International Journal of

Molecular Sciences

Article
Innovative Integration of Arrayan (Luma apiculata) Extracts in
Chitosan Coating for Fresh Strawberry Preservation
Sergio Benavides 1,2, * and Wendy Franco 3,4, *

1 School of Nutrition and Dietetics, Faculty of Health Care Sciences, Universidad San Sebastián,
Concepción 4080871, Chile
2 Agro-Food and Applied Nutrition Research Center, Adventist University of Chile, Chillan 3780000, Chile
3 Chemical Engineering and Bioprocess Department, Pontificia Universidad Católica de Chile, Av. Vicuña
Mackenna 4860, Macul, Santiago 7820436, Chile
4 Departamento de Ciencias de la Salud, Carrera de Nutrición y Dietética, Pontificia Universidad Católica de
Chile, Av. Vicuña Mackenna 4860, Macul, Santiago 7820436, Chile
* Correspondence: sbenavides@uss.cl (S.B.); wfranco@uc.cl (W.F.)

Abstract: Strawberries are a rich source of vitamins and antioxidants, among other nutrients, but
they are highly susceptible to mechanical injuries, dehydration, and microbial spoilage, and thus
have a limited post-harvest shelf-life. Bioactive edible coatings have been studied to decrease or
prevent these damages. In this study, ethanolic extracts of Arrayan (Luma apiculata), a traditional
berry from the south of Chile, were used to enrich a chitosan-based edible film and coat fresh
strawberries. A long-term storage (10 ◦ C) study was conducted to determine the strawberries’ weight
loss, microbial stability, fruit firmness impact, and antioxidant activity. Later, a sensory panel was
conducted to determine overall consumer acceptance. Our results show that the bioactive coating
inhibited the growth of different pathogenic bacteria and spoilage yeast. In the stored strawberries,
the weight loss was significantly lower when the bioactive coating was applied, and the samples’
firmness did not change significantly over time. Microbial growth in the treated strawberries was
also lower than in the control ones. As expected, the antioxidant activity in the coated strawberries
was higher because of the Arrayan extract, which has high antioxidant activity. Regarding sensory
qualities, the covered strawberries did not show significant differences from the uncoated samples,
Citation: Benavides, S.; Franco, W.
with an overall acceptance of 7.64 on a 9-point scale. To our knowledge, this is the first time an edible
Innovative Integration of Arrayan
(Luma apiculata) Extracts in Chitosan
coating enriched with Arrayan extracts has been reported as able to prevent strawberries’ decay
Coating for Fresh Strawberry and spoilage.
Preservation. Int. J. Mol. Sci. 2023, 24,
14681. https://doi.org/10.3390/ Keywords: edible coating; chitosan; Arrayan; strawberries; shelf-life
ijms241914681

Academic Editors: Giovanni Pallio

and Letteria Minutoli
1. Introduction
Received: 29 July 2023 Fruits constitute an important part of the human diet. They are a source of several
Revised: 7 September 2023
minerals, vitamins, sugars, and fiber, among other biomolecules. Huang et al., 2012,
Accepted: 14 September 2023
reported that strawberries, for instance, are a rich source of antioxidants, including vitamin
Published: 28 September 2023
C, folate, and polyphenolic compounds [1]. However, the fruit is very perishable since it is
susceptible to microbial decay, dehydration, and mechanical injury [2]. Strawberries are
particularly sensitive to storage, with the mean shelf-life of the fruit 3 to 10 days at 10 ◦ C.
Copyright: © 2023 by the authors.
An alternative to alleviate this problem is using bioactive coatings made with natural
Licensee MDPI, Basel, Switzerland. compounds. Bioactive coatings can be elaborated with different edible biopolymers and
This article is an open access article can incorporate plant extracts with antimicrobial and/or antioxidant properties. These
distributed under the terms and coatings can inhibit microbial growth, inactivate spoilage enzymes, and limit dehydra-
conditions of the Creative Commons tion [3–5]. A common biopolymer used in the production of food coatings is chitosan [6,7].
Attribution (CC BY) license (https:// This biopolymer has been extensively used for the decay protection of several fresh food
creativecommons.org/licenses/by/ products [8], and it has also been reported as an effective barrier in strawberries [9–12].
4.0/). Chitosan is a polysaccharide formed by β(1-4)-D-glucosamine and N-acetyl-D-glucosamine,

Int. J. Mol. Sci. 2023, 24, 14681. https://doi.org/10.3390/ijms241914681 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023, 24, 14681 2 of 16

