factors, including the quality of the cloves, the fineness of the powder, the method of preparation, and the presence of other ingredients [84]. On the contrary, recently via desorption kinetic studies of the EG@NZ nanohybrid, it was shown that (i) up to 95% desorption of EG is the adsorbed amount and (ii) there is a low release rate of EG molecules due to their strong intramolecular interaction [47]. Thus, it seems that NZ, with its unique porous structure, succeeded at encapsulating higher amounts of EG with lower control release rates than physically encapsulated EG in ClP [73,85]. Overall, this study concludes that the BSG/Gel/Gl/15EG@NZ film is a novel, edible, monolayer, zero oxygen barrier active packaging material which could potentially be applied for the flexible packaging of meat products.

5. Conclusions

This study, by following circular economy and sustainability trends, presents the successful development and characterization of BSG/Gel/Gl/xEG@NZ (where x = 5, 10, and 15 %wt.) and BSG/Gel/Gl/xClP (where x = 5, 10, and 15 %wt.) active films via the extrusion molding compression method. Simultaneously, a comparison study between EG@NZ nanohybrid and clove powder as both reinforcement and antioxidant/antibacterial agents in BSG/Gel/Gl-based active packaging films is, for first time, reported, too. Overall, this study concludes that (i) there is the successful development of such BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP films as promising monolayer, edible, high oxygen barrier active packaging materials with a high potential to be used as a flexible packaging materials in the food industry, and (ii) there is an advantage of nanotechnology as a method of encapsulating bioactive EO components such as ΕΓ in bio-based active food packaging materials. A prospect of the current study could be the investigation of the potential for industrial-scale production of such films via the extrusion–compression method. Limitations of the extrusion–compression method, as well as ingredient availability, should also be studied in the future.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app15179282/s1, Figure S1: Gallic acid obtained calibration curve and linear equation; Figure S2: Linear plots used for the calculation of average values of EC60; Table S1: Experimental data used for the calculation of obtained average EC60 values; Table S2: Statistical Analysis of Tensile properties of BSG/Gel/Gl, BSG/Gel/Gl/xEG@NZ, and BSG/Gel/Gl/xClP films; Table S3: Statistical Analysis of the Oxygen Barrier properties of pure BSG/Gel/Gl film as well as all BSG/Gel/Gl/xEG@NZ, and BSG/Gel/Gl/xClP films; Table S4: Statistical Analysis of the Total Phenolic Content (TPC) of pure BSG/Gel/Gl film as well as all BSG/Gel/Gl/xEG@NZ, and BSG/Gel/Gl/xClP films; Table S5: Statistical Analysis for the In Vitro Antioxidant Activity Determination of pure BSG/Gel/Gl film as well as all BSG/Gel/Gl/xEG@NZ, and BSG/Gel/Gl/xClP films; Table S6: Statistical Analysis of the Total Viable Count (TVC) of minced pork wrapped with commercial packaging (control sample), BSG/Gel/Gl/15EG@NZ films and BSG/Gel/Gl/15ClP film. Table S7: Statistical Analysis of Lipid Oxidation with Thiobarbituric Acid Reactive Substances of minced pork wrapped with commercial packaging (control sample), BSG/Gel/Gl/15EG@NZ films and BSG/Gel/Gl/15ClP film; Table S8: Statistical Analysis of Heme Iron Content Measurements of minced meat wrapped with commercial packaging (control sample), BSG/Gel/Gl/15EG@NZ films and BSG/Gel/Gl/15ClP film; Table S9: Statistical Analysis of Cytotoxic and genotoxic effects of BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP films in Human Lymphocytes.

Author Contributions

Conceptualization—C.E.S. and A.E.G.; data curation—Z.N., A.K., A.A.L., T.A., A.V., P.S., K.Z., M.D., G.B., C.P., A.E.G. and C.E.S.; formal analysis—Z.N., A.K., A.A.L., A.V., M.D., P.S., C.P. and A.E.G.; investigation—Z.N., A.K., A.A.L. and A.E.G.; methodology—A.K., A.A.L., T.A., P.S., M.D., P.K. and A.E.G.; project administration—C.E.S. and A.E.G.; resources—A.K., A.A.L., C.E.S. and A.E.G.; software—Z.N., A.A.L., K.Z., P.K., G.B. and A.E.G.; supervision—C.E.S. and A.E.G.; validation—A.K., A.A.L., T.A., A.V., K.Z., M.D., P.S., G.B. and A.E.G.; visualization—A.K., C.E.S. and A.E.G.; writing—original draft—A.A.L. and A.E.G.; writing—review and editing—A.A.L., P.S., P.K., K.Z., C.P., C.E.S. and A.E.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The datasets generated for this study are available on request to the corresponding author.

