Novel LDPE/halloysite nanotube films with sustained carvacrol release for broad-spectrum antimicrobial activity

Abstract

The emergence of antibiotic resistance of pathogenic bacteria has led to renewed interest in exploring the potential of plant-derived antimicrobials e.g., essential oils (EOs), as an alternative strategy to reduce microbial contamination. However, the volatile nature of EOs presents a major challenge in their incorporation into polymers by conventional high-temperature processing techniques. Herein, we employ halloysite nanotubes (HNTs) as efficient nano-carriers for carvacrol (a model EO). This pre-compounding encapsulation step imparts enhanced thermal stability to the carvacrol, allowing for its subsequent melt compounding with low-density polyethylene (LDPE). The resulting polymer nanocomposites exhibit outstanding antimicrobial properties with a broad spectrum of inhibitory activity against Escherichia coli, Listeria innocua in biofilms, and Alternaria alternata. Their antimicrobial effectiveness is also successfully demonstrated in complex model food systems (soft cheese and bread). This superior activity, compared to other studied carvacrol containing films, is induced by the significantly higher carvacrol content in the film as well as its slower out-diffusion from the hybrid system. Thus, these new active polymer nanocomposites presents an immense potential in controlling microbial contamination and biofilm related adverse effects, rendering them as excellent candidate materials for a wide range of applications.

---

Introduction

Polymeric films are susceptible to bacterial colonization and biofilm formation.¹ Therefore, there is an immense need for developing methodologies for the incorporation of natural antimicrobial molecules in order to control microbial contamination and biofilm related adverse effects in the biomedical, food and industrial fields. Moreover, these polymeric films may also have high potential for use in the food packaging industry in order to reduce, inhibit or prevent the development of microorganisms that are present in the packed food or on the packaging material.²

Antimicrobial function in polymers can be achieved by adding active antimicrobial ingredients into the polymeric matrix or by using polymers with intrinsic antimicrobial properties.³ The latter are suffering from limited applicability as a packaging for solid and semi-solid foods and their properties are inferior in comparison to common synthetic polymers.³

Antimicrobial agents incorporated into polymers have to be selected based on their spectrum of activity, mode of action and chemical composition in order to be compatible with the polymer material and its specific application, as well as effectively manage the target microorganism.^2,4

Different chemicals, such as metal ions, organic acids and their salts have been successfully incorporated as antimicrobial substances into plastic materials.^5,6 However, the emergence of antibiotic resistance in pathogenic bacteria has led to renewed interest in exploring the potential of plant-derived antimicrobials as an alternative strategy to combat microbial infections.⁷ Essential oils (EOs) are natural substances, recognized as GRAS (Generally Recognized as Safe), derived from plants that possess antimicrobial activity against a wide range of microorganisms, including bacteria, yeast and molds.^8–14 Historically, these compounds have been used as a safe, effective and natural remedy for diseases in traditional medicine.⁷ Nowadays, both regulation agencies and consumers demands are tending towards utilization of natural compounds over synthetic substances that their long term effects are unknown.¹⁵

One main disadvantage of EOs is their volatile character, which until recently limited their incorporation into polymers to mainly cold-coating technologies.^16,17 Once the EOs are compounded with polymers by conventional melt-processing technologies, antimicrobial activity may be achieved.^{13,16,18–25} However, this activity is evaluated, in most cases, immediately after films production,^25–28 while the challenges related to longstanding activity of the EO within the polymeric matrix are not addressed.

In our recent studies,^29,30 we have demonstrated that organoclays can be used as active carriers for EOs to preserve them during the high-temperature compounding of the polymer, while preserving high antimicrobial properties for a required shelf life. The organo-modification of clay was found as crucial for the EOs intercalation in between the clay galleries and its enhanced thermal stability.²⁹ The resulting nanocomposites displayed much higher EOs content in the final film in addition to lowered diffusion of the EOs in comparison to the control films with no organoclays carriers.³⁰

In recent years halloysite nanotubes (HNTs), which are naturally occurring members of the kaolin family of aluminosilicate clay and characterized by a hollow tubular structure, emerge as promising nanoscale containers for the encapsulation of different active molecules.^31–37 HNTs are natural nanomaterials and are considered as environmentally friendly.^38–42 Importantly, they can be used as nanocapsules without further chemical modification and exhibit a high level of biocompatibility and very low toxicity.^43–45 In addition, the HNTs excellent mechanical properties and high thermal stability combined with their abundance provide immense potential for designing new biomaterials^46,47 and high-performance polymers composites.^48–51

In this work, we employ HNTs as nano-carriers for carvacrol, a model antimicrobial EO. Taking into consideration their unique hollow tubular structure, we use a pre-compounding step in which HNTs/carvacrol hybrids are produced, in order to promote loading and encapsulation of the carvacrol into the nanotubes, as schematically demonstrated in Scheme 1. Subsequently, the HNTs/carvacrol hybrids are melt-compounded with low-density polyethylene (LDPE) and films are produced. We validate the ability of the HNTs carriers to retain the volatile carvacrol under the high-temperature compounding and their ability to sustain carvacrol release from the films. The resulting LDPE films exhibit outstanding antimicrobial properties with a broad spectrum of inhibitory activity against Gram-negative (Escherichia coli) bacterium, Gram-positive (Listeria innocua) bacterium in biofilm, as well as model pathogenic fungus, Alternaria alternata. To demonstrate the potential applicability of these films for active food packaging, we show their outstanding performance in inhibiting fungi growth on bread and antibacterial effect in soft cheese. Thus, these films can minimize the addition of preservatives into food formulations to extend their shelf life and enhance food quality and safety.

Scheme 1 A schematic illustration of halloysite nanotubes (HNTs) loaded with carvacrol molecules (a model antimicrobial EO) as achieved by a pre-compounding step in which HNTs/carvacrol hybrids are produced.

