plants

Article
Effect of Chitosan-24-Epibrassinolide Composite Coating on the
Quality Attributes of Late-Harvested Pomegranate Fruit under
Simulated Commercial Storage Conditions
Sbulelo Mwelase and Olaniyi Amos Fawole *

Postharvest Research Laboratory, Department of Botany and Plant Biotechnology, University of Johannesburg,
P.O. Box 524, Auckland Park, Johannesburg 2006, South Africa; sbulelom@uj.ac.za
* Correspondence: olaniyif@uj.ac.za

Abstract: This study evaluated the efficacy of chitosan (CH) functionalized with 24-epibrassinolide
(EBR) coating in terms of preserving the postharvest quality of late-harvested pomegranate (cv.
Wonderful) fruit. Late-harvested pomegranate fruit were immersed for 3 min in different surface
treatment solutions—CH 1.5% (w/v), CH + 2 µM EBR, CH + 5 µM EBR, CH + 10 µM EBR and
CH + 15 µM EBR—and distilled water was used as a control treatment. The fruit were air-dried
and subjected to long storage duration at 5 ◦ C with 85 ± 5 RH for 12 weeks. At 4-week sampling
intervals, a batch of fruits was placed at 21 ± 2 ◦ C and 65–70% RH for a further 3 d period to simulate
retail conditions before measurements were taken. Fruit physiological responses, physico-chemical
properties, phytochemical contents, antioxidant capacity and physiological disorders were monitored
during storage. The results showed that the CH-EBR composite edible coatings significantly (p < 0.05)
delayed degradative processes due to senescence. The CH-EBR treatments delayed colour, texture





 and total soluble solids (TSS) degradation and reduced weight loss, respiration, electrolyte leakage
Citation: Mwelase, S.; Fawole, O.A.
and spoilage compared to the control and CH treatment. The treatment effect was more noticeable
Effect of Chitosan-24-Epibrassinolide on fruit treated with CH + 10 µM EBR, which exhibited lower weight loss (18.19%), respiration rate
Composite Coating on the Quality (7.72 mL CO2 kg−1 h−1 ), electrolyte leakage (27.54%) and decay (12.5%), and maintained higher
Attributes of Late-Harvested texture (10.8 N) and TSS (17.67 ◦ Brix) compared to the untreated fruit with respective values of
Pomegranate Fruit under Simulated 24.32%, 18.06 mL CO2 kg−1 h−1 , 43.15%, 37.5%, 8.32 N and 17.03 ◦ Brix. This was largely attributed to
Commercial Storage Conditions. the significantly higher antioxidant content, including the ascorbic acid content, total phenol content,
Plants 2022, 11, 351. https:// total anthocyanin content and DPPH (radical scavenging activity), of the coated fruit compared to the
doi.org/10.3390/plants11030351 control fruit. Therefore, CH + 10 µM EBR treatment is recommended as a postharvest management
Academic Editors: Romina Pedreschi strategy to improve the quality preservation of late-harvested pomegranate fruit during storage.
and Reinaldo Campos-Vargas
Keywords: phytohormone; brassinosteroids; electrolyte leakage; respiration; vitamin C
Received: 26 December 2021
Accepted: 25 January 2022
Published: 27 January 2022

Publisher’s Note: MDPI stays neutral 1. Introduction

with regard to jurisdictional claims in
Pomegranate is amongst the most important crops produced globally because of its
published maps and institutional affil-
countless nutritional and medicinal benefits [1]. The functional benefits of pomegranates
iations.
have resulted in increased consumption and demand for the fruit; consequently, the produc-
tion and market opportunities have also expanded to meet consumer demand [1,2]. This
means that harvest time will also increase due to higher volumes of fruit being harvested.
Copyright: © 2022 by the authors.
While the global demand for pomegranate is increasing, quality issues remain a concern
Licensee MDPI, Basel, Switzerland. in most producing regions, leading to substantial postharvest losses and waste [3]. This
This article is an open access article is a result of the highly perishable nature of pomegranates due to high susceptibility to
distributed under the terms and physiological disorders and decay after harvest. This leads to the overall fruit quality
conditions of the Creative Commons declining, including the loss of functional properties of the fruit.
Attribution (CC BY) license (https:// Cold storage is commonly used to maintain postharvest quality and prolong the
creativecommons.org/licenses/by/ storability of fruits. However, pomegranates are highly susceptible to chilling injury (CI)
4.0/). when stored at the recommended refrigeration temperatures, with fungal attacks and husk

Plants 2022, 11, 351. https://doi.org/10.3390/plants11030351 https://www.mdpi.com/journal/plants

Plants 2022, 11, 351 2 of 24

scald also being postharvest problems encountered during cold storage [4]. The quality,
ripening behaviour and susceptibility to physiological disorders of pomegranate are also
closely related to harvest maturity [5]. According to Fawole and Opara [5], late-harvested
pomegranate fruit have a relatively short postharvest life and tend to develop off-flavours
without any visible postharvest physiological disorders if stored for an extended time.
Furthermore, Defilippi et al. [6] observed that ‘Wonderful’ pomegranate fruits harvested
later in the season were characterized by a higher incidence of decay and scald.
Contrary to Defilippi et al. [6], Kashash et al. [7] reported that early-season ‘Wonderful’
pomegranate fruit were more susceptible to chilling injury and aril browning compared
with late-season fruit. This may have resulted from the different molecular mechanisms
involved in the development of different physiological disorders. Nonetheless, the high
susceptibility to scald and decay in late-harvested pomegranate fruit, as reported by
Defilippi et al. [6], suggests the need for postharvest technologies to maintain quality and
extend the storage life of the late-harvested pomegranate fruit.
Edible coatings constitute a promising technology that is currently used to extend
the storage life and maintain the quality of various minimally processed and whole fruits
and vegetables [8–15]. The potential ability of coatings to maintain the quality and extend
the postharvest life of horticultural produce is due to the barrier applied for the perme-
ability of gases, mainly CO2 and O2 , which reduces metabolic processes of the fruit and
vegetables [16–18]. Edible coatings have attracted much interest, mainly because they are
derived from natural sources and are, therefore, eco-friendly and biodegradable [19]. Thus,
edible coatings provide alternatives to synthetic chemicals and polyethylene packaging
bags in postharvest management, which impose an environmental threat. Edible coatings
also constitute an ideal carrier of active agents, such as antioxidant, antimicrobial and
flavouring compounds, with the advantage of providing improved coating properties
for the improved postharvest management of horticultural crops by combining several
polymers [20,21].
Chitosan (poly β-(1, 4)-2-amino-2-deoxy-D-glucan) (CH) (Figure 1A) is an exceptional
polysaccharide coating material that is widely used in the postharvest quality management
of horticultural crops [22–24]. The popularity of this biopolymer is attributed to its excellent
film-forming properties, biodegradability, nontoxicity and antimicrobial properties [24–26].
However, the application of CH is restricted due to its low antioxidant capacity [22]. As
with the other coating materials, the nature of CH also allows for the incorporation of
different bio-compounds to improve the coating functionality. Hence, our study is focused
at the incorporation of CH with brassinosteroids (BRs).

Figure 1. Chemical structures of chitosan (A) and 24-Epibrassinolide (B).