which is obtained from mushrooms or the shells of crustaceous [13]. The compound is
non-toxic (Generally Recognized as Safe, GRAS), biodegradable, and shows antimicrobial
properties [6]. In several studies, chitosan coatings are enriched with bioactive natural
molecules (essential oils, botanical extracts, and organic acids, among others) to enhance
their bioactive properties [14–16].
Different bioactive films composed of chitosan and other molecules have been re-
ported as able to control or prevent decay in fruits. A blend of chitosan and pullulan in
equal parts (50:50), combined with an extract from pomegranate peel (at a concentration
of 0.02 g/mL), was utilized as an edible coating in mango fruits. This coating proved
effective in minimizing the loss of weight and preserving various quality attributes of the
mango fruits over a span of 18 days during postharvest storage at a temperature of 4 ◦ C.
The characteristics that were maintained include total soluble solids (TSS), pH, firmness,
phenolic content, and the antioxidant activity of the fruits. [17].
In another study, a coating composed of chitosan (at a concentration of 1.5% by
weight) infused with an extract from hairy fig (Ficus hirta Vahl.) was used to coat “Newhall”
navel oranges. This treatment exhibited the lowest decay rate (5.2%), minimized weight
loss (5.16%), and reduced the malondialdehyde content. Furthermore, it bolstered the
function of protective enzymes like superoxide dismutase, peroxidase, chitinase, and -1,3-
glucanase throughout a span of 120 days in cold storage [18]. Furthermore, when guavas
(Allahabadsafeda variety) were coated with a mixture of chitosan (at a concentration of 1%
by weight) and alginate (at a concentration of 2% by weight), along with pomegranate peel
extract (at a concentration of 1% by weight), noticeable reductions were observed in the
losses of ascorbic acid (29%), total phenolics (8%), total flavonoids (12%), and antioxidant
activity as measured through DPPH (12%) and FRAP (9%) assays. These effects were
sustained over a period of 20 days while the coated guavas were stored at a temperature of
10 ◦ C [18].
Bioactive chitosan coatings have been also used to prevent strawberry decay, especially
when the coating is enriched with high-antioxidant plant extracts.
Plant extracts, especially those having a high antioxidant composition, such as berries,
are an interesting alternative as a food additive for preserving fresh food products [17].
Different wild berries are grown in the south of Chile. Among them, Arrayan (Luma
apiculata) is an endemic fruit with interesting properties to be considered as a natural food
additive. Arrayan belongs to the Myrtaceae family and is an endemic tree cultivated in
Chile from the Valparaíso to the Aisén Regions (latitude 33◦ to 45◦ ). The tree has red-brown
stems, with regular leaves, and black to purple edible fruits with a mild and pleasant
taste [18,19] (Figure 1). The fruit is commonly used for infusions, meal preparations, and
ointments [20].
Little information is available in relation to Arrayan extract properties and/or ap-
plications. The fruits have been described as a rich source of phenolic compounds with
high antioxidant activity [19,21–23]. Polyphenols are a group of naturally occurring com-
pounds found in plants, known for their antioxidant and antimicrobial properties and
potential health-promoting effects. They are responsible for the vibrant colors of many
fruits, vegetables, and herbs. Anthocyanins are the most abundant polyphenols in Arrayan
extracts. Flavanols have been also reported, but in lower concentrations [18,20,24]. Arrayan
polyphenols have been reported as able to inhibit pathogenic bacteria such as Pseudomonas
sp. and Staphylococcus sp. [18,19].
Despite these interesting properties, to our knowledge, little information has been
reported regarding potential food applications of Arrayan in terms of the decay protection
of fruits. Therefore, this work aimed to design a bioactive coating based on a chitosan
solution enriched with Arrayan fruit ethanolic extract to improve the shelf-life of fresh
strawberries during cold storage.
Int.
Int.J.J.Mol.
Mol.Sci. 2023,24,
Sci.2023, 24,14681
x FOR PEER REVIEW 33 of
of1617

Figure1.1.Arrayan
Figure Arrayanfruits.
fruits.

2.2.Results
Resultsand
andDiscussion
Discussion
2.1.
2.1. Phenolic CompositionofofArrayan
Phenolic Composition ArrayanExtracts
Extracts
Polyphenols
Polyphenolsmake
makeup upthe
themajority
majorityofofsecondary
secondarycompounds
compoundsfoundfoundininberries.
berries.The
The
most
most abundant and varied categories of phenolic substances discovered in berriesinclude
abundant and varied categories of phenolic substances discovered in berries include
anthocyanins,
anthocyanins,followed
followedby byflavones
flavonesandandflavonoids
flavonoids[25].
[25].Six
Sixtypes
typesofofanthocyanins
anthocyaninshavehave
been
been identified in berries, including cyanidin, delphinidin, malvidin, pelargoni-
identified in berries, including cyanidin, delphinidin, malvidin, peonidin, peonidin,
din, and petunidin
pelargonidin, and [20]. For Arrayan,
petunidin in specific,
[20]. For Arrayan,delphinidin,
in specific,petunidin, peonidin,
delphinidin, and
petunidin,
malvidin have been reported before [18,22,23]. In our study, Peonidin-3-galactoside
peonidin, and malvidin have been reported before [18,22,23]. In our study, Peonidin-3- was
the major anthocyanin present (438 ± 7.00 mg/L) in the Arrayan extracts, followed by
galactoside was the major anthocyanin present (438 ± 7.00 mg/L) in the Arrayan extracts,
Petunidin-3-arabinoside (121 ± 3.00 mg/mL), while Peonidin-3-arabinoside and Malvidin-
followed by Petunidin-3-arabinoside (121 ± 3.00 mg/mL), while Peonidin-3-arabinoside
3-arabinoside were encounter found in concentrations lower than 100 mg/L (Table 1). As
and Malvidin-3-arabinoside were encounter found in concentrations lower than 100 mg/L
for the flavonoids, quercetin has been also reported for in Arrayan extracts [20,24], however,
(Table 1). As for the flavonoids, quercetin has been also reported for in Arrayan extracts
in lower concentration than the one reported here (718 mg/L mg/L).
[20,24], however, in lower concentration than the one reported here (718 mg/L mg/L).
Table 1. Phenolic composition of the ethanolic Arrayan extracts.
Table 1. Phenolic composition of the ethanolic Arrayan extracts.
Compound
Compound Formula
Formula PubChem CID CID Concentration
PubChem Concentration(mg/L)
(mg/L)
Flavonoids
Flavonoids
Quercetin-3-rutinoside C27 H30 O
Quercetin-3-rutinoside C16
27H30O16 5280805
5280805 ± 63.0
718718 ± 63.0
Anthocyanins
Anthocyanins
Petunidin-3-arabinoside
Petunidin-3-arabinoside C21 H21 ClO1121ClO11 91810653
C21 H 91810653 121121 ± 3.00
± 3.00
Peonidin-3-galactoside C22 H23 ClO
Peonidin-3-galactoside C22H1123ClO11 91810512
91810512 438438 ± 7.00
± 7.00
Peonidin-3-arabinoside
Peonidin-3-arabinoside C21 H21 ClO
C21H1021ClO10 91810651
91810651 65.0 ± 2.00
65.0 ± 2.00
Malvidin-3-arabinoside 592 ± 5.00
Malvidin-3-arabinosideC22 H23 ClO
C22H1123ClO11 91810654
91810654 592 ± 5.00

These
Thesemolecules
moleculeshavehavebeen
beenpreviously reported
previously as having
reported functional
as having bioactivities
functional that
bioactivities
include the prevention
that include or inhibition
the prevention of microorganisms.
or inhibition Quercetin
of microorganisms. has been
Quercetin reported
has been as a
reported
broad
as a antimicrobial agent capable
broad antimicrobial agentofcapable
inhibiting
of both bacteriaboth
inhibiting and fungi [26].and
bacteria Anthocyanins
fungi [26].
are a class of natural
Anthocyanins pigments
are a class foundpigments
of natural abundantly in various
found berries.
abundantly These water-soluble
in various berries. These
compounds are responsible for the vibrant red, purple, and blue colors
water-soluble compounds are responsible for the vibrant red, purple, and blue exhibited by these
colors
fruits. Beyond their role in coloration, anthocyanins have garnered significant
exhibited by these fruits. Beyond their role in coloration, anthocyanins have garnered interest
significant interest in scientific research due to their potential health benefits, particularly
their antibacterial activity [27,28].
Int. J. Mol. Sci. 2023, 24, 14681 4 of 16

in scientific research due to their potential health benefits, particularly their antibacterial
activity [27,28].

