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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Chitosan nanoparticles loaded with clove essential oil: Characterization,
antioxidant and antibacterial activities
Milad Hadidi
a
, Shiva Pouramin
b
, Fateme Adinepour
c
, Shaghayegh Haghani
b
, Seid Mahdi Jafari
c,
*
a
Department of Food Technology, University of Lleida, Lleida, Spain
b
Department of Food Science and Technology, Khazar University, Mazandaran, Iran
c
Department of Food Materials and Process Design Engineering, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran
ARTICLE INFO
Keywords:
Clove essential oil
Nanoencapsulation
Nanoparticles
Chitosan
Antibacterial activity
ABSTRACT
One of the recent trends in the food industry is application of natural antioxidant/antimicrobial agents. In this
study, essential oil of clove buds was extracted and encapsulated in chitosan nanoparticles using a two-step
technique of emulsion-ionic gelation. A good retention rate (55.8–73.4 %) of clove essential oil (CEO) loaded in
chitosan nanoparticles was confirmed. Also, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction
(XRD) and differential scanning calorimetry (DSC) analyses revealed the success of CEO encapsulation. Scanning
electron microscopy (SEM) images illustrated regular distribution and spherical shape of nanoparticles with a
size range of 223−444 nm. The antioxidant activity of CEO-loaded chitosan nanoparticles was higher than free
CEO. Similarly, CEO-loaded chitosan nanoparticles had a high antibacterial activity against L. monocytogenes and
S. aureus (inhibition halo diameter of 4.80–4.78 cm). This technique could improve the efficiency of CEO in food
products and a delivery system for novel applications such as active packaging.
1. Introduction
Essential oils (EOs), obtained from medicinal and herbal plants, are
recognized for their antioxidant and antimicrobial properties. EOs of
different herbs have been used as food flavorings, medicines and in the
preparation of fragrances (Asbahani et al., 2015;Bilia et al., 2014).
Eugenia caryophyllata, a member of the Myrtaceae family and commonly
known as clove, is used as an herbal plant. Cloves are the dried flower
buds of clove tree and contain a number of bioactive compounds which
some of them are highly effective antioxidants and antimicrobials.
Clove essential oil (CEO) contains mainly phenylpropanoids such as
eugenol and its derivatives, along with minor levels of β-caryophyllene
and α-humulene organic compounds (Asbahani et al., 2015;Gülçin,
Elmastaş, & Aboul-Enein, 2012). CEO has various applications in the
food, sanitary, biomedical, pharmaceutical, active packaging and cos-
metics industries due to its biological properties including antioxidant,
antimicrobial, antiseptic, pesticide, analgesic and anticarcinogenic ac-
tivities (Chen, Ren, Qian, Fan, & Du, 2017). CEO is used commonly as a
natural preservative, colorant and spice in various food products
(Aguilar-González, Palou, & López-Malo, 2015).
EOs contain both labile and volatile compounds being able to de-
compose or evaporate easily under processing conditions, during utili-
zation and storage, or when incorporated into foods or packaging
materials as a result of high temperatures, low pressures, presence of air
and light, etc. (Donsì, Annunziata, Sessa, & Ferrari, 2011). Similarly,
the antibacterial and antioxidant properties of CEO are significantly
limited due to its extremely volatile and poor water-soluble compo-
nents, like eugenol (Sebaaly, Jraij, Fessi, Charcosset, & Greige-Gerges,
2015). Encapsulation of bioactive compounds such as EOs is not only
one of the most effective techniques to protect them against degrada-
tion under adverse environmental conditions, but also encapsulation
can be used to extend the shelf life EOs and result in controlled release
delivery systems (Vahedikia et al., 2019). Several nanoencapsulation
techniques for bioactive compounds have been studied (Assadpour &
Jafari, 2019). Ionotropic gelation is one of the most efficient en-
capsulation methods which leads to a considerable stability, long useful
life, high loading capacity, good dispersibility in water, as well as the
controlled release of encapsulated bioactive compounds (Hosseini,
Zandi, Rezaei, & Farahmandghavi, 2013).
Chitosan is a linear copolymer that is comprised of B-(1–4) linked D-
glucosamine and N-acetyl-D-glucosamine units and has been used in
medicinal, pharmaceutical, food and feeding applications in recent
years (Hosseinnejad & Jafari, 2016). It is a cationic polysaccharide
which presents the potential to be used for the production of nano-
particles due to its favourable characteristics such as ample sources to
obtain it, controllable and easy extraction, biocompatibility,
https://doi.org/10.1016/j.carbpol.2020.116075
Received 24 November 2019; Received in revised form 10 February 2020; Accepted 25 February 2020
⁎
Corresponding author.
