Biodegradable carboxymethyl cellulose–polyvinyl alcohol composite incorporated with Glycyrrhiza Glabra L. essential oil: Physicochemical and antibacterial features

Abstract Glycyrrhiza glabra L. root essential oil (GGEO) has well‐known antimicrobial and therapeutic features. In this study, a new antimicrobial carboxymethyl cellulose–polyvinyl alcohol (CMC‐PVA) binary film was developed using GGEO as an active compound. The effects of various concentrations of GGEO (0.25%, 0.50%, and 0.75%) were scrutinized on the physicochemical and antibacterial properties of composites. It was discovered that GGEO significantly reduced the composite ultimate tensile strength from 17.01 to 3.86 MPa. Further, by increasing the concentration of GGEO to 0.75%, the water vapor permeability and moisture content increased to 13.61 × 10−9 g/m s−1 Pa−1 and 41.06%, respectively. The results indicated that the active films possessed good inhibitory effects against the gram‐positive bacteria (L. monocytogenes and Staphylococcus aureus) and were less powerful against gram‐negative bacteria (Escherichia coli and S. typhimurium). Finally, the results highlighted that GGEO can act as an excellent antimicrobial agent in combination with CMC‐PVA composite.

Nevertheless, the films formed by it have poor mechanical and barrier properties. The combination of decomposable polymers with different properties and structures seems to be an appropriate solution to overcoming these shortcomings (Cazón et al., 2018;Fattahi et al., 2020).
Polyvinyl alcohol (PVA) is one of the semi-crystalline synthetic polymers whose backbone is composed predominantly of carbon chains and is thus completely biodegradable. It is a nontoxic polymer with a zigzag structure. Various studies have shown its good chemical and mechanical stability, transparency, thermal stability in a wide range of temperatures, processability, and film-forming properties (Muppalla et al., 2014).
Our previous studies with 1.5% CMC showed that it had excellent film-forming properties; however, the formed films had weak mechanical properties (Fattahi & Seyedain-Ardabili, 2021;Fattahi et al., 2020). The mechanical properties of CMC films can be improved by blending with other polymers. PVA is a versatile polymer with many industrial applications, and it may be the only synthesized polymer whose backbone is mainly composed of C-C bonds that is absolutely biodegradable. Although being a synthetic polymer, PVA has aroused great interest due to its good mechanical properties.
So, in order to improve some of the CMC defects and achieve the desired properties, the mentioned polymers are combined to form composite films (Muppalla et al., 2014). However, unlike some other biopolymers such as chitosan, this composite does not have any antimicrobial activities (Raeisi et al., 2015). Yet, this type of packaging can be used as a carrier of active compounds to enhance the safety of foodstuff (Bahrami et al., 2019). Active packaging increases the shelf life of the food without affecting its freshness characteristics.
Over the past few years, the use of essential oils as a natural antimicrobial agent in active packaging has received much attention from scientists (Jahed et al., 2017;Raeisi et al., 2015).
Several studies have suggested that incorporation of essential oils into the composite structure may not only yield antimicrobial activity in the film, but they can also increase water vapor resistance (Jahed et al., 2017;. The first point in the selection of essential oils is their antimicrobial activities. Additionally, aromatic compounds in essential oils are another important point to be considered as these compounds may affect the organoleptic features (Konuk Takma & Korel, 2019).
Glycyrrhiza glabra L. originates from the Iranian plateau and other warm regions of the world. Concerning the constituent compounds, the roots of this plant possess a wide range of triterpenes, flavones, isoflavones, saponins, chalcones, glycoside compounds, etc (Siracusa et al., 2011). The unique sweet taste of Glycyrrhiza roots is due to glycyrrhizin, which is significantly sweeter than sugar. Nowadays, the roots of G. glabra are extensively applied in foods and pharmaceutical industry because of its sweetening, strong antimicrobial, antioxidant, anti-allergic effects, and other effects (Fatima Khattak & James Simpson, 2010).
However, to the best of our knowledge, the effect of G. glabra L. essential oil (GGEO) on the properties of such film-forming has not been investigated so far. Given the clear importance of the use of natural compounds as an essential oil with strong antimicrobial activity in food packaging, the first objective of this study is to produce a new active composite with the mentioned biodegradable polymer and GGEO. Thereafter, the second goal is to improve the rheological, mechanical, and antimicrobial characteristics of the novel composite by adding a different concentration of essential oils for their potential application as active food packaging.