Experimental

Materials

Halloysite nanotubes (HNTs) are supplied by NaturalNano (USA) and are characterized by a tubular form with an external diameter typically smaller than 100 nm, internal diameter of 20 nm and length of 0.2 to 2 μm. Low-density polyethylene (LDPE), Ipethene 320, is supplied by Carmel Olefins Ltd. (Haifa, Israel) with melt flow rate of 2 g/10 min. Carvacrol (98%), Bacto agar, Nutrient Broth (NB) medium, Tryptic Soy Broth (TSB), Potato Dextrose Agar (PDA), and Triton X-100 are purchased from Sigma Aldrich (Israel). NB Bacto-agar is purchased from Becton Dickinson (USA).

Preparation of HNTs/carvacrol hybrids

Halloysite nanotubes/carvacrol hybrids are prepared by shear mixing carvacrol with HNTs at a weight ratio of 2 : 1 (respectively) followed by ultrasonication at room temperature for 20 min at a constant amplitude of 40% using Vibra cell VCX 750 instrument (Sonics & Materials Inc., USA) to achieve a uniform HNTs/carvacrol dispersion (see Scheme 1).

Preparation of LDPE-based films

Low-density polyethylene is melt-compounded with HNTs/carvacrol hybrids using a 16 mm twin-screw extruder (Prism, England) L/D ratio of 25 : 1 with a screw speed of 150 rpm and feeding rate of 2 kg h⁻¹ at 140 °C. Table 1 specifies the composition of the investigated systems. Following the compounding process, 150 μm thick films are prepared by compression molding at 140 °C.

Table 1 Composition of studied films

Sample	Composition (wt%)
	LDPE	HNTs	Carvacrol
a Note that the HNTs are used as a conventional filler and are dry blended with carvacrol following compounding with LDPE. The pre-compounding step, in which the HNTs/carvacrol hybrid is prepared, is not performed.
Neat LDPE	100	0	0
LDPE/HNTs	98	2	0
LDPE/carvacrol	96	0	4
LDPE/HNTs/carvacrol^a	94	2	4
LDPE/(HNTs/carvacrol hybrid)	94	(2/4)

---

Characterization

Thermogravimetric analysis (TGA). Hybrids are characterized by thermal gravimetric analysis (TGA) using TGA-Q5000 system (TA instruments, USA) at a heating rate of 20 °C min⁻¹ under nitrogen atmosphere, beginning at room temperature up to 600 °C.

The accelerated release of carvacrol from the LDPE-based films is investigated by isothermal gravimetric analysis using TGA Q5000 system at a constant temperature of 60 °C under nitrogen atmosphere for a duration of ∼10 h, until the mass of the film becomes steady. This method is used to quantify carvacrol release from the films to the atmosphere, mimicking the films mode of use in real applications e.g., food packaging. Several methods have been reported to characterize the diffusion of active agents and other additives in polymer substrates.^52–56 In this work, we derive the diffusion coefficient from the initial linear slope of the fractional mass release ratio vs. t^1/2 (eqn (1)).^52,56

where m_t and m_∞ are the amounts of additive (carvacrol) released from the film at time t and at equilibrium t = ∞, respectively. D (m² s⁻¹) is the diffusion coefficient and l is the thickness of the film.

High-resolution scanning electron microscopy (HRSEM). The nanostructure of neat HNTs and films is studied using a Carl Zeiss Ultra Plus high-resolution scanning electron microscope (HRSEM) operated at 1 keV accelerating voltage. Films are cryogenically fractured in liquid nitrogen prior to observation.

Infrared spectroscopy. Fourier-transform infrared spectroscopy (FTIR) is used to characterize the chemical structure of carvacrol following compounding. Spectra are recorded using a Thermo 6700 FTIR instrument, equipped with a Smart iTR Attenuated Total Reflectance (ATR) diamond plate, and OMNIC v8.0 software. Spectra are collected from at least three different locations on the films.

Antibacterial activity

Escherichia coli. The antibacterial activity of the different films is evaluated by inhibition of Escherichia coli (E. coli, ATCC 8739) growth in liquid media as previously reported.²⁹ Briefly, loop full of E. coli stored at −80 °C is cultured overnight in 3 mL nutrient broth (NB) medium at 37 °C under agitation (250 rpm). In the following day, the culture is diluted in fresh NB medium and incubated for an additional ∼1.5 h, allowing the cells to enter their logarithmic stage. As the culture reached an optical density value of 0.6, it is diluted by 1 : 100 with 1% NB to obtain a bacterial stock solution at a concentration of 10⁵ CFU (colony forming unit) mL⁻¹. Antibacterial activity tests are performed in 24-well plates where a film sample (disks of 1.2 cm diameter) is placed in a well together with 1 mL of the E. coli stock solution. The plates are incubated at 37 °C under continuous agitation (100 rpm) for 24 h. Incubation is followed by serial dilutions with in 1 : 100 NB (performed in 96-well plates). Viable cell counts are assessed by the drop-plate technique; 20 μL drops are incorporated into NB Bacto-agar in 9 cm Petri plates. Plates are incubated at 37 °C for 24 h; CFU are counted and log reduction is calculated in comparison to E. coli cultured in NB 1 : 100 medium (10⁸ CFU mL⁻¹), used as a control. All measurements, including the growth controls, are performed in triplicates.

Listeria innocua. The potential of the various LDPE films containing carvacrol to inhibit biofilm formation is evaluated using the Gram-positive Listeria innocua (ATCC 33090), as the model microorganism. Bacteria are grown overnight in tryptic soy broth (TSB) medium. On the following day, bacterial cells are diluted in TSB to obtain a stock solution with an optical density value at 595 nm (OD₅₉₅) of 0.3 (corresponding to ∼3 × 10⁸ CFU mL⁻¹). 1 mL from the stock solution is taken into each well in a 24-well plate. Each of the different films (discs of 1 cm in diameter) is added to the well. The plates are then incubated at 25 °C under gentle agitation (100 rpm) for 20 h. In the day after, the films are rinsed 3 times with distilled water to remove the unattached bacteria (i.e. planktonic cells) and subsequently the attached cells are scraped from the films using 250 μL of Tris–HCl (0.1 M, pH 7.2) and cell scrapers. 200 μL out of the 250 μL, used for scrapping the cells, are transferred into the first line of a 96-well plate, while the rest of the lines are filled with 180 μL of Tris–HCl (0.1 M, pH 7.2). Serial dilutions are carried out and the cells are spotted onto NB agar plates, which are then incubated at 37 °C for 20 h. Cell growth is monitored and determined by a viable cell count. The experiments are conducted in triplicates, with internal duplicates.

