Accepted Manuscript

Title: Characterization of citrus pectin films integrated with

clove bud essential oil: Physical, thermal, barrier, antioxidant
and antibacterial properties

Authors: Tanzeela Nisar, Zi-Chao Wang, Xi Yang, You Tian,

Muneeb Iqbal, Yurong Guo

PII: S0141-8130(17)32217-1
DOI: http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.08.068
Reference: BIOMAC 8060

To appear in: International Journal of Biological Macromolecules

Received date: 19-6-2017

Revised date: 17-7-2017
Accepted date: 10-8-2017

Please cite this article as: Tanzeela Nisar, Zi-Chao Wang, Xi Yang,
You Tian, Muneeb Iqbal, Yurong Guo, Characterization of citrus pectin
films integrated with clove bud essential oil: Physical, thermal, barrier,
antioxidant and antibacterial properties, International Journal of Biological
Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.08.068

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Characterization of citrus pectin films integrated with clove bud essential oil:

Physical, thermal, barrier, antioxidant and antibacterial properties

Tanzeela Nisar a, Zi-Chao Wang a, Xi Yang a, You Tiana, Muneeb Iqbal b, Yurong Guo a,*
a
College of Food Engineering and Nutritional Science, Shaanxi Normal University,

Xi’an 710062, P.R. China

b
School of Basic Medical Sciences, Xi'an Jiaotong University Xi'an 710061, China

*Corresponding author:

Dr. Yurong Guo, College of Food Engineering and Nutritional Science, Shaanxi Normal

University

E-mail: yrguo730@snnu.edu.cn,

Telephone: +86 029 85310471

Highlights:

 The effects of clove bud essential oil on properties of the citrus pectin films were
investigated
 CEO improved the mechanical and physicochemical properties
 Pectin/CEO films were more resistant, more elongable and less hydrophilic
 Antioxidant and antimicrobial properties of pectin film were enhanced by CEO
 The interactions between CEO and pectin were confirmed by FTIR analysis

Abstract:

The increasing demand for bio-based materials to be used in food packaging has stimulated

the development of novel, environmentally-friendly edible films. Antimicrobial films were

developed by incorporating different levels of clove bud essential oil (0.5%, 1.0%, and

1.5%) into the citrus pectin in order to modify the functional properties of the films.

Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry analysis

(DSC) and X-ray diffraction (XRD) were performed, together with the determination of
physical, optical, mechanical, antioxidant and antimicrobial properties of pectin emulsified

films. The inclusion of oil significantly enhanced the water barrier properties of the films.

Addition of oil leads to more opaque films with relatively heterogeneous microstructure,

resulting in an increase in film opacity. The composite films were more resistant to

breakage and more flexible than the control films. Differential scanning calorimetry (DSC)

demonstrated that films incorporating CEO exhibited improved heat stability with slightly

higher degradation temperature, compared with control films. The inhibitory effect of

pectin films with CEO was also evaluated on three common foodborne bacteria. These

results revealed that clove oil has a good potential to be incorporated into citrus pectin to

make antimicrobial edible films or coatings for various food applications.

Abbreviations used

ATR-FTIR, Attenuated total reflectance-Fourier transform infrared spectroscopy; CEO,

Clove essential oil; DSC, Differential scanning calorimetry; EB, elongation at break; EM,

elastic modulus; EOs, essential oils, SEM, Scanning electron microscopy; SI , Swelling

index; TS, tensile strength; WVP, water vapor permeability; XRD, X-ray diffraction

Keywords: Active packaging; Citrus pectin film; Clove essential oil; Film

characterization; Antioxidant; antimicrobial activityIntroduction:

In recent years, edible films have received greater attention because of their advantages

over synthetic packaging materials. Active food packaging is one of the innovative concept

that have been emerged as a response to the continuous variations in current consumer
demands for natural, minimally processed and preservative-free food products. It can carry

various antimicrobial agents in packaging systems, which can be more effective than

applying antimicrobial substances directly to the food due to providing continuous

migration of active substances into the food and therefore remain at high concentration for

prolonged period of time [1]. Antibacterial substances incorporated into edible films may

control contamination by reducing the microbial growth rate, extending the lag-phase of

the target microorganism, or inactivating microorganisms by direct contact [2].

Pectin is one of the versatile biomaterial considered as an effective biopolymer for the

production of edible films due to its biocompatibility, biodegradability and non-toxicity.

Several studies have proposed the feasibility of pectin as a biopolymer to formulate edible

films due to its ability to form gels [3]. Pectin films and coatings reveal crystalline or

amorphous areas, which can be appropriate for integration of additives and also for

immobilization of water molecules in the film structure, to facilitate retention of

hydrophilic compounds [4] . Pectin has the potential to carry functional substances, as it is

suitable for integration of phytochemical compounds for developing nutritionally fortified

edible films.

EOs are categorized as GRAS (generally recognized as safe) by U.S. Food and Drug

Administration and can be potential alternatives to synthetic additives [5]. Antimicrobial

packaging carrying essential oils has high potential for food applications. The antibacterial

activity of various essential oils over a wide range of microorganisms has been studied

extensively [6]. The antimicrobial potential of EOs is allocated to a number of and phenolic

compounds (thymol, carvacrol, eugenol) and small terpenoids. They have the ability to
interrupt and penetrate into bacterial cell wall, leading to destruction of cell membrane,

denaturation of cellular proteins, cytoplasmic leakage and eventually cell death [7].

Clove bud essential oil appears as interesting natural compound with great potential use in

food preservation. The high concentrations of eugenol in clove oil is mainly responsible

for its biological and antimicrobial activity as it can denature cellular proteins and reacts

chemically with cell membrane phospholipids therefore changing their permeability. Clove

bud oil also has numerous therapeutic effects, including analgesic, antiphlogistic, anti-

vomiting, anticarminative, antispasmodic, kidney reinforcement and antiseptic [8].

