International Journal of Biological Macromolecules 219 (2022) 812–823

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International Journal of Biological Macromolecules

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

Bio-nanocomposite edible coatings based on arrowroot starch/cellulose

nanocrystals/carnauba wax nanoemulsion containing essential oils to
preserve quality and improve shelf life of strawberry
Josemar Gonçalves de Oliveira Filho a, *, Beatriz Regina Albiero b, Ítalo Henrique Calisto c,
Mirella Romanelli Vicente Bertolo b, Fernanda Campos Alencar Oldoni a,
Mariana Buranelo Egea d, Stanislau Bogusz Junior b, Henriette Monteiro Cordeiro de Azeredo e,
Marcos David Ferreira e
a
São Paulo State University (UNESP), School of Pharmaceutical Sciences, Araraquara, SP, Brazil
b
University of São Paulo (USP), São Carlos Institute of Chemistry (IQSC), São Carlos, SP, Brazil
c
Federal University of São Carlos (UFSCAR), São Carlos, SP, Brazil
d
Goiano Federal Institute of Education, Science and Technology, Campus Rio Verde, GO, Brazil
e
Brazilian Agricultural Research Corporation, Embrapa Instrumentation, São Carlos, SP, Brazil

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

Keywords: This study investigated the effects of bio-nanocomposite coatings developed using arrowroot starch (AA), cel­
Active coatings lulose nanocrystals (CNC), carnauba wax nanoemulsion (CWN), and Cymbopogon martinii and Mentha spicata
Nano reinforcement essential oils (CEO and MEO, respectively) on the physicochemical, microbiological, bioactive, antioxidant, and
Spearmint
aromatic characteristics of strawberries cv. ‘Oso Grande’ in refrigerated storage for 12 days. The coatings
improved the shelf life and stability of strawberries, minimizing their weight loss (2.6–3.9 %), as well as changes
in color and texture (except for those coated with CEO), titratable acidity, pH, soluble solids, anthocyanins,
phenolic compounds, ascorbic acid content, and antioxidant activity compared with uncoated control straw­
berries. The bio-nanocomposite coatings containing MEO and CEO also exhibited antimicrobial activity, reduced
visible fungal deterioration (40–60 %), and reduced microbial load (3.59–4.03 log CFU g− 1 for mesophilic
aerobic bacteria and 4.45–5.22 log CFU g− 1 for fungi and yeast) during storage. They also significantly reduced
the severity of decay caused by inoculation with Botrytis cinerea or Rhizopus stolonifer. The coatings altered the
volatile profile of the fruits during storage, decreasing aldehyde and alcohol concentrations and increasing ester
concentrations. Thus, these bio-nanocomposite coatings, especially those containing MEO, can be used as anti­
microbial coating materials to preserve the post-harvest quality of fresh strawberries.

1. Introduction
The strawberry (Fragaria × ananassa Duch.) is an economically
important fruit worldwide and is highly valued for its unique flavor [1].
Strawberries are a source of beneficial bioactive compounds, including
vitamins (mainly vitamin C), anthocyanins, carotenoids, and minerals.
However, the strawberry is a perishable fruit due to its high respiration
rate, soft texture, and high susceptibility to fungal phytopathogens. All
of these factors can result in important changes in its quality parameters,
including color, texture, and levels of bioactive compounds [2].
In recent years, interest in the use of edible coatings to preserve the
quality and reduce the spoilage of strawberries during the post-harvest
period has grown [1,3–5]. Edible coatings preserve fruit quality by
forming a semi-permeable barrier on the fruit surface, reducing gas and
moisture exchange. This results in reduced water loss, respiration, and
ripening rates [6].
Various biomolecules, including proteins, lipids, and poly­
saccharides, have been used as matrices for edible coatings for fruits.
Among the polysaccharides, starch is a promising natural biopolymer
because of its low cost, effective coat-forming properties, and biode­
gradability [7]. Arrowroot (Maranta arundinacea) starch (AA) is an
inexpensive starch product, but it has not yet been commercially

* Corresponding author.
E-mail address: Josemar.gooliver@gmail.com (J.G. Oliveira Filho).

