Development and characterization of biocomposite films made from kefiran, carboxymethyl cellulose and Satureja Khuzestanica essential oil

Food Chemistry 289 (2019) 443–452

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Development and characterization of biocomposite films made from kefiran, carboxymethyl cellulose and Satureja Khuzestanica essential oil

T

Seyedeh-Maryam Hasheminyaa, Reza Rezaei Mokarrama, , Babak Ghanbarzadeha,c, ⁎ Hamed Hamishekarb, Hossein Samadi Kafilb, Jalal Dehghannyaa, ⁎

a

Department of Food Science and Technology, University of Tabriz, Tabriz 51666-16471, Iran Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran c Department of Food Engineering, Faculty of Engineering, Near East University, P. O. Box 99138, Cyprus, Mersin 10, Nicosia, Turkey b

ARTICLE INFO

ABSTRACT

Keywords: Antioxidant properties Biodegradable films Hydrogen bonds Satureja Khuzestanica essential oil Phenolic compounds

Kefiran-carboxymethyl cellulose biocomposite films incorporated with Satureja Khuzestanica essential oil were developed and characterized. Results indicated that increase in the concentration of the essential oil increased ultimate tensile strength and contact angle but decreased elongation at break, moisture content and water vapor permeability. It also significantly altered color parameters and the percentage of light transmission in the visible and ultraviolet range. Fourier transform infrared spectroscopy revealed the formation of hydrogen bonds between polymer matrix and essential oil. Scanning electron microscopy showed that the surface structure of the films was homogeneous without porosity. Increase in storage modulus and glass transmission temperature in films incorporated with the essential oil was observed through dynamic mechanical thermal analysis. Moreover, significant increase in antioxidant properties and phenolic compounds were noticed. Ultimately, results obtained from evaluation of antimicrobial characteristics of films indicated their inhibitory effects against Staphylococcus aureus and Escherichia coli bacteria.

1. Introduction Food quality and safety are of major concerns in the food industry. Food producers strive to reduce or eliminate microorganisms in food products. Survival of microorganisms in food materials can result in spoilage, loss of food quality, infection and pathogenicity (ShojaeeAliabadi et al., 2013). Due to postponing microbial spoilage in food and reduction of the risk of contamination, biodegradable antimicrobial films have been of great interest to researchers. These films offer several advantages such as environmental compatibility, nontoxic nature and cheap price (Shojaee-Aliabadi et al., 2013; Dashipour et al., 2015; Yu et al., 2018). Therefore, due to nontoxic nature and biodegradability of the edible films, their production has gained more and more importance (Hasheminya et al., 2018). Kefiran is a new resource with appropriate properties of a biopolymer among natural biopolymers. Due to its capacity for film formation in food packaging, this biopolymer has attracted the attention of many researchers (Motedayen et al., 2013; Zolfi et al., 2014). Kefiran is a water-soluble polysaccharide which is obtained from kefir grains or through pure culture of Lactobacillus kefiranofaciens under aerobic condition or mixed culture of Lactobacillus kefiranofaciens and ⁎

Saccharomyces cerevisiae under anaerobic condition (Cheirsilp et al., 2003). In addition to kefiran, carboxymethyl cellulose (CMC) is also widely used as another important polymer in food packaging. This polymer is a water-soluble linear polysaccharide which is cheap, nontoxic and biodegradable. Films and coatings obtained from CMC are edible and have high flexibility and transparency (Dashipour et al., 2015). In order to improve mechanical properties of natural biopolymers, they are combined with other polymers. For instance, in order to improve mechanical and inhibitory properties, kefiran-starch or kefiran-CMC compositions are used (Motedayen et al., 2013; Hasheminya et al., 2018). Furthermore, in order to improve antibacterial characteristics of films, various natural and synthetic compounds including essential oils, metallic ions, organic acids, benzoates, sorbates, isothiocianates, bacteriocins, flavonoids, and tertiary butylhydroquinone (TBHQ) are used (Shojaee-Aliabadiet al., 2013; Dashipour et al., 2015). Considering the fact that synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) have side effects like carcinogenicity, and due to growing public awareness on food quality and safety, consumers prefer that natural additives such as essential oils be added to food (Dashipour et al., 2015; Robledo et al., 2018). Essential

Corresponding authors. E-mail addresses: [email protected] (R.R. Mokarram), [email protected] (J. Dehghannya).

https://doi.org/10.1016/j.foodchem.2019.03.076 Received 20 November 2018; Received in revised form 8 March 2019; Accepted 17 March 2019 Available online 18 March 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.

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oils are volatile, natural, complex and strong-smelling materials which contain high concentrations of phenolic compounds such as carvacrol, eugenol, and thymol that are produced by plants as secondary metabolites. Advantages such as medicinal, antioxidant and antibacterial characteristics have made essential oils as potential replacements for synthetic antioxidants and antimicrobials. In other words, use of essential oils in edible films with the purpose of obtaining antioxidative and antimicrobial stability as well as improving mechanical properties results in production of safer food products (Shojaee-Aliabadiet al., 2013; Dashipour et al., 2015). Satureja Khuzestanica is a native, medicinal and fragrant plant which belongs to the Lamiaceae family that grows in west and south of Iran and is known as Satureja Khuzestanica Jamzad. So far, scant research has been conducted on the chemical composition, antimicrobial and antioxidative properties of Satureja Khuzestanica (Saei-Dehkordi et al., 2012; Farzaneh et al., 2015). Satureja Khuzestanica is used as a taste and flavor agent in food and its antimicrobial, appetizing, tranquilizing, and antioxidative properties have been mentioned in conventional medical texts. Of principal components of Satureja Khuzestanica is its carvacrol phenolic compound (Saei-Dehkordi et al., 2012). Application of biocomposite films containing antioxidant and antimicrobial agents in active packaging protects food from physical, chemical, and biological damages and can act as a barrier against exchange of gas, moisture and microorganisms and, consequently, improves food quality and safety. Based on available resources, no research has yet been conducted on kefiran-CMC films incorporated with Satureja Khuzestanica essential oil or on its effects on various properties of the film. Therefore, the purpose of the present research was to produce kefiran-CMC biocomposite film containing Satureja Khuzestanica essential oil and analyze the effect of various concentrations of the essential oil on antimicrobial characteristics including Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) bacteria as well as films’ physical, antioxidant, mechanical, morphological, optical, color, and dynamic mechanical thermal properties.

