Chitosan and Clove Oil for Strawberry Preservation
Chitosan and Clove Oil for Strawberry Preservation
1
Universidade Federal do Amazonas, Faculdade de Ciências Agrárias, Departamento de Engenharia Agrícola e de Solos,
Manaus, Amazonas, Brasil; 2Universidade Federal da Grande Dourados, Faculdade de Ciências Biológicas e Ambientais,
Dourados, Mato Grosso do Sul, Brasil; 3Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos,
Departamento de Engenharia de Alimentos, Campinas, São Paulo, Brasil; 4Universidade Federal da Grande Dourados,
Faculdade de Engenharia, Departamento de Engenharia de Alimentos, Dourados, Mato Grosso do Sul, Brasil.
HIGHLIGHTS
• The applied coatings were efficient to increase the preservation time of strawberries.
• T3 treatment presented better results in retarding the growth of molds and yeasts.
•
Abstract: The work aimed to evaluate the effects of applying chitosan edible coating with the addition of
different concentrations of clove essential oil in minimally processed strawberries. The strawberries were
selected and sanitized, had their leaves removed, and were submerged in the coatings. Three treatments were
obtained: T1 - control (uncoated strawberries); T2 - 2% chitosan + 1% clove essential oil; T3 - 2% chitosan +
1.5% clove essential oil. After receiving the coatings, the strawberries were placed in PET containers and
stored at 5±1°C for 12 days. Physical, chemical and microbiological analysis were performed. T2 and T3
samples were more efficient in reducing mass loss (13.78% and 13.51%) when compared the control sample
(24.19%). They were also effective in maintaining texture and color for longer. The treatment T3 was more
effective to increasing the shelf life and slowing growth, especially of molds and yeasts. The use of different
concentrations of clove oil prolonged the quality of strawberries during refrigerated storage.
INTRODUCTION
Strawberry (Fragaria ananassa) is a non-climacteric fruit containing a great variety of bioactive compounds
including phenolic constituents, anthocyanins, vitamins and minerals [1]. The changes in fruit composition are
mainly due to the ripening process which may take place after harvesting and/or even when the fruit is attached
to the mother plant. The continuous active metabolism and high physiological activities after harvesting the
strawberries leads to reduction in senescence and ripening periods [2,3]. In addition, the mechanical injuries,
excessive texture softening, spoilage caused by microbial growth, moisture loss, and physiological disorders
also contributes to heavy damage to strawberries during growth, harvesting and storage. Due to these factors,
the shelf life of strawberries is very short (1–2 days) at normal storage temperature [4].
Edible coatings are being used in minimally processed food as a strategy to reduce the effects the
minimally process causes in vegetable tissues [5]. Besides, edible coatings can contribute to the increase the
life of minimally processed fruits, reducing its moisture and solute migration, respiration and oxidation reactions
and can also be applied in the liquid form of the film-forming dispersion, or as thin sheets (films) used to wrap
food products. Both are well considered for food preservation purposes due to their ability to improve food
quality [6].
In order to control the decay and improve the shelf life of strawberries, different postharvest treatments
have been studied, such calcium chloride treatment combined with nano-chitosan [7], chitosan, gelatin, starch,
and sorbitol with or without monoterpenes (geraniol and thymol) [8], alginate-based with natural antimicrobials
[9], polysaccharide-based enriched with essential oils [10], sodium alginate and sodium alginate in combination
with ascorbic acid [11].
Chitosan is a natural non-toxic polymer, biodegradable and economic available, which are reasons for its
use in a variety of applications in food industry, counting on the quality control as a substitute for non-
biodegradable polymers [6]. In previous reports, chitosan coating functioned delaying loss of fruit quality,
including inhibiting the growth of microbes, water loss, loss of nutrition value and postharvest softening in
strawberry fruit during ripening [12,13]. Chitosan, as an edible coating, has advantages such as improving
color, firmness, maintaining fruit freshness, decreasing the respiratory rate, oxidative stress and the growth of
pathogenic microorganisms [14].
