International Journal of Biological Macromolecules 186 (2021) 702–713

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Assessing the efficiency of essential oil and active compounds/poly (lactic

acid) microcapsules against common foodborne pathogens
Priscila Almeida Lucio Campini a, Éder Ramin de Oliveira a, Paulo Henrique Camani a,
Cristina Gomes da Silva b, Eliana Della Coletta Yudice c, Sueli Aparecida de Oliveira a,
Derval dos Santos Rosa a, *
a
Engineering, Modeling, and Applied Social Sciences Center (CECS), Federal University of ABC, Santo André, SP, Brazil
b
Institute of Exact Sciences, Federal University of Amazonas, Manaus, AM, Brazil
c
Adolfo Lutz Institute CLR VIII, Rua Ramiro Colleone, 240, Santo André CEP: 09040-160, SP, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Essential oils’ active compounds present great potential as a bactericidal agent in active packaging. The
Microcapsules encapsulation in polymeric walls promotes their protection against external agents besides allowing controlled
PLA release. This work produced PLA capsules with three different active compounds, Cinnamomum cassia essential
Antibacterial activity
oil (CEO), eugenol (EEO), and linalool (LEO), by emulsion solvent evaporation method. Characterizations
included SEM, Zeta potential, FTIR, TGA, and bactericidal activity against E. coli, S. aureus, L. monocytogenes, and
Salmonella. The active compounds showed microbiological activity against all pathogens. CEO capsules showed
superior colloidal stability. The active compounds’ presence in all capsules was confirmed by FTIR analysis, with
possible physical interaction between CEO, EEO, and the polymeric matrix, while LEO had a possible chemical
interaction with PLA. TGA analysis showed a plasticizing effect of active compounds, and the loading efficiency
was 39.7%, 50.7%, and 22.3% for CEO-PLA, EEO-PLA, and LEO-PLA, respectively. The capsules presented two
release stages, sustaining activity against pathogens for up to 28 days, indicating a satisfactory internal
morphology. This study presented methodology for encapsulation of antimicrobial compounds that can be
suitable for active food packaging. CEO-PLA capsules regarding stability and antibacterial activity achieved the
best results.

1. Introduction mixtures of volatile active compounds obtained from aromatic plants,

Foodborne pathogens are the primary cause of human infections
after consuming contaminated products, most cases caused by bacteria,
characterized mainly by gastrointestinal symptoms like nausea, vomit­
ing, and diarrhea. Staphylococcus aureus, Salmonella species, Campylo­
bacter species, Listeria monocytogenes, and Escherichia coli are the most
common causes of foodborne diseases globally [1].
Over the years, many food preservation techniques have been stud­
ied, preventing the spread of pathogens, food spoilage, and waste gen­
eration [2]. Active packaging has been explored against food spoilage
and foodborne illnesses, absorbing or releasing substances to prolong
shelf life [3]. Previously, shelf life was extended by adding synthetic
antioxidants to packaging, conflicting with consumers’ new profile,
attentive to sustainability issues [4]. Essential oils (EOs) are complex
such as ginger, oregano, mint, thyme, cinnamon, among others [4].
They have been studied as natural additives, designated as Generally
Recognized as Safe (GRAS) by the Food and Drug Administration (FDA)
[3,5]. The natural extracts’ ability to preserve food is attributed to their
composition, divided into terpenes/monoterpenes and aromatics
groups. Monoterpenes are common molecules present in 90% of the EOs
[6]. These compounds are phytochemicals responsible for suspending
the biochemical process of development and multiplication of micro­
organisms. Their hydrophobic properties allow its diffusion through the
bacterial cell membrane and mitochondria, leading to a rupture in the
bacterial cell’s plasmatic membrane and consequent lysis [7]. Active
phytochemicals are also efficient against multi-resistant pathogenic
bacteria due to aromatic compounds, hydrocarbons with one or more
benzene rings in their structure [8].

* Corresponding author at: Av. dos Estados, 5001, A 732-1, Santa Terezinha, Santo André, SP, Brazil.
E-mail address: derval.rosa@ufabc.edu.br (D.S. Rosa).

