Scientia Horticulturae 238 (2018) 343–349

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Effectiveness salicylic acid blending in chitosan/PVP biopolymer coating on T

antioxidant enzyme activities under low storage temperature stress of
‘Banati’ guava fruit
⁎
A.A. Lo’aya, , Mohamed A. Taherb
a
Pomology Department, Faculty of Agriculture, Mansoura University, El-Mansoura, P.O. Box 35516 El-Mansoura, Egypt
b
Agricultural Chemistry Department Faculty of Agriculture Mansoura University, El-Mansoura, P.O. Box 35336 El-Mansoura, Egypt

A R T I C LE I N FO A B S T R A C T

Keywords: The performance of chitosan/poly-vinyl-pyrrolidine (CS/PVP) blending in salicylic acid (SA) to increase chilling
Guava injury tolerance of ‘Banati’ guava fruit under cold otherwise the impacts of CS/PVP-SA on antioxidant enzymes
Cold storage were assessed. The guava fruits were coated with CS/PVP biopolymer with SA at different concentration (0,1,
Antioxidant enzymes and 2 mM) and stored at low temperature (6 °C and 90.95% RH) for fifteen days. Fruit samples were picked every
Chilling injury
three days interval. The physical measurements weight loss%, chilling injury index (CI-index), and fruit skin
color hue angle (h°) were assessed. The chemical analysis for instance soluble solid content (SSC%), titratable
acidity (TA%), SSC/AT-ration, total chlorophyll content, fruit firmness. The antioxidant enzyme activities were
evaluated such as catalase (CAT, EC: 1.11.1.6), peroxidase (POD, EC:1.11.1.7), ascorbate peroxidase (APX, EC:
1.11.1.11), and superoxide dismutase (SOD, EC: 1.15.1.1). Also, plasma cell membrane compartments such as
lipid peroxidation (malondialdehyde, MDA), protein oxidation (protein carbonyl group, PCG), cell membrane
ion leakage (IL%), O2%− and H2O2 production rate were measured. CS/PVP-SA 2mM significantly reduced water
loss, CI-index, h°, maintained fruit skin chlorophyll content, firmness, and delayed the qualitative changes SSC%,
TA% and SSC/TA-ratio due to increase the CS/PVA and SA at 2 mM. Moreover, changes in the antioxidant
capacity of CS/PVP-SA 2mM coated guava fruits were activated. The activation of antioxidant enzymes, alle-
viating CI-index and reducing plasma cell membrane damage. SA bending in Chitosan/PVP biopolymer coating
and storage fruit under cold temperature was obtained using parameters to detect the effect of CS/PVP-SA
treatments. CS/PVP-SA 2mM exhibited a reduction of chilling injury incidences compared to uncoated fruit in all
in overall measurements.

1. Introduction
Guava (Psidium gujava L. cv ‘Banati’) is viewed and a primary sub-
tropical fruit. it is grown extensively in tropical and subtropical regions
of the world (Silva et al., 2018). There are several guava varieties
planted in Egypt. The cultivated area and the crop yield of guava are
according to the latest statistics about 20.6 thousand hectares produ-
cing 350 thousand tons annually according to the Egyptian Ministry of
Agriculture, (E. M. A., 2016). Nutritionally, It contains highly nutri-
tional compounds such as vitamin C, a wide range of types of car-
otenoids (Uddin et al., 2002). However, the main problem of guava
fruit has shown rapid ripening processes after harvesting. It exhibits a
high respiration rate and ethylene production (Hong et al., 2012). Since
guava fruit is classified a climacteric fruit (Kumar et al., 2015). Con-
sequently, the fast ripening forced fruit to enhance deterioration which
leads to short shelf-life, limits transportation and storage period (Hong
et al., 2012). Numerous investigations were led to keeping fruit quality
characteristics. Many different procedures were implemented to control
shelf life or storage. These techniques are cold storage, controlled at-
mosphere, pre and postharvest chemical substances, and biopolymer
coating treatment (Anggarwulan et al., 2015). Therefore, it is essential
to make sense of a probable answer for limit fruit quality decay and
enhance fruit quality after harvesting.
Fundamentally, the cold storage is one of the main which is used to
slow down the physiological processes such as respiration and ethylene
production after harvesting. Despite, the low cold storage temperature
generates active oxygen species (AOS) which are produced in different
organs in plant cells. These are mitochondria, plasma cell membrane,
cytoplasm, and cell wall (Lo’ay, 2005). Moreover, AOS generates under
different stresses, for instance, drought, salinity, reoxygenation and low

⁎
Corresponding author.
E-mail addresses: loayaa2016@gmail.com (A.A. Lo’ay), Mohamedtaher@mans.edu.eg (M.A. Taher).