E-mail address: smjafari@gau.ac.ir (S.M. Jafari).
Carbohydrate Polymers 236 (2020) 116075
Available online 26 February 2020
0144-8617/ © 2020 Elsevier Ltd. All rights reserved.
T
biodegradability and non-toxicity (Hu & Luo, 2016;Younes & Rinaudo,
2015). Moreover, chitosan possesses several functional groups on its
polymer chains which extend the possibility of chemical modification
and thus provides the opportunity for obtaining novel variants having
useful/appropriate physicochemical properties (Arteche Pujana, Pérez-
Álvarez, Cesteros Iturbe, & Issa, 2013;Ramimoghadam, Bagheri, &
Hamid, 2014).
The ionic gelation technique has been widely used for na-
noencapsulation of food components by chitosan via a nontoxic and
multivalent material such as Tripolyphosphate, TPP (Rajabi, Jafari,
Rajabzadeh, Sarfarazi, & Sedaghati, 2019;Akbari-Alavijeh, Shaddel, &
Jafari, 2020). In this regard, concentration, ratios of ingredients, way of
mixing, and pH are the affective factors in gelation process (Akbari-
Alavijeh, Shaddel, & Jafari, 2019). Although the large size of the ob-
tained nanoparticles (200−1000 nm) provides high loading capacity
(up to 80 % for bovine serum albumin), the particle sizes, pH sensi-
tivity, and high polydispersity are considered as the drawbacks of this
method (Katouzian & Jafari, 2016). Paula, Sombra, Abreu, and Paul
(2010)) proposed nanoencapsulation of Lippia sidoides EO by spray
drying using Angico gum/chitosan as wall material ranging from 10 to
60 nm in diameter and encapsulation efficiency of 77.8 %. They also
proved the great larvicidal effect of the nanoparticles against larvae of
Stegomyia aegypti or Aedes which is the dengue vehicle responsible for
many diseases (Paula et al., 2010). Due to the harsh conditions of spray
drying, most of further studies applied emulsification or ionic gelation
techniques to nanoencapsulate EOs.
Oregano EO, eugenol, carvacrol, Carum copticum EO, and Satureja
hortensis L. EO, have been nanoencapsulated via TPP/STPP-Chitosan to
form nanoparticles using emulsion-ionic gelation technique (Esmaeili &
Asgari, 2015;Feyzioglu & Tornuk, 2016;Hosseini et al., 2013;
Keawchaoon & Yoksan, 2011;Woranuch & Yoksan, 2013). These ex-
periments cover a particle size range of 30–200 nm with a wide range of
encapsulation efficiency from 14 % to 47 %. Antimicrobial and anti-
oxidant effect and also the release profile of EOs were studied in all
experiments which shows significant effect of the nanoencapsulation
method on the stability of EOs. The highest encapsulation efficiency in
this area has gained by Jamil et al. (2016) to encapsulate cardamom EO
by TPP-chitosan using ionic gelation technique. They formed the na-
nocomposites in size range of 50–100 nm with a remarkable anti-
microbial effect against S. aureus and E. coli (Jamil et al., 2016).
To the best of our understanding, there are no published papers
reported to date on the antioxidant and antibacterial activities of CEO
encapsulated in chitosan nanoparticles. Hence, the objectives of the
present work were (i) to prepare chitosan nanoparticles containing
CEO; (ii) to investigate the physiochemical properties and structural
characteristics of CEO-loaded chitosan nanoparticles; and (iii) to eval-
uate the antibacterial and antioxidant activities of CEO encapsulated
into chitosan nanoparticles as a natural additive for food products and
active packaging.
2. Materials and methods
2.1. Materials
Low molecular weight chitosan (50–190 kDa) with 75–85 % degree
of deacetylation was supplied by Sigma-Aldrich (St. Louis, Mo, USA).
Four bacterial strains, Staphylococcus aureus,Listeria monocytogenes,
Salmonella typhi and E. coli were purchased from clinical isolations in
Tehran University of Medical Sciences (Tehran, Iran). All other che-
micals utilized in this project were of analytical grade
2.2. Extraction and characterization of CEO
Essential oil of air-dried clove buds was extracted by water dis-
tillation technique using a Clevenger apparatus (British type) for 3 h,
according to the method of Dadalioglu and Evrendilek (2004). The
hydrosol obtained was centrifuged (Avanti 5-26XP, Mervue, USA) in
order to separate the organic and aqueous phases. The CEO was dried
over anhydrous sodium sulfate and stored at 4 °C in a dark glass bottle
until further analysis.