Previous studies demonstrated that potential activity of pectin, could be improved through

integration of natural additives, like pomegranate juice added into pectin matrix [9],

cinnamaldehyde essential oil and papaya puree [10], Mexican lime essential oil [3] and

cranberry extract [11]. However, to the best of our knowledge, no studies have been found

on incorporation of clove oil into pectin polymeric matrix. Therefore, the aim of present

study was to develop active composite pectin based films, and to verify the influence of

clove essential oil addition on microstructure, mechanical and barrier properties of

produced films. Characterization involved the understanding of potential changes in

transport, structural properties and thermal properties of films when CEO was

incorporated.

2. Materials and methods

2.1 Materials

Pectin from citrus fruits (galacturonic acid ≥74%, methoxy groups ≥ 6.7%, MW = 30-100

kg mol-1) was purchased from Sigma Aldrich (St. Louis, MO, USA). Clove bud essential
oil, Anhydrous glycerol, calcium chloride, magnesium nitrate hexahydrate and

Polyoxyethylene (20) sorbitan monooleate (Tween 80) were purchased from Jingbo

biological technology Co., Ltd. (Xi’an, China). Bacterial strains, Escherichia coli were

obtained from China General Microbiology Culture Collection Center (Beijing, China).

Staphylococcus aureus and Listeria monocytogenes were collected from the College of

Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an, China.

2.2 Preparation of inoculum:

E. coli, S. aureus and L. monocytogenes were cultured in 10 ml of Nutrient broth, Luria-

Bertani broth and Trypticase Soy Broth (TSB) supplemented with yeast extract (TSBYE)

respectively, and kept in oscillating incubator at 37 oC for 24 h. 1 ml of each bacterial

suspension was re-cultured in fresh 10 ml of respective broth media and again incubated at

37◦ C for 24 h. The optical density of the bacterial suspensions was adjusted to 108-1010

CFU/mL using McFarland standard.

2.3 Film Preparation

Pectin films were prepared by some modifications in the method used by Galus, Uchański

and Lenart [12]. Film forming solution (3% w/v) was prepared by rehydrating pectin in

sterile deionized water for 12 h at 20 ºC. After complete dissolution of pectin, glycerol as

a plasticizer at level of 30% (w/w of pectin) and calcium chloride as cross linking agent at

a level of 1% (w/w of pectin) were thoroughly mixed with magnetic stirring at 70 ºC. CEO

was mixed with Tween 80 at 0.2% (v/v) of essential oil to assist stable and uniform

essential oil dispersion in the film solution; it was then incorporated into film solution at

concentrations of 0% (as control), 0.5%, 1% and 1.5% (v/v) of solution. Then film mixture

was homogenized at 13,500 rpm by using a Rotor-stator Homogenizer (Ultra Turrax IKA,
T25, Werke, Germany) for 3 min in order to obtain a smooth emulsion. Finally, air bubbles

in the solutions were removed by ultra-sonification under vacuum for 20 min. The solutions

were cast onto sterile plastic petri dishes (diameter 15 cm) and dried at ambient temperature

in hot air oven for 24 hours. The quantity of film solution poured onto each plate was 70

ml. Finally the obtained pectin films were peeled-off and conditioned at 25 oC and 53 ±

1% RH and for 3 days prior to testing.

2.4 Determination of Physical properties of films

2.4.1 Film thicknesses

The thickness films samples was measured by using a digital micro caliper (Mitutoyo

Absolute, Tester Sangyo Co. Ltd., Tokyo, Japan) with an accuracy of 0.001 mm at ten

random locations on each film sample.

2.4.2 Moisture Content and Swelling Ratio

To determine the water content (Eq. 1) and swelling ratio (Eq. 2) of pectin films, film

samples were cut into 2 cm×2 cm squares and weighed (W1). The water content was

determined by drying the film samples in hot air oven at 105 oC for 24 h and weighing

them after cooling to room temperature (W2). The degree of swelling of the film specimens

was determined by immersing them into distilled water for 24 h. Extra moisture was

removed by placing the film pieces between two layers of filter paper, followed by

weighing the residual film squares (W3). The degree of swelling (DS) was calculated

according to Eq (2).

𝑊1 − 𝑊2
Moisture content % = × 100 1
𝑊1
 𝑊3 − 𝑊1
Swelling index (SI) = 2
𝑊1

2.4.3 Solubility assays:

The solubility of film specimens was measured in triplicate according to the method of

Hosseini, Razavi and Mousavi by immersion assays under constant agitation [13]. First

film samples (2 cm×2 cm) were dried at 105oC in a hot oven for 24 h and weighed (W1).

Then the film pieces were put in 50 mL of distilled water with constant agitation for 6 h at

25ºC. Filter paper (Whatman No.1) was used to separate residual film pieces from the

water. The remaining film mass was dried at 105 oC for 24 h until constant weight (W2).

Degree of solubility was calculated as the difference of initial and final weight of film

squares with respect to the initial weight of film samples. Film solubility was calculated

using the following equation.

𝑊1 − 𝑊2
Film solubility % = × 100 3
𝑊1

2.5 Water Vapor Permeability (WVP)

The WVP of the pectin films was measured gravimetrically according to the method of

[1]with some modifications. Special aluminum circular cups (internal diameter: 6 cm,

exposed area: 28.27 cm2, and depth: 1.3 cm) were used. The cups were filled with fully

dried anhydrous CaCl2 (0% RH) and sealed tightly with film specimens, then placed at 25
o
C in a desiccator containing distilled water (100% RH). The cups were weighed

periodically for 24 h with a precision of 0.001 mg. The WVPR and WVP were measured

according to following equations.