Acknowledgments

The authors would like to express their acknowledgements to Katerina Govatsi (Laboratory of Electron Microscopy and Microanalysis, University of Patras) for her assistance with electron microscopy images (SEM).

Conflicts of Interest

Author Alexios Vardakas was employed by the company GAEA Products S.M. S.A. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Applsci 15 09282 g001
Figure 1. (a) XRD plots of (1) BSG/Gel/Gl film, (2) BSG/Gel/Gl/5EG@NZ film, (3) BSG/Gel/Gl/10EG@NZ film, and (4) BSG/Gel/Gl/15EG@NZ film. (b) (1) BSG/Gel/Gl film, (2) BSG/Gel/Gl/5ClP film, (3) BSG/Gel/Gl/10ClP film, and (4) BSG/Gel/Gl/15ClP film.
Figure 1. (a) XRD plots of (1) BSG/Gel/Gl film, (2) BSG/Gel/Gl/5EG@NZ film, (3) BSG/Gel/Gl/10EG@NZ film, and (4) BSG/Gel/Gl/15EG@NZ film. (b) (1) BSG/Gel/Gl film, (2) BSG/Gel/Gl/5ClP film, (3) BSG/Gel/Gl/10ClP film, and (4) BSG/Gel/Gl/15ClP film.
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Figure 2. FTIR-ATR plots of (1) EG@NZ nanohybrid and (2) pure ClP.
Figure 2. FTIR-ATR plots of (1) EG@NZ nanohybrid and (2) pure ClP.
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Figure 3. FTIR-ATR plots of (1) pure BSG/Gel/Gl film, (2) BSG/Gel/Gl/5ClP active film, (3) BSG/Gel/Gl/10ClP active film, (4) BSG/Gel/Gl/15ClP active film, (5) BSG/Gel/Gl/5EG@NZ active film, (6) BSG/Gel/Gl/10EG@NZ active film, and (7) BSG/Gel/Gl/5EG@NZ active film.
Figure 3. FTIR-ATR plots of (1) pure BSG/Gel/Gl film, (2) BSG/Gel/Gl/5ClP active film, (3) BSG/Gel/Gl/10ClP active film, (4) BSG/Gel/Gl/15ClP active film, (5) BSG/Gel/Gl/5EG@NZ active film, (6) BSG/Gel/Gl/10EG@NZ active film, and (7) BSG/Gel/Gl/5EG@NZ active film.
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Figure 4. Representative SEM images of the cryo-cut cross-section (left column) and of the surface (right column) of all prepared films. The magnification level was set at 300× (BSG/Gel/Gl, BSG/Gel/Gl/15EG@NZ, BSG/Gel/Gl/5ClP, and BSG/Gel/Gl/15ClP) and 400× (BSG/Gel/Gl/5EG@NZ, BSG/Gel/Gl/10EG@NZ, and BSG/Gel/Gl/10ClP) for the cross-section images and at 100× for the surface images.
Figure 4. Representative SEM images of the cryo-cut cross-section (left column) and of the surface (right column) of all prepared films. The magnification level was set at 300× (BSG/Gel/Gl, BSG/Gel/Gl/15EG@NZ, BSG/Gel/Gl/5ClP, and BSG/Gel/Gl/15ClP) and 400× (BSG/Gel/Gl/5EG@NZ, BSG/Gel/Gl/10EG@NZ, and BSG/Gel/Gl/10ClP) for the cross-section images and at 100× for the surface images.
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Table 1. Sample names of the films’ weighed masses of their composites (BSG, Gel, Gl, H2O, EG@NZ, and ClP) and twin extruder operating conditions (temperature, rotating speed, and operating time).
Table 1. Sample names of the films’ weighed masses of their composites (BSG, Gel, Gl, H2O, EG@NZ, and ClP) and twin extruder operating conditions (temperature, rotating speed, and operating time).
Sample NameBSG
(g)		Gel
(g)		Gl
(g)		H2O
(g)		EG@NZ
(g)		ClP
(g)
BSG/Gel/Gl3111.6--
BSG/Gel/Gl/5EG@NZ3111.60.347-
BSG/Gel/Gl/10EG@NZ3111.60.733-
BSG/Gel/Gl/15EG@NZ3111.61.160-
BSG/Gel/Gl/5ClP3111.6-0.347
BSG/Gel/Gl/10ClP3111.6-0.733
BSG/Gel/Gl/15ClP3111.6-1.160
Table 2. Obtained mean values of Elastic Modulus (E), ultimate strength (σuts), and % elongation at break (%ε) for all tested films.
Table 2. Obtained mean values of Elastic Modulus (E), ultimate strength (σuts), and % elongation at break (%ε) for all tested films.