BR is a natural, nontoxic, non-genotoxic, biosafe and eco-friendly phytohormone

that represents an alternative to improve the properties of edible coatings [27]. As a
phytohormone, BRs endogenously exists in all plant species at very low concentrations
and are integrated in plants’ responses against stress [28,29]. Several BRs have been
Plants 2022, 11, 351 3 of 24

identified to date; however, among these, brassinolide, 24-epibrassinolide (EBR) and 28-
homobrassinolide are, so far, the most biologically active forms, with EBR (Figure 1B) being
the most extensively used BR analog in physiological studies [30,31]. This is attributed
to the greater bioactivity of EBR compared to the BR active analogs. Recently, the United
States (US) and the European Union (EU), the major importing regions, have exempted
residue limits of EBR from foods and declared its nontoxicity as a plant growth regulator
in agriculture [32,33]. For this reason, several researchers have attempted to develop
alternative safe postharvest preservation strategies using BRs for the improved quality
management of horticultural crops.
Exogenous postharvest applications of BRs have been reported to enhance bio-active
compounds [34–37], delay ripening and senescence [35], and regulate defence-related en-
zymic activities, which develop robust defence mechanisms against different physiological
disorders [34,35,37–39]. The mechanism of BRs further involves (1) the upregulation of
membrane protein genes (leading to higher membrane integrity) [29,31], (2) the enhance-
ment of phenylalanine ammonia lyase (PAL) and suppressed polyphenol oxidase (PPO)
enzymatic activities, leading to higher phenol and anthocyanins accumulation [39–42],
and (3) higher arginine pathway activity, leading to higher polyamines and proline accu-
mulation [39,43,44], resulting in the improved antioxidant capacity of the crop. Notably,
single phytohormone treatment is the main formulation of BRs’ exogenous postharvest
applications for the preservation of horticultural crops. While the use of BRs alone has
shown satisfactory effects in maintaining the quality and delaying the senescence of fruit
and vegetables during storage, its effect cannot be sustained due to the poor adhesion
ability of BRs on fruit surfaces. Therefore, incorporating BRs with CH is hypothesized to
show synergistic effects resulting in improved coating functionality and effectiveness. Such
an effect has been previously reported, but with other phytohormones. Zhang et al. [45]
reported that incorporating chitosan (CH) with salicylic acid remarkably preserved the
quality and antioxidant capacity of cucumber compared to the use of chitosan and salicylic
acid alone during postharvest storage. In addition, Sayyari et al. [46] observed enhanced
performance of salicyloyl chitosan treatment on pomegranate fruit compared to the use of
salicylic acid and chitosan treatment alone during storage at 2 ◦ C for 5 months. Therefore,
the aim of this study was to develop and evaluate the efficacy of CH functionalized with
EBR in effectively maintaining the quality and delaying the senescence of late-harvested
pomegranate fruit subjected to commercial storage conditions.

2. Results and Discussion

2.1. Physiological Responses
2.1.1. Weight Loss
The weight loss of pomegranate fruit increased progressively (p < 0.05) during storage
regardless of treatment (Figure 2A). The highest weight loss was observed in control fruit
and the lowest weight loss was observed in fruit treated with CH + 10 µM, followed by
the CH + 15 µM EBR, CH + 5 µM EBR, CH + 2 µM EBR and CH treatments. It was noted
that CH in combination with EBR significantly (p < 0.05) reduced pomegranate fruit weight
loss compared to CH treatment. Similar results were reported by Zhang et al. [45] and
Sayyari et al. [46], where CH-incorporated salicylic acid significantly lowered the weight
loss of cucumber and pomegranate fruit, respectively, compared with the use of CH and
salicylic acid alone.
Loss of fruit weight is mainly due to water loss caused by transpiration, and edible
coatings form a semi-permeable layer on fruit surfaces, which become a protective barrier
to reduce transpiration rates [47]. However, this is highly influenced by the nature of
the edible coating; for instance, CH is hydrophilic, resulting in poor water vapor barrier
properties [24]. This agrees with our results where CH treatment showed reduced efficacy
in retarding fruit weight loss during storage. However, it could be hypothesized that
incorporating EBR into the coating matrix improved the water vapor barrier properties of
Plants 2022, 11, 351 4 of 24

the coating through its preventive effect against membrane damage [35], which results in
high membrane integrity and, thus, further reduced transpiration rates.

Figure 2. Weight loss (A) and respiration rate (B) of uncoated (control) and coated pomegranate fruit
during storage at 5 ◦ C for 12 weeks plus 3 days at 20 ◦ C. Each bar represents mean ± error bars.
Error bars represent standard error (SE) of the mean values, and different letters on bars represent
statistical differences (p < 0.05) according to Duncan’s multiple range test.

2.1.2. Respiration Rate

The process of respiration is essential and is a good index of fruit quality during
postharvest storage. During this process, the stored carbohydrates are broken down using
oxygen as a co-substrate to produce the energy (ATP) necessary for the activity of the
enzymes involved in metabolic processes. Thus, lower respiration rates are crucial to
maintaining quality and extending fresh produce’s postharvest life. According to the
Plants 2022, 11, 351 5 of 24

factorial analysis, changes in fruit respiration were driven by the combination of treatment
(p < 0.001) and storage duration (p < 0.001) (Figure 2B).
The rate of respiration of all the treated fruit remained steady and lower than that
of the control fruit throughout storage (Figure 2B). The control fruit exhibited an increase
in respiration rates and reached a peak at week 8 of storage; inversely, the EBR-treated
fruits showed a declining rate with storage. While the CH also exhibited a declining rate,
an increase was observed at week 8 of storage. Nevertheless, the peak was significantly
lower than that of the control fruit. This was primarily due to the excellent gas permeability
properties of CH, resulting in high carbon dioxide and low oxygen within the fruit, thus
lowering respiration rates [13].
The enhanced effectiveness of CH + EBR coatings, in terms of reducing the respiration
rates, compared to CH alone could be attributed to the coating being more efficient in terms
of restricting gaseous exchange due to EBR maintaining higher membrane integrity [35].
In agreement with our findings, Zhu et al. [48] and Wang et al. [35] reported reduced
respiration rates of EBR-treated Jujube and kiwifruit, respectively, during storage.

2.1.3. Fruit Texture and Aril Hardness

Fruit firmness decreased significantly (p < 0.001) in all treatments during storage
(Table 1), and both factors (treatments: p < 0.001; storage period: p < 0.001) contributed
to the changes observed in fruit firmness. However, the firmness decreased more slowly
in treated fruits, with slight significant differences observed between CH treatment and
in combination with EBR bioactive compound during storage. At the end of storage
(week 12), CH + EBR (10 µM) treatment showed an optimum effect in retaining fruit
firmness compared to all the treatments. In agreement with our findings, Gao et al. [39] and
Wang et al. [35] reported higher firmness of EBR-treated peach and kiwifruit, respectively,
during storage. The delay in loss of fruit firmness, as influenced by both CH and EBR,
may be attributed to the modified fruit gas atmosphere by the coating, which results
in reduced activity of cell wall degradation enzymes, including polygalacturonase (PG),
pectinmethylesterase (PME), galactosidase and cellulase [46,49]. Reduced activity of these
enzymes results in higher levels of pectin, the key substances involved in the mechanical
strength of the cell wall [49].

Table 1. Fruit firmness, aril hardness and electrolyte leakage of uncoated (control) and coated
pomegranate fruit during storage at 5 ◦ C for 12 weeks plus 3 days at 20 ◦ C.

