This document summarizes a study that investigated the effects of controlled atmosphere storage on pericarp browning, bioactive compounds, and antioxidant enzymes in litchi fruits. Litchi fruits were stored under ten different combinations of oxygen and carbon dioxide levels at 5±1°C for 35 days. Fruits stored under 1% oxygen and 5% carbon dioxide showed reduced weight loss, browning, membrane damage, and lipid peroxidation compared to controls. These fruits also maintained higher levels of bioactive compounds and antioxidant enzyme activities. In contrast, activities of enzymes linked to browning were lower in fruits under this storage condition. Overall, 1% oxygen and 5% carbon dioxide storage best maintained quality and delayed browning of litchi

1. Accepted Manuscript
Effect of controlled atmosphere storage on pericarp browning, bioactive com-
pounds and antioxidant enzymes of litchi fruits
Sajid Ali, Ahmad Sattar Khan, Aman Ullah Malik, Muhammad Shahid
PII: S0308-8146(16)30372-7
DOI: http://dx.doi.org/10.1016/j.foodchem.2016.03.021
Reference: FOCH 18905
To appear in: Food Chemistry
Received Date: 12 December 2015
Revised Date: 7 March 2016
Accepted Date: 8 March 2016
Please cite this article as: Ali, S., Khan, A.S., Malik, A.U., Shahid, M., Effect of controlled atmosphere storage on
pericarp browning, bioactive compounds and antioxidant enzymes of litchi fruits, Food Chemistry (2016), doi:
http://dx.doi.org/10.1016/j.foodchem.2016.03.021
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2. 1
Title
1
Effect of controlled atmosphere storage on pericarp browning, bioactive compounds and
2
antioxidant enzymes of litchi fruits
3
Running title
4
Response of litchi fruit under controlled atmosphere storage
5
Authors
6
Sajid Alia
7
a
Postharvest Research and Training Centre, Institute of Horticultural Sciences, University of
8
Agriculture Faisalabad (38040), Pakistan
9
ch.sajid15@yahoo.com
10
Ahmad Sattar Khana*
11
a
Postharvest Research and Training Centre, Institute of Horticultural Sciences, University of
12
Agriculture Faisalabad (38040), Pakistan
13
ahmad_khan157@yahoo.com
14
*
Corresponding author: Tel: +92-333-8364813; fax: +92-41-9201086
15
Aman Ullah Malika
16
a
Postharvest Research and Training Centre, Institute of Horticultural Sciences, University of
17
Agriculture Faisalabad (38040), Pakistan
18
malikaman1@gmail.com
19
Muhammad Shahidb
20
b
Department of Chemistry and Biochemistry, University of Agriculture Faisalabad (38040),
21
Pakistan
22
mshahiduaf@yahoo.com
23

3. 2
Abstract
24
‘Gola’ litchi fruits were stored under ten different CA-combinations at 5±1°C to investigate its
25
effects on pericarp browning, biochemical quality and antioxidative activities. Control fruit
26
turned completely brown after 28 days of storage and were excluded from the study. Fruit-stored
27
under CA7-combination (1% O2 + 5% CO2) showed reduced weight loss, pericarp browning,
28
membrane leakage and malondialdehyde contents. Soluble solid contents, titratable acidity and
29
ascorbic acid contents were higher in CA7-stored fruit. Activities of catalase and superoxide
30
dismutase enzymes, levels of total anthocyanins, DPPH radical-scavenging-activity and phenolic
31
contents were significantly higher in CA7-stored litchi fruit. In contrast, activities of polyphenol
32
oxidase and peroxidase enzymes were substantially lower in fruit kept under CA7-combination.
33
Fruit subjected to CA7-conditions also maintained higher organoleptic quality. In conclusion, 1%
34
O2 + 5% CO2 CA-conditions delayed pericarp browning, maintained antioxidative activities and
35
biochemical characteristics along with better organoleptic quality of litchi fruit for 35 days.
36
Keywords: Antioxidant enzymes, Lipid peroxidation, Peroxidase, Polyphenol oxidase, Skin
37
discolouration
38
1. Introduction
39
Litchi (Litchi chinensis Sonn.) is a tropical to subtropical fruit, highly admired for its
40
characteristic appealing bright red colour, delicious taste, and attractive aroma (Underhill &
41
Simons, 1993; Holcroft & Mitcham, 1996). Export of this premium quality fruit in the
42
international markets is restricted due to different postharvest problems such as pericarp
43
browning and fruit decay. Besides different other issues, pericarp browning of litchi fruit is one
44
of the major postharvest constraints which greatly reduces its market value around the globe
45

4. 3
(Underhill & Critchley, 1995; Jiang, Zauberman, & Fuchs, 1997). Different factors have been
46
found associated with litchi pericarp browning; however, water loss/desiccation is considered
47
one of the leading causes of this problem. Desiccation of litchi pericarp tissues leads to different
48
physiological and biochemical changes that ultimately results in the direct contact of peel
49
phenolics with polyphenol oxidase (PPO) and peroxidase (POD) enzymes (Zauberman, Ronen,
50
Akerman, Weksler, Rot, & Fuchs, 1991). Oxidation of peel phenolics by PPO and POD enzymes
51
finally leads to pericarp browning of litchi fruit (Zauberman et al., 1991; Zhang, Pang, Zuoliang,
52
& Jiang, 2001).
53
Several approaches such as sulphur dioxide fumigation (Swarts, 1985); modified
54
atmosphere packaging (Chen, Wu, Ji, & Su, 2001); hot-water dips (Olesen, Nacey, Wiltshire, &
55
O’Brian, 2004); CA-storage (Mahajan & Goswami, 2004); cold-storage (Khan, Ahmad, Malik,
56
& Amjad, 2012); postharvest exogenous application of oxalic acid (Zheng & Tian, 2006);
57
ascorbic acid (Sun, Liang, Xie, Lei, & Mo, 2010) and antioxidants (Kumar, Mishra,
58
Chakraborty, & Kumar, 2013) have been used for the management of pericarp browning, overall
59
quality retention and storage-life extension of litchi fruit. However, among these approaches,
60
CA-storage has been found more suitable to slow down the rapid rate of skin colour loss of litchi
61
fruit (Jiang & Fu, 1999). CA-storage with 3.5% O2 and 3.5% CO2 gaseous conditions maintained
62
sensory and biochemical characteristics of ‘Bombay’ litchi fruit (Mahajan & Goswami, 2004).
63
Duan, Liu, Zhang, Su, Lin and Jiang (2011) also reported reduced pericarp browning, lipid
64
peroxidation and reactive oxygen species along with higher activities of antioxidative enzymes
65
under pure O2 conditions in ‘Huaizhi’ litchi fruit for 6 days. Similarly, fruit of litchi cv. ‘Heiye’
66
kept under CA-storage (CA-I = 5% O2 + 5% CO2; CA-II = initially at 70% O2 + 0% CO2 for 7
67
days and later on 5% O2 + 5% CO2 combination) showed reduced pericarp browning (Tian, Bo-
68