The Gas Chromatography (GC) analysis of CEO was performed by a
GC instrument (Model 7890, Agilent Technologies, CA, USA) equipped
with DB- 5 column (30 m ×0.32 mm, film thickness of 0.25 μm),
working from 50 to 250 °C with increasing rate of 5 °C/min. An auto-
sampler with 0.5 μL of volume capacity was used for injection of CEO.
The temperature of injector and detector was 250 and 280 °C, respec-
tively. Nitrogen, as the carrier gas, had a flow rate of 1.1 mL/min
(Adams, 2007;Gasparetto et al., 2017).
2.3. Preparation of CEO-loaded chitosan nanoparticles
Encapsulation of CEO into chitosan nanoparticles (NPs) was done in
a two-step process: oil-in-water emulsification and then, ionic gelation
method described by Keawchaoon and Yoksan (2011) with some
modifications. Chitosan solutions at 1% w/v were prepared through
dissolving chitosan in acetic acid 1% v/v by 60 min sonication Kunshan
Instrument Co., Ltd., Jiangsu, China. Then, undissolved chitosan was
eliminated from this solution by filtering through 1 μm pore size filter.
Tween 80 80 mg was added into the 50 mL of chitosan solution and its
pH was adjusted to 4.2 by NaOH solution 2 N. The mixture was stirred
at 50 °C for 90 min to achieve a homogeneous solution. Different
amounts of CEO were added into this mixture to obtain various chit-
osan/CEO ratios 1:0, 1:0.25, 1:0.5 and 1:1. CEO phase was dropped
constantly into the 50 mL of chitosan aqueous solution during homo-
genization T25, IKA, Germany at a speed of 13,000 rpm for 10 min
under an ice-bath condition for the purpose of achieving an oil-in-water
emulsion. Finally, sodium tripolyphosphate TPP was added to induce
ionic gelation of chitosan. Agitation was continuously done for 40 min.
The formed particles were collected by centrifugation at 9000×g for
30 min at 4 °C, and subsequently washed several times with deionized
water. These suspensions were immediately dried at −32 °C for 42 h by
a freeze dryer (Cryodos 50/230 V, Telstar, Madrid, Spain).
2.4. Determination of retained CEO in chitosan NPs
The amount of loaded CEO in chitosan NPs was determined by
UV–Vis spectroscopy. The prepared suspension in 200 μL water was
mixed with aqueous hydrochloric acid solution (2 M, 5 mL) and re-
fluxed at 95 ◦C for 30 min. After cooling down, 2 mL ethanol (96 % v/v)
was added to this mixture and centrifuged at 10,000 rpm for 5 min in 25
◦C(Hasheminejad, Khodaiyan, & Safari, 2019). The CEO content in the
supernatant was analysed by a UV–Vis spectrophotometer (s2150,
UV–vis Unico, USA) at a wavelength of 279 nm. Chitosan NPs without
CEO were also prepared as blank samples in the same manner. The
amount of CEO was calculated by calibration curve of free CEO in
ethanol (R
2
= 0.998). The retention of CEO in chitosan nanocarriers
was determined by the following equation:
=×
R
etention of CEO Total amount of loaded CEO
Inital amount of CEO
(%) 100
(1)
2.5. Instrumental analysis of CEO-loaded chitosan NPs
The particle size, zeta potential and polydispersity index (PDI) of
chitosan NPs containing CEO were determined by Dynamic light scat-
tering (DLS) using a Zetasizer (3000HS, Malvern Ltd, Worcestershire,
UK); it was equipped with a laser operation of He–Ne at 633 nm and
4.0 mW. Samples were scattered with high-purity water (1:100 v/v) at
the room temperature.
Fourier-transform infrared (FTIR) spectroscopy was applied for
chemical structure analysis of chitosan NPs, CEO-loaded NPs with a
chitosan/CEO ratio of 1:0.5, and pure CEO in a wave number range of
M. Hadidi, et al. Carbohydrate Polymers 236 (2020) 116075
2
400–4000 cm
−1
by a spectrometer (Equinox 55-LSI 01, Bruker, UK).
The dried NPs and potassium bromide were grinded well and pressed to
form pellets. Also, 16 scans at 4 cm
−1
resolutions were achieved for
each spectrum.
X-ray diffraction (XRD) patterns of samples were scanned over a 2θ
range of 5 to 60° using a X-R diffractometer (Model D5000, Siemens,
Munich, Germany) with a speed angle of 0.05 °/min. Differential
scanning calorimetry (DSC) analysis was performed to investigate the
thermal stability of chitosan NPs, CEO-loaded NPs and pure CEO using
a DSC (Model 822, Mettler Toledo, Switzerland) with 10 °C min
−1
in-
creasing heat rate (30–320 °C). Surface morphology and shape of
freeze-dried chitosan NPs containing CEO were studied by Scanning
electron microscopy, SEM (XL 30 Philips, Netherlands). Samples were
glued onto an adhesive tape mounted on the specimen stub and parti-
cles were covered with a layer of platinum by Emitech sputter coater at
15 kV.