| Materials and bacterial strains
Carboxymethyl cellulose (molecular weight of around 90,000) and PVA (molecular weight range of 89,000-98,000, and 99% hydrolyzed grade) were purchased from Sigma-Aldrich Company. Glycerol and the salts used to condition the specimens (calcium sulfate, sodium chloride, and magnesium nitrate) were purchased from the Merck.
Four pathogenic bacteria, two gram-positive, S. aureus PTCC 1,114 and Listeria monocytogenes PTCC 1,163, and two gramnegative, E. coli PTCC 1533 and Salmonella typhimurium ATCC 14,028, were selected for the present study. The pure cultures of these bacteria were obtained from the Persian-type culture collection, Iran. They were all subcultured on tryptic soy broth media (Sigma-Aldrich).

| Preparation of composite films
The solution-casting method was adopted to prepare binary films.
Emulsion mixtures contained 0.25, 0.50, or 0.75% GGEO (dry basis, db), and the ratios of biopolymers-glycerol (plasticizer) and  were kept constant at 0.75% w/v and 50% v/v respectively during the course of the study (Dashipour et al., 2015). The range of GGEO used was based on its effect on gram-positive and gram-negative bacteria in the previous study (Luís et al., 2019).
Each emulsion solution was prepared individually in deionized water. Accordingly, at first, CMC was diluted in distilled water at 1.5% (w/v) with continuous magnetic stirring for 50 min. Further, the PVA solution was prepared by dissolving 5% (w/v) polymer in distilled water under mechanical stirring at 90°C for 120 min. Once the PVA solution was cooled down to the room temperature of 25°C, aqueous solutions were mixed at 50:50 mass ratio under vigorous stirring for 30 min at room temperature of 25°C.
Then, Tween 80 (50% v/v) was added to the composite solutions and mixed at 20,000 rpm for 1 min using an ultra-homogenizer (Ultraturrax, Janke & Kunkel). Later on, the GGEO was added gradually into the solutions and sonicated through ultrasound probe for 5 min with a working power of 100 W and cycle of 70%. Duty cycle controls the length of each pulse when the sonicator is not in continuous mode. A typical setting is 70%, which means on for 0.7 s, then off for 0.7 s, repeated. Prior to film formation, in order to remove air bubbles from the emulsion solution, a vacuum was used for 10 min. After this step, 80 ml of the resulting emulsion solution was cast into Teflon plates of 10 cm diameter, which was allowed to dry using air convection oven at 40°C for 24 hr. Subsequently, the composites were slowly peeled off from the plates. Once the samples were fabricated, to achieve a relative humidity of 53%, they were conditioned by a saturated solution of magnesium nitrate in a desiccator.

| Morphological analysis
The morphology and microstructural analysis of the film samples was examined by scanning electron microscopy (SEM). Prior to examining the cross section, the filmstrips were cryofractured under liquid nitrogen. Thereafter, the samples were coated with a layer of gold and observed by a scanning electron microscope (MIRA3 -FEG Tescan) operating at an acceleration beam voltage of 100 kV (Bahrami et al., 2019). The cross-sectional viewpoints of images were captured at slope angles of 90° in relation to the electron beam. Digital micrographs of the surface of composites and cross section were taken at different magnifications whereby the best visualization was chosen (Nisar et al., 2018).
Atomic force microscopy (AFM) experiments were used for topography characterization of composite films previously conditioned by Mg (NO 3 ) 2 . In doing so, the Nanosurf, Switzerland atomic force microscope was used. The roughness was estimated using the data from the 3-D images by Nanosurf Mobile S software.