5. 4
Qiang, & Xu, 2005). Similarly, reduced pericarp browning has also been reported with
69
application of 1-methylcyclopropene under CA-storage (17% O2 + 6% CO2) in fruit of litchi cv.
70
‘McLean’s Red’ for 21 days (Sivakumar & Korsten, 2010). Although, they reported reduced
71
pericarp browning and maintained quality; but, these studies did not describe detailed changes in
72
postharvest physiology of litchi fruit. Moreover, information about changes in different
73
enzymatic (catalase, peroxidase and superoxide dismutase) and non-enzymatic (DPPH radical
74
scavenging activity and phenolic contents) antioxidants in both peel and pulp tissues in relation
75
to development of litchi pericarp browning under CA-storage is also lacking. The knowledge
76
about occurrence of pericarp browning under prolonged CA storage could further enhance our
77
understanding to devise most appropriate strategies for its management and quality control
78
during the supply chain operations of litchi fruit. Hence, objectives of the current research were
79
to investigate the effects of different concentrations of O2 and CO2 on pericarp browning,
80
membrane integrity, lipid peroxidation, bioactive compounds, antioxidative activities,
81
biochemical attributes and organoleptic quality of ‘Gola’ litchi fruit under extended storage
82
conditions.
83
2. Materials and methods
84
2.1. Fruit source
85
Fruits of litchi cv. ‘Gola were harvested at anticipated commercial maturity [colour = 85-
86
100% red, SSC = 18.5 °Brix, TA = 0.70% and SSC/TA = 26.42 (as per local maturity standards)]
87
from the “Government Fruit Farm Nursery (34o
00.114’N, 72o
56.779’E), Haripur, Khaybar
88
Patkhtun Khaw (KPK), Pakistan. After harvest, uniform sized fruits free from visual defects
89

6. 5
were pre-cooled in refrigerated reefer van (10±1ºC) and shifted to Postharvest Research and
90
Training Center, University of Agriculture Faisalabad, Pakistan.
91
2.2. Storage conditions and treatments
92
The experiment comprised of ten treatments viz; control (air), CA1 = 1% O2 + 3% CO2,
93
CA2 = 2% O2 + 3% CO2, CA3 = 3% O2 + 3% CO2, CA4 = 1% O2 + 4% CO2, CA5 = 2% O2 + 4%
94
CO2, CA6 = 3% O2 + 4% CO2, CA7 = 1% O2 + 5% CO2, CA8 = 2% O2 + 5% CO2 and CA9 = 3%
95
O2 + 5% CO2. Air, N2 and CO2 were mixed via pressure regulator to establish initial O2 and CO2
96
gases levels in CA-pallistores by a continuous flow through system (VPSA-6 Van CA
97
Technology, Amerongen, the Netherlands) automatically regulated, and controlled by the
98
analyzer (S-904 Van CA Technology, Amerongen, the Netherlands). Fruits were stored in CA-
99
pallistores (L × W × H = 43 × 33 × 150 cm) at 5±1ºC with 90±5% RH for 35 days. Gas mixtures
100
of all CA-pallistores were monitored thrice daily to ensure the anticipated gaseous compositions.
101
Fruits were removed from CA-pallistores at 7 days interval, and evaluated for different quality
102
characteristics. Fruit weight loss, decay incidence, and pericarp browning index were assessed
103
from whole fruit; while, soluble solid contents, titratable acidity, sugar: acid ratio, and ascorbic
104
acid contents were determined only from the pulp tissues. DPPH radical scavenging activity,
105
total phenolic contents, activities of catalase, peroxidase and superoxide dismutase enzymes
106
were determined from both peel, and pulp tissues. Membrane leakage, malondialdehyde, total
107
anthocyanins, and activities of polyphenol oxidase enzyme were determined only from the peel
108
tissues. The experiment was conducted under completely randomized design with factorial
109
arrangement. Fruits were placed in plastic crates (L × W × H = 39 × 29 ×11 cm) and stored
110
under CA-pallistores. At each sampling interval, every treatment contained three independent
111

7. 6
crates and each crate contained 25 fruits as a single replication. Overall the experiment
112
comprised of 174 crates having total 3675 fruits.
113
2.3. Fruit weight loss, decay incidence, pericarp browning and browning rate
114
Fruits were weighed on the digital weight balance (ELB-1200, Shimadzu, Kyoto Inc.,
115
Japan) and loss of weight was calculated with the following equation and expressed in terms of
116
percentage.
117
ℎ

8. =
х 100
118
Decay incidence was assessed by dividing the number of decayed/rotten fruits over total
119
number of fruits in each replicate, and expressed as percentage. Pericarp browning was assessed
120
by evaluating the extent of browned area on the surface of fruit as reported by Sivakumar and
121
Korsten (2010) and calculated according to the formula of Zhang and Quantick (1997).
122
Browning rate was calculated by dividing the number of brown fruits over total number of fruits
123
in each replicate, and expressed in percentage.
124
2.4. Pericarp pH, membrane leakage and malondialdehyde (MDA) contents
125
Pericarp pH of litchi fruit was determined by the method as reported previously by Joas,
126
Caro, Ducamp and Reynes (2005). Membrane leakage of the pericarp tissues was determined as
127
described by Jiang and Chen (1995). Membrane leakage was finally expressed in terms of
128
percent of the initial electrolytes to the total electrolytes. MDA contents of litchi pericarp tissues
129
were determined as described by Zheng and Tian (2006), and expressed as nmole g-1
FW.
130

