Characterization and Antimicrobial Activity Studies of Agave Sheets with Thymol and Clove Oil for Active Packaging

ABSTRACT Antimicrobial active sheets based on agave ultimate fibers were prepared by incorporating essential oils (thymol and clove oil) at different concentrations. A complete structural, mechanical, and antimicrobial characterization of treated samples was carried out. SEM proved the presence and the deposit of additives on the surface of the sheet. FTIR spectra confirm the incorporation of essential oils into treated papers. Mechanical properties reveal a decrease in elastic modulus from 31.1 kN/m for raw sheet to 17.8 kN/m for thymol and to 26.7 kN/m for clove oil for treated sheets, and tensile strength from 28.2 N to 24.1 N for thymol and to 14.1 N for clove oil was obtained for the active sheets compared with raw sheets. While the presence of additives increased slightly the elongation at yield of treated samples from 2.3% to 2.6% for thymol and to 2.8% for clove oil. Regarding antibacterial activity, thymol and clove oil showed inhibition against strain present in food. Thymol had the higher inhibition against S. aureus (4.08 cm), and clove oil had the best inhibition against E. coli. (4.13 cm). The obtained results proved that active agave sheets are able to be used as antimicrobial packaging.


Introduction
Due to the increasing concern for environmental protection and food safety, emerging novel packaging materials based on edible biodegradable polymers have received considerable interests worldwide (Alessandroni et al. 2022;Kalkan, Remzi Otağ, and Soner Engin 2020;Mendes et al. 2020;Pellá et al. 2020) To replace the non-degradable counterparts (e.g., plastics) used in conventional packaging, several natural edible films as nontoxic and edible substances with unique physicochemical properties are currently used in the form of proteins (such as zein, gelatin, whey protein, and casein), polysaccharides (such as starch, chitosan, and fiber), and lipids (such as wax, fatty acid and acylglycerol) or their Clove oil Eugenia spp. (oil of cloves) was purchased from Sigma-Aldrich. It has the following characteristics: CAS number: 8000-34-8; chemical formula: C 7 H 12 ClN 3 O 2 ; molecular weight: 205.642; bp: 251°C (lit.); fp: 115°C closed up; density: 1.04 g/ml at 25°C (lit.); refractive index: n20/D = 1.532 (lit.); purity: 98%.

Ultimate agave fibers
To extract ultimate agave fibers, the extraction of technical ones was carried out first, which are shown in Figure 1(a) and were extracted from the leaves using a hydrolysis treatment. The high-temperature treatment (120°C) was carried out in a Mathis autoclave for 90 minutes. This method consists in cutting a part of agave leaf and immersing it in distilled water. At the end of the treatment, the fibers were separated from the matrix by calendaring the leaf, and the individual fibers were rinsed with water.
After that, to obtain individual fibers, shown in Figure 1(b), a mixed treatment involving NaOH (sodium hydroxide) and H 2 O 2 (hydrogen peroxide) was carried out since it gives the best separation results as demonstrated by Jaouadi et al. The Agave ultimate cellulose fibers obtained had a crystalline index of 0.84, the capacity absorption was about 10.5 g/g, and the average diameter was 32.4 μm with an arithmetic length about 941 μm (Jaouadi, Msahli, and Sakli 2011). All chemical products used for extraction were purchased from Chimitex, Tunisia. The treatments were made with a dyeing machine (Mathis) under pressure and during continuous mixing. After each treatment, individual fibers were rinsed with tap water and dried at room temperature.

Sample preparation
These Ultimate agave fibers, in the form of pulp, were used to form hand sheets (our samples). The "sheet Maker" is an automatic machine (Regmed) composed by a mixing cylinder, a doser and a column where a sheet will be formed. The ultimate agave fibers suspension were prepared in the mixing cylinder. Then the doser allows to extract the desired quantity of susupension from mixing cylinder and to transfer it to the colum where a rectangular sheet were formed from a pulp suspension on a wire screen under suction. This machine is well described in the works of the operation of this machine is well described in the works of Zannen S. et al. who used the salme machine (Zannen et al. 2022). Then, the sheet is subjected to a manual pressure before being dried in ambient air. Many formulations were obtained: sheets containing 2, 6, and 10 wt % of thymol (T2, T6, and T10) and 2.5, 5, 7.5, and 10 wt% clove oil (CO2.5, CO5, CO7.5, and CO10). An additional sample without any active compound was also prepared and used as control (Ag0). The active antimicrobial sheets were obtained by impregnation of the sheets in thymol or clove oil solutions containing ethanol (emulsifying agent 50 mg/ml) under continuous stirring with different concentrations of thymol and clove oil. Impregnation was carried out at 50°C for thymol and at ambient temperature for clove oil, for 5 min then samples were dried at ambient temperature. Clove oil (98%) and thymol (99.5%) were purchased from Sigma -Aldrich. Clove oil is a mixture of different compounds (eugenol, methyleugenol, and isoeugenol). The major constituent of clove oil is eugenol (about 70% of clove oil). The chemical structures of thymol and eugenol are shown in Figure 2.