Antibacterial assay with inoculated soft cheese

White soft cheese (5% fat, Tara Dairy, Israel), purchased in a local supermarket, is diluted 1 : 10 in saline according to the FDA's Bacteriological Analytical Manual (BAM), detection and enumeration of Listeria monocytogenes in foods.⁵⁷ L. innocua is added to final concentration of 10⁴ CFU mL⁻¹. 1.2 cm diameter films are incubated in 1 mL culture (per well) in a 24-well plate for 22 h at 26 °C under agitation (100 rpm). For testing antibacterial activity of volatiles, films are placed on top of a Petri plate (no direct contact between the film and the bacterial solution) containing 10 mL L. innocua culture in a 1 : 10 diluted soft cheese as mentioned above, followed by incubation for 22 h at 26 °C under agitation (100 rpm). Both direct and indirect assays are followed by drop test enumeration in duplicates onto oxford-modified plate and incubated for 48 h at 30 °C.

Antifungal activity

Fungal cultures. The phytopathogenic, clinical and food spoilage fungi Alternaria alternata (A. alternata), originating from the surface of tomato and Penicillium italicum (P. italicum), Penicillium digitatum (P. digitatum), Penicillium roqueforti (P. roqueforti), isolated from naturally rotten oranges, are cultured on 1% potato dextrose agar (PDA: 10 g L⁻¹; Bacto-agar: 11 g L⁻¹ in 1000 mL of deionized water) and are utilized as model fungi for the purpose of the study.

Direct contact assay. A modified ISO 16869:2008 protocol is used to evaluate antifungal efficacy the films, as we previously reported.²⁹ Briefly, the ISO protocol is modified by employing 1% PDA instead of nutrient salt agar. Discs (area of ∼7.1 cm²) are removed with a manual puncher from film samples and placed onto 1% PDA that serves as base agar layer, in the center of 9 cm Petri plates. A. alternata conidial suspension is prepared by harvesting conidia from 5 days old cultures with sterilized de-ionized water containing 0.01% w/w of Triton X-100. The spore suspension is adjusted with the aid of a hemocytometer and is then transferred into soft agar (pre-solidified 1% PDA cooled to 45 °C), producing an inoculum suspension within the soft agar at a final conidial concentration of 10⁵ conidia per mL. The inoculum is poured over the film disc and the base agar layer, covering the specimen with a thin layer of inoculum. The Petri plates are left at room temperature for 1 h to allow the inoculum layer to solidify. Then, the plates are sealed with Para-Film and incubated at 25 °C in the dark for 7 days. Neat LDPE films are used as negative controls. Following incubation, the antifungal activity of the films is quantified with the aid of an optical stereomicroscope (Axio Scope.A1; Carl Zeiss, Oberkochen, Germany) using a non-parametric ordinal scale, as follows: 4 = Very High (VH) activity – no fungal growth; 3 = High (H) activity – limited mycelial growth; 2 = Moderate (M) activity – mycelial growth present with sporulation covering up to 10% of the tested sample; 1 = Low (L) activity – mycelial growth present with sporulation covering up to 30% of the tested sample; and 0 = no activity (none) – severe fungal sporulation. All tests are carried out in triplicates and the antifungal efficacy of the tested films is reported as the median of the three replications.

Bread storage assay. Commercial sliced wheat bread (Davidovich bakery Ltd., Kiryat Ata, Israel) is purchased from a local supermarket. The bread contains the following antibacterial and antifungal preservatives: sorbic acid, calcium propionate and potassium sorbate. A preservative-free sliced wheat bread, purchased from a local bakery (French bread, Shemo Bakery, Haifa, Israel), is studied for comparison. Penicillium fungal conidial suspension is prepared as described earlier. A slice of bread is aseptically placed into a sterile 12 cm Petri dish and inoculated in one (for preservative-free bread) or two points (for commercial bread) with approximately 10³ spores per point. Films with an area of ∼25 cm² are attached to the center of the Petri dish lid, assuring no contact between the studied film and the bread. The plates are tightly sealed and incubated for 11 days at 25 °C. The test is carried out in triplicates and the fungal growth is monitored and recorded.

Results and discussion

Preparation and characterization of HNTs/carvacrol hybrids

Fig. 1 depicts the morphology of the neat HNTs, showing that the particles have a typical cylindrical shape. Some of the particles are observed to be hollow and open-ended. The length of the HNTs is non-uniform and varies significantly from 0.2 to 2 μm. In order to load carvacrol within the HNTs, the HNTs are dispersed in carvacrol at a weight ratio of 1 : 2 (respectively) using shear mixing and ultrasonication. The resulting HNTs/carvacrol hybrids are characterized by thermal gravimetric analysis (TGA) and compared to neat carvacrol (Fig. 2). The thermogram for the neat carvacrol shows one clear weight loss process, ascribed for carvacrol evaporation, which is completed at ∼165 °C. For the HNTs/carvacrol hybrid, carvacrol loss occurs at significantly higher temperature of ∼220 °C. Using the thermograms, we can determine the inorganic content within the hybrid to be 33 wt%, matching to the original HNTs content in the dispersion, which indicate that the resulting hybrids are uniform in their composition. Importantly, the TGA results prove that the HNTs drastically improve the thermal stability of the volatile carvacrol molecules, ascribed to the successful carvacrol loading mainly into the lumen and partially onto the external surface of the nanotubes.^34,58,59 It should be noted that additional hybrid compositions were studied in an attempt to further enhance carvacrol's stability. Yet, we found that the composition of 1 : 2 (HNTs : carvacrol) is optimal, in terms of both the processability of the hybrids as well as the achieved thermal stability. Increasing the HNTs concentration, beyond this composition, has a negligible effect on the carvacrol's thermal stability, while considerably impeding with the hybrid preparation process due to the high viscosity of the HNTs–carvacrol dispersion.

Fig. 1 HRSEM images (a and b) of neat HNTs (prior to hybrid formation) at two different magnitude scales.