https://doi.org/10.1016/j.scienta.2018.05.005
Received 27 November 2017; Received in revised form 30 April 2018; Accepted 2 May 2018
Available online 08 May 2018
0304-4238/ © 2018 Elsevier B.V. All rights reserved.
A.A. Lo’ay, M.A. Taher
temperature (Chan et al., 2016). So, enhancing AOS generation leads to
cell compartments damage and the death (Foyer et al., 2017), by which
it reduces fruit quality (Cheng et al., 2010). Accordingly, the alternative
technique that use to reduce AOS formation and increase/improve fruit
quality after harvesting can be important for both producer and con-
sumer (Silva et al., 2018). The implemented technique has been used to
increase storage ability or shelf life of fruit such biopolymer coating
(chitosan) has been affected by fruit quality during shelf life. Chitosan is
highly carbohydrate molecular weight, soluble in an organic acid, and
polysaccharide (Lo’ay and Dawood, 2017a). Also, it is considered a
natural biodegradable polymer and non-toxic material (Drevinskas
et al., 2017). Plus, it could be leading in chitosan with another polymer
such poly-vinyl-pyrrolidine or organic acid (Lo’ay and Dawood, 2017b).
Additionally, it becomes profitable on protection thickness, absorbency,
solubilization, and crystallization. Moreover, it has low toxicity and it is
utilized in board rage of areas such as medical, food cosmetic, and
health-related domains (Drevinskas et al., 2017). Despite, salicylic acid
(SA) is considered an endogenous phenolic nature synthesis that it plays
as the plant growth regulator. Mainly, SA has optimistically impacted
on fruit respiration and ethylene biosynthesis rates (Srivastava and
Dwivedi, 2000). It is integrating fruit water loss, microbial infection,
and maintaining fruit firmness during fruit handling or marketing
(Lo’ay, 2017).
The target of this our study evaluated the capability of chitosan/
PVP blend with salicylic acid as a coating treatment, on the increase of
capacity storage life of 'Banati' guava fruit. Also, the effect of various
concentrations of SA mixing in biopolymer chitosan/PVP on post-
harvest life and fruit quality properties during low-temperature stress.
2. Material and methods
2.1. Fruit materials
The study was done on guava (Psidium guajava L. cv 'Banati')
planted in topsoil soil in a commercial orchard close to Damietta Gov.,
Egypt. The investigation was conducted during tow seasons 2016–2017.
Fruit samples were picked at yellow-green development arrange (fruits
have more yellow than green color) as indicated by color (hue angle)
estimation (Lo’ay and EL-Khateeb, 2011). The fruit was transported in
the cooler van at 10 °C for three hours. Upon arriving at Lab, fruit
samples (480 natural products) were separated into two major groups.
The first batch contains 240 fruits that distributed on four treatments.
Every treatment contains 60 fruits which are distributed on three re-
plicates (20 fruits), for estimating water loss rate, chilling injury index
and fruit skin color hue angle. Be that as it may, the second batch 240
fruits additionally organized as represented previously general treat-
ments for chemical estimations.
2.2. Biopolymer chitosan/PVP leading to salicylic acid preparation
The chemical materials were utilized as a part of this examination:
PVP (K-30 polymer; Ashland organization, China), chitosan (CS) (MW
71.3 = kDa, the level of deacetylation = 94%; Merck, Darmstadt
Germany), salicylic acid was the explanatory review. chitosan (CS)
solution (750 ml, 1% w/v) was prepared by dissolving 7.5 g of CS in
750 ml of 2% (v/v) aqueous CH3COOH anhydrous solution for 8 h
under magnetic stirring. The arrangements of PVP and CS were delib-
erately blended at a proportion of (1:1) and mixed for 2 h. At last, the
resultant arrangement (1500 mL) was similarly subdivided into three
sets (500 mL in everyone). The control set was not supplemented with
any added substance. Salicylic acid was disintegrated and blended into
the mixed arrangements of set 2 and 3 under magnetic stirring for 4 h at
25 °C at two SA concentrations (2 and 4 mM), separately. The mixed
arrangements were put in funnel-shaped carafes and stored at 4 °C until
further examination.
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Scientia Horticulturae 238 (2018) 343–349
2.3. Treatments protocol
The treatments were prepared for the following control, CS/PVP-SA
0mM, CS/PVP-SA 1 mM, and CS/PVP-SA 2 mM. Fruits were immersed in
CS/PVA-SA coating for 5 min, and they were stored at cold temperature
(6 ± 1 °C and air humidity percentage average during the storage
period 90.95% RH) for 15 days
2.4. Non-distractive measurements
Quality elements were proposed, guava fruits were randomly picked
from every treatment and were isolated into three replicates to gauge
weight loss rate based on underlying the initial weight of fruit at har-
vest time and it exhibits in rate. Chilling injury symptoms was tried by
judging the degree the chilling damage of fruit peel as per the accom-
panying scale: 1 = no injury; 2 = light injury; 3 = direct injury (25%
surface influenced); 4 = extreme injury (26-half surface influenced)
and 5 = exceptionally serious harm (> 50% surface influenced) as
depicted (Lo’ay, 2005).The chilling damage file is then figured utilizing
the accompanying formula:
Chilling injury index
5
(Chilling injury index)*(Number of fruits at the level)
= ∑
1→5 Total number of fruit
Fruit skin color profile was recorded (Lo’ay and EL-Khateeb, 2011),
from that point, all pictures were investigated by utilizing programming
ImageJ Ver.1.43 u the USA to get RGB signs to determine fruit skin
color hue angle (Khojastehnazhand et al., 2010). Fruit firmness was
recorded on fruit using Effegi-penetrometer supplemented with a
plunger 8 mm diameter penetrator and it was presented an N unit
(Samaan et al., 2012).
2.5. Chemical measurements
Total soluble solid content (SSC%) was measured utilizing Carlzeiss
hand refractometer and total acidity percentage (TA) was dictated by
titration with 0.1 N NaOH (A.O.A.C., 1995). So, the SSC/TA ratio was
computed as a characterized development record. In term of aggregate
chlorophyll was extricated by drenched 2 g of fruit skin in 20 mL of N,
N-dimethylformamide (DMF). Samples were stored at 4 °C for 16 h to
permit the DMF to separate the chlorophyll. At long last, samples were
centrifuged for 5 min at 15,000 rpm, at that point an clear supernatant
example was proposed chlorophyll A at 663 nm and B at 646 nm wa-
velength utilizing spectrophotometer (UV–vis auto). It exhibited in mg
100 g−1 FW. As to carotene pigment was recorded at 452 nm and it
exhibited in mg 100 g−1 FW (Lo’ay, 2005).
2.6. Antioxidants enzyme activities assessment
Fruit sample (10 g) from every treatment was picked and homo-
genized in 25 mL of ice-cold extraction buffer and 0.5 g poly-vinyl-poly-
pyrrolidone (PVPP) with a Kinematica tissue processor (Crl-6010,
Kriens-LU, Switzerland). For CAT and SOD examines, the extraction
buffer was 50 mM sodium phosphate (pH 7.8). For POD, 100 mM so-
dium phosphate cradle (pH 6.4) was utilized. The homogenate was
centrifuged at 27,000 rpm for 50 min at 4 °C and the subsequent su-
pernatants were utilized shortly for examining of CAT and SOD enzyme
activities (Wang et al., 2005).
In term of, CAT, the response blend comprised of 2.8 mL H2O2
(40 mM, in 50 mM sodium phosphate buffer, pH 7.0) and 0.2 mL en-
zyme extract. The deterioration of H2O2 was estimated by the decrease
in absorbance at 240 nm amid 120 s. The activity was reported as Unit
g−1 FW, where one unit of catalase changes over l moL of H2O2 per
mass of fruit per min at 30 °C.
For SOD, the response blend (3 mL) contained 65 mM sodium
A.A. Lo’ay, M.A. Taher
phosphate cradle (pH 7.8), 13 mM methionine, 75 μM nitro-blue tet-