https://doi.org/10.1016/j.ijbiomac.2022.08.049
Received 22 May 2022; Received in revised form 22 July 2022; Accepted 7 August 2022
Available online 11 August 2022
0141-8130/© 2022 Elsevier B.V. All rights reserved.
J.G. Oliveira Filho et al.
exploited. The high amylose content in AA can help facilitate the
development of edible coatings with good tensile properties [8]. How­
ever, its hydrophilic nature detracts from its water barrier capacity [9].
Nanotechnology has been used to improve the properties of coatings,
giving rise to new materials, known as bio-nanocomposites, which
display better properties. The incorporation of nanostructures (with at
least one dimension ≤100 nm), such as nanoemulsions, nanotubes,
polymeric nanoparticles, and nanocrystals, can significantly improve
the barrier, mechanical, optical, and thermal properties of a coating
and/or provide it with active (e.g., antimicrobial) properties [10].
Carnauba wax is extracted from the Brazilian palm Copernicia pru­
nifera. A carnauba wax nanoemulsion (CWN) has been shown to
improve the water-barrier capacity of starch-based coatings with mini­
mal impact on its mechanical properties and microstructure compared
to conventional emulsions [11]. Moreover, the tensile and barrier
properties of the coatings can be improved using cellulose nanocrystals
(CNCs), which can help improve the adhesion of the coating to the fruit
surface [12].
To confer antimicrobial properties to coatings, natural antimicrobial
agents, including essential oils, have been added [3,13]. Essential oils
contain compounds produced by plant secondary metabolism. They are
especially useful because they are non-toxic, biodegradable, and
generally considered safe for consumption (GRAS) [14].
In our previous study [11], a new antimicrobial bio-nanocomposite
coating material was developed by combining AA, CWN, CNCs, and
Mentha spicata or Cymbopogon martinii essential oil (MEO and CEO,
respectively). The resulting coating materials displayed improved
water-barrier, mechanical, thermal, microstructural, and antifungal
properties against Rhizopus stolonifer and Botrytis cinerea, the primary
fungi that cause decay in strawberries. This study aimed to evaluate the
effect of AA-based edible coatings containing CWN, CNCs, and either
MEO or CEO on the post-harvest quality of strawberries over a 12-day
refrigerated storage period.
2. Materials and methods
2.1. Materials
The composition of AA has been previously described by Oliveira
Filho et al. [15], and the physical characteristics of CNCs were previ­
ously described by Oliveira Filho et al. [16]. Carnauba wax type I (99 %
purity; CAS No.: 8015-86-9) was provided by Pontes Indústria de Cera
(Parnaíba, PI, Brazil). MEO was purchased from Ferquima Ind. e Com.
Ltda. (Vargem Grande Paulista, SP, Brazil), and CEO was purchased
from Laszlo Aromaterapia (Belo Horizonte, MG, Brazil). The chemical
compositions of CEO and MEO (Supplementary File 1) have been pre­
viously described by Oliveira Filho et al. [11]. The fungi used for the
antifungal tests were Botrytis cinerea strain CCT 1252 and Rhizopus sto­
lonifer strain CCT 0276 (Andre Tosello Foundation, Campinas, SP,
Brazil). All other reagents used in this study were of analytical grade.
2.2. Preparation of bio-nanocomposite coatings
AA and CNCs were used directly in the preparation of the nano­
composite coatings. The CWN (droplet size: 39.3 ± 0.7 nm; zeta po­
tential: − 40.32 ± 1.0 mV) was prepared as described by Campos et al.
[17]. The nanoemulsion was characterized in a previous study per­
formed by our group [15].
The CNCs and AA (5:95, w/w) were first dissolved in water under
agitation with a magnetic stirrer (150 rpm) in a thermostatic bath (TE-
2005, TECNAL, Piracicaba, Brazil) at 85 ± 2 ◦ C for 5 min, yielding a 2 %
(w/w) aqueous dispersion. Then, the CWN (15 % on a dry starch basis)
was added to the aqueous mixture, and the suspension was homoge­
nized. Glycerol was used as a plasticizer and added until it reached a
level of 0.17 mL g− 1 of AA [15]. After cooling the dispersions to 40 ◦ C,
the essential oil (MEO or CEO) was added until it reached a
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International Journal of Biological Macromolecules 219 (2022) 812–823
concentration of 0.2 or 0.3 % (v/v), respectively. Mixtures were then
stirred using a high-performance dispersing instrument (UltraTurrax
T25, IKA Werke GmbH & Co, Staufen, Germany) for 5 min at 7000 rpm.
2.3. Contact angle and viscosity of bio-nanocomposite coating
The macroscopic surface wettability was examined using a CAM 101
optical contact meter (KSV Instruments) with a KSV-5000 CCD digital
camera and KSV CAM2008 software. A drop of distilled water (3 μL) was
pipetted onto a film surface, and 60 images were captured in 60 s.
Analysis was performed on the upper surface of the films (that is, the
surface of the film-air interface during drying). Ten replicates were
conducted for each film sample.
Solution viscosity was determined as a function of the shear rate
applied (0.1 to 1500 s− 1) at 25 ◦ C using an AR-1000 N controlled-strain
rheometer (TA Instruments, New Castle, DE, USA). The geometrical
setup was a 20-mm diameter stainless steel plate cone with an angle of
2◦ and a gap of 69 μm. The temperature was controlled using a Peltier
system, and curves were modeled according to the Cross model using the
TA Universal Analysis software.
2.4. Application of bio-nanocomposite coatings on strawberries
Strawberries cv. “Oso Grande” were acquired from a local market
(São Carlos, SP, Brazil). The fruits were visually selected for color, size,
and structural integrity and then washed and sanitized for 10 min in a
2.5 % (v/v) sodium hypochlorite solution. The strawberries were
randomly divided into six treatment groups (AA/CWN/CNC, AA/CWN/
CNC/MEO2, AA/CWN/CNC/MEO3, AA/CWN/CNC/CEO2, AA/CWN/
CNC/CEO3, and an uncoated control), with 20 fruits and five repetitions
per treatment.
The fruits were immersed in the bio-nanocomposite coating solutions
for 2 min, dried (2 h, 25 ± 2 ◦ C), placed in plastic boxes, and stored at 7
± 1 ◦ C and 70–75 % relative air humidity (RH) for 12 days. Strawberries
were evaluated for quality attributes (as described in Sections 2.5–2.9)
at the beginning of the experiment (0 days) and after 3, 6, 9, and 12 days
of storage.
2.5. Physicochemical parameters of strawberries
The rate of strawberry weight loss was determined using the stan­
dard method of the Association of Official Analytical Chemists (AOAC)
[18]. The fruits were weighed on day 0 (the beginning of the experi­
ment) and on days 3, 6, 9, and 12 of the storage period. The percent
difference between the initial and the final weight on each day was used
to calculate weight loss.
The soluble solids (SS) content was determined using an Atago RX-
5000cx digital refractometer and expressed as %Brix [19]. The pH of
the samples was measured using a PHS-3B digital pH meter according to
the standard method. Titratable acidity was determined using 0.1 M
NaOH, with phenolphthalein as the indicator. The results were
expressed as g of citric acid per 100 g of fruit.
