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Food Packaging and Shelf Life 34 (2022) 100938
Available online 5 September 2022
2214-2894/© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Active packaging lm of chitosan and Santalum album essential oil:
Characterization and application as butter sachet to retard lipid oxidation
María Fl´
orez , Patricia Caz´
on , Manuel V´
azquez
*
Department of Analytical Chemistry, Faculty of Veterinary, University of Santiago de Compostela, 27002 Lugo, Spain
ARTICLE INFO
Keywords:
Film
Sandalwood essential oil
Chitosan
Antioxidant activity
UV-barrier properties, sachets
ABSTRACT
Chitosan with sandalwood (Santalum album) essential oil (SEO) using malic acid as solvent were evaluated as an
active packaging lm. It was applied as sachet to butter packaging. The effects of SEO concentration (0.5 %, 1 %
and 2 % v/v) on chitosan lm properties were studied by measuring the equilibrium moisture content, solubility,
water vapor permeability (WVP), mechanical, optical, heat sealability, antioxidant properties, surface
morphology and thermostability. SEO showed a signicant effect on the lm properties except for the puncture
properties and the equilibrium moisture values. SEO promoted a signicant decrease in tensile strength from
5.78 to 2.99 MPa, Young’s Modulus (YM) from 35.74 to 6.81 MPa, WVP from 6.70⋅10
−11
to 3.34⋅10
−11
g/m⋅s⋅Pa,
and sealability from 195.20 to 107.94 N/m. The antioxidant properties of the lms were improved with the
presence of SEO. The addition of SEO signicantly improved the UV-barrier of the lms. The color and trans-
parency of the samples showed signicant variations by the addition of SEO. The active packaging lm was
evaluated as butter sachet. After 3 months of butter storage, a signicant decrease of the thiobarbituric acid
reactive substances was observed, showing a 36 % decrease in the lipid oxidation compared to unpackaged
samples. The lms are completely water-soluble and can be easily removed from foodstuffs after use without
generating solid wastes.
1. Introduction
Food packaging design is one of the most important phases in the
food chain. A perfect food packaging material should protect food
quality over time, being transportable, convenient to use, economical,
and renewable or biodegradable (Priyadarshi & Rhim, 2020). Moreover,
if the packaging has a positive effect on the food, as in the case of active
packaging, its use becomes even more interesting. Active lms are lms
deliberately enriched with active components. These components
release or absorb chemicals in order to prolong the shelf life of the food
while maintaining its sensory and quality characteristics (Yildirim et al.,
2018). Natural and renewable materials are gaining interest due to the
major environmental problems generated by the uncontrolled use of
petroleum derivatives, but also because the consumer has undergone a
trend towards eco-friendly habits.
A wide variety of polysaccharides are being studied for development
as lms intended for food use, such as cellulose (Sirvi¨
o et al., 2014),
starch (Abreu et al., 2015), alginate (Parreidt et al., 2018) and chitosan
(Priyadarshi et al., 2018). Chitosan is the second most abundant
polysaccharide in the world. The exoskeleton of crustaceans is the main
source to obtain chitin and then chitosan by deacetylation. Chitosan and
its derivatives can inhibit the growth of a wide range of molds, yeasts
and bacteria. Chitosan properties like solubility, lm forming ability,
viscosity, chelating ability, antimicrobial properties, among others,
make it an interesting material to apply in multiple areas, including food
packaging (Priyadarshi & Rhim, 2020). Chitosan is soluble in dilute
organic acid which facilitates its application as a lm or coating. The
properties of the chitosan matrix are highly dependent on the type of
solvent used (Rhim et al., 1998). The correct selection of the acid used as
solvent will allow to adapt the properties of the matrix to its purpose. For
example, the use of malic acid results in water-soluble lms that can be
easily removed from foodstuff (Caz´
on et al., 2021). Water soluble lms
can be applied in foods with high fat content and low moisture content,
such as butter, margarine, lard, and oil.
On the other hand, several natural substances, as essential oils are
often added to chitosan lms to enhance their active properties.
Essential oils are aromatic oily liquids obtained from various parts of
plants, such as owers, seeds, roots, wood, herbs, leaves or shoots (Burt,
* Corresponding author.
E-mail address: manuel.vazquez@usc.es (M. V´
azquez).
Contents lists available at ScienceDirect
Food Packaging and Shelf Life
journal homepage: www.elsevier.com/locate/fpsl
https://doi.org/10.1016/j.fpsl.2022.100938
Received 18 May 2022; Received in revised form 4 August 2022; Accepted 22 August 2022
Food Packaging and Shelf Life 34 (2022) 100938
2
2004). Sandal is a type of wood from trees of the genus Santalum. San-
dalwood essential oil (SEO) is clear to yellowish in hue and thick. Its
main use is in the perfume industry, due to its aroma and xative
properties. However, it is also used in the food industry as a avor
ingredient. This essential oil is made up of two main components known
as sesquiterpene alcohols: cis-
α
-santalol (7–60 %) and cis-β-santalol
(7–33 %). These alcohols are responsible of the characteristic odor of
sandalwood. Nevertheless, there are minor constituents that should be
taken into account, such as sesquiterpene hydrocarbons (6 %), esters
(2–4 %), phenols, lactones, terpenes, volatile compounds and fragrant
substances (Burdock & Carabin, 2008; Subasinghe et al., 2013).