https://doi.org/10.1016/j.ijbiomac.2021.07.071
Received 5 February 2021; Received in revised form 7 July 2021; Accepted 11 July 2021
Available online 14 July 2021
0141-8130/Published by Elsevier B.V.
P.A.L. Campini et al.
The composition of each natural extract determines its model of
action against bacteria or fungi. Encapsulation methods are an alter­
native to preserve extracts’ properties and control their activity in the
medium where inserted. The literature presents some examples of
encapsulation processes, such as emulsion systems, spray drying, freeze-
drying, lipid-based nanoencapsulation, biological and non-biological
polymeric nanocarriers [9,10]. In this study, encapsulation was car­
ried out by the emulsion solvent evaporation method. It consists of
dripping the polymer solution into the emulsion, resulting in the oil
micelle’s involvement, and precipitation as capsules [11,12].
Poly (lactic acid) (PLA) is a biodegradable polymer successfully
applied for drug encapsulation purposes, tissue engineering, medical
devices, and biomedical products due to its non-toxicity, biodegrad­
ability, and biocompatibility [13]. Capsules of PLA containing EOs or
their major isolated components promote the active compounds’
controlled release against foodborne pathogens, which can be moni­
tored by antifungal and antimicrobial assays [14–16].
The review presented by Hosseini and Jafari [17] lists recent studies
regarding the importance of encapsulation techniques, evidencing the
superiority of encapsulated bioactive compounds compared to their free
form. The review includes the study reported by Hossaina and co-
workers [18] concerning essential oils encapsulated in nanoemulsions,
incorporating three different EOs (thyme with oregano, tea tree, and
peppermint) in cellulose nanocrystal chitosan reinforced films. The
process increased the stability of the EOs, combined with antifungal
activity and slow release of active compounds.
The research conducted by Cabral et al. [19] confirms the impor­
tance of encapsulation for protecting the antioxidant activity of phenolic
extracts from Plinia cauliflora applied to a chitosan polymeric matrix,
enhancing their preservation. Unfortunately, when it comes to PLA
microcapsules containing EOs or its purified isolate compounds, not too
many papers are available, which can be considered an immense op­
portunity to explore this type of microcarrier’s behavior.
Little information is available on the Web of Science™ (Clarivate
Analytics™, EUA) database about the subject proposed in this work
[20–22]. Therefore, this work aimed to develop three distinct PLA
capsules containing Cinnamomum cassia essential oil (CEO), eugenol
(EEO), and linalool (LEO) to evaluate and compare their antimicrobial
efficiency against the pathogens Escherichia coli (E. coli), Staphylococcus
aureus (S. aureus), Listeria monocytogenes (L. monocytogenes), and Sal­
monella enterica subsp. enterica serovar Choleraesuis (Salmonella).
2. Materials and methods
2.1. Materials
Materials used in the encapsulation process were PLA Ingeo 2003D
(lot 653-89-01, Agricultural Cargill S.A., Minnetonka, EUA), Chloroform
P.A. (Synth Products, Brazil), sodium lauryl ether sulfate (Synth Prod­
ucts, Brazil), and sodium bicarbonate (Tayuyna Laboratory-ADV com­
pany, Brazil). Cinnamomum cassia essential oil (at least 80% in
cinnamaldehyde content) and eugenol (99% purity) were purchased
from Ferquima® (Brazil). Linalool (98.7% purity) was provided by
Quinarí® (Brazil). All reagents and auxiliaries were acquired and used
as received. Biological assays were performed using four different
microorganism cultures: Escherichia coli (E. coli, ATCC 11229), Salmo­
nella enterica subsp. enterica serovar Choleraesius (Salmonella, ATCC
10708), Staphylococcus aureus (S. aureus, ATCC 6538), and Listeria
monocytogenes (L. monocytogenes, ATCC 19117), kindly provided by
Adolfo Lutz Institute-Centre of Interdisciplinary Procedures (Culture
Collections of Microorganisms), affiliated to World Federation Culture
Collections (WFCC) #282, Depository Collection - #017/09 – SECEX/
CGEN/MMA.
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International Journal of Biological Macromolecules 186 (2021) 702–713
2.2. Methods
2.2.1. Evaluation of the Minimum Inhibitory Concentration (MIC) and the
Minimum Bactericidal Concentration (MBC) for the preliminary
determination of active compounds antibacterial concentration
Minimum Inhibitory Concentration (MIC) and the Minimum Bacte­
ricidal Concentration (MBC) are essential parameters to determine the
active compound activity against bacteria and fungi [23]. The dilution
method determined the MIC and the MBC of CEO, EEO, and LEO,
screening their different antimicrobial activities, was carried out ac­
cording to Clinical and Laboratory Standards Institute (CLSI) standard
M07: Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria
That Grow Aerobically [24].
The serial dilution method was applied in microplates with 96-wells
and flat bottom, with 100 μL of Mueller Hinton broth (KASVI) solution
and polysorbate surfactant (Tween 20®, Sigma Aldrich) filled each well
until the 24th. 200 μL of the active compound was added to the first well,
half of this amount was added to the second well, and the same pro­
cedure was adopted until the last well, successively. Then, 100 μL of the
bacterial suspension of each culture was added to all the wells. The
plates were sealed using Parafilm® and maintained in the oven at 37 ◦ C
for 24 h. The procedure was carried out for the CEO, EEO, and LEO.
The solutions were submitted to positive controls for the culture
medium (Mueller Hinton broth with polysorbate surfactant) and each
bacterial culture (culture medium and inoculum) and negative control
for each active compound.
MIC determines the minimum concentration capable of inhibiting
bacteria’s growth after 48 h, and the MBC was found at the previous
well, which is the active compound’s minimal lethal concentration.
These results revealed the amount necessary to kill or inhibit each
bacterium used in this study, which allowed a direct comparison be­
tween the three different active compounds’ actions.
2.2.2. Cinnamomum cassia EO and isolated active compounds
encapsulation
The encapsulation process started with 100 mL of distilled water,
followed by lauryl ether sulfate (0.03 g) and sodium bicarbonate (0.08
g) addition. The solution was then kept under magnetic stirring for 15
min at a low speed. Next, the active compound was added at 0,5% v/v
concentration and kept under stirring for 10 min. The procedure was
adopted for the CEO, EEO and LEO separately.
In the next step, an organic solution of PLA (5 wt%) solubilized in
chloroform at 25 ◦ C under magnetic stirring and kept at ~5 ◦ C until use
was added to the system using a syringe with a blunt tip needle of 0.05
mm of outer diameter. 10 mL of the PLA solution for every 100 mL of the
aqueous solution was added, drop by drop, and kept under stirring for
24 h, followed by filtration and drying of the capsules at room tem­
perature of approximately 25 ◦ C. Fig. 1 illustrates the capsules’
preparation.
2.3. Capsules characterization
2.3.1. Scanning electron microscopy (SEM)
FEI Quanta 250 equipment (Thermo Fisher Scientific, USA) investi­
gated the capsules’ walls and surface morphology, using the accelerating
voltage of 20 kV and spot size of 4 nm. For samples’ preparation, a thin
layer of gold (~20 nm) covered the PLA capsules on the top, using 0.3
mbar pressure at 1.5 kV by sputtering equipment Edwards Scancoat six
Pirani 501 (Edwards Laboratories, USA). SEM images provided the
morphology and size distribution characteristics. The size distribution
was studied on ImageJ software (version 1.52a).
2.3.2. Zeta potential
The capsules were assessed on Zeta potential using a ZetaSizer Nano
NS equipment (Malvern Panalytical, UK). Before the assays, the capsules
were dispersed in distilled water and sonicated using a tip ultrasound
P.A.L. Campini et al. International Journal of Biological Macromolecules 186 (2021) 702–713