Material characterization
The active sheets were characterized by using different techniques to study their physical and mechanical properties as well as their antimicrobial activity.

Scanning Electronic Microscopy (SEM)
The sheet surfaces were analyzed by using a JEOL model JSM-5400 (ETAP Inc., Tunisia) microscope operated at 15 kV. Samples were coated with a gold layer prior to analysis in order to increase their electrical conductivity. Images were registered at 2000× of magnification in order to study their surface.

Determination of mechanical properties
Ten rectangular specimens (dimensions: 15 × 100 mm) are stretched to rupture at a constant rate of loading (20 mm/min) using a tensile hydraulic apparatus Lloyd LRX 2.5 model (Packtec Inc, Tunisia) equipped with a 2.5 kg load cell that measures tensile force. Average tensile strength, elongation at yield and elastic modulus were calculated from the resulting stress-strain curves according to the ASTM D882-09 Standard procedure (ASTM D882-09. 2009). Results are the average of five measurements (±standard deviation).

Infrared analyses
Attenuated total reflection (ATR) is a technique used in conjunction with infrared spectroscopy that enables samples to be passively examined in the solid or liquid state without any preparation. ATR-FTIR is used in many fields since it is a nondestructive, direct, and rapid method. A spectrophotometer of type PerkinElmer UATR (Single Reflection Diamond) with an attenuated total reflectance (ATR) attachment was used to undertake infrared spectra analysis. One hundred scans were taken per sample with a resolution of 2 cm −1 . The infrared spectra were recorded in the range of 4000-500 cm −1 . Preprocessing of IR spectra Spectra normalization. Normalization helps give all samples an equal impact on the model. Without normalization, some samples may have such severe multiplicative scaling effects that will not be significant contributors to the variance and, as a result, will not be considered important by many multivariate techniques.
In this work, the multiplicative signal correction (MSC) method was carried out to normalize infrared spectra, the basic concept of which is to remove non-linearities in the data caused by scatter from particulates in the sample.
Noise removal and baseline correction. Noise represents random fluctuations around the signal that may originate from the instruments or environmental laboratory conditions. In this work, the Fast Fourier Transform (FFT) algorithm available in Origin 6.0 was used. Besides, spectra were baseline corrected with a segmented linear baseline linking the following frequencies: 380, 1185, 1515, 2790, 3660 cm −1 . Processing of spectra, baseline corrections, and derivative spectra were generated using the software resolution program (Origin 6.0) (Turki et al. 2018).

Determination of antimicrobial activity
The evaluation of the antimicrobial activity of the sheet containing thymol and clove oil was carried out by using two test microorganisms: E. coli (gram-negative, ATCC 25,922) and S. aureus (grampositive, ATCC 6538P). The Ag 0 sample was also tested as control. Overnight cultures of E. coli (Escherichia coli) and S. aureus (Staphylococcus aureus) were grown in Tryptic Soy Broth at 35°C for 24 h. The strain selection represented typical spoilage organism groups commonly occurring in various kinds of food products.
Antimicrobial activity tests were carried out by using the agar disk diffusion method. Tenmillimeter circular disks cut from sheets were placed on Petri dishes containing Mueller-Hinton agar (MHA) supplied by INSULAB S.L. (Valencia, Spain), previously spread with 0.1 mL of each inoculum. The bacterial culture concentration in the inoculums was 10 6 CFU mL −1 , corresponding to the concentration that could be found in contaminated food, and standardized in the McFarland scale 0.5, as it has been indicated by others (El-Naggar et al. 2020;Panuwat et al. 2011). The Petri dishes were then incubated at 37°C for 24 h. The antimicrobial activity of each material was evaluated by observing the growth inhibition zone and measuring the diameter (cm) by a ruler. The results were expressed in cm ± standard deviation.

