foods

Article
Characterization and Antimicrobial Activity of
Biodegradable Active Packaging Enriched with
Clove and Thyme Essential Oil for Food
Packaging Application
Shubham Sharma 1,2,3 , Sandra Barkauskaite 1 , Brendan Duffy 3 , Amit K. Jaiswal 1,2, * and
Swarna Jaiswal 1,2
1 School of Food Science and Environmental Health, College of Sciences and Health, Technological University
Dublin—City Campus, Grangegorman, D07H6K8 Dublin, Ireland; [email protected] (S.S.);
[email protected] (S.B.); [email protected] (S.J.)
2 Environmental Sustainability and Health Institute (ESHI), Technological University Dublin—City Campus,
Grangegorman, D07H6K8 Dublin, Ireland
3 Centre for Research in Engineering and Surface Technology (CREST), FOCAS Institute, Technological
University Dublin—City Campus, Kevin Street, D08NF82 Dublin, Ireland; [email protected]
* Correspondence: [email protected] or [email protected]; Tel.: +353-1220-5661




Received: 15 July 2020; Accepted: 11 August 2020; Published: 13 August 2020


Abstract: Bioactive packaging contains natural antimicrobial agents, which inhibit the growth of
microorganisms and increase the food shelf life. Solvent casting method was used to prepare the Poly
(lactide)-Poly (butylene adipate-co-terephthalate) (PLA-PBAT) film incorporated with the thyme oil
and clove oil in various concentrations (1 wt%, 5 wt% and 10 wt%). The clove oil composite films
depicted less green and more yellow as compared to thyme oil composite films. Clove oil composite
film has shown an 80% increase in the UV blocking efficiency. The tensile strength (TS) of thyme oil
and clove oil composite film decreases from 1.35 MPs (control film) to 0.96 MPa and 0.79, respectively.
A complete killing of S. aureus that is a reduction from 6.5 log CFU/mL to 0 log CFU/mL was observed
on the 10 wt% clove oil incorporated composite film. Clove oil and thyme oil composite film had
inhibited E. coli biofilm by 93.43% and 82.30%, respectively. Clove oil composite film had exhibited
UV blocking properties, strong antimicrobial activity and has high potential to be used as an active
food packaging.

Keywords: active food packaging; antimicrobial activity; antibiofilm activity; clove essential oils;
thyme essential oil; poly (lactide); poly (butylene adipate-co-terephthalate)

1. Introduction
Active packaging is a novel method used to prolong the shelf-life of perishable foods, maintain or
improve the quality and safety of prepared foods due to its interaction with the product. Besides, active
packaging for consumable goods has the potential to reduce the addition of active compounds into foods,
reduction in the movement of particles from film to food and localisation activities could introduce the
pathogenic microorganism into the product [1]. Also, the world focus has now shifted to biodegradable
and non-biodegradable polymers such as poly-hydroxy alkanoates (PHA), polyethylene (PE), poly
(lactide) (PLA), bio-based polyethylene terephthalate (PET), poly (butylene adipate-co-terephthalate)
(PBAT) based packaging. Poly(lactide) (PLA) and Poly (butylene adipate-co-terephthalate) (PBAT) are
the biodegradable polymers, and various studies were conducted on the blend of PLA and PBAT [2].

Foods 2020, 9, 1117; doi:10.3390/foods9081117 www.mdpi.com/journal/foods

Foods 2020, 9, 1117 2 of 16

Biodegradable polymer incorporated with an active compound could ensure the food safety, quality
and monitor environmental health [3].
Depending on the types of additives incorporated into the film, active packaging can be categorised
into chemoactive and bioactive. Chemoactive packaging has an impact on the chemical composition of
the food product and gaseous atmosphere inside a pack. It includes ethylene scavengers, modified
atmosphere packaging, and moisture control systems. On the other hand, bioactive packaging contains
antioxidant and antimicrobial agents within the package that interact with biological molecules and
may inhibit the growth of various microorganisms [3] A study by Azadbakht et al. [4] used Eucalyptus
globulus essential oil in chitosan to examine the antimicrobial activity in the packaging of sliced
sausages. The results showed that the log reduction value could be improved by increasing essential
oil concentration. The application of essential oils such as cinnamon oil, clove oil, thyme oil in active
packaging can be used in the forms of films and coatings. Films are usually thin sheets that are made
beforehand and can be used as covers, wrappers, layer separation or packaging for various foods.
On the other hand, coatings are defined as films that can be applied onto the surface of an edible
product [5].
In recent years, there is a growing concern towards chemical active packaging due to the use of
synthetic additives and materials that can cause adverse health effects or make packaging unsustainable
for recycling leading to high waste volume. Due to growing consumer demand for natural products,
synthetic additives become replaced by substance of natural origin, such as essential oils and plant
extracts [6]. Natural antioxidants can interact with the food product and package headspace resulting
in the protection of the food products from oxidation.
Antioxidant active packaging can either release antioxidants into the food and the package or
absorb oxygen and other compounds from the food or its surroundings. Also, active packaging
containing natural essential oil, antioxidants like clove oil, green tea oil, thyme oil is a cost-saving
alternative that also has the potential to eliminate food safety risks [7]. Therefore, the natural plant
extracts and essential oils play a significant role in the antimicrobial/antioxidant activity of the
active packaging. The effectiveness of active packaging containing thyme essential oil/β-cyclodextrin
ε-polylysine nanoparticles (TCPNs) was tested [8]. The results showed that TCPNs incorporated
into gelatin nanofibers significantly improved the antimicrobial properties against bacteria such as
Campylobacter jejuni.
There are several examples of essential oils and their constituents incorporated into active
films. Chitosan films containing Eucalyptus globulus essential oil were developed for the packaging
of sliced sausages that have a high potential to reduce the antimicrobial activity and control the
food-borne contamination in food systems [4]. Another study carried out by Perdones et al. [9] showed
chitosan-based coatings containing lemon essential oil were very effective in delaying the ripening
process in strawberries due to their reduced respiration rate. It was also determined that after seven
days of storage, the aroma of lemon essential oil did not have any impact on the organoleptic properties
of strawberries. The antibacterial activity of these newly developed packaging system was attributed
to the chemical composition of used essential oil [4,9].
Clove (Syzygium aromaticum) is commonly used in the seasoning of food. Recent research showed
that clove essential oil can kill numerous bacteria and some fungi, and the antimicrobial activity is
attributable to eugenol, oleic acids and lipids found in its essential oils. Jirovetz et al. [10] found that
clove essential oil comprises 23 constituents, among them eugenol (76.8%), followed by β-caryophyllene
(17.4%), α-humulene (2.1%), and eugenyl acetate (1.2%) were the main components. Pinto et al. [11] had
also evaluated clove oil by GC-MS and the found a total of 19 components. The oil contains 85.3% of the
phenylpropanoid compound (eugenol) [11]. Moreover, Shrivastava et al. [12] had analysed clove oil
from India and Madagascar. They found 35 constituent in the Madagascar clove oil and 28 constituent
in the Indian clove oil where the major content was eugenol (82.6%), β-caryophyllene (7.2%), eugenyl
acetate (6%), α-bergamontene (0.2%), α-humulene (0.8%), Υ-cadinene (0.2%), iso-eugenol-I (0.1%),
selinene (0.3%) and allo-aromadendrene (0.1%) [12]. Razafimamonjison et al. [13] had evaluated the
Foods 2020, 9, 1117 3 of 16