Fig. 1. Schematic illustration of the PLA capsules’ preparation, containing the active compounds: CEO, EEO, or LEO.

(Vibra Cell VCX 500, Sonics, USA), with an amplitude of 40%.

Table 1
Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concen­
2.3.3. Fourier transform infrared spectroscopy (FTIR)
tration (MBC) percentage of the active compounds against GN and GP bacteria.
An infrared spectrometer Frontier (94942-PerkinElmer, USA) was
used to analyse the samples of CEO, EEO, LEO, PLA, and PLA capsules Bacteria MIC (%) MBC (%)

loaded with active compounds, using attenuated total reflectance. The CEO EEO LEO CEO EEO LEO
samples were carefully deposited to cover the entire detection region. E. colia
0.024 0.19 0.095 0.048 0.38 0.19
Spectra were recorded in the range of 4000 cm− 1 to 500 cm− 1, as an Salmonellaa 0.012 0.19 0.095 0.024 0.38 0.19
average of 32 scans, using 4 cm− 1 resolution. S. aureusb 0.095 0.75 3.12 0.19 1.5 6.25
L. monocytogenesb 0.0015 0.38 0.75 0.0030 0.75 1.5

2.3.4. Thermogravimetry analysis (TGA) a

Thermal stability analysis of CEO, EEO, and LEO, as well as PLA and
the capsules produced, were carried out in a thermal analyser STA6000
(PerkinElmer, USA), heating in the range of 30 ◦ C to 600 ◦ C, with a
heating rate of 10 ◦ C min− 1, under a nitrogen atmosphere with a flow
rate of 20 mL min− 1.
From the TGA results, the encapsulation loading efficiency (LE%)
was evaluated. LE% was calculated by Eq. (1) [25], where is correlated
the ratio between WEO and Wcapsule, corresponding to the essential oil
weight loss and the initial capsules weight, respectively.
LE (%) = WEO /Wcapsule × 100
2.3.5. Evaluation of the antibacterial activity of the capsules
Isolated suspensions and standard bacterial strain cultures were
prepared 24 h before the tests, according to CLSI standard M07 [24].
An amount of 0.1 g of CEO-PLA, EEO-PLA, and LEO-PLA capsules
was placed in the Petri dishes center containing the inoculated cultures
(Gram-negative (GN) cultures: E. coli and Salmonella; Gram-positive
(GP) cultures: S. aureus and L. monocytogenes), occupying an area of
approximately 1 cm2 and incubated at 37 ◦ C. After periods of 7, 14, and
28 days of incubation, it was measured the inhibition zone’s diameter,
which is the zone’s diameter around the capsules that did not present
visual growth of microorganisms [26]. The measurements were per­
formed with the assistance of the ImageJ software (version 1.52a).
2.3.6. Statistical analysis
PAST statistics software (version 4.03) was used for statistical anal­
ysis, applying the one-way ANOVA method for an accurate mathemat­
ical comparison of the samples. In case of non-compliance with the
ANOVA test, the Tukey test was employed.
3. Results and discussions
3.1. Evaluation of MIC and MBC for the preliminary determination of the
active compound antibacterial concentration
To preliminarily evaluate the antimicrobial action of the CEO, EEO,
and LEO according to active compound concentration, Table 1 presents
(GN).
b
(GP).
MIC and MBC values.
Firstly, it was verified that all active compounds presented positive
MIC and MBC values, suggesting a significant inhibition, resulting in an
antimicrobial effect. The results showed that CEO was the most efficient
among the three active compounds tested against all bacteria, especially
for GN pathogens (E. coli and Salmonella), with the best efficiency
against L. monocytogenes (GP). While CEO and EEO presented satisfac­
tory results for both GN and GP bacteria, LEO has proved to be a good
(1)
efficiency only against GN species, not being suitable for use against GP
pathogens due to the low efficiency. According to Riaz and co-workers,
Berthold-Pluta and co-workers [27,28], GN cultures are more resistant
to EOs due to their chemical structure. Their cell wall composition
presents lipopolysaccharides, which partially block the active com­
pounds’ penetration through the cell walls, while GP cultures do not
have that type of protection, making them less resistant to the EO active
compounds [27,28].
However, this study observed the opposite behavior; LEO has proved
to be helpful against the GN bacteria assayed, i.e., E. coli and Salmonella.
Regarding EEO, the same results were obtained by Hyldgaard et al. [29],
observing a higher activity against GN bacteria. Wen and co-workers
[30] successfully incorporated the CEO and β-cyclodextrin complex in
a PLA nanofiber biodegradable matrix, obtaining results against GP and
GN bacteria cultures. The results reinforced the initial proposition of
CEO effectiveness against both cultures, mainly when the EO is associ­
ated with delivery systems such as capsules or nanofibrous structures.
EEO had its antibacterial characteristics confirmed by Piletti et al.
[26], testing this active compound against both GN (E. coli) and GP
(S. aureus) bacteria, with broad inhibition results against both species.
Microbiological assays proved this active compound’s antibacterial ac­
tivity, but it demands EEO higher concentrations than CEO to inhibit
and kill the same bacteria species.
LEO, the main component present on several EOs, was reported by
many authors as a potent antibacterial and antioxidant [31,32].
Nevertheless, its volatility and low redox stability properties revealed
the necessity of an efficient delivering system to preserve its active
compounds [31]. Xue Liu et al. [32] obtained MBC and MIC values