Fig. 2 TGA curves of neat HNTs, carvacrol and the HNTs/carvacrol hybrid, demonstrating the superior thermal stability of carvacrol in the hybrid.

These TGA studies suggest that the loaded HNTs may function as carriers for carvacrol to allow its incorporation into polymers via high-temperature melt compounding processes. Our previous studies^29,30 with organoclays as carriers for carvacrol have also shown enhanced thermal stability of the volatile molecules due to carvacrol intercalation; nevertheless, their performance was inferior compared to the HNTs. These results suggest that in the case of HNTs/carvacrol hybrids, the cylindrical nanostructure allows the carvacrol molecules to enter the lumens of the HNTs (as schematically demonstrated in Scheme 1), delaying mass transport^58,60 and significantly improving their thermal stability.⁵⁹

Characterization of LDPE-based films

Following melt compounding of the HNTs/carvacrol hybrid with LDPE using a twin-screw extruder, films are produced by compression molding at 140 °C. In order to assess the role of HNTs as carriers for the volatile carvacrol, two reference systems are studied. In the first, neat carvacrol is melt-compounded with LDPE. In the second system, HNTs and neat carvacrol, at a weight ratio of 1 : 2 (respectively), are directly melt-compounded with LDPE. Note that in the later, no pre-compounding for HNTs/carvacrol hybrid preparation is performed. The composition of the studied films is presented in Table 1.

The nanostructure of the films is studied by HRSEM. Fig. 3 displays micrographs of cryogenic cross-sectioned LDPE/(HNTs/carvacrol hybrid) films. The HNTs appear to be individually dispersed within the LDPE matrix (Fig. 3a). Some of the tubes are observed to protrude from the surface (marked with white arrows), while some were pulled out during fracturing, leaving behind empty circular holes (marked with black arrows). Higher-magnification image (Fig. 3b) allows observing the interface region between the polymer and HNTs. The adhesion between the nanotubes and the polymer is rather poor, shown by the gaps between the individual nanotubes and the matrix.

Fig. 3 HRSEM images of freeze-fractured LDPE/(HNTs/carvacrol hybrid) film at (a) low and (b) high magnification, showing that the majority of the HNTs are individually dispersed within the LDPE matrix. Some of the HNTs are observed to protrude from the surface (marked with white arrows), while some were pulled out during fracturing, leaving behind empty circular holes (marked with black arrows).

Carvacrol content and distribution within the polymer matrix are highly important parameters, crucial in determining the resulting antimicrobial performance of the nanocomposite systems. These parameters are studied using TGA, Table 2 summarizes the carvacrol and HNTs content in different films (following melt compounding and film production). The inorganic residues, following heating to 600 °C, are attributed to the HNTs content within the LDPE-based films. The neat HNTs contain ∼83 wt% inorganic content, as determined by TGA (see Fig. 2). Accordingly, for all relevant films, HNTs contents of 1.7 wt% are obtained (Table 2). These results are consistent with the inorganic content, determined for neat HNTs, suggesting that the nanotubes are uniformly distributed within the LDPE matrix. The carvacrol content in the LDPE/carvacrol films is 1.8 wt%, implying that more than half of initial carvacrol concentration is lost during the high-temperature processes. The LDPE/HNTs/carvacrol films contain a slightly higher carvacrol content of 2.1 wt%, demonstrating that addition of HNTs (as a conventional filler) is not sufficient in order to preserve the volatile carvacrol molecules during the high-temperature processing. In contrast, the LDPE/(HNTs/carvacrol hybrid) films contain a greater carvacrol content of 3.1 wt%, validating that 80 wt% of the volatile carvacrol molecules are maintained within the film. Furthermore, FTIR measurements (data not shown) performed to verify and quantify the carvacrol content within the films (as described in our previous work³⁰) show similar results.

Table 2 Carvacrol and HNTs content in different LDPE-based films, determined by TGA. Original carvacrol content, pre-processing, is 4 wt% and original HNTs content is 2 wt%

Film sample	Content (wt%)
	Carvacrol	HNTs
LDPE/HNTs	0 ± 0.1	1.7 ± 0.1
LDPE/carvacrol	1.8 ± 0.1	0 ± 0.1
LDPE/HNTs/carvacrol	2.1 ± 0.1	1.7 ± 0.1
LDPE/(HNTs/carvacrol hybrid)	3.1 ± 0.1	1.7 ± 0.1

---

Accordingly, these results confirm that the HNTs/carvacrol hybrid has a crucial role in holding the highly-volatile molecules within the polymer during processing at high temperatures, as observed for the HNTs/carvacrol hybrids (see Fig. 2). Therefore, supporting our hypothesis that the HNTs particles in the hybrid serve as encapsulating carriers, protecting the carvacrol and allowing its preservation during high-temperature compounding processes. Importantly, FTIR studies (see Fig. S1, ESI†) show that the chemical structure of carvacrol is unaffected by the high-temperature compounding, suggesting retention of its antimicrobial properties. Thus, to the best of our knowledge, this is the first study that demonstrates enhanced thermal stability of an encapsulated EO i.e., carvacrol, while retaining its biological function after high-temperature processing, as will be discussed in the next sections.

Many studies have demonstrated the potential of HNTs as nano-carriers for sustained release of different active ingredients, such as drugs,^33,35,46,58 anticorrosion agents^46,61 and dyes.⁶² Hence, we investigate the effect of HNTs on the release of antimicrobial agent (i.e. carvacrol) from the films. Fig. 4a shows isothermal thermograms (at 60 °C to accelerate release) of the three different carvacrol-containing films: LDPE/carvacrol, LDPE/HNTs/carvacrol and LDPE/(HNTs/carvacrol hybrid). The observed weight loss as function of time is attributed to the release of volatile carvacrol moiety (also confirmed by gas chromatography, data not shown). The data of Fig. 4a is used to calculate the diffusion coefficient by plotting the fractional mass loss ratio vs. t^1/2, according to eqn (1).^52,56 The diffusion coefficient is determined from the initial linear slope of Fig. 4b. For the LDPE/carvacrol film, carvacrol content is observed to diminish i.e., the film attains a constant mass value, more rapidly in comparison to the other systems. LDPE/HNTs/carvacrol films were observed to retain a slightly higher carvacrol content than the LDPE/carvacrol film (Table 2); however, a similar profile of carvacrol release is observed, see Fig. 4b. Indeed, both systems have similar calculated diffusion coefficient values of 6.83 × 10⁻¹¹ and 6.65 × 10⁻¹¹ m² s⁻¹, respectively. This result indicates that addition of HNTs as conventional filler has a negligible effect on the carvacrol release rate from the films. On the contrary, for the LDPE/(HNTs/carvacrol hybrid) films a diffusion coefficient of 4.22 × 10⁻¹¹ m² s⁻¹ is calculated. Thus, the slower out-diffusion kinetics of carvacrol from the LDPE/(HNTs/carvacrol hybrid) nanocomposite is ascribed to the effective role of HNTs as nano-carriers, hindering the release of confined carvacrol molecules.

