MIL-100(Fe)-Based Composite Films for Food Packaging

A biocompatible metal–organic framework MIL-100(Fe) loaded with the active compounds of tea tree essential oil was used to produce composite films based on κ-carrageenan and hydroxypropyl methylcellulose with the uniform distribution of the particles of this filler. The composite films featured great UV-blocking properties, good water vapor permeability, and modest antibacterial activity against both Gram-negative and Gram-positive bacteria. The use of metal–organic frameworks as containers of hydrophobic molecules of natural active compounds makes the composites made from naturally occurring hydrocolloids attractive materials for active packaging of food products.


Introduction
Materials for active food packaging are among the most trending research topics in the food industry [1]. Increasing the shelf life of food products [2] by inhibiting food spoilage through direct or indirect interactions with the active agents in the packaging, these composite materials are sought to solve global environmental problems, such as plastic and food waste [3]. The sustainability of their production from renewable resources, often waste [4], is crucial in the era of the depletion of natural resources, especially fossil fuels [5].
The sustainability and biocompatibility of such materials motivated the researchers to consider natural compounds both as matrices and active agents in active food packaging. The focus of this research is the materials produced from natural hydrocolloids, polysaccharide-, and protein-based water-soluble polymers able to form strong three-dimensional networks in aqueous solutions. They include starch [6], cellulose derivatives [7], chitosan [8], carrageenans [9], and others [10][11][12][13] that feature high biocompatibility [14], biodegradability [15], availability and variability [16], and good barrier properties [17]. However, the low affinity of hydrocolloids to hydrophobic molecules of most natural active compounds, such as plant essential oils (EOs) [18], hinders their industrial use in active food packaging.
Of the popular active agents in functional food packaging, EOs are complex mixtures of bioactive molecules that often act in synergy and are hard to isolate [19]. Tea tree essential oil (TTO) from Melaleuca alternifolia, which contains monoterpenes, sesquiterpenes, and their alcohol derivatives [20], is known for its antimicrobial and antioxidant properties that

Scanning Electron Microscopy (SEM)
High-resolution surface images of the composite film samples were obtained using the Zeiss CrossBeam 550 Scanning Electron Microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). Images were collected using an in-lens detector operating at 5 kV.

Thickness
The thickness of the composite film samples was measured in 10 random places using a digital micrometer. The mean values were also used to probe the mechanical and optical properties of the films.

Mechanical Properties
Mechanical properties, such as tensile strength, elastic modulus, and elongation at break, were measured for the composite film samples (750 mm long and 13 mm wide) prepared in accordance with ASTM D882 [38]. Measurements were conducted using a universal testing machine (Zwick/Roell Z2.5, Zwick GmbH & Co., Ulm, Germany) operating with a 50 N load cell with a traction speed of 50 mm/min. All assays were performed at 30 ± 2 • C with a relative humidity of 50 ± 2%. Each sample was tested at least 5 times in parallel. The elastic modulus was obtained as the slope of an elastic (linear) region of the stress/strain curves. The tensile strength (TS) (Equation (1)) and the elongation at break (EAB) (Equation (2)) were obtained as follows: where F is the breaking force exerted (N), S is the cross-sectional film area (mm 2 ), ∆L is the film elongation at the end of the procedure (mm), and L 0 is the initial film elongation (mm).

Water Vapor Permeability (WVP)
The water vapor permeability of the composite film samples was evaluated using the gravimetric method in accordance with ASTM E96 adapted to hydrophilic films [39]. The film samples were cut into circles and fixed in a glass vial containing 6 g of dry silica gel Nanomaterials 2023, 13, 1714 4 of 12 (0% RH) with the distance between the silica gel and the composite film about 2 cm. The vials were kept in a desiccator containing a saturated solution of NaCl with an excess of undissolved NaCl at 25 ± 2 • C and 75% RH for 2 h. After the initial incubation period, a steady state had been achieved and periodic weighing of vials without film samples provided the amount of water permeated through the film for 24 h. WVP was defined using Equation (3): where WVP is the water vapor permeability (g·mm/Pa·h·m 2 ), A (g/h) is the slope of linear regression of the vial weight vs. the time graph, ∆x (mm) is the thickness of the film sample, ∆p (2.377 kPa) is the difference in partial pressure, and S (m 2 ) is the exposed surface area of the film. Each experiment was repeated at least three times.