 𝑚
WVPR = 4
𝑡×A

𝑊𝑉𝑃𝑅 × d
WVP = 5
△𝑝

Where WVP is the water vapor permeability (g﹒m-1s-1 Pa-1), m is the mass of water

permeated through the film (g), t is the time of permeation (s), A is effective film area (m2),

d is the thickness of the film samples (m) and △p is the saturation vapor pressure of water

at the assay temperature (pa).

2.6 Film Color

The color of the film specimens was determined with color difference meter (Shanghai

Precision & Scientific instrument Co., Ltd., Shanghai, China) using the CIE Lab scale

chromaticity parameters: L* from black (0) to white (100), a* specified red (+) to green (-

), and b* specified yellow (+) to blue (-). A white color tile was used as a background

during color calibration. Before evaluation of color, film samples were conditioned at 53%

RH at 25ºC for 48 hours in a desiccator containing Mg (NO3)2 saturated solutions. Total

color difference (△E) was calculated by using the following equation:

△ 𝐸 = [(△ 𝐿)2 + (△ 𝑎)2 + (△ 𝑏)2 ]0.5 6

Where △L, △a and △b are the differentials between color parameters of sample and

standard used as the film background.

2.7 Light transmission and transparency of films

The light barrier properties of pectin films against visible light and ultraviolet (UV) were

determined according to the method proposed by Peng and Li [1] using UV–visible
spectrophotometer (UV 2100, Unico Instruments Co., Ltd.,Shanghai, China) at

wavelengths between 400 and 800 nm. Rectangular film strips were placed in

spectrophotometer test cell and absorbance was checked at the wavelength of 600nm. For

each treatment, four replicates were measured. The opacity of the films was calculated by

means of the following equation:

𝐴𝑏𝑠600
𝑂= 7
𝐿

whereas O is the opacity of films, Abs is the value of absorbance at 600 nm and L

is the film specimen thickness (mm).

2.8 Mechanical properties of films

Tensile strength (TS), percentage of elongation at break (EB) and elastic modulus (EM) of

the test films were determined by using Universal Testing Machine (Model 5655, Instron

Corporation) according to the method proposed by Giosafatto et al. [14]. The film

specimens of 10cm×1cm were stretched at a cross head speed of 1 mm/s with initial grip

separation of 50mm. Tensile strength (TS) was measured by dividing the maximum force

required by the cross-sectional area of film sample and percentage of elongation at break

(%E) was determined by dividing the film’s length at the time of breakage by its original

length of film strip and multiplying by 100.

2.9 Attenuated total reflectance-Fourier-transform infrared (ATR-FTIR)

spectroscopy

The infrared (IR) spectra of the pectin films were determined with Fourier-transform

infrared spectrometer (FTIR) (EQUINX55, Brucher, Germany), using Attenuated Total

Reflectance (ATR) mode to study the interactions of pectin and oil in the film. Samples

were put on a Golden Gate single reflection diamond ATR accessory and the empty crystal

was used for background spectrum. All FTIR spectra were carried out at resolution of 4

cm−1 with 16 scans in the spectral range of 4000 to 400 cm−1.

2.10 Differential scanning calorimetry Analysis (DSC)

Differential scanning calorimetry (DSC) analysis was done in order to determine the

thermal properties of pectin films by using Q1000 DSC system (TA instruments, New

Castle, USA). Films samples were initially conditioned at 50% RH for 24 h. 10mg of film

pieces were subjected to increasing temperature from -50 C to 350 oC in a standard

aluminum pan at a constant heat rate of 10°C/min and the chamber was purged with

nitrogen gas at a flow rate of 30 cm3/min. Thermal parameters such as enthalpy (ΔH), glass

transition temperature (Tg), melting point (Tm) of the film sample were determined

corresponding to endothermic peak areas on DSC thermograms. Analyses of the DSC

thermograms were done by using TA Universal Analysis Software.

2.11 X-ray diffraction (XRD)

X-ray diffraction (XRD) patterns were investigated by X-ray Diffractometer (Rigaku

Corporation, Tokyo, Japan) equipped with a copper tube, operating at 40 kV and 40 mA.

Film specimens were immobilized on the glass surface at the scanning speed of 5°/min.

Scattered radiation in samples were analyzed over a diffraction angular range of 2θ=0-60°

with step size of 2θ = 0.02◦. Diffractograms were fitted by ORIGIN PRO 8 software.

2.12. Scanning electron microscopy (SEM)

The surface and cross sectional morphology of pectin film samples with and without clove

oil incorporation were examined by using anTM3030 scanning electron microscope

(Hitachi Co. Ltd., Kyoto, Japan), working in high vacuum mode, with 30 KV accelerating

voltage, 60 Pa pressure and 10 mm working distance. Dried fragments of film were fixed

on specimen stub with carbon tape and then coated with a thin layer of gold by DC sputter

coater (AGAR B7340, Agar Scientific, Stansted, UK). The films were imaged at a voltage

of 15 kV. Digital micrographs of film surface and cross section were collected at

magnifications of 5000× and 3000 × respectively.

2.13 Total Phenolic Contents

Total phenolic content (TPC) of the pectin film samples were estimated according to the

method described by Siripatrawan and Harte [15]. For this purpose, 25 mg of film specimen

was dissolved into the 5 mL of water, then 0.1 mL of film extract solution was mixed with

7 mL water and 0.5 mL F-C reagent and kept at room temperature for 8-10 min before the

addition of 1.5 ml of sodium carbonate solution (2% w/v) and 0.9 ml of distilled water.

Then the solution was mixed thoroughly and kept in a dark chamber for 2 h at room

temperature. The absorbance of the solution was measured at 765 nm using a

spectrophotometer (UV 2100, Unico Instruments Co., Ltd.,Shanghai, China). The

outcomes were calculated as mg gallic acid equivalents (GAE)/g of dried sample using

following equation:

𝐶×𝑉
𝑇= 8
𝑀
Where T is total phenolic content (milligram/g dried film) in GAE, C is the concentration

of gallic acid attained from the standard curve (mg/ml), V is the volume of film extract

solution (mL) and M is the weight of dried film sample (g).