Weeks of Cold Storage + 3 Days at 20 ◦ C

Parameter Treatment Harvest
4 8 12
12.86 ± 0.29
Control 10.81 ± 0.64 b–f 10.56 ± 0.48 b–f 8.32 ± 0.34 a
CH 1.5% 9.77 ± 0.53 bc 10.24 ± 0.48 b–e 9.79 ± 0.44 bc
Fruit firmness (N) CH + EBR (2 µM) 10.02 ± 0.47 b–d 11.5 ± 0.45 d–f 9.55 ± 0.44 ab
CH + EBR (5 µM) 11.71 ± 0.42 ef 11.72 ± 0.30 ef 10.33 ± 0.38 b–e
CH + EBR (10 µM) 11.42 ± 0.40 d–f 11.11 ± 0.45 c–f 10.8 ± 0.39 b–f
CH + EBR (15 µM) 11.73 ± 0.47 ef 11.93 ± 0.43 f 9.91 ± 0.61 bc
12.55 ± 0.3
Control 10.77 ± 0.19 a–d 10.57 ± 0.62 a–c 9.86 ± 0.54 a
CH 1.5% 11.48 ± 0.48 cd 10.94 ± 0.64 a–d 9.87 ± 0.5 a
Aril hardness (N) CH + EBR (2 µM) 11.52 ± 0.30 cd 10.97 ± 0.49 a–d 9.94 ± 0.32 ab
CH + EBR (5 µM) 11.32 ± 0.47 a–d 10.95 ± 0.45 a–d 10.45 ± 0.17 a–c
CH + EBR (10 µM) 12.13 ± 0.24 d 11.4 ± 0.55 b–d 11.3 ± 0.57 a–d
CH + EBR (15 µM) 11.67 ± 0.24 cd 11.44 ± 0.48 cd 10.71 ± 0.41 a–c
Plants 2022, 11, 351 6 of 24

Table 1. Cont.

Weeks of Cold Storage + 3 Days at 20 ◦ C

Parameter Treatment Harvest
4 8 12
3.91 ± 0.88
Control 7.64 ± 1.24 ab 27.52 ± 2.57 fg 43.15 ± 1.97 i
Electrolyte leakage
CH 1.5% 10.61 ± 1.97 bc 15.61 ± 2.03 cd 35.1 ± 2.26 h
(%) CH + EBR (2 µM) 16.44 ± 3.76 cd 27.23 ± 1.01 fg 29.19 ± 3.71 f–h
CH + EBR (5 µM) 3.1 ± 0.06 a 19.64 ± 2.85 de 22.86 ± 1.96 ef
CH + EBR (10 µM) 5.38 ± 1.31 ab 14.64 ± 0.04 cd 27.54 ± 0.79 fg
CH + EBR (15 µM) 3.4 ± 0.2 a 14.7 ± 0.43 cd 30.46 ± 0.93 gh
Significance level (p) Coating Treatment Storage period A×B
(A) (B)
Fruit firmness <0.001 <0.001 0.105
Aril hardness 0.023 <0.001 0.990
Electrolyte leakage <0.001 <0.001 <0.001
Data presented as mean ± SE. Different letters in each column indicate significant differences between the
interaction effect of storage time and treatments at p < 0.05 according to Duncan’s multiple range test. SE—
standard error.

As observed for fruit firmness, aril hardness decreased significantly (p < 0.001) with
prolonged storage, with significant differences observed amongst coating treatments
(p < 0.001) (Table 1). The arils’ hardness was also considerably (p < 0.05) affected by
the coating treatments. The overall decrease in the arils’ textural property during storage
shows that CH + EBR treatments significantly lowered the loss of the arils’ textural property
compared to the control and CH treatments. Fawole and Opara [5] reported that high aril
hardness is partly due to higher juice contents, resulting in higher turgor pressure within
the arils. Thus, the efficacy of CH and EBR treatments to effectively reduce transpiration
could be attributed to the observed effect on aril hardness. CH + EBR (10 µM) treatment
showed a good effect in terms of reducing the loss of aril hardness, and delayed senescence
better compared to the other treatments.

2.1.4. Electrolyte Leakage

Electrolyte leakage (EL) is generally accepted as an index that quantifies membrane
damage and the loss of the membrane integrity of fresh produce [50]. The damage is
mediated by increased production of H2 O2 and the bursting of other reactive oxygen
species (ROS), and thus, increasing plant oxidative stress causes the leakage of membrane
intracellular ions and metabolites [51]. The EL of control and treated pomegranate fruit
during storage is shown in Table 1. The EL of pomegranate fruit increased with storage
in all treatments; however, EBR treatments significantly (p < 0.001) inhibited the leakage
increase (Table 1). The highest EL was observed in the control fruit, followed by CH
treatment, with CH + EBR (5 µM) showing the optimum effect in terms of limiting leakage
ions and membrane damage.
The suppression of EL by EBR treatments was also reported by Aghdam and Moham-
madkhani [37] on tomato, Pakkish et al. [42] on grapes and Wang et al. [35] on kiwifruit.
The literature suggests that the EBR hormone alleviates membrane damage by regulating
the activity of antioxidant defence systems, which inhibit the burst in accumulation of toxic
ROS. Ghorbani and Pakkish [41] and Pakkish et al. [42] observed higher catalase (CAT)
activity in BR-treated orange (1.5 ppm) and grapes (10 µM), respectively. Aghdam et al. [40]
noted that postharvest EBR treatment enhanced the PAL activity of tomato fruit, which
was significantly higher than in control fruit, an effect associated with higher membrane
integrity and lower EL.
Plants 2022, 11, 351 7 of 24

2.2. Physico-Chemical Properties

2.2.1. Colour Attributes
Colour is an important parameter that plays a significant role in the acceptability of
foods. The colour changes of pomegranate peel and arils, as determined by CIELAB values
(a*: redness; C*: chroma (colour intensity); h◦ : hue angle), are presented in Figure 3A–F.
There was a significant interaction between the main factors for all the investigated colour
attributes except for the aril hue angle. Noticeably, coated fruit retained higher peel
redness, as indicated by a* values throughout storage, compared with control fruit, which
showed the lowest values of a*. However, during the last week of storage (week 12),
significantly lower peel redness was observed for CH + EBR (10 µM)-treated fruits. Colour
intensity decreased gradually with storage and was also highly affected (p < 0.001) by the
coating treatments (Figure 3B). A similar effect was observed where the coating treatments
maintained significantly (p < 0.05) higher chroma values throughout the storage period as
compared to the control treatment. In agreement with our findings, Fawole and Opara [1]
reported decreasing peel redness and colour intensity over the storage period. The hue
angle behaviour was unclear; nonetheless, insignificant differences were observed between
most treatments on different sampling days (Figure 3C).

Figure 3. Changes in colour parameters; (A) peel redness (a*), (B) peel chroma (C*), (C) peel hue angle
(h◦ ), (D) aril redness (a*), (E) aril chroma (C*) and (F) aril hue angle (h◦ ) of pomegranate fruit during
storage at 5 ◦ C for 12 weeks plus 3 days at 20 ◦ C. Each bar represents mean and error bars. Error bars
represent SE of the mean values, and different letters on bars represent statistical differences (p < 0.05)
according to Duncan’s multiple range test. Dotted lines represent measurements at harvest.

With regard to the aril colour appearance, there were inconsistent fluctuations dur-
ing storage; however, the overall trend showed that aril redness decreased significantly
Plants 2022, 11, 351 8 of 24

(p < 0.001) with storage (Figure 3D). However, storage period did not have a significant
effect (p = 0.077) on the colour intensity of the arils (Figure 3E). This is in line with the report
by Fawole and Opara [1], who observed non-significant changes in the aril colour intensity
of ‘Bhagwa’ pomegranate cultivar during storage. It was suggested that pomegranate’s
aril colour is relatively stable during storage. Neither the storage period nor the coating
treatments had a significant effect on the hue angle (treatments: p < 0.722; storage period:
p < 0.083), which suggests that the arils’ colour saturation remained stable during storage.
Notably, CH + EBR (10 µM) treatment maintained higher values of redness and chroma.
The retention of the colours of arils from fruit subjected to CH + EBR (10 µM) treatment
could be attributed to the ability of the coating to reduce the activity of enzymes associated
with the degradation of anthocyanins in pomegranate fruit.