Color measurements were performed on the external surface of the
fruits (on the peel) using a Konica Minolta CR-400 colorimeter (Konica
Minolta, Osaka, Japan). The colorimeter was equipped with a C illu­
minant and used the CIELAB scale. The hue angle (h◦ ), chroma (C*), and
total color difference (ΔE) were calculated using Eqs. (1), (2), and (3),
respectively:
h0 = tan− 1 (b* /a* ) (1)
( )1/2
C* = (a* )2 + (b* )2 (2)
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
ΔE* = (Lt * − Lt0 * )2 + (at * − at0 * )2 + (bt * − bt0 * )2 (3)
J.G. Oliveira Filho et al.
where subscripts t and 0 correspond to parameters evaluated at the time
and the beginning of the study, respectively.
Strawberry firmness was evaluated using a digital TA.XTplus Texture
Analyzer (Stable Micro Systems Ltd., England, UK) with a 4-mm diam­
eter probe, 10-mm s− 1 velocity, 2-mm penetration distance, and 12-mm2
contact area. The results are expressed in Newtons (N) and calculated
based on three penetrations in the distal region of each fruit.
2.6. Bioactive compounds and antioxidant activity of strawberries
The total phenolic content (TPC) was measured using the
Folin–Ciocalteu method. Extracts were prepared by mixing 20 mL of
homogenized sample and 50 mL of methanol in an automatic water bath
shaker for 2 h at 25 ◦ C. After the extracts were concentrated using a
rotary evaporator, the volume was adjusted to 10 mL using methanol.
Then, 0.2 mL of extract was mixed with 2.6 mL of deionized water and
0.2 mL of the Folin–Ciocalteu reagent. The mixture was incubated at
25 ◦ C for 6 min, and 2 mL of 7 % (w/w) sodium carbonate solution was
added to stop the reaction. Samples were protected from light and
incubated for 90 min. The absorbance was measured at 750 nm (Shi­
madzu 1600, Portland, EUA). The results were expressed as mg gallic
acid equivalents (GAE) per g of strawberry [20].
The anthocyanin content was determined using the single pH
method based on a pH of 2 and absorbance at 535 nm, as described by
Bromberger et al. [21].
Ascorbic acid levels were measured using high-performance liquid
chromatography (HPLC) according to a method described by et al. [22].
Briefly, 0.1 g of the extract was transferred into 10-mL graduated flasks,
and metaphosphoric acid at a concentration of 3 % (w/v) was added.
The mixture was then filtered (0.45 μm) and placed in amber flasks.
Samples (30 μL) were injected into a Varian HPLC with dual pumps (Pro
Star 210) and a UV–Vis detector (Pro Star 325) set to 245 nm. The
mobile phase was a phosphate buffer at pH 2.5, with a flow rate of 1.0
mL min− 1. Separation was performed on an Agilent C18 column (2.5 ×
25 mm, 5 μm). L-ascorbic acid (purity ≥99.0 %) was used as a standard.
The radical scavenging activity of the extracts against 2,2-diphenyl-
1-picrylhydrazyl (DPPH) was evaluated according to the methods
described by Ejaz et al. [23]. The test was performed by adding 30 μL of
the extract (prepared as described for TPC analysis) and 2.97 mL of
DPPH (0.4 mg mL− 1) methanolic solution. After 15 min, the absorbance
was measured at 517 nm using a UV-VIS spectrophotometer. Eq. (4) was
used to estimate activity, with methanol used as a blank and methanol
+0.2 mM DPPH solution as a control sample.
2.10. Control of Rhizopus stolonifer and Botrytis cinerea on inoculated
%DPPH discoloration = [1 − (absorbance sample/absorbance control ) ] × 100 strawberries with and without bio-nanocomposite coating
(4)
2.7. Respiration rate of strawberries
The respiration rate was determined according to Martins et al. [24]
using a respirometer (model 6600, Illinois Instruments, Inc., USA).
Approximately 300 g of the strawberry samples were placed in her­
metically sealed 700 mL glass containers with a silicone septum in the
lid. At each measurement point, the concentrations of O2 (paramagnetic
sensor) and CO2 (infrared sensor) were evaluated after collecting air
samples from the containers.
2.8. Decay percentages and microbiological parameters of strawberries
The presence or absence of mold growth on the strawberries during
storage was visually evaluated, and fruits with any visible spoilage were
considered decayed. The decay percentage was based on the number of
decayed strawberries per treatment (each treatment group contained 20
strawberries). Subsequently, 25 g of fruit samples from each treatment
was mixed with 225 mL sterile saline solution (8.5 % NaCl) and ho­
mogenized under aseptic conditions. Serial dilutions were prepared, and
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International Journal of Biological Macromolecules 219 (2022) 812–823
appropriate dilutions were used for microorganism analysis. Aerobic
bacteria counts were determined using Petrifilm™ Rapid Aerobic Count
Plates (LuQiao Co., Beijing, China) after incubation at 37 ◦ C for 48 h.
Mold and yeast counts were determined using Petrifilm™ Rapid Test
Yeast and Fungi (LuQiao Co., Beijing, China) after incubation at 28 ◦ C
for 5 days.
2.9. Determination of volatile compounds in strawberries
To determine the volatile compound profile of the strawberries, a
headspace solid-phase microextraction procedure (HS-SPME) was
developed. Prior to the extraction, strawberries were blended with
saturated NaCl in a 1:1 ratio on days 0, 6, and 12 to obtain a puree. Then,
a DVB-CAR-PDMS fiber (Supelco®) was exposed to the headspace of the
puree samples (3 g) under these randomly established extraction con­
ditions: 30 ◦ C, 10 min of equilibrium between the sample and the fiber,
and 30 min of extraction. The HS-SPME was conducted in a bath using a
jacketed beaker for better temperature control.
After extraction, the fibers were immediately introduced into the gas
chromatography injector, and the desorption of the analytes was carried
out in the splitless mode at 220 ◦ C for 10 min. After desorption, the fibers
were reconditioned at 250 ◦ C for 15 min according to the manufacturer's
recommendations to ensure the absence of peaks in the blanks [25]. All
extractions were performed in triplicate.
All volatile compound analyses were performed using gas chroma­
tography–mass spectrometry (GC–MS) (model GC-2010Plus, Shimadzu,
Kyoto, Japan) coupled to a quadrupole mass spectrometric detector. The
separation of the analytes took place in a DB-5 fused silica capillary
column (30 m × 0.25 mm id. × 0.25 μm). The chromatographic con­
ditions used were as follows: injector at 220 ◦ C; splitless mode; carrier
gas helium at 1.0 mL min− 1; temperature ramp from 40 ◦ C to 170 ◦ C,
with an increment of 2 ◦ C min− 1 from 40 to 90 ◦ C, and an increment of
3 ◦ C min− 1 from 90 to 170 ◦ C; interface at 250 ◦ C; and electron ioni­
zation source at +70 eV (35–350 m/z).
Volatile compounds were tentatively identified based on compari­
sons of the mass spectra obtained from the samples and the Willey 229
mass spectra, with a minimum similarity of 80 %. A solution of n-alkanes
(C6-C20) (Alltech, PA, USA) was injected into the GC–MS equipment
under the same conditions to obtain the Van den Dool and Kratz pro­
grammed temperature retention indices (LTPRI – linear temperature