2.14 DPPH radical scavenging assay

The free radical scavenging activity of pectin based films was evaluated by using stable

radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) according to method used by Martins,

Cerqueira and Vicente with slight modifications [16]. The UV absorbance of the mixture

was measured at 517nm by using Perkin-Elmer spectrophotometer and DPPH free radical

scavenging activity was calculated using the following equation:

𝐴𝑠𝑎𝑚𝑝𝑙𝑒
DPPH radical scavenging effect (%) = (1 − ) × 100 9
𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙

Whereas, Asample is the absorbance value of sample solution and Acontrol is the absorbance

of DPPH solution at 517 nm. All experiments were done in triplicates.

2.15 Evaluation of Antimicrobial activity of films

The antibacterial effect of the pectin films with clove essential oil was determined by using

the disk diffusion method, according to the method of Jouki, Yazdi and Mortazavi [17].

The films were cut into 10 mm diameter discs with a hole-puncher and placed on specific

agar plates, which had been previously spread with 0.1 mL of broth cultures comprising

approximately 108-1010 CFU/mL of respective bacterial strain. The agar plates were

incubated at 37 ± 1 0C for 24 h. The diameter of inhibition zones around the film discs was

measured by using a digital calliper (VWR, USA). The bacterial growth under the discs
(area of contact with the agar surface) was examined visually. The experiments were made

in triplicate for each film.

2.16 Statistical analysis

The experimental data were analyzed by one-way analysis of variance (ANOVA) using

SPSS 13.0 (SPSS Inc., USA) and expressed as means ± standard deviation. The significant

differences between mean values were calculated by Duncan’s multiple test at a

significance level of p < 0.05.

3. Results and Discussion:

3.1 Physical Properties of film:

Preliminary experiments revealed that the maximum concentration of lipids that could be

added to pectin appeared to be 1.5% (v/v) of the film forming solution. Films containing

higher amounts showed an irregular lipid distribution, which might be arose from poor

stability of the emulsion at higher lipid concentrations or from the limited dispersion

capacity of the lipids. Table 1 shows the impact of incorporating CEO on the physical

properties of pectin films. The addition of CEO into the pectin film-forming solution

led to an increase in thickness of the film specimens, which ranged between 0.057

and 0.094 mm. Norajit, Kim, and Ryu [18] also reported that the incorporation of ginseng

extract in alginate based films increased the thickness of films significantly. Film thickness

is influenced by the amount of solid contents in film forming emulsion when compared

with control films.

3.2 Moisture content and film solubility:

Water resistance or insolubility is usually essential for potentially commercial edible films

[19]. Cross-linking pectin with CEO resulted in decreased affinity of the polysaccharide

polymer toward water molecules and produced films with low moisture content and low

solubility in water, which is valuable for integrity and water resistance of food products.

The Incorporation of CEO into pectin decreased moisture content (%) of the films from

18.43% to 10.14% and film solubility from 25.37 % to 17.44 % by increasing concentration

of oil from 0.5% to 1.5% (Table 1) This probably happened due to the hydrophobic nature

of lipid and interaction among the components of CEO and hydroxyl groups of pectin.

Similar outcomes were obtained by Ghasemlou, et al. for kefiran films incorporating oleic

acid and for k-carrageenan based films containing Satureja hortensis [20]. According to

them, these outcomes were attributed to the decrease in hydrophilic properties of the edible

films and also due to interaction of oil components with hydroxyl groups of film matrix,

which could limit the interaction of hydroxyl groups with water molecules due to their less

availability, consequently leading to formation of more water-resistant film. In our study

lowest moisture content with least solubility (10.14% and 17.44% respectively) was

attained for edible films formulated with 1.5% clove essential oil. These observations

are also in agreement with the data reported by [21], they observed that a-tocopherol

addition in chitosan films significantly decreased the water content of films.

3.3 Swelling Index (SI):

The resistance of edible films to water is important for the potential application of biofilms.

The swelling capacity of edible films is significant when coated materials are in contact

with water. The values of the swelling index of pectin films containing CEO were lower

than those for the control film (Table 1).This trend is again credited to the hydrophobic
nature of lipid, which causes intermolecular interaction between pectin matrix and oil,

resulting in low swelling ratio. A similar trend was noticed for other biodegradable films

altered with hydrophobic substances [22].

3.4 Water vapor permeability (WVP)

Water vapor permeability is essential parameter to estimate the permeation of water vapors

through film matrix at a certain temperature. WVP of pectin based films incorporated with

different concentration of CEO are shown in Table. WVP of CEO incorporated films were
−11
significantly (P < 0.05) decreased from 13.22 × 10 to 6.52 × 10−11 by increasing the

amount of oil up to 1.5% in the film matrix. The Polymeric chains become less mobile due

to addition of oil in the film matrix leading to reduce diffusibility of water vapors via the

interface with the polysaccharide chains which lead to decrease in WVP [23]. Therefore,

the lower values of WVP obtained with CEO addition could be due to the development of

an interconnecting lipid network in film matrix.