Gas chromatography (Model GC-17A, Version 3, Shimadzu, Japan) coupled with mass spectrometry (Model QP-5050A, Shimadzu, Japan) equipped with DB-5 column (polydimethylsiloxane, 60 m × 0.25 mm i.d, film thickness 0.25 μm) was used to analyze the chemical composition of SEO. Helium was utilized as carrier gas with the flow rate of 1.3 mL/min. The oven temperature was first maintained at 50 °C for 3 min and was then increased to 240 °C at the rate of 3 °C/min and was finally stabilized at this temperature for 9 min. Injector temperature and detector temperature were 220 and 260 °C, respectively. In addition, ionization potential, volume injected and scan speed were, respectively, 70ev, 1 μL essential oil in n-hexane (2%) and 2000 amu/s (3.32 × 10-21 g/s) (Nazemiyeh et al., 2009). Essential oil composition was identified by using Kovats retention index, investigating obtained mass spectral and comparing them with retention time of standard compounds as well as computer databases (Nist 107, Nist 21). 2.3. Extraction of kefiran polysaccharide Piermaria et al. (2011)’s method was used to extract kefiran polysaccharide. In order to grow and proliferate, kefir grains were first cultured at 22 °C in pasteurized skim milk in a viscobator equipped with temperature controller. After 48 h, grains were separated using a plastic strainer basket and were blended for 1 h using a blender in boiling water with the ratio of 1:10 (kefir grains-boiling water). Then, the resulting mixture was centrifuged (Model 2-16KL, Sigma Laborzentrifugen GmbH, Germany) at 10,000×g for 15 min. In order for polysaccharide sedimentation to occur in the mixture, 96% ethanol at −18 °C was added with the ratio of 1:1 to the supernatant obtained from centrifugation and the resulting compound was kept at −18 °C overnight. Subsequently, centrifugation at 10,000×g was used at 4 °C for 20 min. Sediment resulting from centrifugation was dissolved in distilled water again and was centrifuged under similar condition (10,000×g at 4 °C for 20 min). Then, washing with distilled water was done in three steps and finally, kefiran polysaccharide was obtained as white sediment. The extraction yield of kefiran was 2.01% which was determined as the weight of lyophilized sample (Z) relative to the fresh weight of the kefir grains (X) (Zhi et al., 2018):

2. Materials and methods 2.1. Materials

Y=

In this study, carboxymethyl cellulose (1500–3000 cp, SigmaAldrich, USA), calcium chloride, calcium nitrate, anhydrous sodium sulfate, Folin-Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), sodium carbonate, gallic acid (Sigma-Aldrich, USA), glycerol, Tween 80, tryptic soy broth, Mueller Hinton agar (MHA) and nutrient agar (Merck, Germany) were used. Also, kefir grains and Satureja Khuzestanica were bought from a local market to produce kefiran polysaccharide and extract essential oil, respectively. Staphylococcus aureus ATCC 25,923 and Escherichia coli ATCC 25,922 bacteria were also obtained from the microbial collection of the Department of Microbiology and the Drug Applied Research Center of Tabriz University of Medical Sciences, Iran.

Z × 100 X

(1)

2.4. Preparing kefiran-CMC biocomposite films incorporated with Satureja Khuzestanica essential oil Kefiran-CMC biocomposite film incorporated with Satureja Khuzestanica essential oil was prepared by Hasheminya et al. (2018)’ method with some minor modifications. First, 1% kefiran solution was prepared in distilled water (w/v) by using a magnetic blender at 40 °C for 30 min. On the other hand, 1% CMC solution was prepared in distilled water (w/v) by using a magnetic blender at 80 °C for 1 h. In order to produce kefiran-CMC biocomposite films, the two aforementioned solutions were combined with the ratio of 50:50 and glycerol was added as a plasticizer. Glycerol magnitude was 50% of the CMC dry weight. Blending continued for another 15 min after the addition of the plasticizer. Subsequently, Satureja Khuzestanica essential oil was added to the solution in concentrations of 0 (control), 1, 1.5, and 2% (v/v). Then, Tween 80 (0.5% v/v based on the essential oil) was added to solutions as emulsifier. Afterwards, a rotor-stator homogenizer (IKA T25-Digital Ultra Turrax, Staufen,Germany) with the rotational speed of 1500 rpm was used at 70 °C for 5 min in order to homogenize the obtained solution. Solutions were then cooled up to 50 °C and finally, 50 mL of each solution was added to 9-cm-diameter glass plates and dried at 25 °C for 72 h. All the prepared films conditioned at 25 °C and relative humidity of 55% in a desiccator containing saturated calcium nitrate, 48 h before all the experiments.

2.2. Extraction and evaluation of Satureja Khuzestanica essential oil compositions The aerial (leaves and stems) parts of Satureja Khuzestanica were dried in an oven (model UNB 400, Memmert, Germany) at 50 °C for 72 h and powdered in mortar. The dried material (100 g) were immersed in distilled water (500 mL) in a 1000 mL round bottom flask. Satureja Khuzestanica essential oil (SEO) was obtained through hydrodistillation in clevenger-type apparatus for 4 h. Yield of essential oil was 1.1% (w/w based on dry weight). The extraction of essential oils was carried out several times. The obtained essential oil was dehydrated by anhydrous sodium sulfate and after sterilizing through a PVDF 0.22 μm syringe filter (Millipore Iberica SA, Madrid, Spain), it was kept in dark glass bottles at 4 °C. 444

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2.5. Measurement of physical properties

et al., 2014):

2.5.1. Thickness Samples’ thickness was measured by calculating their thickness at six random points, using a digital micrometer (Model IP 65, Mitutoyo Manufacturing, Japan), and then reported the mean (Zolfi et al., 2014).

TS =

2.5.2. Contact angel In order to determine the hydrophobicity of the samples, contact angle of water droplets on the surface of each film was examined. First, samples were cut into 20 mm × 50 mm pieces using a manual cutter. Then, using a Hamilton syringe (Model 701 RN SYR, Hamilton Company, Switzerland), 10 μL distilled water was added to the surface of the specimens from the height of 1 cm. Images were taken from both sides of the droplet by using a digital camera (Model PowerShot SD890 IS Digital ELPH, Canon, Japan). Calculation of the contact angle was done, using Adobe Acrobat 9.0 Professional software, as the angle between the tangent line on the droplet in the point of contact and the line drawn along the surface of the film (Hasheminya et al., 2018).