9. 7
2.5. Total anthocyanins, DPPH radical scavenging activity and total phenolic contents
131
Total anthocyanin contents of litchi pericarp tissues were extracted according to the
132
method as described by Zheng and Tian (2006), and calculated as: ∆A g-1
FW = (A530 – A620) –
133
0.1(A650 – A620). Total antioxidant activity of 2, 2–diphenyl–1–picrylhydrazyl–radical (DPPH)
134
was assessed by bleaching purple–coloured–solution (stable–DPPH–radical) of methanol, and
135
expressed in terms of percent (Brand-Williams, Cuvelier, Berset, 1995). Total phenolic
136
contents were determined with Folin–Ciocalteu reagent (Ainsworth Gillespie, 2007), and their
137
concentration was expressed as gallic acid equivalent (mg GAE 100 g-1
FW).
138
2.6. Soluble solid contents (SSC), titratable acidity (TA), SSC: TA ratio and ascorbic acid
139
contents
140
SSC of litchi fruit juice were determined by a digital refrectometer (RX-5000 Atago,
141
Japan), and expressed in terms of ºBrix. TA was determined by juice titration against 0.1 N
142
NaOH and expressed as percent (%) malic acid. SSC: TA ratio was determined by dividing the
143
SSC with their corresponding TA values. Ascorbic acid contents were determined by titrating 5
144
mL of litchi juice aliquot (obtained from 10 mL juice and 90 mL of 0.4% oxalic acid) against
145
2,6-dichlorophenolindophenol and expressed as mg 100 mL-1
of fruit juice.
146
2.7. Enzymes assays
147
Litchi peel and pulp tissues (1 g each) were homogenized in 2 mL phosphate buffer [for
148
pulp (pH 7.2)] and citrate buffer [for peel (pH 4) in pre-chilled mortar, and pestle. After thorough
149
homogenization, samples were taken in eppendorfs, and centrifuged at 10,000 × g for 10 min
150
with micro-centrifuge machine (235-A, Pegasus Scientific Inc., USA) at 4°C. Finally, obtained
151

10. 8
supernatant was collected with the help of micropipettes, and used for the further assays of the
152
following enzymes.
153
Activity of catalase (CAT) enzyme (EC 1.11.1.6) in peel and pulp tissues of litchi fruit
154
was assayed according to the method of Liu, Zou, Meng, Zou and Jiang (2009) with few
155
modifications. Enzyme extract (100 µL) of peel and pulp tissues was mixed with freshly
156
prepared 100 µL H2O2 (5.9 mM) solution to start reaction of the enzyme. Absorbance was noted
157
on ELX-800 Micro-plate Reader (Bio-Tek Instruments, Inc., USA) at 240 nm wavelength.
158
Activity of CAT enzyme was expressed as U mg −1
protein; where, one unit (U) of enzyme
159
activity was defined as “absorbance change in 0.01 unit min−1
”.
160
Superoxide dismutase (SOD) enzyme (EC 1.15.1.1) activity was assessed by 50%
161
inhibition of photochemical-reduction of the nitro-blue-tetrazolium (NBT) as outlined by Stajner
162
and Popovic (2009) with slight modifications. Distilled water (800 µL), phosphate buffer with
163
pH 5 [500 µL (50 mM)], 100 µL NBT (20 µM), 200 µL methionine (22 µM), 200 µL Triton-X
164
(0.1 µM), 100 µL riboflavin (0.6 µM) as an enzyme-substrate were mixed with enzyme extract
165
(100 µL) in test tubes. Test tubes containing reagents and enzyme extract were wrapped with
166
aluminum foil, and exposed to UV-light lamp for 15 min. Finally, absorbance was taken at 560
167
nm on Micro-plate Reader. Activity of SOD enzyme was expressed as U mg−1
protein; where,
168
one unit of enzyme activity was defined as “quantity of enzyme that inhibited 50% NBT
169
photochemical-reduction”.
170
Peroxidase (POD) enzyme (EC 1.11.1.7) activity in peel and pulp tissues of litchi fruit
171
was determined as described previously by Liu et al. (2009) with few modifications. Fresh
172
reaction mixture was prepared by adding 800 µL phosphate buffer [pH 5 (50 mM)], 100 µL
173

11. 9
H2O2 (40 mM), and 100 µL of guaiacol (20 mM). Finally, 100 µL reaction mixture was taken
174
and reacted with 100 µL enzyme extract, and absorbance was noted on Micro-plate Reader at
175
470 nm. The activity of enzyme was expressed in terms of U mg−1
protein; where, one unit (U)
176
of CAT enzyme activity was defined as “the absorbance change in 0.01 unit min−1
”.
177
Activity of polyphenol oxidase (PPO) enzyme (EC 1.14.18.1) was determined by the
178
method as reported by Waite (1976) with some modifications. One g peel tissues (1 g) were
179
homogenized in 10 mL of 100 mM citrate buffer (pH 4) comprising insoluble
180
polyvinylpyrollidon in falcon tubes (50 mL). Falcon tubes consisting peel tissues and
181
polyvinylpyrollidon were vortexed for 1-2 min, and incubated on ice for 30 min. After the
182
incubation period, falcon tubes were centrifuged at 10,000 × g for 5 min at 4°C. Supernatant was
183
mixed with the freshly prepared 1.45 mL of 100 mM citrate buffer (pH 6.8), and 0.50 mL of 4-
184
methylcatechol (100 mM) as an enzyme substrate. Finally, absorption was taken at 412 nm on
185
Micro-plate Reader, and enzyme activity was expressed as U mg−1
protein; where, one unit (U)
186
of PPO activity was defined as the amount of enzyme that causes “increase in the absorbance of
187
0.001 unit min-1
”.
188
Protein contents were determined by using bovine serum albumin as a standard
189
(Bradford, 1976).
190
2.8. Organoleptic quality
191
Eating quality was assessed by panel test [panelists were 24-30 years old postgraduate
192
students (both male and female) of postharvest science]. Fruits were manually peeled, and placed
193
in plates. Each fruit sample plate was tagged by a random four-digit code, and order of the
194
presentation of fruit samples was randomized for each panelist. The evaluation was carried out at
195