Thermogravimetric analysis (TGA) analysis was carried out with a
Q50 Thermogravimetric Analyzer (TA, USA). For this purpose, 10 mg of
freeze-dried sample was placed in the platinum pan of TGA furnace and
the measurements were performed with a heating rate of 10 °C min
−1
from 20 to 600 ◦C under nitrogen atmosphere.
2.6. Determination of antioxidant activity
The antioxidant activities of chitosan NPs, CEO-loaded chitosan NPs
and pure CEO were examined by DPPH free radical scavenging method
(Keawchaoon & Yoksan, 2011). About 10 mg of each sample was mixed
with ethanolic DPPH solution (100 mL, 0.1 M) and let stand at ambient
temperature in the absence of light for 1 h. The absorbance was mea-
sured at 517 nm using a UV-Vis spectrophotometer (s2150, UV–vis
Unico, USA). A control sample was also prepared without CEO/particles
and ethanol was used for the baseline correction. The antioxidant ca-
pacity was determined as a percentage of DPPH radical scavenging
capacity using the following formula:
Radical scavenging activity (%) = [(A
control
–A
sample
)/A
control
] × 100
(2)
where, A
control
and A
sample
are the absorbances of control and target
samples, respectively.
2.7. Determination of antibacterial properties
Four bacterial strains, S. aureus,L. monocytogenes,S. typhi and E. coli
were cultured for 24 h at 37 °C in nutrient agar. The inhibitory activity
of chitosan NPs, CEO-loaded chitosan NPs with a chitosan/CEO ratio of
1:0.5 and pure CEO against the growth of mentioned bacterial strains
were examined. The nutrient agar (10 mL) was placed in Petri dishes.
Previously, 20 μL of bacterial suspensions (10
8
CFU mL
−1
) mixed with
10 mL semisolid agar were incorporated homogeneously on the Petri
dishes. After solidification of agar, a hole was made with a tunneler
(5 mm in diameter). Then, 32 μL of a solution containing different
samples were injected in each well. The Petri dishes were incubated for
24 h at 37 °C. In the end, the inhibition zones were determined by a
digital Vernier Caliper (Sotelo-Boyása, Correa-Pachecoa, Bautista-
Banosa, & Gómez, 2017). For in vitro assessment of the effect of CEO-
loaded NPs on bacterial growth, volumes ranging from 2 to 32 μL were
examined using the agar plate method and the Minimum Inhibitory
Volume (MIV) was established.
2.8. Statistical analysis
One-way analysis of variance (ANOVA) was utilized for a com-
pletely randomized statistical analysis; also Tukey’s multiple compar-
ison test was applied at a significance level of P < 0.05 to determine
the significant differences among means. The SPSS software (version
23.0) was used for all statistical analyses.
3. Results and discussion
3.1. Chemical composition of extracted CEO
Table 1 shows that twenty-three compounds were detected in CEO
by GC/MS. The main compounds were eugenol (89.86 %) and β-car-
yophyllene (5.40 %) that are in agreement with previous data published
in literature (Chaieb, Hajlaoui et al., 2007;Sebaaly et al., 2015). The
high level of antiradical activity in CEO probably derives from its hy-
drogen donating ability exhibited by a wide range of constituents in it
such as: eugenol, eugenyl acetate, β-caryophyllene, 2-heptanone
(Chaieb, Zmantar et al., 2007), acetyl-eugenol, α-humulene, methyl
salicylate, iso-eugenol, methyl-eugenol (Yang, Lee, Lee, Choi, & Ahn,
2003), phenylpropanoids, dehydrodieugenol, trans-coniferyl aldehyde,
biflorin, kaempferol, rhamnocitrin, myricetin, gallic acid, ellagic acid
and oleanolic acid (Khaleque et al., 2016).