programmed retention index). A maximum variation of LTPRI of ±10
was allowed.
Previously selected and sanitized strawberries were inoculated with
30 μL of a spore suspension (105 spores mL− 1) of an R. stolonifer or
B. cinerea strain [26]. After inoculation, strawberries were incubated at
25 ◦ C for 12 h, and the coatings were applied by immersing the fruits in
the solution and allowing them to dry at room temperature for 2 h. The
control samples were uncoated. The strawberries (20 per treatment
group) were placed in transparent 157 × 130 × 40-mm plastic con­
tainers (Galvanotec, GA 92, Carlos Barbosa, RS, Brazil) and stored at
25 ◦ C ± 1 ◦ C and 70–75 % RH for 7 days, with a photoperiod of 12 h.
The antifungal activity of the treatments was evaluated by the severity
of the disease in the fruit, as determined using a score scale of 0–6 (0 =
absence of symptoms; 1 = 1–20 % affected area; 2 = 21–40 %; 3 =
41–60 %; 4 = 61–80 %; and 5 = 81–100 %) [27].
2.11. Statistical analysis
The data were presented as means ± standard deviation. All data
were analyzed using analysis of variance (ANOVA) and subsequently by
Duncan's multiple amplitude test to assess significant differences be­
tween treatments (p = 0.05). Statistical analyses were performed using
J.G. Oliveira Filho et al.
Sigma Plot 12.0.
3. Results and discussion
3.1. Contact angle and viscosity of bio-nanocomposite coatings
The coated surfaces presented contact angle values ranging from
77.08◦ to 80.63◦ (Fig. 1A). Because they were higher than 65◦ , they
could be considered hydrophobic [28]. Their hydrophobic behavior
mainly resulted from the presence of carnauba wax, as has been
observed in other studies [29]. Coatings with hydrophobic characteris­
tics can effectively reduce moisture loss (and, consequently, weight loss)
in post-harvest fruits and vegetables [6]. Moreover, all contact angle
values were significantly equal (p > 0.05) to each other, which shows
that neither changing the essential oil nor increasing its concentration
was a determining factor for the hydrophobicity of the emulsions, but
the presence of carnauba wax was, as predicted. Apart from hydro­
phobicity, which is related to the barrier property of the coating against
water loss, viscosity is another physicochemical parameter that must be
evaluated prior to its application in food, particularly with how it
changes according to the coating composition. The viscosity of the film-
forming dispersions was evaluated at 25 ◦ C as a function of the shear rate
(Fig. 1B). All dispersions showed a decrease in viscosity with increasing
applied shear, which is a characteristic of polymeric pseudoplastic
behavior. Such behavior can be explained by the movement of poly­
meric chains until they are randomly oriented, leading to lower vis­
cosities [30].
The initial viscosity (η0) of the dispersions ranged from ~1.09 to
~1.86 Pa s− 1 for the coatings. The coated control sample (AA/CWN/
CNC) showed the highest viscosity, which tended to decrease with the
addition of essential oils to the polymeric matrix. Moreover, samples
that contained CEO showed that an increase in essential oil concentra­
tion resulted in a significant decrease (p < 0.05) in the viscosity of the
dispersions, from 1.60 ± 0.24 Pa s− 1 in AA/CWN/CNC/CEO2 to 1.09 ±
0.16 Pa s− 1 in AA/CWN/CNC/CEO3. This behavior of polymeric emul­
sions containing essential oils was also observed by Sánchez-González
et al. [31], who reported that the addition of lemon, bergamot, and tea
tree essential oils to chitosan and hydroxypropyl methylcellulose dis­
persions resulted in less-viscous systems with more stable droplets that
are less sensitive to changes in applied shear.
Viscosity values between 1 and 10 Pa s− 1 are the most suitable
conditions for experiments involving food-coating, stirring, spreading,
and dripping [32]. Outside this range, low-viscosity dispersions may not
completely settle on the fruit surface and can be more susceptible to
dripping, whereas high-viscosity dispersions impair surface wetting
A B
a
a a a a
Fig. 1. Contact angle (A) and viscosity (B) of bio-nanocomposite coatings.
815
International Journal of Biological Macromolecules 219 (2022) 812–823
[33]. Thus, the initial viscosity values obtained for the dispersions
developed in this study were considered appropriate for use as food-
coating materials. Furthermore, it was evident that the composition of
the essential oil and its concentration in the emulsions were greater
determinants of the viscosity of the coatings than its hydrophobic
character. Once more, the presence of carnauba wax explains the pre­
dominant hydrophobicity, and the new interactions formed between the
wax and the essential oil components explain the change in the poly­
meric network and the consequent change in its initial viscosity.
The viscosity of the coatings did not negatively affect the respiration
rate or post-harvest quality of the strawberries (as discussed later in
3.2.4 Respiration rate). The viscosity of the film-forming solution is
important because it can influence the thickness of the coating formed
on the fruit surface [34]. Highly viscous film-forming solutions can
completely block the flow of O2 and CO2, damaging fruit quality.
3.2. Effect of bio-nanocomposite edible coatings on post-harvest quality
parameters of strawberries
3.2.1. Weight loss and physicochemical parameters
Fig. 2A shows the weight loss of coated and control strawberries after
12 days of storage at 7 ± 1 ◦ C and 70–75 % RH. All treatment groups
displayed weight loss with storage time, but coated strawberries showed
reduced weight loss (7.5–8.8 %) compared to those in the control
treatment group (11.4 %). Similar results were previously reported in
strawberries coated with CMC- and chitosan-based solutions containing
MEO [3], as well as strawberries coated with a nanoemulsion of pullulan
and cinnamon essential oil [1]. According to Guerreiro et al. [35], edible
coatings act as hydrophobicity barriers (corroborating the contact angle
results in Fig. 1A), mainly on the stomata of fruits, protecting them
against moisture loss and reducing fruit transpiration.
The soluble solid content (SS) of strawberries decreased with
increasing storage time (Fig. 2B). However, the SS of coated strawberries
was relatively stable over the storage period, indicating that the coat­
ings, mainly AA/CWN/CNC/MEO2, AA/CWN/CNC/MEO3, and AA/
CWN/CNC/CEO3, considerably reduced the conversion rate of sugars
compared to the other treatments. Similar results have been reported in
other studies involving the application of coatings to strawberries [1,3].
The pH of the strawberries also decreased throughout the storage
period (Fig. 2C), resulting in lower pH values in the control than in the
coated strawberries. After 12 days, the fruits coated with AA/CWN/
CNC/MEO2 and AA/CWN/CNC/MEO3 showed the highest pH values
(3.1 and 3.3, respectively). Post-harvest reductions in strawberry pH
occur mostly owing to fungal action, which acidifies the fruit as it decays
[36].
J.G. Oliveira Filho et al. International Journal of Biological Macromolecules 219 (2022) 812–823

