Journal of Agriculture and Food Research 7 (2022) 100260

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Journal of Agriculture and Food Research

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Effect of biopolymer coatings made of zein nanoparticles and ε-polylysine

as postharvest treatments on the shelf-life of avocados (Persea americana
Mill. Cv. Hass)
Franciela Garcia a, Wei-Jen Lin b, Valerie Mellano c, Gabriel Davidov-Pardo a, *
a
Nutrition and Food Science Department, California State Polytechnic University Pomona, Pomona, 3801 West Temple Ave, Pomona, CA, 91768, USA
b
Biological Sciences Department, California State Polytechnic University Pomona, Pomona, 3801 West Temple Ave, Pomona, CA, 91768, USA
c
Plant Science Department, California State Polytechnic University Pomona, Pomona, 3801 West Temple Ave, Pomona, CA, 91768, USA

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

Keywords: Avocados (Persea americana) are a popular fruit produced and consumed all around the world. Due to their
Edible coatings climacteric nature, they are predisposed to rapid loss of moisture and firmness after harvest and prior to sale. The
Colletotrichum spp. prevalence of the fungal phytopathogen Colletotrichum spp. Exacerbates the rate of decay by causing anthracnose,
Active packaging
resulting in relatively short shelf-lives, increased waste, and economic losses. The objective of this study was to
Antimicrobial
Nanotechnology
assess the effect of coatings made of zein nanoparticles (ZNP), zein and ε-polylysine nanoparticles (ZPL), zein
solution (ZS), and ε-polylysine solution (PL) on the weight loss, respiration rate, firmness, color, and fungal decay
of avocados compared to a control (C). By day 15 in ambient storage, control avocados had scarred surfaces,
significant color change, overripe pulp, and clear signs of fungal decay. At this time, avocados treated with ZPL
lost the least weight, 20.14%, compared to the control, 28.05%. The lowest respiration rates were seen in ZPL
treatment with values of 107.83 mg of CO2/kg/h. The flesh of the ZPL- and ZNP- treated fruit remained firmer,
5.01 and 4.07 N, respectively, compared to the control, 3.67 N. For pulp color, ZPL-treated fruit showed the
smallest color difference as measured by total color change, ΔE, 3.67 compared to the control, 15.45. At the
conclusion of a 15-day Colletotrichum spp. inoculation study, ZPL treated avocados had the significantly lowest
fungal severity score of 3.33, while ZNP-treated fruit and the control had scores of 4 and 4.7, respectively. The
results of this project support biopolymeric coatings as a novel technique with the potential to extend shelf-life
and reduce avocado waste both at retail and household levels to possibly mitigate economic losses.

1. Introduction cardiovascular disease, diabetes and high cholesterol [5–8]. Neverthe­

less, avocados have relatively short shelf-lives, which is partially a result
Avocados (Persea americana) are tropical, climacteric fruits with from their physiological climacteric nature. As climacteric fruits,
high production demand across the globe. In 2018, 6.4 million metric avocados undergo a climacteric peak after harvest and before ripening
tons were produced around the world, equaling 12.9 Billion US Dollars, when cellular respiration, ethylene gas production and enzyme activity
a 23% increase from the previous year [1]. Hass avocados are the most of pectinases and polygalacturonases are comparably high [9–11]. This
consumed and commercially cultivated avocados [2]. Avocados are a climacteric peak results in softening of the fruit, an increase in respira­
unique fruit due to their high lipophilic fraction, about 15%, that is not tion rate, and water migration from the fruit to its environment. As all of
commonly seen in other fruits [3]. From that percent, about 60–70% is these metabolic activities reach their peak simultaneously, avocados
comprised of monounsaturated fatty acids (MUFAs), with the remaining ripen and spoil rapidly. The short shelf-life of this fruit is further exac­
percent pertaining to polyunsaturated and saturated fatty acids [4]. The erbated by the prominent presence of the fungal phytopathogen Colle­
consumption of foods with significant levels of MUFAs has been totrichum spp. That causes anthracnose. This disease is characterized by
consistently linked to the reduction of health problems like black spot lesions on the skins of the fruit that will eventually penetrate

Abbreviations: ZNP, Zein nanoparticles; ZPL, Zein and ε-polylysine nanoparticles; ZS, Zein solution; PL, ε-polylysine solution; C, Control.
* Corresponding author.
E-mail address: gdavidov@cpp.edu (G. Davidov-Pardo).

https://doi.org/10.1016/j.jafr.2021.100260
Received 14 August 2021; Received in revised form 30 November 2021; Accepted 18 December 2021
Available online 21 December 2021
2666-1543/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
F. Garcia et al. Journal of Agriculture and Food Research 7 (2022) 100260

Fig. 1. Mycelia of Colletotrichum spp. (A) front, (B) back, (C) Colletotrichum spp. conidia at 50×, (D) Colletotrichum spp. mycelia at 50×.