Fig. 4 Accelerated release experiments of carvacrol from different films. (a) Isothermal thermograms at 60 °C, depicting carvacrol release vs. time from the three different carvacrol-containing films: LDPE/carvacrol, LDPE/HNTs/carvacrol and LDPE/(HNTs/carvacrol hybrid). (b) Plot of m_t/m_∞ (fractional mass loss ratio) vs. t^1/2 for the three different carvacrol-containing films.

Antibacterial activity of the films

In order to assess the biofunctionality of the carvacrol post processing, the antibacterial activity of the different films is characterized by incubating the films with E. coli suspensions (10⁸ per mL, for 24 h, at 37 °C), after which cell viability is determined and log reductions are calculated in comparison to growth of E. coli exposed to the control film. These experiments are carried out periodically to evaluate the effect of storage time at room temperature on the antimicrobial efficacy of the films (Fig. 5); thus, replicating industrial systems where bulk materials are kept stored in the factory or in an external storage facility until used. Both reference films, without carvacrol, exhibit no antibacterial activity; while films containing carvacrol reduced E. coli counts to undetectable levels, indicating a durable bactericidal efficacy of carvacrol within the films. Nevertheless, storage time has a significant influence on the antibacterial property of the films. LDPE/carvacrol films lose their bactericidal property within three weeks of production, while LDPE/HNTs/carvacrol films preserve their bactericidal efficacy for an additional week. In contrast, the longest bactericidal efficacy is recorded for LDPE/(HNTs/carvacrol hybrid) films, exhibiting activity for up to six weeks, after which their performance decays. These results are consistent with both the effective carvacrol content (Table 2) and its out-diffusion kinetics (Fig. 4).

Fig. 5 Antibacterial activity against E. coli of neat LDPE, LDPE/HNTs, LDPE/carvacrol, LDPE/HNTs/carvacrol and LDPE/(HNTs/carvacrol hybrid) films vs. storage time at room temperature.

Next, we determined the ability of the various films to compromise the occurrence of L. innocua biofilm and its viability. L. innocua is a Gram-positive bacterium and an appropriate indicator for the pathogenic Listeria monocytogenes.^63,64 A biofilm is a multicellular layer of adherent bacteria surrounded by a matrix of extracellular polysaccharides, which has the potential to act as a chronic source of microbial contamination, leading to significant health hazards.⁶⁵ Biofilm formation represents serious challenges specifically to the food industry, since these may lead to cross-contamination of the products, resulting in lowered-shelf life and transmission of diseases.⁶⁶ Thus, L. innocua was selected owing to its ability form true, three-dimensional biofilms under static conditions.^65,67 Table 3 summarizes the L. innocua biofilm viability on the different films, as determined by viable count measurements. Both control films, neat LDPE and LDPE/HNTs, show insignificant effect on the biofilm viability. In contrast, the LDPE/carvacrol film managed to reduce the viability by 1 log, and the LDPE/HNTs/carvacrol and LDPE/(HNTs/carvacrol hybrid) films exhibit enhanced antibiofilm efficacy, with 2.2 and 3.2 log reductions, respectively. These results confirm that the LDPE/(HNTs/carvacrol hybrid) film has the best antibiofilm activity against L. innocua, and that it is superior to all films tested in this regards. Previous studies^27,68 have also shown that EOs-containing polymeric films can inhibit biofilm formation. Nevertheless, in these studies, antibiofilm efficacy was measured by an optical density technique, which provides only biomass information and not viability. Thus, it is inappropriate to compare the antibiofilm performance of our films to previous studies due to the differences in the protocols.

Table 3 Log reduction of biofilm viability of Listeria innocua when exposed to the studied films

Film sample	Log reduction by viable cell count
Neat LDPE	None
LDPE/HNTs	None
LDPE/carvacrol	1.0 ± 0.3
LDPE/HNTs/carvacrol	2.2 ± 0.3
LDPE/(HNTs/carvacrol hybrid)	3.2 ± 0.3

---

It is a fact that there is a difference between antimicrobial activity in vitro and in real food systems. Interactions of antimicrobial agents with different food components (such as fats, proteins and sugars) often result in inferior antimicrobial activity.⁶⁹ Thus, to evaluate the antibacterial characteristics of the carvacrol-containing polymer films in a relevant food system, we used soft cheese as a food model.⁷⁰ We adapted the FDA's BAM for detection and enumeration of L. monocytogenes⁵⁷ and inoculated the cheese with L. innocua to contain 4 log CFU per mL bacteria. The cheese is then incubated (at 26 °C for 22 h) with the different films (in direct contact and indirect-headspace contact), followed by Listerial count. The results (shown in Fig. S2, ESI†) reveal that LDPE/(HNTs/carvacrol hybrid) films retard the growth of L. innocua; generating a profound and reproducible decrease of 1.5 log CFU per mL and 1 log CFU per mL in bacteria population for indirect and direct contact assays, respectively.

These two test methods simulate packaged food scenarios during storage or freight, in which the food is in both direct and indirect contact with the packaging material. Thus, representing the high efficacy of the LDPE/(HNTs/carvacrol hybrid) films to manage bacteria development on packaged food.