2.2. Antimicrobial Activity

Foodborne bacteria represent a constant risk for human consumption. The presence of
coliforms such as Salmonella sp. and Escherichia coli has been associated with ready-to-eat
fruits [29]. In 2011 and 2016, outbreaks associated with E. coli O157:H7 in strawberries
were reported [30]. Additionally, there have been connections to Salmonella sp. outbreaks,
as well [31]. On the other hand, Staphylococcus aureus and Listeria sp. contaminations are
associated with poor manufacturing practices of ready-to-eat fruits [32].
Microbial strawberry decay is also associated with fungi growth, in which yeasts play
an important spoilage role [33]. Therefore, the antimicrobial capability of free extracts and
the BC coating were tested against selected foodborne bacteria and yeasts.
The antimicrobial activity of the free extract (FE), a 1% w/v chitosan solution (CH),
and the bioactive coating (1% w/v chitosan + 0.8% w/v free extract, BC) were determined
in vitro. All the treatments inhibited the growth of bacteria and yeasts (Table 2). As
expected, the FE showed, in general, significantly greater antimicrobial effects than the
CH or BC. The antimicrobial activity was transversal to either Gram-positive (St. aureus
and L. inoccua) or Gram-negative (S. typhimurium and E. coli) bacteria, as well as yeasts
(P. kluyverii and M. pulcherrima).

Table 2. Antimicrobial activity of free Arrayan extract, chitosan solution, and bioactive coating
against common foodborne bacteria and spoilage yeast.

Inhibition Zone Diameter (mm)

Microorganism
FE CH BC
Foodborne bacteria
St. aureus 21.70 ± 0.03 a 12.30 ± 0.11 b 19.33 ± 0.31 c
L. innocua 25.00 ± 0.05 a 13.00 ± 0.11 b 17.17 ± 1.33 c
E. coli 22.60 ± 0.04 a 10.30± 0.10 b 17.85 ± 0.21 c
S. typhymurium 21.60 ± 0.01 a 13.30 ± 0.10 b 18.32 ± 0.10 c
Yeasts
P. kluyverii 27.10 ± 0.02 a 19.60 ± 0.12 b 19.50 ± 0.03 b
M. pulcherrima 28.40 ± 0.04 a 23.50 ± 0.08 b 21.40 ± 0.05 b
Data are presented as mean values ± standard deviations. Different letters within a row represent significant
differences (p < 0.05). FE: free extract (0.8% w/v), CH: chitosan solution (1% w/v), and BC: bioactive coating (1.0%
w/v chitosan + 0.8% w/v free extract).

Viktorová et al. (2020) studied the antimicrobial effects of a methanolic Arrayan

extract over Pseudomonas aeruginosa and St. aureus, establishing for the latter an IC50
(median inhibitory concentration) between 0.354 and 0.385 mg/mL [20]. On the other
hand, Treulen et al. (2019) established the MIC of an Arrayan extract for St. aureus
between 1 and 10 mg/mL, consistent with our study, where a dose used for BC of 0.8%
w/v (8 mg/mL) was established [34]. In a similar manner, Araya-Contreras et al. (2019)
determined that hexane Arrayan extracts showed antibacterial activity against St. aureus,
St. epidermidis, I. saprophyticus, Enterococcus sp., A. baumanii, Pseudomona aeruginosa, and E.
coli at concentrations greater than 100 µg/mL [19].
The presence of bioactive compounds, such as flavonoids, tannins, and essential oils,
in the extract is thought to contribute to this antibacterial effect [18]. These compounds have
demonstrated the ability to disrupt bacterial cell membranes, inhibit enzymatic activities
crucial for bacterial survival, and interfere with bacterial quorum sensing, leading to
reduced bacterial growth and virulence [35].
The BC’s antimicrobial activity was significantly lower than the FE but higher than
the CH alone. This can be explained by the slowing effect generated by the chitosan matrix
on the bioactive agent (FE) release. This effect has already been identified in other studies,
Int. J. Mol. Sci. 2023, 24, 14681 5 of 16

where a polymeric matrix affects the release rate of an antimicrobial or antioxidant agent,
decreasing its initial impact, but maintaining a certain persistence over time [36–40]. This
can be advantageous in terms of ensuring a regulated activity over time, especially during
food storage. However, a minimum release threshold must be established to generate the
expected bioactive effect.

2.3. Physical and Chemical Evolution during Cold Storage

The effect of the BC on fresh strawberries’ shelf life was assessed by coating fresh
strawberries with the FE (0.8% w/v), the CH (1.0% w/v chitosan), or the BC (1.0% w/v
chitosan + 0.8% w/v FE), and then storing at 10 ◦ C for 21 days. During this time, different
parameters were determined every 7 days.

2.3.1. Weight Loss

During storage, water loss, and therefore weight loss, is a very common problem
that impairs the quality of the strawberries. Figure 2A shows the weight loss experienced
by the experimental strawberries for 21 days (3 weeks). Overall, the water loss in all the
treated samples increased with time. However, the samples treated with the bioactive
coating showed a significantly lower weight loss percentage than the other treatment and
the control samples. The greatest weight loss occurred for the control samples (without
treatment), reaching a loss of 8% in the first week, close to 15% in the second, and over
20% in the third, while the strawberries coated either with the CH or the BC showed lower
weight loss trends. The lowest weight loss was observed when the strawberries were
coated with the BC, where, after 3 weeks of storage, weight loss was close to 7%, with
no significant differences being observed with the control group at week 0, suggesting
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 6 of 17
that the addition of the FE in a polymeric matrix significantly increases the stability of
fresh strawberries.