The clove essential oil is obtained from the shoots and leaves of the Syzygium aromaticum plant,
presenting several functions due to the high content of eugenol, a phenolic compound that has anti-
inflammatory, healing and analgesic effects [15]. In addition to eugenol clove essential oil it contains carvacrol,
both acting as antioxidants and antimicrobials [16].
Essential oils are aromatic and volatile liquids extracted from plant components e.g. roots, flowers, stems,
leaves, seeds, fruits and the whole plant, and are one of the compounds with these characteristics [17]. The
essential clove oil, e.g., has eugenol in its composition substance responsible for analgesic, anti-inflammatory
and antioxidant activity [18]. The use of clove essential oil aims to reduce the microbial growth of minimally
processed fruits [19].
Thus, the objective of the present work was to evaluate the effect of an edible coating based on chitosan
with the addition of essential clove oil to increase the shelf life of minimally processed strawberries.
Preparation of strawberries
Fresh strawberries (Fragaria ananassa), chitosan (degree of deacetylation around 89%, and molecular
mass between 150 and 170 kDa) and clove oil were obtained from the local market in Dourados-MS, Brazil
were used. The fruits were selected and classified according to their skin color, without physiological defects,
injuries and detectable infections from the presence of microorganisms. The strawberries were transported to
the bioengineering laboratory of the Federal University of Grande Dourados in plastic boxes, free of solar rays
and at a temperature of approximately 10-12ºC. They were then stored at 5±1°C until processing.
The leaves and stalk of the strawberry were removed with the help of a stainless steel knife, then they
were sanitized in an organic chlorine solution at 2 g.L-1, for 5 minutes, the water was drained for 2 to 3 minutes
on sieves.
Brazilian Archives of Biology and Technology. Vol.65: e22210278, 2022 [Link]/babt
Pizato, S. et al. 3
The chitosan solution was prepared by dissolving it in distilled water containing 1% (v/v) acetic acid and
agitated for 30 minutes until complete dissolution at a temperature of 25ºC, using a propeller agitator (Fisaton
313D), then the clove essential oil (CEO) was added and the agitation continued for another 5 minutes. The
strawberries were totally submerged in the different coverings for three minutes and then drained with the help
of sieves. Three treatments were obtained: T1 - control (uncoated strawberries); T2 - 2% chitosan + 1% clove
essential oil; T3 - 2% chitosan + 1.5% clove essential oil.
After applying the treatments, the strawberries were stored in a quantity of nearly 50 grams per package
in PET (polyethylene terephthalate), with lid (SANPACK) and with external measurements of 15x10x4cm, and
these packages were stored under refrigeration at 5±1°C for a period of 12 days.
Table 1. Weight loss (%) of strawberries coated with chitosan and different concentration of clove essential oil at 5±1ºC,
for 12 days.
Treatments
Days
T1 T2 T3
0 0gA 0gA 0gA
1 0.47 ± 0.16 fA 0.53 ± 0.24 fA 0.49 ± 0.11fA
3 2.61 ± 0.12 eA 1.94 ± 0.33 eB 2.05 ± 0.14eB
5 4.94 ± 0.22 dA 3.31 ± 0.24 dB 3.17 ± 0.08dB
7 10.54 ± 0.48 cA 7.41 ± 0.43 cB 7.26 ± 0.19cB
9 19.11 ± 0.09 bA 11.76 ± 0.07 bB 11.59 ± 0.13bB
12 24.19 ± 0.27aA 13.78 ± 0.65aB 13.51 ± 0.31aB
Means of 3 repetitions ± standard deviation, followed by the same lowercase letter in the column and uppercase in the
line do not differ from each other by the Tukey test (p<0.05). (T1) control (uncoated strawberries); (T2) 2% chitosan + 1%
clove essential oil; (T3) 2% chitosan + 1.5% clove essential oil.