into the pulp, speeding up natural ripening [12]. Anthracnose disease is the shelf-life of Hass avocados (Persea americana Mill. Cv. Hass). The
the most severe postharvest disease, ultimately having adverse conse­ effects on the shelf-life were quantified by assessing weight loss, respi­
quences on quality and marketability [13]. ration rate, firmness, color change, and fungal decay.
Refrigeration is the most commonly used method to slow down both
ripening behavior and fungal decay [14]. Despite its advantages, 2. Materials and methods
refrigeration has limited effectiveness, as avocados are no longer
refrigerated at retail locations and households. Furthermore, industrial 2.1. Materials
solutions to these challenges include the use of fungicides like Pro­
chloraz, Fludioxonil, and Copper Hydroxide [15,16]. Although the use Recently harvested Hass avocados (Persea americana Mill. Cv. Hass)
of fungicides has helped control fungal incidence and allowed the in­ of uniform size (less than 24 h from harvest) were kindly donated by
dustry to move forward, consumer concerns regarding the long-term Henry Avocado Corporation (Escondido, Ca, USA) for the shelf-life study
safety of these chemicals has pioneered the need for novel and safe al­ and by Mellano and Co. Farm (San Luis Rey, Ca, USA) for the fungal
ternatives to reduce avocado fungal prevalence and extend fruit inoculation study. Zein was kindly donated by Kobayashi Perfumery Co.
longevity [17]. Ltd (Tokyo, Japan). ε-PL was purchased from Pro Food International Inc.
Biopolymeric coatings and edible films have been utilized to extend (Naperville, IL, USA). All other reagents were of analytical grade and
the shelf-life of other fruits and vegetables. Biopolymeric coatings and were purchased from VWR International (Radnor, PA, USA).
films are made from biological ingredients such as proteins and poly­
saccharides that usually create a physical barrier, keeping internal car­ 2.2. Methods
bon dioxide inside while keeping external oxygen outside. Coatings
made using emulsions, waxes or prolamines are hydrophobic in nature 2.2.1. Colletotrichum spp. Isolation and culturing
that can also reduce the loss of moisture from the fruit to the environ­ The procedure to isolate and culture Colletotrichum spp. was based on
ment [18,19]. Active components can be incorporated into coatings and the work of Kimaru et al. [32] with some modifications. Hass avocados
films to offer additional advantageous properties, such as antioxidant that were significantly ripe and showing anthracnose signs were chosen
and antimicrobial activities, besides the physical safeguard. In the past, for fungal isolation. Avocados were disinfected with 1% sodium hypo­
avocados have been coated with chitosan, candelilla wax, pectin, car­ chlorite solution for 30 s, then rinsed with DI water and allowed to air
boxymethyl cellulose with and without essential oils and polyphenols dry. Pieces of 3 mm2 of the avocado skins with lesions were aseptically
[20–22]. Coated avocados resulted in a reduction of fungal prevalence cut and placed on 9 cm potato dextrose agar (PDA) petri dishes, three
and reductions in respiration rate, softening, color change and weight pieces per plate. Tetracycline at a concentration of 10 μg/ml was added
loss. during the molten state of the PDA to inhibit bacterial growth on the
Zein, a hydrophobic corn prolamine, has gained significant attention media. The PDA plates were incubated for 2–3 days at room temperature
as it has repeatedly proved its ability to hinder gas and water transfer (25 ◦ C). The growing fungi were further sub-cultured three times into
when applied to a multitude of produce. Guavas coated with zein and individual PDA plates with 10 μg/ml tetracycline to obtain pure cul­
tannic acid had lower respiration rates, less loss of firmness, and reduced tures. The pure culture plates were incubated at room temperature
ethylene production [23]. Similarly, apples coated with zein and pro­ (25 ◦ C) for 10 days. After 10 days, the fungi were characterized ac­
pylene glycol had decreased weight loss and respiration rates [24]. As a cording to macroscopic and microscopic morphological traits such as
result of its hydrophobic composition, zein is only soluble in 60–90% conidia shape, growth on PDA plates and growth rate using an optical
ethanol solutions [25]. This limits its application and use in food microscope at 50× (Olympus BX53 Olympus Corporation, Center Val­
products. An effective technique to mitigate this challenge is antisolvent ley, PA, USA).
precipitation. In this technique, a component known as the antisolvent is A careful morphological characterization of the isolated fungus
introduced to the solution, in this case water, causing zein to supersat­ revealed white, uniform and fuzzy growth on the PDA plate with a
urate, create nucleation sites and form nanoparticles via hydrophobic green-grey circle in the center (Fig. 1A and B). Furthermore, conidia
interactions [26]. Once nanoparticulated, zein could remain in suspen­ were cylindrical and rounded at both ends (Fig. 1C), and mycelia were
sion in water-based solvents. formed of thin, long septate hyphae with slight branching (Fig. 1D).
Furthermore, ε-polylysine (ε-PL) is a natural peptide that has shown These morphological characteristics have previously been recorded for
a wide range of antimicrobial functionality. Many studies have revealed Colletotrichum spp. [12,32,33], and therefore, the isolate was classified
its antibacterial ability against Staphylococcus aureus [27], Salmonella as presumptive Colletotrichum spp. with confidence.
spp. and Listeria monocytogenes [28], and Escherichia coli [29]. The
antifungal activity of ε-PL is limited but has provided successful results 2.2.2. Inhibitory concentration (IC) of ε-polylysine (ε-PL)
against Penicillium digitatum in citrus [30] and against Botrytis cinerea in In-vitro tests were performed in order to find the inhibitory con­
jujube fruit [31]. centration of ε-PL as described by Liu et al. [30] with some modifica­
The present study was carried out with the objective of assessing the tions. Increasing concentrations of ε-PL were dissolved in sterile water
effects of biopolymeric coatings made of zein nanoparticles and ε-PL on (0, 0.25, 0.5, 1, 1.25, 1.5, and 2 mg/ml) and were incorporated into the