3.2. Successful loading of CEO within chitosan nanoparticles (NPs)
The retention rate of CEO loaded into chitosan nanoparticles ranged
from 55.8 to 73.4% which was significantly (P < 0.05) affected by the
ratio of chitosan/CEO as shown in Table 2; the retention was increased
up to 1:0.5 ratio of chitosan/CEO. As the amount of CEO increases to
above 1:0.5, retention rate shows a reduction which could be due to
saturated CEO level in chitosan NPs. The highest encapsulation effi-
ciency of Coriandrum sativum EO in chitosan particles was reported to
be in 1:0.6 ratio of chitosan/EO (Das et al., 2019). The findings of
Matshetshe, Parani, Manki, and Oluwafemi (2018) revealed that en-
capsulation efficiency of cinnamon EO in chitosan NPs ranged from
10.12 to 20.04% and it was decreased at higher EO levels. Sotelo-Boyas
et al. (2017) determined encapsulation efficiency of thyme EO and
carvacrol in chitosan nanocapsules to be 72 and 81.4 %, respectively. In
another study by Hasani, Ojagh, and Ghorbani (2018), encapsulation
efficiency values for lemon EO in chitosan-Hicap nanogels varied from
68.09–85.43%, depending on the relative proportions of chitosan and
EO. Our results are consistent with the studies cited above.
Table 1
Chemical components of extracted clove essential oil determined by GC/MS.
Amount (%) Retention time Component
0.1 1029 Limonene
0.1 1032 1,8–Cineole
t*1137 Cis -Limonene oxide
t*1142 Trans-Limonene oxide
0.1 1190 Methyl Salicylate
0.2 1195 Methyl chavicol
0.1 1248 Chavicol
89.8 1359 Eugenol
t*1403 Methylugenol
t*1407 Cis –isoeugenol
5.4 1415 Caryophylleneβ
0.1 1449 Trans–isoeugenol
t*1452 Cloveneα
t*1455 Cis –methylisoeugenol
2.1 1458 Humulene-α
t*1491 Trans-methylisoeugenol
t*1506 Farnesene-α
1.2 1522 Eugenyl acetate
t*1566 Cis-isoeugenyl acetate
0.4 1583 Caryophyllene oxide
0.1 1593 Caryophyllene alcohol
0.1 1607 Humulene epoxideα
t*1614 Trans- isoeugenyl acetate
* trace.
M. Hadidi, et al. Carbohydrate Polymers 236 (2020) 116075
3
3.3. Size and surface charge of CEO-loaded chitosan NPs
The results of particle size, zeta potential and polydispersity index
(PDI) of CEO-loaded chitosan NPs with different ratio of chitosan/CEO
are presented in Table 2. Chitosan NPs without CEO were found to have
a mean diameter of 223.2 nm. The size of NPs was increased by loading
of CEO and increment of chitosan/CEO ratio, which accords with the
results of Keawchaoon and Yoksan (2011).Hosseini et al. (2013) also
observed enlargement of oregano EO-loaded chitosan NPs; they re-
ported that chitosan NPs and oregano EO-loaded chitosan NPs had a
particle diameter of 281.5 and 309.8–402.2 nm, respectively.
Zeta potential is a measure of the magnitude for the electrostatic or
charge repulsion/attraction between particles which is a major factor in
phenomena like dispersion, flocculation or aggregation and hence a key
parameter for evaluating the stability of dispersions, emulsions and
suspensions (Dickinson, 2009). In the present work, CEO-loaded chit-
osan NPs had a positive zeta potential (Table 2) whereas the highest
value of zeta potential was found in chitosan NPs without CEO
(+34.5 mV). In a study by Hasani et al. (2018), the values of zeta po-
tential for chitosan-Hicap nanocapsules containing lemon EO ranged
from +10.58 to +44.23 mV. In another research, chitosan NPs and
cinnamon EO-loaded chitosan NPs have positively charged surfaces due
to the presence of amine groups; zeta potential values were +24.0 mV
and +26 to +30.5 mV, respectively (Matshetshe et al., 2018). On the
contrary, chitosan NPs loaded with different levels of Summer savory
EO had negative zeta potential values varying from -7.54 to -21.12 mV;
a significant reduction was observed in the zeta potential value with
increasing EO concentration (Feyzioglu & Tornuk, 2016).
Lertsutthiwong, Rojsitthisak, and Nimmannit (2009) also measured
negative zeta potential values ranging from -21.8 to -23.1 mV for tur-
meric EO loaded into chitosan nanocapsules; their results indicated that
interactions between EO and NPs influence the structure and charge of
these nanocarriers.
As a rule, PDI is employed to examine the particle size distribution
in suspensions. A lower PDI indicates a more homogenous particle size
distribution and consequently, reflects diameter uniformity (Hasani,
Elhami Rad, Hosseini, & Shahidi Noghabi, 2015). Our data revealed
that PDI of samples was in the range of 0.117 to 0.337 (Table 2) which
is indicative of a monodisperse and stable particle population of uni-
form distribution.