704
P.A.L. Campini et al.
between 0.5 and 1 μL/mL against GN Pseudomonas aeruginosa, similar to
the values obtained against GN cultures evaluated in this paper. LEO was
more effective against GN cultures than EEO, demanding half of the
concentration against those bacteria.
The hydrophobicity of the plant secondary metabolites (PSMs) that
constitute the EOs makes it possible to accumulate around the microbial
cell membrane, affecting the lipidic membranes’ structure and function,
disturbing microorganisms’ biological function, leading chemiosmotic
control to failure [33]. According to Hyldgaard et al. [34], the functional
groups present in the chemical structure and how they interact with
bacteria cells attribute the mode of bactericidal action to essential oils.
CEO has aldehyde groups in their structure that affect the membrane
integrity, releasing cellular content; this leads to crosslinking with DNA
and proteins, potentially contributing to broader bactericidal activity.
EEO and LEO activity occurs due to the -OH functional group, leading to
membrane permeability, promoting coagulation of cell contents and
inhibiting the ATP production. Additionally, it causes ATP leakage,
affecting protein properties, contributing to an inhibitory effect even at
sub-lethal concentrations [33].
3.2. Scanning electronic microscopy (SEM) and zeta potential of capsules
Fig. 2 reports the size distribution histogram and capsules’ mor­
phologies for CEO-PLA, EEO-PLA, and LEO-PLA obtained by SEM im­
ages. The average size values for CEO-PLA, EEO-PLA, and LEO-PLA
capsules were (80.1 ± 2.6) μm, (23.4 ± 1.6) μm, and (75.2 ± 2.3) μm,
respectively. 12% of the CEO-PLA capsules presented diameter of 50 μm
Fig. 2. (Left) Histogram of the size distribution; (center) SEM images; and (right) zoom of SEM images for a) CEO-PLA capsules, b) EEO-PLA capsules, and c) LEO-
PLA capsules, respectively.
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International Journal of Biological Macromolecules 186 (2021) 702–713
or less (minimum diameter at 36.6 μm and the maximum at 142.7 μm);
EEO-PLA capsules presented 55% of the capsules measured under 50 μm
(minimum size at 4.7 μm and maximum at 79 μm), and LEO-PLA cap­
sules presented 84.2% of the capsules with a diameter measured above
50 μm (minimum at 20 μm and maximum at 99.4 μm). Moreover, EEO-
PLA and LEO-PLA showed higher size regularity, while CEO-PLA pre­
sented a polydisperse distribution.
Studies conducted by Marson and co-workers described that using
anionic surfactants respecting its CMC is crucial to the obtention of
capsules with high sphericity, consistent with the highly spherical cap­
sules obtained in this study, once lauryl ether sulfate CMC is of 0.8
mmol.L− 1 [35,36].
According to capsules size, Hu, Liu & Deng [31] observed that spe­
cific natural oily extractions’ hydrophobic profile, as CEO and LEO,
promotes capsules with a larger size. Besides, fixed polymer concen­
tration was used, which may not respect the interactions between the
polymer matrix and the surfactant. Budinčić and co-workers [37] ob­
tained capsules with mean diameters of 5 to 6.85 μm respecting the
interactions between the polymer-surfactant and using spray-drying
technique and a deagglomeration step. High values of hydrophilic-
lipophilic balance (HLB) of the surfactant also contribute to the larger
size of the capsules, as described by Antonioli and co-workers [38], who
obtained PLA capsules containing lemongrass with diameters ranging
from 97 to 158 nm using a low HLB surfactant. The size may also be
attributed to the solvent evaporation technique, resulting in water
remaining inside the capsules, as indicated by thermogravimetric anal­
ysis in Section 3.3. Noghabi and Molaveisi [39] obtained smaller CEO
P.A.L. Campini et al.
microcapsules between 0.550 and 2.956 μm dimensions, applying the
spray drying technique, while simple solvent evaporation could lead to
larger capsules with higher polydispersity [40].
Small agglomeration may be attributed to the active compound
properties, as pointed by Mikulcová, Bordes, and Kašpárková, where the
polar character of the oils may contribute to the formation of larger sizes
and aggregates [41]. Another factor that could affect capsules’ poly­
dispersity was the shear rate of the mixing process during the Oil/Water
system emulsification combined with a high ionic surfactant concen­
tration. That explains the size and the polydispersity of the capsules
[42,43].
The ANOVA test revealed an outcoming p-value of less than 0.05,
which indicates a non-compliance between the three different capsule
sizes. Tukey’s test was then applied to compare similarities and differ­
ences between the three different capsules. The results are displayed in
Table 2.
Tukey’s test results revealed conformity between CEO-PLA and LEO-
PLA capsules, with a p-value higher than 0.05. However, compared to
EEO-PLA capsules, the two other systems have no similarity, confirming
the results obtained by SEM images since the particle sizes of EEO-PLA
capsules were at least two times smaller than the values obtained for the
other capsules.
Zeta potential measurements were performed to describe the
colloidal stability of the capsules in an aqueous medium. A value of
+33.3 ± 1.0 mV for CEO-PLA was observed, while EEO-PLA and LEO-
PLA capsules had charges of +10.2 ± 1.2 mV and + 10.6 ± 1.6 mV,
respectively. According to the literature, Zeta potential values above 30
mV indicate capsules’ good stability because of the balance between
