全 文 ：※基础研究 食品科学 2015, Vol.36, No.01 19
Mechanisms of Chlorophyll Breakdown in the Drying of Green Prickleyashes
(Zanthoxylum schinifolium Zucc.) with Damaged Oil Vacuoles
KAN Jianquan1,2, CHEN Kewei1, ZHANG Fusheng1,2, ZHENG Jiong1,2
(1. College of Food Science, Southwest University, Chongqing 400715, China;
2. Laboratory of Quality and Safety Risk Assessment for Agro-products on Storage and Preservation (Chongqing),
Ministry of Agriculture, Chongqing 400715, China)
Abstract: Fast drying could restrain chlorophyll (Chl) degradation in green prickleyashes (GPs) drying except for GPs
with damaged oil vacuoles (GP-DOV). To explore the possible mechanisms behind this phenomenon, the variations in Chl
pigments and related enzymes were investigated. Compared with GPs with intact oil vacuole, the variations in Chl pigments
in GP-DOV were more serious and complicated, that is, only 6% of the total Chl was retained in GP-DOV. This damage
seemed to inhibit the formation of pyrochlorophyll pigments after the formation of pheophorbide-a in the pheophorbide-a
oxygenase pathway and resulted in the accumulation of pheophorbide a, which accounted for 18% of the total Chl pigments
in dried GP. During the slow drying process, the damage gradually induced the activity of chlorophyll-degrading peroxidase, resulting in
the accumulation of C132 oxidized Chl pigments. The activities of related enzymes such as chlorophyllase and pheophorbidase showed a
downward trend whereas chlorophyll-degrading peroxidase and metal-chelating substances was fluctuated.
Key words: green prickleyash; drying; chlorophyll breakdown
油胞破坏性青花椒干燥过程中叶绿素降解机制
阚建全1,2，陈科伟1，张甫生1,2，郑  炯1,2
（1.西南大学食品科学学院，重庆  400715；
2.农业部农产品贮藏保鲜质量安全风险评估实验室（重庆），重庆  400715）
摘  要：青花椒在快速干燥过程中可以抑制叶绿素的降解，但油胞破坏性青花椒除外。为了探讨这个现象背后的
发生机制，本实验将对油胞破坏性青花椒的叶绿素和相关酶的变化进行研究。结果表明，与油胞完整性青花椒相
比，干燥后和油胞破坏性青花椒的叶绿素变化更加严重和复杂，干燥后的叶绿素仅剩下原来的6%。油胞破坏抑制
了通过脱镁叶绿酸a氧化酶途径形成的焦脱植基叶绿素，并导致脱镁叶绿酸a的在青花椒体内积累（占总叶绿素的
18%）。在慢速干燥过程中，逐渐增加的叶绿素降解过氧化物酶活力使得C132氧化型叶绿素的含量不断增加，相关
的酶、叶绿素酶和脱镁叶绿素酸酶呈现出下降趋势，而叶绿素降解过氧化物酶和脱镁螯合物则出现波动变化。
关键词：青花椒；干燥；叶绿素降解
中图分类号：TS255.1                    文献标志码：A 文章编号：1002-6630（2015）01-0019-08
doi:10.7506/spkx1002-6630-201501004
收稿日期：2014-01-13
基金项目：国家自然科学基金面上项目（31071599）
作者简介：阚建全（1965—），男，教授，博士，研究方向为食品化学与营养学、食品安全与质量控制。E-mail：kanjianquan@163.com
The green prickleyash (GP) Zanthoxylum schinifolium
Zucc., characterized by a strong spicy and astringent taste,
is a traditional spice found in China and other subtropical
areas. Its fruit presents a bright green color because of the
high content of chlorophyll (Chl) pigments in the peel. Dried
GPs are the main products for commercial trade[1]. During
harvest seasons, when farmers pick bunches of GPs from
the trees and place them on the ground to dry under the
sun, oil vacuoles should be given extra care because they
are vulnerable to damage. Mechanical destruction of oil
vacuoles causes quick degradation of the greenness of GPs,
resulting in an undesirable brownness that greatly affects its
marketability. “Brown prickleyashes” have poor quality and
sold at a very low price.
Chl pigments are responsible for the typical green
color of vegetables and fruits. They are also easy to
20 2015, Vol.36, No.01 食品科学 ※基础研究
degrade into colorless compounds during leaf senescence
and fruit ripening. A study reported that Chl-a follows the
pheophorbide (Pheide)-a oxygenase (PAO) degradation
pathway to become non-colored linear tetrapyrroles[2]. The
reaction is initiated by converting Chl-a to chlorophyllide
(Chlide)-a using chlorophyllase (Chlase) and then removing
magnesium from the porphyrin ring of Chlide-a to generate
pheophorbide (Pheide)-a in the presence of a metal-chelating
substance (MCS), which may be non-enzymatic in nature[3-5].