Figure 2. Physical and chemical changes in the treated strawberries (FE, CH, and BC) and control
Figure 2. Physical and chemical changes in the treated strawberries (FE, CH, and BC) and control
(without treatment) during storage (21 days at 10 °C). (A): Weight loss; (B): total soluble solids; (C):
(without treatment) ◦
firmness; (D): pH. during storage (21 days at 10 C). (A) Weight loss; (B) total soluble solids;
(C) firmness; (D) pH.
Muley and Singhal (2020) demonstrated that by using chitosan coatings, it is possible
to control weight loss in strawberries [10]. In a test carried out at 5 °C, they managed to
reduce the weight loss by approximately 50% compared to the control group on day 5 of
storage. By day 7, the coated strawberries had only lost 13% of their initial weight, a higher
loss than our results, in which, on day 7, the samples with BC had only lost 7% in weight
at 10 °C [10], while Petriccione et al. reported a 7–9% weigh loss when different
strawberries were coated with chitosan [41] after 10 days of storage. The bioactive coating
Int. J. Mol. Sci. 2023, 24, 14681 6 of 16

Muley and Singhal (2020) demonstrated that by using chitosan coatings, it is possible
to control weight loss in strawberries [10]. In a test carried out at 5 ◦ C, they managed to
reduce the weight loss by approximately 50% compared to the control group on day 5 of
storage. By day 7, the coated strawberries had only lost 13% of their initial weight, a higher
loss than our results, in which, on day 7, the samples with BC had only lost 7% in weight at
10 ◦ C [10], while Petriccione et al. reported a 7–9% weigh loss when different strawberries
were coated with chitosan [41] after 10 days of storage. The bioactive coating used in our
study was enriched with a rich polyphenolic extract that can also aid in the retention of
water in the fruit, and therefore contribute to the control of weight loss. Riaz et al. (2020)
suggested that slower weight losses can be attributed to a decrease in the hydrophilicity
nature of CH, due to the incorporation of polyphenol-rich extracts [42].

2.3.2. Firmness
Figure 2C shows the results observed regarding firmness during cold storage. The
control samples showed higher firmness loss, that is, the strawberries without treatment.
On the other hand, in the treated samples, little difference was observed, showing values
between 28 to 30 N of rupture force. There is a close relationship between the water
content in fruit and its resistance to rupture. A higher water content increases turgor. In
the opposite direction, a lower water content implies less turgor and, consequently, less
firmness [43].
The samples treated with the BC showed lower weight loss values, which allowed
them to maintain greater firmness. In percentage terms, the control group lost 35.5%
firmness, while the BC treatment only lost 2.3%. Saleh and Abu-Dieyeh (2022) studied
the effects of a chitosan film containing Prosopis juliflora extract to increase the shelf-life of
strawberries [14]. They concluded that their control group (strawberries without treatment)
showed up to 30.2% firmness loss, whereas the strawberries treated with the coating had
no difference in terms of firmness between days 0 and 21. These results are consistent
with those reported in this study. Riaz et al. (2020) reported that when strawberries are
coated with a polyphenol-rich chitosan coating, the fruits retain firmness after six days of
cold storage. This can be possible because the coating decreases the respiration rate, and
therefore decreases the fruit ripening and senescence [44].

2.3.3. Total Soluble Solids (TSS)

Figure 2B shows the variations in total soluble solids during the storage period. The
results indicate that the control group significantly increased the concentration of TSS,
while no significant variations were observed for the FE, CH, or BC treatments. The
variation in the control group might also be related to the weight loss of the strawberries
[Figure 2A] since, during storage, the strawberries lose water by exudation, and sugars and
other solids are concentrated, resulting in increases in ◦ Brix. In a similar study by Saleh
and Abu-Dieyeh (2022), the same phenomenon was evidenced between control groups
of strawberries (without treatment) and strawberries treated with an extract of Prosopis
juliflora and chitosan [14]. They demonstrated that the control group increased TSS by 8.9%,
while the coated group only increased by 1.5%. They also associated the increase in TSS
with the weight loss of the strawberries during storage.
Changes in soluble solids can also be attributed to the respiratory rates of the stored
strawberries. Ayala-Zavala et al. (2004) reported that, in storage at 10 ◦ C, a higher loss
in TSS was observed, compared to storage at 0 and 5 ◦ C. The authors attributed that, at
10 ◦ C, the respiratory rate of the fruits was increased, explaining in part the changes in
TSS [45]. When strawberries are treated with bioactive coatings, the respiratory rate of the
fruit decreases. Esghi et al. (2014) reported slower respiration when strawberries were
coated with a nano-chitosan suspension [46]. Garcia et al. (2010, 2011) used a cassava starch
edible coating and verified a decrease in the respiration rate and an increase in water vapor
resistance, as well as reduced weight loss and mechanical property loss [47,48].
Int. J. Mol. Sci. 2023, 24, 14681 7 of 16

2.3.4. pH
During the cold storage, the samples treated with the FE, the CH solution, and the BC
showed little variation in pH, maintaining values close to the initial values (3.25, 3.75, and
3.67, for the FE, the CH solution, and the BC, respectively), while, in the control samples,
decreases in pH were observed (Figure 2D). Changes in pH during the storage of fresh fruits
are commonly associated with the spoilage process, in which bacteria produce organic
acids due to bacterial proliferation [49]. This result correlates with the microbial quality
observed during the storage, in which the control samples showed greater microbial loads
(Table 2).

2.4. Microbial Stability during Strawberries Cold Storage

The shelf-life of a food is determined in part by its microbiological stability, that is, the
ability to limit or prevent deteriorating microbial proliferation. In that sense, the BC and its
components (FE and CH) were evaluated regarding their ability to restrict the growth of
aerobic mesophilic microorganisms and fungi (molds and yeasts). The effect was measured
after 3 weeks of cold storage, and the results are shown in Table 3.

Table 3. Microbiological stability.