SEO has been approved as generally recognized as safe (GRAS) for
use in food as a avoring component by the Flavor and Extract Manu-
facturers Association (No. 3005). It also has been approved by the FDA
as a natural avoring substance that can be used in combination with
other avors. Council of Europe added sandalwood to the list of sub-
stances, spices, and seasonings (Burdock & Carabin, 2008).
The chitosan lms enriched with essential oils have been tested in
multiple foodstuffs. However, no studies have been carried out on water-
soluble chitosan-based active lms for extending the shelf-life of foods.
The main advantage of these lms is that they are easily disposable
without environmental impact, but their soluble nature limits their ap-
plications in foods with high moisture content. Nevertheless, they can be
an useful tool for preserving foods with high lipid content. This type of
food tends to suffer easily from lipid oxidation, which causes off-avors
and degradation of colors and nutrients (Vieira et al., 2017).
To our knowledge, SEO-chitosan lms have not been studied.
Considering the potential application of SEO in food packaging and the
suitability of chitosan as a matrix to carry active agents, the objectives of
this study were to develop and characterize the physic-chemical prop-
erties of a novel SEO-chitosan lm. The effect of SEO at different ratio on
the mechanical, water vapor permeability (WVP), optical, equilibrium
moisture content (%W), solubility, sealability (S), and antioxidant
properties of chitosan lms were evaluated. The surface morphology,
compatibility, and thermostability of the lm were also evaluated using
scanning electron microscope (SEM), Fourier transform infrared spec-
troscopy (FT-IR) and simultaneous thermal analysis (TGA/DSC).
Considering the water-soluble nature of chitosan lm from malic acid
solutions (Caz´
on et al., 2021), its antioxidant and lipid oxidation
retarding capacity has been evaluated in high-fat foods, by selecting
butter as a model food. Its application in butter has been tested by
analysing the antioxidant and optical properties of the lms as well as
the thiobarbituric acid reactive substances (TBARS) of the butter sam-
ples after 90 days of storage.
2. Experimental
2.1. Materials
Chitosan (M
w
100000–300000 and CAS number 9012–76–4) was
purchased from Acros organics (Geel, Belgium) and DL-malic acid extra
pure (CAS number 6915–15–7) was provided by Scharlau Microbiology
(Barcelona, Spain). Soy lecithin supplied by Ynsadiet (Madrid, Spain)
and pure sandalwood essential oil supplied by Pranarˆ
om (Ghislenghien,
Belgium) were used to prepare the chitosan lm-forming solution. The
food packaging properties of the developed lms were tested on butter
purchased from Mantequería Arias (Madrid, Spain).
2.2. Preparation of lms
Chitosan lms were made by dissolving 1 % (w/w) chitosan in an
aqueous solution of 2 % (w/v) malic acid (Table 1). The acid concen-
tration was adjusted until the chitosan was completely dissolved,
yielding a clear solution. The stock solution was kept stirred overnight at
room temperature. As a natural emulsier, soy lecithin (0.1 % w/v) was
added to the solution. Chitosan-soy lecithin solution was homogenized
at 15,000 rpm for 5 min (Ultra Turrax®, IKA, Staufen, Germany). The
foam formed in the mixture was removed with the aid of an ultrasonic
bath for 15 min. Subsequently, the SEO was added until a nal con-
centration ranged 0–2% (v/v), and homogenized at 15,000 rpm for 5
min following by a degassed step in an ultrasonic bath for 15 min.
From the resulting lm-forming solution, 40 ml were poured in a
Petri dishes of 210 mm diameter and dried for 48 h at room temperature.
The lms were cut to a specic size and stored for 5 days at determined
conditions depending on the test. The thickness (mm) was measured at
ve random locations using a Thickness Meter ET115S (Etari GmbH,
Stuttgart, Germany).
2.3. Scanning electron microscope (SEM)
SEM pictures were used to examine and characterize the morphology
of SEO-chitosan lms made with malic acid as solvent. High-vacuum
microscope (JEOL JSM-6360LV, Jeol Ltd, Tokyo, Japan) was used to
photograph dried and gold-coated samples at an accelerating voltage of
20 kV. Using conductive double-sided carbon tape, samples were
adhered to slides.
2.4. Fourier transform infrared spectroscopy (FT-IR)
FT-IR ABB Bomen 102 (ABB Ltd, Zurich Switzerland) tted with a
Universal Attenuated Total Reectance (UATR) accessory (SPECAC
Golden Gate) with diamond crystal was used to examine the presence of
certain chemical groups and crosslinking in the lms. The selected
samples were conditioned for 48 h at 65 ±2 % relative humidity and 21
±1 ºC. FTIR spectra in the range (400–4000) cm
−1
were recorded at a
spectral resolution of 4 cm
−1
.
2.5. Water solubility, equilibrium moisture and water vapor permeability
Water solubility of the samples was measured using the gravimetric
method of immersion in a known volume of distilled water, as described
elsewhere (Caz´
on et al., 2019). The equilibrium moisture content (%W)
was estimated using the gravimetric method by comparing the weights
of conditioned samples at 33 % relative humidity (% RH) and dried
samples (Caz´
on et al., 2019). Water vapor permeability (WVP) was
measured following the ASTM Standard Test Method E96 (https://www.
astm.org/Standards/E96.htm). The WVP of the samples was determined
at 30 ºC and considering 50 % RH, as an intermediate value between the
two levels which dene the RH gradient utilized in the WVP determi-
nation. The test was run at least 4 h, enough time to reach a dynamic
equilibrium in the vapor ux. Each test was carried out by triplicate.