Elastic Modulus E (MPa)
Average ± Stdev		σuts (MPa)
Average ± Stdev		Elongation at Break—%ε
Average ± Stdev
BSG/Gel/Gl17.57 ± 5.62 d1.83 ± 0.29 c27.25 ± 8.12 a
BSG/Gel/Gl/5EG@NZ43.97 ± 7.76 cd3.80 ± 0.96 bc17.71 ± 3.17 bc
BSG/Gel/Gl/10EG@NZ52.56 ± 7.75 bc5.07 ± 0.52 ab16.51 ± 1.32 bc
BSG/Gel/Gl/15EG@NZ77.01 ± 13.76 a5.82 ± 0.47 a14.28 ± 1.50 c
BSG/Gel/Gl/5ClP59.77 ± 7.45 abc5.74 ± 0.70 a23.08 ± 2.63 a
BSG/Gel/Gl/10ClP71.17 ± 12.60 a5.55 ± 0.72 a17.77 ± 1.59 b
BSG/Gel/Gl/15ClP70.29 ± 15.92 ab5.54 ± 0.59 a15.84 ± 2.57 bc
abcd The letters are used to show the significant difference in values in the same column for each film. The use of the letters is further explained in the Supplementary document.
Table 3. Oxygen transmission rate (OTR) mean values, as well as the calculated oxygen permeability PeO2 mean values, of all tested films.
Table 3. Oxygen transmission rate (OTR) mean values, as well as the calculated oxygen permeability PeO2 mean values, of all tested films.
Sample NameAverage Thickness (mm)O.T.R.
(mL·m−2·day−1)		PeO2
(cm2·s−1)
BSG/Gel/Gl0.27 ± 0.01 a8301.2 ± 450.3 a2.59 × 10−7 ± 0.19 × 10−7 a
BSG/Gel/Gl/5EG@NZ0.25 ± 0.02 a0 b0 b
BSG/Gel/Gl/10EG@NZ0.27 ± 0.01 a0 b0 b
BSG/Gel/Gl/15EG@NZ0.25 ± 0.02 a0 b0 b
BSG/Gel/Gl/5ClP0.27 ± 0.02 a0 b0 b
BSG/Gel/Gl/10ClP0.25 ± 0.02 a22.8 ± 1.5 b5.87 × 10−10 ± 0.42 × 10−10 b
BSG/Gel/Gl/15ClP0.25 ± 0.01 a16.8 ± 1.1 b5.17 × 10−10 ± 0.32 × 10−10 b
ab The letters are used to show the significant difference in values in the same column for each film. The use of the letters is further explained in the Supplementary document.
Table 4. EC60 antioxidant activity values and TPC of all tested films.
Table 4. EC60 antioxidant activity values and TPC of all tested films.
Sample NameEC60
mg/mL		TPC 1
(mg GAE/L)
BSG/Gel/Gl23.46 ± 3.13 a17.28 ± 0.86 d
BSG/Gel/Gl/5EG@NZ2.80 ± 0.55 bc95.28 ± 4.76 bcd
BSG/Gel/Gl/10EG@NZ1.83 ± 0.68 c256.15 ± 14.59 ab
BSG/Gel/Gl/15EG@NZ2.70 ± 0.40 bc291.77 ± 12.81 a
BSG/Gel/Gl/5ClP5.02 ± 0.25 ab67.28 ± 3.36 bcd
BSG/Gel/Gl/10ClP2.50 ± 0.26 c135.07 ± 6.75 abcd
BSG/Gel/Gl/15ClP3.00 ± 0.12 abc184.28 ± 9.21 abc
abcd The letters are used to show the significant difference in values in the same column for each film. The use of the letters is further explained in the Supplementary document. 1 Results are expressed as Milli Gram Gallic Acid Equivalents (GAE) per 1 L.
Table 5. Comparison of antimicrobial activities from BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP films on different bacteria strains.
Table 5. Comparison of antimicrobial activities from BSG/Gel/Gl/xEG@NZ and BSG/Gel/Gl/xClP films on different bacteria strains.
SamplesZOI (mm)
S. aureusE. coli
BSG/Gel/GlN.D.N.D.
BSG/Gel/Gl/5EG@NZN.D.N.D.
BSG/Gel/Gl/10EG@NZ2520
BSG/Gel/Gl/15EG@NZ4530
BSG/Gel/Gl/5ClPN.D.N.D.
BSG/Gel/Gl/10ClPN.D.N.D.
BSG/Gel/Gl/15ClP25N.D.
ZOI: Zone of Inhibition; N.D.: Not Detected.
Table 6. Percentages of MN, BNMN, cytostasis, and CBPI in human lymphocytes treated with BSG/Gel/Gl/15EG@NZ and BSG/Gel/Gl/15ClP films.
Table 6. Percentages of MN, BNMN, cytostasis, and CBPI in human lymphocytes treated with BSG/Gel/Gl/15EG@NZ and BSG/Gel/Gl/15ClP films.
Concentrations (μg/mL)BNMN ± S.E. (‰)CBPI ± S.ECytostasis (%)
Control *10002 ± 01.65 ± 0.030
MMC (0.05) *100013 ± 2.81.48 ± 0.0225.6 ± 2
BSG/Gel/Gl
5010001 ± 0 A,a1.64 ± 0.01 A,a2.3 ± 2 A,a
10010001 ± 0 A,a1.63 ± 0.00 AB,a3.1 ± 0 A,a
50010002 ± 0 A,ab1.54 ± 0.01 B,ab17.6 ± 1 A,ab