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F. Garcia et al. Journal of Agriculture and Food Research 7 (2022) 100260

PDA media. Mycelial disks of 7 mm in diameter of 7-day old Colleto­ took place in ambient storage, 25 ◦ C at 44% RH, to simulate market and
trichum spp. cultures were placed in the center of PDA plates with the household storage conditions. At this stage 4 avocados per treatment
varying ε-PL concentrations and incubated at room temperature (25 ◦ C). were assessed every 3 days [17].
The diameter of mycelial growth was measured after 7 days, and the
antifungal ability of ε-PL was determined according to the growth of 2.2.4.1. wt. All avocados were weighed on day 0 of the shelf-life study
Colletotrichum spp. The percent of mycelial growth inhibition was using an analytical balance. Weight was further recorded on the day of
calculated using equation (1) [34], assessment and weight loss was calculated as a percentage loss from the
( ) initial original weights (Eq. (2)) [21].
dt
% ​ inhibition = 1 − ∗100, 1
dc (initial weight − final weight) x 100
Weight loss % = 2
initial weight
where, dt was the mycelial diameter of the treatment and dc was the
mycelial diameter of the control. 2.2.4.2. Firmness. The firmness was analyzed with a texture analyzer
(TA-XT2, Stable Micro Systems, Godalming, Surrey, UK). A 5 mm
2.2.3. Preparation of coating solutions stainless steel cylindrical probe applied pressure and punctured each
Four different coating solutions namely zein nanoparticle suspension avocado 15 mm at the horizontal equatorial region to assess the firmness
(ZNP), zein-ε-PL nanoparticle suspension (ZPL), zein solution (ZS), ε-PL [18]. Macro settings were adjusted to identify the peak force that was
solution (PL), and a control were made to assess their effect on the shelf- considered the firmness. Results were given in units of grams and then
life of Hass avocados. Zein nanoparticles (ZNP) were made using zein converted to Newtons.
dissolved in 85% (v/v) ethanol solution at 30 g/L as the solvent phase
and adjusted to pH 3. The solvent phase was then mixed into the anti-
2.2.4.3. Respiration rate. Individual avocados were placed in airtight 2-
solvent phase, water at pH 3, at a 1:5 ratio at 460 rpm for 2 min. The
Liter plastic jars for 5 min immediately after being weighted. A CO2 gas
final zein concentration in the system was 5 g/L and the final ethanol
sensor (Vernier Software and Technology, Beaverton, Oregon, USA) was
concentration was 14% (v/v) [35]. Zein-ε-PL nanoparticles (ZPL) were
used; a probe passed through the lid of the sealed jar containing the
formulated following the same procedure as ZNP apart from the anti­
avocado. The respiration rate was given in ppm, and values were then
solvent phase. To find the highest concentration of ε-PL that could be
translated to mg/kg/h to take into account the size of the fruit, size of
incorporated in the system, increasing concentrations of ε-PL (0–7
the CO2 chamber and the time required to obtain the readings [17].
mg/ml) were dissolved in the antisolvent phase before performing the
antisolvent precipitation. As shown in section 3.2 the highest concen­
2.2.4.4. Color. To assess color, a Hunter LAB colorimeter (CR-400
tration of ε-PL that the system supported was 5 mg/ml in the antisolvent
Chroma Meter, Konica Minolta, Chiyoda, Japan) was used. First, mea­
for a final concentrations of 4 mg/ml ε-PL, 5 g/L of zein, and 14% (v/v)
surements of the skin color were taken at four cardinal points; top,
ethanol.
bottom, right, and left. To measure the color of the pulp, avocados were
A zein solution (ZS) was made by dissolving 5 g/L of zein in 85% (v/
cut in half and the seed was removed. Each half was measured on the top
v) ethanol solution and adjusting to pH 3. A ε-PL solution was made by
and bottom of the pit and then sliced in the back to measure color behind
dissolving 4 mg/ml of ε-PL in 14% (v/v) ethanol solution and adjusting
the pit. This procedure was taken from Maftoonazad & Ramaswamy
to pH 3. Control solution (C) was a 14% (v/v) ethanol solution at pH 3 in
[18] with modifications. Equation (3) was used to determine overall
order to keep the ethanol concentration and pH constant throughout all
color change, ΔE, throughout the shelf life study [14]:
treatments.
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
( )2 ( )2
L* − L*i + (a* − a*i ) + b* − b*i 3
2
ΔE =
2.2.3.1. Characterization of nanoparticle suspension. Nanoparticle
diameter size and polydispersity index were measured through dynamic
where, L*, a*, and b* are the color coordinates at time t, and Li*, ai*, and
light scattering with a Zetasizer Nano ZS90 (Malvern Panalytical, Mal­
bi* are the initial color coordinates.
vern, UK). The zeta potential of the nanoparticles was determined
through particle electrophoresis using the Zetasizer Nano ZS90 (Malvern
2.2.5. Fungal inoculation study
Panalytical, Malvern, UK). To measure the electrophoretic mobility, the
A total of 30 avocados were disinfected with 1% sodium hypochlorite
Smoluchowsky model was used and 1.49 as the refractive index of zein.
solution for 30 s and then rinsed with DI water. Three 5 mm deep holes
Samples were diluted in water at pH 3, at a 1:50 ratio, to prevent back
were made per avocado using a 5 mm probe [37,38]. Through aseptic
light scattering [36]. Stability of the nanoparticles were assessed ac­
technique, 5 mm, 7-day-old mycelial disks were inserted into each hole.
cording to these characteristics on day of production, day 3 and day 7.
Inoculated avocados were stored at room temperature (25 ◦ C) for 24 h to
give the fungi time to colonize [39]. A total of 10 avocados per treatment
2.2.3.2. Avocado coating application. A total of 220 avocados were were coated in either zein-ε-PL nanoparticle suspension (ZPL), zein
coated for the shelf-life study. Each fruit was coated in one of the five nanoparticle suspension (ZNP), or control (C). ZPL and ZNP were chosen
coating solutions: zein and ε-PL nanoparticles (ZPL), zein nanoparticles to move forward with the inoculation study due to their significantly
(ZNP), ε-PL solution (PL), zein solution (ZS), or control (C). All avocados better performance in the shelf-life study. Avocados were coated two
were dipped and immediately taken out of the coating suspension and times, the same as in the shelf-life study [17]. Disease incidence was
then allowed to air dry for 1 h. This process was conducted twice on each measured every day for 15 days using equation (4) [40]:
avocado [17]. Fruits were separated according to treatment in separate
uncovered containers for the longevity of the shelf-life study. Disease incidence % =
number of infected wounds x 100
. 4
total number of inoculation sites
2.2.4. Shelf-life study Furthermore, to assess the severity of fungal deterioration and the
During the shelf-life study, avocados were assessed for weight loss, degree of fungal disease, the surface of each fruit was visually rated, and
firmness, respiration rate, and color. The study was six weeks long, infection was classified according to the following scores: Clean fruit
beginning with three weeks in refrigerated storage, 4 ◦ C and 79% RH, to (rating 0), 1–10% surface area (rating 1), 11–20% (rating 2), 21–40%
simulate postharvest storage conditions. In this stage of the shelf-life (rating 3), 41–70% (rating 4), 71–100% (rating 5) [41].
study, 4 avocados per treatment were assessed for the 5 aforemen­
tioned attributes every seven days [22]. The last three weeks of the study