Weeks
Treatment
0 1 2 3
Control 2.12 ± 0.01 aA 1.14 ± 0.03 aB 3.39 ± 0.04 aC 4.26 ± 0.02 aD
Mesophilic
aerobic
FE 1.83 ± 0.04 bA 1.05 ± 0.03 aA 2.23 ± 0.06 bB 3.46 ± 0.04 bC
(log CFU/mL) CH 2.32 ± 0.02 aA 1.85 ± 0.01 aB 2.52 ± 0.02 bA 3.17 ± 0.01 bC
BC 1.63 ± 0.06 bA 1.25 ± 0.02 aA 1.37 ± 0.03 cA 1.62 ± 0.01 cA
Control 1.35 ± 0.12 aA 2.11 ± 0.08 aB 3.15 ± 1.10 aC 4.32 ± 0.17 aD
Yeast and
Mold
FE 1.41 ± 0.08 aA ≤0.1 bB 1.21 ± 0.11 bA 3.34 ± 0.18 bC
(log CFU/mL) CH 1.37 ± 0.11 aA ≤0.1 bB 1.67 ± 0.08 bA 2.78 ± 0.05 cC
BC 1.32 ± 0.09 aA ≤0.1 bB 1.13 ± 0.02 bA 1.05 ± 0.01 dA
Data are presented as mean values ± standard deviations. Values with different lowercase letters are significantly
different (p < 0.05) in the same column. Mean values with different capital letters are significantly different
(p < 0.05) in the same line. FE: free extract (0.8% w/v); CH: chitosan solution (1% w/v); BC: bioactive coating (0.8%
w/v FE in 1% w/v CH solution).

It can be seen that both aerobic mesophilic microorganisms and fungi were able to
increase freely in the control group, reaching 4.32 log CFU/g at the end of the storage,
well above the concentrations observed in the treated strawberries. The application of BC
was significantly more effective than the rest of the treatments since, in the third week, the
microbial population did not exceed the concentration of 1.7 log CFU/g, with no significant
differences from the control group on day 0. Similar results were observed for yeast growth,
in which the BC-coated samples showed 1.05 log CFU/g after 3 weeks of storage, with
no significant differences from the concentrations at the beginning of the experimentation
while, in the control sample, the final yeast concentrations reached values higher than
4 log CFU/g (Table 3). The use of BC has been reported as an efficient way to prevent
microbial spoilage in different food products since the bioactive compound embedded in
the polymer allows for a controlled release of the antimicrobial, which prevents microbial
proliferation [49,50]. Berry extracts, and especially their anthocyanins, have been reported
as an interesting alternative for the development of bioactive films, which enable the control
of microorganisms (both fungi and bacteria) to maintain freshness and extend food shelf-
life [51]. The Arrayan extract studied here is rich in these molecules, therefore making it a
desirable additive for the development of bioactive coatings.

2.5. DPPH Radical Scavenging Assay and ORAC Antioxidant Activity

Berries are commonly a good source of antioxidants responsible for the oxidation of
free radicals, acting as oxygen scavengers. As the fruit ripens, the antioxidant activity
increases; however, this activity is lost as the fruit decays. In our study, we propose to
Int. J. Mol. Sci. 2023, 24, 14681 8 of 16

use a BC enriched with Arrayan extract, which is rich in polyphenols, anthocyanin, and
flavonoids [24], as a means to protect against antioxidant loss in the strawberries.
Two techniques were used to determine the oxygen-scavenging activity of the antioxi-
dants DPPH and ORAC. We measured these two in the stored strawberries at the beginning
and end of the storage (Table 4).

Table 4. Antioxidant capacity in strawberries after 21 days of storage at 10 ◦ C.

(µmol of TE·100 g/dw) *

Treatment DPPH ORAC
Initial Week 3 Initial Week 3
Control 167.2 ± 1.14 aA 49.50 ± 1.14 aB 75.1 ± 0.12 aC 61.1 ± 0.34 aD
BC 315.4 ± 1.10 bA 264.1 ± 1.24 bB 306.2 ± 2.31 bC 289.1 ± 3.22 bC
Data presented as mean values ± standard deviations. * Micromoles of Trolox equivalents (TE) per 100 g of dried
weight. Mean values with different lowercase letters are significantly different (p < 0.05) in the same column.
Mean values with different capital letters are significantly different (p < 0.05) in the same line.

In the case of the control group, the ability to control DPPH decreased from 167.2 to
49.50 µmol of TE·100 g/dw, that is, a 70% decrease in the antioxidant activity of strawberries.
On the other hand, for the group with the BC treatment, the ability to control DPPH only
decreased by 16% (from 315.4 to 264.1 µmol of TE·100 g/dw). Something similar occurred
in the ORAC measurement, where the control group had a fall of 18% in Trolox equivalent
terms, against only 5% of the group of strawberries treated with BC. On the other hand, it
was possible to show that the antioxidant capacity of the control reached a Trolox equivalent
of 61.1 at the end of 3 weeks, compared to the treated group of 289 at the same time. These
results are consistent with those reported by Muley and Singhal (2020), who showed a 27%
drop in the control capacity of DPPH in 8 days of cold storage [10]. It should be noted that
they used a coating based on chitosan and wheat protein, without adding any bioactive,
which would indicate that the barrier effect of the biopolymers can protect and retain the
antioxidants present in the fruit.
Our results differ from those reported by Popescu et al. (2022) [12] and Saleh and Abu
(2022) [14], which reported increases in the antioxidant activity of strawberries coated with
bioactive films. The increases were associated with fruit maturity [12,14]. In our study,
the storage was carried out at 10 ◦ C, which could have prevented the fruit ripening.

2.6. Total Phenolic Content (TPC), Ascorbic Acid, Flavonoids, and Anthocyanins
Antioxidant activity is usually related to the presence of molecules such as polyphenols,
anthocyanins, flavonoids, and ascorbic acid, compounds in which strawberries are very rich.
Protecting these functional molecules during storage is an important factor in maintaining
the nutritional characteristics of the strawberries. In that sense, the ability of BC to prevent
the loss of antioxidant agents was evaluated. Table 5 shows the variation in flavonoids,
polyphenols, anthocyanins, and ascorbic acid in strawberries coated with the BC after
3 weeks of cold storage. A drop in concentration was observed over time for all the
molecules, which is to be expected given that they progressively oxidize due to contact
with air. However, there are significant differences in losses between the control group
(without treatment) and strawberries treated with BC.
Regarding the flavonoids, it can be seen that the control group showed a loss of up
to 49% after 21 days of storage, while in the BC-treated strawberries, it was only 14%.
Regarding the polyphenols, the drop in the control group was greater, losing up to 72%
of these antioxidant agents, compared to the treated strawberries, which lost only 18%.
Something similar occurred with the ascorbic acid, where the control group lost 45% of the
compounds, compared to the treated strawberries, with losses of only 17%. Finally, the
anthocyanins show losses of 16% for the control group and less than 1% for the treated
strawberries. This might be due to the protective barrier of the BC (chitosan + Arrayan
extract) against oxidative reactions, which reduces the loss of the antioxidant compounds.
Int. J. Mol. Sci. 2023, 24, 14681 9 of 16

Different studies have shown similar behavior when using films or coatings with the
incorporation of antioxidant agents for the preservation of food [10,52–54].