a bb
bc
c
c
a bb
bc
c
c
b
bc b b
a c
b b b bb

B C

a a a aa a a a
aab a a aa aa a a aa a aa aa
b ab b a aa a ab ab b a aa a a a a aa bb
b a a ab b a b
b b b b c
c
c

D E

a
a b
a bb b
c b
aaa aa bbbb
bc
c
a a a a a ac
b c bc
b a a
a a aa a a a a
bb a a
a a a
bb a a a
b b
bc c
c c d c

Fig. 2. A) Weight loss (%), B) SS (%), C) pH, D) TA (g citric acid /100 g), and E) firmness (N) of strawberries during storage for 12 days at 7 ± 1 ◦ C and 70–75 % RH.
Different uppercase letters indicate significant differences (p ≤ 0.05) among treatments on the same day.

The titratable acidity (TA) (Fig. 2D) was stable in all coated fruits
during the first six days of storage. By contrast, control strawberries
showed elevated reductions in TA content from day 3 onwards. This
reduction in acidity is related to the metabolism of strawberries, which
consumes organic acids during respiration [1]. However, the TA of all
treatment groups increased from day 6 and was significantly higher for
the control strawberries. In this case, the increase in acidity during
storage was likely attributable to the increased fungal burden, which
produces metabolites and acidifies the environment [1].
On the last day of storage, the fruits in the treatment groups,
including MEO, had the lowest TA. This suggests that MEO is especially
effective against fungal growth. This result agrees with the observed pH
values, as strawberries coated with MEO displayed the highest pH values
at the end of the storage period.
3.2.2. Physical parameters
Firmness is a determining factor in the post-harvest quality of
strawberries, mainly because it affects the ability to transport the fruit.
The coated strawberries did not lose their firmness as quickly during
storage as did the control fruits. An exception was the fruits with

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J.G. Oliveira Filho et al. International Journal of Biological Macromolecules 219 (2022) 812–823

coatings that contained CEO (Fig. 2E). The main reason for the loss of
firmness (softening) of strawberries is the degradation of the cell wall,
especially the middle lamella of the cells. This mainly occurs because of
tissue damage caused by microorganisms such as fungi, but fruit respi­
ration and water loss also contribute to cell wall degradation [37].
Fruits with coatings that contained CEO showed the highest loss of
firmness during storage compared to fruits with other coatings. This loss
probably resulted from the action of geraniol, the main component of
CEO (Supplementary File 1). Geraniol acts on the cellular tissue of the
fruit, causing structural changes that lead to softening and an increased
release of enzymes or substrates that favor this process. This increase in
softening was also observed in fresh-cut melons with alginate-based
coatings that contained geraniol [38].
Using coatings containing MEO can provide a barrier that reduces
water loss, respiration rates, and fungal contamination, thereby
reducing the rate of fruit softening.
Color is one of the most important and intuitive parameters that
consumers use to assess the quality of fresh fruits and vegetables [39].
Fig. 3 shows the color parameters (L*, C *, h◦ , and ΔE) of the control and
coated strawberries as measured over the storage period. In general, the
application of the edible coatings did not affect the color of the straw­
berries. The main color changes in all treatments were observed after
day 6 of the storage period when a decrease in L* and C* values and an
increase in h◦ and ΔE values were observed.
The control strawberries showed the lowest L* values (23.14) at the
end of the storage period (Fig. 3A). In the present study, the decrease in
L* values could be related to the loss of moisture from the surface of the
fruits [40], which would explain the weight loss results (Fig. 2A). In
coated strawberries, the highest percentage of fresh weight loss during
the storage period and higher L* values correlated with a significant
increase in the intensity of color (h◦ values close to 40◦ ) (Fig. 3C).
The color saturation (C*) of the control and coated strawberries
decreased after day 6 of the storage period. From the 6th day onwards,
control strawberries showed a less intense color (lower C* values) than
coated strawberries (Fig. 3B). Hernández-Carrillo et al. [41] and Guer­
reiro et al. [35] also observed an increase in C* values in coated and
control strawberries during storage, which was attributed to deteriora­
tion and microbial degradation of anthocyanins.
The control strawberries showed a significant increase in color in­
tensity after day 6 of the storage period, with h◦ values close to 40◦
(Fig. 3C). This reduction may be related to strawberry decay caused by
fungi, which probably resulted in accelerated degradation of anthocy­
anins relative to that in the coated strawberries. A similar result was
described by Hernández-Carrillo et al. [41] in strawberries coated with
pectin and reuterin, with an increase in h◦ values due to anthocyanin
degradation and fungal spoilage.
These changes in color parameters resulted in an increase in the total
color difference (ΔE) of the films after day 6, highlighting the differences
in color intensities compared to day 0 in the control strawberries
(Fig. 3D). Edible coatings can slow down physiological processes in the