12. 10
room temperature under uniform laboratory conditions. Each panelist washed his/her mouth
196
before evaluation of the next sample. The following characteristics were selected for the
197
characterization of organoleptic/eating quality of litchi fruit; taste, presence of any off-flavours,
198
aroma, and overall acceptance assessed on a hedonic scale that comprised of 9 = extremely like,
199
5 = neither like nor dislike, and 1 = extremely dislike.
200
2.9. Statistical analysis
201
The research was conducted under completely randomized factorial design. The factors
202
were treatments and storage periods. Each treatment was replicated thrice with 25 fruits as a
203
single replication. Data were subjected to two-way analysis of variance (ANOVA) by
204
generalized linear model with Statistical Analysis System software program version-9.0 (SAS
205
Institute Inc., Cary, NC, USA). Effects of treatments, storage periods and their interactions on
206
different physical, enzymatic, biochemical and sensory quality characteristics were analyzed by
207
using Tukey’s test at P ≤ 0.05. All the assumptions of ANOVA were checked to ensure the
208
validity of statistical analysis. All the data are reported as the means ± standard error.
209
3. Results
210
3.1. Fruit weight loss, decay incidence, pericarp browning and browning rate
211
Fruit weight loss increased gradually with the advancement of storage period (Fig. 1A).
212
However, weight loss was significantly higher in control (15.37%), than CA-stored fruit (3.48%)
213
up to 28 days of storage. Among different CA-combinations, fruit stored under 1% O2 + 5% CO2
214
conditions exhibited substantially (P ≤ 0.05) about 2.71-fold less weight loss during 35 days of
215
storage, as compared to other treatments (Fig. 1A).
216

13. 11
Decay incidence continuously increased with the increase in storage periods, irrespective
217
to the treatments. However, fruit subjected to CA-storage showed significantly (P ≤ 0.05)
218
53.51% less decay incidence on day-28, than control (Fig. 1B). Among the various CA-
219
treatments, fruit stored under 1% O2 + 5% CO2 conditions exhibited significantly (P ≤ 0.05)
220
about 59% less decay incidence, in contrast to other treatments after 35 days of storage (Fig. 1B).
221
Pericarp browning showed continuous increasing trend as storage period progressed.
222
Control fruit were completely turned brown during 28 days of storage; whereas, CA-storage
223
exhibited significantly less pericarp browning of litchi fruit (Fig. 1C). On an average, litchi fruit
224
kept under 1% O2 + 5% CO2 CA-combination showed significantly (P ≤ 0.05) reduced pericarp
225
browning (2.33 score), in contrast to all other treatments after 35 days of storage (Fig. 1C).
226
Browning rate of litchi fruit significantly increased with the increase in storage periods.
227
Control fruit exhibited significantly higher browning rate throughout the storage periods (Fig.
228
1D). On the other hand, fruit subjected to CA-storage showed significantly (P ≤ 0.05) 4.12-fold
229
less browning rate on day-28, than control (Fig. 1B). Among CA-treatments, fruit stored under
230
1% O2 + 5% CO2 conditions exhibited substantially (P ≤ 0.05) 2.54-fold less browning rate, than
231
other CA-combinations after 35 days of storage (Fig. 1D).
232
3.2. Pericarp pH, membrane leakage and MDA contents
233
As storage period progressed, pericarp pH gradually increased irrespective to the
234
treatments. However, on day-28, CA-stored fruit exhibited substantially (P ≤ 0.05) 75% more
235
acidic pericarp pH, as compared to control fruit (Fig. 2A). Among the different CA-treatments,
236
litchi fruit kept under 1% O2 + 5% CO2 conditions showed significantly (P ≤ 0.05) about 89%
237
more acidic pericarp pH, in contrast to all other treatments after 35 days of storage (Fig. 2A).
238

14. 12
Membrane leakage and MDA contents of litchi pericarp tissues gradually increased with
239
the progress in storage period (Fig. 2B and C). However, fruit subjected to CA-storage showed
240
significantly (P ≤ 0.05) about 1.50- and 2.71-fold less membrane leakage, and MDA contents,
241
respectively, than control up to 28-days of storage. As far as different CA-combinations are
242
concerned, fruit stored under 1% O2 + 5% CO2 conditions exhibited significantly (P ≤ 0.05)
243
reduced membrane leakage (71%), and MDA contents (60%), than other treatments,
244
respectively, after 35 days of storage (Fig. 2B and C).
245
3.3. Total anthocyanins, DPPH radical scavenging activity and phenolic contents
246
Total anthocyanin contents gradually decreased throughout the storage period regardless
247
of the treatments. However, averaged over the control, CA-stored fruit showed significantly (P ≤
248
0.05) 4.47-fold higher total anthocyanin contents during 28 days of storage (Fig. 3A). As far as
249
different CA combinations are concerned, fruit kept under 1% O2 + 5% CO2 maintained
250
significantly (P ≤ 0.05) 1.44-fold more total anthocyanin contents having reddish colour, as
251
compared to other CA gases combinations after 35 days of storage (Fig. 3A).
252
DPPH radical scavenging activity and total phenolic contents in peel tissues of litchi fruit
253
progressively and continuously decreased with increased storage period in all treatments (Fig. 3B
254
and C). On an average, control fruit showed more pronounced decrease in DPPH radical
255
scavenging activity and phenolic contents, as compared to fruit subjected to CA-storage. Fruit
256
stored under CA-atmosphere had significantly (P ≤ 0.05) higher DPPH radical scavenging
257
activity, and phenolic contents (1.53- and 1.90-fold) in peel tissues of litchi fruit, than control on
258
day-28 (Fig. 3B and C). Among the different CA-treatments, fruit stored under 1% O2 + 5% CO2
259
gaseous composition showed significantly (P ≤ 0.05) higher DPPH radical scavenging activity
260
(76%), and phenolic contents (82%) in peel tissues of litchi fruit, than other treatments,
261

15. 13
respectively, after 35 days of storage (Fig. 3B and C). Similarly, DPPH radical scavenging
262
activity and total phenolic contents of pulp tissues were also significantly (P ≤ 0.05) 1.51- and
263
1.96-folds higher in CA-stored litchi fruit, than control up to 28 days of storage period
264
(Supplementary Fig. 2A and B). Among different CA-treatments, fruit stored under 2% O2 + 3%
265
CO2 exhibited substantially (P ≤ 0.05) 1.63-fold higher DPPH radical scavenging activity;
266
whereas, total phenolic contents were found to be 1.21-fold higher in fruit kept under 1% O2 +
267
5% CO2 , than all other CA-conditions, after 35 days of storage (Supplementary Fig. 2A and B).
268
3.4. SSC, TA, SSC/TA ratio and ascorbic acid
269
SSC showed continuous decrease from day-7 to day-35 regardless of the treatments. Control
270
fruit exhibited most pronounced decrease in SSC, as compared to CA-stored fruit (Fig. 4A).
271
Averaged over the control, fruit subjected to CA-storage had substantially (P ≤ 0.05) 79% higher
272
mean SSC, as compared to control during 28 days of storage (Fig. 4A). Among the different CA-
273
treatments, fruit stored under 1% O2 + 4% CO2 maintained significantly (P ≤ 0.05) 85% higher
274
SSC up to 28 days of storage; however, on day-35, SSC were substantially (P ≤ 0.05) 89%
275
higher in fruit subjected to 1% O2 + 5% CO2 conditions (Fig. 4A).
276
Litchi fruit exhibited significant (P ≤ 0.05) linear decline in TA from day-7 to day-35 in all
277
treatments (Fig. 4B). However, control fruit showed highest decrease in TA, as compared to fruit
278
stored under CA-conditions on day-28 (Fig. 4B). On an average, fruit kept under CA-
279
environment had significantly (P ≤ 0.05) 2.0-fold more TA, in contrast to control fruit. As far
280
different treatments are concerned, fruit subjected to 1% O2 + 5% CO2 CA-conditions maintained
281
significantly (P ≤ 0.05) 1.25-fold higher TA, than all other treatments after 35 days of storage
282
(Fig. 4B).
283