Antifungal activity of the films

The antifungal efficacy of the films is examined utilizing the cosmopolitan fungus Alternaria alternata as model fungus. A. alternata is used due to its wide range of associations with food and human health. It is a phytopathogen, causing diseases on agronomic and horticultural crops such as, potato, oil seed, olive and tomato. It can develop and sporulate at low temperatures (<10 °C), making it a postharvest decay causing organism of fresh produce, spoiling commodities during refrigerated transport and storage.^9,71,72 Moreover, A. alternata is an important allergen,⁷³ producing mycotoxins such as alternariol, tenuazonic acid, altertoxin, alternuene and AAL-toxins,⁷² making it a clinical fungus that may cause disease in immune-compromised patients.

Table 4 summarizes the results of the direct in vitro analyses conducted with A. alternata, showing the capability of the studied films to maintain antifungal activity following the high-temperature processes. Additionally, Fig. 6 depicts images of the studied LDPE-based films after 7 days exposure to A. alternata. For the neat LDPE film, a substantial and homogenous fungal growth is observed to occur all over the Petri dish (Fig. 6a). In contrast, incubation of the fungus with LDPE/(HNTs/carvacrol hybrid) films resulted in a complete eradication of the fungus (Fig. 6d). In the case of LDPE/carvacrol system, no fungal growth is observed above the film disk and less dense growth can be seen throughout the dish (Fig. 6b). The LDPE/HNTs/carvacrol films exhibit a similar growth pattern; however, a pronounced pigment change of the fungal conidia from the common brown-black to light brown-yellow is observed (Fig. 6c). The dark pigment in A. alternata conidia is an outcome of melanin accumulation, which shields the conidia from the harmful effect of UV irradiation. In his report, Durrell⁷⁴ indicated that conidia lacking melanin were eradicated by UV light exposure of four minutes or less. Thus, exposure of the fungi to this film affected the durability and survivability of A. alternata.

Table 4 Antifungal activity of studied films in a direct contact tests against A. alternata

Film sample	Antifungal activity
Neat LDPE	0 = none
LDPE/carvacrol	1 = low
LDPE/HNTs/carvacrol	2 = moderate
LDPE/(HNTs/carvacrol hybrid)	3 = very high

---

Fig. 6 Effect of direct contact assay on the development of A. alternata following 7 days of incubation at 25 °C in the dark with the following films: (a) neat LDPE (b) LDPE/carvacrol (c) LDPE/HNTs/carvacrol and (d) LDPE/(HNTs/carvacrol hybrid). Conidial suspensions are mixed in pre-solidified agar and poured over the tested film. The film margins are marked for clarity.

As spoilage and poisoning of foods by fungi is a major problem,⁷⁵ we explore the effect of the studied films on fungal growth in bread models. Bakery products (such as bread) are susceptible to contamination by a variety of molds (mainly Penicillium and Aspergillus species) and thus provide highly relevant food model systems.⁷⁶ In order to mimic a realistic bread storage scenario, an indirect-headspace assay is employed to evaluate the effect of the different films on the development and fungi growth. We used several types of bread: fungi-inoculated commercial sliced bread (with preservatives), fungi-inoculated sliced bread (preservative-free) and natural sliced bread (preservative-free). The LDPE/(HNTs/carvacrol hybrid) films show an outstanding performance of complete inhibition of fungal growth for all bread types.

For the Penicillium-inoculated preservative-free bread, a significant fungal growth is observed for the reference slices (Fig. 7a), while in the presence of the LDPE/carvacrol film, a sparse and weak fungal growth is observed (Fig. 7b). Storage of the bread with the LDPE/(HNTs/carvacrol hybrid) films (Fig. 7c) results in complete eradication of the fungus. Without inoculation, different fungi populations are observed to develop onto the bread slices during storage (Fig. S3, ESI†). However, no fungus is detected when the bread is stored with the LDPE/(HNTs/carvacrol hybrid) film. Similar behavior is observed for the fungi-inoculated commercial bread, which contains fungal inhibitors (sorbic acid, calcium propionate and potassium sorbate) to extend its shelf life, see Fig. S4 (ESI†). Thus, bread storage experiments demonstrate the ability of LDPE/(HNTs/carvacrol hybrid) films to effectively prevent the growth of a variety of fungal contaminants and extend the shelf lives of foods. These results are consistent with both antibacterial and antibiofilm properties of the studied films, emphasizing the critical role of carvacrol encapsulation in the HNTs carriers, which in turn hinder the substantial carvacrol loss during processing and delay its out-diffusion from the nanocomposite films.

Fig. 7 Storage experiment of fungi-inoculated sliced bread (preservative-free). Indirect headspace assay examining the effect of different films on the development of fungi following 11 days of incubation at 25 °C in the dark: (a) none (b) LDPE/carvacrol film (c) LDPE/(HNTs/carvacrol hybrid) film. Penicillium growth regions are circled in red for clarity.

Conclusions

The present study demonstrates LDPE-containing carvacrol films with sustained and efficient bacteriocidal, as well as fungicidal activity. This is attained by encapsulation of the highly-volatile carvacrol into the HNTs, hindering the carvacrol loss during the polymer processing. The LDPE/(HNTs/carvacrol hybrid) system exhibits significantly higher carvacrol content in the film, in addition to reduced carvacrol diffusion compared to LDPE/carvacrol or LDPE/HNTs/carvacrol films. This is additionally confirmed by excellent and extended antibacterial and antibiofilm properties against E. coli and L. innocua, respectively. The LDPE/(HNTs/carvacrol hybrid) films also show superb antifungal activity against A. alternata in vitro and against a variety of other fungi, involved in food spoilage. We successfully demonstrate the significant effect of these films on extending the shelf life of bread and soft cheese, as model food systems. Thus, revealing their immense potential as active food packaging systems, minimizing the addition of preservatives into food formulations and extending food shelf life and safety. This newly developed technology, in which EOs can be effectively incorporated into plastic films, presents a significant step towards achieving antimicrobial polymeric systems with long lasting and wide range of antimicrobial ability. Such systems are of great importance for various applications: food, medical, personal care and hygiene, where tailor-made materials with tunable antimicrobial properties can be designed.

Acknowledgements

This study was supported by Magnet Program of the Israeli Ministry of Economy and the Israeli P^3 Consortium.