clove essential oil from bud, lead and stem. In all essential oils, they also found the major constituent
as eugenol in bud (72.08–82.36%), stem (87.52–96.65%) and leaf (75.04–83.58%). Eugenyl acetate was
also found in bud (8.61–21.32%), leaf (0–1.45%) and stem (0.07–2.53%). Moreover, β-caryophyllene are
found more in leaf (11.65–19.53%), than in bud essential oils (2.76–8.64%) and in stem essential oils
(1.66–9.72%) [13].
Thyme (Thymus vulgaris) essential oil is used as flavor enhancer for foods and beverages and known
to possess antimicrobial and antioxidant properties, which are due to presence of various constituents
such as thymol, carvacrol and cymene [14]. In a study carried out by Porte et al. [15] reported
that thyme oil has thirty-nine constituents, where major constituents of the oil were thymol (44.7%),
p-cymene (18.6%) and γ-terpinene (16.5%). Various studies have analysed thyme essential oil and
found the main components as thymol (55.3%), p-cymene (11.2%), carvacrol (8.7%, and β-caryophyllene
(4.2%). The other contents found were isoborneol (2.3%), γ-terpinene (3.4%), acetovanillone (1.7%)
and linalool (1.7%) [16,17]. Tohidi et al. had analysed thyme oil by gas chromatography–mass
spectrometry (GC–MS) and found 38 compounds having major elements as thymol (12.4–79.74%),
carvacrol (4.37–42.14%), p-cymene (0.8–12.86%) and geraniol (0.3–22.44%) [18].
In the present study, essential oils (thyme oil and clove oil) have been incorporated as an
antimicrobial agent in a PLA/PBAT (poly (lactide)/poly (butylene adipate-co-terephthalate) blend film
for food packaging application. Effect of thyme oil and clove oil on the structural, functional, mechanical,
antimicrobial and biofilm inhibition properties of the composite films was studied. To the best of
author’s knowledge, this is the first report to incorporate thyme oil and clove oils into the biodegradable
polymer (PLA/PBAT) blend film and investigate their potential as an active food packaging.

2. Materials and Methods

2.1. Materials
PBAT (poly (butylene adipate-co-terephthalate) (m.p. 110–120 ◦ C, the density of 1.26 g/cm3,
Ecoworld PBAT003;) and Poly (lactide) (PLA, the weight-average molecular weight of 200 kDa, Synterra
BF 2004) were purchased from Helian Polymer (Belfeld, Netherland). Ethanol was purchased from
Merck KGaA (Germany) and Chloroform was obtained from Sigma Aldrich (Ireland). Clove essential
oil (Mol wt. 150.2 g/mol, relative density 1.04 g/cm3 at 25 ◦ C) and Thyme essential oil (Mol wt.
164.2 g/mol, relative density 0.917 g/cm3 at 25 ◦ C) of natural origin (100% purity) were purchased from
a local supermarket, Dublin, Ireland. Tryptic Soy Broth (TSB) and Tryptic Soy Agar [8] powder were
purchased from Sigma Aldrich (Ireland). Foodborne pathogenic bacteria, Staphyloccocus aureus (ATCC
25923) and Escherichia coli (NCTC 9001) were used in this study. All of these strains were grown in TSB
media and stored at 4 ◦ C before further testing.

2.2. Preparation of Composite Films

By using a solvent casting method, the essential oils (thyme and clove) were incorporated into
biodegradable films of PLA-PBAT [19]. The blending ratio of PLA:PBAT was taken as 98:2 [20].
In 100 mL chloroform various concentrations of essential oil (1 wt%, 5 wt%, and 10 wt% of polymer
resin) were pipetted out. To make up the equal volume in all the beaker ethanol was added in the
samples accordingly. 4 g of polymer resin (3.92 g of PLA and 0.08 g of PBAT) and 50 wt% of glycerol [21]
were added and mixed using a magnetic stirrer at 23 ± 2 ◦ C for 24 h. A control sample of the PLA/PBAT
film was prepared using the same method, followed by the addition of only ethanol (without any
essential oil).
After the stirring of 24 h the solubilised mixture was poured onto a Teflon coated glass plate
(24 cm × 30 cm) and spread evenly with a bend glass rod and allowed to dry at the room temperature
for 24 h. The prepared films were peeled off from the glass plates and placed into the incubator
at 25 ◦ C and 50% relative humidity (RH) for 48 h. The Control films prepared was named as Poly
(lactide)-Poly (butylene adipate-co-terephthalate) (PLA-PBAT) [22]. The thyme containing films were
Foods 2020, 9, 1117 4 of 16

PLA/PBAT-Thyme 1% , PLA/PBAT-Thyme 5% , and PLA/PBAT-Thyme 10% . Clove containing films were

named as PLA/PBAT-Clove 1% , PLA/PBAT-Clove 5% , and PLA/PBAT-Clove 10% .