EB =

WVTR × X P

(5)

2.7. Measurement of color and optical properties To measure color parameters of the film samples, first, a digital camera was used to image the films (Model PowerShot SD890 IS Digital ELPH, Canon, Japan) in proper light. In order to simulate daylight luminosity, the light system included two Commission Internationale d’Eclairage (CIE) source D65 lamps. After placing the digital camera on a white trapezoidal-cross section chamber, images of the samples were taken at a distance of 30 cm. Then, color properties were assessed by using Hunter color parameters in terms of the degree of lightness (L), redness-greenness (a) and yellowness-blueness (b) by using Adobe Photoshop CC 2017 software. Color properties were measured in 5 different points in films and their mean was considered for calculation of the total color difference (ΔE), yellowness index (YI), whiteness index (WI) and chroma (C*) (Yam and Papadakis, 2004):

E=

YI =

(L

* = Cab

L ) 2 + (a

a ) 2 + (b

b) 2

142.86 × b L

WI = 100

(100

(6) (7)

L)2 + (a)2 + (b) 2

(a ) 2 + (b ) 2

(8) (9)

In the equations above, L, a, and b are values related to color parameters of the films and L*, a* and b* are values related to standard color parameters obtained from a white surface (L* = 93.21, a* = −0.45, and b* = 0.77). In addition, inhibitory effects of films against light were examined by using a UV–vis spectrophotometer (Model 802106-00, PharmaciaBiotech Co., England) in the wavenumber range of 200–800 nm and their opacity was calculated through Eq. (10) (Arfat et al., 2014):

(2)

Opacity =

Afterwards, water vapor permeability (WVP) of the films was calculated through Eq. (3):

WVP =

L max × 100 L0

In Eq. (5), Lmax is the highest elongation at break and L0 is the primary film length.

2.5.4. Water vapor permeability (WVP) Films’ permeability to water vapor was measured according to ASTM E96 standard (ASTM, 1995). To this purpose, 15-mm-diameter, 45-mm-tall vials with pierced lids with the mean of 4 mm were used. First, the films were cut into a circular shape and 4 g of calcium chloride (0% relative humidity) was added to vials. The films were then placed on top of vials and their lids were closed shut. Later, vials were placed in a desiccator containing saturated sodium chloride solution (75% relative humidity) at 25 °C. Such a difference in relative humidity resulted in difference in water vapor pressure of 1753.55 kPa and the consequent driving force led to transfer of water vapor from desiccator into the vial. Vials were weighed by using a digital scale with the precision of 0.0001 g periodically for 7 days. The increasing vial weight curve was drawn during time and the slope (S) was calculated using linear regression. Water vapor transmission rate (WVTR) was obtained through dividing the slope by the surface area of the film (A).

S A

(4)

In Eq. (4), Fmax is the highest tensile strength and Amin is the minimum cross section area.

2.5.3. Moisture content Moisture content of the films was determined by measuring films’ weight loss in an oven (Model UNB 400, Memmert, Germany) at the temperature of 110 °C until reaching constant dry weight (Dashipour et al., 2015).

WVTR =

Fmax Amin

A600 X

(10)

where A600 is the absorbance in the wavenumber of 600 nm and X is the thickness of the film (mm).

(3)

2.8. Fourier transform infrared spectroscopy (FTIR)

In Eq. (3), X is the thickness of the film (mm) and ΔP is the difference in water vapor pressure (1753.55 kPa).

In order to carry out FTIR test, samples of the films were first cut into pieces of 3 × 3 cm2 and were then directly placed on the sample holder of the instrument (Kanmani & Rhim, 2014). The range of the infrared spectroscopy of the films in the wavenumber of 400–4000 cm−1 was obtained through Fourier transform infrared spectroscopy (Model Tensor 27, Brucker, Germany).

2.6. Measurement of mechanical properties Ultimate tensile strength and elongation at break of the biocomposite films were measured using a mechanical testing instrument (Model DBBP-20, Microelectronics Universal Testing Instrument, South Korea) at room temperature (25 °C) with the standard method of ASTM D882-02 (ASTM, 2002). First, samples were cut into 10 × 1 cm2 pieces and were placed in a desiccator containing calcium nitrate with 50 ± 5% relative humidity for 48 h. Then, the films were placed between the two grips of the instrument. The primary distance between the two grips and the moving speed of the upper grip were 50 mm and 5 mm/min, respectively. Parameters of ultimate tensile strength and elongation at break were calculated based on Eqs. (4) and (5) (Zolfi

2.9. Dynamic mechanical thermal analysis (DMTA) Dynamic mechanical thermal analysis was performed on 5 × 1 cm2 rectangular samples using DMTA instrument (Triton Technology, UK). Testing was done in the frequency of 1 Hz, strain of 0.02% and temperature range of −120 to 120 °C with the heating rate of 5 °C/min. The diagrams of storage modulus (E') and the loss tangent (tan δ) were obtained as functions of temperature (Motedayen et al., 2013). 445

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2.10. Scanning electron microscopy (SEM)

2.13.2. Viable cell count method In this method, the two bacterium of Staphylococcus aureus and Escherichia coli were first inoculated in 20 mL of tryptic soy broth before incubating at 37 °C for 16 h. Then, cultured bacteria were centrifuged at 2516×g for 10 min. Next, 100 mL of sterile new tryptic soy broth was added to the resulting pellet and diluted 10 times using sterile distilled water. Then, the film samples were cut into pieces of 5 × 5 cm2 and separately placed in a conical flask containing 50 mL of the prepared culture containing 106 –107 CFU/mL of Staphylococcus aureus and Escherichia coli. The samples were incubated at 37 °C under mild shaking (60 rpm) and assessed after 24 h through colony count on nutrient agar culture medium (Hasheminya et al., 2018).

A scanning electron microscope (Model MIRA3 FEG-SEM, Tescan, Czeck Republic) was used for electron microscopy tests. First, samples were cut into small pieces of 0.5 × 0.5 cm2 and were placed on the sample holder. Then, the sample holder was covered with gold in the sputter coater (Model SBC-12, KYKY, China). Following this phase, samples were imaged using SEM at the voltage of 10 kV (Kanmani & Rhim, 2014). 2.11. Measurement of DPPH radical-scavenging activity Antioxidative activity of kefiran-CMC biocomposite films was measured through Dashipour et al. (2015)’s method. In this method, 25 mg of each biocomposite sample was dissolved in 5 mL distilled water through continuous stirring. Then, 0.1 mL of the obtained biocomposite extract solution was added to 3.9 mL of DPPH solution (0.1 mM methanol solution). Afterwards, samples were kept in the dark at room temperature for 60 min and finally, the absorbance was read at wavenumber of 517 nm and DPPH radical-scavenging activity was calculated by equation (11):

DPPH scavenging activity (%) =

Ablank

Asample

Ablank

× 100

2.14. Statistical analysis To examine the effect of the concentration of Satureja Khuzestanica essential oil (0 (control), 1, 1.5 and 2%) on the measured properties, statistical analysis based on completely randomized design (CRD) was performed with three replicates for all the tests. The means were compared by Duncan’s multi range test in probability level of 0.05. SAS software (version 9.4) was used to analyze and evaluate ANOVA. 3. Results and discussion

(11)

where Ablank is the absorbance rate of the control and Asample is the absorbance rate of the sample.