Light Transmittance and Transparency
Light transmittance of the composite film samples was measured at the ultraviolet and visible ranges (220-500 nm) using a UV-vis spectrophotometer UV-2600 (Shimadzu, Kyoto, Japan). The transparency value (TV) of the film was calculated using Equation (4): where T 500 is the fractional transmittance at 500 nm and ∆x (mm) is the film thickness. The higher transparency value corresponds to the lower transparency of the film.

Indentification of the Components of TTO in MIL-100(Fe)-TTO
MIL-100(Fe)-TTO powder (10 mg) was placed in a 1M HCl solution (2 mL) and diluted with distilled water (8 mL). After the complete hydrolysis of MOF, the aqueous phase was separated from the precipitate and extracted with methylene chloride (10 mL); the organic fraction was separated and analyzed using a GCMS-QP2020 (Shimadzu, Kyoto, Japan) with a quadrupole detector. Analysis conditions: sample volume-1 µL; injector temperature −250 • C; column SH-RTx-5MS (30 m, 0.25 mm, 0.25 µm); thermostat temperature-40 • C, 1 min -> heating to 290 • C, 30 • C/min -> 290 • C, 2 min; carrier gas-helium, column flow rate-1 mL/min; ion source temperature-200 • C; and interface temperature-250 • C. The contents of the TTO components in the test sample were estimated by comparing their retention time (min), peak area, peak height, and mass spectra patterns with those from the database of authentic compounds stored in the library of the National Institute of Standards and Technology (NIST).

Antibacterial Properties of Composite Films
The antibacterial activity of different composite films samples was probed against Gram-negative bacteria, Escherichia coli (E. coli ATCC 25922), and Gram-positive bacteria, Staphylococcus aureus (S. aureus ATCC 6538p), using the standard colony counting method [30]. Fresh bacterial cultures in the Mueller-Hinton (MH) broth were obtained via incubation at 37 • C. Bacterial suspensions were then adjusted to~10 7 CFU/mL with sterilized 9% NaCl solution. Different samples were cut into circles with a diameter of 5 cm and immersed into bacterial suspensions (30 mL) in the culture dish. The supernatant was taken out at 1, 3, and 5 h after the addition of the samples and it was diluted to a proper concentration with a 9% NaCl solution. The resulting solutions (100 µL) were then spread on the surface of MH agar solid media. The solid media were cultured at 37 • C for 18 h, and the viable numbers of E. coli and S. aureus colonies were counted via visual observation. The E. coli and S. aureus suspension without any samples was cultured on an MH agar solid medium as a control. Each experiment was repeated at least three times.

Statistical Analysis
Statistical analysis was performed using DSAASTAT, ver. 1.541, by Andrea Onofri (Perugia, Italy). The data were analyzed using the analysis of variance (ANOVA), and multiple comparisons of the means were made using the Tukey's test (p < 0.05).

Results and Discussion
3.1. Characterizations of MIL-100(Fe) and MIL-100(Fe)-TTO MIL-100(Fe) was synthesized via the sustainable protocol in water at room temperature as described previously [37] to produce a precipitate with high crystallinity in the absence of any inorganic corrosive acid, such as HF and HNO 3 that are conventionally used to induce crystal growth [40]. The use of the iron(II) salt prevents the formation of semi-amorphous Fe-BTC (a MOF material, commercialized as Basolite F300, with the same metal ions and organic linkers) that occurs under the same conditions from an iron(III) salt. Although Fe-BTC can be reconstructed in water for high crystallinity using the recently emerged approach [41], the process is time-consuming. The formation of the target MIL-100(Fe) under the chosen conditions was confirmed using powder X-ray diffraction ( Figure 1a). In agreement with the data reported earlier [40], the XRD pattern featured both groups of the characteristic peaks of MIL-100(Fe) at 5-7 • and 10-11 • 2θ ranges. multiple comparisons of the means were made using the Tukey's test (p < 0.05).