2.3. Characterisation of Films

2.3.1. Surface Colour and Optical Properties

ColorQuest XE (Hunter Lab) spectrophotometer was used to determine the surface colour of
composite films. The colour values were taken in terms of L (+lightness, −darkness), a (+redness,
−greenness), and b (+yellowness, -blueness). White colour plate (L = 93.97, a = −0.88, b = 1.21)
was used as a standard background. For each film the readings are taken in triplicate from different
locations. The mean value with the standard deviation of the readings was considered. The total
colour difference which determines the difference between the colour values of standard tile and film
samples (∆E) was considered using an equation:

∆E = [(∆L)2 + (∆a)2 + (∆b)2 ] 1/2

where ∆L, ∆a, and ∆b are the differences between standard colour plate, each colour value and the
colour value of the film samples, respectively [22,23].
The optical properties such as the light transmittance spectrum was observed using UV-Vis
spectrophotometer. The rectangular films of (3 cm × 7 cm) were cut and mounted between two
spectrophotometer magnetic cells. The light transmittance spectrum of the composite film was
measured between the wavelength of 200–700 nm. UV-light barrier property was obtained by the
percent transmittance at 280 nm (T280 ) and transparency of the films were determined at 600 nm (T600 ).
Fourier-transform infrared spectrometer (FTIR) (Thermo Scientific) was used to determine the
infrared spectrum of absorption of the composite films. FTIR was operated at the resolution of 4 cm−1 .
The film sample of 4 cm × 4 cm were cut and positioned on the ray exposing stage (crystal plate).
The spectrum was recorded at wavenumber of 4000–400 cm−1 . The readings were taken in triplicates
from the surface. The functional groups present in the composite films were determined by the peak at
specific wavenumber.

2.3.2. Thickness and Tensile Properties

The thickness of the composite films was determined by Digital micrometre (Mitutoyo, Japan)
having 0.001 mm resolution. Five readings were taken from the random positions. The mean values
and standard deviation were considered of these readings.
Mechanical properties such as tensile strength (TS) and elongation at break (EB) of the composite
films were measured by using standard ASTM D 882–88 method [24] by Instron Universal Testing
(Model 5565, Instron Engineering Corporation, Canton, MA, USA). The composite films were cut into
rectangular strips of 3 cm × 15 cm. Grip length of 50 mm and a crosshead speed of 50 mm/min using a
500 N load cell set in Instron Instrument were used to operate at room temperature until the sample
broke at a certain point. The Tensile Strength was calculated using the Equation:

TS = F/A

where F is the maximum force (N) required to separate the sample, and A is the initial cross-sectional
area (m2 ) of the composite films [22].
Percentage elongation at break (EB) is the ratio between changed film length and initial length of
the samples [25]. The EB was calculated using the following formula:

EB (%) = [(Xf − Xo )/Xo ] × 100

Foods 2020, 9, 1117 5 of 16

where Xo is the initial grips separation (50 mm) of samples, Xf is the film elongation at the moment
of failure.

2.3.3. Surface Hydrophobicity

Water contact angle (WCA) determines the interaction of the film surface with the liquid interphase
by using a dynamic contact angle analyser (FTA-200 system) [26]. It evaluates the surface is hydrophobic
or hydrophilic in nature. Rectangular films of 3 cm × 8 cm placed on the stainless-steel platform having
the water contact angle analyser attached. With the help of a micro-syringe a drop of distilled water
approximately 10 µL was dropped on the film surface. The interaction of the drop on the surface of
the film was observed by taking a picture with a high-speed camera and analysed it by the image
processed by computer. Three readings were taken from the different location. Also, the experiment
was performed in triplicates.

2.3.4. Antibacterial Activity

The Japanese Industrial Standard (JIS Z 2801:2000) [27] was used to determine the antibacterial
efficacy of the composite films incorporated with thyme oil and clove oils against Staphylococcus aureus
(ATCC 25923) and Escherichia coli (NCTC 9001). The kinetic study was performed using square pieces
of the composite films (5 cm × 5 cm). Each film was sterilised each side using UV light for 20 min.
Films were then placed on the aluminium plate (5.5 cm × 5.5 cm) and aseptically transferred into
a sterilised petri dish containing filter paper which was wetted with autoclaved water. The initial
bacterial inoculum 106 CFU/mL was prepared aseptically for the test. 200 µL of the test inoculum
was then pipetted onto three pieces of each film, leaving one film as a positive control. Each petri
dish containing the sample was covered with a lid and then incubated with the test inoculum at the
temperature of 37 ◦ C and relative humidity (RH) of >90%. Films were tested at 0 h, 4 h, 8 h, 12 h
and 24 h. One set of inoculated samples was immediately tested after 0 h by placing the samples
in a sterilised stomacher bag. 20 mL of Maximum Recovery Diluent (MDR) was poured and mixed
gently by the stomacher (AGB Scientific-Lab blender 400) for 40–45 s. For the viable cell counts the
samples were taken from the MRD culture, appropriately diluted and plated on Tryptic Soy Agar
plates. The above process is repeated to determine the viable cell count after 4 h, 8 h, 12 h and 24 h
samples and the control sample after their incubation.