razolium (NBT), 10 μM EDTA, 2 μM riboflavin and 0.1 mL of the en-
zyme extract. The blends were lit up by light (60 moL m−2 s−1) for
10 min and the absorbance was then determined at 560 nm. The
Mixture held oblivious filled in as clear. Turf action was communicated
as unit g−1, where one unit was characterized as the measure of che-
mical that caused a 50% decreases of the SOD inhabitable NBT di-
minishment per mass of natural product mash every hour.
Ascorbate peroxidase (APX) action was defined and marked one
unit of APX as the measure of catalyst that oxidized 1 μmoL ascorbate
min−1 at 30 °C. The particular movement was communicated as Unit
g−1 FW (Zhang et al., 2013).
POD activity was measured at 30 °C and determined by monitoring
the increase rate of absorbance at 398 and 460 nm. POD activity was
expressed as unit g−1, where one unit was expressed as the increase rate
of absorbency per mass of fruit pulp per min (Tian et al., 2005).
2.7. Lipid and protein of plasma cell membrane degradation and
permeability
Fruit sample was ground in a mortar and mixed with 25 ml of 5%
(w/v) metaphosphoric acid, 500 μl of 2% (w/v) butylated hydro-
xytoluene in ethanol, and finally homogenized by a mixer. The cali-
bration curves made by measuring 1,1,3,3-tetraethyoxypropane
(Sigma) in the range 0–2 mM (TBARS) which was equivalent to 0–1 mM
malondialdehyde (MDA). 1,1,3,3-Tetraethyoxypropane is stoichiome-
trically converted into MDA during the acid-heating step of the assay
(Iturbe-Ormaetxe et al., 1998). The amount of TBARS present is ex-
pressed as MDA equivalents. Protein carbonyl group as a terminal
product of protein oxidation was measured according to (Levine et al.,
1994). Samples from fruit peel about 2.5 g were ground in a mortar and
mixed with 10 ml of 20 mM potassium phosphate buffer (pH 7.0) to
extract soluble proteins. Afterward, the spectrum was measured
spectro-photochemically (UV–vis spectrophotometer) against the com-
plementary blank in case of cured (without sample) samples or against
water in case of purified proteins. The carbonyl content of the protein
was calculated from the absorbance of the 2,4-dinitrophenylhydrazine
measured at 390 nm and assuming an extinction coefficient of
22,000 M−1 cm−1.
Samples of 6 fruits were collected from the peel tissue. The disks
were washed three times with demineralized water and placed in 10 mL
0.4 M mannitol in demineralized water at 24 °C for 3 h. The electrical
the conductivity of the aqueous phase was measured using conductivity
meter, after which the tissue samples were killed by heating in water
bath at 100 °C for 20 min. This cooking process allows the release of all
electrolytes from the tissue. Once cooled to room temperature the
conductivity was re-measured and the relative electrolyte leakage from
the uncooked peel samples was calculated as a percentage (Lo’ay,
2005).
2.8. Measurements of O2%− rate and H2O2 production
Fruit peel samples (1.0 g) were homogenized in 3 mL of cold 50 mM
potassium phosphate buffer (pH 7.8) containing 1% (w/v) poly-vinyl-
pyrrolidone (PVP) and then centrifuged at 5000×g at 4 °C for 15 min.
The O2%− production rate was measured by monitoring the nitrite
formation from hydroxylamine in the presence of O2%− (Yang et al.,
2011). A standard curve with NO2 was used to calculate the O2%−
production rate from the reaction equation of O2%− with hydro-
xylamine. The O2%− production rate was expressed as nmol
min−1 g−1 FW. The content of H2O2 was assayed (Xu et al., 2012). Peel
sample (1.0 g) was homogenized in 6 mL of 100% acetone and then
centrifuged at 5000×g for 10 min at 4 °C. The clear supernatant (1 mL)
was mixed with 0.1 mL of 5% Ti (SO4)2 and 0.2 mL of concentrated
NH4OH solution. The titanium-peroxide complex precipitated and this
sediment was dissolved in 4 mL of 2 M H2SO4 after centrifugation at
345
Scientia Horticulturae 238 (2018) 343–349
3000×g for 10 min. The absorbance was measured at 415 nm. The
H2O2 content was calculated from a standard curve prepared in a si-
milar way and expressed as nmol g−1 FW.
2.9. Statistical analysis
Data of means from the experiment for evaluation of water loss %,
chilling injury index, and the fruit skin color hue angle was analyzed
using one-way analysis of ANOVA with complete block randomize de-
sign when CS/PVP-SA treatments as a factor were considered. However,
the difference between treatments means was compared using two-way
analysis for chemical measurements when shelf life time (days) and CS/
PVP-SA treatments were studied. Means of all studied treatments were
compared using Duncan’s Multiple Range Test at P < 0.05 using the
CoStat software package, Version 6.303 (789 lighthouse Ave PMB 320,
Monterey, CA, 93940, USA).
3. Results and discussion
3.1. Physical quality attributes
The physical quality features as a function of storage time (days) for
the CS/PVP mixed with various SA concentrations appeared in Table1.
CS/PVP-SA treatments show a significant impact when there were
considered as a factor. Considering, the CS/PVP-SA 2 mM treatment
presents an essentially more reduction in water loss rate (11.82%),
slightly chilling injury indications showing up on fruit skin (1.08 nearly
CI-index 1, no symptoms), fruit color hue angle was kept up
(108.07 h°), and the total chlorophyll content (4.99 mg 100 g−1 FW)
compared with control and different CS/PVP-SA treatments. Be that as
it may, the control fruits treatment introduced a more rapid loss in
physical properties during the storage time. It is recorded more loss in
water (31.11%), more chilling injury indications (4.84 CI-list more than
4 particularly severity injured), fruit color hue angle (77.92 h°), and
more quick loss in all-out chlorophyll (2.92 mg 100 g−1 FW) at 15th
day of the cold storage period. This could be because of the mixing SA
in CS/PVP biopolymer blend which gives more successful than CS/PVP
only (Petriccione et al., 2015). Likewise, the procedure of SA in bio-
polymer coating, give an insurance or quenching active oxygen species
(AOS) during cold storage (Sayyari et al., 2011). Also, it is under-
standable that the coating fruit by CS/PVP-SA 2mM reducing water loss
by limiting water transpiration from fruit skin surface by supporting the
common wax layer on fruit skin surface which it was changed during
fruit growth (Lo’ay, 2011). Additionally, the mixture of SA in CS/PVP
biopolymer coating blend could be limited cell wall degrading enzyme
activities, for example, polygalacturonase (PG), xylanase (XYL), and
pectinase (PT) during storage (Lo’ay and Dawood, 2017b). In this way,
less water loss from stored fruit (Champa et al., 2015), decreasing
chilling injury occurrence (Sayyari et al., 2011), and keeping up fruit
color hue angle and total chlorophyll content (Srivastava and Dwivedi,
2000). Additionally, it may be the case that the part of SA to expand
cancer prevention agent catalyst exercises amid cool stockpiling worry
by searching for AOS (Pal et al., 2004)
3.2. Chemical quality attributes
Table 2 depicts a significant interaction at P ≤ 0.001 between sto-
rage time (days) and CS/PVP-SA treatments. Noticeably, total soluble
solid content increased slightly and gradually overall treatments up to
end of cold storage period compared to the initial value at harvest time.
However, TA% decreased during storage time which recorded much
higher decreases than the initial values at harvest time. Detectably, the
coated treatment CS/PVP-SA 2mM provides more stability on these
parameter compared to control fruits and CS/PVP-SA treatments during
cold storage. It was recorded SSC%, 11.61; TA, 0.557; and SSC/TA-
ration, 20.84% compared to the control fruits (SSC%, 12.69; TA, 0.398;
A.A. Lo’ay, M.A. Taher Scientia Horticulturae 238 (2018) 343–349