Characterizations of MIL-100(Fe) and MIL-100(Fe)-TTO
MIL-100(Fe) was synthesized via the sustainable protocol in water at room tempera ture as described previously [37] to produce a precipitate with high crystallinity in th absence of any inorganic corrosive acid, such as HF and HNO3 that are conventionall used to induce crystal growth [40]. The use of the iron(II) salt prevents the formation o semi-amorphous Fe-BTC (a MOF material, commercialized as Basolite F300, with th same metal ions and organic linkers) that occurs under the same conditions from a iron(III) salt. Although Fe-BTC can be reconstructed in water for high crystallinity usin the recently emerged approach [41], the process is time-consuming. The formation of th target MIL-100(Fe) under the chosen conditions was confirmed using powder X-ray di fraction ( Figure 1a). In agreement with the data reported earlier [40], the XRD pattern fea tured both groups of the characteristic peaks of MIL-100(Fe) at 5-7° and 10-11° 2θ range TTO was encapsulated into MIL-100(Fe), which was activated under vacuum at 12 °C for 24 h to remove all the solvent molecules from the pores, using vacuum distillatio through the layer of TTO was encapsulated into MIL-100(Fe), which was activated under vacuum at 120 • C for 24 h to remove all the solvent molecules from the pores, using vacuum distillation through the layer of powdered MIL-100(Fe), as this technique ensures better adsorption of TTO as compared to the conventional post-synthetic incorporation via soaking. The XRD pattern of the powder sample of the resulting MIL-100(Fe)-TTO (Figure 1b) showed no changes in the position of the diffraction peaks upon the encapsulation of TTO; however, the change in relative intensities of both groups of the characteristic peaks (an increase for the group at 5-7 • 2θ and a decrease for the group at 10-11 • 2θ) might hint some minor changes in the crystal structure of MIL-100(Fe) upon the encapsulation of the chosen guest.
The components of TTO in MIL-100(Fe)-TTO were identified by analyzing the composition of the organic matter remaining from acid hydrolysis using GC-MS ( Figure 2). The comparison to the sample of TTO prepared under the same conditions showed that all the main components of TTO, except for terpinen-4-ol, were present in MIL-100(Fe)-TTO (Table 2). A higher content of p-cymene in the latter may result from stacking interactions of its aromatic ring with BTC 2linkers that facilitate its encapsulation in the pores of MIL-100(Fe).
The components of TTO in MIL-100(Fe)-TTO were identified by analyzing the composition of the organic matter remaining from acid hydrolysis using GC-MS ( Figure 2). The comparison to the sample of TTO prepared under the same conditions showed that all the main components of TTO, except for terpinen-4-ol, were present in MIL-100(Fe)-TTO (Table 2). A higher content of p-cymene in the latter may result from stacking interactions of its aromatic ring with BTC 2-linkers that facilitate its encapsulation in the pores of MIL-100(Fe).  Table 2. Terpinen-4-ol 97

Characterizations of Composite Films
The composite films containing MIL-100(Fe) or MIL-100(Fe)-TTO as a filler at different concentrations (0.5, 1, 2, and 5 wt% of the total hydrocolloid weight) and a mixture of Kc and HPMC in a 4:1 ratio as a polymer matrix were fabricated using the Dr. Blade technique via a home-built coating machine to produce a layer of the film-forming solution with the same thickness for all compositions. The MIL-100(Fe) and MIL-100(Fe)-TTO powders were ultrasonically pre-treated to evenly disperse the particles of these MOFs in the  Table 2. Terpinen-4-ol 97