2.3.5. Biofilm Inhibition

To study the biofilm inhibition of the essential oil composite film common food borne pathogen
E. coli (NCTC 9001) was used. Three square pieces of 4 cm × 4 cm of each film (one set for positive control
and two sets for biofilm inhibition) were cut and sterilised using UV light for 20 min. Films were then
placed on the aluminium plate (5 cm × 5 cm) and transferred into a sterilised petri dish containing filter
paper which was wetted with autoclaved water. 400 µL of the prepared test inoculum (106 CFU/mL)
was put on the film and incubated at 37 ◦ C and relative humidity (RH) of >90% for 72 h. After the
incubation, each film was rinsed with sterile water three times. The films were then stained with 1%
(w/v) crystal violet (500 µL) for 45 min [28]. The test film samples were again washed thrice with the
sterile distilled water. Biofilms were quantified by eluting crystal violet with 95% ethanol (500 µL) for
10 min and transferring 100 µL aliquots to 96 well plates. The absorbance was then determined at
600 nm to determine the percent inhibition of E. coli biofilm.

2.4. Statistical Analysis

All measurements are determined in triplicates. Statistical differences between multiple sample
comparisons were evaluated by analysis of variance (ANOVA) and multiple comparisons (Fischer’s
least significant difference test) using STATGRAPHICS Centurion XV software (Stat Point Technologies
Inc. Warrenton, VA, USA). Differences were considered to be significant if the value of p < 0.05.
All results are stated as mean ± standard deviation.
Foods 2020, 9, 1117 6 of 16

3. Results and Discussion

3.1. Surface Colour

To make the food product look more attractive, is played by the appearance of the active food
packaging. The surface colour and transparency are the major factor to adopt the food packaging.
The film appeared to be homogeneous, smooth and flexible. L (+lightness, −darkness), a (+redness,
−greenness), and b (+yellowness, −blueness) values of the composite films incorporated with the
essential value steadily increased (Tables 1 and 2). With the increase in the concentration of both the
essential oils (thyme and clove) the lightness was observed to decrease significantly (p < 0.05). Also,
the a-value of the thyme oil composite film significantly decreased by 1.2 folds while, for the clove oil
composite film decreased by 2.07 folds as the oil concentration increases from 1 wt% to 10 wt%.

Table 1. Surface colour, UV barrier and transparency of composite films with Thyme oil.

Total Colour
Redness, Yellowness, Transmittance Transmittance
Film Lightness (L) Difference
Greenness (a) Blueness (b) (T280 (%)) (T600 (%))
(∆E)
PLA/PBAT 91.56 ± 0.36 a −1.08 ± 0.02 c 1.34 ± 0.03 a 2.42 ± 0.35 d 0.05 ± 0.00 b 72.46 ± 0.63 a
a a a 73.43 ± 1.45 a
PLA/PBAT-Thyme1% 93.21 ± 0.08 b −1.17 ± 0.02 b 1.31 ± 0.04 0.97 ±0.24 0.04 ± 0.01
PLA/PBAT-Thyme5% 92.93 ± 0.05 c −1.27 ± 0.01 a 1.69 ± 0.03 b 1.22 ± 0.04 b 0.04 ± 0.01 a 81.28 ± 2.63 b
a c c a
PLA/PBAT-Thyme10% 92.95 ± 0.03 d −1.30 ± 0.02 1.89 ± 0.07 1.29 ± 0.05 0.04 ± 0.01 81.28 ± 3.32 b
The letters (a–d) at each concentration specifies groups that are significantly different (p < 0.05).

Table 2. Surface colour, UV barrier and transparency of composite films with Clove oil.

Redness, Yellowness, Total Colour Transmittance Transmittance

Film Lightness (L)
Greenness (a) Blueness (b) Difference ∆E (T280 (%)) (T600 (%))
PLA/PBAT 91.56 ± 0.54 a −1.08 ± 0.02 d 1.34 ± 0.03 a 2.42 ± 0.03 b 0.05 ± 0.00 c 72.46 ± 1.07c
PLA/PBAT-Clove1% 93.12 ± 0.96 a −1.41 ± 0.01 c 2.08 ± 0.05 b 1.33 ± 0.04 a 0.02 ± 0.00 b 71.63 ± 0.35c
a c c a
PLA/PBAT-Clove5% 92.55 ± 0.79 −2.06 ± 0.03 b 4.23 ± 0.25 3.55 ± 0.04 0.01 ± 0.00 69.63 ± 0.68 b
a a a 65.53 ± 0.93a
PLA/PBAT-Clove10% 92.14 ± 1.18 −2.99 ± 0.02 7.78 ± 1.35 d 7.15 ± 0.04 d 0.01 ± 0.00
The letters (a–d) at each concentration specifies groups that are significantly different (p < 0.05).

In addition, the yellowness (b-value) of films increased significantly in the clove oil composite
film. It was observed that the yellowness of the thyme composite film increased by 1.4 folds with the
increase in the concentration of the oil, whereas, in the clove oil composite oil increased by 3.7 folds.
The greenness and yellowness values of the clove oil composite film (PLA/PBAT-clove oil films) are
significantly different from each other and depicted the highest a and b values, which means less
green and more yellow as compared to PLA/PBAT-thyme oil films. The phenolic compounds present
in the clove oil could be a possible reason for the increase in the yellowness in the PLA/PBAT-clove
oil films [29]. The thyme oil composite film had shown decrement in the total colour difference (∆E
values) while, of PLA/PBAT-clove blend film ∆E values increased as the oil concentration increased
(Tables 1 and 2). Similar results have been observed by various studies [30], after the incorporation
of the clove oil in polymer film has observed the transformation of colour from bluish to yellowish.
Furthermore, Ejaz [31] et al., had observed the decrease in transparency with the increase in the
concentration of clove oil in the film matrix due to the presence of the colouring elements in essential
oil and heterogenous network of the film.