Table 1
Effect of postharvest of the CS/PVP with salicylic acid application at different concentration on water loss%, fruit skin chilling injury index, fruit color hue angle, and
total chlorophyll content of fruit mature color guava ‘Banati’ fruit during 15 days under cold storage (6 °C ± 1).
Measurements Storage time (days)

D0 D3 D6 D9 D12 D15

Water loss %
a
Control 0.00 5.18 ± 0.549 12.00 ± 0.887 a 21.38 ± 1.296 a 28.46 ± 0.815 a 31.11 ± 0.158 a

b
CS/PVP-SA 0 mM 0.00 2.95 ± 0.039 9.58 ± 0.356 b 14.35 ± 0.586 b 18.17 ± 0.555 b 19.49 ± 0.692 b

b
CS/PVP-SA 1 mM 0.00 2.54 ± 0.043 8.05 ± 0.462 c 10.43 ± 0.468 c 12.10 ± 0.398 c 14.31 ± 0.414 c

b
CS/PVP-SA 3 mM 0.00 2.28 ± 0.024 5.91 ± 0.327 d 8.95 ± 0.211 c 9.93 ± 0.303 d 11.82 ± 0.320 d

Chilling injury-index
a a a a a
Control 1.00 1.27 ± 0.014 1.58 ± 0.046 2.50 ± 0.089 3.37 ± 0.080 4.84 ± 0.085
b b b b b
CS/PVP-SA 0 mM 1.00 1.00 ± 0.000 1.17 ± 0.080 1.75 ± 0.030 1.96 ± 0.036 2.08 ± 0.012
b c c c c
CS/PVP-SA 1 mM 1.00 1.00 ± 0.000 1.00 ± 0.000 1.05 ± 0.014 1.13 ± 0.011 1.23 ± 0.011
b c c c d
CS/PVP-SA 3 mM 1.00 1.00 ± 0.000 1.00 ± 0.000 1.00 ± 0.000 1.02 ± 0.003 1.08 ± 0.017

Fruit skin color hue angle (h°)

a c
Control 124.38 ± 1.929 116.46 ± 1.588 97.81 ± 1.269 d 91.08 ± 1.804 d 82.41 ± 0.549 d 77.92 ± 1.391 d
a b
CS/PVP-SA 0 mM 124.38 ± 1.929 120.38 ± 0.338 110.89 ± 1.266 c 99.19 ± 0.841 c 93.33 ± 1.362 c 88.29 ± 1.016 c
a ab
CS/PVP-SA 1 mM 124.38 ± 1.929 122.01 ± 0.330 117.88 ± 0.371 b 105.88 ± 2.771 b 102.86 ± 1.443 b 101.55 ± 0.633 b
a a
CS/PVP-SA 3 mM 124.38 ± 1.929 123.69 ± 1.061 120.46 ± 0.568 a 113.18 ± 0.612 a 109.90 ± 1.370 a 108.07 ± 1.553 a
−1
Total chlorophyll content (Chl ab mg 100 g FW)
Control 5.83 ± 0.306 a 5.17 ± 0.087 cde
4.72 ± 0.186 fg
3.89 ± 0.320 h
3.06 ± 0.033 i
2.92 ± 0.037 i