2.6. Mechanical properties analysis
Tensile and puncture tests were performed on the lms with a tex-
turometer (TA-XT plus, Stable Micro System, UK) and the accessories
recommended for each test. Tensile strength (TS, MPa), percentage of
elongation at break (%E, %) and Young’s Modulus (YM, MPa) were
measured by the tensile test. The test was performed using the ASTM
standard method D-882 (https://www.astm.org/d0882–18.html), with
the initial grips spacing set to 40 mm and the load rate at 1 mm/s. The
Table 1
Films composition of lecithin and sandalwood essential oil (SEO). Samples
contains also chitosan 1 % (w/w) and malic acid 2 % (w/v).
Film samples Lecithin SEO
% (w/v) % (v/v)
SEO-chitosan_0 0 0
SEO-chitosan_0.5 0.1 0.5
SEO-chitosan_1 0.1 1.0
SEO-chitosan_2 0.1 2.0
M. Fl´
orez et al.
Food Packaging and Shelf Life 34 (2022) 100938
3
samples were cut to 15 ×100 mm and conditioned for 5 days at room
temperature and 57 % RH before being clamped between the grips of the
texturometer. Seven replicates were tested for each batch (Caz´
on et al.,
2018).
In the puncture test, a lm holder (Reference HDP/FSR, Stable Micro
System, UK) was used with the texturometer to hold the lm sample.
Sample squares for each batch were cut to 30 ×30 mm and conditioned
at 57 % RH for at least 5 days before analysis. A cylindrical probe (d =3
mm) with a speed of 1 mm/s was used and the force was recorded until
rupture. The burst strength (BS) and distance to burst (DB) were
computed using the force (g) against strain (mm) curves.
2.7. Seal strength property
Film samples were cut into 15 ×100 mm. Two lm were placed
together and an area of 10 ×10 mm at the edge of the lms was heat-
sealed using a vacuum heat-sealer (Model PH7 Food Machinery,
Madrid, Spain) and a sealing time of 2.5 s. Seal strength of the heat-
sealed lms was determined according to the ASTM standard method
F88/F88M-21 (https://www.astm.org/f0088_f0088m-21.html) using a
texturometer (TA-XT plus, Stable Micro System, UK). Each tail of the
sealed lm was clamped to the opposing grips and the seal remains
unsupported while the test is being conducted (technique A: unsup-
ported) (Fig. 1). The distance between the grips was set to 50 mm. A 5 kg
load cell and a test speed of 30 mm/min were used. The seal strength (N/
m) was given as the highest force required to cause seal failure. Seal
strength was calculated by the following Eq. 1 (Prateepchanachai et al.,
2019):
Seal strength (N/m) =peak force/film width (1)
2.8. Antioxidant properties of lms
Two procedures were used to determine the antioxidant capacity of
the pure SEO and the SEO-chitosan lms: 1,1-diphenyl-2-picrylhydrazyl
radicals (DPPH
•
) and the 2,2′-azino-bis (3-ethylbenzothiazoline-6-
sulfonic acid) (ABTS
•+
) free radical scavenging assay. The aliquots were
obtained by the methanolic extracts of the lms, using 1 g of sample in
24 ml of methanol and left overnight in darkness.
UV-Vis spectrophotometer V-670 (Jasco Inc, Japan) at 515 nm was
used to examine the radical scavenging on DPPH
•
, and at 734 nm to
analyze the ABTS
•+
radical cation scavenging activity following the
method described elsewhere (Antoniewska et al., 2018; Rutkowska
et al., 2020). The results were expressed as % DPPH
•
and % ABTS
•+
scavenging activity.
2.9. Optical and thermal (TGA/DSC) properties of lms
Spectrophotometer V-670 (Jasco Inc, Japan) was used to measure
the UV-Vis spectra of the lm samples in the UV-Vis light ranges
(190–800 nm) and optical properties of the samples were determined as
described elsewhere (Caz´
on et al., 2019).
TGA/DSC (Mettler Toledo, Switzerland) thermogravimetry and dif-
ferential scanning calorimetry equipment were used to analyze the
thermal stability properties of the lms. The samples were placed in
hermetic aluminum pans, and the test was carried out at a heating rate of
10 ºC/min from 50 to 400 ºC, in a nitrogen atmosphere (50 ml/min).
2.10. Storage stability of butter packed in SEO-chitosan sachets
Rectangular samples of butter (6 g) were packed in chitosan sachets
with different SEO concentration (0–2 %). Unpackaged butter samples
were considered as control sample. The samples were stored at 5 ◦C for 3
months. The storage time was selected based on previous studies which
indicate that there are signicant changes in the quality of butter after 3
months in the refrigerator (Krause et al., 2008; Okturk et al., 2001).
The DPPH
•
and ABTS
•+
parameters were analysed in unpacked and
packed butter samples after 3 months of storage. The procedure fol-
lowed was as aforementioned and the analysis was by triplicated.
The TBARS values in the butter samples were determined using a
spectrophotometric method (Zeb & Ullah, 2016). Butter samples (1 g)
was mixed with 5 ml of acetic acid glacial. Then, the solution was added
to 0.5
μ
L butylhydroxytoluene (BHT). Extraction was performed with
funnel and lter paper. Aliquot of 1 ml of extract was mixed with 1 ml of
thiobarbituric acid solution. The mixture was incubated at 95 ºC for 1 h,
then the absorbance was measured at 532 nm. The results were
expressed as mg malondialdehyde (MDA)/kg butter.