3.2. Optical Properties

Transparency of the active food packaging plays a very important role in the acceptability of film
by the consumers. Another optical property which plays a significant role in the food industry is the
UV barrier property of the film. The film must be able to prevent the oxidation of the food induced by
the UV light and increase the shelf life of the food. The transmission spectrum of light was measured
at 280 nm (T280), which indicates the superior UV barrier property of the film significantly (p < 0.05).
The decrease in the transmittance at 280 nm was due to the interruption in the passage of light
through the film surface [32,33]. The T280 (for UV light) depicted that for the PLA/PBAT-Thyme10% UV
light blocking property increased by 20% with respect to the control film while, of the PLA/PBAT-
Clove10% increased by 80% (Tables 1 and 2). A very high concentration of the phenolic compound in
Foods 2020, 9, 1117 7 of 16
the clove oil absorbed UV light which results for the best UV blocking properties for the clove oil
composite film. These results align with various studies in which a UV barrier property had been
observed
at 280–700by nmtheto incorporation
determine the of cloveproperties
optical essential oil in the
of the film matrix.
composite Mulla 1a,b).
film (Figure [33] etThe
al., control
observedfilma
significant
(PLA/PBAT) increase
showed inthe
thehigher
UV barrier property after
transmittance at 300the incorporation
nm, whereas theof clove oil.
essential oilSimilarly, Ejaz film
incorporated [31]
et al., had
showed noobserved the improvement
or low transmittance at 300innm.
the UV blocking property of the composite film.

90

80

70 Transparency at 600 nm
Transmittance (%)

60

50

40

30

20

10 UV light barrier at 280 nm (a)

0
280 330 380 430 480 530 580 630 680
Wavelenght (nm)
PLA/PBAT PLA/PBAT-Thyme 1%
PLA/PBAT-Thyme 5% PLA/PBAT-Thyme 10%

90
80
70
Transparency at 600 nm
Transmittance (%)

60
50
40
30
20
10 (b)
0
280 330 380 430 480 530 580 630 680
Wavelenght (nm)
PLA/PBAT PLA/PBAT-Clove 1%
PLA/PBAT-Clove 5% PLA/PBAT-Clove 10%

Figure 1. Light
Light transmittance
transmittance spectra
spectra of
of PLA/PBAT
PLA/PBAT incorporated
incorporated with
with (a) thyme
thyme oil and (b) clove oil
composite film.

TAs
600 seen in the light)
(for visible Figuredepicts
1b, the
thetransmittance
transparency of
of the PLA/PBAT-thyme
the composite films. Nooilsignificant
films haddifference
shown a
remarkable
was observed increase
between above 280film
control nmand
whereas,
1 wt%the PLA/PBAT-clove
thyme oiland
composite film, films increased
between above
5 wt% and 380 nm.
10 wt%
Furthermore, the incorporation of the essential oil (clove and thyme oil) decreased the transmittance
at 280 nm (T280 ), which indicates the superior UV barrier property of the film significantly (p < 0.05).
The decrease in the transmittance at 280 nm was due to the interruption in the passage of light through
the film surface [32,33]. The T280 (for UV light) depicted that for the PLA/PBAT-Thyme10% UV light
blocking property increased by 20% with respect to the control film while, of the PLA/PBAT-Clove10%
increased by 80% (Tables 1 and 2). A very high concentration of the phenolic compound in the clove
oil absorbed UV light which results for the best UV blocking properties for the clove oil composite
film. These results align with various studies in which a UV barrier property had been observed by the
incorporation of clove essential oil in the film matrix. Mulla [33] et al., observed a significant increase
Foods 2020, 9, 1117 8 of 16

in the UV barrier property after the incorporation of clove oil. Similarly, Ejaz [31] et al., had observed
the improvement in the UV blocking property of the composite film.
T600 (for visible light) depicts the transparency of the composite films. No significant difference
was observed between control film and 1 wt% thyme composite film, and between 5 wt% and 10 wt%
thyme composite film. The transmittance of light at 600 nm of the control film is observed to be 72.46%.
On the incorporation of thyme oil (10 wt%) transmittance value is observed as 81.28% (Table 1) whereas
by the incorporation of clove oil (10 wt%) it becomes 65.53% (Table 2). The value observed suggests that
on the incorporation of thyme oil the transparency of the film increased, while clove oil incorporation
had decreased the transparency of the film. Teixeria [34] et al., had also observed the decrease in the
transparency due to the incorporation of clove oil. Sanuja [35] et al., had also observed the increase in
the opaqueness and decrease in the transparency of the packaging film due to the incorporation of
the clove oil. The transparency of the PLA/PBAT-clove oil films is observed to decrease due to the
presence of phenolic compounds which led to the yellowish tint composite film. Therefore, due to the
high property of UV blocking PLA/PBAT-clove films could be used as an active packaging material.

3.3. FTIR Analysis

For the qualitative analysis of the chemical properties of the composite film Fourier transform
infrared spectroscopy (FTIR) was performed. The FTIR spectra has shown the peaks demonstrating
the chemical composition of the composite film (Figure 2). The overlapping peaks at 727,884 and
1445 cm−1 , demonstrated the presence of the aromatic compounds with C-H and C-C stretch (in the
ring). The peaks determined in the range of 1073–1079 cm−1 stands for the aliphatic amines with C-N
stretch. Range of 1176–1180 cm−1 stands for the C-O vibrational stretching, which depicts the presence
of alcohols, carboxylic acids, esters and ethers. The existence of alkanes in the composite films was
determined by their C-H vibrational stretching in the range of 1353–1364 cm−1 . The occurrence of O-H
stretching resulted in a wide peak at 3265–3316 cm−1 wavelength range indicating the presence of
alcohols and phenols. It was observed that as the concentration of the essential oil in the composite film
increased the functional groups like O-H had shown shorter peaks, whereas, the peaks exhibiting C-N,
C-O, -CHO had become sharper (Figure 2). Correa-Pacheco [36] et al., had mentioned that essential
oils are unstable compounds and are susceptible to transformation, polymerization and oxidation,
which could be a possibility of the chemical changes occurring in the PLA/PBAT composite films.
Foods 2020, 9, 1117 9 of 16
Foods 2020, 9, x FOR PEER REVIEW 9 of 16