Table 5. Concentrations of antioxidant agents in treated strawberries over time.

Flavonoids Polyphenols Anthocyanins Ascorbic Acid

(mg eq/100 g) (mg EAF/100 g) (mg/100 g) (mg/100 g)
Treatment
Initial Week 3 Initial Week 3 Initial Week 3 Initial Week 3
aA aB aA aB aA
Control 1.31 ± 0.12 0.67 ± 0.19 25.2 ± 0.12 7.35 ± 0.09 89.6 ± 0.14 bB
75.2 ± 0.12 bA 97.1 ± 0.12 52.5 ± 0.17 aB
Int. J.BC
Mol. Sci. 2023,
2.2724, x FOR
± 0.12 bA PEER REVIEW
1.95 ± 0.16 bA 25.3 ± 0.13 aA 20.6 ± 0.04 bB 101.2 ± 0.21 aA 100.2 ± 0.15 aA 97.2 ± 0.11 aA 10 ±
80.2 of0.15
17 bB

Data presented as mean values ± standard deviations. Values with different lowercase letters are significantly
different (p < 0.05) in the same column. Mean values with different capital letters are significantly different
(p < 0.05) in the same line. Control: without treatment; BC: bioactive coating (0.8% w/v free extract in 1% w/v
Different
chitosan studies have shown similar behavior when using films or coatings with the
solution).
incorporation of antioxidant agents for the preservation of food [10,52–54].
2.7. Sensory Analysis
2.7. Sensory Analysis
Twenty-five panelists analyzed strawberries freshly coated with the BC to determine
Twenty-five panelists analyzed strawberries freshly coated with the BC to determine
the effect on the treated samples’ texture, color, aroma, sweetness, and acidity. The results
the effect on the treated samples’ texture, color, aroma, sweetness, and acidity. The results
showed no significant differences in any of the characteristics between the untreated and
showed no significant differences in any of the characteristics between the untreated and
the treated samples (Figure 3), indicating that the application of the bioactive coating does
the treated samples (Figure 3), indicating that the application of the bioactive coating does
not change the natural characteristics of the fresh strawberries. Furthermore, the overall
not change the natural characteristics of the fresh strawberries. Furthermore, the overall
acceptance of the strawberries was close to 8 for both samples.
acceptance of the strawberries was close to 8 for both samples.

Figure 3.3. Sensory

Figure Sensory appreciation
appreciationofofstrawberries
strawberriescoated
coatedwith
withthethe
bioactive coating
bioactive (0.8%
coating w/vw/v
(0.8% free free
Arrayan extract in 1% w/v chitosan solution). Attributes were evaluated on a 9-point hedonic scale,
Arrayan extract in 1% w/v chitosan solution). Attributes were evaluated on a 9-point hedonic scale,
where 1 was “dislike extremely”, and 9 was “like extremely”.
where 1 was “dislike extremely”, and 9 was “like extremely”.

3.3.Materials
Materials and Methods
and Methods
3.1. Plant
3.1. Plant Material
Material
Arrayan branches
Arrayan brancheswith
withleaves
leavesand
andripe
ripe fruits
fruits were
were collected
collected fromfrom
the the Valparaiso
Valparaiso Region
Region
◦ 0 (33°02′18.58″
00 ◦ S;
0 71°29′48.04″
00 W) and transported to the Microbial Food Laboratory
(33 02 18.58 S; 71 29 48.04 W) and transported to the Microbial Food Laboratory (Pontifical
(Pontifical
Catholic Catholic University
University of Chile) inofcoolers
Chile) in coolers provided
provided with drywith dry iceThe
ice packs. packs. The and
leaves leaves
fruits
and fruits were manually separated at the laboratory, washed with a sodium hypochlorite
were manually separated at the laboratory, washed with a sodium hypochlorite solution
solution (100 mg/L) (J.T. Baker, Mexico D.F., Mexico), and washed twice with distilled
(100 mg/L) (J.T. Baker, Mexico D.F., Mexico), and washed twice with distilled water.
water.

3.2. Extracts and Coatings Preparation

The extracts were prepared as Viktorová et al. (2020) described, with a few
modifications. Briefly, the sanitized fruits were oven-dried (Thermo Scientific PR205045G)
at 60 ± 5 °C for 48 h, finely ground, and pulverized using a food processor to achieve a
Int. J. Mol. Sci. 2023, 24, 14681 10 of 16

3.2. Extracts and Coatings Preparation

The extracts were prepared as Viktorová et al. (2020) described, with a few modi-
fications. Briefly, the sanitized fruits were oven-dried (Thermo Scientific PR205045G) at
60 ± 5 ◦ C for 48 h, finely ground, and pulverized using a food processor to achieve a parti-
cle size of 0.3–0.5 mm [18]. The powder was mixed with 100% ethanol (J.T. Baker, Mexico).
The mixture was shaken (Slow-Speed Orbital Shaker, Cole-Palmer, Vernon Hills, IL, USA)
at 100 rpm at room temperature for 24 h. Then, the extraction mixture was filtered using a
Whatman No. 1 filter (Whatman International, Maidstone, UK). The filtered extracts were
dried under reduced pressure at 40 ◦ C using a rotary evaporator (Büchi, Labortechnik AG,
Flawil, Switzerland). The crude extracts were filter-sterilized (0.45 µm, Nalgene, Millipore,
Milwaukee, WI, USA) and stored at −20 ◦ C until experimentation. For the experiment,
three coating solutions were analyzed: (i) free extract solution (FE: 0.8% w/v), (ii) chitosan
solution (CH: 1.0% w/v), and (iii) bioactive coating (BC: 0.8% w/v free extract in 1.0% w/v
chitosan solution). Based on preliminary studies, the selected concentration showed the
minimal concentration for bacteria and fungi control.

