Journal Pre-proof Development of antimicrobial films based on chitosan-polyvinyl alcohol blend enriched with ethyl lauroyl arginate (

Abstract


Introduction
The current trend in food packaging is mainly oriented towards the substitution of nonbiodegradable petroleum-based polymers by packaging materials that are eco-friendly and also prolong food shelf life (Kanatt, Rao, Chawla, & Sharma, 2012).In this context, considerable research has been conducted involving the fabrication of biodegradable food packaging materials that come from renewable natural resources and agri-food industry wastes (Cazón, Vázquez, & Velazquez, 2018; Sarwar, Niazi, Jahan, Ahmad, & Hussain, 2018).Among bio-based natural polymers, chitosan (CS) has received significant attention for its potential to substitute -partially or totally -petroleum-based polymers (Leceta, Guerrero, & De La Caba, 2013).CS is a cationic linear polysaccharide consisting of poly-β-(1-4)-D-glucosamine units obtained by partial deacetylation of chitin, the major component of the insect's exoskeleton and shells of crustacean such as crab, shrimp, and crawfish.CS is the second most abundant polysaccharide after cellulose with unique biological properties such as biocompatibility, biodegradability and non-toxicity.In addition, this amino polysaccharide has high antimicrobial activity against many pathogenic and spoilage microorganisms, including both Gram-positive and Gram-negative bacteria which makes it an excellent candidate for food packaging applications (Rubilar, Candia, Cobos, Díaz, & Pedreschi, 2016).However, there are some limitations which are associated with CS such as low mechanical strength, low thermal stability, rigid crystalline structure and high production cost.A simple and effective alternative to overcome these drawbacks could be blending of CS with synthetic polymers.Films formed by the blending of natural and synthetic polymers represent a new class of material with modified physical and mechanical properties compared to films made of individual components.Blending CS with polyvinyl alcohol (PVA) has been intensively investigated by many researchers to gain biodegradable and antimicrobial films for food packaging applications with new and desired properties ( PVA is a synthetic, low cost, non-toxic and water-soluble polymer commercially obtainable from hydrolysis of polyvinyl acetate with excellent film forming properties.Despite its synthetic character, this polymer was recognized as biodegradable and it shows high tensile strength, flexibility, gas barrier properties and good resistance to acid and alkali media (Aloui et al., 2016).PVA has been evaluated for safety by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2003 at the 61st meeting (Bellelli, Licciardello, Pulvirenti, & Fava, 2018) and it has also been approved for use in packaging meat and poultry products by the USDA (Kanatt et al., 2012).PVA is highly miscible with other hydrophilic polymers such as CS, owing to the formation of intermolecular hydrogen bonds between hydroxyl groups of PVA and hydroxyl and amine groups of CS.Due to the high compatibility of CS and PVA, the resulting films show homogeneous structure.Moreover, blending PVA with CS is a promising strategy to reduce the production cost and improve the mechanical property and stability of CS films.
Antimicrobial packaging as a part of active packaging systems is intended to extend the shelf life of food products and assure the safety and quality of packaged foods (Tripathi, Mehrotra, & Dutta, 2008).Ethyl lauroyl arginate (LAE) is considered as an effective antimicrobial substance among novel food additives (Rubilar et al., 2016).LAE remains stable at pH 3-7 and is odorless and colorless as well (Kashiri et al., 2016).LAE is a synthetic surfactant consisting of an ethyl esterified arginine head with a lauroyl tail attached to the α-amino group that is highly active against a wide range of food pathogens and spoilage microorganisms including bacteria, yeast and molds with a low-dose application (Becerril, Manso, Nerin, & Gómez-Lus, 2013).This cationic surfactant disrupts the cytoplasmic membrane of microorganisms and inhibits the growth of microorganisms by causing cell

2.
Material and methods

Materials and reagents
Chitosan (CS) with a molecular weight of 100-300 kDa was obtained from Acros Organics

Preparation of film-forming solutions (FFS) and films
Preparation of films was adapted from the procedures of

Scanning electron microscopy (SEM)
The SEM images from the surface and cross-section of the films were obtained with the use of a scanning electron microscope (FEI, Quanta 200, Oregon, USA).Film samples were fixed on a stainless-steel support with a double-sided conductive adhesive.The analysis was conducted in low vacuum (0.6 Torr) at an acceleration voltage of 20 kV.