The conversion of Pheide-a to primary fluorescent Chl
catabolites (FCCs) requires two enzymes: PAO and red
chlorophyll catabolite reductase. Finally, FCCs undergo a
series of modifications to yield colorless non-fluorescent
Chl catabolites[2,6]. Pheide-a can also be converted to
pyropheophorbide (PyroPheide)-a, another methoxycarbonyl-
free Chl catabolite, through chemical and physical
modifications[7-8]. Pheophorbidase (Phedase) is responsible
for the conversion of demethylated Pheide-a to C132-
carboxyl PyroPheide-a. C132-carboxyl PyroPheide-a then
changes to PyroPheide-a by non-enzymatic decarboxylation.
Pheophorbide demethoxycarbonylase (PDC), another enzyme
found in some Chl-b-less mutants, is identified to directly
catalyze Pheide-a to PyroPheide-a without any intermediate[9].
Several studies reported that Chl-a can undergo another
conversion to form C132-hydroxychlorophyll (C132-OH
Chl)-a in the storage of some green vegetables[10-13]. This
conversion is mainly mediated by chlorophyll-degrading
peroxidase (Chl-POD), which facilitates the oxidization of
Chl-a at C132 in the presence of polyphenols and hydrogen
peroxide[14]. Except for common Chl catabolites, several
other types of Chl derivatives, such as C151-hydroxy-lactone
chlorophyll (C151-OH-lactone Chl)-a, pyrochlorophyllide
(PyroChlide)-a, and pyropheophytin (PyroPhy)-a, were also
found in some ripen fruits or processed food[15-16].
However, only a few studies reported on the color
changes and chlorophyll degradation in the post-harvest
processing of GPs. In our previous study, the chlorophyll
breakdown was initiated after the fruits were detached from
the tree, especially during slow drying where GPs were
placed in the shade to dry. Considering that oil vacuoles are
intact, Chl-a and Chl-b are the major Chl pigments in GPs
before and after slow drying[1]. However, the results showed
that these two types of Chl pigments minimally contribute
in dried GPs with damaged oil vacuoles (GP-DOV). In
some extreme cases, dried GP-DOV is totally black, which
is much worse than slow-dried GPs with intact oil vacuoles
(GP-IOV). This study examines the mechanisms behind Chl
degradation in GP-DOV drying, including the changes in peel
color, enzyme activity, and content of chlorophylls and their
derivatives. The relationships among these changes were also
established to explore the possible mechanisms behind color
degradation during GP-DOV drying.
1 Materials and Methods
1.1 Raw materials
Fresh GPs were harvested at commercial maturity
from a local farm in Chongqing, China. They were picked
carefully from the same tree to ensure consistency. Leafstalks
were cut off to leave only subglobose fruits. The broken oil
vacuoles were induced on an oscillator, and 50 g fresh GPs
in a 500 mL Erlenmeyer flask were treated at 200 r/min for
60 s. Two drying modes (slow and fast) were employed to
dry GP-DOV. To avoid the interference of moisture content,
5 g fresh GP-DOV was placed in a polyvinyl plastic open-
tank before drying. Slow-dried samples were placed indoors
in dim light to evaporate water, where an environment of
25 ℃ with 50% relative humidity was maintained by an air-
conditioner. Fast-dried samples were dried in an oven at 50 ℃ 
to obtain greenish products from intact oil vacuoles before
drying. Samples were determined at an interval of 12 h for
slow drying and 1 h for fast drying until the moisture content
dropped below 5%. Finally, the products were obtained after
108 h in slow mode and 10 h in fast mode. The samples were
frozen at －80 ℃ before analysis.
1.2 Determining color and moisture content
An Ultra Scan PRO (Hunterlab, America) was applied
to evaluate the color changes in GPs peel. The procedure was
repeated 20 times to minimize inaccuracy. The samples were
dried in an oven at 100 ℃  to constant weight to measure
moisture content.
1.3 Acetone powder preparation
Acetone powder of GPs was prepared according to the
methods described by Roca et al.[15]. Deseeded GPs were
homogenized with 20 volumes of cold acetone (－20 ℃), and
the supernatant was filtrated and discarded. The residue was
continually washed with 5 volumes of cold acetone to achieve
a colorless percolate. Finally, the residue was dried by cold
wind to obtain dry powder. Approximately 0.12 g acetone
powder was obtained from 1 g deseeded GPs (fresh weight).