BSG/Gel/Gl/15ClP
5010002 ± 0 AB,a1.57 ± 0.01 A,a12.4 ± 2 B,a
10010001 ± 0 B,a1.55 ± 0.01 AB,a15.3 ± 1 AB,a
50010003 ± 0 A,a1.51 ± 0.01 B,b22.1 ± 2 A,a
BSG/Gel/Gl/15EG@NZ
5010001.5 ± 0.7 A,a1.65 ± 0.03 A,a0.00 ± 5 A,a
10010002 A,a1.63 ± 0.02 A,a3.5 ± 3 A,a
50010001.5 ± 0.7 A,b1.55 ± 0.01 A,a15.6 ± 2 A,b
BN: Binucleated lymphocytes; MN: total number of micronuclei in lymphocytes; S.E.: Standard Error; MMC: mitomycin C (positive control); and AB the letters are used to show the significant difference in values in the same column for each film, while ab are used to show the significant difference in values in the same column for each concentration. The use of the letters is further explained in the Supplementary document. * Control and MMC materials are chemical agents used to provide the initial values for film samples’ measurements comparisons. Such values are excluded from the statistical analysis.
Table 7. TVC values of minced pork wrapped with the control, BSG/Gel/Gl/15EG@NZ, and BSG/Gel/Gl/15ClP films with respect to storage time.
Table 7. TVC values of minced pork wrapped with the control, BSG/Gel/Gl/15EG@NZ, and BSG/Gel/Gl/15ClP films with respect to storage time.
Sample0th Day2nd Day4th Day6th Day
TVC (mg/kg)
CONTROL4.30 ± 0.05 C,a5.7 ± 0.04 BC,a6.92 ± 0.06 AB,a8.18 ± 0.05 A,a
BSG/Gel/Gl/15EG@NZ3.55 ± 0.04 C,b4.70 ± 0.04 BC,b5.71 ± 0.05 AB,b6.67 ± 0.10 A,b
BSG/Gel/Gl/15ClP3.74 ± 0.04 C,ab4.959 ± 0.04 BC,ab6.01 ± 0.05 AB,ab7.18 ± 0.05 A,ab
ABC The letters are used to show the significant difference in TVC values between different days for the same film, while ab are used to show the significant difference in values between different films for the same day. The use of the letters is further explained in the Supplementary document.
Table 8. TBA and heme iron content mean values of minced pork wrapped with the control, BSG/Gel/Gl/10EG@NZ, and BSG/Gel/Gl/10ClP films with respect to storage time.
Table 8. TBA and heme iron content mean values of minced pork wrapped with the control, BSG/Gel/Gl/10EG@NZ, and BSG/Gel/Gl/10ClP films with respect to storage time.
Sample0th Day2nd Day4th Day6th Day
TBARS (mg/kg)
CONTROL0.46 ± 0.01 C,a0.59 ± 0.02 BC,a0.75 ± 0.02 AB,a0.81 ± 0.02 A,a
BSG/Gel/Gl/15EG@NZ0.46 ± 0.01 B,a0.47 ± 0.02 B,b0.60 ± 0.02 AB,b0.65 ± 0.02 A,b
BSG/Gel/Gl/15ClP0.46 ± 0.01 C,a0.53 ± 0.02 BC,ab0.68 ± 0.02 AB,ab0.73 ± 0.02 A,ab
Sample0th Day2nd Day4th Day6th Day
Heme iron (μg/g)
CONTROL7.67 ± 0.16 A,a6.26 ± 0.21 AB,b5.55 ± 0.15 BC,b4.20 ± 0.25 C,b
BSG/Gel/Gl/15EG@NZ7.67 ± 0.16 A,a7.51 ± 0.25 AB,a6.67 ± 0.18 ABC,a5.04 ± 0.30 C,a
BSG/Gel/Gl/15ClP7.67 ± 0.16 A,a6.89 ± 0.23 AB,ab6.11 ± 0.17 BC,ab4.62 ± 0.28 C,ab
ABC The letters are used to show the significant difference in TBARS or heme iron values in each film between different days, while ab are used to show the significant difference in values in the same day between different films. The use of the letters is further explained in the Supplementary document.
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Ntari, Z.; Kechagias, A.; Leontiou, A.A.; Vardakas, A.; Dormousoglou, M.; Angelari, T.; Zaharioudakis, K.; Stathopoulou, P.; Karahaliou, P.; Beligiannis, G.; et al. Eugenol@Natural Zeolite Nanohybrid vs. Clove Powder as Active and Reinforcement Agents in Novel Brewer’s Spent Grain/Gelatin/Glycerol Edible, High Oxygen Barrier Active Packaging Films. Appl. Sci. 2025, 15, 9282. https://doi.org/10.3390/app15179282