CS/PVP-SA 0 mM 5.83 ± 0.306 a 5.32 ± 0.006 bcd

5.09 ± 0.008 cdef
4.92 ± 0.017 ef
4.46 ± 0.026 g
4.06 ± 0.031 h

CS/PVP-SA 1 mM 5.83 ± 0.306 a 5.41 ± 0.020 bc

5.16 ± 0.025 cde
4.99 ± 0.003 def
5.00 ± 0.000 def
4.87 ± 0.058 ef

CS/PVP-SA 3 mM 5.83 ± 0.306 a 5.66 ± 0.170 ab

5.31 ± 0.063 bcd
5.09 ± 0.008 cdef
5.02 ± 0.015 def
4.99 ± 0.003 def

CS/PVP-SA treatments mean in a column are significantly different at (P < 0.05), using Duncan’s Multiple Rang Test, each value represents mean and ± SE (n = 3)
replicates. The superscript letters, indicated significantly between effect of treatments.
Total chlorophyll content: the interaction between storage time (days) and CS/PVP-SA treatments.
Chilling injury index classes: (1 = no symptoms healthy; 2 = slightly symptoms (1–20%); 3 = moderately (21-50%); 4 = sever (51-80%), and 5 = very severe injury
(81-100%).

and SSC/TA-ration, 31.85%). The observed data could be associated
with the effect of biopolymer coating treatment loaded with salicylic
acid at 2 mM it reduces water transpiration (Sun et al., 2014), and the
presence of SA to biopolymer coating delays fruit ripening/senescence
(Srivastava and Dwivedi, 2000), and activities antioxidant enzymes
during cold temperature storage stress (Lo’ay, 2017), so, more protec-
tion to fruit tissue/cell against oxidative reaction (Foyer et al., 2017). In
term of fruits firmness, it decreased gradually during the cold storage
period. The obtained results could be due to more inhibition occurred of
cell wall degradation enzymes activities such as PG, CEL, and XYL
which are correlated to fruit firmness (Pasanphan et al., 2010). The
mixture CS/PVP with SA at 2 mM enhances the antioxidant enzymes
system activities which lead to less lipid peroxidation and protein
oxidation accumulation (Lo’ay and Dawood, 2017b).

Table 2
Effect of postharvest of the CS/PVP with salicylic acid application at different concentration on total soluble solid content % (SSC%), total acidity % (TA%), SSC/TA-
ratio, and fruit firmness (N) of fruit mature color guava ‘Banati’ fruit during 15 days under cold storage (6 °C ± 1).
Measurements Storage time (days)

D0 D3 D6 D9 D12 D15

Total soluble solid content %

hi abc abcd abcd ab a
Control 11.46 ± 0.325 12.49 ± 0.206 12.37 ± 0.030 12.49 ± 0.041 12.62 ± 0.018 12.69 ± 0.046
hi cdef cdef cde bcde abcd
CS/PVP-SA 0 mM 11.46 ± 0.325 12.13 ± 0.067 12.17 ± 0.017 12.17 ± 0.020 12.21 ± 0.010 12.33 ± 0.036
hi hi gh fgh efgh defg
CS/PVP-SA 1 mM 11.46 ± 0.325 11.49 ± 0.049 11.68 ± 0.110 11.73 ± 0.131 11.85 ± 0.120 12.05 ± 0.118
hi i hi hi hi hi
CS/PVP-SA 3 mM 11.46 ± 0.325 11.18 ± 0.035 11.50 ± 0.040 11.50 ± 0.052 11.52 ± 0.073 11.61 ± 0.063

Total acidity %
a fg j k l m
Control 0.591 ± 0.005 0.555 ± 0.003 0.506 ± 0.003 0.483 ± 0.002 0.452 ± 0.005 0.398 ± 0.002
a def i j k l
CS/PVP-SA 0 mM 0.591 ± 0.005 0.562 ± 0.001 0.528 ± 0.002 0.506 ± 0.003 0.479 ± 0.006 0.450 ± 0.003
a cd def g h i
CS/PVP-SA 1 mM 0.591 ± 0.005 0.569 ± 0.003 0.562 ± 0.000 0.551 ± 0.001 0.538 ± 0.002 0.527 ± 0.001
a b bc cd de efg
CS/PVP-SA 3 mM 0.591 ± 0.005 0.578 ± 0.001 0.572 ± 0.000 0.569 ± 0.000 0.564 ± 0.002 0.557 ± 0.001

SSC/TA-ratio
jk ef d c b a
Control 19.38 ± 0.545 22.48 ± 0.225 24.43 ± 0.219 25.86 ± 0.220 27.94 ± 0.401 31.85 ± 0.348
jk gh e d c b
CS/PVP-SA 0 mM 19.38 ± 0.545 21.57 ± 0.169 23.02 ± 0.133 24.05 ± 0.188 25.50 ± 0.340 27.37 ± 0.304
jk ij hi gh fg e
CS/PVP-SA 1 mM 19.38 ± 0.545 20.17 ± 0.155 20.75 ± 0.226 21.27 ± 0.295 22.03 ± 0.326 22.86 ± 0.288
jk k jk ij i hi
CS/PVP-SA 3 mM 19.38 ± 0.545 19.33 ± 0.095 20.08 ± 0.101 20.20 ± 0.109 20.42 ± 0.210 20.84 ± 0.545