2.2.2. Titratable Acidity, Total Soluble Solids and BrimA

The changes in the total soluble solids (TSS) and titratable acidity (TA) of treated and
control pomegranate fruits during storage are presented in Table 2. These two attributes
are important in determining the taste quality of pomegranate fruit. In this study, TA
decreased significantly (p = 0.011) with prolonged storage. As shown in Table 1, the overall
effect of coating treatments on TA was not significant (p = 0.279). However, control fruit
were characterized by lower TA from week 8 of storage until the end of the experiment as
compared to the treated fruits, which showed the highest TA. On the last day of storage, the
TA values in fruit treated with CH, CH + EBR (2 µM), CH + EBR (5 µM), CH + EBR (10 µM)
and CH + EBR (15 µM) were 0.85, 0.97, 0.96, 0.93 and 1% citric acid, respectively, which
were all higher than the control values (0.81% citric acid). The literature has shown that TA
is directly correlated with the concentration of organic acids in the fruit, which are the main
respiratory substrates in pomegranates [52]. Thus, the gradual decline in TA with storage
indicates the ongoing metabolic processes in the fruit during storage. Therefore, higher TA
during storage indicates a prolonged storage life and high quality of the fruit. The high TA
in the coated fruits could result from the enhanced effectiveness of the coatings in terms of
reducing respiration rates, as previously explained. The lower respiration rates result in
fewer organic acids being used up.

Table 2. Titratable acidity (TA), total soluble solids (TSS) and BrimA of uncoated (control) and coated
pomegranate fruit during storage at 5 ◦ C for 12 weeks plus 3 days at 20 ◦ C.

Storage Period (Weeks of Cold Storage + 3 Days at 20 ◦ C)

Parameter Treatment Harvest
4 8 12
1.68 ± 0.24
Control 1.01 ± 0.14 a–c 0.85 ± 0.09 ab 0.81 ± 0.04 a
CH 1.5% 1.12 ± 0.10 a–c 0.99 ± 0.06 a–c 0.85 ± 0.04 ab
TA (% citric acid) CH + EBR (2 µM) 0.96 ± 0.04 a–c 0.97 ± 0.08 a–c 0.97 ± 0.10 a–c
CH + EBR (5 µM) 1.19 ± 0.18 a 1.05 ± 0.03 a–c 0.96 ± 0.08 a–c
CH + EBR (10 µM) 1.12 ± 0.01 bc 0.93 ± 0.07 a–c 0.93 ± 0.08 a–c
CH + EBR (15 µM) 1.07 ± 0.06 ab 0.97 ± 0.07 a–c 1 ± 0.03 a–c
18.03 ± 0.15
Control 17.47 ± 0.15 cd 17.3 ± 0.06 a–c 17.03 ± 0.07 a
CH 1.5% 17.43 ± 0.17 b–d 17.37 ± 0.09 b–d 17.1 ± 0.12 ab
TSS (◦ Brix) CH + EBR (2 µM) 17.93 ± 0.03 e 17.93 ± 0.67 ef 17.1 ± 0.06 ab
CH + EBR (5 µM) 17.43 ± 0.03 b–d 17.4 ± 0.15 b–d 17.33 ± 0.03 a–d
CH + EBR (10 µM) 17.4 ± 0.06 b–d 17.33 ± 0.15 b–d 17.67 ± 0.09 de
CH + EBR (15 µM) 17.43 ± 0.19 b–d 17.33 ± 0.12 b–d 17.13 ± 0.07 a–c
Plants 2022, 11, 351 9 of 24

Table 2. Cont.

Storage Period (Weeks of Cold Storage + 3 Days at 20 ◦ C)

Parameter Treatment Harvest
4 8 12
15.09 ± 0.71
Control 15.44 ± 0.24 ab 15.6 ± 0.23 a–d 15.41 ± 0.07 a–c
CH 1.5% 15.22 ± 0.05 ab 15.39 ± 0.16 ab 15.4 ± 0.17 ab
BrimA CH + EBR (2 µM) 16.01 ± 0.1 cd 16 ± 0.12 cd 15.16 ± 0.26 a
CH + EBR (5 µM) 15.05 ± 0.33 a 15.34 ± 0.02 ab 15.4 ± 0.14 a–c
CH + EBR (10 µM) 15.17 ± 0.15 a 15.54 ± 0.15 a–d 15.8 ± 0.24 b–d
CH + EBR (15 µM) 15.29 ± 0.2 ab 15.5 ± 0.04 a–d 15.13 ± 0.14 a
Coating treatment
Significance level (p) Storage period (B) A×B
(A)
TA 0.279 0.011 0.932
TSS <0.001 <0.001 <0.001
BrimA 0.038 0.131 0.042
Data presented as mean ± SE. Different letters in each column indicate significant differences between the
interaction effect of storage time and treatments at p < 0.05 according to Duncan’s multiple range test. SE—
standard error.

The TSS also decreased gradually with storage (Table 2). Our findings agree with those
of Fawole and Opara [1], who reported a decrease in TSS content in ‘Ruby’ pomegranate
fruit with an increased storage period. TSS are sugars that are also used as an energy source
in pomegranate for respiration [53]. This helps to explain the declining TSS content with
storage. Notably, the coating treatments maintained significantly (p < 0.001) higher levels
of TSS than the control during storage. At the same time, there were slightly significant
differences between CH coating alone and in combination with EBR. During the last week
of storage, the highest level of TSS (17.67 ◦ Brix) was recorded in fruits coated with CH
+ EBR (10 µM) and the lowest levels (17.03 ◦ Brix) were observed in control fruits. The
BrimA index did not change significantly (p > 0.131) with storage (Table 1). Contrary to
our results, Fawole and Opara [5] and Fawole et al. [17] reported a decrease in the BrimA
index for ‘Bhagwa’ and ‘Wonderful’ pomegranate fruit with prolonged storage durations.
Arendse et al. [54] observed an increase in the BrimA index of ‘Wonderful’ pomegranate
fruit during storage. Notably, the BrimA index was influenced by the coating treatments
(p = 0.038) and, overall, treating fruit with CH + EBR (10 µM) resulted in a better BrimA
index compared to other treatments.

2.3. Phytochemical Content and Antioxidant Properties

2.3.1. Ascorbic Acid Content
Generally, the changes in ascorbic acid content were influenced by the combined
effects of treatment (p < 0.001) and storage period (p < 0.001). The ascorbic acid content
initially declined from harvest to week 4 of storage, thereafter peaked at week 8 of storage,
and then declined. Thus, overall, the ascorbic acid contents of pomegranate fruit decreased
with storage. Noticeably, the ascorbic acid levels were higher in the treated fruit and lower
in the control fruit (Figure 4). At the end of storage (week 12), the ascorbic acid content
of the control fruit was 33.28 mg AA/100 mL. For the CH, CH + EBR (2 µM), CH + EBR
(5 µM), CH + EBR (10 µM) and CH + EBR (15 µM) treatments, the ascorbic acid content
reached 37.79, 33.79, 35.77, 39.04 and 38.63 mg AA/100 mL, respectively, which were
all significantly higher than control. Ascorbic acid is reported to be the most important
water-soluble antioxidant that directly protects the plant against oxidative stress [55].
Further, a decrease in ascorbic acid content was found to result from the enzyme
activity of ascorbate oxidase (AO) using oxygen as the primary substrate, which caused
degradation in ascorbic acid [55]. Thus, the excellent gas barrier properties of CH resulted
in lower O2 within the fruit and consequently lower oxygen availability for AO activity.
The high ascorbic acid content in fruit treated with CH + EBR (10 µM) and CH + EBR
Plants 2022, 11, 351 10 of 24

(15 µM), respectively, could be attributed to the direct involvement of EBR in detoxifying
ROS in cells [35]. Ascorbic acid breaks down H2 O2 to water [35], an effect associated
with reduced fruit susceptibility to physiological disorders, delayed senescence, and high-
quality maintenance.

Figure 4. Ascorbic acid content of uncoated (control) and coated pomegranate during storage at 5 ◦ C
for 12 weeks plus 3 days at 20 ◦ C. Each bar represents mean ± error bars. Error bars represent SE of
the mean values, and different letters on bars represent statistical differences (p < 0.05) according to
Duncan’s multiple range test. Dotted line represent measurement at harvest.