repulsion forces among all the emulsion particles, avoiding agglomera­
tion [44]. Similar results were obtained by Budinčić and co-workers
[37], which may be attributed to the usage of anionic surfactant in the
presence of positive ions, as described by Kundu and co-workers [45].
CEO-PLA capsules present higher colloidal stability between all the
capsules developed in this study, suggesting that the CEO had a prom­
inent effect on changing the polymer’s superficial charges compared to
the other compositions. These results corroborate with other works in
the literature. Soto-Chilaca and co-workers [46] prepared N-acylated
chitosan nanoparticles containing cinnamaldehyde, with Zeta potential
values from +24.5 to +29.9 mV and small agglomeration between
nanoparticles. The Zeta potential values found in this work for CEO-PLA
capsules were close to the values found by Soto-Chilaca and co-workers.
Therefore, a hypothesis to explain the better colloidal stability is the
possibly changed in superficial charges of PLA by CEO’s molecules with
higher efficiency, compared to other capsules. Another possible expla­
nation comes from the affinity of PLA with positively charged ions in the
emulsion system, which was saturated with Na+ ions from lauryl sodium
sulfate and sodium bicarbonate, as noticed by Kim et al. [47] and Pan
et al. [48], who observed interactions between oxygen with unpaired
electrons in PLA and positively charged particles. The better colloidal
stability could improve the capsules’ dispersion when added into the
polymeric matrix, contributing to a uniform controlled release.
3.3. Fourier transform infrared spectroscopy (FTIR) and
thermogravimetry analysis (TGA)
Fig. 3 presents the spectra of PLA, the active compounds (CEO, EEO,
and LEO), and the different capsules prepared from PLA containing
active compounds.
Table 2
Tukey’s test results comparing, individually, each group of capsules.
Interaction
CEO-PLA ∕
= EEO-PLA
CEO-PLA = LEO-PLA
EEO-PLA LEO-PLA
=
∕
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International Journal of Biological Macromolecules 186 (2021) 702–713
From FTIR spectra, presented in Fig. 3a and b, some considerations
about raw materials were performed to comprehensive their chemical
structure and particularities. The PLA spectrum showed some charac­
teristic peaks, as an intense peak at 1749 cm− 1 and others among 1452
cm− 1 and 1359 cm− 1, associated with the stretching of carbonyl (C=O)
groups and bending and deformation vibrations in –CH3 groups,
respectively [49,50]. Other peaks at 1180 cm− 1, 1081 cm− 1, 1043 cm− 1,
870 cm− 1, and 754 cm− 1 were also observed, being related to C-O-C
stretching, C–O deforming, and O–H bending. [49–51].
Considering active compounds, the CEO spectrum presented char­
acteristic peaks and bands of Cinnamomum cassia essential oil, covering
peaks at 1671 cm− 1 and 1625 cm− 1 attributed to carbonyl groups and
unsaturated vibrations of benzene rings [30,52]. Other peaks were also
found at 1120 cm− 1 and 688 cm− 1, related to C-OH of phenolic com­
pounds and C– – C bonds of aromatic compounds, and a band among 829
and 745 cm− 1, associated with C–H bonds in aromatic rings [30,52].
The EEO spectrum presented a peak at 3000 cm− 1 related to the
stretching of the CH2 and CH3 groups. Bands were also observed at
3100–3600 cm− 1, 1511 cm− 1, and 1432 cm− 1, addressed to stretching of
–OH groups, C– – C bonds in the aromatic ring, and -OH groups in
phenolic compounds, respectively [53]. At last, LEO spectrum presented
a band at 3381 cm− 1 and two peaks at 2973–2868 cm− 1, corresponding
to -OH symmetric stretching vibration and aliphatic C–H stretching,
respectively [54]. Some critical peaks were found at 1114 cm− 1 and
1000 cm− 1, addressed to aromatic and alcohol structures, and other
peaks as at 1453 cm− 1, 1116 cm− 1, 917 cm− 1, and 639 cm− 1 to asso­
ciated with C–H groups, alcohol groups, stretching of C–O groups, and
aromatic compounds, respectively [54].
Capsule’s spectra were analysed to understand interactions between
active compounds and PLA polymeric wall. All capsules’ compositions
presented a peak at 1753 cm− 1, associated with stretching of carbonyl
groups, indicating that the chemical structure of PLA has not changed
during the capsules’ preparation, even with the presence of oils and
surfactants [55]. CEO-PLA capsules showed peaks at 1674 cm− 1 and
1626 cm− 1, addressed to stretching and vibrations of –C=O groups on
aldehydes, as well as vibrations of unsaturation in the benzene ring, like
the ones detected in the CEO spectrum [51]. Other peaks at 1451 cm− 1
and 749–689 cm− 1 related to bending out of the C–H plane and the
presence of C– – C groups in aromatic rings were also observed [50].
Analysing the curves for PLA and CEO-PLA capsules, some peaks pre­
sented small shifts around 1750 cm− 1, 1450 cm− 1, 1180 cm− 1, 1040
cm− 1, and 870 cm− 1. Regarding EEO-PLA and LEO-PLA capsules, the
same behavior was observed, with the presence of the active compound
characteristics peaks and neat PLA peaks slights shifted for higher
wavenumbers. It possibly occurred due to the interaction between the
active compounds and PLA structure. Da Silva and co-workers reported
that a slight shift in some peaks was an indication of interaction between
essential oil structures and functional groups presents in capsule’s
structure material [56]. The occurrence of similar bands and peaks be­
tween the active compounds, the polymer, and the active compounds
loaded capsules was evidenced. Therefore, it suggested an interaction