Samples of packaged butter with the lms were analyzed for color
(L*, a*, and b* values) after 3 months of refrigerated storage at 5ºC using
a colorimeter ColorStriker (Mathai, Hannover, Germany). Two measures
were carried out for control, each treatment and replicate.
2.11. Statistical analysis
The results obtained were statistically analyzed employing Microsoft
Excel® software by one-way analysis of variance (ANOVA). The Tukey
Post Hoc test was used to analyze differences between pairs of values
based on condence intervals. The least signicance difference was
p<0.05.
3. Results and discussion
The formulations of the experiments are shown in Table 1. The lm-
forming solution was prepared at room temperature, which resulted in a
low-energy procedure. For complete dissolution of chitosan, a malic acid
concentration of 2 % was required. During the incorporation of soy
lecithin and SEO, some foam was formed under stirring. An ultrasonic
bath was applied to remove the foam, resulting in a bubble-free solution
with a milky appearance. The lms were peeled off the Petri dishes.
They were easy to handle. It was detected a certain adhesion charac-
teristic that could be of interest for their application as food packaging.
Fig. 1. SEO-chitosan lm running sealability test. SEO is sandalwood essen-
tial oil.
M. Fl´
orez et al.
Food Packaging and Shelf Life 34 (2022) 100938
4
3.1. Scanning electron microscopy (SEM)
The SEM images of the bottom, top and cross section are shown in
Fig. 2. Pure chitosan lms showed a smooth and at surface. Never-
theless, a discernible change in the chitosan lm microstructure was
detected when the concentration of SEO in the lms increased. The
bottom SEM images, which correspond to the side in direct contact with
the Petri dish, suggested that the addition of soy lecithin allowed a ho-
mogeneous distribution of the oil in the lms. No irregularities or breaks
were detected on the lms that could indicate that the ultrasonic bath
was effective to remove any bubbles (Caz´
on et al., 2021).
However, the top SEM images indicated that when SEO was incor-
porated into the chitosan lm, the uniformity of the lm’s morphology
was compromised. The top surface suggested that there were certain
areas of heterogeneity. This is attributed to oil droplets trapped in the
polysaccharide network. When the SEO concentration reached 2 %, the
formation of larger oil droplets was observed, this could be due to a
higher frequency of collisions between oil droplets, resulting in a
probable coalescence (Peng & Li, 2014). These oil droplets were not
exactly spherical, as is usual in other emulsions such as oil/water. This
could be related to the traction forces generated by the chitosan network
when the solvent evaporates (Hafsa et al., 2016).
On the other hand, the cross-section images conrm the presence of
an oil phase in the lm, which also causes the lm to cut less cleanly
compared to pure chitosan lm. This was also noticeable when handling
the lms, an increase in adhesiveness could be observed as the oil
concentration increased. Similar behavior has been described for chi-
tosan lms with Citrus limonia essential oil (Gonçalves De Oliveira Filho
Fig. 2. Scanning electron microscopy images of the top, bottom and cross section of the SEO-chitosan lms using malic acid as solvent. SEO is sandalwood
essential oil.
M. Fl´
orez et al.
Food Packaging and Shelf Life 34 (2022) 100938
5
et al., 2020) or Eucalyptus globulus essential oil (Hafsa et al., 2016).
3.2. Fourier transform infrared spectroscopy (FT-IR)
Fig. 3a shown the FT-IR spectra in the range between 4000 and
800 cm
−1
of SEO-chitosan lms. This analysis was done with the aim of
identifying possible molecular interactions between the functional
groups of chitosan, malic acid, soy lecithin and SEO in the lm structure.
The FT-IR spectra of pure chitosan samples showed all the characteristic
bands. The stretching vibrations of -OH and -NH are represented by the
absorption bands at 3500–3400 cm
−1
. The stretching vibrations of the
C-H bond in the -CH
2
group give the absorption peaks at 2928 cm
−1
(Kalaycıo˘
glu et al., 2017). Whereas the amide-I vibrations of stretching
C
–
–
O are represented by the absorption at 1700 cm
−1
, the amide-II vi-
brations of stretching NH
2
are represented by the absorption at
1554 cm
−1
(Priyadarshi et al., 2018). The vibrations of the C-C y C-O
stretches and the bending of C-H bonds in polysaccharide structure can
explain the spectrum absorption between 1200 and 800 cm
−1
(Liu et al.,
Fig. 3. a) FT-IR spectra of SEO-chitosan lms using malic acid as solvent, b) UV–VIS spectra prole of SEO-chitosan lms using malic acid as solvent. SEO is
sandalwood essential oil.
Table 2
Physical and mechanical properties of chitosan lms. Film composition is described in Table 1.