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F. Garcia et al. Journal of Agriculture and Food Research 7 (2022) 100260

Fig. 2. Average Colletotrichum spp. colony diameter growth ±-SD from three replicates at different ε-polylysine concentrations on day 7. Different letters per
treatment are significantly different for each concentration (p < 0.05).

Fig. 3. Average particle diameters (n = 3) ± SD of zein-ε-polylysine nanoparticles with increasing ε-polylysine concentration at pH 3. Different letters are signif­
icantly different at p < 0.05.

2.2.6. Statistical analysis anionic fungal cell membranes, causing cytoplasmic leakage, and ulti­
SPSS software (IBM 23, Armonk, New York, USA) was used for all mately disrupting fungal growth and propagation [42]. Previous studies
data analysis. All parts of the experiments were performed in at least that have used ε-PL as an antifungal agent are in agreement with this
triplicates and results were indicated by mean values with standard interpretation. Liu et al. [30] used ε-PL to control the presence of Peni­
deviations. Analysis of variance (ANOVA) and Tukey post-hoc tests were cillium digitatum in citrus fruit and concluded that PL inhibited mycelial
performed to look for significant differences at a significance level of p growth via membrane rupture and inhibition of germ tube and hyphae
< 0.05. elongation. In addition, Luz et al. [43] successfully used ε-PL to hinder
the growth of Aspergillus parasiticus and Penicillium expansum in bread
3. Results and discussion and concluded that changing membrane permeability was its funda­
mental mode of action. The in vitro effect of other cationic antimicro­
3.1. Inhibitory concentration (IC) of ε-PL bials, such as chitosan, on the growth of Colletotrichum gloeosporioides
has been studied, finding the minimum inhibitory concentration to be
Fig. 2 shows the results from the inhibitory concentration (IC) study 20 mg/ml [44]. At lower concentrations the authors found similar
of ε-PL. As expected, an inverse correlation between ε-PL concentration inhibitory effects compared to this study.
in the PDA plates and the size of mycelial growth was found. Concen­
trations higher than 0.5 mg/ml ε-PL showed no significant difference in 3.2. Nanoparticle suspension characterization and stability
reduction of mycelial diameter growth. At this concentration, mycelial
diameter was reduced by 50% compared to the control at day 7. ε-PL Bare zein nanoparticles were made successfully with a size of 102.95
mechanism of action corresponds to its cationic composition targeting ± 8.88 nm, PDI score of 0.141 ± 0.028 and a zeta potential of 36.95 ±

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F. Garcia et al. Journal of Agriculture and Food Research 7 (2022) 100260

Fig. 4. Average weight loss percentages (n = 4) ± SD of all 5 coating treatments during (A) the entire shelf-life study (shaded area indicates refrigerated storage), (B)
refrigerated storage, and (C) ambient storage. Different letters are significantly different (p < 0.05) at day 15 of ambient storage.