between the structures of the active compounds and PLA.
The deconvolution of spectra from one characteristic peak related to
functional groups of CEO, EEO, and LEO (highlighted in some regions of
spectra in Fig. 3c, d, and e) and their respective capsules were performed
to understand in-depth the interaction between these compounds. Fig. 4
presents deconvoluted curves from FTIR spectra for active compounds
and their capsules.
According to deconvoluted curves in Fig. 4a and b for CEO, one peak
at 1671 cm− 1 attributed to carbonyl groups from the leading functional
group was considered to calculate the area. Deconvoluted peak area of
CEO presented a total of 2.07, while for CEO-PLA capsules, the area
decreased to 1.50. This reduction of 0.57 may indicate a weak interac­
tion between PLA and CEO. Gomes, Moreira, and Castell-Perez [40]
observed that due to cinnamaldehyde’s chemical structure, the major
component in CEO, oil diffusivity from polymer walls onto the capsule’s
P.A.L. Campini et al.
Fig. 3. a) FTIR spectrum with range of 4000 to 500 cm− 1; b) region’s zoom between 2000 and 500 cm− 1 for all samples; c), d) and e) zoom for CEO (range of
1800–1000 cm− 1), EEO (range of 1800–1100 cm− 1), and LEO (range of 1200–600 cm− 1), and its respective PLA capsule, respectively.
structure was favoured. The same behavior was observed for EEO
(Fig. 4c and d), considering one peak at 1511 cm− 1 attributed to C– –C
bonds from an aromatic ring, which presented an area reduction of 0.41
between EEO and EEO-PLA capsules curves. This result indicates a weak
interaction between the CEO and EEO molecules and the polymer
chemical structure. However, for LEO (Fig. 4e and f), one peak at 917
cm− 1, corresponding to C–O stretching, lead to an area difference of
5.92 between LEO and LEO-PLA capsules curves. Thus, one hypothesis
to explain this area reduction is the strong interaction between LEO and
PLA structure, affecting the active compound’s migration outside of
capsules’ structure. Therefore, CEO-PLA and EEO-PLA capsules pre­
sented possibly a physical interaction among these compounds, even
facilitating oil exudation. In opposite, LEO-PLA capsules presented a
strong interaction among their compounds, suggesting a chemical
interaction.
Thermogravimetric analysis was applied to verify the influence of
active compounds on the polymeric wall. The results are shown in Fig. 5
707
International Journal of Biological Macromolecules 186 (2021) 702–713
and Table 3.
As shown by the curves in Fig. 5b, the occurrence of single peak
curves demonstrates the volatile nature of the active compounds, which
was the case for the three alternatives assayed (CEO, EEO, and LEO).
Comparing the DTG curves, all active compounds presented a similar
profile, with a single step curve related to thermal degradation, repre­
sented by a single peak with Tmax1 at 189 ◦ C, 194 ◦ C, and 149 ◦ C for CEO,
EEO, and LEO, respectively. Onset and offset temperatures were verified
at 146 ◦ C and 197 ◦ C for EEO, 149 ◦ C and 200 ◦ C for CEO, and 117 ◦ C
and 152 ◦ C for LEO, which is in accordance with the literature [57–61].
Therefore, when the temperature reached around 240 ◦ C, all essential
oils were degraded, and the weight loss is 100%. DTG also showed that
neat PLA capsules had a curve with one-step weight loss, with Tmax2 at
344 ◦ C, related to the polymer thermal degradation, also noticed by
Herrera-Kao and co-workers [49].
Analysing the DTG curves (Fig. 5c) of EEO-PLA and LEO-PLA cap­
sules, it presented a peak around 60 ◦ C, generated by the evaporation of
P.A.L. Campini et al.
Fig. 4. Deconvoluted peaks from FTIR curves, comparing active compound types (a) CEO, (c) EEO, (e) LEO; and its respective capsules (b) CEO-PLA, (d) EEO-PLA, (f)
LEO-PLA.
the excess water present in these samples. The same behavior was re­
ported by Tovar and co-workers [62], attributing this decomposition
peak to structural bound water and some degradation of low molar mass
compounds of EOs. While CEO-PLA, this event was not detected the peak
water release, only a single peak event at 164 ◦ C is correlated to the
released EO, followed by the second event of degradation of the polymer
itself. The Tmax1 peaks, which are correlated to the release of EOs present
in the capsules, show a shift to lower temperatures, comparing the peaks
in Fig. 5b and c. The results suggest higher thermal stability for capsules
with lower moisture content due to water presence acting as a polymer
plasticizer, also notice by Toledo Hijo and co-workers [63]. As
mentioned before, a better drying process may impact capsule
708
International Journal of Biological Macromolecules 186 (2021) 702–713
properties, which needs further investigation. Besides, thermal degra­
dation at lower temperatures suggests an exposed active compound,
probably adsorbed on the capsule surface. In contrast, the degradation in
higher temperatures suggests an effective encapsulation, with the
polymer walls retarding the thermal degradation. Therefore, EEO and
LEO capsules had not presented an efficient thermal stabilization of the
active compounds, while CEO-PLA capsules had different behavior,
presenting a better thermal stabilization of the active compound, as
shown in Fig. 5a.
The discrete reduction of Tmax2 of capsules is related to the interac­
tion of active compounds with the polymeric walls, indicating the active
compounds’ action as plasticizers into the polymer structure, as also
P.A.L. Campini et al. International Journal of Biological Macromolecules 186 (2021) 702–713