3.4. Chemical structure of CEO-loaded chitosan NPs
Fig. 1 illustrates FTIR spectra of chitosan NPs, CEO-loaded chitosan
NPs and pure CEO. The spectrum of chitosan NPs without incorporation
of CEO (A) displays peaks at 3445 cm
−1
(OeH), 3298 cm
−1
(NeH2
stretching), 2991 cm
−1
(CeH stretching), 1546 cm
−1
(N–H
2
of amide
II), 1367 cm
−1
(CeN stretching), 1201 cm
−1
(β-(1 −4) glycosidic
linkage), 1065 cm
−1
(CeOeC stretching of glucose ring), 997 cm
−1
(CeO stretching) and 904 cm
−1
(vibration of the pyranose ring). There
was no peak for C]O stretching of amide I and two peaks appeared at
1065 cm
−1
(CeOeC stretching of glucose ring) and 1546 cm
−1
(amide
II), indicating electrostatic associations between PO
43-
group of TPP and
NH
3+
group of chitosan (Hosseini et al., 2013;Yoksan,
Jirawutthiwongchai, & Arpo, 2010). The various peaks seen in pure
CEO (C) are consistent with the presence of a variety of compounds in
this EO, as detected by GC/MS (Table 1). Peaks in CEO-loaded chitosan
NPs (B) show the successful encapsulation of CEO within chitosan na-
nocarriers; an intense peak was observed at 2991 cm
−1
also seen in
CEO (2999 cm
−1
). By comparison to unloaded chitosan NPs (A),
greater amounts of alcohols, ethers, carboxylic acid esters, anhydrides,
alkanes and CH
3
groups were found in loaded chitosan NPs (B) and pure
CEO (C). It can be seen from the FTIR spectra that the addition of CEO
to chitosan NPs led to a significant increase in the intensity of CH
stretching peak at 2991 cm
−1
, demonstrating an increment in the
content of ester groups arising from CEO compounds. All CEO peaks
appeared in the spectrum of CEO-loaded chitosan NPs at the same wave
number implying no alteration or interaction between chitosan and
CEO. To conclude, FTIR results indicated encapsulation of CEO into
chitosan NPs.
3.5. Thermal properties of CEO-loaded chitosan NPs
DSC is a method of thermal analysis to examine the formation of
solid state complexes. The DSC curves of chitosan NPs, CEO-loaded
chitosan NPs and pure CEO are depicted in Fig. 2. The thermogram of
chitosan NPs without loading CEO (A) shows an endothermic peak at
81.8 °C, indicating water evaporation. The DSC curve for pure CEO (C)
presents a very narrow endothermic peak from 80.5–91 °C (peak at
85.5 °C) which is related to the change of phase (melting and eva-
poration) of the EO components. DSC data also reveal the successful
encapsulation of CEO within carrier polymer (chitosan). It has been
reported that if the encapsulated bioactive material is not being ade-
quately incorporated into the encapsulating polymer, individual peaks
of each compound (the bioactive substance and the polymer) will ap-
pear in the DSC thermogram. Encapsulated peak characteristics will not
be observed if there is no acceptable interaction between EOs and NPs
(Cocero, Martín, Mattea, & Varona, 2009;Mezzomo et al., 2012). As
can be seen in Fig. 2, there are neither endothermic nor exothermic
peaks for the CEO-loaded chitosan NPs with a chitosan/CEO ratio of
1:0.5 (B). Thus, the absence of characteristic melting and crystalline
peaks in the thermograms of CEO (C) loaded into chitosan NPs reveal
encapsulation of CEO in the chitosan polymer matrix.
TGA thermogram of CEO (Fig. 3) showed one-step mass loss starting
at 124 °C (peak at 177 °C). The temperature at which a material is
subjected to a highest rate of weight loss is known as the temperature of
maximum degradation rate (T
d
) which is recognized from the first de-
rivative of the TGA curve at the maximum slope of weight change
known as derivative thermogravimetry (DTG) thermogram, as plotted
in (Fig. 3). Chitosan NPs presented two steps of weight loss with two T
d
values. The first one from 70 to 100 ◦C was attributed to the evapora-
tion of moisture and the second one at 244 °C which is attributed to the
dehydration and decomposition of the chitosan NPs. CEO-loaded chit-
osan NPs showed a new one-step mass loss from 320 to 380 °C (Fig. 3C
(left)) with a new degradation temperature detected at 363 °C (Fig. 3C
(right)) that represents a significant enhancement of the thermal sta-
bility of encapsulated CEO in chitosan by about 2 folds. This finding is
in agreement with the results reported by Shetta, Kegere, and Mamdouh
(2018). These authors confirmed that the encapsulated peppermint and
green tea EOs in chitosan particles decomposed at a higher temperature
Table 2
Particle size, zeta potential, polydispersity index (PDI), and retention value of CEO-loaded chitosan nanoparticles.