Fruit firmness (N)

a
Control 11.69 ± 0.388 9.24 ± 0.426 abc 7.18 ± 0.243 bcd 5.56 ± 0.275 cd 5.12 ± 0.266 cd 4.17 ± 0.131 d

a
CS/PVP-SA 0 mM 11.69 ± 0.388 10.34 ± 0.115 abc 9.11 ± 0.362 abc 8.67 ± 0.063 abc 7.58 ± 0.211 abcd 7.62 ± 0.177 abcd

a
CS/PVP-SA 1 mM 11.69 ± 0.388 11.08 ± 0.259 ab 10.50 ± 0.151 ab 9.87 ± 0.325 ab 9.23 ± 0.409 abc 9.17 ± 0.303 abc

a
CS/PVP-SA 3 mM 11.69 ± 0.388 11.58 ± 0.175 a 11.26 ± 0.236 ab 10.74 ± 0.137 ab 10.38 ± 0.175 ab 9.87 ± 0.151 ab

The interaction between storage time (days) and CS/PVP-SA treatments mean in a column are significantly different at (P < 0.05), using Duncan’s Multiple Rang
Test, each value represents mean and ± SE (n = 3) replicates. The superscript letters, indicated significantly between effect of treatments.

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A.A. Lo’ay, M.A. Taher Scientia Horticulturae 238 (2018) 343–349

Table 3
Effect of postharvest of the CS/PVP with salicylic acid application at different concentration on antioxidant enzyme activities such as catalase (CAT), peroxidase
(POD), ascorbate peroxidase (APX), and superoxide dismutase (SOD) of fruit mature color guava ‘Banati’ fruit during 15 days under cold storage (6 °C ± 1).
Measurements Storage time (days)

D0 D3 D6 D9 D12 D15

Catalase activity (CAT, unit g−1 FW)

g ef g h i i
Control 16.37 ± 0.406 18.49 ± 0.112 16.40 ± 0.040 14.87 ± 0.313 12.87 ± 0.120 12.41 ± 0.283
g cde f ef g g
CS/PVP-SA 0 mM 16.37 ± 0.406 19.22 ± 0.249 18.30 ± 0.524 18.46 ± 0.060 16.75 ± 0.334 16.08 ± 0.044
g bc cd cde def f
CS/PVP-SA 1 mM 16.37 ± 0.406 19.85 ± 0.156 19.34 ± 0.027 19.18 ± 0.135 18.94 ± 0.328 18.30 ± 0.156
g a b b bcd cd
CS/PVP-SA 3 mM 16.37 ± 0.406 21.41 ± 0.670 20.37 ± 0.554 20.39 ± 0.577 19.68 ± 0.214 19.35 ± 0.035

Peroxidase activity (POD, unit g−1 FW)

Control 8.71 ± 0.147 m 8.94 ± 0.119 l 9.57 ± 0.068 k 9.93 ± 0.081 j 10.09 ± 0.110 ij
10.21 ± 0.096 i

CS/PVP-SA 0 mM 8.71 ± 0.147 m 9.40 ± 0.054 k 10.29 ± 0.104 i 10.56 ± 0.079 h

10.81 ± 0.052 fg
10.98 ± 0.008 ef

CS/PVP-SA 1 mM 8.71 ± 0.147 m 10.21 ± 0.1467 i 10.81 ± 0.249 fg 11.28 ± 0.218 d

11.17 ± 0.133 de
11.82 ± 0.044 c

CS/PVP-SA 3 mM 8.71 ± 0.147 m 10.63 ± 0.046 gh 11.35 ± 0.024 d 12.05 ± 0.128 b

12.10 ± 0.008 b
12.49 ± 0.008 a

−1
Ascorbate peroxidase activity (APX, unit g FW)
Control 16.39 ± 0.544 l 29.06 ± 1.355 g
22.49 ± 0.660 i
19.40 ± 0.537 jk
15.31 ± 0.401 l
12.48 ± 0.473 m

CS/PVP-SA 0 mM 16.39 ± 0.544 l 33.87 ± 0.813 e

28.92 ± 0.281 g
25.32 ± 0.569 h
20.36 ± 0.589 j
18.76 ± 0.254 k

CS/PVP-SA 1 mM 16.39 ± 0.544 l 37.77 ± 0.738 d

32.24 ± 0.336 f
31.25 ± 0.260 f
26.59 ± 0.494 h
23.53 ± 1.553 i

CS/PVP-SA 3 mM 16.39 ± 0.544 l 46.30 ± 1.589 a

43.87 ± 0.052 b
41.96 ± 0.311 c
38.28 ± 0.554 d
31.67 ± 0.914 f

−1
Superoxide dismutase activity (SOD, unit g FW)
Control 344.66 ± 0.278 g 370.00 ± 1.000 e
331.00 ± 1.154 h
295.33 ± 2.603 j
278.66 ± 4.977 k
235.33 ± 2.728 l

CS/PVP-SA 0 mM 344.66 ± 0.278 g 381.00 ± 1.154 d

356.00 ± 3.214 f
330.00 ± 1.527 h
306.33 ± 3.282 i
289.33 ± 5.607 j

CS/PVP-SA 1 mM 344.66 ± 0.278 g 396.33 ± 3.179 bc

391.33 ± 0.881 c
380.66 ± 0.881 d
346.66 ± 2.962 g
332.33 ± 2.403 h

CS/PVP-SA 3 mM 344.66 ± 0.278 g 418.33 ± 1.452 a

403.00 ± 4.041 b
400.33 ± 3.929 b
384.33 ± 1.763 d
378.00 ± 0.577 d

The interaction between storage time (days) and CS/PVP-SA treatments mean in a column are significantly different at (P < 0.05), using Duncan’s Multiple Rang
Test, each value represents mean and ± SE (n = 3) replicates. The superscript letters, indicated significantly between effect of treatments.