Atomic force microscopy (AFM)
The surface morphology of the films was analyzed using an atomic force microscope (Park Scientific Instruments, South Korea).Films were fixed onto AFM specimen metal discs using a double-sided tape and then placed to a magnetic sample holder located on the top of the scanner tube.The images were scanned in no-contact mode under ambient condition.The surface roughness (R a ) of the films was calculated on the basis of the root mean square (R q ) deviation from the average height of peaks after subtracting the background using ProScan software (version 1.51b).All samples were analyzed in triplicate.

Spectroscopy
The infrared spectra of different films were obtained using an ATR/FT-IR spectrometer (type Alpha, Bruker Optik GmbH, Ettlingen, Germany).Spectra were collected from two different locations from the top and bottom side of the same samples in the 4000-400 cm −1 wavenumber range by accumulating 64 scans with a spectral resolution of 4 cm -1 .

Thickness and mechanical properties
Film thickness was measured with a digital micrometer (Model IP65, SAMA Tools, Viareggio, Italia) at five different random positions (one at the center and four at the edges).The means of these five separate measurements were recorded.
The tensile strength (TS), elongation at break (E%) and elastic modulus (EM) were determined using a dynamometer (Z1.0,Zwick/Roell, Ulm, Germany) according to ASTM standard method D882 (ASTM, 2001a).The films with known thickness were cut into rectangular strips (9.0 x 1.5 cm 2 ).Initial grip separation and cross-head speed were set at 70 mm and 50 mm/min, respectively.Measurements were repeated 10 times from each type of film.The software TestXpert® II (V3.31) (Zwick/Roell, Ulm, Germany) was used to record the TS curves.TS was calculated by dividing the maximum load to break the film by the crosssectional area of the film and expressed in MPa.The E% was calculated by dividing film elongation at rupture by the initial grip separation expressed in percentage (%).EM was calculated from the initial slope of the stress-strain curve and expressed in MPa.

UV barrier, light transmittance, opacity value and color
The barrier properties of films against UV and visible light were determined at the UV (200, 280 and 350 nm) and visible (400, 500, 600, 700 and 800 nm) wavelengths.These optical characteristics were estimated with a VWR®Double Beam UV-VIS 6300PC spectrophotometer (China) using square film samples (2 × 2 cm 2 ).The opacity of the films was calculated by Eq. ( 1): Opacity value= where T 600 is the fractional transmittance at 600 nm and x is the film thickness (mm).The greater opacity value represents the lower transparency of the film.For each film, four readings were taken at different positions and average values were determined.
The color of films was measured with a CR-400 Minolta colorimeter (Minolta Camera, Co., Ltd., Osaka, Japan) at room temperature, with D65 illuminant and 10° observer angle.The instrument was calibrated with a white standard (L* = 99.36,a* = -0.12,b* = -0.07)before measurements.Results were expressed as L* (lightness), a* (red/green) and b* (yellow/blue) parameters.The total color difference (∆E * ) was calculated using the following Eq.( 2): where ∆L * , ∆a * and ∆b * are the differences between the corresponding color parameter of the samples and that of a standard white plate used as the film background.For each film, five readings were taken at different positions and the average values were determined from the top and bottom sides.

Moisture content and water solubility
Moisture content (MC) of the films (2 × 2 cm 2 ) was determined as the percentage of weight loss upon drying to constant weight (Md) in an oven at 105 ± 2 °C and the initial weight (Mw) according to the following Eq.( 3): MC (%): The solubility of films (2 × 2 cm 2 ) in water was determined by drying to constant weight in an air-circulating oven at 105± 2 °C (Wi) and then each film was immersed in 50 mL distilled water at 25 °C following Gontard, Guilbert, & Cuq, (1992) with slight modifications.After 24 h, the film samples were dripped and dried to constant weight at 105± 2 °C (Wf) to determine the weight of dry matter which was not solubilized in water.The measurement of water solubility (WS) was determined according to the following Eq.( 4): All measurements for MC and WS were made in triplicate.