3.1. Chemical compositions of the Satureja Khuzestanica essential oil Chemical compositions of Satureja Khuzestanica essential oil, Kovats retention index and the percentage of each compound are shown in Table 1. From the total of 24 identified compounds in Satureja Khuzestanica essential oil, the highest amounts belonged to carvacrol (80.55%), p-cymene (6.43%), beta-bisabolene (3.25%), citronellal (1.8%) and linalool (1.35%). In a study conducted by Saei-Dehkordi et al. (2012) to identify chemical compositions of Satureja Khuzestanica essential oil, compounds such as carvacrol, thymol, p-cymene, alphaterpinene, cis-caryophyllene, and beta-bisabolene were identified as having the highest amount. In addition, in another study, compounds such as carvacrol, p-cymene, and alpha-terpinene were introduced as major identified compounds in Satureja Khuzestanica essential oil (Farzaneh et al., 2015). Minor differences in chemical compositions of Satureja Khuzestanica essential oil are attributed to genetic variety and the climatic conditions of its growth area (Farzaneh et al., 2015).

2.12. Measurement of total phenolic content Total phenolic content was measured by using Folin-Ciocalteu reagent on the basis of gallic acid standard (Dashipour et al., 2015). First, 25 mL of each biocomposite film sample was dissolved in 5 mL distilled water. Then, 0.1 mL of the obtained biocomposite extract solution was mixed with 7 mL distilled water and 0.5 mL Folin-Ciocalteu. After keeping the compound at room temperature for 8 min, 1.5 mL of sodium carbonate solution (2% w/v) and 0.9 mL of distilled water were added to the compound. The mixture was then kept in the dark at room temperature for 2 h. Then, the absorbance at wavenumber of 765 nm was read through using a UV–visible spectrophotometer (Model 802106-00, Pharmacia-Biotech Co., England). The total phenolic content was calculated by Eq. (12):

T=

C×V M

3.2. Physical properties

(12)

where T is the total phenolic content (mg gallic acid/g dried film), C is the concentration of gallic acid obtained from standard calibration curve (mg/mL), V is the volume of biocomposite extract (mL), and M is the weight of dried film (g) (Dashipour et al., 2015).

3.2.1. Thickness Biopolymers became significantly thicker after the addition of essential oils (Table 2). This result corresponded with the findings of Table 1 Chemical composition of Satureja Khuzestanica essential oil (SEO).

2.13. Measurement of antimicrobial activity Agar diffusion and viable cell count methods were used to evaluate antimicrobial activity of films on Staphylococcus aureus and Escherichia coli bacteria. 2.13.1. Agar diffusion method In this method, both bacterial strains were first incubated at 37 °C overnight in tryptic soy broth. Then, 100 μL microbial suspension (equal to 0.5 McFarland standard) of each bacterium was inoculated to the plate containing Mueller Hinton agar. Afterwards, biocomposite films were cut in sterile condition into 10 mm-diameter circles and were taken to the abovementioned medium. Finally, plates containing biocomposite films infected with microbial suspension were incubated at 37 °C for 24 h. Caliper was used to measure the diameter of the growth inhibition zone (Dashipour et al., 2015).

Compounds

KIa

Area (%)

Compounds

KI

Area (%)

α-pinene Myrcene δ-3-Carene p-cymene Limonene

933 981 1007 1014 1019

0.5 0.98 0.39 6.43 0.28

1263 1289 1330 1348 1421

0.4 80.55 0.3 0.25 0.42

1,8 Cineole Trans-sabinene hydrate Linalool Citronellal Borneol Terpinen-4-ol L-Carvone

1023 1072

0.32 0.27

Thymol Carvacrol Eugenol Carvacryl acetate BetaCaryophyllene α-bergamotene α-Humulene

1427 1435

0.19 0.13

1085 1159 1167 1180 1222

1.35 1.8 0.4 0.38 0.29

Beta-Bisabolene α-cadinene Spathulenol Farnesyl acetate 6-Paradol

1503 1534 1577 1817 2232

3.25 0.22 0.27 0.38 0.25

a

446

Kovats indices calculated against n-alkanes on DB-5 column.

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Table 2 Physical and mechanical properties of kefiran-carboxymethyl cellulose biocomposite films as influenced by different concentrations of Satureja Khuzestanica essential oil (SEO).

*

Film

Thickness (mm)

Control 1% SEO 1.5% SEO 2% SEO

0.090 0.113 0.133 0.153

± ± ± ±

0.010d 0.005c 0.003b 0.001a

Contact angle (°) 36.77 37.30 39.66 42.84

± ± ± ±

2.38c 0.12bc 0.84b 1.04a

MC1 (%) 29.73 23.64 21.31 19.98

± ± ± ±

WVP2 (×10 2.68a 1.19b 1.12bc 1.46c

5.75 4.63 3.74 3.04

± ± ± ±

-7

g.m/m2.Pa.h)

0.81a 0.96b 0.91c 0.74d

UTS3 (MPa) 3.24 3.59 3.91 4.17

± ± ± ±

0.13c 0.06bc 0.22ab 0.47a

EB4 (%) 79.97 74.13 69.89 65.27

± ± ± ±

1.66a 3.25b 0.96c 2.39d

Different letters in the same column indicate a significant difference (p < 0.05). 1 Moisture content 2 Water vapor permeability 3 Ultimate tensile strength 4 Elongation at break

Tongnuanchan et al. (2012), Arfat et al. (2014) and Nogueira et al. (2019). Increase in film thickness is attributed to the increase in solid content (Arfat et al., 2014).

(2013) observed that decrease in permeability to water vapor in films was due to the hydrophobic nature of essential oils containing monoterpene hydrocarbons which, as a hydrophobic phase, result in formation and increase of tortuous paths in film matrix. Also, in their study about the effect of adding Carum copticum essential oil to chitosan film, Jahed, Alizadeh Khaledabad, Almasi et al. (2017) proposed that formation of hydrogen bonds between functional groups of film matrix and hydroxyl groups of the essential oil and glycerol was the reason for decrease in the hydrophilic nature of the film matrix, which resulted in decrease in WVP. Various parameters such as the ratio of hydrophobic to hydrophilic groups, number of cracks, porosity, and tortuous paths in the film structure affect films’ permeability to water vapor (Jahed, Alizadeh Khaledabad, Rezazad Bari et al., 2017). Results of this study correspond with the results from FTIR results (Section 3.5) which showed increase in films’ hydrophobicity and the formation of hydrogen bonds between Satureja Khuzestanica essential oil and kefiranCMC.