A B

a
a a a a a a aa
a aa ab a
a a a a aa a ab aa aaa a
a aa b aaa a a a a aa
aa b a
a
c a ababab aa
c b
aa
b
c

C D

a
a
b
a bc
bc c b
aaa a b d
aa a aa a a bc bc
aa a
aa aa c c
a
c c
b d d
bc
c c c
a a a a a
aa a a a a a

Fig. 3. Color parameters A) L*, B) C* C) h◦ , and D) Delta E (ΔE) of control and coated strawberries stored for 12 days at 7 ± 1 ◦ C and 70–75 % RH. Different
uppercase letters indicate significant differences (p ≤ 0.05) among treatments on the same day.

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J.G. Oliveira Filho et al. International Journal of Biological Macromolecules 219 (2022) 812–823

fruit, reducing gas exchange, slowing respiration and transpiration, and
lowering microbial growth on the fruit surface, thereby helping to delay
color changes during ripening [42]. In general, in the present study,
coatings based on AA, CWN, and CNC associated with either MEO or
CEO were able to preserve strawberry color during storage. This was
demonstrated by a more intense red coloration and a lower difference in
color when comparing the fruits to day 0 of the storage period.
3.2.3. Bioactive compounds and antioxidant activity
The total phenolic compounds content (TPC) decreased in all sam­
ples over the storage period (Fig. 4A). However, the coatings slowed
down these losses because they provided a barrier against gas exchange.
Such a barrier results in reduced activity of the polyphenol oxidase
enzyme and, consequently, a lower rate of phenolic degradation during
storage [5]. Similar results on the effect of coatings on the TPC of
strawberries have been observed in other studies [5,20].
Among the treatments evaluated in the present study, strawberries
coated with MEO or CEO showed the least TPC reduction (1.43–1.52 to
1.03–1.11 mg GAE g− 1 of fruit and 1.57–1.68 to 1.07–1.13 mg GAE g− 1
of fruit, respectively) compared to treatments without essential oils
(1.72 to 0.78 mg GAE g− 1 of fruit). This may correlate with the slower
fungal-related deterioration of strawberries during storage (Fig. 4B) due
to the antimicrobial action of essential oils, which impairs the degra­
dation of these compounds. A similar result was reported by Quintana
et al. [13] for strawberries coated with chitosan with rosemary and
thyme essential oils. The coatings containing essential oils and extracts
showed lower losses of TPC compared to the control or coatings con­
taining only chitosan.
The anthocyanin content also decreased in all strawberry samples
during storage (Fig. 4B). From day 6 of the storage period, control
samples showed the highest reduction in anthocyanin content (4.57 to
4.11 mg cyanidin-3-O-glucoside 100 g− 1 of fruit) compared to coated
samples (4.47–4.72 to 4.4–-4.54 mg cyanidin-3-O-glucoside 100 g− 1 of
fruit). This reduction in anthocyanin content over storage time may be
the result of senescence and loss of anthocyanin synthesis capacity [43].
In addition, anthocyanin degradation occurs faster as oxygen levels
increase [44]. The retardant effect of the coatings on anthocyanin
degradation was probably due to the reduction in strawberry respiration
and will be discussed further in Section 3.2.4. A similar effect on the
anthocyanin content of strawberries was reported by Khodaei et al. [45]
for strawberries with an edible coating of CMC, low methoxyl pectin,
Persian gum, and tragacanth gum.
Overall, ascorbic acid content decreased with storage time in
strawberries, but the application of the coatings effectively retarded this
decrease in ascorbic acid content compared to control fruits (Fig. 4C).
Other studies using strawberry coatings have observed similar outcomes
[5,46].
The irreversible oxidation of ascorbic acid to dehydro-L-ascorbic acid
and then to keto-L-gluconic acid causes the ascorbic acid level to drop
during fruit storage [47]. In the present study, the edible coatings
probably created a barrier to gases on the surface of strawberries, pre­
venting the entry of oxygen and reducing enzymatic activity,

A B

a a a
a a aa a a a aa a a aaaa a a aa a a a a aa
a a a aa a a a aa b
aa a b a a aa
a b
b a a aa
b c
c c
b
c

C D

a a a aa a
a a aa aa
a a a aa a aa aa
a a a a aa a aa a a
b aa a a
a aaa a a aaa
b b a a c
b b aa b b
c
c

Fig. 4. A) Total phenolic content (mg GAE g− 1 of fruit), B) anthocyanin content (mg cyanidin-3-O-glucoside 100 g− 1 of fruit), C) ascorbic acid (mg kg− 1 of fruit), and
D) antioxidant activity (% DPPH scavenging) of control and coated strawberries during storage for 12 days at 7 ± 1 ◦ C and 70–75 % RH. Different uppercase letters
indicate significant differences (p ≤ 0.05) among treatments on the same day.