Notes and references

1. J. W. Costerton, P. S. Stewart and E. P. Greenberg, Science, 1999, 284, 1318–1322.
2. P. Appendini and J. H. Hotchkiss, Innovative Food Sci. Emerging Technol., 2002, 3, 113–126.
3. J. H. Han, Food Technol., 2000, 54, 56–65.
4. A. Sorrentino, G. Gorrasi and V. Vittoria, Trends Food Sci. Technol., 2007, 18, 84–95.
5. Z. Liang, M. Zhu, Y.-W. Yang and H. Gao, Polym. Adv. Technol., 2014, 25, 117–122.
6. P. Suppakul, J. Miltz, K. Sonneveld and S. W. Bigger, J. Food Sci., 2003, 68, 408–420.
7. A. Upadhyay, I. Upadhyaya, A. Kollanoor-Johny and K. Venkitanarayanan, BioMed Res. Int., 2014, 2014, 1–18.
8. S. Burt, Int. J. Food Microbiol., 2004, 94, 223–253.
9. Q. Chen, S. Xu, T. Wu, J. Guo, S. Sha, X. Zheng and T. Yu, J. Sci. Food Agric., 2014, 94, 2441–2447.
10. H. J. D. Dorman and S. G. Deans, J. Appl. Microbiol., 2000, 88, 308–316.
11. M. Elgayyar, F. A. Draughon, D. A. Golden and J. R. Mount, J. Food Prot., 2001, 64, 1019–1024.
12. K. A. Hammer, C. F. Carson and T. V. Riley, J. Appl. Microbiol., 1999, 86, 985–990.
13. M. Ramos, B. A. Valdes, M. Peltzer and A. Jimenez, J. Bioequivalence Bioavailability, 2013, 5, 154–160.
14. A. Valdes, A. C. Mellinas, M. Ramos, N. Burgos, A. Jimenez and M. C. Garrigos, RSC Adv., 2015, 5, 40324–40335.
15. S. Pacor, A. Giangaspero, M. Bacac, G. Sava and A. Tossi, J. Antimicrob. Chemother., 2002, 50, 339–348.
16. V. Muriel-Galet, J. P. Cerisuelo, G. López-Carballo, S. Aucejo, R. Gavara and P. Hernández-Muñoz, Food Control, 2013, 30, 137–143.
17. V. Otero, R. Becerril, J. A. Santos, J. M. Rodriguez-Calleja, C. Nerin and M.-L. Garcia-Lopez, Food Control, 2014, 42, 296–302.
18. K. K. Kuorwel, M. J. Cran, K. Sonneveld, J. Miltz and S. W. Bigger, J. Food Sci., 2011, 76, R164–R177.
19. G.-O. Lim, S.-A. Jang and K. B. Song, J. Food Eng., 2010, 98, 415–420.
20. M. D. Sanchez-Garcia, M. J. Ocio, E. Gimenez and J. M. Lagaron, J. Plast. Film Sheeting, 2008, 24, 239–251.
21. J. P. Cerisuelo, J. Alonso, S. Aucejo, R. Gavara and P. Hernández-Muñoz, J. Membr. Sci., 2012, 423–424, 247–256.
22. Y. Peng and Y. Li, Food Hydrocolloids, 2014, 36, 287–293.
23. N. Sanla-Ead, A. Jangchud, V. Chonhenchob and P. Suppakul, Packag. Technol. Sci., 2012, 25, 7–17.
24. J. P. Cerisuelo, V. Muriel-Galet, J. M. Bermúdez, S. Aucejo, R. Catalá, R. Gavara and P. Hernández-Muñoz, J. Food Eng., 2012, 110, 26–37.
25. M. Ramos, A. Jiménez, M. Peltzer and M. C. Garrigós, J. Food Eng., 2012, 109, 513–519.
26. M. V. Dias, H. S. de Medeiros, N. D. F. F. Soares, N. R. D. Melo, S. V. Borges, J. D. D. S. Carneiro and J. M. T. D. A. K. Pereira, LWT--Food Sci. Technol., 2013, 50, 167–171.
27. A. Nostro, R. Scaffaro, M. D'Arrigo, L. Botta, A. Filocamo, A. Marino and G. Bisignano, Appl. Microbiol. Biotechnol., 2012, 96, 1029–1038.
28. A. Valderrama Solano and C. Rojas Gante, Food Bioprocess Technol., 2012, 5, 2522–2528.
29. R. Shemesh, D. Goldman, M. Krepker, Y. Danin-Poleg, Y. Kashi, A. Vaxman and E. Segal, J. Appl. Polym. Sci., 2015, 132, 41261.
30. R. Shemesh, M. Krepker, D. Goldman, Y. Danin-Poleg, Y. Kashi, N. Nitzan, A. Vaxman and E. Segal, Polym. Adv. Technol., 2015, 26, 110–116.
31. M. Liu, Z. Jia, D. Jia and C. Zhou, Prog. Polym. Sci., 2014, 39, 1498–1525.
32. Y. M. Lvov and R. R. Price, in Bio-inorganic Hybrid Nanomaterials, Wiley-VCH Verlag GmbH & Co. KGaA, 2008, ch. 14, pp. 419–441,  DOI:10.1002/9783527621446.
33. N. G. Veerabadran, R. R. Price and Y. M. Lvov, Nano, 2007, 2, 115–120.
34. Y. M. Lvov, D. G. Shchukin, H. Möhwald and R. R. Price, ACS Nano, 2008, 2, 814–820.
35. Y. Lvov and E. Abdullayev, Prog. Polym. Sci., 2013, 38, 1690–1719.
36. E. Abdullayev and Y. Lvov, J. Nanosci. Nanotechnol., 2011, 11, 10007–10026.
37. G. Cavallaro, G. Lazzara, S. Milioto, F. Parisi and V. Sanzillo, ACS Appl. Mater. Interfaces, 2014, 6, 606–612.
38. Y. Zhang, Y. Chen, H. Zhang, B. Zhang and J. Liu, J. Inorg. Biochem., 2013, 118, 59–64.
39. L. Yu, Y. Zhang, B. Zhang and J. Liu, Sci. Rep., 2014, 4, 4551.
40. Y. Chen, Y. Zhang, J. Liu, H. Zhang and K. Wang, Chem. Eng. J., 2012, 210, 298–308.