Characterizations of Composite Films
The composite films containing MIL-100(Fe) or MIL-100(Fe)-TTO as a filler at different concentrations (0.5, 1, 2, and 5 wt% of the total hydrocolloid weight) and a mixture of Kc and HPMC in a 4:1 ratio as a polymer matrix were fabricated using the Dr. Blade technique via a home-built coating machine to produce a layer of the film-forming solution with the same thickness for all compositions. The MIL-100(Fe) and MIL-100(Fe)-TTO powders were ultrasonically pre-treated to evenly disperse the particles of these MOFs in the film-forming solution before the addition of hydrocolloids, as the increase in the viscosity of the solution caused clumping of the powders and an uneven distribution of the filler particles. A 2 mm substrate-blade gap was chosen as the biggest height that the film-forming solution was able to retain at 45 • C.
The resulting composite films containing MIL-100(Fe) and MIL-100(Fe)-TTO featured good transparency with the brown color of MIL-100(Fe) becoming more intense with the increase in the content of the filler (Figure 3a,c). The particles of MIL-100(Fe) or MIL-100(Fe)-TTO (50-400 nm) were uniformly distributed in the films (Figure 3b,d) which kept the microstructure of the matrix at lower concentrations of these filles. At concentrations above 2%, however, the aggregation of the particles (0.5-5 µm) resulted in a rougher surface on the composite films.
film-forming solution before the addition of hydrocolloids, as the increase in the viscosity of the solution caused clumping of the powders and an uneven distribution of the filler particles. A 2 mm substrate-blade gap was chosen as the biggest height that the film-forming solution was able to retain at 45 °C.
The resulting composite films containing MIL-100(Fe) and MIL-100(Fe)-TTO featured good transparency with the brown color of MIL-100(Fe) becoming more intense with the increase in the content of the filler (Figure 3a,c). The particles of MIL-100(Fe) or MIL-100(Fe)-TTO (50-400 nm) were uniformly distributed in the films (Figure 3b,d) which kept the microstructure of the matrix at lower concentrations of these filles. At concentrations above 2%, however, the aggregation of the particles (0.5-5 µm) resulted in a rougher surface on the composite films.
The incorporation of MIL-100(Fe) or MIL-100(Fe)-TTO into the composite films was confirmed using powder X-ray diffraction (Figure 1). The latter showed the presence of the characteristic peaks of MIL-100(Fe) at 10-11° 2θ in the XRD patterns of all the samples and a gradual increase in their intensities with an increase in the content of the filler in the continuous phase of the amorphous matrix. Transparency has a great impact on the appearance of a packaged product. Therefore, UV-vis spectroscopy was used to assess the light transmittance of the obtained films ( Figure 4). The control film that contained no filler featured the lowest transmittance of 2.07 at 500 nm (Table 3), which is the highest transparency of the films. However, it dramatically increased upon the incorporation of MIL-100(Fe) or MIL-100(Fe)-TTO following the above color change with an increase in the content of these fillers. The difference between the composite films containing MIL-100(Fe) and MIL-100(Fe)-TTO may arise from the higher mass and, therefore, a lower amount of the embedded MOF particles when loaded with TTO. The films block the UV light up to 320 nm, which can be useful for reducing its harmful impact on foodstuff during long-term storage. The incorporation of MIL-100(Fe) or MIL-100(Fe)-TTO into the composite films was confirmed using powder X-ray diffraction (Figure 1). The latter showed the presence of the characteristic peaks of MIL-100(Fe) at 10-11 • 2θ in the XRD patterns of all the samples and a gradual increase in their intensities with an increase in the content of the filler in the continuous phase of the amorphous matrix.
Transparency has a great impact on the appearance of a packaged product. Therefore, UV-vis spectroscopy was used to assess the light transmittance of the obtained films ( Figure 4). The control film that contained no filler featured the lowest transmittance of 2.07 at 500 nm (Table 3), which is the highest transparency of the films. However, it dramatically increased upon the incorporation of MIL-100(Fe) or MIL-100(Fe)-TTO following the above color change with an increase in the content of these fillers. The difference between the composite films containing MIL-100(Fe) and MIL-100(Fe)-TTO may arise from the higher mass and, therefore, a lower amount of the embedded MOF particles when loaded with TTO. The films block the UV light up to 320 nm, which can be useful for reducing its harmful impact on foodstuff during long-term storage.  Good mechanical properties are crucial for the applications of composite materials in food packaging. While the film thickness gradually increased with the content of MIL-100(Fe) and MIL-100(Fe)-TTO, the presence of 0.5-1% of these fillers in the composition caused the decrease in the tensile strength, elongation at break, and elastic modulus of the composite films (Table 3). At higher contents of MIL-100(Fe) and MIL-100(Fe)-TTO (to 2% and 5%, respectively), however, these mechanical properties improved, possibly by the compaction of the polymer matrix that occurs along with the aggregation of the filler particles. The tensile strength of the films is comparable to that of high-density polyethylene (HDPE, 15-45 MPa) and low-density polyethylene (LDPE, 7-14 MPa) commonly used in commercial packaging [42].
Water vapor permeability (WVP), which describes the moisture migration between food components and the surrounding atmosphere, is another important characteristic of food packaging. The composite films containing MIL-100(Fe) and MIL-100(Fe)-TTO show different behaviors (Table 3). The addition of MIL-100(Fe)-TTO to the composition resulted in an increase in WVP that correlated with the content of this filler. In contrast, the films with MIL-100(Fe) had better moisture barrier properties at lower concentrations (0.5%). This may be explained by bonding between the sulfate groups of κ-carrageenan and the metal ions on the surface of MIL-100(Fe) NPs that is inhibited by aggregation at higher concentrations (1-5%) and in the presence of a hydrophobic layer of TTO on MIL-100(Fe)-TTO NPs. Table 3. Thickness, mechanical (tensile strength, TS; elongation at break, EAB; elastic modulus, EM), barrier (water vapor permeability, WVP), and optical (transmittance value, TV) properties of the composite films without the filler and with its different concentrations (0.5, 1, 2, and 5 wt% of the total hydrocolloid weight).