Film samples %W WVP TS %E
% g/m⋅s⋅Pa MPa %
SEO-chitosan_0 0.78 ±0.13 6.70⋅10
−11
±7.09⋅10
−12 a
5.78 ±2.62
a
27.68 ±2.91
a
SEO-chitosan_0.5 0.60 ±0.06 4.52⋅10
−11
±5.22⋅10
−12 b
11.40 ±2.70
b
48.86 ±6.38
b
SEO-chitosan _1 0.68 ±0.11 4.26⋅10
−11
±1.56⋅10
−12 bc
2.20 ±0.43
c
74.01 ±10.9
c
SEO-chitosan _2 0.78 ±0.20 3.34⋅10
−11
±8.28⋅10
−12c
2.99 ±0.43
c
94.53 ±9.98
d
Film samples YM BS DB Sealability (S)
MPa g mm N/m
SEO-chitosan_0 35.74 ±12.07
a
3209.49 ±1222.02 7.18 ±0.78 195.20 ±33.74
a
SEO-chitosan_0.5 68.43 ±12.89
b
3147.06 ±404.13 6.34 ±0.67 80.88 ±6.49
bc
SEO-chitosan_1 7.84 ±0.74
c
2680.35 ±543.72 6.36 ±0.89 79.09 ±1.72
b
SEO-chitosan_2 6.81 ±0.80
c
2874.04 ±380.04 6.19 ±0.34 107.94 ±8.45
c
SEO – sandalwood essential oil; %W - equilibrium moisture content; WVP - water vapor permeability; TS - tensile strength; %E - percentage of elongation to break; YM -
Young’s modulus; BS - burst strength (puncture properties); DB - distance to burst (puncture properties);
Values are expressed as mean ±standard deviation. Different letters in the same column indicate signicant differences (p ˂ 0.05).
M. Fl´
orez et al.
Food Packaging and Shelf Life 34 (2022) 100938
6
2013). An important change was found with a new peak at 1745 cm
−1
,
indicating that with increasing SEO concentration there was an increase
in the interaction between CH and the essential oil. The C
–
–
O stretching
vibrations in the ester structures contained in fatty acids were respon-
sible for this band, which is the distinctive band of oils (Priyadarshi
et al., 2018). As a result of the probable interactions between chitosan,
oil and soy lecithin, the intensity of the amide-II peak of chitosan in the
blend lm was reduced.
It can be determined that the addition of SEO to the chitosan lms
exerts modications on the spectra of the chitosan lms. All these
changes could be related to a possible intermolecular interaction and
molecular compatibility between the functional groups of the essential
oil and the hydroxyl and amino groups of the chitosan matrix. Similar
results were observed when various types of fats (Akyuz et al., 2018),
citronella and cedarwood essential oil (Shen & Kamdem, 2015) or
turmeric extract (Kalaycıo˘
glu et al., 2017) were added to chitosan lms.
3.3. Evaluation of the water solubility, equilibrium moisture content and
water vapor permeability (WVP)
To determine the water solubility of the lms, the soluble matter of
the samples was analysed. After a few seconds of immersion in distilled
water, the lms lost the whole structure and is completely water soluble.
This is because malic acid is not a volatile solvent, therefore it remained
in the matrix and improved lm solubility. In addition, it was observed
that the presence of SEO in the matrix did not decrease the solubility of
the lms. This is an interesting because the lm can be easily removed
from foodstuffs avoiding solid wastes.
The equilibrium moisture content (%W) of SEO-chitosan lms
ranged between 0.60 % and 0.78 % as shown in Table 2. The addition of
SEO to the chitosan lms showed no signicant differences (p>0.05) in
equilibrium moisture content.
The WVP values calculated for chitosan lms were ranged between
3.34⋅10
−11
−6.70⋅10
−11
g/m⋅s⋅Pa, as shown Table 2. The addition of
SEO showed a signicant difference on WVP values (p<0.05) as
compared with pure chitosan lms. The presence of hydrophobic
compounds of the essential oil promoted the decrease of WVP, reaching
a reduction of almost 50 % at a 2% SEO concentration. The presence of
the oil droplets observed in the SEM images (Fig. 2) created a tortuous
path that made it difcult for the water molecules to permeate through
the matrix. Moreover, the increased number of hydrogen bonds between
the functional groups of the chitosan and the SEO may decrease the
availability of hydrophilic groups to interact with water molecules,
resulting in a more water-resistant layer (Shen & Kamdem, 2015).
The observed effect of SEO addition on chitosan lms in the present
study is in agreement with the results obtained by Shen and Kamdem
(2015), who found that adding 10 % (w/w) citronella and cedarwood
essential oil into chitosan lms resulted in a considerable reduction in
the WVP of the samples. In a similar way, Yuan et al. (2016) observed a
signicant decrease in the WVP of chitosan lms with cinnamon
essential oil.
It should be noted that certain factors such as chitosan properties,
lm composition, WVP method and measurement conditions or lm
thickness, are important for the subsequent WVP results (Rhim et al.,
1998).
3.4. Evaluation of the mechanical properties
Table 2 shows the results of mechanical properties obtained from the
tensile and puncture tests. The tensile test for SEO-chitosan lms showed
values in the range 2.20–11.40 MPa for TS, 27.68–94.53% for E% and
6.81–68.43 MPa for YM. The addition of SEO to chitosan lm had a
signicant effect (p<0.05) on the three tensile properties analysed.
The values of TS and YM decreased while the %E values increased by
the addition of SEO, indicating a decrease in the resistance to break and
an increase in the deformation capacity by the addition of SEO. The
changes in the mechanical behavior could be explained by the structural
arrangement of the lipid phase in the chitosan matrix, as shown the SEM
images (Fig. 2). The structural arrangements resulted in a discontinuous
and open matrix with unequal physical interactions (Fl´
orez et al., 2022).