Water vapor transmission rate and water vapor permeability
Water vapor transmission rate (WVTR) of the films was determined gravimetrically in triplicate according to the ASTM E96 method (ASTM, 2001b) with some modifications.Films were sealed on top of glass test cups with an internal diameter of 10 mm and a depth of 55 mm filled with 2 g anhydrous CaCl 2 (0% RH).The cups were placed in desiccators containing BaCl 2 (90% RH), which were maintained in incubators at 45 °C.WVTR was determined using the weight gain of the cups and was recorded and plotted as a function of time.Cups were weighed daily for 7 days to guarantee the steady state permeation.The slope of the mass gain versus time was obtained by linear regression (r 2 ≥ 0.99).WVTR (g /day m 2 ) and WVP (g mm/kPa day m 2 ) were calculated according to the following Eqs.( 5) and ( 6): where ∆W/∆t is the weight gain as a function of time (g/day), A is the area of the exposed film surface (m 2 ), L is the mean film thickness (mm) and ∆P is the difference of vapor pressure across the film (kPa).

In vitro antimicrobial activity 2.10.1. Disk diffusion assay
Antibacterial activity test on films was assessed against four typical food bacterial pathogens including Listeria monocytogenes (UNIMORE 19115), Escherichia coli (UNIMORE 40522), Salmonella typhimurium (UNIMORE 14028) and Campylobacter jejuni (UNIMORE 33250) using the disk diffusion assay according to (Haghighi et al., 2019).Films (sterilized with UV light) were cut into a disc shape of 22 mm diameter and placed on the surface of BHIA agar plates, which had been previously streaked with 0.1 mL of inocula containing 10 6 CFU/mL of tested bacteria.The plates were then incubated at 30 °C for 24 h (C.jejuni plates were incubated at 37 °C).The diameter of the inhibition zones was measured with a caliper and recorded in millimeters (mm).All tests were performed in triplicates.

Evaluation of antimicrobial activity in liquid medium
Antimicrobial activity of CS-PVA films enriched with LAE (1-10%) evaluated against L.
A loop of each strain was transferred to 10 mL of BHIB and incubated at 30°C (C.jejuni plates were incubated at 37 °C) for 24 h to obtain stationary phase (optical density of 0.9 at 600 nm).Then, cells were diluted in BHIB and incubated at 30 °C (C.jejuni tube was incubated at 37 °C) to obtain exponential phase (optical density of 0.2 at 600 nm).One hundred µL of microorganism in exponential phase was inoculated into tubes with 10 mL of BHIB.A 0.025 g portion of film (1.5 x 1.5 cm 2 ) was added to each tube in sterile conditions.
The tubes were incubated at 30°C for 24 h.As a control, CS-PVA film without LAE was used.
Depending on the turbidity of each tubes, serial dilutions with NaCL were made and plated on the Petri dishes with BHIA culture medium.Colonies were counted after incubation at 30 °C for 24 h.

Statistical analysis
The statistical analysis of the data was performed through analysis of variance (ANOVA) using SPSS statistical program (SPSS 20 for Windows, SPSS INC., IBM, New York).The experiment was performed in 3 replicates and the number of repeats varied from one analysis to another and was reported in each subsection.The differences between means were evaluated by Tukey's multiple range test (p<0.05).The data were expressed as the mean ± SD (standard deviation).