Mechanical properties
Tensile tests were conducted to explore the impact of essential oils addition to sheets on the tensile properties compared with untreated ones. All the results are summarized in Table 1. The addition of clove oil and thymol to sheet resulted in a slight modification of tensile properties. The elastic modulus was remarkably decreased from 31.1KN/m for raw sheets to 17.8KN/m for sheets treated with thymol and to 26.7KN/m for sheets treated with clove oil. As found by Robledo et al., Nordin et al. and Othman et al., the addition of thymol and clove oil at a certain concentration formed a non-miscible phase in the starch matrix that caused segregation of the starch chains. Hence, the chain mobility reduces, and this causes a reduction in the elasticity of the films (Nordin et al. 2020;Othman et al. 2021;Robledo et al. 2018).
There are a slight decrease in tensile strength for sheets treated with thymol from 28.2 N to 24.1 N and an important reduction for sheets treated with clove oil from 28.2 N to 14.1 N. The interaction between the starch biopolymer matrix and phenolic compounds of essential oils resulted in the heterogeneity of the film structure, thus reducing the tensile strength of the films. A similar trend of findings was revealed by (Marina et al. 2012) where they found a slight modification of tensile strength when thymol was incorporated into the polypropylene films. The reduction in tensile strength was attributed to the complex structures formed between the lipids from the phenolic compounds and the starch polymers, which reduced the cohesion of the starch network forces, thus decreasing the films' resistance to breakage (Jiménez et al. 2013). The addition of essential oils resulted in lowered interaction between biopolymer and hindered polymer chain-to-chain interactions and reduced crosslinking. This finding is also consistent with the work of (Nordin et al. 2020) who incorporated thymol and glycerol in CS films and found a decrease in tensile strength by 96% compared to neat CS films. A similar trend was also observed by (Mehran et al. 2013) who investigated the tensile strength of starch films incorporated with ZEO or MEO. Tawakkal et al. (Tawakkal, Cran, and Bigger 2018) reported that polylactic acid (PLA) composites incorporated with 30% w/w untreated kenaf or treated kenaf fibers with 0, 5, and 10% w/w thymol loadings experienced a reduction in tensile strength values. They reported that a localized plasticizing effect between the PLA and the thymol occurred, which facilitated thymol molecule diffusion into the bulk of the matrix between the PLA chains. Othman et al. (Othman et al. 2021) reported that the addition of thymol interfered with the interaction between the polymer matrix and the CNP in the presence of the applied stress and subsequently reduced the tensile strength (Davoodi, Kavoosi, and Shakeri 2017).
The treatment enables improving slightly the elongation at yield for treated samples from 2.3% to 2.6% for thymol treatment and to 2.8% for clove oil treatment. This behavior could be explained by some plasticizing effect caused by the addition of both additives to the sheets resulting in the increase in ductile properties, which would also result in changes in the materials crystallinity. This behavior has also been reported for LDPE-based samples with carvacrol (Persico et al. 2009), antimicrobial active films based on polypropylene (PP) prepared by incorporating thymol and carvacrol at three different concentrations (Marina et al. 2012), and polyethylene (PE) loaded by clove essential oil (CEO), which showed better mechanical (Shen et al. 2021). Figure 3 presents the normalized FTIR spectra related to the different samples treated with clove oil and thymol, respectively. The variation in the intensity of several peaks present in the spectrum and the appearance of new peaks in the processed samples confirm the incorporation of clove oil and thymol in the sheet structure.

Fourier transform infrared spectroscopy (FTIR)
We clearly notice modifications of the band between 3000 and 3600 cm −1 , related to the vibration of the group O-H of the cellulose and of functionalized sheets (an increase in the intensity with clove oil treatment and a slight decrease with thymol treatment). We also observe the appearance of a band at approximately 2917 cm −1 attributed to the stretching of the symmetrical and asymmetric CH groups in the aromatic methoxyl groups for the treated samples. The band at 2850 cm −1 attributed to cellulose Iβ for the attraction of CH groups in the methyl and methylene groups of the side chains increases in intensity after treatment of the paper. The absorption peak at 1638 cm −1 from the C=C aromatic skeletal vibration of the benzene ring is not characteristic of cellulose. Its presence in the functionalized papers confirms the presence of the latter in the treated paper. New peaks existed at about 1512 cm −1 with the addition of clove oil and thymol. This could be due to the interaction between the functional groups of essential oils and sheet. An increase in the intensity of the band at 1413 cm −1 assigned to the stretching of the CH 2 groups was signaled. Incorporation of essential oils slightly altered the intensity of the bands at around 1370 cm −1 corresponding to the stretching of the alcohol groups of the cellulose and leads to the appearance of bands localized at about 680 cm −1 characteristics of the deformation vibration of the C-OH bond occurred after paper processing (Palai, Kumar Sarangi, and Shankar Mohapatra 2020;Turki et al. 2018). Some of these different variations of many bands were reported in the literature (Milanovic et al. 2020;Palai, Kumar Sarangi, and Shankar Mohapatra 2020;Shen et al. 2021).
All these variations can confirm the incorporation of clove oil into sheet. Table 2 and Figure 4 show the results of the antimicrobial tests performed against S. aureus and E. coli of active sheets with 10 wt% of essential oils by the agar disk diffusion method. This method is very simple, and it is based on the measurement of the clear zone caused by growth inhibition produced by a sheet disk containing the antimicrobial agent when putting in direct contact with a bacterial culture (Palai, Kumar Sarangi, and Shankar Mohapatra 2020;Weerakkody et al. 2010) The presence and size of the inhibition zone indicate the susceptibility of the bacteria to the essential oil. When inhibition zones are smaller than 0.7 cm, a sample is considered as a non-active against bacteria. Inhibition zone diameter greater than 1.2 cm indicates satisfactory inhibitory effect. As it can be seen in Table 2, the inhibition zones of treated sheets with clove oil were 1.53 cm, 1.87 cm, 2.48 cm, and 2.95 cm for CO2.5, CO5, CO7.5, and CO10, respectively, against S.aureus and were 2.77 cm, 3.05 cm, 3.46 cm, and 4.13 cm for CO2.5, CO5, CO7.5, and CO10, respectively, against E. coli. On the other hand, the inhibition zones of treated sheets with thymol were 2.71 cm, 3.35 cm, and 4.08 cm  for T2, T6, and T10, respectively, against S. aureus and were 1.74 cm, 2.65 cm, and 3.28 cm for T2, T6, and T10, respectively, against E. coli. It was clear that the antibacterial activity of treated sheets with clove oil and thymol increased significantly with the concentration of both additives. The same effect was observed in the work of Shen et al. (Shen et al. 2021). It was also clear that sheets containing 10 wt% of thymol were the most effective against S. aureus showing the largest inhibition zone. Otherwise, sheets containing 10 wt% of clove oil were the most effective against E. coli showing the largest inhibition zone.