Our findings supports the results obtained by Nonsee, Supitchaya and Thawien [2], who

also observed that WVP values tended to decrease when encapsulated clove oil was

incorporated into hydroxypropyl methylcellulose (CMC) based films. According to them

clove oil could entered into the structural matrix of films, which further limit the passage

of liquid molecules through the film. Guillén et al. [24] observed the influence of sunflower

oil on cod gelatin based films. They also observed a decrease in WVP values and an

improvement in hydrophobic properties of the resulted films. Likewise Teixeira et al. [25]

also reported that the permeability of fish protein based films to water vapors was

significantly reduced after the assimilation of clove, garlic and oregano essential oils in the

films.
Pelissari, Grossmann, Yamashita and Pineda [26] stated that transfer of water vapors

mostly occurs through the hydrophilic portions of the film structure and generally

depends on the hydrophobic- hydrophilic ratio of the film components. But some

studies related to incorporation of natural extracts and essential oils have not revealed

progress in WVP [27] [16] [28]. This could be due to difference in hygroscopic properties

of essential oils used by which they attract water molecules. The specific composition of

essential oil could be responsible for the transfer of water vapors across the film matrix. It

cannot be supposed that Water vapor transmission rate can be reduced only by adding a

hydrophobic substance in the film matrix, but the distinct effect of lipid incorporation on

the microstructure of the film matrix is the determining factor [29]. As SEM images show

pectin films embedded with CEO showed a smooth texture with uniform pores distributed

throughout the film matrix (Fig. 3). These structures could explain the observation of

improved WVP of pectin films with higher CEO concentration.

3.5 Film color and opacity:

Color and transparency of the edible film are two significant indexes in terms of overall

appearance and consumer acceptance. The effect of CEO addition on color coordinates

(L*, a* and b*), total color difference (ΔE) and opacity of pectin (O) films are recorded in

Table 2. Edible films without incorporation of oil appeared more clear and transparent. The

addition of oil affected the color and transparency of biofilms. Control films were lighter

in color as indicated by higher L* values. L* and a* values of the film specimens were

decreased from 87.80 to 85.84 and 0.51 to 0.26 respectively while b* values were

increased from 3.52 to 7.26, as the CEO concentration was increased from 0% to

1.5%. Pectin films integrated with a concentration, higher than 0.5% of CEO showed
higher ΔE than control film. This observation was probably due to an increase observed in

colorimetric coordinate and the decrease in brightness value of the film specimens. This

phenomenon was attributed to the phenolic compounds of CEO, which might show light

absorption at lower wavelengths. The variance in color parameters appears to be the

consequence of natural yellow of essential oils or lipids [29]. Similar variations in color

parameters were observed by Benavides et al. [30] after addition of oregano essential oil

into alginate based films.

Pectin films without CEO were more transparent (indicated by lower opacity value) as

compared to films combined with CEO (Table 2). The results demonstrated that the opacity

of the films markedly increased from 0.75 to 3.15 by increasing oil concentration up to

1.5%, giving more-opaque films. This behavior could be attributed to the coalescence,

light-scattering and creaming effect provoked by the distribution of lipid particles during

the drying process of the films which promoted the surface coarseness of the film samples

due to the presence of large lipid molecules [23]. Reduction in transparency of composite

films containing essential oils has also been detected by other authors [31] [32].

3.6 Mechanical Strength:

Tensile strength (TS) is recognized as the maximum stress supported by film before break

and percent elongation at break (EB) is a mechanical property that provides information

about deformation of a material prior to breakage. If the material is proposed for some food

packaging applications, certain deformation is mandatory before fracturing [33, 34]. The

mechanical properties of films are summarized in Table 3. Compared with the control,

Incorporation of CEO increased the tensile strength, young’s modulus and percentage

elongation at break (E %) of the pectin films significantly.

Pure pectin film had a tensile strength of 14.78 MPa. When the amount of clove oil in the

film-forming mixture was increased from 0 to 1.5% w/w, the TS significantly increased

from 14.78 MPa to 33.78MPa and EB increased from 6.37% to 11.75%. TS and EB were

usually related to the network of film microstructure and the intermolecular force among

them [29]. Matrices or particles with chemical resemblance interact more strongly when

dispersed; the particles behave as setting centers, decrease chain mobility, and bring an

improvement in tensile strength. The core chemical components of clove oil are eugenol,

eugenol acetate, iso-eugenol, and caryophyllene. The chemical resemblance between CEO

(particle) and pectin (matrix) is primarily due to hydroxyl groups present in the

arrangement structures that form these materials [35].

Our findings were comparable with the Ghanbarzadeh et al. [23], who also noticed an

increase in film strength after incorporating oleic acid in CMC edible films. A strong

interaction between the carbohydrate polymer and essential oil produced a cross linking

effect. Similar observations were previously noticed by other researchers by adding oleic

acid in chitosan films [36] [37].

On the other hand, there are also some reports demonstrating that a reduction of tensile

parameters of polysaccharide based films is the expected output when a lipid is integrated

in the system [38] [39], they attributed the behavior to the existence of structural

discontinuities in the film network triggered by the lipid dispersed phase.

EB was increased by adding CEO because oils are liquid at room temperature and they will

exist in film matrix in the form of oil droplets which can be easily deformed, improving

the film’s extensibility [30]. Materials with high EB values propose good extensibility and

flexibility due to the cohesion among polymer molecules [40]. A strong interaction among
the polymer and clove oil showed a crosslinking effect, which reduced the free volume and

molecular mobility of the polysaccharide polymer. Extensibility of quince Seed mucilage

films was also increased after addition of thyme and oregano essential oils [17]. This trend

is also consistent with the outcomes obtained by [41] and [27].

3.7 Attenuated total reflectance-Fourier-transform infrared (ATR-FTIR)

spectroscopy

ATR-FTIR spectra have been used to monitor intermolecular interactions and structural

changes within the film matrix at molecular level through a detailed spectral analysis. From

the FTIR spectrum of pure pectin film (Fig. 1(A)) broad area of absorption between 3400

and 2500 cm-1 are allocated to the stretching vibrations due to intermolecular interactions

through O-H bonds among pectin monomers [42]. The peak around 2933cm-1 refers to C-

H vibrations of methyne groups in polymer chains and methyl group of the methyl ester

[43]. The band elongations between 1760–1730 correspond to the C=O and C-O of ester

bonds. Furthermore, strong vibration peaks at 1630–1600 cm-1 and 1400 cm-1 are attributed

to asymmetric and symmetric vibrations of carboxyl functional, respectively [44]. Besides,

the absorption bands at 1100 and 1040 cm-1 are assigned to C-O-C stretching vibrations of

the polymer chain structure [45].