Free Extract Polyphenolic Composition

The quantification of specific polyphenols was carried out following the protocol
reported by Allcca-Alca (2019). Briefly, using Millipore filter type GV (0.22 µm), twenty
µL of the filtered extract (0.22 µm, Millipore filter) was analyzed in an HPLC system
(Waters 2695, Waters, Milford, MA, USA) equipped with a photodiode array detector (PAD)
(Waters, Milford, MA, USA). An X-terra RP18 (5 µm, 250 mm × 4.6 mm) column (Waters,
Milford, MA, USA) was used for phenolic separation at 30 ◦ C. The mobile phase was
made from water:formic acid (95:5, v/v, pH 2) and acetonitrile, used in a gradient. A flow
rate of 0.5 mL/min was used. Polyphenols were identified and quantified by comparing
the retention time and area under the curve of different standards. The analyses were
performed in triplicate, and the results were expressed as mg of specific polyphenol per
gram of dried sample [55].
The anthocyanin identification was conducted following the protocol reported by
Fuentes et al. (2016) in methanol and methanol–HCl extract of Arrayan berries with an
elution gradient of water/glacial acetic acid/phosphoric acid [56]. Detection wavelengths
were 520 and 280 nm, respectively. The flow rate was 1 mL/min, column temperature was
40C, and injection volume was 20 mL. The quantification was done by comparison with
standards [56].

3.3. Coatings’ In-Vitro Antimicrobial Activity

Four food-born bacteria: Staphylococcus aureus (ATCC 6538), Listeria innocua (ATCC
33090, Escherichia coli (ATCC 25922), and Salmonella typhimurium (ATCC 14028), and two
deteriorative yeasts (Pichia kluyverii and Metchsnikowia pulcherrima) were used for the an-
timicrobial susceptibility test. The bacteria were obtained from the American Type Culture
Collection (ATCC); while the spoilage yeasts were isolated from spoiled strawberries and
stored in the culture collection at the Microbiology and Food Fermentation Laboratory at
Pontificia Universidad Católica de Chile, (Santiago, Chile).
The disk diffusion method was followed [57]. Briefly, the microorganisms were
overnight cultured in Mueller Hinton Broth (MHB, Oxoid, Thermofisher, Hampshire,
England) to achieve a cell concentration of about 6 log CFU/mL. Then, 500 µL of the active
culture was spread onto Mueller Hinton Agar (MHA, Oxoid, Thermofisher, Hampshire,
England). Blank discs (BBL, Dilaco, Santiago, Chile.) were impregnated with 20 µL of each
coating solution and placed in the inoculated agar plates. A blank disc (negative control),
penicillin, and chloramphenicol discs were used as positive controls for the bacterial
experiments, and a tioconazole disc was used for the yeast cultures (Oxoid, Thermofisher,
Hampshire, UK). Inhibition halos were measured after a one-day incubation at 37 ◦ C for
the bacterial experiment and two days at 25 ◦ C for the yeast ones.
Int. J. Mol. Sci. 2023, 24, 14681 11 of 16

3.4. Shelf-Life Study (Physical, Chemical, and Microbiological Changes)

Fresh strawberries were purchased at “La Vega” Farmer´s Market (Santiago, Chile).
The strawberries were screened for decay and/or bruises upon arrival at the laboratory.
Those that showed visual defects were discarded. The experimental design reported by
Saleh et al. (2022) was followed [14]. Briefly, the decay-free strawberries were divided into
four batches of 30 individual berries.
• Control: Untreated strawberries.
• Free extract (FE): Strawberries sprayed with free extract solution 0.8% w/v.
• Chitosan solution (CH): Strawberries dipped in 1.0% w/v chitosan solution.
• Bioactive coating (BC): Strawberries dipped in the bioactive coating (1.0% w/v chitosan
+ 0.8% w/v free extract).
A randomized design was used for the shelf-life study in which five samples made one
experiment unit (one replicate), and each experimental condition had six replicates. The
coated strawberries were left to air-dry over a sterilized rack at room temperature. Later,
each replicate was placed in a sanitized plastic clamp container. The samples were stored for
21 days at 10 ± 2 ◦ C and an ambient humidity of 60 ± 5% (Hygrometer with datalogging,
Fisherbrand, Waltham, MA, USA) and analyzed at the beginning of the experiment and,
after that, every 7 days. Weight loss, firmness, total solids concentration, pH, and microbial
proliferation were measured to determine the impact on shelf-life.

3.4.1. Weight Loss

The strawberry samples were weighed at the beginning of each experiment (day 0),
and then the weight was measured at the time of sample collection up to 21 days of storage.
The difference between the initial and final weights determined the weight loss. An average
percent loss was calculated [58].

3.4.2. Firmness
The firmness of the treated strawberries was evaluated in terms of the force required
to rupture the rind of the fruits. A texture analyzer (TA-XT2, TA Instruments, Crawley, UK)
was used for this. The measurement was carried out in the central upper quadrant area
of the strawberries using a cylindrical probe (aluminum, diameter: 5 mm). The test speed
was fixed at 600 mm/min [59].

3.4.3. pH and Titratable Acidity (TTA)

The pH was measured with a pH meter (Mettler Toledo, Columbus, ON, USA), and
TTA was determined using the amount of NaOH 1 N needed to achieve a pH value of 8.2
in the samples. The acidity was calculated, taking ascorbic acid as the predominant acid in
the strawberries using the mathematical relationship (1).

w N × V1 × Eq
% ascorbic acid = × 100 (1)
v V2 × 1000

where
N = Standard concentration NaOH (normality)
V1 = Volume NaOH used
V2 = Sample volume
Eq = Ascorbic acid equivalent weight (0.00277).

3.4.4. Total Soluble Solids (TTS)

The samples of treated and untreated strawberries (control) were blended into juice,
and large particulates were separated from the supernatant by decantation. Total soluble
solids were determined using a refractometer (Atago ATC-1, Tokyo, Japan) at a temperature
of 20 ± 1 ◦ C. Three drops of strawberry juice were taken to obtain the measurements. The
refractometer was calibrated to zero with distilled water between each measure [52].
Int. J. Mol. Sci. 2023, 24, 14681 12 of 16

3.4.5. Microbial Stability

At all the sample points, the strawberries were blended into juice. The juice was left to
sit for the decantation of the particulates. The supernatant was transferred into a sterilized
flask, serially diluted with peptone water 1% (3M, Kamen, Germany), and spread-plated
on Yeast and Mold Agar (YMA, Millipore, Milwaukee, WI, USA) for yeast and mold counts,
and Plate Count Agar (PCA, Merck, UK) for the total mesophilic aerobic count. The plates
were incubated at 25 ◦ C for 24 to 48 h or until colonies were observed [14].