Many studies show a reduction weight loss of strawberries coated with chitosan-based polymers such as
chitosan-whey protein isolate conjugate coated (16.15%) after 8 days [3], nano-chitosan 0.2% with CaCl2 3%
[7].
The weight loss in strawberries is associated to thin skin and rate of transpiration making prone to rapid
water loss, further causing deterioration and shriveling [25]. Chitosan coating act as a semipermeable barrier
creating a suitable atmosphere for strawberries delaying a water loss. Coatings with essential oil reduce the
water loss in many fruits and vegetables. Similar results were reported by Perdones [26] where strawberries
coated with chitosan 1% and lemon essential oil 3% showed a weight loss of 30% after 11 days. The
incorporation of essential oil enhanced the moisture barrier of the composite coating, resulting from its
hydrophobic nature which increased the distance travelled through the film by water molecules [27,28].
Table 2 shows the total soluble solids (ºBrix) of strawberries coated with chitosan and different
concentration of clove essential oil at 5±1ºC, for 12 days.
Table 2. Total soluble solids content (ºBrix) values of strawberries coated with chitosan and different concentrations of
clove oil at 5±1ºC, for 12 days.
Treatments
Days
T1 T2 T3
0 7.26±0.09dA 7.24±0.12dA 7.26±0.11dA
1 7.28±0.12dA 7.25±0.08dA 7.27±0.05dA
3 7.57±0.19 dA 7.35±0.16 dA 7.30±0.11dA
5 8.20±0.05 cA 7.92±0.09 cB 7.87±0.08cB
7 8.83±0.21bA 8.22±0.11bB 8.02±0.07cB
9 9.08±0.04bA 8.86±0.23aAB 8.56±0.13bB
12 9.30±0.11aA 9.06±0.06aB 8.94±0.03aC
Means of 3 repetitions ± standard deviation, followed by the same lowercase letter in the column and uppercase in the
line do not differ from each other by the Tukey test (p<0.05). (T1) control (uncoated strawberries); (T2) 2% chitosan + 1%
clove essential oil; (T3) 2% chitosan + 1.5% clove essential oil.
Sugar and organic acids compose much of the total soluble solids in strawberries [25]. In respect to the
total soluble solids (TSS), there were no significant statistical differences between the treatments after three
days of storage at 5±1ºC (Table 3).
During 12 days of storage, the TSS of strawberry fruit increased in 2.04, 1.82 and 1.68, for the treatments
T1, T2 and T3 respectively, which seems to be due to break down of organic matters to sugars and their
involvement in the respiration cycle [29,30]. Once, strawberry's initial metabolic process converts
carbohydrates to sugars and others soluble compounds, which increases total soluble solids [31]. However, it
is possible verify that the coated strawberries had the lowest TSS values.
The color attributes of the fruit tissue are generally expressed in terms of L* (Luminosity), a* (green–red
chromaticity), and b* (yellow–blue chromaticity) and are shown in the Table 3 for strawberries minimally
processed.
Table 3. Color obtained of strawberries coated with chitosan and different concentrations of clove oil at 5±1ºC, for 12
days.