Figure 2. FTIR spectra of PLA/PBAT incorporated with (a) thyme oil and (b) clove oil composite film.
Figure 2. FTIR spectra of PLA/PBAT incorporated with (a) thyme oil and (b) clove oil composite film.
3.4. Thickness of the Films
3.4. Thickness of the Films
Digital micrometer was used to measure the thickness of the composite films. It was observed
Digital
that the micrometer
thickness was usedfilms
of the composite to measure
increasesthe thickness
with of the
the increase incomposite films. Itofwas
the concentration observed
the essential
that
oil the thickness
(thyme of theoil).
oil and clove composite films increases
The thickness withoil
of the thyme theincreased
increase byin 3.08
the concentration
folds (113.33 µmof the
of
essential oil (thyme10%oil and clove oil). The thickness of the thyme oil increased
PLA/PBAT-Thyme ) with respect to the control PLA/PBAT film (36.71 µm) while, the thickness of the by 3.08 folds (113.33
μm of
clove oil PLA/PBAT-Thyme
10%) with respect to the control PLA/PBAT film
composite film increased by 2.90 folds (106.67 µm of PLA/PBAT-Clove10%(36.71 μm) significant
) depicting while, the
thicknessin
difference ofthe
thevalues
clove (Tables
oil composite
3 and 4).film increased
Similar resultsby
are2.90
seenfolds (106.67
in studies μm of
where thePLA/PBAT-Clove
thickness increased )
10%

depicting
with significant difference
the incorporation in the
of essential oil values (Tablespackaging
in the active 3 and 4). Similar resultsThis
film [37,38]. are seen
couldin be
studies where
due to the
the thickness
distribution increased
pattern of thewith the incorporation
essential of essential
oil in the matrix oil in the active packaging film [37,38].
of the film.
This could be due to the distribution pattern of the essential oil in the matrix of the film.

3.5. Tensile Properties of the Films

Foods 2020, 9, 1117 10 of 16

Table 3. Thickness, tensile properties and hydrophobicity of the thyme oil composite films.

Water Contact
Tensile Strength Elongation at
Film Thickness (µm) Angle (WCA
(TS (MPa)) Break (EB (%))
(Degree))
PLA/PBAT 36.71 ± 4.16 a 1.35 ± 0.03 b 5.63 ± 0.41 b 61.61 ± 2.82 a
PLA/PBAT-Thyme1% 46.67 ± 5.73 a,b 1.52 ± 0.08 b 3.10 ± 0.38 a 72.57 ± 1.54 b
PLA/PBAT–Thyme5% 63.33 ± 10.17 b 1.26 ± 1.26 b 5.84 ± 0.25 b 74.06 ± 3.00 b
PLA/PBAT–Thyme 10% 113.33 ± 14.59 c 0.96 ± 0.08 a 16.51 ± 0.47 c 80.57 ± 2.28 c
The letters (a–d) at each concentration specifies groups that are significantly different (p < 0.05).

Table 4. Thickness, tensile properties and hydrophobicity of the clove oil composite films.

Water Contact
Tensile Strength Elongation at
Film Thickness (µm) Angle (WCA
(TS (MPa)) Break (EB (%))
(Degree))
PLA/PBAT 36.71 ± 4.58 a 1.35 ± 0.03 c 5.63 ± 0.41 a 61.61 ± 2.82 a
PLA/PBAT-Clove1% 46.67 ± 3.59 a 0.94 ± 0.08 b 39.97 ± 0.09 d 62.24 ± 5.75 a
PLA/PBAT-Clove5% 73.33 ± 7.07 b 0.89 ± 0.06 a, b 27.58 ± 0.36 c 64.69 ± 2.15 a
PLA/PBAT-Clove 10% 106.67 ± 16.68 c 0.79 ± 0.03 a 25.67 ± 0.52 b 74.74 ± 4.11 b
The letters (a–d) at each concentration specifies groups that are significantly different (p < 0.05).

3.5. Tensile Properties of the Films

Universal Testing Machine (Zwick/Roell-A730271) was used to determine the mechanical
properties (tensile strength (TS) and elongation at break (EB)) of the essential oil incorporated composite
film. The tensile strength of the composite films was observed to decrease with the incorporation of
the essential oils (thyme oil and clove oil). The tensile strength of the control PLA/PBAT film was
observed as 1.35 MPa (Tables 3 and 4). No significant difference in the control film, 1 wt% and 5 wt%
thyme oil composite film was observed. As the thyme oil in the composite film increases from 1 wt%
to 10 wt% the TS decreases from 1.52 MPa to 0.96 MPa, respectively, whereas, the increase in the clove
oil concentration in the composite film the TS decreases from 0.94 MPa to 0.79 MPa. This shows a
significant difference in the 10 wt% thyme oil composite film and 10 wt% clove oil composite films from
other films. The increase in the concentration of the oil in the composite oil could weaken the structure
as the stronger polymer-polymer interaction is replaced by a weaker polymer-oil interaction [39,40].
The flexibility of the film is depicted by the elongation at break (EB). The EB is observed to increase
with the increase in the concentration (Tables 3 and 4). A significant difference between the 10 wt%
thyme oil, 10 wt% clove oil composite film and the control film was observed. The EB of the control
film PLA/PBAT was observed to be 5.63%. With the increase in the thyme oil concentration in the
composite film (PLA/PBAT-Thyme10% ) the EB increases to 16.51%, while, for the increase in the clove
oil concentration in the composite film (PLA/PBAT-Clove10% ) the EB increases to 25.67%. The improved
flexibility of the composite films is due to a better interaction between PLA and PBAT polymers and a
presence of the essential oil.
Many studies show that the incorporation of the essential oil influences the TS depending on the
interaction of the oil with the composite film matrix [39]. Bonilla [41] et al., had incorporated basil and
thyme essential oil in chitosan film and studied that the film had become more fragile with increased
strechability [41]. Furthermore, Chen [42] et al., had studied 14.13% decrease in TS and 26.64% increase
in EB after the incorporation of clove oil in poly(vinyl alcohol) active film. Hosseini [43] et al., had also
stated that the destabilization process could take place during the drying of the film and could lead to
the reduction in TS.
Foods 2020, 9, 1117 11 of 16