Scanning electron microscopy (SEM)
The surface and cross-section images of CS-PVA blend film (control) and CS-PVA film enriched with different concentrations of LAE (1-10%) (active films) are presented in Fig. 1.
The film microstructure greatly affects the final physical, mechanical and barrier properties.This is mainly due to the interaction between the film components and LAE.The surface of the control film was smooth and homogenous and did not show pores or cracks indicating good compatibility between CS and PVA to form a blend (Fig. 1a).This could be explained by strong molecular interaction between functional groups of chitosan and PVA.Similar results were reported by Ghaderi, Hosseini, & Gómez-Guillén (2019) and Jahan, Mathad, & Farheen (2016) who noticed that the surface of CS-PVA blend film was homogenous without pores.Addition of LAE up to 1% did not affect the surface morphology of active films (Fig. 1c) indicating LAE was evenly distributed and well dispersed in the film matrix.Small particles and aggregations were observed at the surface of films with increasing concentration of LAE up to 10% (Fig. 1e, 1g and 1i).A compact and continuous structure without irregularities like air bubbles and pores and any evidence of phase separation can be observed in the crosssection of the control film (Fig. 1b).The cross-section of active films containing LAE up to 2.5% showed similar results (Fig. 1d and 1f).However, active films containing 5 and10% LAE showed irregular and sponge shape structure (Fig. 1h and 1j).This effect was more obvious in CS-PVA film containing 10% LAE.This might be related to the agglomeration of LAE in the film matrix at high concentrations which resulted in disrupted structures.The interaction between the polymer chains was disturbed by interactions with functional groups of LAE, producing films with less integrity.Gaikwad, Lee, Lee, & Lee (2017) also reported that the order of low-density polyethylene (LDPE) films was interrupted by the addition of high amount of LAE powder (5 and 10%) mainly due to the inhomogeneous distribution of LAE inside the matrix and the low interfacial interaction between the LDPE and LAE powder.

Atomic force microscopy (AFM)
AFM was further performed to characterize the surface morphology of control and active films.Typical 3D surface topographic AFM images and root mean square (R q ) and roughness (R a ) values are presented in Fig. 2. The surface of control film was relatively smooth and homogenous as indicated by lower R q and R a values (17.1 and 12.0 nm, respectively).Increasing concentration of LAE up to 10% led to the increase in the roughness of the films, as indicated by higher R q and R a values.The difference in roughness value between control and active films was in accordance with the film microstructure observed by SEM analysis.

Mechanical properties
The tensile strength (TS), elongation at break (E%) and elastic modulus (EM) are three important parameters for evaluation of mechanical properties.Values are given as mean ± SD (n = 3).Different letters in the same column indicate significant differences (p<0.05).

UV barrier, light transmittance and opacity value
Protecting food from the effect of UV-Vis radiation is one of the desired characteristics of packaging material due to their influence on product performance and consumer acceptance.The transmission of visible light (400-800 nm) was higher than 80% for the control film.The active films containing 1 and 2.5% LAE showed similar results while active films containing 5 and 10% LAE showed lower values.Thus, once a critical LAE concentration is exceeded, aggregates are formed, that were large enough to scatter the light and thereby interfere with its transmission (Bonnaud, Weiss, & McClements, 2010).The light barrier property is also an important factor for food preservation to avoid photo-oxidation of organic compounds and degradation of vitamins and other pigments.In addition, it provides a clear view of the food content and its condition (Figueroa-Lopez, Andrade-Mahecha, & Torres-Vargas, 2018; Yadav & Chiu, 2019).In this study, all films can be considered as transparent due to the opacity value lower than 5 at 600 nm (Tab.2).The higher value of this parameter represents the lower transparency of the film.Values are given as mean ± SD (n = 3).Different letters in the same column indicate significant differences (p<0.05).

Moisture content, water solubility, water vapor transmission rate and water vapor permeability
One of the major drawbacks of biodegradable films for food packaging applications is their sensitivity to water.Due to the hydrophilic nature of CS and PVA, when these films are exposed to high relative humidity conditions, water molecules are absorbed by the polymeric chains, exerting a plasticizing effect and resulting in changes of the mechanical and barrier  Values are given as mean ± SD (n = 3).Different letters in the same column indicate significant differences (p<0.05).