3.2.2. Contact angle Results indicated that contact angle of the films significantly increased with increase in the concentration of the essential oils (Table 2). Similar results have been reported by Ojagh et al. (2010). This phenomenon, which reduces the hydrophilic property of the films, can be attributed to the presence of hydrophobic compounds as well as to the decrease in hydroxyl groups in biopolymer (Ojagh et al., 2010). The hydrophobic nature of the essential oil added to films and the decrease in hydroxyl groups – as the result of formation of hydrogen-bonding interactions between essential oil and kefiran-CMC biopolymer (section 3.5) – increased the contact angle (Ojagh et al., 2010). 3.2.3. Moisture content Results indicated that through increase in the concentration of the essential oil, moisture content of the films significantly decreased (Table 2). These findings corresponded with the results obtained by Ojagh et al. (2010), Ghasemlou et al. (2011), Shojaee-Aliabadi et al. (2013) and Dashipour et al. (2015). Ghasemlou et al. (2011) attributed the decrease in moisture content of kefiran film, which was caused by the addition of oleic acid, to increase in hydrophobic nature of the films. Ojagh et al. (2010) also attributed the reason of decrease in moisture content of the chitosan films incorporated with cinnamon essential oil to the creation of compact structure of film as a result of covalent bonds formation between functional groups of chitosan chains. In addition, interaction between the essential oil components and hydroxyl groups of film matrix can, due to decrease in availability of hydroxyl groups, lead to decrease in hydrogen-bonding interactions between kefiran-CMC polysaccharide and water and, ultimately, result in decrease in moisture content of the films (Ojagh et al., 2010; Ghasemlou et al., 2011; Shojaee-Aliabadi et al., 2013; Dashipour et al., 2015).

3.3. Mechanical properties The effect of increase in concentration of the essential oil on ultimate tensile strength and elongation at break is shown in Table 2. Increase in the concentration of the essential oil significantly increased ultimate tensile strength and decreased elongation at break. These findings corresponded with results obtained by Prodpran et al. (2007), Ojagh et al. (2010), Nie et al. (2015) and Jahed, Alizadeh Khaledabad, Almasi et al. (2017) and contradicted with the results found by Tongnuanchan et al. (2012), Shojaee-Aliabadi et al. (2013) and Dashipour et al. (2015). Increase in ultimate tensile strength can be attributed to the formation of new hydrogen bonds between hydroxyl groups of kefiran-CMC and Satureja Khuzestanica essential oil (Section 3.5), leading to rearrangement of the polymer matrix (Ojagh et al., 2010; Atares and Chiralt, 2016). On the other hand, scanning electron microscopy (SEM) images indicated a uniform and homogeneous structural rearrangement in films incorporated with essential oil (Section 3.7), similar to the results reported by Jahed, Alizadeh Khaledabad, Almasi et al. (2017). The structural rearrangement was indicative of increase in polymer matrix continuity which reduced elongation at break (Ojagh et al., 2010). In addition, the reduction in moisture content of the films containing essential oil (Section 3.2.3.) can increase ultimate tensile strength and reduce elongation at break, a phenomenon attributed to the reduction in the plasticizing effect of water (Prodpran et al., 2007; Ojagh et al., 2010). Prodpran et al. (2007) investigated the influence of palm oil and chitosan incorporation on properties and microstructure of proteinbased films obtained from round scad (Decapterus maruadsi) muscle. Addition of palm oil increased ultimate tensile strength and decreased elongation at break. The authors contributed the increased ultimate tensile strength to arrangement and coherence of the polymer resulting in the strengthening of the film matrix with tighter and more compact structure. Additionally, reduction in the elongation at break was related

3.2.4. Water vapor permeability (WVP) Water vapor permeability of various films with different concentrations of Satureja Khuzestanica essential oil is shown in Table 2. Increase in concentration of the essential oil resulted in significant decrease in WVP. Results corresponded with findings of Ojagh et al. (2010), Shojaee-Aliabadi et al. (2013) and Jahed, Alizadeh Khaledabad, Almasi, and Hasanzadeh (2017), Jahed, Alizadeh Khaledabad, Rezazad Bari, and Almasi (2017). Addition of essential oils containing monoterpene hydrocarbons, as a hydrophobic phase, to kefiran-CMC results in discontinuities in the hydrophilic phase, leads to increase in tortuosity factor in film matrix and consequently decreases WVP (Atares, De Jesús, Talens, & Chiralt, 2010). Overall, tortuous paths play a major role in the degree of permeability to water vapor in various films (Shojaee-Aliabadi et al., 2013). In their study about addition of Satureja hortensis essential oil to carrageenan films, Shojaee-Aliabadi et al. 447

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Table 3 Color parameters and opacity of kefiran-carboxymethyl cellulose biocomposite films as influenced by different concentrations of Satureja Khuzestanica essential oil (SEO). Film

L1

Control 1% SEO 1.5% SEO 2% SEO

90.20 87.33 82.93 81.53

a2 ± ± ± ±

0.60a 0.61b 0.42c 0.95c

−2.67 −3.33 −4.13 −4.73

± ± ± ±

0.31a 0.42b 0.12c 0.12d

b3

ΔE4

WI5

1.87 ± 0.23d 7.20 ± 0.20c 9.93 ± 0.12b 12.00 ± 0.80a

3.90 ± 0.63d 9.19 ± 0.43c 14.26 ± 0.24b 16.79 ± 0.17a

89.67 85.05 79.82 77.45

± ± ± ±

0.65a 0.53b 0.30c 0.36d

YI6

C7

Opacity

2.96 ± 0.37d 11.78 ± 0.28c 17.11 ± 0.15b 21.02 ± 1.17a

3.26 ± 0.30d 7.94 ± 0.28c 10.76 ± 0.15b 12.90 ± 0.74a

0.39 0.93 1.14 3.21

± ± ± ±

0.09d 0.09c 0.03b 0.12a

*Different letters in the same column indicate a significant difference (p < 0.05). 1 Lightness 2 Redness and greenness 3 Yellowness and blueness 4 Total color change 5 Whiteness index 6 Yellowness index 7 Chroma

result was ascribed to a highly porous microstructure as confirmed by SEM micrographs of the surfaces. In general, the reason for these changes have been attributed to the plasticizing effect of essential oils, the change in the initial continuous structure of polymers to a discontinuous and heterogeneous structure and the rearrangement of polymer matrix in the presence of essential oils (Tongnuanchan et al., 2012; Shojaee-Aliabadi et al., 2013; Dashipour et al., 2015; Atares and Chiralt, 2016). Overall, mechanical properties including tensile strength and elongation at break depend on films’ microstructural network, constituents, nature of filler, matrix-filler interaction, their relative proportions, preparation conditions and existing intermolecular force (Atares and Chiralt, 2016). In general, changes in the mechanical properties may be attributed to particular interactions of polymer matrix with essential oil components affected by relative humidity, presence of surfactants and temperature (Jahed, Alizadeh Khaledabad, Almasi et al., 2017). In fact, mechanical property can improve as a result of rearranging the polymer network. Essential oils contain various chemical compositions with different properties which can, depending on their nature, create links with polymer matrix, rearrange the polymer and, consequently, improve mechanical properties (Atares and Chiralt, 2016).