16. 14
SSC/TA ratio substantially increased from day-7 to day-14 and then decreased from day-
284
21 to day-28, and once again increased slightly on day-35 (Fig. 4C). However, CA-stored fruit
285
showed about 63% reduced SSC/TA ratio during 28 days of storage, as compared to control (Fig.
286
4C). Among the different CA-combinations, fruit stored under 2% O2 + 4% CO2 maintained
287
significantly (P ≤ 0.05) 72% lower SSC/TA ratio during first 28 days of storage; but, after 35
288
days, SSC/TA ratio was about 84% lower in fruit kept under 1% O2 + 5% CO2 conditions, as
289
compared to other treatments (Fig. 4C).
290
Ascorbic acid contents continuously decreased from day-7 to day-35, irrespective to the
291
treatments (Fig. 4D). Nevertheless, control fruit showed substantial (P ≤ 0.05) decline, as
292
compared to fruit subjected to CA-storage which had about 2.04-fold higher mean ascorbic acid
293
contents on day-28 (Fig. 4D). As far as different treatments are concerned, fruit subjected to 1%
294
O2 + 5% CO2 CA-combination exhibited about 1.56-fold higher ascorbic acid contents, than all
295
other treatments after 35 days of storage (Fig. 4D).
296
3.5. Activities of PPO, POD, CAT and SOD enzymes
297
Activities of pericarp PPO enzyme showed gradual increase with the progress in storage
298
period. Control fruit exhibited more rapid increase in PPO enzyme activities, and reached to
299
maximum level on day-28 (Fig. 5A). On an average, mean PPO activity of control fruit was
300
significantly (P ≤ 0.05) 3.34-fold higher, than CA-stored fruit on day-28 of storage (Fig. 5A).
301
Among the treatments, fruit subjected to 1% O2 + 5% CO2 conditions exhibited significantly (P ≤
302
0.05) 1.54-fold reduced activities of PPO enzyme in peel tissues of litchi fruit, than other CA-
303
treatments after 35 days of storage (Fig. 5A). Activities of POD enzyme in fruit pericarp tissues
304
exhibited continuous increasing trend with increased storage periods, regardless of the treatments
305
(Fig. 5B). However, control fruit exhibited significant (P ≤ 0.05) increase in the activities of
306

17. 15
POD enzyme, than CA-stored fruit. Averaged over the control, fruit stored under CA-storage
307
exhibited 68% reduced activities of POD enzymes, than control during 28 days of storage (Fig.
308
5B). Among various CA-conditions, fruit kept under 1% O2 + 5% CO2 showed 61% less
309
activities of POD enzyme activities in peel tissues of litchi fruit, in contrast to other
310
combinations after 35 days of storage (Fig. 5B). On the other hand, POD enzyme activities in
311
pulp tissues of litchi fruit were significantly (P ≤ 0.05) 53.34% less under CA-conditions, than
312
control during 28 days of storage. Among different CA-treatments, fruit subjected to 1% O2 +
313
5% CO2 showed significantly (P ≤ 0.05) 52.53% reduced activities of pulp POD enzyme, after
314
35 days of storage (Supplementary Fig. 3A).
315
Activities of CAT and SOD enzymes in peel tissues exhibited gradual decrease from day-
316
7 to day-35, irrespective to the treatments (Fig. 5C and D). However, activities of CAT and SOD
317
enzymes in control fruit were declined more quickly than CA-stored fruit. On an average, fruit
318
subjected to CA-conditions showed significantly (P ≤ 0.05) about 40- and 53% higher activities
319
of CAT and SOD enzymes, than control, respectively, during 28 days of storage (Fig. 5C and D).
320
Among different CA-treatments, fruit stored under 1% O2 + 5% CO2 conditions maintained
321
significantly higher activities of CAT and SOD enzymes (66% and 87%), in contrast to all other
322
CA-conditions after 35 days of storage (Fig. 5C and D). Similarly, activities of CAT and SOD
323
enzymes in pulp tissues of litchi fruit were also substantially (P ≤ 0.05) 1.85- and 1.97-folds
324
higher under CA-conditions, as compared to control, on day-28 of storage period
325
(Supplementary Fig. 3B and C). Among different CA-combinations, fruit stored under 1% O2 +
326
5% CO2 showed significantly (P ≤ 0.05) 1.38- and 1.23-folds higher activities of pulp CAT and
327
SOD enzymes, after 35 days of storage period (Supplementary Fig. 3B and C).
328