| Fourier transform infrared (FT-IR) spectroscopy
Fourier transform infrared spectra of all composite samples without and with the addition of GGEO were captured using FT-IR spectroscopy (TENSOR 27 spectrophotometer and OPUS data collection program, USA). For this purpose, the specimens were cut in 2 cm × 2 cm and then exposed to radiation. The corresponding spectra were assessed within a wavelength range of 4,000-400 cm −1 .

| Thickness
The thickness of the composite films was determined by a hand-held digital micrometer (Guanglu Instruments Co.), with a precision of 0.01 mm. The values were taken at 10 random positions for each film. The averages were used to determine the water vapor permeability and mechanical properties.

| Dynamic mechanical thermal analysis (DMTA)
Dynamic mechanical thermal analysis analysis was conducted by a dynamic mechanical thermal analyzer (Triton Technology). The test was operated at 1 Hz constant frequency and a strain lower than 0.1 within the temperature range of −100-100°C with a heating rate of 5°C/min. Before the analysis, all films were preconditioned at 53% relative humidity with Mg (NO 3 ) 2 for 24 hr. The storage modulus (E') and loss factor (tan δ) of composite films were obtained as a function of temperature.

| Mechanical properties
The tensile properties of the composite film were measured using a tensile tester (Zwick/Roell model FR010) according to the ASTM standard method D882-97 (Song et al., 2018). The specimens were cut in a dumbbell shape (80 × 5 mm). Prior to testing, samples were conditioned in a desiccator containing saturated Mg (NO 3 ) 2 solution.
The separation of the primary knob and crosshead speed was adjusted to 50 mm and 5 mm/min, respectively. Finally, ultimate tensile strength (MPa) and strain at break (%) were measured for each sample. The experiment was performed in three replicates and the mean was reported as the result.

| Moisture content and water vapor permeability (MC and WVP)
To measure the moisture content (MC), the samples that were cut into squares of 3 × 3 cm were placed in Petri dishes. Then, the samples were dried in an oven at 105°C for 24 hr. The average of three replicates of different weights of samples was reported as the percentage of moisture content.
A gravimetric method, which was a modified form of ASTM E96-00, was used to determine the water vapor permeability (WVP) of composite films (Otoni, Moura, et al., 2014). Small cups containing 3 g anhydrous calcium sulfate (0% RH) were sealed by each specimen film (1.7 × 10 −6 m 2 exposed film area). The cups were placed in a desiccator containing a saturated salt solution (75% RH). Note that the difference in RH (75% RH) between sodium chloride and calcium sulfate acts as a motive force for water vapor. The cups were then weighed individually every 24 hr. This process lasted for 1 week until the steady state was reached. The weight change of the cups was recorded as a function of time. Water vapor transmission rate (WVTR) was calculated as the slope (g/s) divided by the transfer-exposed film area (m 2 ).
The WVP was measured according to the following equation (Nisar et al., 2018): where P is the saturation vapor pressure of water (Pa) at the test temperature (25°C), R 1 is the RH in the desiccator, R 2 is the RH in the cup, and X is the film thickness (m).

| Optical
The color value of composite specimens including lightness (L), redness (a), and yellowness (b) was assessed using a Hunter lab (CR-

| Antibacterial activity
The antimicrobial properties of the samples were evaluated by disk diffusion method. In this method, the inhibition zone around the disk on solid media was used to determine the antimicrobial activity of the film samples against typical selected bacteria. For this purpose, the mentioned bacteria were smeared on Mueller Hinton agar medium at a 10 5 CFU/ml concentration. In the next step, the specimen films were punched in the form of a disk, 6 mm in diameter, and placed on the seeded solid agar surface. The plates were then incubated at 37°C for overnight.

| Statistical analysis
The present study was performed based on a completely randomized design (CRD). Experiments on all treatments were measured in three replications and were expressed as means and standard deviations (mean ± SD). Data analysis was performed with the oneway analysis of variance (ANOVA) using SPSS software version 18.0 (SPSS Inc). Following this step, Duncan's multiple range tests were applied to check the significant difference between treatments at the 95% confidence level.