Besides, the incorporation of essential oil into chitosan lms could
disrupt polymer-polymer interactions, leading to weaker essential
oil-polymer interactions, resulting in a plasticizing effect of the essential
oil (Hosseini et al., 2015). The new interactions gave a less stiff structure
due to the reduction of the cohesion of the polymer network forces,
decreasing the TS and YM values and increasing the %E. Same trend in
the mechanical properties was observed when Origanum vulgare (Hos-
seini et al., 2015) or Melaleuca alternifolia (Caz´
on et al., 2021) essential
oil were added to chitosan lms.
The puncture test results revealed a statistically non-signicant ef-
fect of SEO concentration on BS and DB values, despite a slightly
decrease of the values was observed (Table 2). The samples’ deformation
and how the polymer chains must arrange under deformation forces in
several directions differ from the restructuring and deformation produce
Table 3
Antioxidant properties of the developed chitosan-based lms and SEO. Film
composition is described in Table 1.
Samples DPPH
•
(%)
ABTS
•+
(%)
SEO 78.23 ±4.87 15.65 ±0.86
SEO-chitosan_0.5 7.34 ±1.64
a
2.54 ±0.27
a
SEO-chitosan_1 12.74 ±3.38
a
8.75 ±0.10
b
SEO-chitosan_2 23.69 ±6.03
b
19.22 ±0.57
c
SEO - sandalwood essential oil
Values are expressed as mean ±standard deviation.
Different letters in the same column indicate signicant differences (p ˂ 0.05).
Table 4
Optical properties and color parameters of the lms. Film composition is described in Table 1.
Film samples UV-C UV-B UV-A Transparency Opacity
%T %T %T
SEO-chitosan_0 6.01 ±0.78 16.53 ±1.28 35.88 ±1.84 34.90 3.87
SEO-chitosan_0.5 1.59 ±0.01 6.54 ±0.04 19.73 ±0.08 37.40 8.38
SEO-chitosan_1 0.33 ±0.00 2.45 ±0.01 7.87 ±0.03 32.22 19.39
SEO-chitosan_2 0.16 ±0.18 1.14 ±0.95 3.50 ±2.09 18.01 22.13
Samples L* a* b*
SEO-chitosan_0 84.73 ±3.81
a
-0.48 ±0.27
a
6.18 ±0.45
a
SEO-chitosan_0.5 66.62 ±8.17
b
1.67 ±1.01
b
15.1 ±4.45
b
SEO-chitosan_1 59.87 ±11.14
a
1.21 ±0.31
b
8.40 ±1.27
a
SEO-chitosan_2 33.88 ±6.52
a
1.62 ±0.71
b
8.15 ±1.50
a
SEO - sandalwood essential oil; UV-C (200–280 nm); UV-B (280–315 nm); UV-A (315–400 nm); %T - percentage of transmittance; L* , lightness: black =0 and white
=100; a* , green =-a* and red = +a* ); b* , blue =-b* and yellow= +b* .
Values are expressed as mean ±standard deviation.
Different letters in the same column indicate signicant differences (p ˂ 0.05).
M. Fl´
orez et al.
Food Packaging and Shelf Life 34 (2022) 100938
7
by a single horizontal force. Hence, under tensile or puncture defor-
mation forces, the same matrix can exhibit different resistance and
deformation behavior (Caz´
on et al., 2021).
In the manufacture of lms, heat sealability of the material and its
durability is important. In addition, if these lms are intended for food
use, they should be strong enough to keep the product sealed and not
release their contents during storage and handling. The SEO-chitosan
samples showed a sealability values ranged from 79.09 to 195.20 N/m
(Table 2). The addition of SEO to the chitosan lms had a signicant
effect (p<0.05) on the sealability of the lms, resulting in a decrease in
sealability values as the SEO concentration increased. It was noted that
the lm without SEO showed the highest values of sealability
(195.20 N/m). This could be explained by the different active compo-
nents of sandalwood essential oil, which could exert a modication on
the network of the lm. Results are in the range obtained for lms of
chicken protein/sh skin gelatin mixtures containing gallic acid or
tannic acid where values of 64.83–113.5 N/m in the sealability of the
lms were observed (Nilsuwan et al., 2021).
3.5. Evaluation of antioxidant properties
Antioxidant activity of pure SEO and SEO-chitosan lms was eval-
uated by two methods: ABTS
•+
and DPPH
•
(Table 3). Pure sandalwood
results showed values of 78.23 % for DPPH
•
and 15.65 % for ABTS
•+
verifying the antioxidant capacity of the essential oil and its suitability
for application in food lms. The results obtained from the lm extracts
showed values from 7.34 % to 23.69 % DPPH
•
and the scavenging
ABTS
•+
activity ranged from 2.54 to 19.22 %. Both scavenging activities
increased signicantly (p<0.05) with increasing oil concentration.
After the addition of SEO (from 0.5 % to 2 %), the antioxidant capacity
of the lms increased signicantly. Sandalwood essential oil contains a
great variety of sesquiterpenoid alcohols called santalols (Demole et al.,
1976).
α
-santalol is the main bioactive principle of the oil, therefore
most of the antioxidant activities are attributed to it (Misra & Dey,
2013). This behavior can be attributed to the presence of these active
components from the essential oil, which can exert their antioxidant
activity by several possible mechanisms: free-radical scavenging activ-
ity, hydrogen donors, transition-metal-chelating activity, and/or
singlet-oxygen-quenching capacity (Liyana-Pathirana & Shahidi, 2006).