2.3.2. Total Phenolic Content

Changes in the total phenolic content (TPC) of coated and control pomegranate fruit
during storage are presented in Figure 5. Generally, the TPC significantly (p < 0.001)
decreased with storage. However, significant enhancements were observed at week 8 of
storage but thereafter declined to the end of storage. Our findings agree with those of
Fawole and Opara [1], who reported a decline in TPC in ‘Ruby’ and ‘Bhagwa’ pomegranate
fruit during storage at 5 ◦ C for 16 weeks. Similarly, Sayyari et al. [30] reported a decline in
TPC in pomegranate fruit (cv. Malase) coated with 1–2% CH and stored at 4.5 ◦ C for 120
days. Noticeably, the coating treatments significantly suppressed (p < 0.001) the decline in
TPC during storage. At the end of the experiment (week 12), fruits coated with chitosan +
EBR (5 µM) significantly maintained a higher TPC than other treatments.
The changes in TPC during cold storage may be related to fluctuations in the PAL
and PPO enzyme activities [56]. PAL is the key enzyme in the phenylpropanoid pathway
that is directly involved in the biosynthesis of phenolic compounds. In contrast, PPO
activity results in the oxidation of phenolic substances using oxygen as a co-substrate.
The consequently lower oxygen within coated fruit could be attributed to the higher TPC
in treated fruit due to reduced PPO activity. The inhibition of PPO activity by coating
treatments has been largely observed in the literature [57–60]. Furthermore, BRs have been
reported to induce higher PAL activity [40,46], which is integrated with the BR-induced
plant defence mechanism. Therefore, the lower PPO activity and higher PAL activity in CH
+ EBR-coated pomegranate resulted in the observed higher TPC.
Plants 2022, 11, 351 11 of 24

Figure 5. Total phenolic content of uncoated (control) and coated pomegranate during storage for
12 weeks at 5 ◦ C plus 3 days at 20 ◦ C. Each bar represents mean ± error bars. Error bars represent SE
of the mean values, and different letters on bars represent statistical differences (p < 0.05) according
to Duncan’s multiple range test. Dotted line represent measurement at harvest.

2.3.3. Total Anthocyanin Content

Total anthocyanin content (TAC) changes in coated and uncoated (control) pomegranate
fruit during storage are presented in Figure 6. TAC significantly increased with storage
in the coated fruit. In contrast, the TAC significantly declined in the uncoated fruit with
storage. Thus, significantly lower TAC was observed in the uncoated fruit on the last week
of storage, followed by the CH treatment with CH + EBR (5 µM) treatment showing high
TAC. Anthocyanins are regarded as non-enzymatic antioxidants in plants, suggesting that
the EBR-mediated accumulation in anthocyanins is part of the mechanisms employed to
maintain the quality of pomegranate fruit. As previously explained, the induced higher
PAL enzyme activity and suppressed PPO activity in EBR-treated fruit is suggested to have
resulted in higher TAC. In agreement with our findings, Sayyari et al. [46] reported an
enhanced accumulation of TAC in ‘Mallas Saveh’ pomegranate fruit coated with salicylic acid
+ chitosan during storage at 2 ◦ C for 5 months.

Figure 6. Total anthocyanin content of uncoated (control) and coated pomegranate during storage for
12 weeks at 5 ◦ C plus 3 days at 20 ◦ C. Each bar represents mean ± error bars. Error bars represent SE
of the mean values, and different letters on bars represent statistical differences (p < 0.05) according
to Duncan’s multiple range test. Dotted line represent measurement at harvest.
Plants 2022, 11, 351 12 of 24

2.3.4. DPPH Radical-Scavenging Activity

The radical-scavenging activity of pomegranate fruit significantly (p < 0.05) declined
gradually with storage in all the treatments (Figure 7). The coating treatments also signifi-
cantly (p < 0.05) affected the radical-scavenging activity of pomegranate fruit. However,
insignificant differences (p > 0.05) were observed between control, CH alone, and CH in
combination with EBR at 2 and 5 µM treatments at week 8 and week 12 of storage. Further,
the treatments of CH + EBR (10 µM) and CH + EBR (15 µM) were statistically similar during
the last week of storage. However, these treatments maintained higher radical-scavenging
activity throughout the storage period of the fruit. The higher radical-scavenging activity
of CH + EBR-treated fruit may be attributed to the enhanced antioxidant enzyme activity
induced by EBR, thereby improving the fruit’s overall antioxidant capacity and scavenging
ability [61]. In support of our findings, previous studies have demonstrated enhanced CAT,
superoxide dismutase (SOD) and ascorbate peroxidase (APX) antioxidant enzymes activi-
ties in mandarins [62], mushrooms [63] and grapefruits [42] treated with EBR. The authors
further reported that enhanced antioxidant enzyme activities were correlated with reduced
H2 O2 production, confirming the improved radical-scavenging ability of treated fruit.

Figure 7. DPPH radical-scavenging activity of uncoated (control) and coated pomegranate fruit
stored for 12 weeks at 5 ◦ C plus 3 days at 20 ◦ C. Each bar represents mean ± error bars. Error
bars represent SE of the mean values s, and different letters on bars represent statistical differences
(p < 0.05) according to Duncan’s multiple range test. Dotted line represent measurement at harvest.

2.3.5. Ferric Reducing Antioxidant Power (FRAP)

Generally, the changes in ascorbic acid content were influenced by the combined
effects of changes in the FRAP of uncoated and coated pomegranate fruits during storage,
as presented in Figure 8. The FRAP assay has been widely applied as an index of the
total antioxidant content of foods. In our study, the FRAP of both coated and uncoated
pomegranate fruit significantly declined during storage up to week 8, which thereafter
increased. The changes in the antioxidant power of pomegranate fruit were highly influ-
enced by the interaction between the main factors investigated (p < 0.001). The coated fruit
showed significantly (p < 0.001) higher antioxidant power throughout storage than the
control fruit. The reason for the later increase in the antioxidant power of pomegranate fruit
remains unclear; however, it can result from the induced accumulation of anthocyanins and
enhanced antioxidant enzyme activities, thereby improving the fruit’s overall antioxidant
content. On the contrary, Fawole and Opara [1] reported an early increase in FRAP after
4 weeks of storage, which then declined for the rest of the storage period.
Plants 2022, 11, 351 13 of 24

Figure 8. Ferric reducing antioxidant power (FRAP) of uncoated (control) and coated pomegranate
fruit stored for 12 weeks at 5 ◦ C plus 3 days at 20 ◦ C. Each bar represents mean ± error bars. Error
bars represent SE of the mean values, and different letters on bars represent statistical differences
(p < 0.05) according to Duncan’s multiple range test. Dotted line represent measurement at harvest.

2.4. Physiological Disorders

The internal decay in the late-harvested pomegranate fruit was attributed mainly
to aril browning, while the external decay resulted from shrivelling (Figure 9A,B). The
incidence of both the physiological disorders increased with storage in the control fruit;
however, inconsistent changes were observed in the coated fruit. In support of our findings,
Fawole et al. [17] reported increased severity and incidence of the internal decay of ‘Won-
derful’ pomegranate fruit with extended storage. The reduction in physiological disorder
incidence and susceptibility by the EBR treatment might be attributed to the stimulated
activity of antioxidant enzymes, which results in enhanced antioxidant capacity of fruit
and, in turn, reduced ROS production, which mainly results in physiological disorders.
The visual appearances of fruit during storage showing the marked increase in shrivelling
are shown in Figure 10. The appearances showed an increase in shrivelling disorder with
storage. The apparent increases were observed in the control and CH 1.5% treatments,
which showed significantly higher shrivelling after 8 weeks of storage. Further, the CH
+ EBR (2 µM) and CH + EBR (5 µM) treatments showed a high increment in shrivelling
after 12 weeks of storage, whereas the score of the other treatments was still lower. This
suggests that the CH + EBR (10 µM) and CH + EBR (15 µM) treatments significantly inhib-
ited shrivelling and better maintained the visual appearance of fruit until the last week
of storage.
Plants 2022, 11, 351 14 of 24

Figure 9. Internal decay (A) and shrivel incidence (B) of uncoated (control) and coated pomegranate
fruit stored for 12 weeks at 5 ◦ C plus 3 days at 20 ◦ C.