5.24 mV. As seen in Fig. 3, when incorporating ε-PL into the antisolvent, repulsion to prevent particle aggregation [46]. In this case the zeta
6.5 mg/ml was the highest concentration that could be added before potential of the system at a concentration of 5 mg/ml ε-PL was 47.3 ±
flocculation and aggregation were observed. A 7-day storage stability 0.93 mV. It is speculated that at higher ε-PL concentrations (above 5
study determined that ε-PL concentrations above 5 mg/ml resulted in mg/ml), the system experienced depletion flocculation due to the higher
nanoparticle solutions with increased particle size, increased PDI, and concentration of dissolved ε-PL in the system, causing the aggregation of
lower zeta potential values (data not shown). Size distribution for the zein particles [47]. Due to their stability over short-term storage,
nanoparticle solution with 5 mg/ml ε-PL remained monomodal, with zein-PL nanoparticles at 5 mg/ml of ε-PL in the antisolvent were chosen
diameters of 195.77 ± 25.96 and 280 ± 24.98 nm and PDI scores of to coat the avocados for the shelf-life study.
0.023 ± 0.017 and 0.056 ± 0.023 for day 0 of fabrication and day 7,
respectively. PDI scores under 0.05 are indicative of monodisperse size 3.3. Shelf-life study
distribution; revealing a stable solution after 7 days [45]. Multimodal
size distributions were seen with higher ε-PL concentrations, repre­ 3.3.1. wt loss
senting multiple particle sizes and particle destabilization. The stabili­ All 220 avocados with their respective coatings were weighed at Day
zation mechanism of the system is most likely due to the electrostatic 0 of the shelf-life study, presenting no significant difference among
repulsion experienced by the zein nanoparticles. It is known that at weights. The 4 avocados per treatment analyzed at each time point were
minimum of ±30 mV is necessary to ensure sufficient electrostatic weighed prior to firmness, respiration rate, and color measurements,

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F. Garcia et al. Journal of Agriculture and Food Research 7 (2022) 100260

Fig. 5. Average respiration rates (n = 4) ± SD of all 5 coating treatments during the entire shelf-life study (shaded area indicates refrigerated storage). Different
letters are significantly different (p < 0.05) at day 15 of ambient storage.