3.6. Water Contact Angle (WCA)

Water Contact angle (WCA) is used to determine the surface hydrophobicity. WCA indicates the
interaction of the film with the liquids which is important for the application of the film in the food
industry [44]. The surface is determined to be hydrophobic if the contact angle higher than 65◦ [22,45].
The control PLA/PBAT film has shown WCA as 61.61◦ (Tables 3 and 4). No significant difference was
observed in the WCA value of the control, 1 wt% and 5 wt% of the essential oil (thyme oil and clove
oil) incorporated films, whereas the WCA value of higher concentration (10 wt% of thyme and clove
oil) composite film showed significant difference. As the thyme oil concentration in the composite
film increased from 1 wt% to 10 wt% the WCA increased from 72.57 to 80.57◦ , whereas as the clove oil
concentration increased from 1 wt% to 10 wt% in the composite film the WCA increased from 62.24 to
74.74◦ . Therefore, the incorporation of the clove oil (10 wt%) and thyme oil (1 wt%, 5 wt% and 10 wt%)
had increased the hydrophobicity of the film, which could play a significant role in the increase in the
quality of the active packaging.

3.7. Antibacterial Activity

Antibacterial active packaging prevents the growth of bacteria on the surface of the food due
to direct contact of the packaging material. The antibacterial efficiency of the essential oil (thyme
and clove oil) incorporated composite film was determined against E. coli (Gram negative bacteria)
and S. aureus (Gram positive bacteria). The incorporation of the clove oil in the composite film had
demonstrated significant antibacterial activity. It was also observed in Figure 3c,d that, as the thyme oil
composite film doesn’t demonstrate antibacterial activity for 24 h. Though 5 wt% thyme oil composite
film or the thymol content shows antimicrobial activity for the first 4 h against E. coli whereas, 10 wt%
thyme oil composite film shows antibacterial activity for 8 h against S. aureus and 12 h against E. coli.
Thyme oil composite film (10 wt%) slightly reduces the growth of E. coli from 6.5 log CFU/mL to
6.32 log CFU/mL in 12 h after that it reaches to 6.84 log CFU/mL.
As shown in Figure 3a, the 1 wt% clove oil composite film has shown antibacterial effect against
E. coli and S. aureus in the first 4 h, whereas 5 wt% clove oil composite have shown antibacterial activity
against E. coli up till 12 h, after that it has not shown antibacterial activity. However, in the presence of
10 wt% clove oil composite film (PLA/PBAT-Clove10% ) E. coli growth had reduced from 6.5 log CFU/mL
to 4.4 log CFU/mL. However, an exceptional antibacterial activity has been observed by 10 wt% clove
oil composite film against S. aureus. In presence of 5 wt% clove oil composite film the S. aureus growth
had reduced from 6.5 log CFU/mL to 4.5 log CFU/mL. 10 wt% clove oil composite film had shown
a complete killing of S. aureus by reducing growth from 6.5 log CFU/mL to 0 log CFU/mL. It has
been reported that the incorporation of essential oil including clove oil has higher bacterial inhibition
efficacy against Gram positive bacteria [46]. Mulla [33] et al., had also studied strong antibacterial
efficacy of clove essential oil against S. typhimurium and L. monocytogenes. Moreover, Mupalla et al.,
(2014) had observed that the clove oil film had shown promising antibacterial activity. Also, the films
were observed to be more active against S. aureus than B. cereus [47].
Foods 2020, 9, 1117 12 of 16
Foods 2020, 9, x FOR PEER REVIEW 12 of 16

Figure
Figure3.3.Antimicrobial
Antimicrobial efficiency of of
efficiency composite
compositefilm. (a) effect
film. of clove
(a) effect oil composite
of clove film on
oil composite filmE.on
coli,E.(b) effect
coli,
of(b)
clove oilofcomposite
effect film on S. film
clove oil composite aureus
on (c) effect of
S. aureus (c)thyme oilthyme
effect of composite film on E.
oil composite colion
film and (d) effect
E. coli and of
thyme oil composite
(d) effect of thyme oilfilm on S. aureus.
composite film on S. aureus.
 Aggregation of bacteria on the surface led to the formation of the biofilm. Biofilms on the food
contact surfaces causes critical problems in the food industry. A biofilm study was observed against
E. coli for 72 h. As shown in Figure 4, the control film shows minimal biofilm inhibition (5.27%). The
thyme oil composite film (1 wt%) had inhibited biofilm by 55.96%, whereas 1 wt% clove oil composite
Foods 2020, 9, 1117 13 of 16
film had inhibited E. coli growth by 60.31%. As the concentration rises to 5 wt%, thyme oil composite
film had inhibited biofilm growth by 71.39% and clove oil composite film had inhibited biofilm
3.8. Biofilm
growth of E.Inhibition
coli by 75.65%.
However, a significant inhibition of E. coli biofilm growth was observed by clove oil composite
Aggregation of bacteria on the surface led to the formation of the biofilm. Biofilms on the food
film (10 wt%). 10 wt% clove oil composite film had inhibited the biofilm by 93.43%. Moreover, clove
contact surfaces causes critical problems in the food industry. A biofilm study was observed against
oil was more effective than thyme oils as they contain higher concentrations of eugenol, 180 mg
E. coli for 72 h. As shown in Figure 4, the control film shows minimal biofilm inhibition (5.27%).
[48].Therefore, the results signify that the high concentration (10 wt%) of essential oils (thyme oil or
The thyme oil composite film (1 wt%) had inhibited biofilm by 55.96%, whereas 1 wt% clove oil
clove oil) inhibit the formation of the biofilm (E. coli) on all tested composite films, though the
composite film had inhibited E. coli growth by 60.31%. As the concentration rises to 5 wt%, thyme oil
percentage inhibition of clove oil (93.43% inhibition) was higher than thyme oil (82.30% inhibition).
composite film had inhibited biofilm growth by 71.39% and clove oil composite film had inhibited
Cui [49] et al., had also studied that the incorporation of clove oil-loaded chitosan nanoparticle had
biofilm growth of E. coli by 75.65%.
inhibited E. coli biofilm up to 99.99%.