Antimicrobial properties of sheets
Thus, the essential oils evaluated in this study displayed satisfactory inhibitory effect against all bacteria evaluated. However, wide variation of the inhibitory effect of clove essential oil was reported in the literature. In the present study, inhibition zones of T10 and CO10 treated sheets observed against S. aureus were higher than the inhibition zone of 3.2 cm reported by Doninelli et al. (2010) and 2.83 cm reported by Radünz et al. (2019). The inhibition zones for T10-and CO10-treated sheets against E. coli were larger than the inhibition zone of 1.97 cm reported in the literature (Doninelli et   2010) and 2.81 cm reported by Radünz et al. (2019). The presence of the inhibition zone formed by sheets treated with essential oil against studied bacteria is possibly due to lipophilic characteristic of oils, which allows an interaction between the oil and the lipids of the cell membrane of the bacterium, altering its permeability. As a general conclusion, the addition of clove oil and thymol to agave sheets demonstrated antimicrobial activity in bacterial strains potentially present in food (Wang et al. 2021). Figure 5a shows SEM images obtained for non-treated samples used as control (Ag0) to compare with images of treated sheets. The entanglement of agave ultimate fibers among themselves observed in this figure, which gives the sheet its good mechanical and physical properties, is non-uniform throughout the paper giving it a certain random porosity. Figure 5b shows SEM images obtained for samples with 10 wt % of thymol and clove oil additives. In comparison to Figure 5a, there is no modification in the state of the surface as well as the entanglement of the fibers of both treated sheets, but a deposit of additives was observed on their surfaces (surrounded in Figure 5b). This fact was further evaluated by using other techniques.

Conclusion
Clove oil and thymol have demonstrated their potential to be used as active additives in agave sheets for food packaging applications. Characterization of active sheets was carried out by using different analytical techniques to evaluate the effect of these additives in the sheet. The infrared spectra of treated sheets have proven the incorporation of clove oil and thymol into treated sheets. As a result, the mechanical properties were modified by a decrease in elastic modulus of about 43% for thymol treated samples and 15% for clove oil treated samples. The tensile strength also showed a decrease of approximately 15% for thymol and 50% for clove oil. An increase in elongation at yield about 13% for thymol and 21% for clove oil was obtained. A good antimicrobial activity against S. aureus and E. coli was proved. Therefore, it could be concluded that the addition of antimicrobial additives shows some potential to improve product quality and safety aspects in food packaging applications and these treated sheets could be used for sustainable food packaging owing to their biodegradability and antimicrobial properties.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Highlights
• Active films based on agave ultimate fibers were prepared by incorporating thymol and clove oil • Structural, mechanical, and antimicrobial characterization was carried out • SEM micrographs and FTIR spectra confirm the incorporation of essential oils into treated papers • A decrease in elastic modulus and tensile strength and a slight increase in the elongation at yield were obtained for the active formulations. • Thymol had the higher inhibition against S. aureus and clove oil had the best inhibition against E. coli.