18. 16
3.6. Organoleptic characteristics
329
Organoleptic evaluation was carried out to assess any changes in the eating quality and
330
overall acceptance of CA-stored ‘Gola’ litchi fruit. Organoleptic quality was comparatively
331
decreased from day-7 to day-35 in all treatments (Fig. 6A, B, C and D). However, according to
332
the panelists, control fruit exhibited substantially (P ≤ 0.05) lower scores for organoleptic
333
parameters (Fig. 6A, B, C and D). On the other hand CA-stored fruit exhibited substantially (P ≤
334
0.05) about 2.03-, 1.68-, 1.91- and 1.67-fold higher scores for taste, flavour, aroma, and overall
335
acceptance, respectively, than control fruit on day-28 (Fig. 6A, B, C and D). Among the different
336
CA-combinations, fruit kept under 1% O2 + 5% CO2 conditions showed significantly (P ≤ 0.05)
337
1.23-, 1.25-, 1.22- and 1.22-fold higher score for taste, flavour, aroma and overall acceptance,
338
respectively after 35 days of storage period (Fig. 6A, B, C and D).
339
4. Discussion
340
In the present study, weight loss was significantly lower in CA-stored fruit (Fig. 1A).
341
Water is the main component of fruits and vegetables; therefore, reduction of its loss from the
342
commodity is the most critical requirement for maintenance of postharvest quality attributes
343
(Jiang Fu, 1999). Pericarp browning of litchi fruit directly correlates with moisture loss
344
(Underhill Simons, 1993). After harvest at the lower levels of relative humidity, rate of water
345
loss becomes more obvious, and litchi fruit rapidly loses its attractive bright red colour (Jiang
346
Fu, 1999). The lower weight loss in CA-stored fruit could be attributed to minimum moisture
347
loss. CA-stored litchi fruit exhibited significantly less decay incidence throughout the storage
348
period (Fig. 1B). The reduced decay incidence could be due to suppressed micro-cracks of litchi
349
pericarp (Underhill Simons, 1993) because micro-cracks act as major entry points of
350
pathogens. Similarly, Tian et al. (2005) also reported reduced decay incidence in CA-stored
351

19. 17
‘Heiye’ litchi fruit. In the current work, litchi fruit subjected to CA-storage showed significantly
352
less pericarp browning, as compared to control fruit which were completely turned brown (5.0
353
score) after 28 days of storage (Fig. 1C). Reduced pericarp browning may possibly be ascribed to
354
the higher membrane integrity and decreased activities of POD and PPO enzymes (Zauberman et
355
al., 1991). It is now a well-established fact that pericarp browning in litchi fruit is usually
356
ascribed to oxidation of the peel phenolics (Duan et al., 2011). Initiation of the enzymatic
357
browning is also associated with the loss of membrane integrity due to de-compartmentalization
358
of different enzymes like POD and PPO and their substrates (phenolics) (Jiang, Li, Jianrong,
359
2004). Similarly, less browning rate in CA-stored fruit may be correlated with reduced pericarp
360
browning (Fig. 1D).
361
Pericarp pH of CA-stored litchi fruit remained more acidic, than control (Fig. 2A).
362
Anthocyanin degradation strongly depends upon the pericarp pH of litchi fruit (Zauberman et al.,
363
1991). Pericarp pH also plays critical role in the activation of PPO and POD enzymes as when
364
pH changes from acidic to basic it substantially increase activities of these enzymes and it
365
ultimately affects the flavylium-cations-carbinol ratio of anthocyanins. Under the increased pH
366
(acidic to basic) conditions, anthocyanins rapidly transform into the colorless-carbinol-
367
compounds (Underhill Critchely, 1995; Joas et al., 2005).
368
In the current work, CA-stored litchi fruit showed substantially less membrane leakage
369
(Fig. 2B). Membrane permeability which is usually expressed in terms of relative leakage rate is
370
considered as most important critical index of membrane integrity of litchi fruit (Sivakumar
371
Korsten, 2010). Senescence of fruit results in increased electrolyte leakage. CA-storage
372
suppressed the fruit senescence which ultimately resulted in reduced electrolyte leakage as found
373
previously in ‘Blackamber’ Japanese plums (Singh Singh, 2013). In the present study, MDA
374

20. 18
contents were lower in CA-stored ‘Gola’ litchi fruit (Fig. 2C). MDA contents are known as one
375
of the major lipid per-oxidized products which reflect the actual extent of membrane-lipid-
376
peroxidation induced by reactive oxygen species (ROS) (Shewfelt del Rosario, 2000).
377
Increased production of ROS generally leads to enhanced lipid peroxidation, causes membrane
378
deterioration, and ultimately leads to reduced storage potential, loss of quality, and marketability
379
of the horticultural products (Marangoni, Palma, Stanley, 1996; Shewfelt del Rosario,
380
2000). The lower MDA production in CA-stored fruit could be attributed to reduced ROS
381
accumulation (Hodges Forney, 2000); therefore, inhibiting the peroxidation of the membranes
382
of litchi fruit.
383
The higher anthocyanin contents in CA-stored fruit (Fig. 3A) may be attributed to acidic
384
pericarp pH which inhibited the activities of PPO and POD enzymes. DPPH radical scavenging
385
activity was significantly higher in pulp (Supplementary Fig. 2A) and peel (Fig. 3B) tissues of
386
CA-stored litchi fruit. Antioxidants of fruits are believed to be contributed by several bioactive
387
compounds such as ascorbic acid, anthocyanins, and phenolics (Barman, Siddiqui, Patel,
388
Prasad, 2014). So, higher DPPH radical scavenging activity in CA-stored fruit may be due to
389
suppressed oxidation of ascorbic acid, anthocyanins, and phenolic compounds (Stewart, Oparka,
390
Johnstone, Iannetta, Davies, 1999). Fruit subjected to CA-storage also showed substantially
391
higher total phenolics in litchi pulp (Supplementary Fig. 2B) and peel (Fig. 3C) tissues. Higher
392
peel and pulp TPC of litchi fruit might be ascribed to the inhibited oxidation and decreased
393
membrane leakage because impaired membrane integrity ultimately causes mixing of POD and
394
PPO enzymes with phenolic contents (Zauberman et al., 1991).
395
In the current work, SSC of litchi fruit was significantly higher (Fig. 4A). Higher SSC
396
could be ascribed to suppressed senescence (Barman et al., 2014) of CA-stored fruit. TA was
397