| SEM and AFM
The Atomic force microscopy is further used to clarify the descriptive information obtained by SEM. The calculations of roughness indices can be used to provide quantitative information regarding the surface morphology of composite at the nanometer scale (Jahed et al., 2017). The roughness of the films is often critical to their applications, and in particular, roughness strongly impacts their optical, barrier, and frictional properties. Table 1 reports the results of roughness indices, the average (R a ) plus root-mean-square (R q ) roughness for blend films with and without essential oil. In addition, the corresponding plots (2-D and 3-D) of surface morphologies are depicted in Figure 2.
According to Table 1  Note: Data are given as means and standard deviation. Different superscript letters indicate the significant difference of values in the same column (p < .05).

| FT-IR
The FT-IR spectra of films were used to study the structure of composites and confirm the interaction between the matrix of polymers and GGEO over the wavenumber range 4,000-400 cm -1 . The spectra of neat film and composites containing different concentrations of GGEO are displayed in Figure 3. Both CMC and PVA possess some common functional groups in their structure; therefore, the group areas of FT-IR spectra are in relatively similar locations and their spectra vary generally in the fingerprint regions. As can be seen from the figure, the peaks located within the wavenumber 3,500-3,800 cm -1 region were found in both samples, related to the stretching vibration of wide-ranging hydroxyl groups (-OH) of polymers and GGEO. The sharp peak at 1,500 cm -1 can be attributed to symmetrical stretch of acidic groups (COO-) in GGEO and the combination of polymers. Further, the strong peaks observed around region 1,600 cm -1 might be associated with the C-O of the six-carbon cyclic pyranose of CMC and GGEO. In the sample containing essential oil, the peaks are also integrated into the region within 1,600-1,000 cm -1 . This indicates the interaction between the functional groups of polymers and functional groups of GGEO. Therefore, as shown in the figure in this area, the peak corresponding to these functional groups has been considerably broader.

| Thickness
According to the literature, the thickness of the films is affected by the compatibility between polymer and filler, the number of solid contents, and free volumes inside the film matrix (Chu et al., 2019;Nisar et al., 2018). According to Table 2 They reported that the increase in film thickness could be due to the generation of incompatibilities between the polymer and essential oil, leading to a less dense structure and thus increased film thickness.   for sodium caseinate films plasticized with linseed oil resin (Pereda et al., 2015).

| DMTA
The curves for tan δ as a function of temperature for the composites containing 0.25% and 0.75% GGEO, as well as the control film, are indicated in Figure 4b. Solid-to-liquid state transition temperature can be calculated from the maximum value of tan δ (loss factor) curve (Sahraeian et al., 2019). The higher-temperature peak of loss factor has corresponded to the glass transition temperature (T g ) of CMC-PVA composite. T g is related to the segmental mobility of the molecular chains in amorphous regions. (Salarbashi et al., 2018). In a study, as the GGEO concentration increased, the glass transition temperature decreased, and also compared to the neat CMC-PVA composite the loss peaks tended to broaden. Due to the migration of GGEO droplets, many empty voids were generated in the matrix of the composite. As mentioned earlier, by increasing the number of free volumes, the chain mobility of the blend films was enhanced F I G U R E 4 Storage modulus (a) and tan δ (b) versus temperature curves of CMC-PVA and CMC-PVA-GGEO blend films (Bahrami et al., 2019). These results were consistent with the findings of Zinoviadou et al., (2009) who reported a similar behavior due to the plasticizing action of oregano oil on the thermo-mechanical properties of whey protein isolate films.

| Mechanical properties
The study of mechanical properties such as ultimate tensile strength (UTS) and strain at break (SAB) can help predict the stress tolerance and structural integrity of composites (da Rocha et al., 2018). The effect of GGEO added at different concentrations on the mechanical properties of the blend films is summarized in The changes observed in SAB and UTS in the blend films are related to inter-and intra-molecular forces as well as the network microstructure (Abdollahi et al., 2019;Han et al., 2018). The incorporation of GGEO into the CMC-PVA matrix increases the free volume and chain mobility of the polymers. These results were in agreement with the phenomenon discussed earlier for DMTA analysis. In conclusion, EO due to its lipid nature acts as a plasticizer in the composite matrix and can be easily deformed in the blend film structure (Song et al., 2018). of several studies are consistent with ours (Song et al., 2018;Wu et al., 2017).