3.6. Evaluation of the optical properties
The UV-barrier properties of the SEO-chitosan lms were determined
from the transmittance values in the UV region. The color and opacity of
the lms were determined from the transmittance values in the visible
region.
Table 4 shows the average percentage of transmittance (%T) of the
examined SEO-chitosan lms in the UV-C (200–280 nm), UV-B
(280–315 nm) and UV-A (315–400 nm) regions. Fig. 3b shows the UV
prole spectra of the lms. SEO-chitosan lms showed %T values ranged
6.01–0.16 %, 16.53–1.14 % and 35.88–3.50 % in the UV-C, UV-B and
UV-A regions, respectively. The results concluded that the addition of
SEO to the chitosan lms exerted a signicant decrease in the trans-
mittance values and thus improving the UV blocking capacity of the
lms.
The best results were obtained on lms with 2% SEO, where the
transmittance percentage was below 5 % in the 200–400 nm wavelength
region, which reafrmed the good UV-barrier properties of the lm.
These results showed that chitosan lms containing SEO may retard UV-
induced lipid oxidation.
Other important factors when assessing optical properties are
transparency and opacity, as these have a direct impact on the visual
appearance of the product to be coated. Table 4 shows the values of
transparency ranged 18.01–37.40, while opacity values ranged
22.13–3.87. As can be seen, the addition of SEO promoted the decrease
in the transparency values and an increase in the opacity of the lms. A
decrease in light transmission with the addition of SEO, with a conse-
quent increase in opacity and a decrease in transparency, was also
previously observed by the addition of essential oils in edible lms
(Tongnuanchan et al., 2012).
Table 4 shows the CIE coordinates of the SEO-chitosan lms. Light-
ness (L*) varied from 33.88 to 84.73. The addition of SEO had a sig-
nicant effect (p<0.05) compared to pure chitosan lms. Increasing
SEO concentration, a signicant decrease in L* values was shown,
indicating a tendency of the lm to darken (Zhang et al., 2018).
Fig. 4. Thermogravimetry and differential scanning calorimetry of SEO-
chitosan lms using malic acid as solvent. SEO is sandalwood essential oil.
M. Fl´
orez et al.
Food Packaging and Shelf Life 34 (2022) 100938
8
However, the b* and a* values increased signicantly from 6.18 to 15.1
and from −0.48–1.67 when the oil was at a concentration of 0.5%. The
increase in a* and b* values explains that the samples have a tendency
towards redness and yellowness compared to the pure chitosan lm. The
same trend in the results was observed when Melaleuca alternifolia
essential oil was added to chitosan lms (Caz´
on et al., 2021).
3.7. Evaluation of the thermal properties (TGA/DSC)
The SEO-chitosan lms were evaluated by TGA/DSC for study the
effects of sandalwood essential oil on the thermal physical property.
Fig. 4 shows the thermograms of chitosan lms, pure or enriched with
SEO up to 2 % (v/v).
Thermogravimetric curves indicated similar thermal behaviors with
three steps. There are slight differences related to the composition of the
samples. The weight loss between 50 and 150 ºC is due to the loss of
water linked to the internal structure of the lm (Chu et al., 2019). In
comparison, weight loss was slower in pure chitosan lms in agreement
with the data obtained for %W. It must be taken into account that some
of the water present in the lm may evaporate before the TGA/DSC
analysis is carried out and the temperature of 50 ºC is reached (Caz´
on
et al., 2021). The second weight loss step is due to thermal degradation
of the chitosan (Chen et al., 2008; Lewandowska, 2009). Finally, in the
third step there is a weight loss probably due to the degradation of
by-product generated by chitosan during the thermal degradation
process.
Endothermic peaks were found between 140 and 240 ºC. These peaks
correspond to chemisorbed water via hydrogen bonds, and the degra-
dation of the chitosan amino group and the malic acid (Bonilla et al.,
2014). At 225 ºC, the total weight losses of the samples increased with
the SEO concentration, being 38.01 %, 33.68 %, 35 % and 40 %. Ac-
cording to that, the lms incorporated with SEO up to 1.5 % showed a
decreased weight loss compared to the control lm, indicating greater
thermal stability. However, when the SEO addition is up to 2 % there
was a slight decrease in this property. This could be explained due to the
presence of high concentrations of SEO in the lms leads to an increase
in the discontinuity of the lm matrix. This generated a decrease in the
compatibility of the multiphase system, which causes the lms to lose
more weight and therefore have lower heat resistance (Noshirvani et al.,
2017). Similar results were obtained by Xu et al. (2019) when cinnamon
and clove essential oil were added to gum arabic-chitosan lms.
The results obtained suggest that the interactions produced in the
matrix between chitosan and SEO (from 0.5 % to 1 %) increases the
thermal stability of the lms by reducing their weight. On the other
hand, these lms were stable until they reached 120ºC. This is an
interesting nding, because it means that as the developed SEO-chitosan
lms could have applicability in pasteurization treatments.
3.8. Storage stability of butter packed in SEO-chitosan sachets
The antioxidant properties of the chitosan lms as sachets for butter
were tested after 3 months at 5 ºC refrigerated storage. Fig. 5 shows the
butter samples sealed in SEO-chitosan lms at day 90 of storage at 5 ºC.