Figure 10. Visual appearances of uncoated (control) and coated pomegranate fruit during storage at
5 ◦ C for 12 weeks plus 3 days at 20 ◦ C.

2.5. Correlation Matrix and Principal Component Analysis

Pearson’s correlation was used to reveal the degree of correlation between selected
variables during the last week of storage (Table 3). Weight loss was negatively correlated
with fruit texture (r = −0.87) and aril hardness (r = −0.94). This relationship clearly
showed that an increase in fruit weight loss decreased fruit texture and aril hardness,
Plants 2022, 11, 351 15 of 24

which indicates senescence. A strong and significant negative correlation was recorded
for fruit texture and electrolyte leakage (r = −0.86), supporting our previous discussion
that increased leakage of intracellular ions and metabolites results in reduced membrane
integrity. Furthermore, electrolyte leakage was negatively and strongly correlated with
TPC (r = −0.97), TAC (r = −0.95) and FRAP (r = −0.95), suggesting that enhancements in
the antioxidant capacity of the fruit, induced by CH + EBR coatings, inhibited the leakage
of intracellular ions. Moreover, this suggests that total phenolics, including anthocyanins,
and FRAP mainly influenced the fruit’s antioxidant capacity since ascorbic acid and DPPH
showed a poor correlation to electrolyte leakage. This was further supported by the
observed significant strong negative correlation of internal decay with TPC (r = −0.94)
and TAC (r = −0.87). In addition, shrivelling disorder was negatively correlated with
TPC (r = −0.9), TAC (r = −0.92) and FRAP (r = −0.96). Moreover, TA was positively and
strongly correlated with TPC (r = 0.88), TAC (r = 0.91) and FRAP (r = 0.86), suggesting
that limiting the consumption of organic acids through respiration, and hence, delayed
senescence, could be attributed to high antioxidant systems in the fruit, as evidenced by
the high TPC, TAC and FRAP in the treated fruit. Significant positive correlations were
recorded for TAC and TPC (r = 0.98), FRAP and TPC (r = 0.94), and FRAP and DPPH
(r = 0.95).
A principal component analysis (PCA) bootstrap hulls and PC×iplot were generated to
demonstrate distinctness between the coating treatments based on the evaluated parameters
on the last week of storage (Figure 11A,B). The first and second components explained
49% and 27.52% of the variation, respectively. Thus, the studied principal components
explained 76.51% of the total variation. From the bootstrap hulls plot (Figure 9A), six
detectable groupings were evident, which include control, CH 1.5%, CH + 2 µM EBR, CH
+ 5 µM EBR, CH + 10 µM EBR and CH + 15 µM EBR. A PCA biplot was used to identify
the features responsible for the clustering patterns (Figure 11B). Cluster 1 (Control) was
characterized by higher respiration, electrolyte leakage, decay and shrivelling. Cluster
2 (CH 1.5%) was closely related to cluster 1 and was characterized by high weight loss.
Cluster 3 (CH + 2 µM EBR) was characterized by high colour intensity and redness of the
peel. Cluster 4 (CH + 15 µM EBR) was characterized by high TA, TAC, TPC and FRAP.
These parameters were negatively correlated with the parameters in cluster 1. Cluster 5 (CH
+ 5 µM EBR) was a crosslink between cluster 4 and cluster 6 and did not have individual
characterization. Cluster 6 (CH + 10 µM EBR) was characterized by high colour intensity of
the arils, TSS, aril hardness, ascorbic acid, DPPH and fruit texture.
Plants 2022, 11, 351 16 of 24

Table 3. Pearson’s correlation matrix between quality attributes of uncoated (control) and coated pomegranate fruit stored for 12 weeks at 5 ◦ C plus 3 days at 20 ◦ C.
Values in bold are different from 0 with a significance level alpha = 0.05. Abbreviations: weight loss (WL), respiration rate (RR), fruit texture (FT), aril hardness (AH),
redness (a*), colour intensity (C*), hue angle (h◦ ), titratable acidity (TA), total soluble solids (TSS), ascorbic acid (AA), total phenolic content (TPC), total anthocyanin
content (TAC), 2,2-diphenyl-1-picryl hydrazyl (DPPH) free radical scavenging activity, and ferric ion reducing antioxidant power (FRAP).

A* C* h◦
a* C* h◦
WL RR FT AH EL (Ar- (Ar- (Ar- TA TSS BrimA AA TPC TAC DPPH FRAP Decay Shrivel
(Peel) (Peel) (Peel)
ils) ils) ils)
WL 1
RR 0.612 1
FT −0.867 −0.774 1
AH −0.941 −0.456 0.769 1
EL 0.749 0.496 −0.860 −0.566 1
a*
0.016 −0.406 0.196 −0.291 −0.357 1
(Peel)
C*
−0.016 −0.429 0.206 −0.270 −0.368 0.998 1
(Peel)
h◦
−0.225 −0.501 0.117 −0.053 −0.188 0.614 0.665 1
(Peel)
a*
−0.515 −0.092 0.613 0.673 −0.489 −0.373 −0.403 −0.690 1
(Arils)
C*
−0.606 −0.221 0.664 0.775 −0.446 −0.407 −0.429 −0.612 0.976 1
(Arils)
h◦
−0.196 −0.533 0.016 0.166 0.316 0.019 0.063 0.610 −0.477 −0.275 1
(Arils)
TA −0.743 −0.416 0.611 0.508 −0.814 0.438 0.478 0.603 0.050 0.054 0.027 1
TSS −0.789 −0.428 0.804 0.872 −0.596 −0.418 −0.413 −0.328 0.843 0.895 −0.080 0.281 1
BrimA −0.314 −0.166 0.409 0.541 −0.076 −0.682 −0.700 −0.688 0.788 0.839 −0.086 −0.344 0.805 1
AA −0.639 −0.688 0.661 0.695 −0.273 0.104 0.101 0.029 0.462 0.610 0.413 0.178 0.535 0.416 1
TPC −0.679 −0.498 0.777 0.438 −0.974 0.466 0.485 0.369 0.283 0.236 −0.248 0.875 0.442 −0.111 0.145 1
TAC −0.628 −0.356 0.673 0.386 −0.945 0.467 0.487 0.373 0.230 0.163 −0.316 0.906 0.350 −0.221 0.039 0.984 1
DPPH −0.915 −0.605 0.674 0.911 −0.431 −0.142 −0.102 0.307 0.320 0.478 0.528 0.572 0.662 0.299 0.716 0.372 0.317 1
FRAP −0.666 −0.374 0.739 0.468 −0.954 0.509 0.515 0.241 0.391 0.330 −0.368 0.856 0.400 −0.142 0.246 0.942 0.953 0.352 1
Decay 0.695 0.673 −0.865 −0.477 0.930 −0.353 −0.374 −0.327 −0.330 −0.318 0.111 −0.737 −0.580 −0.111 −0.232 −0.941 −0.870 −0.421 −0.807 1
Shrivel 0.770 0.448 −0.730 −0.568 0.896 −0.502 −0.522 −0.414 −0.277 −0.265 0.129 −0.944 −0.376 0.218 −0.351 −0.902 −0.917 −0.543 −0.959 0.759 1
Plants 2022, 11, 351 17 of 24

Figure 11. Principal component analysis (PCA) biplot showing the correlation between the measured
parameters and coating treatment during the last week (week 12) of storage. Bootstrap hulls (A) and
biplot (B). (a*) redness, (C*) colour intensity, (h◦ ) hue angle, (TA) titratable acidity, (TSS) total soluble
solids, (TAC) total anthocyanin content, (TPC) total phenolic content, (DPPH) 2,2-diphenyl-1-picryl
hydrazyl and (FRAP) ferric ion reducing antioxidant power.