The spectral differences between different film samples were largely attributed to altered

conformation and orientation of polypeptide chains as affected by incorporation of CEO.

No significant difference (no new absorption peaks) was found in the pectin film spectra

when CEO were added to the film. Generally, films with and without addition of CEO

showed the similar major peaks but the amplitude of peaks varied.
It was observed that the broad peak ranged between 3500 and 3100 cm-1, related to the O-

H stretching vibration, become more flattened and shifted to 3324 cm-1 with increasing the

concentration of CEO, demonstrating the decreased stretching of free O–H bonds due to

the binding interactions between oil and pectin [46]. The presence of clove oil also leads

to changes in the band region ranging 2800-2900 cm-1, showed the shift of band at 2936

cm-1 to 2928 cm-1., associated with the stretching vibration of the aliphatic C-H group

(CH2) [47].

By other side, amplitude of peak at wavenumbers 1741 cm-1 decreased slightly when the

films were incorporated with oil. This peak is attributed to the C=O stretching vibration,

that can be described by the existence of the carbonyl radical in the ester functional group

of triglycerides, associated with the presence of clove oil [47]. Similar spectral alterations

were also observed by Cerqueira, Souza, Teixeira and Vicente [48] by adding corn oil into

the galactomannan edible films.

3.8 Differential scanning calorimetry (DSC)

The information about intermolecular structural changes caused by temperature variations

is necessary in order to determine the thermal resistance of a packaging material. The

thermal stability of pectin edible films incorporated with clove oil was studied by

thermogravimetric analysis to further understand the structural interaction between

polymers and CEO. Differential scanning calorimetric (DSC) curves exhibiting the

thermally-induced endothermic changes of pectin films between -70°C to 300°C are shown

in Figure 1(B) and table 4.Thermal properties of pectin depend mainly on its chemical

composition and state transition happened during processing as well as on interdependence

of both these factors [49]. Heat flow alterations in thermal transition occurring between 80-
100 °C, were mainly associated to water evaporation linked with the hydrophilic groups in

the polymeric structure. [43] [50]. After this, a decomposition step observed around 160°C

was associated to decomposition and depolymerization of pectin, which showed the

maximum decomposition rate at 228.86 °C for pure pectin films [51].

Addition of clove essential oil did not show significant (p> 0.05) influence on the

endothermic peaks; however, some heat transitions shifted slightly to higher temperatures

and the enthalpy of melting (Δhm) related to evaporation was decreased by the inclusion of

oil. The DSC degradation peaks for 0.5%, 1% and 1.5% CEO/pectin films appeared at

231.53 oC, 234.88 oC, and 232.21 oC respectively, which shows that CEO has increased the

thermal stability of films slightly. However, enthalpy of the melting (Δhm) in endothermic

peak is reduced, which shows that clove oil decreased the amount of heat generated by the

decomposition of pectin, thus confirming the thermal stability of the films. The increased

heat stability could be attributed to the more hydrophobic nature and larger molecular

weight of CEO. This outcome are consistent with those obtained by [5] [20]. They observed

that the inclusion of lipids in the kefiran and gelatin based films increased their thermal

stability significantly. Ma, et al. [52] also noticed that the addition of olive oil in film matrix

resulted in increased melting point (Tm) of the helix–coil transition of gelatin.

The results of DSC measurements indicated that control films possessed only two steps of

weight loss, while CEO-loaded pectin films at higher concentration exhibited three steps

of weight loss. DSC curve of films containing 1%, 1.5% of CEO showed emergence of

another small peak before major exothermic peak at 213 oC and 217 oC respectively. These

exothermic peaks may be related to melting of CEO. Oils are complex mixture of

triacylglycerols (TAGs) acting also as a solvent for minority components, such as vitamins,
phenolic compounds, pigments, phospholipids, free fatty acids, and mono- and

diacylglycerols. Thus, the first endothermic peak is probably associated with the fusion of

free fatty acids. While the new small peak before the emergence of major exothermic peak

is probably associated with the decomposition of the TAGs and fatty acids into smaller

chains present in CEO.

Jouki, Yazdi, Mortazavi, and Koocheki [17] also noticed the appearance of a new peak

with increasing the concentration of oregano essential oil up to 2% in quince seed mucilage

films. Similarly, Aliheidari, et al. [53] observed that DSC curve of the casein-based films

integrated with Matricaria recutita essential oil (MEO) showed two Tm peaks. This result

coincides closely with those from the SEM studies, where a partial separation of the two

phases may have occurred at higher concentration.

3.9 X-ray diffraction analysis

XRD analysis was used to explore crystalline structure and evaluate the compatibility of

pectin and clove oil. It has been reported well defined peaks at 12.72o, 16.30o, 18.45 o,

25.32 o and 40.14 o of 2θ associated to pure pectin crystallinity [54] [55]. In the current

study, pectin films exhibited a broad peak at 15.72o of 2θ confirming the presence of some

crystalline structure (Fig. 2). Inclusion of CEO into pectin films did not alter the peak

positions in the XRD pattern significantly. The illustrated diffractograms of composite

films proposed good compatibility between both polymers as the basic crystalline fraction

of pectin matrix was not diminished in composite films. As, no new diffraction peaks were

noticed in composite CEO/pectin films, suggesting no chemical interactions between

pectin and CEO.

3.10 Scanning Electron Microscopy (SEM):

Microstructure of pectin films containing CEO was studied to get some understandings on

oil droplets organization along the biopolymer film matrix, and its possible influences on

the film properties. SEM was used to evaluate the film morphology and distribution of lipid

droplets in the film matrix. Microstructural examination of the films reveals relevant

information about the spatial arrangement of its components, providing us a better

understanding of mechanical properties and water vapor transmission mechanisms [37].