and compared to their original weights on day 0. During refrigerated stayed similar from 115.09 ± 45.63 to 100.5 ± 23.04 mg of CO2/kg/h
storage, weight loss throughout all treatments started to significantly and 125.91 ± 43.06 to 129.82 ± 48.2 mg of CO2/kg/h, respectively
differ at a marginal rate. (Fig. 4). When moved to ambient storage, all (Fig. 5). At day 15 in ambient storage, ZPL and ZNP avocados signifi­
treatments experienced a more rapid increase in weight loss over time. cantly exhibited the lowest rates observed, 107.83 ± 19.46 mg of CO2/
By the end of refrigerated storage, ZPL and ZNP avocados significantly kg/h and 135.36 ± 50.4 mg of CO2/kg/h, respectively.
(p < 0.05) exhibited the lowest weight loss throughout the entire study. As mentioned above, cold temperatures are known to slow physio­
By day 15 in ambient storage, ZPL and ZNP significantly (p < 0.05) lost logical activity of fruit including their respiration rate [48]. The hy­
the least weight, 20.14 ± 1.83% and 21.80 ± 6.41, respectively. On the drophobicity of the zein coatings due to their predominantly nonpolar
other hand, C avocados lost 28.05 ± 4.66%. ZPL avocados reached amino acid composition can also explain the lower respiration rates of
29.29 ± 4.51% weight loss by day 21 in ambient storage, similar to the the avocados coated with zein nanoparticles. Zein films have shown to
weight loss seen with C avocados on day 15. reduce oxygen transmission rates from the fruit to the environment [50,
Cold temperatures are known to slow physiological activity of fruit 51]. Maftoonazad and Ramaswamy [18] obtained similar respiration
such as oxygen and water transfer and enzymatic activity, resulting in rates when they applied methyl cellulose coatings to avocados; rates
reduced weight loss and respiration rate [48]. The results obtained from were about 110 mg/kg/h by day 6 in ambient storage. Moreover, Tesfay
the weight loss studies corroborate with Tesfay et al. [22], who reported et al. [22] reported a similar decrease in moisture loss and respiration
noticeable increases in weight loss from cold to ambient storage in their rates after coating avocados with moringa leaf extract, chitosan and
shelf-life study of Hass and Fuerte avocados. This escalation is supported carboxymethyl cellulose edible coatings. Saidi et al. [53] found that
by the change to a higher temperature where metabolic activities related similar differences in respiration rates (~3× lower) between avocados
to ripening resume and speed up the rapid aging of the fruit [49]. Aside coated with chitosan and/or carboxymethyl cellulose and control
from the physical temperature change, another explanation for the avocados with no increase in the production of ethanol or acetaldehyde.
dramatic increase in weight loss observed could be due to changes in
relative humidity (RH) from refrigerated and ambient storage condi­ 3.3.3. Firmness
tions. Vapor-phase diffusion is the main driver for moisture loss in fruits During cold storage, avocados across treatments lost a large fraction
and vegetables due to the gradient of water vapor pressure between the of their original firmness (Fig. 6). When moved to ambient storage,
inside and outside the fruit [18]. The RH percentages of refrigerated and avocados continued to lose firmness but in a more gradual manner. By
ambient storage were 79% and 44%, respectively. At 44% RH the dif­ day 21 of cold storage, ZPL and ZNP avocados were the firmest treat­
ference in moisture between the outside and inside of the fruit is larger ments at 12.35 ± 4.28 N and 11.09 ± 3.43 N, respectively. At this same
than at 77% RH, leading to a higher rate of moisture migration. The point in the study, the firmness of C avocados was about half as much.
significant reduction in weight loss of the coated treatments with zein ZPL and ZNP significantly (p < 0.05) retained the highest firmness
nanoparticles (ZNP and ZPL) can be explained by the predominantly throughout the study, and on day 15 of ambient storage they displayed
nonpolar amino acid composition of zein, which is responsible for its superior firmness values of 5.01 ± 0.43 N and 4.07 ± 1.7 N, respectively,
hydrophobicity, thus blocking the moisture migration from the fruit to compared to the control at 3.35 ± 0.23 N. In the present study, there was
the environment [50,51]. The presence of ε-PL is not expected to have an no evident firmness trends nor a significant difference between C, ZS,
effect on the moisture migration from the fruit due to is hydrophilic and PL treatments.
nature. Other studies have shown the effectiveness of zein coatings in Firmness is an attribute with direct correlation to ripening. Increased
preventing weight loss for tropical fruits such as mangos or guavas [23, enzyme activity of pectinmethylesterase, polygalacturonase, and
52]. galactosidase facilitates the depolymerization of structural components
like pectin weakening the cell wall [9,11]. Cenobio-Galindo et al. [54]
3.3.2. Respiration rate observed analogous findings to those presented in this study when
The respiration rates of avocados in all treatments remained similar coating avocados with orange essential oil and xoconostle fruit nano­
for the first three weeks of storage (Fig. 5). When moving the avocados emulsions. Their avocados were of similar firmness (~80 N) at the begin
to room temperature, C avocados rates increased the most from 148.64 of the study, and by day 20 of refrigerated storage, control group had a
± 33.02 to 181.2 ± 22.67 mg of CO2/kg/h. ZPL and ZNP avocados both firmness of ~ 6 N, corresponding to the values seen in this study. As

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F. Garcia et al. Journal of Agriculture and Food Research 7 (2022) 100260

Fig. 6. Average firmness values (n = 4) ± SD of all 5 coating treatments during (A) the entire shelf-life study (shaded area indicates refrigerated storage), (B)
refrigerated storage, and (C) ambient storage. Different letters are significantly different (p < 0.05) at day 15 of ambient storage.

biopolymeric coatings create a physical barrier around the fruit, it could All avocados had initial L values between 33.44 ± 1.04 and 38.34 ±
be argued that the retention of firmness of ZPL and ZNP treatments was 3.01 (Fig. 7A). Throughout the study, values decreased, indicating a
expected with the reduction of respiration rate and retention of mois­ transition from dark green to dark brown-black. For all treatments, a*
ture, improving cell wall integrity [55]. Moreover, the presence of PL values increased, representing the avocados becoming less green
did not have an effect when comparing ZPL and ZNP (Fig. 6), because PL (Fig. 7B). Moreover, b* values decreased as the study progressed, indi­
was not effective in preventing moisture or reducing respiration rate cating that avocados were becoming less yellow (Fig. 7C). Throughout
(Figs. 4 and 5), likely due to its hydrophilic nature. In previous work, refrigerated storage, all avocados within each treatment had comparable
avocados coated with pepper tree essential oil and chitosan particles, ΔE values (Fig. 7D). C and PL avocados presented the highest values,
also resulted in higher firmness values by ~10 N compared to the con­ while ZNP, ZPL, and ZS the lowest. At room temperature, ΔE values
trol [56]. increased for all treatments. ZNP, ZPL, and ZS maintained the lowest
values of 18.29 ± 0.53, 17.08 ± 10.19, and 17.06 ± 4.09, respectively.
3.3.4. Skin color change Although discernible differences were observed, no statistical difference
Color of avocado skins was evaluated according to lightness (L was shown (p > 0.05).
value), blue/yellow coordinates (b* value), green/red coordinates (a* This shift in color could be explained by a decrease in chlorophyll
value) and overall color change, ΔE. and an increase in the anthocyanin cyanidin-3-O-glucoside as avocados

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F. Garcia et al. Journal of Agriculture and Food Research 7 (2022) 100260

Fig. 7. Skin color averages (n = 4) ± SD of all 5 coating treatments throughout the entire shelf-life study (shaded area indicates refrigerated storage). (A) L* values
(B) a* values (C) b* values (D) Δ E.