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J.G. Oliveira Filho et al. International Journal of Biological Macromolecules 219 (2022) 812–823

contributing to greater retention of the ascorbic acid content of the
samples, as well as greater antioxidant activity [46].
The antioxidant activity of strawberries in all treatment groups
decreased over the storage period (Fig. 4D), especially in control
strawberries (from 77.90 to 33.24 %). Similar results were observed by
Wani et al. [46], who coated strawberries with arabic, carrageenan, and
xanthan gums with added lemongrass essential oil. They reported that
the coatings maintained the DPPH scavenging activity of strawberries.
At the end of the storage period, strawberries with coatings that
contained essential oils retained the highest antioxidant activity.
Therefore, coatings that contained MEO or CEO showed the best ability
to preserve the antioxidant activity of strawberries during refrigerated
storage.
Bioactive compounds present in strawberries, such as ascorbic acid,
anthocyanins, flavonoids, and phenolic compounds, play an important
role in the antioxidant activity of this fruit [48]. The increased antiox­
idant levels in fruits with coatings containing essential oils are due to
their improved ability to preserve these aforementioned bioactive
compounds in strawberries during storage.
3.2.4. Respiration rate
Fig. 5A shows the respiration rate values in terms of CO2 production
of control and coated strawberries after 12 days of storage at 7 ± 1 ◦ C.
Respiration rates in all treatment groups increased during the storage
period. Coated strawberries displayed significantly reduced respiration
rates (25.21–26.40 mL CO2 kg− 1 h− 1) compared to control strawberries

A a
B
a a

b
a
b b c
a b a
cc c c d
a b c
bb bb cc c
bb b b b e
a a aa aa c f
a d d
e

C D

E F
a a b b b b a a b b b b

Fig. 5. A) CO2 production rate (mL kg− 1 h− 1), B) visible decay rate, C) aerobic mesophilic bacteria, D) yeasts and molds, E) severity of B. cinerea symptoms, and F)
severity of R. stolonifer symptoms of both control and coated strawberries during storage for 12 days at 7 ± 1 ◦ C and 70–75 % RH. Different uppercase letters indicate
significant differences (p ≤ 0.05) among treatments on the same day.