3.5. Antioxidant Activity

3.5.1. Total Phenolic Content (TPC) and Ascorbic Acid
Following the manufacturer’s instructions, the TPC and ascorbic acid were determined
using an enzymatic autoanalyzer photometer (Y15, Biosystem, Barcelona, Spain).

3.5.2. Flavanol and Anthocyanin Determination

The determination of total flavonoids was conducted following the method described
by Zhishen, Mengcheng, and Jianming (1999), with some modifications. Briefly, 1 mL of
strawberry ethanolic extract was mixed with 6.4 mL of deionized water, 300 µL of NaNO2 at
0.05 g/mL, 300 µL of AlCl3 at 0.10 g/mL, and 2 mL of NaOH. The mixture was left to settle
for 30 min, and then absorbance was measured at 415 nm in a UV-Vis spectrophotometer
(Spectroquant, modelPharo 300, Darmstadt, Germany) [60]. The concentration of total
flavonoids was calculated based on a quercetin standard curve, and the results were
expressed as mg of quercetin equivalents per g of fresh weight of the sample (mg EQ/g).
Monomeric anthocyanins were determined by a spectrophotometric method reported
by [61]. Briefly, 2 mL of the strawberry juice was diluted in 25 mL of a solution composed
of 15 mL of 0.2M KCl and 375 mL of 0.2M HCl to achieve a pH of 1. Another 2 mL was
diluted up to 25 mL with a buffer solution at pH 4.5 (400 mL of 1M CH3 CO2 Na, 240 mL of
1M HCl, and 360 mL of H2 O). The absorbance of the solutions was measured at 510 nm.
The concentration of anthocyanins was determined by:


AbspH1 − AbspH4.5 ∗ 484.82 ∗ 1000
Monomeric anthocyanins = ∗ DF (2)
ε
where 484.22 is the molecular weight of Cyanidin-3-glucoside chloride, ε is the molar
absorptivity at 510 nm in the pH 1 solution, and DF is the dilution factor.

3.6. DPPH Radical Scavenging Assay

One hundred µL of diluted juice was mixed with 1 mL of 2,2-diphenyl-1-picrylhydrazyl
(DPPH) (Calbiochem, Merck, Darmstadt, Germany) (100 mg/L). The mixture was incu-
bated in the dark at 37 ◦ C for 45 min. After this time, the samples were centrifuged, and the
change in the supernatant color was measured using a spectrophotometer (Spectroquant,
modelPharo 300, Darmstadt, Germany) at 517 nm; 100 µL of methanol in 1 mL DPPH was
used as the control, and methanol alone was used as the blank. The radical scavenging
activity (RSA) was calculated with the formula:

100
% RSA = (absorbance of control − absorbance of sample) ∗
absorbance of control

3.7. ORAC Antioxidant Activity

The ORAC activity was determined using 2,20 -azobis(2-amidinopropane) dihydrochlo-
ride (AAPH) and fluorescein, as described by [62]. Briefly, 45 µL of strawberry juice (diluted
in 75 mM phosphate buffer, pH 7.4) was transferred to microplates containing 75 µL of
APPH (18 mM) and 200 µL of fluorescein (108 nM). The plates were placed in a Multi-
Mode Microplate Reader (Fluoroskan Ascent, Thermo Scientific, Waltham, MA USA) and
incubated for 60 min at 37 ◦ C, shaking every 3 min. During incubation, the fluorescence
(485 nm Ex/538 nm Em) was monitored every 3 min throughout the experiment. The
Int. J. Mol. Sci. 2023, 24, 14681 13 of 16

ORAC activity was expressed as micromoles of Trolox equivalents (TE) per 100 g of dried
weight (µmol of TE·100 g/DW).

3.8. Sensory Analysis

Twenty-five untrained panelists (average age 25 years old, 13 female and 12 male)
were recruited to conduct a 9-point hedonic scale sensory test by scoring the following
attributes: texture, color, aroma, sweetness, acidity, and overall acceptance. Fresh decay-free
strawberries were washed with sterile water, air-dried, and then dipped in the bioactive
coating solution (1% chitosan with 8 mg/mL free extract). After dipping for 1 m, the
samples were air-dried and used for evaluation. Strawberries without treatment (non-
coated) were used as controls. The samples were presented in white plastic disposable
dishes labeled with a 3-digit random code. The panelists were asked to rate each sample
on a scale from 1 to 9, where 1 represents “dislike extremely”, and 9 “like extremely”.

3.9. Statistical Analysis

All the experiments were carried out in triplicate and two experiment runs. The
data were expressed as the mean ± standard deviation. A one-way analysis of variance
(ANOVA) was performed to determine significant differences, and for multiple compar-
isons, the post hoc t-test with Bonferroni correction was used. Statistically significant
differences were considered at p values < 0.05.

4. Conclusions
Our results suggest that coating fresh strawberries with a bioactive coating composed
of Arrayan extracts embedded in a chitosan matrix significantly improves the shelf-life
of the fruits when stored at 10 ◦ C. After three weeks of storage, the BC prevented signifi-
cant weight loss, while the strawberries maintained their turgor. The fruits showed little
microbial spoilage (without any visible mold formation). The strawberries were able to
preserve their antioxidant activity, as reflected by the minor variations in polyphenols,
flavonols, anthocyanins, and ascorbic acid concentrations. Finally, the treated strawberries
did not change in appearance or flavor, conserving the natural fruits’ sensory characteristics.
These results suggest that Arrayan extracts can be used as natural additives to improve the
shelf-life of strawberries when embedded in a polymeric chitosan matrix.

Author Contributions: Conceptualization, W.F. and S.B.; methodology, W.F.; formal analysis, W.F.
and S.B.; resources, W.F.; writing—original draft preparation, W.F. and S.B. All authors have read and
agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki and approved by the Ethics Committee of Pontifical Catholic University of Chile (protocol
code 170728007, 17 May 2018).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Available at request to the corresponding authors.
Conflicts of Interest: The authors declare no conflict of interest.

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