Fig. 5. (a) TGA curves of (1) PLA, (2) CEO, (3) EEO, (4) LEO, (5) CEO-PLA capsules, (6) EEO-PLA capsules, and (7) LEO-PLA capsules, respectively; (b) DTG curves of
(2) CEO, (3) EEO, (4) LEO, (c) DTG curves of (1) PLA, (5) CEO-PLA capsules, (6) EEO-PLA capsules, and (7) LEO-PLA capsules, respectively.

encapsulation rate and encapsulation efficiency [65], the values are

Table 3
considered satisfactory. Finally, PLA capsules loaded with CEO pre­
Thermal decomposition temperatures and loading efficiency of encapsulation.
sented the best encapsulation into PLA capsules among the assays, once
Sample Tonset1 (◦ C) Tmax1 (◦ C) Tonset2 (◦ C) Tmax2 (◦ C) LE (%) these capsules presented high loading efficiency and the best effect of
1– PLA 103.5 137.0 247.5 344.0 – thermal stabilization. Despite the high loading efficiency of EEO-PLA
2– CEO 149.0 189.0 – – – capsules, high water content was noticed, as for LEO-PLA capsules,
3– EEO 146.0 194.0 – – –
evidencing the necessity of improvements on drying methods.
4– LEO 117.0 149.0 – – –
5– CEO-PLA 84.0 164.0 288.5 338.0 39.7
6– EEO-PLA 88.5 138.0 287.0 346.0 50.7
7– LEO-PLA 74.0 107.0 288.5 346.0 22.3 3.4. Capsules’ antimicrobial assays

Fig. 6 shows the antimicrobial evaluation of the capsules incubated

noted by Pereira and co-workers [64], who observed a decrease of
thermal stability for zein films containing garlic and thyme essential oils
due to intermolecular interactions between the polymeric matrix and
essential oils. Thus, FTIR and TGA results suggest that the interaction
between the active compounds and PLA does not promote relevant
changes in the polymeric walls. However, a strong interaction between
LEO and PLA was detected by FTIR and confirmed by TGA analysis, as
shown in Fig. 5a, where the thermogram for LEO-PLA capsules shows a
different profile, not presenting a well-defined double-stepped curve,
which significantly affected the thermal stabilization.
The loading efficiency (LE %) was evaluated through the mass loss
events according to Eq. (1), resulting in 39.7%, 50.7%, and 22.3% for
CEO-PLA, EEO-PLA, and LEO-PLA capsules. Wen et al. [30] obtained a
loading content of 10,8% for capsules with cinnamon essential oil con­
tent, while Barbosa et al. [25] obtained a maximum loading efficiency of
29%. While PLA was reported as a material with a low drug
with the bacteria cultures listed in an overall bar chart of the inhibition
zones along 7, 14, and 28 days from the preparation.
In general, all capsules presented a certain degree of inhibition
against at least three types of bacteria, highlighting that the inhibition
halo ranged from 22 to 90 mm, among the best results. Fig. 6 evidences
the essential oils’ inhibition zone, highlighted by a yellow circle. CEO-
PLA and EEO-PLA presented a broader inhibition zone than LEO-PLA
in all periods evaluated. LEO-PLA capsules did not present activity
against L. monocytogenes (GP) for long periods of interaction (14 and 28
days); however, it proves to be active for all other bacteria after seven
days stored. Rehab and Zeinab [66] observed similar behavior to LEO
and EEO, with LEO presenting less activity against the same bacteria.
The best results were verified to CEO-PLA capsules for all periods and
bacteria tested, as displayed in the infographics of Fig. 6. Among these
results, CEO-PLA capsules showed a more relevant effect against Sal­
monella (GN) and L. monocytogenes (GP), with halo formation at 80 mm

709
P.A.L. Campini et al. International Journal of Biological Macromolecules 186 (2021) 702–713

Fig. 6. The bars represent the inhibition zone growth (mm) of the capsules (CEO-PLA, EEO-PLA, or LEO-PLA) after periods of 7-, 14-, and 28-days storage, and
correspondent images of the antimicrobial assay.

on the 28th day and 90 mm on the 14th day, respectively. The inhibition expressive against Salmonella (GN) (70 mm on the 7th day). The activity
zone graphics showed the capsules’ sustained-release behavior against against S. aureus (GP) promoted a halo formation of 70 mm on the 7th
both bacteria types. CEO-PLA capsules presented a relatively stable in­ day, while against L. monocytogenes (GP) the inhibition zone was 68 mm
hibition zone against E. coli (GN) (46.5 mm on the 7th day) and more on the 7th day.