to the water content of films containing palm oil. Due to the fact that water in hydrophilic films can act as a plasticizer, lower water content can reduce the flexibility of the films (Prodpran et al., 2007; Bourtoom and Chinnan, 2009). In another study, Ojagh et al. (2010) developed a biodegradable film made from chitosan and cinnamon essential oil with increased ultimate tensile strength and decreased elongation at break. The authors ascribed this result to a strong interaction between polymer and cinnamon essential oil due to a cross-linker effect, which, in turn, decreased free volume and molecular mobility of the polymer. This phenomenon led to a sheet-like structure and arrangement of stacking layers with a compact structure which increased continuities within the polymer matrix and decreased elongation at break. Furthermore, given that water acts as a plasticizer for most hydrocolloid films, reducing moisture content is associated with reduced film flexibility. In addition, Nie et al. (2015), observed that addition of grape seed procyanidins and green tea polyphenol to edible myofibrillar protein-based film increased ultimate tensile strength and reduced elongation at break. This phenomenon was related to the formation of cross-links between phenol and matrix polymer and its consequent polymer inter-chain space reduction, which was confirmed by reduced water vapor permeability of the polymer. Phenol-matrix polymer inter-chain interaction decreased plasticizing influence of grape seed procyanidins and green tea polyphenol and, therefore, increased polymer’s rigidity. In general, type of phenolic compounds and their concentrations as well as plasticizers magnitudes change mechanical properties of films. In another study, Pereda et al. (2012) developed and characterized edible chitosan/olive oil emulsion films. Ultimate tensile strength increased by adding olive oil to the films. Increase in ultimate tensile strength was attributed to the formation of crosslinks between olive oil and polymer matrix. Formation of a structure with more crosslinks in the films decreased free volume and molecular mobility of the polymer. On the contrary, in some other studies (Tongnuanchan et al., 2012; Shojaee-Aliabadi et al., 2013; Dashipour et al., 2015), addition of essential oils reduced tensile strength and increased elongation at break. Tongnuanchan et al. (2012), investigated properties of fish skin gelatin film incorporated with citrus essential oil. In their study, addition of essential oil decreased interaction between gelatin molecules, leading to decrease in rigidity and increase in extensibility/elasticity of films. Addition of essential oil in films could obstruct polymer chain-to-chain interactions and provide flexible domains. Shojaee-Aliabadi et al. (2013), also noticed reduced tensile strength and increased elongation at break by adding Satureja hortensis essential oil to κ-carrageenan films. This finding was attributed to replacing a part of the stronger polymerpolymer interactions by the weaker polymer-oil interactions in the network of films. This phenomenon can weaken the polymer structure and reduce the tensile strength of the films. Moreover, Dashipour et al. (2015), reported ultimate tensile strength reduction by addition of Zataria multiflora essential oil to carboxymethyl cellulose films. This

3.4. Color and optical properties Color and transparency of food packaging plays an important role in its appearance and acceptability by consumers. Effects of addition of Satureja Khuzestanica essential oil on color parameters such as lightness (L), redness-greenness (b), blueness-yellowness (a), total color difference, chroma, whiteness and yellowness indexes of various films as well as their opacity are shown in Table 3. Lightness (L) and rednessgreenness (a) parameters significantly decreased through increase in concentration of the essential oil, compared to the control film. However, yellowness-blueness parameter (b) significantly increased through increase in concentration of the essential oil, compared to the control film. Furthermore, through increase in concentration of essential oil in films, total color difference (ΔE), yellowness index (YI), and chroma (C) significantly increased. On the other hand, whiteness index (WI), significantly decreased through increase in concentration of the essential oil. Results of this test corresponded with findings of Shojaee-Aliabadi et al. (2013) and Atef et al. (2015). Overall, films incorporated with essential oil had a yellowish appearance and increase in the total color difference (ΔE) and yellowness-blueness (b) as well as decrease in whiteness (WI), lightness (L) and redness-greenness (a) were indicative of the yellowish appearance of the films. This phenomenon can be attributed to the phenolic compounds which exist in the essential oil (Shojaee-Aliabadi et al., 2013; Dashipour et al., 2015; Jahed, Alizadeh Khaledabad, Rezazad Bari et al., 2017). Overall, the color of edible films is directly dependent on the kind and concentration of the 448

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Table 4 Visible-ultraviolet light transmission percentages for kefiran-carboxymethyl cellulose biocomposite films as influenced by different concentrations of Satureja Khuzestanica essential oil (SEO). Film

Control 1% SEO 1.5% SEO 2% SEO *

Light Transmission (%) at 200–800 nm 200

280

350

37.98 ± 4.34a 22.74 ± 2.04b 13.46 ± 3.18c 7.21 ± 1.20d

44.46 ± 0.06a 29.04 ± 1.10b 16.38 ± 2.27c 9.24 ± 1.75d

73.11 56.28 51.33 23.62

400 ± ± ± ±

0.48a 2.22b 2.18c 0.66d

86.90 63.97 60.68 27.44

500 ± ± ± ±

3.61a 4.17b 2.69c 2.36d

91.75 68.06 65.00 29.53

600 ± ± ± ±

2.39a 0.47b 0.89c 1.21d

92.27 78.44 70.42 32.22

700 ± ± ± ±

0.76a 1.83b 0.56c 1.43d

93.33 79.52 73.70 34.28

800 ± ± ± ±

2.33a 1.29b 1.95c 3.04d

95.10 84.09 77.94 36.78

± ± ± ±

1.03a 0.04c 2.09c 3.01d

Different letters in the same column indicate a significant difference (p < 0.05).