| MC and WVP
Characteristics such as moisture content (MC) and water vapor permeability (WVP) are important physical properties to assess water retention by the composite matrix and the water diffusion through the composite, respectively . Generally, the lower the values of these parameters, the higher the efficiency of the film is as a moisture barrier (Bastos et al., 2016). The MC and WVP values of the control and GGEO incorporated blend films are reported in GGEO. These results are in agreement with the data reported by Jouki et al., (2014). According to them, these results were attributed to the breakdown of the film structure by the presence of EO. In this way, more water molecules are confined among polymer chains via hydrogen bonding.
Likewise, the WVP values of the blend films increased by incorporation of GGEO in the composite matrix ( Table 2). The WVP of the CMC-PVA films significantly increased (p < .05) from 7.25 × 10 −9 g/m s −1 Pa −1 to 12.68 × 10 −9 g/m s −1 Pa −1 as the concentration of GGEO rose from 0% to 0.25%. In addition, the WVP of the blend films increased slightly (p > .05) from 12.68 × 10 −9 g/m s −1 Pa −1 to 13.61 × 10 −9 g/m s −1 Pa −1 at GGEO contents over 0.25%. According to the SEM images (Figure 1), this result can be explained by the discontinuities occurring in the composite structure by improper dispersion of essential oil droplets. This could increase the amount of water vapor transfer, resulting in the WVP increase .
A similar result has been reported by Dashipour et al., (2015) where the effect of Zataria multiflora essential oil on WVP of CMC

| Optical
Colorimetric parameters of the blend films, including Hunter Lab color values (L, a, b), the total difference in color (ΔE), whiteness index values (WI), and yellowness index values (YI), are provided in Table 3. As shown, the addition of essential oil into the composite matrix led to a marked drop in both the WI and Hunter L-values of the blend films. However, YI, Hunter a-, and b-values significantly rose with an increase in essential oil. Therefore, the incorporation of GGEO increased the darkness, yellowness, and redness of blend films as compared with the control film. These optical changes can be related to the yellowish and reddish color of GGEO, absorbing light at a lower wavelength. This property could help protect the packaged foods from visible light and ultraviolet rays, thus reducing the photo-oxidation reactions, discoloration, and nutrient loss .
In some studies, a similar trend has been reported with the addition of essential oils to packaging films, for example chitosan with turmeric essential oil (Li et al., 2019), soy protein with clove essential oil (Ortiz et al., 2018), and chitosan with Perilla frutescens (L.) Britt. essential oil . Generally, the incorporation of GGEO at different concentrations in a composite matrix intensified the color parameters, such that the ΔE of blend films significantly increased. Such a behavior has also been reported in previous studies

| Antibacterial activity
The antimicrobial activity of the fabricated films was measured using a disk diffusion method and measuring the inhibition zone around each sample in media disk. Figure

| CON CLUS IONS
In present study, the fabrication and characterization of CMC-PVAbased composite films for food antimicrobial packaging application were carried out. Generally, chemical structure and morphological properties of CMC-PVA-based films were affected by GGEO (as active agent), which was confirmed by FT-IR, SEM, and AFM testing.
Furthermore, ultimate tensile strength, storage modulus, and glass transition temperature of CMC-PVA-based films were decreased by addition of EO and these films showed a tendency to yellowing, with a significant increase in darkness and less WI than the control film. Microbial tests represented superior antibacterial activity of composite films especially at the highest concentrations of GGEO (0.75%). Nevertheless, further studies are required to improve the hydrophobicity and moisture barrier of derived films.

CO N FLI C T O F I NTE R E S T
There is no conflict of interest to declare.