The scanning electron micrographs of the surface and cross sections of pectin films

incorporated with CEO essential oil are shown in Fig. 3. When oil was incorporated into

the film formulation, the surface micrograph suggested a dense sheet like structure, while

the cross sectional images revealed that the sheets stacked in compact layers, which

indicated CEO incorporated uniformly in the film matrix. The SEM images also exhibited

that oil droplets were homogenously entrapped in continuous network of polysaccharide.

Clove essential oil impregnated pectin films at a level of 0.5% had relatively smoother

surfaces without any phase separation. However, the rough surface and looser texture with

sponge-like structure and micro-cavities were obtained in the films incorporated with 1 and

1.5% CEO. These structural discontinuities could be related to the formation of two phases

(oil and polymer) in the film matrix at higher percentage of essential oil. This roughness

appears as a consequence of the migration of aggregates or droplets to the top of the film

during film drying, which leads to surface irregularities. Flocculation and creaming of oil

droplets occurred during film drying [39]

Films prepared with 0.5% and 1% w/w CEO showed relatively smaller size micropores as

compared to those prepared with 1.5% w/w CEO and the number of lipid droplets also
increased as the concentration of oil in the film solution increased. The increased droplet

concentration may enhance the possibility of oil droplet coalescence during drying

phenomenon and resulted in the formation of large size pores.

The size reduction of oil droplets during homogenization led to increase in interactions

between oil particles and polymer matrix [28]. Different surface morphology of films is

possibly resulted by structural changes of components of micro-emulsions during drying

process because same amount of pectin was used to prepare all films. Similar observations

were reported for chitosan films incorporated with cinnamon essential oil [33].

3.11 Total phenolic (TP) content and antioxidant activity:

Total phenolic contents and free radical scavenging activity of pectin films with and

without CEO incorporation are shown in Fig 4.Antioxidant activity of films was

determined by using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay. DPPH is a stable free

radical and when dissolved in alcohol it exhibits a characteristic absorption at 517 nm.

Antioxidant molecules act as hydrogen donor and scavenge the free radicals by turning the

color of DPPH assay solution from dark purple to light yellow, resulting in a reduction in

absorbance value. Films with CEO exhibited a higher level of radical scavenging activity

with values of 5.80, 34.10, 51.00 and 64.73% for 0, 0.5, 1 and 1.5% of CEO containing

films, respectively. In accordance with previous data, the quince seed mucilage films

containing oregano essential oil also showed the greatest TPC as compared to control [17].

The Folin–Ciocalteu reagent method was used for the determination of total phenolic

compounds in film samples. The results revealed that total phenolic content in the pectin

based films were increased significantly (P < 0.05) with the inclusion of clove essential oil
into pectin films. The lowest TP content (0.49 mg gallic acid/g), was observed for control

film (pure pectin) and the highest TP content was 13.52 mg gallic acid//g that were found

in the film with 1.5%% CEO. Our findings were in line with the previous findings of

Shojaee-Aliabadi et al. [56], who also reported that antioxidant capacity of kappa-

carrageenan films increased after the addition of Satureja hortensis essential oil.

3.12 Antimicrobial Activity:

Plants can produce various antimicrobial compounds in order to protect themselves from

biotic attacks that could be significant for resistance against microbial infections [57]. The

antibacterial activities of pectin films incorporated with CEO against S. aureus, E. coli and

L. monocytogenes bacteria are shown in Table 5. Pure pectin film was used as a control to

investigate potential antimicrobial effect of the pectin without additives. The control film

did not show any inhibition against three pathogenic bacteria, indicating that pectin has no

antimicrobial properties against the tested bacteria. As the concentration of CEO increased,

the diameter of inhibition zones increased significantly for all bacterial strains tested. The

zones of inhibitions against S. aureus, E. coli and L. monocytogenes were increased from

18.50 mm to 30.27mm, 12.53 mm to 21.20 mm and 14.67mm to 26.43 mm respectively

with increasing the concentration CEO from 0.5 to 1.5 %. For the films with the same clove

oil concentration, the inhibition zone diameters were significantly (p<0.05) different

among the bacteria and followed the increasing order of E. coli< L. monocytogenes< S.

aureus.

These outcomes are in accordance with the work of Hosseini, Razavi, Mousavi, Yasaghi,

and Hasansaraei [58], who also observed inhibitory effects of clove oil against E. coli, L.

monocytogenes and S. aureus in chitosan based edible films. The major inhibitory
components of clove oil are eugenol, ß-caryophyllen and acettaugenol [59]. EOs have the

potential to interact with the bacterial cell membrane or other intracellular components,

and subsequently, disruption of cell configuration, ions exchange, leakage, alteration of

permeability, inhibition of respiration and death could be occur [60].

Inhibition of L. monocytogenes by edible films with thyme and CEO was also observed by

Hosseini, Razavi and Mousavi [13].The antimicrobial agents were more effective against

gram positive (L. monocytogenes and S. aureus) bacteria as compared to gram negative (E.

coli). The main cause is the difference in structure of cell wall of gram positive bacteria. In

gram positive bacteria the major component of cell wall is peptidoglycan and small amount

of proteins. On the other hand the cell wall of gram negative bacteria possesses more

complex structure with addition of various polysaccharides, proteins and lipid based

peptidoglycan [10]. Nonsee, Supitchaya and Thawien [2] reported the powerful inhibitory

effect of encapsulated clove oil on the growth of E. coli , L. monocytogenes, and S. aureus

in HPMC based films.

Gram-negative bacteria possess an additional hydrophilic membrane embedded with

lipopolysaccharide molecules that serve as hurdle to hydrophobic compounds. This

decreases the penetration of hydrophobic compounds through cell membrane. Due to

absence of additional lipophobic outer membrane, Gram positive bacteria allow easy

penetration of active compounds with greater inhibitory effect. Comparable observations

were also demonstrated by Fisher and Phillips [61].