100
90
Percentage of biofilm inhibition (%)

80
70
60
50
40
30
20
10
0
PLA-PBAT 1wt% Essential oil 5wt% Essential oil 10wt% Essential oil

Composite film

Thyme oil composite film Clove oil composite film

Figure 4. Biofilm inhibition of PLA/PBAT-Thyme oil composite film and PLA/PBAT-Clove oil composite
Figure 4. Biofilm inhibition of PLA/PBAT-Thyme oil composite film and PLA/PBAT-Clove oil
film against E. coli.
composite film against E. coli.

However, a significant inhibition of E. coli biofilm growth was observed by clove oil composite
4. Conclusions
film (10 wt%). 10 wt% clove oil composite film had inhibited the biofilm by 93.43%. Moreover, clove
Thyme
oil was moreoil and clove
effective thanoils wereoils
thyme incorporated into the
as they contain biodegradable
higher polymer
concentrations (PLA/PBAT)
of eugenol, 180 mgblend[48].
film and their
Therefore, potential
the results as anthat
signify active
thefood
highpackaging
concentration was(10
investigated. Incorporation
wt%) of essential of clove
oils (thyme oil oroilclove
and
thyme
oil) oil had
inhibit the aformation
very highof influence on the
the biofilm composite
(E. coli) film properties
on all tested composite such as optical,
films, though morphological,
the percentage
mechanical
inhibition ofproperties, antibacterial
clove oil (93.43% efficacy
inhibition) wasand
higherbiofilm
than inhibition
thyme oil property. The clove Cui
(82.30% inhibition). oil composite
[49] et al.,
filmsalso
had depicted
studiedpale yellow
that the film with ofless
incorporation transparency
clove and high
oil-loaded chitosan UV-light barrier
nanoparticle property
had inhibited as
E. coli
compared to thyme
biofilm up to 99.99%. oil composite films. Clove oil composite film has shown 80% UV barrier property.
Significant decrease in the tensile strength by the incorporation of the essential oil has been observed.
4.
TheConclusions
tensile strength decreases from 1.35 (control film) to 0.96 MPa (PLA/PBAT-Thyme10%) and 0.79
MPaThyme
(PLA/PBAT-Clove 10%). However, as the concentration of essential oil increases, the water contact
oil and clove oils were incorporated into the biodegradable polymer (PLA/PBAT) blend
angle (WCA) of the composite
film and their potential as an active films has
foodincreased
packaging significantly. Also, a very
was investigated. high antibacterial
Incorporation of clove
property had been shown by clove oil composite film. Clove oil
oil and thyme oil had a very high influence on the composite film properties such composite film had reduced E. coli
as optical,
growth by 2.1 log
morphological, CFU/mL.
mechanical Also, a complete
properties, killing
antibacterial of S.and
efficacy aureus thatinhibition
biofilm is, a reduction from
property. The6.5 log
clove
oil composite films depicted pale yellow film with less transparency and high UV-light barrier property
as compared to thyme oil composite films. Clove oil composite film has shown 80% UV barrier
property. Significant decrease in the tensile strength by the incorporation of the essential oil has been
observed. The tensile strength decreases from 1.35 (control film) to 0.96 MPa (PLA/PBAT-Thyme10% )
and 0.79 MPa (PLA/PBAT-Clove10% ). However, as the concentration of essential oil increases, the water
Foods 2020, 9, 1117 14 of 16

contact angle (WCA) of the composite films has increased significantly. Also, a very high antibacterial
property had been shown by clove oil composite film. Clove oil composite film had reduced E. coli
growth by 2.1 log CFU/mL. Also, a complete killing of S. aureus that is, a reduction from 6.5 log CFU/mL
to 0 log CFU/mL was observed by 10 wt% clove oil composite film. The thyme oil composite film
(10 wt%) had inhibited biofilm by 82.30% against E. coli whereas, clove oil composite film (10 wt%) had
inhibited the E. coli biofilm by 93.43%. Thus, the clove oil composite film has demonstrated strong UV
barrier, antibacterial and biofilm inhibition property, which can avoid the undesirable photochemical
reaction, adherence and growth of pathogenic bacteria which can increase the shelf life of the packed
food. Therefore, clove oil composite film can be used as an active packaging.

Author Contributions: S.S.: Conceptualization, Formal analysis, Data curation, Investigation, Methodology,
Writing-original draft, Writing-review & editing. S.B.: Laboratory work, Data curation, Writing-original draft.
B.D.: Resources, Funding acquisition, Supervision. S.J.: Methodology, Formal analysis, Validation, Resources,
Supervision, Writing-review & editing. A.K.J.: Funding acquisition, Project administration, Conceptualization,
Resources, Supervision, Writing-review & editing. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Acknowledgments: Authors would like to acknowledge the funding from TU Dublin—City Campus, Ireland
under the Fiosraigh Scholarship programme (2017).
Conflicts of Interest: The authors declare no conflict of interest.

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