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A Green Technology for Control of Avocado Necrotic Fungi Using Bioactive
Coatings
Article in International Journal of Green Energy · January 2018
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International Journal of Green Technology, 2018, 4, 24-28
24
A Green Technology for Control of Avocado Necrotic Fungi Using
Bioactive Coatings
Diana Nieto-Oropeza, Jorge Aguirre-Joya, Raúl Rodríguez Herrera, Leonardo SepúlvedaTorre and Cristóbal Noé Aguilar*
Food Research Department, School of Chemistry, Universidad Autónoma de Coahuila, Saltillo 25280
Coahuila, México
Abstract: Bioactive films and coatings have been used as carriers for food additives such as antioxidant or antimicrobial
agents. Larrea tridentata polyphenols and Aloe vera gel have demonstrated to act as efficient antimicrobial agents on
food systems and can be incorporated into a coating matrix. In this research, bioactive coatings were prepared on a
candelilla/pectin-based formulation. Bioactive coating antifungal activity against necrotic pathogenic fungi (Botritis
cinerea and Fusarium oxysporum) was investigated by determining fungal radial growth, and the best in vitro fungicide
concentration (MIC50) of Larrea tridentata polyphenols was determined. The highest fungal growth control was observed
on B. cinerea (88%). Bioactive coatings presented MIC50 of 558 and 612 mg/L for F. oxysporum and B. cinerea,
respectively. Candelilla wax-pectin-Aloe vera bioactive coatings incorporated with L. tridentata polyphenols inhibited the
growth of necrotic pathogenic fungi and can be useful for preservation of avocado and increase of its shelf life. After all,
avocado (Persea americana Mill.) is a susceptible fruit because of extreme environment conditions and invasion of
microorganisms which may induce a negative reaction on quality. Technology of bioactive coatings is an alternative for
extend fruit shelf-life and keeping organoleptic characteristics of Avocado cv. Hass.
Keywords: Avocado, bioactive coating, shelf-life quality, necrotic fungi.
1. INTRODUCTION
Persea Americana Mill. (Avocado) is a food with
origin in Central America and South of Mexico [1]. This
fruit has high nutritional value being its principal
compounds: ascorbic acid, carotenoids, phenols,
palmitic, oleic, and linoleic acids, A, B, C, and E
vitamins, and fiber [2, 3]. The most common and
commercial cultivar is Hass, a cross between
Guatemalan and Mexican races [4]. The principal
quality parameter for avocado commercialization is
maturity, because fruit has large harvest periods and
only maturates when it is cut from the tree [2].
Worldwide, the principal producer of avocado cv.
Hass is Mexico [5] with 30% of global production, which
is mainly sold to USA, France, Japan and Canada. In
2017, Mexico produced 1,997,629 tons of avocado [6].
Nevertheless, its commercialization and global
distribution stills facing some problems such as: fruit
short shelf life, mediated mainly by two factors; a
natural
accelerated
respiration
task
and
phytopathogenic fungi spoilage [7]. Average time of
avocado shelf life at room temperature is between 5 to
7 days and 2 to 4 weeks under optimum conditions of
temperature and relative humidity [5].
Some methods for disease control are: fungicides, cold
treatment and careful handling to avoid fruit
*
Address correspondence to this author at the Food Research Department,
School of Chemistry, Universidad Autónoma de Coahuila, Saltillo 25280
Coahuila, México; Tel: +52844 4155752, 45161238; Fax: *52 844 4159534;
E-mail: cristobal.aguilar@uadec.edu.mx
damage [8]. However, Botrytis cinerea and Fusarium
oxysporum are common necrotic pathogens found
under field conditions and can damage to agricultural
commodities, decreasing crop quality and production
[9]. As it was described previously [10], the main factor
responsible for crop losses around the world is
microbial deterioration.
A green alternative to control avocado spoilage is
using natural based coatings added with antimicrobial
compounds [11]. Natural coatings are an easy and
environmental friendly technology, in this case, coating
cover the food with fine films (0.3 mm or less) which
can regulating vapor and carbon dioxide permeability,
reducing food quality losses and extending shelf life
[12, 13]. Natural bioactive coatings represent a primary
packaging that helps to maintain moisture and firmness
[14] and are designed to intentionally interact with food
and/or the surrounding environment [15].