Table 5 shows DPPH
•
and ABTS
•+
values in the lms used for packaging
butter after 90 days. Results revealed a signicant increase (p<0.05) of
the DPPH
•
values from 10.70 % to 23.14 % for chitosan lms with 1 %
and 2 % SEO, respectively. ABTS
•+
values ranged from 6.68 % to 19.34
% with a signicant increase by the addition of any concentration of SEO
compared with the chitosan lms without SEO. DPPH
•
and ABTS
•+
re-
sults suggested that the active lms successfully kept the antioxidant
properties after 3 months of storage.
The TBARS value was used to indicate that the second stage of lipid
auto-oxidation is occurring. This assay detects malonaldehyde (MDA),
an unfolded product of endoperoxides resulting from the oxidation of
Fig. 5. Butter samples sealed in SEO-chitosan lms stored at 4ºC for 90 days. SEO is sandalwood essential oil.
Table 5
Antioxidant and optical properties of chitosan lms applied on butter after 3
months of storage. TBARS of butter after 3 months of storage. Film composition
is described in Table 1.
Samples DPPH
•
ABTS
•+
TBARS
(%) (%) mg MDA / kg
butter
Butter w/o lm – – 616.90 ±9.58
a
Butter with SEO-chitosan_0 10.70
±2.46
a
6.68 ±0.31
a
417.91 ±4.43
b
Butter with SEO-chitosan
_0.5
6.68 ±1.65
a
11.42
±0.72
b
414.66 ±5.60
b
Butter with SEO-chitosan
_1
12.11
±3.40
a
11.87
±0.38
b
410.77 ±8.27
b
Butter with SEO-chitosan
_2
23.14
±6.07
b
19.34
±0.12
c
393.25 ±6.22
c
Samples L* a* b*
Butter w/o lm 83.74 ±0.29 -1.03
±0.07
bc
36.26 ±0.64
Butter with SEO-chitosan_0 80.28 ±2.22 1.54 ±1.01
a
38.81 ±4.73
Butter with SEO-chitosan
_0.5
81.83 ±0.99 1.09
±0.55
ac
37.14 ±2.71
Butter with SEO-chitosan
_1
78.17 ±2.19 1.96 ±0.72
a
45.17 ±2.24
Butter with SEO-chitosan
_2
78.47 ±1.51 1.13 ±0.39
a
44.01 ±1.49
SEO - sandalwood essential oil; TBARS – Thiobarbituric reactive substances
Values are expressed as mean ±standard deviation (SD).
Different letters in the same column indicate signicant differences (p ˂ 0.05).
Values of L* and b* showed no signicant differences.
M. Fl´
orez et al.
Food Packaging and Shelf Life 34 (2022) 100938
9
unsaturated fatty acids (Han Lyn et al., 2021). Table 5 shows TBARS
values of the unpackaged (control) and packaged butter after 3 months
of refrigerated storage. The TBARS values for control sample was
616.90 mg MDA/kg butter and for packaged butter ranged from 417.91
to 393.25 mg MDA/kg butter. TBARS values showed a signicant effect
of packaged samples (p<0.05) compared to the control. After 3 months
of butter storage, a 36 % decrease in the lipid oxidation was observed
due to the SEO-chitosan lms. This behavior can be explained by the
radical scavenging properties of the lms, as supported by the results of
the antioxidant assays (Han Lyn et al., 2021). On the other hand, butter
samples packaged with SEO-chitosan lms decreased lipid oxidation
compared to butter packed with control lms without oil. Therefore, the
presence of sandalwood essential oil further decreases the oxidation of
the butter. Same trend was observed in chitosan/graphene oxide bio-
composite lms that stored palm-oil based margarine (Han Lyn et al.,
2021).
3.9. Optical properties of butter wrapping lms
Table 5 shows the CIE coordinates of the unwrapped and wrapped
butter samples with SEO-chitosan lms. In this case, the lightness (L*)
varied from 80.28 to 78.47 and the b* values were from 38.81 to 44.01.
For both values, no signicant difference was observed (p>0.05).
However, the a* (redness parameter) values varied from 38.81 to 44.01,
producing a signicant effect (p<0.05) compared to butter without
lm. Results suggested that the brightness of the lms and the yellowish
of the samples were not affected by the presence of the lm that wraps
around the butter, only the redness color of the lms was slightly
modied. Hence, the lms had sufcient transparency to maintain the
visual appearance of the butter and the consumer could observe it
without signicant alterations.
4. Conclusions
The incorporation of SEO into chitosan matrix was successfully
performed to obtain antioxidant lms. The lms are completely water-
soluble and can be easily removed from foodstuffs after use without
generate solid wastes. The lms can be heat sealed and used as sachets.
This makes them a good option for use in the food packaging industry
partially replacing plastic containers. Regarding the optical properties,
the color and transparency of the lms applied on butter allowed to see
the real color of the butter. The concentration of essential oil present in
the lm did not inuence the visual appearance of the wrapped product
under study. Considering the TBARS results, it can be stated that the
application of SEO on chitosan lms successfully delayed butter
oxidation.
CRediT authorship contribution statement
Maria Fl´
orez: Writing- Original draft preparation. Patricia Caz´
on:
Conceptualization, Supervision, Writing- Reviewing and Editing. Man-
uel V´
azquez: Methodology, Writing- Reviewing and Editing, Project
administration.
Declaration of Competing Interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Acknowledgments
The authors appreciate the funding support of Xunta de Galicia,
within the postdoctoral fellowship granted to Patricia Caz´
on Díaz (No.
ED481B-2021–040). The use of RIAIDT-USC analytical facilities is
acknowledged.
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