Moreover, the peak at the 900–700 cm−1 was linked to bending vibration of CeH (Piermaria et al., 2011). Overall, FTIR analysis showed that peaks observed in the films incorporated with essential oil were similar to control films. Addition of essential oil to polymer matrix shifted the peak position to lower wavenumbers. For instance, peaks of 3406, 2931, 2852, 1620, 1423, 1101, and 883 cm−1 in control film shifted to 3394, 2927, 2848, 1612, 1412, 1099, 877 cm−1 in the film containing 2% essential oil. In other words, peaks’ shifting to lower wavenumbers in the films incorporated with essential oil indicated the formation of new physical (hydrogen) bonds between kefiran-CMC matrix and essential oil (Hasheminya et al., 2018). Furthermore, wavenumbers of 2927 cm−1 and 2848 cm−1 in the film containing 2% essential oil pointed to the presence of lipids, which was intensified through addition of essential oil to film matrix (Arfat et al., 2014). In other words, increase in peak intensity is indicative of increase in the amount of essential oil which contains hydrocarbons. Similar results were reported by Arfat et al. (2014) in their study of fish protein isolate/fish skin gelatin film containing basil leaf essential oil. Increase in peak intensity related to the essential oil in this study can be attributed to increase in hydrophobicity in films incorporated with essential oils. This phenomenon corresponded with findings from the contact angle test (Section 3.2.2) (Tongnuanchan et al., 2012).

essential oil which is added to the film (Atares and Chiralt, 2016). In addition, results indicated that increase in concentration of the essential oil increased opacity significantly (Table 3). Similar findings have been reported by Shojaee-Aliabadi et al. (2013). Also, the percentage of light transmission in various concentrations of essential oil facing ultraviolet-visible light in the range of 200–800 nm is shown in Table 4. Increase in concentration of the essential oil significantly decreased the percentage of light transmission in films in both ultraviolet range (200–800 nm) and the visible range (400–800 nm) (Hasheminya et al., 2018). These findings corresponded with Atef et al. (2015)’s research on examining optical properties of agar-cellulose biocomposite films incorporated with savory essential oil. Significant increase in opacity and decrease in the percentage of light transmission in films incorporated with essential oil can be attributed to light scattering resulting from distribution of lipid droplets with refractive index which is different from the continuous phase in the film matrix and also, to primary properties of emulsion such as volume fraction and concentration of lipid phase (Shojaee-Aliabadi et al., 2013). Overall, improving light barrier properties can prove useful through retarding lipid oxidation, preventing nutrient destructions, discoloration and off-flavors in food systems (Atef et al., 2015). 3.5. Fourier transform infrared spectroscopy (FTIR)

3.6. Dynamic mechanical thermal analysis (DMTA)

The results of FTIR as influenced by the concentration of the essential oil in all films are presented in Fig. 1. Peaks at the wavenumber range of 3700–3100 cm−1 demonstrated stretching modes of OeH in carbohydrates and water. In addition, peaks at the 3000–2800 cm−1 were related to CeH stretching vibrations (Piermaria et al., 2011). Peaks at the 1620 and 1423 cm−1 were, respectively, indicative of strength vibrations of carboxyl groups (COO–) and bending vibration (scissoring vibration) of CH2 (Hasheminya et al., 2018). In addition to the abovementioned, peaks at the 1135–1070 cm−1 were associated to stretching modes of carbohydrate rings and CeOeC, CeOH, and CeH side groups and confirmed the presence of kefiran polysaccharide.

Fig. 2a shows the effect of increase in essential oil concentration on the loss tangent of films in the thermal range of −120 to 120 °C. In the diagram of loss tangent, two peaks that showed two relaxation phases were observed. The first relaxation phase (left peak) was related to the kefiran-CMC-poor phase and glycerol-rich phase and the second relaxation phase (right peak) was related to glass transition temperature of the kefiran-CMC-rich phase (Zinoviadou et al., 2009; Lomelí-Ramírez et al., 2014). As shown in Fig. 2a, both relaxation phases increased through increase in concentration of the essential oil. First relaxation phase changed from −44.55 °C in the control film to, respectively, –33.63, –22.46 and 0.63 °C in films containing 1, 1.5 and 2% essential oil. Second relaxation phase in the control film, compared to films containing 1, 1.5 and 2% essential oil, increased from 0.3 °C to 18.67, 23.95, and 35.61 °C, respectively. Increase in glass transition temperature is related to restriction or reduction of mobility of polymer chains, which results from molecular interactions between essential oil and polymer matrix (Belhassen et al., 2009; Yu et al., 2009; Liang et al., 2011). Our findings did not correspond with results of Zinoviadou et al. (2009). In the aforementioned study, glass transition temperature in films containing oregano oil in whey protein isolate films decreased, compared to control film; this result was attributed to decrease in ultimate tensile strength and increase in elongation at break. Furthermore, Fig. 2b shows the effect of increasing concentration of the essential oil on storage modulus of the films in the temperature range of −120 to 120 °C. Increase in temperature decreased storage modulus of all biocomposite films. Also, increase in concentration of the essential oil increased storage modulus of the films in a way that films containing 2% essential oil had the highest storage modulus. This result

Fig. 1. Fourier transform infrared spectroscopy (FTIR) of kefiran-carboxymethyl cellulose biocomposite films as influenced by different concentrations of Satureja Khuzestanica essential oil (SEO). 449

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Fig. 3. Scanning electron microscopy (SEM) images of kefiran-carboxymethyl cellulose biocomposite films as influenced by different concentrations of Satureja Khuzestanica essential oil (SEO) (a: Control; b: 1%; c: 1.5% and d: 2%).

the film forming solution had a stable emulsion system and that its stability was maintained during drying of the solution (Tongnuanchan et al., 2012). In addition, it is possible that through its placement between kefiran-CMC chains and creation of inter-molecular interactions, the essential oil has caused the formation of a more homogeneous structure in the film incorporated with essential oil (Jahed, Alizadeh Khaledabad, Almasi et al., 2017). In a similar research on the effects of addition of Zataria multiflora essential oil nanoemulsion to basil seed gum, Gahruie et al. (2017) pointed out the good compatibility between the essential oil and polymer matrix and mentioned that this phenomenon was the reason for improvements in mechanical properties. Similarly, in the present study, formation of a compact structure and uniform distribution of the essential oil in polymer matrix can be attributed to improvements in films’ permeability to water vapor as well as improvements in their mechanical properties (Ojagh et al., 2010; Tongnuanchan et al., 2012; Gahruie et al., 2017).

Fig. 2. Damping modulus (tan δ) (a) and storage modulus (b) of kefiran-carboxymethyl cellulose biocomposite films as influenced by different concentrations of Satureja Khuzestanica essential oil (SEO).