3. Materials and Methods

3.1. Procurement and Handling of Fruit
Mature pomegranate fruit (cv. Wonderful) were procured from Ubali Pomegranate
Farm, Kameelfontein, Johannesburg (25◦ 380 31.9” S, 28◦ 270 33.0” E), South Africa, during the
Plants 2022, 11, 351 18 of 24

2020/2021 growing season. The fruit were immediately transported in a well-ventilated

vehicle to the postharvest and agroprocessing research laboratory at the University of
Johannesburg (PARL-UJ). Upon arrival, fruit were sorted for uniformity of colour and
size, and fruit with blemishes and external damage were removed. The fruit were then
disinfected with 0.01% sodium hypochlorite and air-dried before treatment application.

3.2. Preparation of Treatments

The polysaccharide-based edible coating, based on chitosan (Sigma Aldrich Chemical
Co., St. Louis, MO, USA) at 1.5% (w/v), was prepared by dissolving 15 g of chitosan in
deionized water containing 1% (v/v) of acetic acid and concentrations of 24-epibrassinolide
(EBR) (Sigma Aldrich Chemical Co. St. Louis, MO, USA) at 2, 5, 10 and 15 µM. EBR
concentrations were prepared by first dissolving the EBR powder in ethanol and then
made to the desired volume with distilled water. The final concentration of ethanol in
the solution was about 0.1%. The chitosan-EBR solution was made up to 1 L after adding
Tween-80 (0.1% w/v), glycerol (1% w/v) and canola oil (1% w/v). The mixture was further
subjected to magnetic stirring for 90 min at a controlled temperature of 40–50 ◦ C to ensure
complete solubility.

3.3. Experimental Design

A completely randomized design was used with five boxes as replicates per treatment
and each box containing ten randomly selected fruits. Coatings were applied by fruit
immersion for 3 min. Distilled water was used as a control treatment. Subsequently,
fruits were left to dry at 21 ± 2 ◦ C and 55 ± 5% RH and packed inside standard open-top
commercially used cartons. Fruits were then stored at 5 ◦ C with 85 ± 5 RH for 12 weeks.
The temperature (◦ C) and relative humidity (% RH) within the cold room were monitored
throughout the storage period using an Ebro EBI300 data logger (Ebro Electronic™, EBI300
data logger, Breisgau, Germany). At each sampling point, a batch of fruit was placed at
21 ± 2 ◦ C and 65–70% RH for a further 3-day period to simulate retail conditions before
measurements were made.

3.4. Physiological Responses

3.4.1. Weight Loss
Ten randomly selected fruit per treatment were individually weighed at 4-week inter-
vals using an electronic weighing scale (RADWAG Electronic PS 4500.R2.M, Poland, 0.01
accuracy). The results were expressed as mass loss percentages based on the initial fruit
mass on the day of harvest [1].

W (%) = [ (Wi − Wf/Wf) × 100] (1)

where W is the weight loss (%) of fruit, Wi (g) is the weight of fruit at the day of harvest and
Wf (g) is the weight of fruit at the given sampling interval, and the results were expressed
as mean ± S.E.

3.4.2. Respiration Rate

The fruit respiration rate was measured using the closed system method previously
described by Fawole and Opara [1]. Briefly, in five replicates, two fruit were placed in a 3 L
glass jar, hermetically sealed with a lid containing a rubber septum in the middle for 2 hrs
at room temperature. After the incubation time, CO2 production inside the glass jar was
measured using an infrared O2 /CO2 gas analyser (Checkmate 3, PBI Dansensor, Denmark).
The results obtained for CO2 production were presented as mean ± S.E. (ml CO2 /kg··h).

3.4.3. Electrolyte Leakage

Electrolyte leakage (EL) was determined using the method described by Mirdehghan
et al. [64], with modification by Sayyari et al. [65]. Briefly, 6 discs (10 mm) of peel tissue
Plants 2022, 11, 351 19 of 24

per replicate were cut using a cork borer and then immersed in 25 mL of 0.4 M mannitol
and incubated for 4 h at room temperature under constant shaking; subsequently, the
initial conductivity (Ci) of the solution was measured using a conductivity meter (Hanna
Instruments 9033, Woonsocket, RI, USA). After readings were taken, the samples were
autoclaved (Medsource TC-459 autoclave, Taichung, Taiwan) at 101.3 KPa and 121 ◦ C for
20 min and allowed to cool down at room temperature for 24 h before the final conductance
(Cf) was measured. EL was expressed as a percentage of total electrical conductivity using
Equation (2), according to Ehteshami et al. [12]

EL (%) = [(Ci/Cf) × 100] (2)

3.4.4. Textural Dynamics

Fruit texture (puncture resistance) was determined following a procedure developed
by Fawole and Opara [66]. This was conducted using a firmness analyser with a 5 mm
diameter cylindrical probe (GÜSS-FTA, Strand, South Africa) programmed to penetrate
8.9 mm into the fruit at the speed of 10 mm.s−1 . Tests were performed on the two equilateral
regions of 10 individual fruit. The peak force required to puncture the fruit skin was taken
as the puncture resistance, and the results were expressed as mean ± S.E.
The aril compression test was performed using a texture analyser meter (Agrosta
texture analyser, Calib, France) with a 35 mm compression probe. A total of 20 arils per
treatment were used, and the test was run per aril aligned horizontally on the compression
platform. The textural profile was interpreted using force (N) and distance (mm) as the
fundamental variables. The instrument’s operating conditions were: 1 mm/s probe test
speed and 0.30 N trigger force.

3.5. Physico-Chemical Properties

3.5.1. Colour Attributes
Colour measurements were assessed in CIELAB coordinates (L*, a*, b*) using a
colourimeter (Konica Minolta Chroma Meter CR-400, Osaka, Japan), which was calibrated
using white standard tile. External colour change was monitored by measuring peel colour
of eight randomly selected fruits per treatment at the two opposite equatorial positions.
Duplicate colour measurements were made on arils placed in a colourless glass Petri dish
for internal colour monitoring. The colour parameters, chroma (C*) and hue angle (h◦ )
were then calculated from the (L*, a*, b*) coordinates using the Equations (3) and (4),
respectively [1].
C* = [ (a*2 + b*2 )]1/2 (3)
h◦ = arctan (b*/a*) (4)

3.5.2. Total Soluble Solids and Titratable Acidity

Total soluble solids (TSS) and titratable acidity (TA) were estimated from pomegranate
juice extracted from the arils of ten fruits per treatment per sampling point. Juice from
arils of each fruit was extracted using a juice extractor (Salton juice extractor, Brunswick,
Canada) without crushing the seeds. TA was measured using a non-automated laboratory
titrator (Thermo scientific, Orion star T910, Chelmsford, MA, USA) according to the method
described by Fawole and Opara [1]. Briefly, 2 mL of fresh was mixed with 90 mL of distilled
water and titrated with 0.1 M NaOH to a pH value of 8.2. The results were expressed as a
percentage of citric acid. TSS was determined using a digital refractometer (Atago, Tokyo,
Japan). BrimA, a criterion for consumer acceptance of juice, was also calculated using
Equation (5).
BrimA = TSS − k (TA) (5)
where k is a constant that reflects the tongue’s higher sensitivity index, ranging from 2 to 10.
In this study k-value of 2 was used to avoid a negative BrimA index of the pomegranate [67].
Plants 2022, 11, 351 20 of 24

3.6. Phytochemical Contents and Antioxidant Properties

3.6.1. Ascorbic Acid Content
Ascorbic acid content was determined using a method described by Kawhena et al. [14].
Briefly, 1 mL of pomegranate juice (PJ) was extracted with 9 mL of 1% metaphosphoric acid
(MPA). The mixture was then vortexed and sonicated (Labotech Sonic Clean; model: 705;
Gauteng, South Africa) for 3 min in ice-cold water. The mixture was centrifuged (Heraeus
Biofuge, Stratos) at 4 ◦ C for 10 min at the speed of 10,000 rpm. A quantity of 500 µL of
the extract was then mixed with 4.5 mL of 2,6-dichlorophenolindophenol (dye) in a dark
room. The samples were further incubated for 30 min in the dark. After incubation, the
absorbance was measured at 515 nm against a blank in a UV-visible spectrophotometer
(United scientific, SP-UV 300, Shanghai, China). The ascorbic acid content of each sample
was calculated based on the calibration curve of standard L-ascorbic acid. The results
were expressed as milligrams of ascorbic acid equivalent per hundred millilitres of crude
pomegranate juice (mg AA/100 mL PJ).