Treatments
Parameter analyzed Days
T1 T2 T3
0 33.84 ± 0.13aA 34.07 ± 0.09aA 34.04 ± 0.11aA
1 33.80 ± 0.07aB 34.05 ± 0.03aA 34.01 ± 0.09aA
3 32.30 ± 0.24 bB 33.68 ± 0.25bA 33.91 ± 0.17aA
L* 5 30.94 ± 0.11 cA 33.11 ± 0.03cA 33.16 ± 0.09bA
7 30.22 ± 0.34 dB 32.73 ± 0.19dA 32.78 ± 0.14cA
9 29.89 ± 0.27 dB 32.14 ± 0.29eA 32.44 ± 0.04dA
12 29.63 ± 0.21dB 31.06 ± 0.05fA 31.54 ± 0.09eA
0 41.83 ± 0.12aA 41.89 ± 0.09aA 41.87 ± 0.14aA
1 41.80 ± 0.06aA 41.85 ± 0.03aA 41.85 ± 0.09aA
3 41.51 ± 0.13bA 41.69 ± 0.11aA 41.81 ± 0.11aA
Chroma a* 5 41.02 ± 0.09 cB 41.31 ± 0.34aAB 41.80 ± 0.19aA
7 40.73 ± 0.16 dC 40.94 ± 0.02bB 41.71 ± 0.08aA
9 40.14 ± 0.28 eB 40.89 ± 0.07bA 41.23 ± 0.42abA
12 39.87 ± 0.21 eB 40.68 ± 0.17bA 40.77 ± 0.09bA
0 30.88 ± 0.26 aA 30.89 ± 0.14aA 30.91 ± 0.08aA
1 30.80 ± 0.21 aA 30.80 ± 0.12aA 30.89 ± 0.17aA
3 29.18 ± 0.12bC 30.29 ± 0.24bB 30.77 ± 0.09aA
Chroma b* 5 28.84 ± 0.09cB 30.01 ± 0.06bA 30.13 ± 0.31bA
7 28.18 ± 0.22dB 29.63 ± 0.16cA 29.91 ± 0.11bA
9 27.37 ± 0.06 eC 29.09 ± 0.05dB 29.73 ± 0.09bA
12 26.11 ± 0.19 fC 28.45 ± 0.09eB 29.22 ± 0.15cA
Means of 3 repetitions ± standard deviation, followed by the same lowercase letter in the column and uppercase in the
line do not differ from each other by the Tukey test (p<0.05). (T1) control (uncoated strawberries); (T2) 2% chitosan + 1%
clove essential oil; (T3) 2% chitosan + 1.5% clove essential oil.
The L* parameter is an indicator of fruit darkening. As can be observed in Table 3, all the strawberries
showed decreasing L* values with storage time. Analyzing parameter L*, from the fifth day, all treatments
showed a significant difference between them. All days, treated strawberries were also significantly brighter
than control samples after of the 12 days (29.63±0.21 vs 31.6±0.05 and 31.54±0.09, respectively), which might
be considered a positive result, considering that decrease in brightness is usually due to the formation of dark
tissues and brown spots, that may be related to inappropriate storage conditions as well as yeast infection [9].
Basaglia and coauthors [32] also found a decrease in L* values when they studied chitosan and cinnamon
essential oil coatings in minimally processed pineapples.
a* values of all samples slightly decreased over time, showing the tendency to have a less saturated red
color. However, significant reductions were detected after 5 days for control and after 7 days for treated fruit.
Clove oil had a significant effect on red color of strawberries all days of storage, showing more saturated color
than control samples.
Explanations for this result may be found in the among red color is relation of a anthocyanin on present in
the strawberry fruit. Anthocyanins are considered the compounds responsible for red color of strawberries [33],
and discoloration of the fruit may be due to anthocyanin degradation by the action of hydrolytic enzymes that,
by breaking down the linkage of the glycosidic substituent in the moieties, lead to loss of color during
postharvest storage [34,9].
Measurements of b* values, representing the chromaticity of blue (negative value) and yellow (positive
value) colors, showed a significant (p≤0.05) reduction b* values for treated strawberries which ranged from the
initial and final value of 30.88±0.26 to 26.11±0.19 vs 30.89±0.14 to 28.45±0.09 and 30.91±0.08 to 29.22±0.15
for control and treated strawberries, respectively, at the days of storage time. Indeed significant differences
were detected from the beginning to the end of storage time, although fruit developed a significantly less vivid
coloration, as evidenced by lower values of Chroma after five days in the treated strawberries.
Coating treatments showed a better firmness than control treatment during storage time (Table 4).
Table 4. Firmness (N) of strawberries coated with chitosan and different concentration of clove essential oil at 5±1ºC, for
12 days.