Some reports [16] indicated that most used
biopolymers to manufacture bioactive coatings and
films are proteins, lipids and saccharides. Coatings are
applied over the food in a liquid form, then it is dried on
the food surface to make a solid and continuous barrier
[17]. With the aim to improve, final characteristics of
packaging, such as moisture retention and gas
migration control, most authors have focused on
composite or multicomponent coatings [18]. One of the
most green promising technologies for traditional
packaging are the bioactive coatings, where active
compounds are added to improve characteristics of
E-ISSN-2414-2077 © 2018 International Journal of Green Technology
A Green Technology for Control of Avocado Necrotic Fungi Using Bioactive Coatings
packaging such as polyphenols to reduce oxidation in
food and/or to control spoilage as antimicrobial agents
[19].
In the present study, candelilla (Euphorbia
antisiphilityca Zucc.) wax (CAS N° 8006-44-8) was
used as lipid source, since it has been reported as an
efficient ingredient for edible and bioactive coatings,
specially to prolong quality and shelf life of fruits and
vegetables [20-22]. Candelilla wax is an edible
nonrestrictive ingredient approved by FDA, widely used
in the food and candy industries. Pectin was employed
as biopolymer for coating since it was recognized as
GRAS (CAS N° 9000-69-5), and has capability of
stabilizing all ingredients and is used by food
industries, without limits other than current good
manufacturing practices [5], with the intention to make
a handle film glycerol was added as plasticizer, due to
its compatibility, stability and food grade (CAS N° 5682-5). Aloe mucilage (Aloe vera) was added according
to previous results of our research group, to improve
characteristics of coating, also Larrea tridentata purified
polyphenols where added as antioxidant/antifungal
component [7]. Based on these antecedents, the main
objective of present work was to evaluate the control of
necrotic fungi of avocado, by applying a green
technology of bioactive coatings made with pectin,
glycerol, Aloe mucilage and candelilla wax
functionalized
with
Larrea
tridentata
purified
polyphenols.
2. MATERIALS AND METHODS
2.1. Reagents and Biological Material
Isolates of Fusarium oxysporum and Botrytis
cinerea where kindly provided by the Food Research
Department at Universidad Autónoma de Coahuila,
Saltillo, Coahuila, Mexico. Avocados (Persea
americana Mill.) cv. Hass, and leaves of Aloe vera
were acquired in a super market at Saltillo, Coahuila,
Mexico. National Forest Commission (CONAFOR) from
Mexico provided leaves of creosote bush (Larrea
tridentata L.) and candelilla wax (Euphorbia
antisyphilitica Zucc.) from communities with legal
permission to exploit non-timber natural resources.
Pectin from citric (at least 65 % galacturonic acids) and
glycerin were obtained from Faga Lab (Favela Pro, SA
de CV, Cerro Agudo, Sinaloa, Mexico). PDA was
purchased from Sigma-Aldrich (Sigma-Aldrich Inc.,
Germany). All other chemicals where from analytical
grade.
International Journal of Green Technology, 2018, Vol. 4,
25
2.2. Extraction of Polyphenols from Creosote Bush
The leaves of creosote bush were dehydrated using
an oven at 60°C by 24 hours and pulverized in a mill;
the obtained powder (12.5 g) of creosote bush leaves
was placed in distilled water (500 ml) at 70°C during 30
min with constant agitation. The obtained extract was
decanted and filtered using a filter paper. The filtered
®
solution was passed through Amberlite-XAD16 with
the conditions described for polyphenols recovery [23]
to obtain a powder rich in total polyphenols from L.
tridentata (PTP).
2.3. Determination in vitro of Fungicide Activity
The poisoned medium method was used for
determinate the fungicide activity. The PDA medium
was sterilized. After that, PTP was added before agar
solidified. The tested PTP concentrations were 250,
500, 1000 and 1250 ppm, and control was a Petri dish
with PDA without polyphenols and each fungal strain.
Botrytis cinerea and Fusarium oxysporum were
activated on Petri plates with PDA. An explant of 5 mm
diameter with necrotic fungal was placed on poisoned
medium with polyphenols. Radial growth was
measured with a Vernier in a kinetical way until the
control plate was completely invaded by the fungal
strain.
2.4. Bioactive Coating Manufacturing
Pectin (1.1% w/v) was dispersed in distilled water at
80°C with constant agitation. Candelilla wax (0.16%
w/v) and glycerol (0.3% v/v) were added when pectin
was completely dispersed at room temperature.
Thereafter, Aloe vera mucilage (5 % w/v) was
incorporated.
TPT
were
added
in
different
concentrations to obtain treatments: C1 (320 ppm), C2
(620 ppm) and C3 (920 ppm), CS represents control
avocado without coating and CC represents a control
coating without bioactive from L. tridentata. Final
dispersion was homogenate at 1000 rpm/15 min [7].
2.5. Application of the Bioactive Coating
Agar PDA medium was used to reactive fungal
strains, then, spores were recovered with Tween 80.