3.9.
In vitro antimicrobial activity

Disk diffusion assay
The antimicrobial activity of control and active films against common bacterial food pathogens, namely C. jejuni, E. coli, L. monocytogenes and S. typhimurium, was evaluated by the disk diffusion assay (Fig. 6) and the details are presented in Tab.

Evaluation of antimicrobial activity in liquid medium
The Antimicrobial activity of control and active films against common bacterial food pathogens including C. jejuni, E. coli, L. monocytogenes and S. typhimurium was also evaluated in liquid medium and the details are presented in Tab. 5. CS-PVA film without LAE used as a control.Among tested microorganisms C. jejuni showed higher log reduction (2) for CS-PVA-LAE 10% films that is in agreement with the DDA result.The incorporation of LAE (1-10%) showed a log reduction against all tested microorganisms.Clearly, the higher the LAE concentration in the film, the greater the antimicrobial efficiency of the CS-PVA film.

Table 5
Antimicrobial activity of films based on a control CS-PVA blend (CS-PVA) and CS-PVA enriched with LAE (1-10% w/w) expressed as logarithm of colony forming units (log CFU/ mL) and log reduction value (LRV).Values are given as mean ± SD (n = 3).Different lowercase letters in the same column indicate significant differences (p<0.05).

Conclusion
In this study, biodegradable active films based on CS-PVA blends enriched with LAE at different concentrations (1-10%, w/w) were developed and their microstructural, physical, optical, mechanical, barrier and antimicrobial properties were evaluated for food packaging applications.The results showed that all films containing LAE were transparent.The incorporation of LAE could improve the UV and light barrier properties of CS-PVA film, which may be useful to protect food from UV degradation and photo-oxidation.The characteristic absorption bands in the ATR/FT-IR spectra of the CS-PVA blends did not show significant band shifts and intensity changes up to 2.5% LAE content, indicating low interactions between the polymer and the LAE.However, at elevated LAE content the C=O, NH 2 and NH functionalities of this additive contribute to competitive molecular interactions with the hydroxyl, amino, ether and residual acetate groups of the CS-PVA film network.The presence of LAE greatly influenced TS and E%.Films with LAE were less resistant and less stretchable than the control film and a significant deterioration of mechanical properties occurred above 2.5% incorporation of LAE.The developed active films, especially those including 5 and 10% LAE, were effective against common bacterial food pathogens.The results suggest that the CS-PVA films enriched with different concentrations of LAE could be considered as environmentally friendly packaging material with antimicrobial properties to extend the shelf life of food products and that might be an alternative to synthetic plastics for certain applications.

Declaration of interest
None.
deformation and affecting their metabolic process negatively (Muriel-Galet, Carballo, Hernández-Muñoz, & Gavara, 2016).LAE has been considered as GRAS (generally recognized as safe) by the U.S. Food and Drug Administration (FDA, 2005) and has been authorized as food preservative by the European Food Safety Authority (EFSA, 2007).Incorporation of LAE as an antimicrobial compound into antimicrobial packaging to improve food safety and quality has been reported in several studies (De Leo et al., 2018; Haghighi et al., 2019a; Higueras, López-Carballo, Hernández-Muñoz, Gavara, & Rollini, 2013; Kashiri et al., 2016; Moreno, Cárdenas, Atarés, & Chiralt, 2017a; Rubilar et al., 2016).However, literature concerning the effects of LAE on the functional properties of CS-PVA blend film is not available.Therefore, the objective of the present study was to develop biodegradable films based on CS-PVA blend enriched with different concentrations of LAE to evaluate microstructural, physical, optical, mechanical and water barrier properties for food packaging applications.Moreover, the antimicrobial activity of films against four common food bacterial pathogens including Campylobacter jejuni, Escherichia coli, Listeria monocytogenes and Salmonella typhimurium, was investigated.