Table 4
Effect of postharvest of the CS/PVP with salicylic acid application at different concentration on lipid peroxidation (malondialdehyde, MDA), protein oxidation
(protein carbonyl group, PCG), and cell membrane permeability percentage of fruit mature color guava ‘Banati’ fruit during 15 days under cold storage (6 °C ± 1).
Measurements Storage time (days)

D0 D3 D6 D9 D12 D15

Lipid peroxidation (malondialdehyde, MDA μM g−1 FW)

Control 2.74 ± 0.092 m 3.94 ± 0.035 jk
6.16 ± 0.027 g
8.41 ± 0.058 d
11.07 ± 0.136 b 15.53 ± 0.752 a

CS/PVP-SA 0 mM 2.74 ± 0.092 m 3.03 ± 0.030 lm

5.94 ± 0.027 g
7.06 ± 0.082 f
7.58 ± 0.212 e 10.17 ± 0.317 c

CS/PVP-SA 1 mM 2.74 ± 0.092 m 2.96 ± 0.028 m

5.19 ± 0.181 h
5.94 ± 0.032 g
6.07 ± 0.074 g 7.00 ± 0.155 f
m m kl j
CS/PVP-SA 3 mM 2.74 ± 0.092 2.81 ± 0.006 3.50 ± 0.048 4.03 ± 0.070 4.19 ± 0.014 ij 4.62 ± 0.020 i
−1
Protein oxidation (protein carbonyl group, PCG mM 100 g FW)
Control 21.94 ± 0.607 l 25.65 ± 0.066 g
28.10 ± 0.360 e
31.98 ± 0.950 c
35.45 ± 1.149 b
38.76 ± 0.862 a

CS/PVP-SA 0 mM 21.94 ± 0.607 l 24.60 ± 0.130 hi

26.91 ± 0.253 f
28.78 ± 0.487 de
31.57 ± 0.520 c
31.77 ± 0.110 c

CS/PVP-SA 1 mM 21.94 ± 0.607 l 24.03 ± 0.063 ij

25.48 ± 0.315 gh
26.96 ± 0.341 f
29.28 ± 0.553 d
28.98 ± 0.350 de

CS/PVP-SA 3 mM 21.94 ± 0.607 l 22.03 ± 0.242 l

22.79 ± 0.333 kl
23.53 ± 0.276 jk
23.69 ± 0.306 ijk
24.03 ± 0.066 ij

Cell membrane permeability percentage (IL%)

Control 11.29 ± 0.632 l 15.81 ± 0.316 h
18.74 ± 0.491 fg
27.55 ± 1.098 c
30.47 ± 0.629 b
38.91 ± 1.213 a

CS/PVP-SA 0 mM 11.29 ± 0.632 l 13.65 ± 0.340 ij

17.39 ± 0.050 g
23.71 ± 0.856 e
25.28 ± 0.260 d
26.29 ± 0.262 cd

CS/PVP-SA 1 mM 11.29 ± 0.632 l 12.92 ± 0.205 ijk

15.82 ± 0.291 h
17.90 ± 0.269 fg
18.11 ± 0.060 fg
18.98 ± 0.290 f

CS/PVP-SA 3 mM 11.29 ± 0.632 l 11.94 ± 0.286 kl

12.43 ± 0.261 jkl
12.79 ± 0.310 ijk
13.60 ± 0.086 ij
13.94 ± 0.032 i

The interaction between storage time (days) and CS/PVP-SA treatments means in a column are significantly different at (P < 0.05), using Duncan’s Multiple Rang
Test, each value represents mean and ± SE (n = 3) replicates. The superscript letters, indicated significantly between effect of treatments.

3.3. Effect of CS/PVP-SA treatments on antioxidant enzyme activities
Table 3 clarifies the variance of antioxidant enzyme activities such
as catalase (CAT, CE: 1.11.1.6), peroxidase (POD, CE: 1.11.1.7), as-
corbate peroxidase (APX, CE: 1.11.1.11), superoxide dismutase (SOD,
CE: 1.15.1.1) activities unit g−1 FW as a function of storage time for
‘Banati’ guava fruit were coated with CS/PVP plus SA at different
concentrations. Apparently, the antioxidant enzyme activities present
significantly interaction at P ≤ 0.001 when storage time and CS/PVP-
SA treatments were studied. Clearly, the data of antioxidant enzyme
activities increased after harvesting and coating by CS/PVP-SA up to
the 3rd day of storage time thereafter they decreased up to end of the
storage period. An exception was observed that the POD activity in-
creased up to end of the experiment in overall treatments. The rates of
antioxidant activities decreased due to the decreased concentration of
salicylic acid after the 3rd day of storage. Fruits were treated by CS/
PVP-SA 2mM presented slightly decreases in antioxidant enzymes ac-
tivities up to end of cold storage time. It was recorded the maximum
activities at 3rd day CAT (24.41), POD (10.63), APX (46.30), and SOD
(418.33 unit g−1 FW) compared to the control fruit (CAT: 18.49, POD:
8.94, APX: 29.06, and SOD: 370.00 unit g−1 FW). Thereafter, they
decreased up to the 15th day of storage time. This could be due to the
effect of CS/PVP on antioxidant enzyme activities (Landi et al., 2014),
and the impact of SA roles on scavenging ROS and maintaining a net-
work of antioxidant system capacity during cold temperature stress
(Champa et al., 2015). So, SA and CS/PVP suggest increasing anti-
oxidant enzymes (Batista Silva et al., 2018). Therefore, the termination
of lipid peroxidation and protein oxidation of plasma cell membrane

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A.A. Lo’ay, M.A. Taher Scientia Horticulturae 238 (2018) 343–349

Table 5
Effect of postharvest of the CS/PVP with salicylic acid application at different concentration on O2•− production and H2O2 content of fruit mature color guava ‘Banati’
fruit during 15 days under cold storage (6 °C ± 1).
Treatments Storage time (days)

D0 D3 D6 D9 D12 D15

O2%− production rate (mmol min−1 g−1 FW)