The number of spores was counted using a
7
Neubauer´s chamber and was adjusted to 1 x 10
spores/mL. Avocados were washed with hypochlorite
solution at 5 % and dried at room temperature, after
that were inoculated with 20 µL droplet of each fungus
7
(Botrytis cinerea and Fusarium oxysporum) at 1 x 10
spores/mL for each one and were let at room
26
International Journal of Green Technology, 2018, Vol. 4
Diana et al.
Table 1: In Vitro Growth Rate for Minimum Inhibitory Concentration
Microorganism
Growth Rate Control (mm/h)
Growth Rate 1000 ppm (mm/h)
Percent Delay (%)
MIC 50 (ppm)
Botrytis cinerea
0.88
0.24
88
612
Fusarium oxysporum
0.4
0.2
50
558
Concentrations of L. tridentata polyphenols used
during the in vivo assay and added to the coatings
were selected according the results obtained from in
vitro assay, in this case, 320, 620 and 920 ppm were
used (C1, C2 and C3, respectively). Results showed
that fungal (B. cinerea and F. oxysoprum) invasion into
avocado mesocarp diminished in comparison to control
100
80
60
40
C
3
C
2
C
1
C
0
C
20
S
With the methodology used for polyphenols
extraction from Larrea tridentata leaves was possible to
achieve 2.98 % of recuperation yield (dried weight),
while some studies reported to being achieved 6.8 % of
recuperation yield [24], differences may be due to plant
physiological state and genotype, soil type,
geographical region and season of tissue recollection.
Determination of fungicide activity in vitro (MIC50) was
evaluated by measuring B. cinerea and F. oxysoprum
growth on poisoned medium with polyphenols of L.
tridentata (Table 1). In the present study, MIC50
concentrations are lower than those reported by [25],
where those authors used 1000-4000 ppm of Larrea
tridentata leaf resin to inhibit 96-99 % of B. cinerea and
42-63 % of F. oxysporum. However, using polyphenols
powder it was not possible to obtain 100% of F.
oxysoprum inhibition. It should be mentioned that, there
are reports where aqueous extracts of L. tridentata
were used and an 40 % of Botrytis sp. inhibition was
observed [26] while 60-90 % of F. oxysoprum inhibition
was determined [27]. Extracts of L. tridentata also has
been used to treat food borne pathogens, in
concentrations from 1000 to 5000 ppm, the best control
for Staphylococcus aureus was observed using 5000
ppm [28].
C
3. RESULTS AND DISCUSSION
(CC) (Figure 1). Treatment C3 (920 ppm) displayed the
lowest fungal invasion, for B. cinerea was observed
15.5 % ± 3.31 and for F. oxysoprum 21.9 % ± 9.99,
comparing with control without and with coating. Other
authors [29] evaluated the efficacy of L. tridentata
extracts against gram negative bacteria with promising
results against Staphylococcus aureus. The present
study demonstrated that it is possible prevent
microorganism infection (B. cinerea and F. oxysoprum)
in avocado by using bioactive coating. Similar results
has been mentioned [30], who consigned that fungal
invasion (1.2 %) by C. gloesporioides with coatings of
candelilla wax and ellagic acid (reagent).
Fungal invasion (%)
temperature (25 °C) for 12 hours. After this time,
avocados were two times immersed completely for 3 s
in the emulsion for each treatment, C1 (320 ppm), C2
(620 ppm), C3 (920 ppm), CC represents a control
coating without bioactive from L. tridentate and CS
represents control avocado without coating. Avocados
were storage at 25°C and 60 % RH [7].
Treatments
Figure 1: Fungal invasion of
Botrytis cinerea and
Fusarium oxysporum. Treatments C1 (320 ppm), C2 (620
ppm) and C3 (920 ppm), CS represents control avocado
without coating and CC represents a control coating without
bioactive from L. tridentate, the line of each bar represents its
standard deviation.
CONCLUSION
Bioactive coating presented antifungal activity
against the two fungal strain. The best treatment to
control fungi invasion was C3 (920 ppm of polyphenols)
with 15.5 and 21.9 % of necrotic invasion in
comparison to controls. These results showed that
bioactive coating functionalized with Larrea tridentata
purified polyphenols had an important antifungal
potential, which might be utilized in the food industry.
A Green Technology for Control of Avocado Necrotic Fungi Using Bioactive Coatings
The bioactive coatings could be an excellent alternative
to preserve avocado for attack of B. cinerea and F.
oxysoprum and extend fruit shelf life.
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ACKNOWLEDGMENTS
The authors acknowledge the financial support
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Accepted on 26-03-2018
Published on 10-05-2018
© 2018 Diana et al.; International Journal of Green Technology.
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