ripen [57,58]. For the Hass cultivar specifically, skin color is closely 3.3.5. Pulp color change
associated with different ripening stages; avocados are dark green when Color of fruit pulp was evaluated according to lightness (L value),
nearly harvested and transition to dark brown-black as they become ripe blue/yellow coordinates (b* value), green/red coordinates (a* value)
[59]. When avocados were moved to ambient storage, an increase in and overall color change, ΔE. As observed with skin L values, as the
overall color change, ΔE, throughout all treatments was expected. Room study advanced, values decreased illustrating that avocado flesh was
temperature favors resumption of metabolic activities, facilitating becoming darker (Fig. 8A). Change in L values across all groups
ripening and promoting different pigment concentrations of chlorophyll remained similar through refrigerated storage and until day 12 in
and anthocyanins. Similar trends have been observed in other studies ambient storage. At day 12, control avocados experienced the largest
that measured the L*a*b* values from skins of avocados during storage and fastest L-value drop relative to all other groups. Across treatments,
[54,60]. a* values increased (Fig. 8B) and b* values decreased (Fig. 8C), indi­
cating that avocado flesh was becoming less green and less yellow,

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F. Garcia et al. Journal of Agriculture and Food Research 7 (2022) 100260

Fig. 8. Pulp color averages (n = 4) ± SD of all 5 coating treatments throughout the entire shelf-life study (shaded area indicates refrigerated storage). (A) L* values
(B) a* values (C) b* values (D) Δ E.

respectively. For overall color change, ΔE remained similar within The pulp color change observed in whole avocados could be
groups until day 12 of room temperature storage, when C and PL explained by the action of the enzyme polyphenol oxidase (PPO). It is
avocados’ color change steeply increased (Fig. 8D). At day 15 of ambient known that during fruit ripening PPO acts on the blemish formation,
storage, C avocados displayed the severest pulp color change (Fig. 8D). causing the grey pulp disorder in avocados [20]. Tesfay et. al. [22] found
At day 21, C avocados had ΔE values of 24.05 ± 11.57 while ZPL had a that coating avocados with chitosan and CMC resulted in lower PPO
value of only 14.11 ± 11.14 and ZNP of 13.09 ± 3.26. By the end of the activity and thus less color change of the pulp compared to non-coated
study, ZPL and ZNP avocados never achieved the degree of color change avocados. Furthermore, Cenobio-Galindo et al. [54], who coated
seen in the control (Fig. 8D) reaching only about half the ΔE of C avocados with orange essential oil and Opuntia oligacantha extract
avocados. Although discernible differences were observed Fig. 9, no nanoemulsions, also found a direct correlation in lowering PPO activity
statistical difference was shown (p > 0.05). with the application of the coatings and a reduction in color change of

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F. Garcia et al. Journal of Agriculture and Food Research 7 (2022) 100260

Fig. 9. Opened avocados from representative samples of the 5 treatment groups on (A) day 15 of ambient storage and (B) day 21.