3.8. Antioxidant activity and total phenolic content Effect of increase in essential oil concentration on antioxidant activity and phenolic compounds content of various films is shown in Fig. 4(a and b). Control film showed no antioxidant activity and through increase in essential oil concentration, antioxidant activity increased significantly (Fig. 4a). This result corresponded with findings of Tongnuanchan et al. (2012), Shojaee-Aliabadi et al. (2013) and Dashipour et al. (2015). Furthermore, results indicated that there were zero phenolic compounds in the control film and through increase in essential oil concentration in films, phenolic compounds significantly increased (Fig. 4b). These results corresponded with the results of antioxidant activity of films and also, with the results obtained by Shojaee-Aliabadi et al. (2013) and Dashipour et al. (2015). Essential oils are rich resources of phenolic compounds and antioxidant power of films depends on the concentration of the added essential oil (Dashipour et al., 2015). Phenolic compounds such as carvacrol in Satureja Khuzestanica essential oil, which were observed using GC–MS, quench free radicals (Burt, 2004). Antioxidative mechanisms of natural antioxidants may be related to their hydrogen-donating ability and their efficiency as hydrogen peroxide, superoxide and free radical scavengers (Gülçin, 2005).

corresponded with the results of Belhassen et al. (2009) and LomelíRamírez et al. (2014). Increase in storage modulus demonstrated the good interaction between essential oil and polymer matrix, which ultimately increased glass transition temperature (Fig. 2a) (LomelíRamírez et al., 2014). This effect can also be attributed to decrease in mobility of polymer molecules as the result of the presence of fillers such as essential oils (Lomelí-Ramírez et al., 2014). This result corresponded with results obtained from evaluation of mechanical properties, indicating the increase in ultimate tensile strength and decrease in elongation at break in films incorporated with essential oil (Yu et al., 2009). 3.7. Scanning electron microscopy (SEM) Scanning electron microscopy (SEM) images from the surface of various films in different concentrations of essential oil are shown in Fig. 3. Surface images of kefiran-CMC film and also, all the films incorporated with essential oil displayed a homogeneous structure without porosity. Similar results have been reported by Ojagh et al. (2010), Tongnuanchan et al. (2012), Jahed, Alizadeh Khaledabad, Almasi et al. (2017), Jahed, Alizadeh Khaledabad, Rezazad Bari et al. (2017) and Gahruie et al. (2017). Formation of a homogeneous structure without pores in films incorporated with essential oil indicated that

3.9. Antimicrobial activity Results obtained through evaluation of antimicrobial activity of 450

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Fig. 4. DPPH-scavenging activity (a), total phenolic content (b) and antimicrobial activity of kefiran-carboxymethyl cellulose biocomposite films against Staphylococcus aureus and Escherichia coli bacteria by agar diffusion method (c), viable cell count method (d) and sample photos of agar diffusion method for Escherichia coli bacteria (e) as influenced by different concentrations of Satureja Khuzestanica essential oil (SEO).

various films in different concentrations of the essential oil through two methods of agar diffusion and viable cell count against Staphylococcus aureus (gram-positive) and Escherichia coli (gram-negative) bacteria are shown in Fig. 4 (c and d). In addition, sample photos of agar diffusion method for Escherichia coli bacterium are also demonstrated in Fig. 4e. Findings indicated that the control film had no antimicrobial property against Staphylococcus aureus and Escherichia coli bacteria (Fig. 4c and 4d). However, increase in essential oil concentration significantly increased antimicrobial characteristic against both bacteria in both methods of agar diffusion (Fig. 4c) and viable cell count (Fig. 4d). Overall, antimicrobial activity of films against Staphylococcus aureus, compared to Escherichia coli, was greater in both methods and this effect became greater through increase in the concentration of essential oil (Fig. 4c and d). Similar results have been reported by Tongnuanchan et al. (2012), Shojaee-Aliabadi et al. (2013) and Dashipour et al. (2015). Of important properties of essential oils and their constituent ingredients is their hydrophobic property which enables them to penetrate into bacterial cell membrane lipids and mitochondria and disturb cell structure. This phenomenon results in loss and leakage of ions and other cell contents. Although leakage up to a certain amount may be tolerated, loss of considerable cell contents or loss of ions and critical molecules will result in cells’ death (Burt, 2004). Overall, the more the phenolic compounds such as carvacrol, eugenol, and thymol in the essential oil, the stronger their antimicrobial activities against food pathogens. Probably, the mechanism of the effect of these compounds, similar to other phenolic compounds, includes disturbing cytoplasmic membrane, disrupting proton motive force (PMF) and electron flow,

active transport and coagulation of cell contents (Burt, 2004). Most of the studies conducted on the effect of essential oils on food pathogens demonstrate that the effect of essential oils on gram-positive bacteria is a little more than their effect on gram-negative bacteria. In other words, gram-positive bacteria are more sensitive to antibacterial effect of essential oils (Burt, 2004, Shojaee-Aliabadi et al., 2013). The reason for lower sensitivity of gram-negative bacteria may be due to the existence of the outer membrane surrounding the cell wall which restricts the diffusion of hydrophobic compounds of the essential oil to lipopolysaccharide layer of bacterial cell membrane. Antibacterial property in this study belongs to carvacrol compound which constitutes most of the essential oil composition (Table 1) (Burt, 2004, ShojaeeAliabadi et al., 2013). In addition to affecting gram-positive bacteria, carvacrol causes cell death through destroying the outer membrane of gram-negative bacteria, freeing lipopolysaccharides and, consequently, increasing permeability of the cytoplasmic membrane to ATP (Burt, 2004). It is obvious that antibacterial property of films depends on various factors such as film type, polarity, concentration of antibacterial agents, and initial bacterial number. 4. Conclusions In this research, kefiran-CMC biocomposite films containing Satureja Khuzestanica essential oil were produced and the effect of the essential oil in different concentrations of 0 (control), 1, 1.5 and 2% on various properties of films was investigated. Results indicated that increase in the concentration of the essential oil improved physical and mechanical 451

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properties. In addition, adding essential oil to films improved color parameters and the percentage of light transmission in visible and ultraviolet ranges. Furthermore, increase in the concentration of the essential oil in films increased storage modulus and glass transition temperature. New hydrogen bonds formed between polymer matrix and the essential oil were observed through Fourier transform infrared spectroscopy (FTIR). Scanning electron microscopy (SEM) also showed that surface structure of films incorporated with essential oil were homogeneous without porosity. Also, improvement in antioxidant properties of films incorporated with essential oil was observed through evaluation of DPPH radical-scavenging activity. In addition, films incorporated with essential oils exhibited antimicrobial characteristic – due to the existence of polyphenolic compounds – against Staphylococcus aureus and Escherichia coli bacteria. Overall, findings of this study demonstrated that due to improvements in their various qualitative properties, such as higher opacity (to retard lipid oxidation, prevent nutrient destructions, discoloration and off-flavors) and antimicrobial properties, the produced novel biocomposite films might have a potential to be used in food packaging.

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