Chitosan/CEO ratio Particle size (nm) Zeta potential (mV) PDI Retention of CEO (%)
1:0 223.2 ± 35.6 +34.50 ± 1.6 0.337 ± 0.018 –
1:0.25 265.1 ± 18.2 +20.14 ± 0.7 0.264 ± 0.013 55.8 ± 3.9
1:0.50 295.8 ± 45.6 +16.50 ± 1.6 0.221 ± 0.005 73.4 ± 4.8
1:1 444.5 ± 63.6 +10.14 ± 0.7 0.117 ± 0.025 63.1 ± 5.5
CEO: clove essential oil.
M. Hadidi, et al. Carbohydrate Polymers 236 (2020) 116075
4
than their free forms, indicating the increased thermal stability of EOs
by encapsulation.
3.6. Crystallinity of CEO-loaded chitosan NPs
Fig. 4 shows the crystallographic structure of chitosan NPs (A), CEO-
loaded chitosan NPs with chitosan/CEO ratio of 1:0.5 (B) and chitosan
powder (C). Diffraction spectrum of chitosan powder depicts a sharp
peak at 2Ɵof 26° which is denoting its high degree of crystallinity due
to its amorphous shape (Hosseini et al., 2013;Shetta et al., 2018). No
peak was appeared in the diffractogram of unloaded chitosan NPs that
may show destruction of chitosan crystalline structure by TPP cross-
linking reaction (Rokhade et al., 2006). As can be seen in the
diffractogram of CEO-loaded chitosan NPs compared with empty (un-
loaded) chitosan NPs, the inclusion of CEO resulted in a change in the
complex structure of TPP-chitosan. The XRD pattern of chitosan NPs is
characteristic of as compared with chitosan NPs; in diffraction spectrum
of CEO-loaded chitosan NPs, the characteristic peak at 2θof 28° con-
firming the presence of CEO within chitosan NPs. This indicated that
the incorporation of CEO might result in a change of the chitosan–TPP
complex structure. Similar data have been reported by Keawchaoon and
Yoksan (2011),Shetta et al. (2018) and Hosseini et al. (2013).
Fig. 1. FTIR spectra of (A) chitosan nanoparticles; (B) CEO-loaded chitosan nanoparticles with chitosan/CEO ratio of 1:0.5; and (C) CEO.
Fig. 2. DSC thermograms of (A) chitosan nanoparticles; (B) CEO-loaded chit-
osan nanoparticles with chitosan/CEO ratio of 1:0.5; and (C) CEO.
Fig. 3. TGA (left) and DTG (right) thermograms of (A) CEO; (B) chitosan nanoparticles; (C) CEO-loaded chitosan nanoparticles with chitosan/CEO ratio of 1:0.5.
Fig. 4. XRD patterns of (A) chitosan nanoparticles; (B) CEO-loaded chitosan
nanoparticles with chitosan/CEO ratio of 1:0.5; and (C) chitosan powder.
M. Hadidi, et al. Carbohydrate Polymers 236 (2020) 116075
5
3.7. Morphology and microstructure of CEO-loaded chitosan NPs
The external morphology and particle size of chitosan particles and
CEO-loaded chitosan particles were evaluated by SEM (Fig. 5). Electron
micrographs of the samples demonstrate presence of spherical struc-
tures, the absence of cracks and the formation of a continuous layer on
the walls of NPs. The SEM images for unloaded chitosan NPs and CEO-
loaded chitosan NPs illustrate regular distribution and spherical shape,
which seem to be well stable and separated during the preparation
process. All particle diameters were observed < 500 nm, verifying the
particle size determined by DLS analysis (Section 3.3 and Table 2).
Although, the NP size differences can be assigned to the conditions of
experiments and NP production method (Kavaz, Idris, & Onyebuchi,
2019;Zhao et al., 2011).
3.8. Antioxidant activity of CEO-loaded chitosan NPs
Encapsulation process not only decreases the evaporation rate of
volatile components in EOs, but also it enhances the antioxidant ac-
tivity of these bioactives compared to their free forms as a result of
protection against the adverse effects including oxygen and tempera-
ture (Pan, Luo, Gan, Baek, & Zhong, 2014;Woranuch & Yoksan, 2013).
The DPPH radical scavenging assay is a method for the assessment of
antioxidant activity. The mechanism of this method is based on the
quenching of the single electron of DPPH by the antioxidant compounds
and subsequently, the solution is decolorized (Sarabandi et al., 2019).