21. 19
progressively decreased with the progress in storage period (Fig. 4B). The continuous decline in
398
TA of control fruit could be due to malate decarboxylation, and the subsequent pyruvate
399
decarboxylation (Hawker, 1969). SSC/TA ratio substantially increased with the advancement of
400
storage period (Fig. 4C). SSC/TA ratio usually increases due to conversion of starch into sugars
401
(Akhtar, Abbasi, Hussain, 2010). Delayed increase in SSC/TA ratio of CA-stored fruit could
402
be due to suppressed conversion of starch into simple sugars. In the present study, CA-stored
403
fruit had significantly higher ascorbic acid contents (Fig. 4D). Ascorbic acid has been known to
404
decline under prolonged-storage conditions probably owing to the utilization of different organic
405
acids during fruit respiration or their likely conversion to the sugars (Kader, 2002). Oxidative
406
deterioration of ascorbic acid contents may also leads to its rapid reduction (Piga, Caro, Pinna,
407
Agabbio, 2003). However, significantly higher ascorbic acid contents in CA-stored fruit could be
408
due to inhibited senescence, and reduced oxidation of various organic acids (Piga et al., 2003).
409
Fruit subjected to CA-storage showed significantly lower activities of peel PPO and POD
410
enzymes (Fig. 5A and B) which could be attributed to the acidic pericarp pH of litchi fruit (Joas
411
et al., 2005). Another possible reason for reduced activities of PPO and POD enzymes may be
412
the suppressed membrane de-compartmentalization. Increased de-compartmentalization usually
413
leads to the mixing of both these enzymes with peel phenolics (Zauberman et al., 1991;
414
Underhill Critchley, 1995). In the current study, CA-stored fruit maintained higher activities
415
of CAT and SOD enzymes in fruit peel (Fig. 5C and D) and pulp tissues (Supplementary Fig. 3B
416
and C). CAT and SOD are antioxidative detoxifying enzymes, and play imperative role in the
417
scavenging of ROS (Duan et al., 2011). CAT defends the cells from hydrogen peroxide (H2O2)
418
by catalyzing its decomposition into corresponding O2 and H2O molecules. Increased activity of
419
CAT enzyme is very critical as far as defensive mechanism against H2O2 damage is concerned.
420

22. 20
Higher activity of CAT in CA-stored fruit suggests the strong ability of the cells to scavenge
421
H2O2 (Duan et al., 2011). On the other hand, SOD enzyme is responsible for the dismutation of
422
superoxide (O-2
) free radicle to O2 and H2O2; thus, it ultimately prevents the cells from O-2
-
423
induced-radical-damage. Increased SOD activity in CA-stored fruit could provide more
424
protection against the oxidative damage caused by O−2
radicals. Higher activities of CAT and
425
SOD enzymes in CA-stored fruit could be due to reduced lipid peroxidation, and electrolyte
426
leakage (Zheng Tian, 2006; Duan et al., 2011; Singh and Singh, 2013). Increased activities of
427
CAT and SOD enzymes have also been reported in CA-stored ‘Blackamber’ Japanese plums
428
(1% O2 + 3% CO2) and Pleurotus eryngii mushroom (2% O2 + 30% CO2) (Singh Singh, 2013;
429
Li, Zhang, Hu, Sun, Wang, Zhao, 2013).
430
In the present study, CA-stored litchi fruit maintained significantly higher organoleptic
431
quality, than control (Fig. 6A, B, C and D). Maintenance of organoleptic quality is very
432
important because prolonged CA-storage may cause development of off-flavours due to ethanol
433
contents (Tian et al., 2005). Significantly lower organoleptic scores for taste, flavour, aroma, and
434
overall acceptance of control fruit could be attributed to the development of off-flavours
435
(Sivakumar Korsten, 2010). Higher sugar: acid ratio also results in reduced organoleptic
436
quality due to excessive sweetness (Sivakumar Korsten, 2010).
437
5. Conclusion
438
In conclusion, CA-storage of ‘Gola’ litchi fruit under 1% O2 + 5% CO2 conditions
439
substantially delayed enzymatic browning, maintained acidic pericarp pH, reduced membrane
440
leakage, MDA contents, and resulted in substantially lower activities of PPO and POD enzymes.
441
CA-stored fruit showed significantly higher peel anthocyanin contents, DPPH radical scavenging
442

23. 21
activity, total phenolic contents and activities of CAT and SOD enzymes. CA-stored litchi fruit
443
also maintained significantly better biochemical and organoleptic characteristics for 35 days.
444
Thus, 1% O2 + 5% CO2 CA-combination could be used to reduce postharvest pericarp browning
445
and to extend the storage life of litchi fruit.
446
Acknowledgements
447
The first author is highly grateful to Higher Education Commission, Pakistan for funding
448
his PhD research under Project-2077 entitled as “Postharvest Storage Life and Quality
449
Management of Litchi”. Technical assistance of Mr. Umair Javid for smooth running of
450
controlled atmosphere system is also acknowledged.
451
Conflict of interest
452
The authors declare no conflict of interest.
453
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454
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29. 27
Figure Captions
563
Fig. 1. Effect of controlled atmosphere (CA) storage on weight loss (A), decay incidence (B),
564
pericarp browning (C), and browning rate (D) of litchi fruit. Vertical bars represent ±
565
S.E. of means, and bars are invisible where values are smaller than the symbols. n = 3.
566
Tukey HSD (P ≤ 0.05) for fruit weight loss: treatments (T) = 0.973, storage periods (SP)
567
= 0.649 and T × SP = 1.947; decay incidence: T = 2.113, SP = 1.408 and T × SP =
568
4.226; pericarp browning: T = 0.365, SP = 0.243 and T × SP = 0.731; browning rate; T =
569
1.889, SP = 1.259 and T × SP = 3.779.
570
Fig. 2. Effect of controlled atmosphere (CA) storage on pericarp pH (A), membrane leakage (B),
571
and malondialdehyde (C) in peel tissues of litchi fruit. Vertical bars represent ± S.E. of
572
means, and bars are invisible where values are smaller than the symbols. n = 3. Tukey
573
HSD (P ≤ 0.05) for pericarp pH: treatments (T) = 0.095, storage periods (SP) = 0.063 and
574
T × SP = NS; membrane leakage: T = 5.595, SP = 3.730 and T × SP = NS;
575
malondialdehyde: T = 1.279, SP = 0.852 and T × SP = 2.558. NS = Not significant.
576
Fig. 3. Effect of controlled atmosphere (CA) storage on total anthocyanins contents (A), DPPH
577
radical scavenging activity (B), and total phenolic contents (C) in peel tissues of litchi
578
fruit. Vertical bars represent ± S.E. of means, and bars are invisible where values are
579
smaller than the symbols. n = 3. Tukey HSD (P ≤ 0.05) for total anthocyanins: treatments
580
(T) = 0.019, storage periods (SP) = 0.013 and T × SP = 0.039; DPPH radical scavenging
581
activity: T = 1.767, SP = 1.178 and T × SP = 3.534; total phenolic contents: T = 7.226,
582
SP = 4.817 and T × SP = 14.453.
583