1.4 Enzyme extraction
The crude enzyme was extracted from acetone powder
※基础研究 食品科学 2015, Vol.36, No.01 21
using the method described by Fukasawa et al.[17]. Briefly,
500 mg acetone powder was treated with 15 mL of sodium
phosphate buffer (10 mmol/L, pH 7.0) to extract Chl-POD
and then with 15 mL of sodium phosphate buffer (50 mmol/L,
pH 7.0) containing 50 mmol/L potassium chloride and
0.24 g/100 mL Triton-X-100 to obtain Chlase, Phedase, and
MCS. This procedure was carried out in an ice bath with 1 h
stirring. The supernatant, applied as the crude enzyme extract,
was acquired by filtration through four layers of cotton gauze
and subsequent centrifugation (Sigma, 4K15, Germany) at
9 000 × g for 15 min at 4 ℃.
1.5 Pigment preparation and analysis
1.5.1 Standard pigment preparation
Chl-a and Chl-b extracted from spinach leaves were
employed to prepare standard pigments[1]. Dephytoled
Chl derivates (Chlide-a and Pheide-a) were acquired
from their corresponding Chl parents by enzymatic
deesterification using a Chlase partially purified (20% to 40%
of ammonium sulfate) from citrus peels[18]. C132-OH Chl
pigments were prepared by selenium dioxide oxidation of
their corresponding Chl pigments with reflux-heating for
4 h in pyridine solution under argon. Carbomethoxy-free Chl
derivatives (pyropheophytin, PyroChlide, and PyroPheide)
were also prepared from their respective parents by reflux-
heating at 100 ℃ in collidine[15]. C151-OH-lacton Chl was
obtained by oxidation in alkaline medium achieved by
mixing Chl acetone solution with 0.5% sodium hydroxide[19].
Sequentially, Chl was oxidized by atmospheric oxygen after
stirring at room temperature for 10 min. The above oxidized
products were transferred to diethyl ether and washed with
sodium chloride (NaCl)-saturated solution. The separation
of C132-OH Chl and C151-OH-lacton Chl from mixtures
was carried out by thin-layer chromatography and sequent
preparative high-performance liquid chromatography (HPLC,
Shimadzu, Japan). All magnesium-free Chl derivatives were
acquired from their corresponding Chl precursors in diethyl
ether using acidification by adding four to five drops of 10%
hydrochloric acid and then washed with distilled water.
1.5.2 Sample pigments analysis
Pigments were extracted by the method described by
Roca et al.[15] with some modifications. The samples (5 g
in fresh weight) were deseeded before homogenization in
30 mL dimethylformamide (DMF) saturated with magnesium
carbonate, and the residue was filtrated and washed with DMF
repeatedly until the filtrates were colorless. The combination
of the DMF extracts was washed thrice with 50 mL hexane to
eliminate lipids and oils, and the Chl pigments were reserved
in DMF phase. Subsequently, 10 g/100 mL NaCl solution
(0 ℃) was added into the DMF phase, and diethyl ether-
hexane (1:1, V/V) was applied to extract the Chl pigments
from the DMF aqueous phase. This extraction was repeated
until the aqueous phase became colorless. The combined
organic phase was continuously washed with distilled water
to eliminate polyphenols and other water-soluble compounds
and then dehydrated with anhydrous sodium sulfate. The
organic phase was evaporated under vacuum below 30 ℃,
and the dryness was dissolved in 5.0 mL acetone.
Separation was performed on a GL Sciences column
(ODS-SP, 4.6 mm × 250 mm, 5 μm) with a two-mobile-phase
eluting system following the method described by Roca
et al. [15]: (A) ion pair reagent/water/methanol (1:1:8) and
(B) methanol/acetone (1:1). The ion pair reagent was
0.05 mol/L tetrabutyl ammonium bromide and 1 mol/L
ammonium acetate in water. The eluting program was
operated as follows: the ratio of B/A mobile phase increased
linearly from 0% to 100% within 35 min. Isocratic B was
held for 15 min to stop the eluting procedure, and mobile
phase A was held for another 10 min to condition the column
before the next injection. The flow rate was kept at 1 mL/min
throughout the separation, apart from the period from 30 min
to 50 min, which had a low flow rate at 0.6 mL/min. The Chl
pigments and their derivatives were determined by HPLC
according to the method described by Fraser and Frankl[20].
1.6 Measurement of chlorophyllase, peroxidase,
pheophorbidase, and MCS dechelating activity
The enzyme activity involved in Chl degradation was
measured through HPLC. For Chlase, the method was
described by Yang et al. [21]. The reaction mixture contained
500 μL of 0.1 mol/L phosphate buffer (pH 7.5) with 0.15%
Triton-X-100, 100 μL of Chl-a (500 μg/mL) acetone solution,
and 500 μL crude enzyme extract. As for Chl-POD, the method
was described by Yamauchi et al. [22] with some modifications.