The free radical scavenging activity of pure CEO has been reported by
others in the literature which is attributed mainly to the presence of
phenolic and terpenic compounds such as eugenol and β-caryophyllene
(Chen et al., 2017;Donsì et al., 2011). The scavenging activity of free
and encapsulated CEO on DPPH radicals was in the range of 15.4–60.4
% and 15.9–71.8 %, respectively (Fig. 6). It can be seen that the anti-
oxidant activity of CEO-loaded chitosan NPs was significantly higher
than that of free (non-encapsulated) CEO. Woranuch and Yoksan
(2013) and Ghahfarokhi, Barzegar, Sahari, and Azizi (2016) reported
enhanced antioxidant activity for encapsulated thyme EO and eugenol
within NPs compared to their free forms.
3.9. Antibacterial potential of CEO-loaded chitosan NPs
The in vitro inhibitory activity of pure CEO, unloaded chitosan NPs
and CEO-loaded chitosan NPs were tested using S. aureus,L. mono-
cytogenes,S. typhi and E. coli. As can be seen in Fig. 7, the greatest
inhibitory activity in all cases was achieved by the encapsulated CEO
into chitosan NPs; for these samples, inhibition halo (IH) values of 4.8,
4.78, 4.49 and 3.95 cm were observed against L. monocytogenes,S.
aureus,S. typhi and E. coli, respectively. The nanoencapsulation of CEO
Fig. 5. SEM images of (A) chitosan nanoparticles; and (B and C) CEO-loaded
chitosan nanoparticles with chitosan/CEO ratio of 1:0.5.
Fig. 6. DPPH radical scavenging activity (%) of chitosan nanoparticles, CEO-
loaded chitosan nanoparticles with chitosan/CEO ratio of 1:0.5, and CEO.
M. Hadidi, et al. Carbohydrate Polymers 236 (2020) 116075
6
provided a great potential for boosting the antimicrobial activity of
pure CEO against four foodborne bacteria. These results agree with the
findings of Esmaeili and Asgari (2015) who illustrated an improvement
of the antibacterial activity of encapsulated Carum copticum EO (CEO)
against two of the most important foodborne bacteria (S. aureus and E.
coli).
Minimum inhibitory volume (MIV) for CEO-loaded chitosan NPs
was 2 μL against the four tested bacteria. In terms of pure CEO, MIV was
2μL for S. aureus,L. monocytogenes,S. typhi; and 4 μL for E. coli. Also for
unloaded chitosan NPs, MIV was 8 μL for the tested bacteria. It has been
documented that nano-sized particles can penetrate through the bac-
terial cell wall and destroy their cell membrane (Elsabee & Abdou,
2013;Seil & Webster, 2012); so they demonstrate a greater anti-
bacterial potential compared to larger particles (Zhang, Jung, & Zhao,
2016). Chitosan NPs which were not loaded with CEO resulted in a
significant (p < 0.05) reduction in bacterial growth compared to the
control, showing antibacterial activity of chitosan. This antibacterial
effect can be associated with factors such as increasing the permeability
of bacterial cell membrane as well as its significant depolarization by
chitosan (Raafat, Bargen, Haas, & Sahl, 2008). Hasheminejad et al.
(2019) reported that CEO encapsulated in chitosan NPs revealed a great
antifungal activity against Aspergillus niger, compared with unloaded
chitosan NPs and free CEO. The results of this study are in agreement
with the report of Feyzioglu and Tornuk (2016) who investigated the
antibacterial activity of Summer savory (Satureja hortensis L) EO loaded
into chitosan NPs. As regards the mechanism of antibacterial properties
of EOs, this has not been related to a specific compound or mode of
effect. The hydrophobicity of EOs is a key property that enables them to
breakdown the lipids in the bacterial cell membrane causing the
leakage of ions and other cell compounds and thereby cell death (Burt,
2004).
4. Conclusion
As the application of EOs in food materials is problematic due to
their low solubility in water, encapsulation techniques have been in-
troduced in recent years as efficient means to increase their dis-
persibility in aqueous media. In the present study, chitosan NPs loaded
with CEO were successfully produced with a high retention rate of CEO.
As shown by FTIR and DSC, CEO encapsulation resulted in a good
compatibility between the essential oil and the capsule wall. The CEO-
loaded chitosan NPs illustrated strong antibacterial and DPPH radical
scavenging activities. The results of this work demonstrate that CEO can
be encapsulated into chitosan NPs, and this strategy could be re-
commended a novel approach for applications in food, active packa-
ging, and other products.
CRediT authorship contribution statement
Milad Hadidi: Resources, Data curation, Investigation. Shiva
Pouramin: Resources, Data curation, Investigation. Fateme
Adinepour: Formal analysis, Writing - original draft. Shaghayegh
Haghani: Methodology, Visualization. Seid Mahdi Jafari:
Conceptualization, Project administration, Validation, Supervision.
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