Sample
Thickness (   Good mechanical properties are crucial for the applications of composite materials in food packaging. While the film thickness gradually increased with the content of MIL-100(Fe) and MIL-100(Fe)-TTO, the presence of 0.5-1% of these fillers in the composition caused the decrease in the tensile strength, elongation at break, and elastic modulus of the composite films (Table 3). At higher contents of MIL-100(Fe) and MIL-100(Fe)-TTO (to 2% and 5%, respectively), however, these mechanical properties improved, possibly by the compaction of the polymer matrix that occurs along with the aggregation of the filler particles. The tensile strength of the films is comparable to that of high-density polyethylene (HDPE, 15-45 MPa) and low-density polyethylene (LDPE, 7-14 MPa) commonly used in commercial packaging [42].
Water vapor permeability (WVP), which describes the moisture migration between food components and the surrounding atmosphere, is another important characteristic of food packaging. The composite films containing MIL-100(Fe) and MIL-100(Fe)-TTO show different behaviors ( Table 3). The addition of MIL-100(Fe)-TTO to the composition resulted in an increase in WVP that correlated with the content of this filler. In contrast, the films with MIL-100(Fe) had better moisture barrier properties at lower concentrations (0.5%). This may be explained by bonding between the sulfate groups of κ-carrageenan and the metal ions on the surface of MIL-100(Fe) NPs that is inhibited by aggregation at higher concentrations (1-5%) and in the presence of a hydrophobic layer of TTO on MIL-100(Fe)-TTO NPs.
The antibacterial properties of the films were tested against E. coli and S. aureus using the standard colony counting method ( Figure 5). The films based on MIL-100(Fe) emerged as more active against both pathogens than those based on MIL-100(Fe)-TTO (Tables 4 and 5). A plausible reason behind the lower antibacterial activity of the latter is the release of the iron(III) ions and BTC 2− anions upon the decomposition of MIL-100(Fe) in a saline environment inhibited by a hydrophobic layer of TTO on the surface of NPs. The hydrophobicity of the components of TTO and its low concentration in MIL-100(Fe)-TTO may also be contributed to the rather poor performance of the composite films based on MIL-100(Fe)-TTO. The antibacterial properties of the films were tested against E. coli and S. aureus using the standard colony counting method ( Figure 5). The films based on MIL-100(Fe) emerged as more active against both pathogens than those based on MIL-100(Fe)-TTO (Tables 4 and  5). A plausible reason behind the lower antibacterial activity of the latter is the release of the iron(III) ions and BTC 2− anions upon the decomposition of MIL-100(Fe) in a saline environment inhibited by a hydrophobic layer of TTO on the surface of NPs. The hydrophobicity of the components of TTO and its low concentration in MIL-100(Fe)-TTO may also be contributed to the rather poor performance of the composite films based on MIL-100(Fe)-TTO.     Table 4. Antibacterial activity of the films with the different concentrations of the filler (0.5, 1, 2, and 5 wt% of the total hydrocolloid weight) against S. aureus.

Sample
Cell Density (lg(CFU/mL)) 0 h 1 h 3 h 5 h Table 5. Antibacterial activity of the films with the different concentrations of the filler (0.5, 1, 2, and 5 wt% of the total hydrocolloid weight) against E. coli.

Conclusions
The use MIL-100(Fe) loaded with the active compounds of tea tree essential oil as a filler for the hydrocolloid-based composite films improved their UV-blocking ability without a deterioration in the mechanical properties. The latter were comparable-at the highest concentration of the filler-to those of commercial LDPE and HDPE, which cannot be achieved upon the direct incorporation of essential oils into the hydrocolloid matrix, as it produces brittle materials based on κ-carrageenan [43,44] and hydroxypropyl methylcellulose [45,46]. The films with MIL-100(Fe) also showed modest antibacterial activity against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria while the films with MIL-100(Fe)-TTO did not have any significant bactericidal effect on these foodborne pathogens. These composite materials, therefore, may emerge as active packaging or active coating on foods. Further studies are, however, needed to assess their effect on the packaged products; they are underway within our group. Data Availability Statement: Data presented in this study are available on request from the corresponding author.