AMA Style

Ntari Z, Kechagias A, Leontiou AA, Vardakas A, Dormousoglou M, Angelari T, Zaharioudakis K, Stathopoulou P, Karahaliou P, Beligiannis G, et al. Eugenol@Natural Zeolite Nanohybrid vs. Clove Powder as Active and Reinforcement Agents in Novel Brewer’s Spent Grain/Gelatin/Glycerol Edible, High Oxygen Barrier Active Packaging Films. Applied Sciences. 2025; 15(17):9282. https://doi.org/10.3390/app15179282

Chicago/Turabian Style

Ntari, Zoe, Achilleas Kechagias, Areti A. Leontiou, Alexios Vardakas, Margarita Dormousoglou, Tarsizia Angelari, Konstantinos Zaharioudakis, Panagiota Stathopoulou, Panagiota Karahaliou, Grigorios Beligiannis, and et al. 2025. "Eugenol@Natural Zeolite Nanohybrid vs. Clove Powder as Active and Reinforcement Agents in Novel Brewer’s Spent Grain/Gelatin/Glycerol Edible, High Oxygen Barrier Active Packaging Films" Applied Sciences 15, no. 17: 9282. https://doi.org/10.3390/app15179282

APA Style

Ntari, Z., Kechagias, A., Leontiou, A. A., Vardakas, A., Dormousoglou, M., Angelari, T., Zaharioudakis, K., Stathopoulou, P., Karahaliou, P., Beligiannis, G., Proestos, C., Salmas, C. E., & Giannakas, A. E. (2025). Eugenol@Natural Zeolite Nanohybrid vs. Clove Powder as Active and Reinforcement Agents in Novel Brewer’s Spent Grain/Gelatin/Glycerol Edible, High Oxygen Barrier Active Packaging Films. Applied Sciences, 15(17), 9282. https://doi.org/10.3390/app15179282

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