Control 0.88 ± 0.040 hi 0.95 ± 0.011 fghi
1.27 ± 0.017 d
2.61 ± 0.058 c
3.64 ± 0.316 b
4.23 ± 0.008 a
CS/PVP-SA 0 mM 0.88 ± 0.040 hi 0.90 ± 0.014 hi
0.96 ± 0.003 efghi
1.04 ± 0.024 efgh
1.12 ± 0.008 def
1.16 ± 0.0005 de
CS/PVP-SA 1 mM 0.88 ± 0.040 hi 0.86 ± 0.008 hi
0.95 ± 0.003 fghi
0.98 ± 0.003 efghi
1.04 ± 0.005 efgh
1.11 ± 0.011 defg
CS/PVP-SA 3 mM 0.88 ± 0.040 hi 0.79 ± 0.008 I
0.86 ± 0.005 hi
0.92 ± 0.005 ghi
0.94 ± 0.003 fghi
0.97 ± 0.018 efghi

H2O2 content (nmol g−1 FW)

mn i g c b a
Control 0.30 ± 0.005 0.34 ± 0.005 0.40 ± 0.005 0.58 ± 0.012 0.68 ± 0.015 0.79 ± 0.011
mn jkl ijk fg e d
CS/PVP-SA 0 mM 0.30 ± 0.005 0.32 ± 0.005 0.33 ± 0.003 0.41 ± 0.014 0.45 ± 0.012 0.52 ± 0.008
mn lmn klm h g f
CS/PVP-SA 1 mM 0.30 ± 0.005 0.30 ± 0.003 0.31 ± 0.003 0.36 ± 0.005 0.40 ± 0.006 0.43 ± 0.005
mn l lmn jkl ijk ij
CS/PVP-SA 3 mM 0.30 ± 0.005 0.29 ± 0.003 0.30 ± 0.003 0.32 ± 0.005 0.32 ± 0.006 0.33 ± 0.003

The interaction between storage time (days) and CS/PVP-SA treatments means in a column are significantly different at (P < 0.05), using Duncan’s Multiple Rang
Test, each value represents mean and ± SE (n = 3) replicates. The superscript letters, indicated significantly between effect of treatments.

was low rates (Lo’ay, 2005), so, plasma membrane function saved
(Batista Silva et al., 2018).
3.4. Lipid peroxidation (malondialdehyde, MDA), protein oxidation
(protein carbonyl group, PCG), plasma cell membrane permeability
percentage
Table 4 shows the variations of plasma cell membrane structure
termination during cold storage as for lipid peroxidation (MDA), pro-
tein oxidations (PCG), and cell membrane leakage (IL%) as a function of
storage time. Clearly, the MDA, PCG, and IL% show a significant in-
teraction at P ≤ 0.001 when storage time (days) and CS/PVP-SA
treatments were considered. Generally, all cell membrane parameters
(MDA, PCG, and IL%) are progressively increased during the storage
period. The increases are independently due to decreases of salicylic
acid presences in biopolymer mixture. Fruits were coated with CS/PVP-
SA 2mM treatment presents more protection for cell membrane com-
partments results in less accumulation of MDA (4.62 μM g−1 FW), PCG
(24.03 mM 100 g−1 FW), and IL% (13.94%) compared to other treat-
ments. However, control fruits show more rapid accumulation in MDA
(15.53 μM g−1 FW), PCG (38.76 mM 100 g−1 FW), and IL% (38.91%) at
15th day of cold storage period. The observed increases in MDA, PCG,
and IL% of control fruits during storage time might suggest that due to
the decreases in antioxidant enzyme activities (Youwei and Yinzhe,
2013). So, more ROS produced which lead to enhancing oxidative re-
action in term of lipid peroxidation and protein oxidation processes
thereafter more dysfunction occurred of cell membrane structure and
more IL% (Lo’ay, 2005), finally, cell death and more visual chilling
injury symptoms (Foyer et al., 2017).
3.5. Effect of CS/PVP-SA on O2%− and H2O2 production rates
Table 5 presents the variations between O2%− and H2O2 production
rates as a function of storage time. The O2•− and H2O2 production rates
present a significant interaction at P ≤ 0.001 when storage time (days)
and CS/PVP-SA treatments were studied. Generally, the O2%− and H2O2
production increased gradually during 15 days of cold storage overall
treatments. control fruit shows more rapid in O2%− and H2O2 produc-
tion compared to other treatments at up to end of storage period.
However, fruits were coated with CS/PVP-SA 2 mM treatment which
presented significantly decreases in O2%− and H2O2 production (0.97
and 0.33 nmol min−1 g−1 FW) compared to control fruits (4.55 and
0.79 nmol min−1 g−1 FW) at 15th day of cold storage. The highest
amount of O2%− and H2O2 production at the 3rd day of storage time
might be related to other metabolic processes such as aerobic respira-
tion (Tang et al., 2012). So, increasing antioxidant enzyme activities
such as SOD could also enhance fruit tissues defense against O2%−.
Consequently, increasing CAT and APX activities would participate
strongly in scavenging O2%− and H2O2 during cold storage (Yang et al.,
2011). These results demonstrated that SA increases the antioxidant
enzyme activities, which not only scavenged H2O2 but mitigated O2%−
generation.
4. Conclusion
Chitosan/PVP- blending in salicylic acid at different concentrations
as a coating for guava fruits provide a protection/alleviation chilling
injury incidence during cold storage. Indeed, CS/PVP-SA 2 mM coating
treatment increased chilling injury tolerance by increasing antioxidants
enzyme activities during cold stress. So, it minimized oxidative reac-
tions by scavenging O2%− and H2O2 generation which it maintained
plasma cell membrane compartments by reducing lipid peroxidation
(MDA) and protein oxidation (PCG) accumulation. Therefore, less
dysfunction of plasma cell membrane and less ion leakage percentage
presented. Positively, less chilling injury symptoms appeared during
cold storage. This means that the treatment of fruits biopolymer CS/
PVP with salicylic acid at 2 mM, is a positive coating treatment for
marketing guava fruit.
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