Treatments
Days
T1 T2 T3
0 5.23 ± 0.19aA 5.31 ± 0.12aA 5.33 ± 0.09aA
1 5.20 ± 0.11aA 5.30 ± 0.11aA 5.33 ± 0.11aA
3 5.03 ± 0.36 abA 5.27 ± 0.09 aA 5.29 ± 0.09aA
5 4.89 ± 0.12 abA 5.24 ± 0.20 aA 5.21 ± 0.23aA
7 4.47 ± 0.26 bB 5.04 ± 0.09 abA 5.12 ± 0.06aA
9 4.11 ± 0.12 bcB 4.96 ± 0.07 bA 5.01 ± 0.17aA
12 3.87 ± 0.09cB 4.81 ± 0.21bA 4.97 ± 0.13aA
Means of 3 repetitions ± standard deviation, followed by the same lowercase letter in the column and uppercase in the
line do not differ from each other by the Tukey test (p<0.05). (T1) control (uncoated strawberries); (T2) 2% chitosan + 1%
clove essential oil; (T3) 2% chitosan + 1.5% clove essential oil.
After 12 days, strawberries coated with chitosan 2% + clove essential oil 1% and with chitosan 2% + clove
essential oil 1.5% showed the greatest firmness with values of 4.81 and 4.97 N respectively; while the control
treatment recorded a firmness of 3.87 N at the end of the storage time.
The loss of firmness on strawberries is associated to degradation of the middle lamella of the cell wall by
depolymerization of the xyloglucan-cellulose matrix and the solubilization of pectins [35,11]. In this study show
that strawberries coated with chitosan 2% and clove essential oil 1.5% showed the best firmness; similar results
were reported by Martínez-González and coauthors [24], which recorded a best firmness for strawberries
coated with nanostructured chitosan-propolis than non-coated strawberries.
Chu and coauthors [28] also showed similar results in the pullulan-coated strawberry incorporated with
cinnamon essential oil, decreasing about 12% of its initial firmness at the end of storage compared to uncoated
strawberries that had a greater loss of firmness. The coating with essential oil help to maintain the firmness by
antioxidant activity and anti-microorganism activity [36].
The presence of Escherichia coli (<102 CFU g-1) and Salmonella sp. (absence in 25 g) was not detected
in minimally processed strawberry samples, confirming the efficiency of good manufacturing practices and the
action of organic chlorine in the disinfection of the samples.
Microorganisms rapidly colonized when the storage went beyond 5 days, especially in control strawberries.
According to Fan and coauthors [37], coatings applied on fresh fruit produce an internal modified micro-
atmosphere that generally affects the growth rate of spoilage and pathogenic microorganisms.
Figure 1 (a) shows statistical difference the growth of psychrotrophic organisms during the 12 days storage
period.
a b
Figure 1. Psychrotrophic (a) and mold and yeast (b) found in strawberries coated with chitosan and different proportions
of clove essential oil at 5±1 ºC, for 12 days. (T1) control (uncoated strawberries); (T2) 2% chitosan + 1% clove essential
oil; (T3) 2% chitosan + 1.5% clove essential oil.
As observed, there was an accentuated growth of these microorganisms with the passing of the storage
days, and the control treatment (T1) presented a higher growth (5.93±0.34 log/CFU/g), differing statistically
from the others at the end of the experiment. The T2 and T3 treatments did not differ from each other during
the evaluated period (4.51 and 4.16 log/CFU/g respectively).
As much as there are no standards in psychrotrophic bacteria in Brazil, it has been suggested that foods
with populations above 105 and 106 CFU/g [38]. In this study, only the control treatment was above these limits
from the 12th day of storage, demonstrating that the use of coatings was efficient to reduce the growth of
psychrotrophic microorganisms. Chevalier and coauthors [5] observed similar results, when they worked with
coatings based on protein isolate of tilapia applied on minimally processed melons, and the control treatment
also showed values above 105 from the 9th.