3.6.2. Total Phenolic Content

Total phenolic compound extraction and quantification was carried out using a Folin
andCiocalteu’s method previously described by Fawole and Opara [67] with slight modifi-
cations. Briefly, 1 mL of pomegranate juice (PJ) was extracted with 29 mL of 50% aqueous
methanol. The resulting mixture was vortexed and then sonicated in ice for 20 min in a cold
water bath. In duplicates, 50 µL of methanolic PJ extract was mixed with 450 µL of 50%
methanol followed by the addition of 500 µL of Folin and Ciocalteu0 s phenol reagent and,
after 2 min, sodium carbonate (2%) was added. The mixture was vortexed and incubated
for 40 min in a dark room. The absorbance was subsequently measured at 725 nm using
a UV–visible spectrophotometer (United scientific, SP-UV 300, Shanghai, China). The
results were expressed as milligrams of gallic acid equivalent per 100 mL of crude PJ (mg
GAE/100 mL PJ) and expressed as mean ± S.E.

3.6.3. Total Anthocyanin Content

The pH differential method was used to determine the total anthocyanin content
using two buffer systems, namely potassium chloride (KCL) buffer for pH 1 and sodium
acetate (C2 H3 NaO2 ) buffer for pH 4.5. Briefly, 0.5 µL of the extracted juice was mixed with
3.5 mL of KCL and C2 H3 NaO2 buffers, respectively, and incubated for 30 min in a dark
environment. Samples were read at 510 nm and 700 nm in a UV–visible spectrophotometer
(United scientific, SP-UV 300, Shanghai, China). The results were expressed as milligram
cyanidin-3-glucoside equivalent per 100 mL of crude PJ (mgC3gE/100 mL PJ) according to
the following Equations (6) and (7):

A = [(A510 − A700) pH 1 − (A510 − A700) pH 4.5] (6)

TAC (mg/100 mL) = [(A × MW × DF × 100/] × L (7)

where A = Absorbance values at 510 nm and 700 nm,  = Cyanidin-3-glucoside molar
absorbance (26,900), MW = Cyanidin-3-glucoside molecular weight (449.2 g/mol), DF =
Dilution factor, L = Cell path length (1 cm) and TAC = total anthocyanin content.

3.6.4. DPPH Radical-Scavenging Activity

The DPPH assay was carried out according to the method reported by Fawole and
Opara [67]. Methanolic extract of PJ (15 µL) was then diluted with 735 µL methanol
followed by the addition of 750 µL methanolic DPPH solution (0.1 mM). The mixture was
vortexed and incubated for 30 min in the dark. Thereafter, the absorbance was measured at
517 nm using a UV–vis spectrophotometer (United scientific, SP-UV 300, Shanghai, China).
The free-radical capacity of PJ was expressed as ascorbic acid (mM) equivalent per 100 mL
of crude PJ (mM AAE/100 mL).
Plants 2022, 11, 351 21 of 24

3.6.5. Ferric Reducing Antioxidant Power (FRAP)

The antioxidant power of fruit was measured according to the method by Benzie and
Strain [68] with modifications by Fawole et al. [69]. A FRAP working solution (300 mM
acetate buffer (50 mL), 2,4,6-tripyridyl-s-triazine (TPTZ) (5 mL) and 20 mM FeCl3 (5 mL))
was freshly prepared prior to the measurements. In duplicate, 150 µL of aqueous methanolic
PJ extracts (150 µL) was mixed with 2850 µL of the FRAP working solution and incubated
in the dark for 30 min. Thereafter, the absorbance was measured at 593 nm using a UV–
vis spectrophotometer (United scientific, SP-UV 300, Shanghai, China). The results were
expressed as Trolox (mM) equivalents per 100 mL of crude PJ (mM TE/100 mL PJ).

3.7. Fruit Physiological Disorders and Decay

The disorders assessed included shrivelling and aril browning. These were visually
inspected at 4-week intervals. The degree of incidence of the disorders was subjectively
determined using a hedonic scale as described by Fawole and Opara [1], where 0 = none
(no symptoms), 1 = trace (1–25%), 2 = slight (26–50%), 3 = moderate (51–75%) and 4 =
severe (76–100%). The disorder index was then calculated using the following equation:

Disorder index = [(value of hedonic scale × no. of fruit at each scale number)/total no. of fruit] (8)

3.8. Statistical Analysis

The data collected were subjected to the analysis of variance (ANOVA) using statistical
software (GenStat 18th Edition, VSN International, Hemel Hempstead, UK). Mean values
were separated by Duncan’s multiple range using the least significant difference (LSD) test
at the 5% level of significance. Pearson’s linear correlation coefficients (r) and principal
component analysis (PCA) were carried out using the statistical software XLSTAT Version
2020.4.1.1020 (Addinsoft, Paris, France).

4. Conclusions
Overall, the results of this study showed that the higher concentrations of EBR (CH + 10
and 15 µM EBR) had a considerable effect on the fruit quality of late-harvested pomegranate
fruit. Based on the correlation and PCA analysis, CH + 10 µM EBR significantly delayed
fruit metabolic changes, resulting in higher fruit texture, aril hardness, DPPH, TSS and
colour intensity. This was mainly attributed to the higher DPPH and ascorbic acid of
the treatment. However, the high phenolics, including the anthocyanins and FRAP of
the CH + 15 µM EBR treatment, showed the best effectiveness in terms of reducing the
susceptibility of late-harvested pomegranate fruit to physiological disorders, particularly
aril browning and shrivelling. However, CH + 10 µM EBR treatment could be recommended
to maintain quality and reduce susceptibility to physiological disorders of the late-harvested
pomegranate fruit due to its extended and broader effect compared to the CH + 15 µM
EBR treatment. Studies investigating the interaction of EBRs with other plant hormones,
growth regulators and a combination of EBR treatment with other postharvest technologies
such as controlled atmosphere, modified atmosphere and edible coating technologies are
still required.
In addition, enzymatic, metabolomic and proteomics-based studies to further under-
stand the mechanism of brassinosteroids’ exogenous postharvest application in fruit and
vegetables are also still required.

Author Contributions: S.M., formal analysis, methodology, investigation, validation, writing—

original draft preparation; O.A.F., conceptualisation, writing—review and editing, visualisation,
supervision, funding acquisition, project administration, software, visualization, resources. All au-
thors have read and agreed to the published version of the manuscript.
Funding: This work is based on the research supported wholly or partially by the National Research
Foundation of South Africa (Grant Number: 129295). The authors are grateful to the University
Research Committee at the University of Johannesburg for financial support received by S.M.
Plants 2022, 11, 351 22 of 24

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author. The data is not publicly available due to an ongoing PhD program.
Conflicts of Interest: The authors declare no conflict of interest.

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