30. 28
Fig. 4. Effect of controlled atmosphere (CA) storage on SSC (A), TA (B), SSC: TA ratio (C),
584
and ascorbic acid contents (D) of litchi fruit. Vertical bars represent ± S.E. of means, and
585
bars are invisible where values are smaller than the symbols. n = 3. Tukey HSD
586
(P ≤ 0.05) for SSC: treatments (T) = 0.770, storage periods (SP) = 0.513 and T × SP =
587
1.540; TA: T = 0.037, SP = 0.025 and T × SP = 0.075; SSC: TA ratio: T = 7.947, SP =
588
5.298 and T × SP = 15.894; ascorbic acid contents: T = 3.527, SP = 2.351 and T × SP =
589
7.055.
590
Fig. 5. Effect of controlled atmosphere (CA) storage on activities of PPO (A), POD (B), CAT
591
(C), and SOD (D) enzymes in peel tissues of litchi fruit. Vertical bars represent ± S.E. of
592
means, and bars are invisible where values are smaller than the symbols. n = 3. Tukey
593
HSD (P ≤ 0.05) for PPO: treatments (T) = 1.015, storage periods (SP) = 0.676 and T ×
594
SP = 2.030; POD: T = 1.429, SP = 0.952 and T × SP = 2.858; CAT: T = 1.178, SP =
595
0.785 and T × SP = NS; SOD: T = 2.109, SP = 1.406 and T × SP = 4.219. NS = Not
596
significant.
597
Fig. 6. Effect of controlled atmosphere (CA) storage on taste (A), flavour (B), aroma (C), and
598
overall acceptance (D) of litchi fruit. Vertical bars represent ± S.E. of means, and bars
599
are invisible where values are smaller than the symbols. n = 3. Tukey HSD
600
(P ≤ 0.05) for taste: treatments (T) = 0.392, storage periods (SP) = 0.261 and T × SP =
601
0.784; flavour: T = 0.508, SP = 0.339 and T × SP = 1.017; aroma: T = 0.436, SP = 0.291
602
and T × SP = 0.873; overall acceptance: T = 0.503, SP = 0.335 and T × SP = NS. NS =
603
Not significant.
604
605

31. 29
Supplementary Figure Captions
606
Supplementary Fig. 1. Effect of controlled atmosphere (CA) storage on disease incidence (A),
607
and disease severity (B) of litchi fruit. Vertical bars represent ± S.E. of
608
means, and bars are invisible where values are smaller than the
609
symbols. n = 3. Tukey HSD (P ≤ 0.05) for disease incidence: treatments
610
(T) = 2.086, storage periods (SP) = 1.390 and T × SP = 4.172; disease
611
severity: T = 0.400, SP = 0.267 and T × SP = 0.801.
612
Supplementary Fig. 2. Effect of controlled atmosphere (CA) storage on DPPH radical
613
scavenging activity (A), and total phenolic contents (B) in pulp tissues
614
of litchi fruit. Vertical bars represent ± S.E. of means, and bars are
615
invisible where values are smaller than the symbols. n = 3. Tukey HSD
616
(P ≤ 0.05) for DPPH radical scavenging activity: treatments (T) = 2.123,
617
storage periods (SP) = 1.415 and T × SP = NS; total phenolic contents: T
618
= 7.227, SP = 4.817 and T × SP = 14.453. NS = Not significant.
619
Supplementary Fig. 3. Effect of controlled atmosphere (CA) storage on activities of POD (A)
620
CAT (B), and SOD (C) enzymes in pulp tissues of litchi fruit. Vertical
621
bars represent ± S.E. of means, and bars are invisible where values are
622
smaller than the symbols. n = 3. Tukey HSD (P ≤ 0.05) for POD:
623
treatments (T) = 1.438, storage periods (SP) = 0.958 and T × SP =
624
2.876; CAT: T = 1.178, SP = 0.785 and T × SP = NS; SOD: T = 2.169,
625
SP = 1.446 and T × SP = 4.338. NS = Not significant.
626
627

32. 30
0
4
8
12
16
Fruit
weight
loss
(%)
Control CA-1
CA-2 CA-3
CA-4 CA-5
CA-6 CA-7
CA-8 CA-9
A
0
10
20
30
40
Decay
incidence
(%)
B
0
25
50
75
100
7 14 21 28 35
Browning
rate
(%)
D
Storage period (Days)
0
1
2
3
4
5
Pericarp
browning
(Score)
C
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
Fig. 1.
654

33. 31
0
1
2
3
4
5
Pericarp
pH
Control CA-1 CA-2 CA-3 CA-4
CA-5 CA-6 CA-7 CA-8 CA-9
A
0
16
32
48
64
Membrane
leakage
(%)
B
0
10
20
30
40
50
7 14 21 28 35
Malondialdehyde
(nmole
g
-1
FW)
C
Storage period (Days)
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
Fig. 2.
679

34. 32
20
45
70
95
DPPH
radical
scavenging
activity
(%)
B
50
150
250
350
7 14 21 28 35
Total
phenolic
contents
(mg
GAE
100
g
-1
FW)
C
Storage period (Days)
0.00
0.20
0.40
0.60
0.80
1.00
Total
anthocyanin
contents
(∆Ag
-1
FW)
Control CA-1
CA-2 CA-3
CA-4 CA-5
CA-6 CA-7
CA-8 CA-9
A
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
Fig. 3.
705
706

35. 33
0
7
14
21
Soluble
solid
contents
(
o
Brix)
Control CA-1 CA-2 CA-3 CA-4
CA-5 CA-6 CA-7 CA-8 CA-9
A
0.0
0.2
0.4
0.6
Titratable
acidity
(%)
B
25
50
75
100
Sugar
acid
ratio
C
10
35
60
85
7 14 21 28 35
Ascorbic
acid
(mg
100
-1
mL)
D
Storage period (Days)
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
Fig. 4.
731

36. 34
0
20
40
60
Catalase
(U
mg
-1
protein)
C
0
10
20
30
40
50
Polyphenol
oxidase
(U
mg
-1
protein)
Control CA-1
CA-2 CA-3
CA-4 CA-5
CA-6 CA-7
CA-8 CA-9
A
0
15
30
45
60
Peroxidase
(U
mg
-1
protein)
B
20
55
90
125
7 14 21 28 35
Superoxide
dismutase
(U
mg
-1
protein)
D
Storage period (Days)
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
Fig. 5.
756

37. 35
1
3
5
7
9
Taste
(Score)
Control CA-1 CA-2 CA-3 CA-4
CA-5 CA-6 CA-7 CA-8 CA-9
A
1
3
5
7
9
Flavour
(Score)
B
1
3
5
7
9
Aroma
(Score)
C
1
3
5
7
9
7 14 21 28 35
Overall
acceptance
(Score)
D
Storage period (Days)
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
Fig. 6.
781