Conclusion:
This study suggested that integration of CEO into biodegradable pectin has a significant

influence on physicochemical properties of the subsequent pectin/CEO films. This

incorporation influenced the barrier, mechanical, antioxidant and antimicrobial properties

of pectin films positively. Emulsified pectin films showed more hydrophobic nature, which

was confirmed by the decrease in moisture content and WVP values, proposing improved

water barrier properties of the resultant films. The addition of CEO significantly modified

the mechanical properties of the films and promoted greater thermal stability for the films

as exhibited by DSC analysis. The microbiological assessment by agar disc-diffusion assay

confirmed the antimicrobial efficiency of pectin films against food-borne pathogens,

particularly Gram-positive S. aureus and L. monocytogenes. The research results revealed

that the pectin films incorporated with clove essential oil have a great potential to be used

as a natural antimicrobial to preserve food. Further investigations are needed to explore the

effects of these pectin-based films on the microbiological quality and safety of food

products, particularly those with high oxidative and microbial-sensitivity.

Acknowledgement

This research is sponsored by China Agriculture Research System (CARS-28) from

Ministry of Agriculture of People’s Republic of China.

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their components on the survival of Campylobacter jejuni, Escherichia coli O157,

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in food systems, J. of Appl. Microb. 101(6) (2006) 1232-40.

Figure Caption
FIGURE 1: (A) ATR-FTIR spectrum of Pectin/CEO films, (B) DSC thermograms of

Pectin/CEO films.

FIGURE 2: XRD spectra of pectin films containing different percentage of CEO.

FIGURE 3: SEM micrographs of films surface: (a.1) control; (b.1) 0.5% CEO; (c.1) 1%

CEO; (d.1) 1.5% CEO and SEM micrographs of films cross-sections: (a.2) control; (b.2)

0.5% CEO; (c.2) 1% CEO; (d.2) 1.5% CEO

FIGURE 4: DPPH scavenging activity and total phenolic contents of pectin films

incorporated with CEO. Values are given as mean ± standard deviation. Different letters

indicate significantly different (p < 0.05) when analyzed by Duncan’s New Multiple

Range Test.

FIGURE 5: Inhibitory effects of CEO-pectin films on the growth of Staphylococcus

aureus (A),
 FIGURE 1

(A)

(B)
FIGURE 2
FIGURE 3
FIGURE 4
 FIGURE 5

TABLE 1: Physical properties of pectin films incorporated with CEO.

Thickness Moisture Solubility in Swelling WVP
Film
(mm) content (%) water (%) degree (%) (×10 g.m-1s-1Pa-
-11
Samples 1
)
Control 0.057±0.003d 18.43±0.40a 25.37±0.40a 3.40±0.22a 13.22±0.15a
0.5% CEO 0.068±0.002c 14.29±0.28b 22.61±0.60b 2.91±0.42b 9.48±0.27b
 1.0% CEO 0.080±0.002b 12.04±0.48c 19.52±0.30c 2.61±0.36c 7.24±0.26c
1.5% CEO 0.094±0.003a 10.14±0.12d 17.44±0.60d 1.86±1.38d 6.52±0.16d
Values are expressed as mean ± standard deviation. Different letters in the same column indicate
significant differences (p<0.05).

TABLE 2: Color and opacity of pectin films incorporated with CEO.

Opacity
Film Samples L* a* b* ΔE
(A·mm-1)
Control 87.80±0.48a 0.51±0.05a 3.52±0.39d 6.79±0.44d 0.75±0.25d
0.5% CEO 87.15±0.37b 0.43±0.13b 5.30±0.69c 8.38±0.85c 1.29±0.19c
1% CEO 86.79±0.42c 0.34±0.26c 6.65±0.64b 9.52±0.82b 2.48±0.31b
1.5% CEO 85.84±0.28d 0.26±0.36d 7.26±0.33a 10.55±0.60a 3.15±0.29a
Values are expressed as mean ± standard deviation. Different letters in the same column
indicate significant differences (p<0.05).

TABLE 3: Tensile strength, % Elongation at break and Elastic Modulus of pectin films
incorporated with CEO
Film samples Tensile Strength (MPa) Elongation at break (%) Elastic Modulus
Control 14.78±0.25d 6.37±0.37d 2.32±1.03d
0.5% CEO 20.98±0.21c 8.96±0.43c 2.34±0.75c
1% CEO 25.7±0.28b 10.68±0.38b 2.41±0.62b
1.5% CEO 33.78±0.22a 11.75±0.67a 2.87±0.67a
 Values are expressed as mean ± standard deviation. Different letters in the same column indicate
significant differences (p<0.05).

TABLE 4: DSC thermal parameters (peak melting temperature (Tm) and enthalpy of
melting (ΔHm) of pectin films incorporated with CEO.

Peak melting
Film samples Enthalpy of melting (ΔHm) (J/g)
temperature (Tm)(°C)
Control 228.86±0.32d 206.9±0.51a
0.5% CEO 231.53±0.27c 151.0±0.22b
1% CEO 234.88±0.41a 140.3±0.17c
1.5% CEO 232.21±0.36b 105.5±0.26d

TABLE 5: Antimicrobial effect of pectin films incorporated with CEO against

Staphylococcus aureus, Escherichia coli and Listeria monocytogenes.
Inhibition zone diameter (mm)
Film Samples
S.aureus E. coli Listeria monocytogenes
Control 0.00 0.00 0.00
c c
0.5%CEO 18.50±0.16 12.53±0.34 14.67±0.46c
1.0%CEO 23.23±0.39b 17.23±0.45b 19.73±0.45b
1.5%CEO 30.27±0.41a 21.20±0.33a 26.43±0.29a
Values are expressed as mean ± standard deviation. Different letters in the same column
indicate significant differences (p<0.05)