710
P.A.L. Campini et al.
The EEO-PLA capsules showed adequate sustained-release, showing
better activity against GN cultures. The most expressive effective was
against Salmonella, reaching a maximum of 56 mm of inhibition zone
after the 14th day, and against E. coli, with an inhibition zone at 27.5
mm after the 14th day produced. EEO-PLA capsules were less effective
against GP cultures than GN cultures, which was also observed by
Nazzaro et al. [67]. An average inhibition zone of 27.8 mm was observed
throughout the test period against S. aureus (GP). In comparison, against
L. monocytogenes (GP), an inhibition zone of 27.5 mm was obtained only
within the first seven days period, not presenting any activity against
this culture in the following days.
As expected by the MIC and MBC results, LEO-PLA capsules were the
less active of the three alternatives assayed, presented effective activity
within seven days after being prepared against the bacteria, excepted
L. monocytogenes (GP), losing the bactericidal capacity after 14 days of
storage. However, after 28 days of storage, capsules exhibited inhibition
against E. coli (GN) at 20.5 mm, indicating that the active component’s
release occurs in two stages, probably due to the formed particles in­
ternal morphology be able to accommodate the active compound, while
an amount probably stayed outside the capsules, promoting the anti­
bacterial activity during the first seven days of tests.
Similarly, CEO and EEO had previously been reported as antimi­
crobials with broad activity against these bacteria set. As previously
discussed, the CEO acts through the cell membranes, due to carbonyl
groups at the cinnamaldehyde molecule. MIC and MBC results
confirmed this CEO’s property, where the lower concentration evi­
denced the best results in comparison to the other active compounds. For
CEO-PLA and EEO-PLA capsules, the results were similar to that ob­
tained in MIC and MBC assays, showing higher activity against
L. monocytogenes (GP) and Salmonella (GN), respectively. Nevertheless,
for LEO-PLA capsules, the results obtained were different from the MIC
analysis, with better activity against S. aureus and lowered for Salmonella
(GN) and E. coli (GN), the opposite of the expected, evidencing positive
aspects of encapsulation technique against foodborne pathogens.
4. Conclusions
The active compounds showed MIC and MBC values that allow
concluding their action against both GN and GP foodborne pathogens.
The methodology adopted to synthesize capsules was satisfactory,
resulting in microcapsules with a minimum size of 4.7 μm for EEO-PLA
and a maximum diameter of 142.7 μm for LEO-PLA, with approximately
spherical and non-porous wall morphology for all samples. Zeta poten­
tial measurements showed that CEO-PLA capsules had outstanding
colloidal stability, indicating a possible modification of superficial
charges in capsules’ polymeric wall promoted by this EO.
FTIR results evidenced that, except for LEO-PLA capsules, the EOs
possibly showed a physical interaction with the polymer, which resulted
in a more significant release of the active principles through the poly­
meric walls. TGA analysis evidenced plasticizing effects of the active
compounds, and loading efficiency of 39.7%, 50.7%, and 22.3% for
CEO-PLA, EEO-PLA, and LEO-PLA capsules, respectively. For CEO-PLA
and EEO-PLA capsules, the inhibition zone increased over time,
evidencing the capsules’ prolonged efficiency against the elected bac­
teria for up to four weeks. Relevant results were achieved with CEO-PLA
capsules regarding the stability and antibacterial activity of other
samples.
The results showed that the encapsulated active compounds pro­
moted higher stability and bacterial activity, suggesting applying the
capsules in a food packaging system to assess the bacterial activity in
loco. Future challenges include joint PLA capsules with other polymeric
biodegradable and renewable matrices to form films, creating complete
biodegradable active food packaging while extending the shelf life of
foods without using hazardous chemical food preservatives.
711
International Journal of Biological Macromolecules 186 (2021) 702–713
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgments
The authors thank the Federal University of ABC (UFABC), REVA­
LORES Strategic Unit, and Multiuser Central Facilities (CEM - UFABC)
for technical support, and BASF S.A for supplying the polymer used in
the development (PBAT). The authors are grateful to the Adolfo Lutz
Institute’s Nucleus of Chemical and Bromatological Sciences – CLR/IAL
– Santo André VIII, (Project: CTC-09-L/2019) for its valuable contribu­
tions with bacterial tests and facilities. Also, the authors thank the
financial support provided by the National Council for Scientific and
Technological Development (CNPq) (305819/2017-8 and 426530/
2016-0) and Coordination for the Improvement of Higher Education
Personnel (CAPES) (1752355 and 88882.451553/2019-01), and the São
Paulo State Research Support Foundation (FAPESP) (2018/11277-7 and
2019/16301-6).
CRediT authorship contribution statement
Priscila Almeida Lucio Campini: Conceptualization, Methodology,
Formal Analysis, Data Curation, Investigation, Writing – Original Draft.
Éder Ramin de Oliveira: Conceptualization, Investigation, Software,
Formal Analysis, Methodology, Writing – Original Draft, Visualization,
Validation;
Paulo Henrique Camani: Writing – Original Draft, Software, Formal
Analysis, Visualization, Validation.
Cristina Gomes da Silva: Project Administration, Writing – Review &
Editing, Supervision, Funding Acquisition.
Eliana Della Coletta Yudice: Resources, Investigation.
Sueli Aparecida de Oliveira: Writing – Original Draft, Software,
Validation.
Derval dos Santos Rosa: Resources, Writing – Review & Editing,
Supervision, Funding Acquisition.
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