41. Y. Lvov, A. Aerov and R. Fakhrullin, Adv. Colloid Interface Sci., 2014, 207, 189–198.
42. J. Zhang, Y. Zhang, L. D. Y. Chen, B. Zhang, H. Zhang, J. Liu and K. Wang, Ind. Eng. Chem. Res., 2012, 51, 3081–3090.
43. V. Vergaro, E. Abdullayev, Y. M. Lvov, A. Zeitoun, R. Cingolani, R. Rinaldi and S. Leporatti, Biomacromolecules, 2010, 11, 820–826.
44. E. Abdullayev, A. Joshi, W. Wei, Y. Zhao and Y. Lvov, ACS Nano, 2012, 6, 7216–7226.
45. W. O. Yah, H. Xu, H. Soejima, W. Ma, Y. Lvov and A. Takahara, J. Am. Chem. Soc., 2012, 134, 12134–12137.
46. Y. M. Lvov, D. G. Shchukin, H. Mohwald and R. R. Price, ACS Nano, 2008, 2, 814–820.
47. A. Sánchez-Fernández, L. Peña-Parás, R. Vidaltamayo, R. Cué-Sampedro, A. Mendoza-Martínez, V. Zomosa-Signoret, A. Rivas-Estilla and P. Riojas, Materials, 2014, 7, 7770–7780.
48. M. Du, B. Guo and D. Jia, Polym. Int., 2010, 59, 574–582.
49. Z. Jia, Y. Luo, B. Guo, B. Yang, M. Du and D. Jia, Polym.-Plast. Technol. Eng., 2009, 48, 607–613.
50. B. Lecouvet, J. G. Gutierrez, M. Sclavons and C. Bailly, Polym. Degrad. Stab., 2011, 96, 226–235.
51. G. Cavallaro, D. I. Donato, G. Lazzara and S. Milioto, J. Phys. Chem. C, 2011, 115, 20491–20498.
52. J. Crank, The Mathematics of Diffusion, Oxford Univ., 1975, p. 414.
53. L.-T. Lim and M. A. Tung, J. Food Sci., 1997, 62, 1061–1062.
54. M. J. Cran, L. A. S. Rupika, K. Sonneveld, J. Miltz and S. W. Bigger, J. Food Sci., 2010, 75, E126–E133.
55. P. Suppakul, K. Sonneveld, S. W. Bigger and J. Miltz, LWT--Food Sci. Technol., 2011, 44, 1888–1893.
56. J. Miltz, Migration of low molecular weight species from packaging materials: theoretical and practical considerations, Technomic Publishing Co, Lancaster, 1987.
57. A. D. Hitchins and K. Jinneman, Bacteriological analytical manual online, US Food and Drug Administration, 2003.
58. N. G. Veerabadran, D. Mongayt, V. Torchilin, R. R. Price and Y. M. Lvov, Macromol. Rapid Commun., 2009, 30, 99–103.
59. G. Gorrasi, Carbohydr. Polym., 2015, 127, 47–53.
60. D. G. Shchukin, S. V. Lamaka, K. A. Yasakau, M. L. Zheludkevich, M. G. S. Ferreira and H. Mohwald, J. Phys. Chem. C, 2008, 112, 958–964.
61. E. Abdullayev, R. Price, D. Shchukin and Y. Lvov, ACS Appl. Mater. Interfaces, 2009, 1, 1437–1443.
62. P. Yuan, P. D. Southon, Z. Liu and C. J. Kepert, Nanotechnology, 2012, 23, 375705.
63. A. Carlez, J.-P. Rosec, N. Richard and J.-C. Cheftel, LWT--Food Sci. Technol., 1993, 26, 357–363.
64. S. Liu, V. M. Puri and A. Demirci, Int. J. Food Sci. Technol., 2009, 44, 29–35.
65. N. Oulahal, W. Brice, A. Martial and P. Degraeve, Food Control, 2008, 19, 178–185.
66. E. Giaouris, E. Heir, M. Hébraud, N. Chorianopoulos, S. Langsrud, T. Møretrø, O. Habimana, M. Desvaux, S. Renier and G.-J. Nychas, Meat Sci., 2014, 97, 298–309.
67. E. J. Marsh, H. Luo and H. Wang, FEMS Microbiol. Lett., 2003, 228, 203–210.
68. A. Nostro, R. Scaffaro, M. D'Arrigo, L. Botta, A. Filocamo, A. Marino and G. Bisignano, Appl. Microbiol. Biotechnol., 2013, 97, 9515–9523.
69. J. Gutierrez, C. Barry-Ryan and P. Bourke, Food Microbiol., 2009, 26, 142–150.
70. J. M. Farber and P. I. Peterkin, Microbiol. Rev., 1991, 55, 476–511.
71. D. Prusky, Y. Fuchs, I. Kobiler, I. Roth, A. Weksler, Y. Shalom, E. Fallik, G. Zauberman, E. Pesis, M. Akerman, O. Ykutiely, A. Weisblum, R. Regev and L. Artes, Postharvest Biol. Technol., 1999, 15, 165–174.
72. J. Alexander, D. Benford, A. Boobis, S. Ceccatelli, B. Cottrill, J. Cravedi, A. Di Domenico, D. Doerge, E. Dogliotti and L. Edler, EFSA J., 2011, 9, 2407–2504.
73. S. H. Downs, T. Z. Mitakakis, G. B. Marks, N. G. Car, E. G. Belousova, J. D. Leuppi, W. E. I. Xuan, S. R. Downie, A. Tobias and J. K. Peat, Am. J. Respir. Crit. Care Med., 2001, 164, 455–459.
74. L. W. Durrell, Mycopathol. Mycol. Appl., Suppl., 1964, 23, 339–345.
75. J. I. Pitt, Br. Med. Bull., 2000, 56, 184–192.
76. P. Lavermicocca, F. Valerio and A. Visconti, Appl. Environ. Microbiol., 2003, 69, 634–640.

---

Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra16583k

‡ Equal contribution.

---