Clove has a high amount of eugenol (45-90%) and is a potent antimicrobial agent, capable of degenerating
proteins. The action mechanism of the essential oil on microorganisms is complex and has not yet been fully
elucidated. Numerous reports have been made about the antimicrobial action mechanisms of oils, which cause
lysis and loss of membrane integrity, due o the output of ions and inhibition of cellular respiration [39].
As a typical lipophilic compound, essential oils cross the cell wall and the cytoplasmic membrane, the
cytotoxic activity seems to be linked to the disruption of the structures of the different layers of polysaccharides,
fatty acids and phospholipids, due to its mechanism of action that reaches several targets to the same time
[40].
Figure 1 (b) shows statistical difference the growth of molds and yeasts in strawberries minimally
processed during 12 days storage period. The growth of mold and yeast increased over the storage days for
all evaluated treatments, with the control treatment (T1) presenting the highest growth (6.19 log CFU g-1). The
growth rate of molds was inhibited in the samples treated with chitosan (2%) and clove essential oil (1 and
1.5%), treatment T2 and T3 (5.14 and 3.85 log CFU g-1) respectively.
Many studies have mentioned the antibacterial and antifungal properties of chitosan. Pizato and coauthors
[41], for example, demonstrated the low microbial growth in minimally processed broccoli for up to 12 days in
refrigerated storage when 1.5% chitosan coating was used. Tokatli and Demirdöven [42] demonstrated the
efficiency of chitosan when working with sweet cherry (Prunus avium L.) coated with edible chitosan films. This
coating inhibited the growth of yeasts and molds for 25 days in refrigerated storage at 4°C. The antimicrobial
action of chitosan is associated with the formation of a polyelectrolyte composition, since its protonated amine
groups selectively bind to the cell surface of microorganisms, negatively charged, consequently causing the
loss of intracellular components and inhibition of microbial growth [43].
Perdones and coauthors [26] worked with chitosan along with lemon essential oil; they obtained a positive
effect in reducing the fungal activity in strawberries stored at 5ºC. Also, Hernández-Carrillo and coauthors [44]
demonstrated that the combination of pectin with lemon essential oil and reuterine was efficient in reducing the
growth of molds and yeasts in strawberries stored under refrigeration, demonstrating a great potential for
preserving the fungal deterioration of these fruits. These results agree with the present study, which also found
a lower growth of molds and yeasts when coatings based on chitosan and clove essential oil were used, which
proves the efficiency of both chitosan and the oil used as an inhibitor of microorganisms.
Figure 2 shows the appearance of strawberries over the days of storage. As we can see, the strawberries
developed a darker color (redder) with the passing of the days of storage; besides, the T1 and T2 treatments
showed apparent mold growth (days 9 and 12). The use of chitosan (2%) and clove oil (1.5%) were efficient to
maintain the quality of the strawberry for longer time. These observations can be confirmed when we evaluate
Figure 1 (b), in which there was greater growth of mold and yeast for these treatments.
T1 T2 T3
Day 0
Day 1
Day 3
Day 5
Day 7
Day 9
Day 12
Figure 2. Appearance of strawberries without and with chitosan coatings containing different concentrations of clove
essential oil over the days of storage.
CONCLUSION
The treatments T2 (2% chitosan and 1% CEO) and T3 (2% chitosan and 1.5% CEO) were the most
efficient treatments in the conservation of minimally processed strawberries, when compared to the control
sample.
The use of chitosan and clove essential oil exhibited a delay in the growth of molds and yeasts, a lower
weight loss and firmness. The T2 and T3 treatments proved to have great potential to be applied as coatings
as they reduced mass loss by approximately 86%, maintained physical and chemical characteristics, and
reduced microbiological changes, especially in mold and yeast growth.
The use of clove oil in its highest concentration (1.5%), kept the minimally processed strawberries safe for
consumption for a longer time when stored at 5±1ºC.
For future trends in relation to this work, clove oil encapsulated in different coatings may be used.
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