Fig. 4 .
Fig. 4. ATR/FT-IR spectra of LAE formulation (Mirenat-D).3.4.Thickness The UV-Vis light transmittance of control and active films in the wavelength range of 200-800 nm is presented in Fig.5.Control film showed a higher UV light transmittance (200-350 nm) compared to the active films.UV light transmittance was reduced at increasing LAE concentration and active films behaved as effective UV barriers at 200 nm since the transmittance value was below 1%.UV barrier property of films is an important parameter for food packaging applications to minimize UV-induced lipid oxidation, to preserve the organoleptic properties of the packaged food, to avoid nutrient losses, discoloration and offflavors, thereby prolonging food shelf life(Hajji et al., 2016; Wu, Sun, Guo, Ge, & Zhang, 2017).

Fig. 6 .
Fig. 6.Disk diffusion results of films based on a control CS-PVA blend (CS-PVA) and CS-PVA enriched with LAE (1-10% w/w).3.9.2.Evaluation of antimicrobial activity in liquid medium Generally, adequate mechanical strength and extensibility are required for the development of biodegradable films for food packaging applications.The mechanical properties of control and active films are presented in Tab. 1.The control film showed the highest TS, E% and EM values.The presence of LAE greatly influenced TS and E% (p<0.05).Films containing LAE were less resistant and less stretchable than the control film (p<0.05).Incorporation of LAE up to 10% decreased the tensile strength from 42.48 to 15.70 MPa and E% from 54.25 to 14.31%.The significant deterioration of mechanical properties above 2.5% incorporation of LAE (Tab. 1) is also consistent with the ATR/FT-IR observation, that band shifts and intensity changes occur above this threshold concentration.This tendency could be explained by the fact that active the molecular mass of the polymer, deacetylation degree of CS, degree of hydrolysis of PVA, pH of the FFS, drying conditions and type of LAE.
films containing a high concentration of LAE are unable to form a cohesive and continuous matrix as it was confirmed by SEM analysis.This can be attributed to the competitive interaction of the functional groups of LAE and the CS-PVA blend that limit cohesion forces within the polymer in the film matrix and consequently decrease the degree of physical crosslinking by weakening the intermolecular hydrogen bonding, thereby resulting in the reduction of mechanical properties.Despite the reduction of TS after incorporation of LAE, it should be noted that the TS values for the active films containing LAE up to 2.5% were comparable with those of plastic films that are used widely in the market, such as high which might be due to the application of Mirenat-G (10% LAE, 90% glycerol) as LAE source.Hence the observed effect could be mainly due to the plasticizing effect of glycerol.Literature data regarding mechanical properties are controversial and are influenced by multiple factors such as (Higueras et al., 2013;Rubilar et al., 2016)ons did not influence the MC.Solubility is defined as the content of dry matter solubilized after 24 h immersion in water.The control film showed the lowest WS value and addition of LAE increased the WS value.CS-PVA film containing 10% LAE showed the highest WS value (p<0.05).The higher solubility values of active films could be explained by the hydrophilic nature of CS-PVA blend film and low oilwater equilibrium partition coefficient of LAE (K ow <0.1), which means that LAE has a high affinity to water molecules(Higueras et al., 2013;Rubilar et al., 2016).

Table 3
4. The control film did not show an inhibition zone against any of the tested microorganisms.The absence of inhibition zone could be explained by the limitation of CS to diffuse in agar medium(Leceta, (Hajji et al., 2016;Tripathi et al., 2009)2013) and incapability of PVA to inhibit bacterial growth as it has been reported by other authors(Hajji et al., 2016;Tripathi et al., 2009), so that only microorganisms in direct contact with the active sites of CS in the CS-PVA film network are inhibited.Active films containing 1% LAE were only effective against C. jejuni.In general, LAE was more effective against C. jejuni compared to the other microorganisms considered, which showed inhibition haloes ranging from 3 to 5-fold wider.No differences were observed in the inhibition zones produced by CS-PVA films incorporating 5 and 10% of LAE against all tested microorganisms.Similar results reported by Muriel-Galet, López-

Table 4
Inhibition zone diameters of the film disks (22 mm diameter) based on a control CS-PVA blend (CS-PVA) and CS-PVA enriched with LAE (1-10% w/w).