819
J.G. Oliveira Filho et al.
(30.22 mL CO2 kg− 1 h− 1) from day 3 of the storage period. On the last
day of the experiment, the CO2 production rate of control strawberries
was 80.22 mL CO2 kg− 1 h− 1, significantly higher than that observed in
coated strawberries (31.32–44.21 mL CO2 kg− 1 h− 1). A reduction in CO2
generation in coated strawberries compared to uncoated control sam­
ples has also been reported in other studies [3,40].
Among treatment groups, strawberries with coatings that contained
MEO or CEO, regardless of concentration, had lower CO2 production
rates (29.21–31.32 mL CO2 kg− 1 h− 1) than coatings without essential
oils (37.21 mL CO2 kg− 1 h− 1) from day 9 of the storage period. This may
be related to the higher microbial load of fruits covered with coatings
without essential oils (as will be shown later in Fig. 5B, C, and D. A
similar result was observed by Perdones et al. [40] in strawberries
coated with chitosan and lemon essential oil, which had lower CO2
generation levels than fruits coated solely with chitosan.
These results demonstrate that AA/CWN/CNC coatings containing
MEO or CEO appear to affect gas exchange during strawberry respira­
tion, reducing the permeability to gases on the surface of fruits. This
result was predicted based on measurements of TPC, anthocyanin con­
tent, and firmness of the fruits.
3.2.5. Fungal decay rate and microbiological parameters
The incidence of fungal spoilage in strawberries increased during
storage (Fig. 5B) and was higher in control fruits than in coated fruits.
Coatings containing MEO and CEO (AA/CWN/CNC/MEO2, AA/CWN/
CNC/MEO3, AA/CWN/CNC/CEO2, and AA/CWN/CNC/CEO3) were
more effective at inhibiting fungal spoilage than was the AA/CWN/CNC
coating. According to Oliveira Filho et al. [26], MEO and CEO exert
strong antimicrobial activity against the main disease-causing fungi in
strawberries post-harvest (R. stolonifer and B. cinerea).
On the last day of storage, the incidence of disease in strawberries
with coatings containing essential oils was significantly lower (15–35 %)
than that of control strawberries or strawberries with coatings that did
not include essential oils (75 % and 50 %, respectively) (Supplementary
File 2). Similar results were reported by Chu et al. [1] in strawberries
treated with nanoemulsion coatings of pullulan containing cinnamon
essential oil and Xue et al. [49] in strawberries coated with phytoglyc­
ogen/zein nanocomplexes containing thymol.
Figs. 5C and D show the results of the microbiological analysis of
strawberries during storage. Strawberries containing MEO and CEO
coatings had the lowest mesophilic bacterial, mold, and yeast count
values during storage. At the end of the storage period, aerobic meso­
philic bacteria counts were 3.63, 3.85, 3.45, and 3.80 log CFU g− 1 for
AA/CWN/CNC/MEO2, AA/CWN/CNC/MEO3, AA/CWN/CNC/CEO2,
Fig. 6. Heatmaps of the relative ester contents of each group of samples (n = 3). The color of the scale bar denotes the extent of the compound content, with green
indicating high content and pink indicating low content.
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International Journal of Biological Macromolecules 219 (2022) 812–823
and AA/CWN/CNC/CEO3, respectively; these values were significantly
lower than those of the control (7.39 log CFU g− 1) and AA/CWN/CNC
(5.96 log CFU g− 1) samples. Fungi and yeast counts showed similar
behavior, with values of 4.45, 483, 4.95, and 5.22 log CFU g− 1 for AA/
CWN/CNC/MEO2, AA/CWN/CNC/MEO3, AA/CWN/CNC/CEO2, and
AA/CWN/CNC/CEO3, respectively, significantly lower than those of the
control (8.17 log CFU g− 1) and AA/CWN/CNC (6.08 log CFU g− 1)
samples. Similar results have been described in strawberries coated with
chitosan and carboxymethylcellulose and M. spicata essential oil [3],
gellan gum and geraniol [36], and a nanoemulsion of pullulan and
cinnamon essential oil [1]. These authors reported that aerobic bacteria,
mold, and yeast counts in coated strawberries were lower than those in
the control or strawberries coated without essential oils. The antimi­
crobial capacity of bio-nanocomposite coatings in the present study may
be due to the excellent antimicrobial capacities of MEO and CEO [26].
3.3. Effect of bio-nanocomposite coatings on strawberry volatile
compound profiles
Figs. 6 and 7 present the volatile compound profiles of control and
coated strawberries during storage. The samples showed noticeable
differences in their volatile compound profiles due to the effects of
coating and storage time.
In general, significant differences were observed in the aromatic
profiles of the coated and control samples. The accumulation of alde­
hydes and alcohols in stored fruits is associated with overripening and
microbial spoilage [50]. During storage, a decrease in ester concentra­
tions (Fig. 6) and an increase in aldehyde and alcohol concentrations
(Fig. 7) were observed in the control fruits. In the coated fruits, a
decrease in the concentration of aldehydes and alcohols was observed
(Fig. 6), as well as an increase in the concentration of esters (Fig. 7). This
may be related to the effect of coatings on delaying the senescence and
deterioration of strawberries during storage. Similar results were re­
ported by Almenar et al. [51] in chitosan-coated strawberries stored
under refrigeration.
Strawberries coated with AA/CWN/CNC/MEO2 and AA/CWN/
CNC/MEO3 on day 0 showed changes in their volatile compound pro­
files due to the incorporation of volatiles from MEO into the samples.
Carvone, limonene, and 1,8-cineole are found in MEO (Supplementary
File 1, Oliveira Filho et al. [26]), and our results show the progressive
loss of MEO compounds over the storage period. Losses of carvone, 1,8-
cineole, and limonene increased during storage, and trans-
dihydrocarvone was completely lost after 6 days of storage. Despite
these high losses of MEO volatile compounds, the remaining amount of
J.G. Oliveira Filho et al.
Fig. 7. Heatmaps of relative contents of terpenes and alcohols (A) and aldehydes and ketones (B) in each group of samples (n = 3). The color of the scale bar denotes
the extent of the compound content, with green indicating high content and pink indicating low content.
oil in the fruits was sufficient to control fungal decomposition in
strawberries (Fig. 5B). Similar behavior was reported by Perdones et al.
[52] for strawberries with a coating containing chitosan and lemon
essential oils.
3.4. Effect of bio-nanocomposite coatings on the control of B. cinerea and
R. stolonifer in artificially inoculated strawberries
Coatings containing MEO and CEO (Fig. 5E and F) effectively
reduced the incidence and progression of spoilage by B. cinerea and
R. stolonifer at 25 ◦ C during seven days of storage. Eleven percent of
fruits inoculated with B. cinerea and treated with coatings containing
essential oils had over 61 % of their area contaminated by the fungus,
whereas control strawberries and those coated with AA/NEC/NCC had
94 and 67 % of their area contaminated by the fungus, respectively
(Fig. 5E).
For fruits inoculated with R. stolonifer, 100 % of control fruits and 94
% of fruits coated with AA/NEC/NCC had over 61 % of their area
contaminated by the fungus. By contrast, only 0–33 % of the fruits
treated with coatings containing essential oils had over 61 % of their
area contaminated. Our findings on the in vivo application of coatings
with essential oils for the control of post-harvest diseases of strawberries
are in agreement with those of da Silva et al. [53] and Oliveira et al.
[54], who demonstrated that coatings based on carboxymethylcellulose
and essential oils were able to reduce the severity of post-harvest dis­
eases, such as soft rot caused by R. stolonifer in strawberries.
The ability of MEO and CEO to control B. cinerea and R. stolonifer has
been demonstrated in previous studies [11,26]. The high antifungal
activity of EOs is related to the presence of compounds such as carvone
in MEO [55] and geraniol, geranyl acetate, linalool, and caryophyllene
in CEO [56]. Therefore, bio-nanocomposite coatings containing MEO
and CEO can act as an alternative to synthetic fungicides for the control
of post-harvest diseases in strawberries.
4. Conclusions
Bio-nanocomposite edible coatings improved the quality and shelf
life of strawberries during storage, minimizing changes in physico­
chemical, microbiological, bioactive, antioxidant, and aromatic pa­
rameters compared to control strawberries (uncoated). The bio-
nanocomposite coatings functionalized with essential oils are able to
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International Journal of Biological Macromolecules 219 (2022) 812–823
control the development of post-harvest diseases in artificially inocu­
lated strawberries. It can be concluded that bio-nanocomposite coatings,
especially those containing MEO, can be used as antimicrobial plant-
based coating materials to preserve fresh strawberries during storage.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijbiomac.2022.08.049.
CRediT authorship contribution statement
Josemar Gonçalves de Oliveira Filho: Methodology, Investigation,
Writing – original draft. Beatriz Regina Albiero: Methodology, Inves­
tigation. Ítalo Henrique Calisto: Methodology, Investigation. Mirella
Romanelli Vicente Bertolo: Methodology, Investigation. Fernanda
Campos Alencar Oldoni: Methodology, Investigation. Mariana Bur­
anelo Egea: Investigation, Writing – original draft. Stanislau Bogusz
Junior: Investigation, Writing – original draft. Henriette Monteiro
Cordeiro de Azeredo: Supervision, Writing – review & editing. Marcos
David Ferreira: Conceptualization, Funding acquisition, Supervision,
Writing – review & editing.
Data availability
Data will be made available on request.
Acknowledgments
The authors are grateful to FAPESP (processes 2018/24612–9 and
2019/18748–8) and CAPES (001). The authors Azeredo and Ferreira
thank CNPq for their research productivity fellowships (308777/2021-2
and 310728/2019-3, respectively), IF Goiano (Process no
23218.001842.2022-52), and Empresa Brasileira de Pesquisa Agro­
pecuária – Embrapa, Rede Agronano, CNPq/MCTI Sisnano (442575/
2019-0) from Brazil.
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