the avocado mesocarp. In further studies, the impact of coating avocados 3.4. Fungal inoculation study
with zein nanoparticles on PPO activity can be evaluated to confirm and
quantify the reduction in activity and the correlation with pulp color Avocados coated with ZNPand ZPL showed the best results in the
change. As observed in the moisture loss, respiration rate and firmness shelf-life study extending avocado viability. Therefore, these two
studies, the treatments with less changes in those parameters are also treatments along with the control were chosen for the inoculation study.
those with less pulp color change. This could be attributed to a slower As shown in Fig. 10A, control avocados displayed 40% fungal incidence
ripening process, leading to less changes in pulp color [20]. by day 6 of the study. Both ZPL and ZNP avocados reached that same
incidence percent until day 12. By day 11, all control avocados showed
3.3.6. Overall comparison of the coating effects on the shelf-life of avocados fungal incidence compared to only 8 avocados in the ZNP group and
In this study, the application of coatings with zein nanoparticles only 5 avocados in the ZPL group. At the culmination of the 15 days, ZPL
presumably hindered physiological activities from taking their normal and ZNP avocados exhibited 60% fungal incidence compared to the
course by slowing processes like respiration rate, moisture loss and 83.33% of the control group. Furthermore, the severity index revealed a
possibly enzymatic activity, which delayed the ripening rate. As seen significant (p < 0.05) difference between ZNP and ZPL coatings, sup­
with results for respiration rate, weight loss, firmness, and color, porting ε-PL as an effective antifungal agent (Fig. 10B). At the end of the
avocados with ZS coatings did not delay ripening as well as ZNP and ZPL inoculation study, control group had a severity score of 4.7 out of 5.
coatings despite having the same amount of zein in solution. There are Following, ZNP treatment had a score of 4 and ZPL group a score of 3.33
two possible explanations for this occurrence. First, ZPL and ZNP coat­ (Fig. 10C).
ings are made of nanoparticulated zein, which brings a higher surface According to the fungal incidence results, ZPL treatment appeared to
area to volume ratio, allowing for more of the zein to interact with the have the same ability as ZNP to prevent the appearance of fungal decay,
avocado surface and have an effect [26]. Second, ZS coating is bare zein although in the latter, the antifungal component, ε-PL, was absent. In
dissolved in 85% ethanol solution, while ZPL and ZNP coatings are other studies that have tested the incidence of fungal diseases in
nanoparticles suspended in a solution with 14% ethanol. During the avocados treated with coatings that did not contain antimicrobial
coating application, nanoparticles in ZPL and ZNP solutions are more agents, it has been shown that the oxygen barriers that coatings provide,
likely to be attracted to the hydrophobic surface of the avocado, due to reduced the available oxygen required by fungal growth, thus reducing
their high hydrophobicity, as opposed to return to the hydrophilic its presence [37,66]. In particular, Maftoonazad et al. [37] saw results
environment in which they are suspended (~85% water). On the other that could explain the efficacy of ZNP when coating avocados. In their
hand, the zein in ZS coating will be more attracted to the ethanol in study of avocados coated with pectin-based coatings to inhibit Lasiodi­
solution than the skin of the fruit. Earlier work exploring the hydro­ plodia theobromae infection, authors made a strong connection between
phobic properties of zein has noted that zein without any plasticizers the high effect coatings had on reduced oxygen uptake of the fruit,
have contact measure angles that shift toward more hydrophobic sur­ directly affecting the oxygen levels and energy required for fungal
faces, resulting in higher water repulsion [50]. Furthermore, it has been metabolic activity and growth.
shown that the use of zein nanoparticles as composites in different ed­ Assaying fungal incidence across all three groups gave information
ibles films increase their hydrophobicity demonstrated by an increase in only about the presence of the fungal phytopathogen but did not address
the water contact angle compared to films without zein nanoparticles the severity of disease presence. Despite both ZNP and ZPL having equal
[61–63]. The zein in ZS coatings may not as effectively attach to the disease incidence, investigating the disease severity demonstrated that
fruit, saturating less of the surface, explaining the higher respirations the fungal lesions in the avocados with ε-PL present in the coatings were
rates, weight loss percentages, lower firmness values and larger color less severe. In-vivo results in this inoculation study agree with in-vitro
changes observed in the results. Control and ε-PL coatings were not results (Section 3.1) showing that PL only has fungistatic and not
expected to halt physiological activities as effectively since none con­ fungicidal activity against the Colletotrichum spp. strain used this study.
tained hydrophobic or film forming components. In avocados coated with chitosan and propolis there was no significant
The results of this study align with several others, in which hydro­ difference in anthracnose incidence when the two coating components
phobic coatings were made with candelilla wax [64], orange essential were combined, compared to avocados coated with chitosan alone, but a
oil nanoemulsions [54], pepper tree essential oil-chitosan loaded significant reduction compared to the control. Regarding the severity of
nanoparticles [56] or thyme essential oil-chitosan [65]. The authors of the disease, similarly to the findings in this study the combination of
the cited studies coincide in attributing the retention of firmness, coating materials significantly reduced the severity of anthracnose dis­
weight, and color to the barrier properties of the coatings against ease [39]. Furthermore, the addition of an antimicrobial to coatings
moisture and gas exchange. made with sodium alginate also increased the in-vivo efficacy of the
coatings against the growth of Colletotrichum spp [66]. As mentioned

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F. Garcia et al. Journal of Agriculture and Food Research 7 (2022) 100260

Fig. 10. Data from inoculation study depicting (A) disease incidence % (n = 10) ± SD and (B) disease severity (n = 10) ± SD across all three treatments. Different
letters are significantly different (p < 0.05) on day 15 of storage. (C) Avocados at day 15 of innoculation study storage, from left to right: Control, ZNP and
ZPL treatments.

above, ε- PL acts as an antifungal molecule by the electrostatic absorp­ 4. Conclusion

tion of its cationic make-up to the negatively charged membranes of
fungal cell walls, causing the destruction of the organism by cytoplasmic As the demand for avocado production increases worldwide, the
leakage [18]. need for solutions to minimize post-harvest losses, due to fungal path­
ogens, and extend avocado shelf-life is heightened. As consumer trends
become predominantly geared toward the use of less synthetic additives,
the use of safe and natural ingredients is favored. The results of this work

11
F. Garcia et al. Journal of Agriculture and Food Research 7 (2022) 100260

show that the application of biopolymeric coatings made of zein nano­ [10] D.A. Brummell, Cell wall disassembly in ripening fruit, Funct. Plant Biol. 33 (2)
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☒ The authors declare that they have no known competing financial Mill.) cv. Hass, Food Packag. Shelf. Life 14 (Dec 2017) 102–107, https://doi.org/
interests or personal relationships that could have appeared to influence 10.1016/j.fpsl.2017.09.003.
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This work was supported by the California Agricultural Research
and carboxymethyl cellulose as edible coatings for enhancing quality and
Institute grant number 19-04-100. Matching funds were provided by the extending postharvest life of avocado (Persea americana Mill.) fruit, Food Packag.
Education/Research grants from the Southern California Institute of Shelf. Life 11 (Supplement C) (2017) 40–48, https://doi.org/10.1016/j.
Food Technologists Section. Research reported in this publication was fpsl.2016.12.001, 2017/03/01/.
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