The standard reaction system (1 500 μL) contained 750 μL of
0.1 mmol/L phosphate buffer (pH 5.5), 50 μL of 1.0 g/100 mL
Triton-X 100, 50 μL of 5 mmol/L p-coumaric acid in ethanol
solution, 50 μL of 0.3% hydrogen peroxide, 100 μL of Chl-a
(500 μg/mL) acetone solution, and 500 μL crude enzyme
extract. Both reaction mixtures were incubated at 25 ℃ for
60 min in dim light. The reactions were stopped by adding
5 mL acetone for Chlase and 6 mL for Chl-POD. The assay
of Phedase activity was carried out following the method
described by Suzuki et al.[9] with some modifications. The
22 2015, Vol.36, No.01 食品科学 ※基础研究
reaction mixture in a total volume of 800 μL consisted of
20 mmol/L phosphate buffer (pH 7.0) with 4.9 μg Pheide-a
and 200 μL crude enzyme extract. The mixture was incubated
at 25 ℃ for 2 h to finish the spontaneous decarboxylation.
The reaction was stopped by adding 3.2 mL acetone. The
samples were stored at －20 ℃ for approximately 18 h prior
to HPLC. To measure MCS dechelating activity, the reaction
mixture with 800 μL of 10 mmol/L phosphate buffer (pH
7.5), 250 μL of Chlide-a (6.2 μg) in water, and 200 μL crude
enzyme extract was incubated at 37 ℃ and then immediately
analyzed by HPLC. One unit of Chlase, Chl-POD, Phedase,
and MCS activity was defined as the formation of 1 μg of
Chlide-a, C132-OH-Chl-a, PyroPheide-a, and Pheide-a per
min, respectively. Protein content was assayed by the method
described by Brandford[23] using bovine serum albumin as
standard. Each measurement was performed in triplicate.
1.7 Statistical analysis
GPs were selected randomly, and each sample was
determined thrice, except for the color measurement, which
was determined 20 times. Analysis of variance was performed
using Duncan’s test from SAS 6.0 statistical software package
with significance at the 0.05 level. All results are represented
as mean ± standard deviation (SD).
2 Results and Analysis
2.1 Peel color and Chl content
The values of H* and a* were recorded to monitor color
changes in the peels of GP-DOV. The a* value, correlating
the green color of peel, increased rapidly from below －2 to
over 4 in the two drying modes (Fig.1). This result agreed
with our visual observation that the green color degraded
quickly after oil vacuoles were destroyed. The final products
dried at 50 ℃ showed a slightly lower a* value than those
dried at 25 ℃ but still showed an undesirable degreening.
However, the H* value showed a sharply decreasing trend.
Moreover, the final products showed no conspicuous
difference between the two drying conditions. Compared
with the samples of intact oil vacuoles whose green color can
be retained by fast drying[1], the destruction of oil vacuoles
prompted a rapid degradation in color, i.e., conversion from a
typical green to an undesirable brown in a short time.
The associated Chl changes were also coincidental with
the degreening process. Chl variations in the two drying
modes were compared in Fig. 2. At 50 ℃, Ch1-a and Chl-b
decreased by approximately 88% and 76%, respectively,
with the total Chl content descending from 210.50 μg/g
FW to 31.49 μg/g FW. This Chl degradation can explain
why fast drying could not retain the green color when oil
vacuoles were damaged. At 25 ℃, a larger amount of Chl was
degraded, where Ch1-a and Chl-b declined by approximately
97% and 86%, respectively, with only 13.06 μg/g FW of the
total Chl left in the dried GP. This decomposition proportion
of Chl in GP-DOV (94%) is more than two times higher
than that in GP-IOV (44% in the total Chl, data not shown)
under the same drying condition (25 ℃). The degradation
rate in GP-DOV was also significant. Approximately 73%
and 78% of the total Chl-a were decomposed at 50 ℃  and
25 ℃ within 3 h and 24 h, respectively. However, no significant
difference in Chl content was observed between the dried GP-
DOVs acquired from different drying temperatures. Fast drying
failed to limit Chl degradation in GP-DOV.
8 A
6
4
2
0ˉ2ˉ4
70 60 50 40 30 20 10 0
Moisture content/%
a*
50 ć,10 h
25 ć,108 h
110 B
100
80
60
50
30
20
40
70
90
70 60 50 40 30 20 10 0
Moisture content/%
H
*
50 ć,10 h
25 ć,108 h
Fig.1 Changes in peel color of GP-DOV
220 A
200
160
100
80
40
20
0
60
140
120
180
70 60 50 40 30 20 10 0
Moisture content/%
C
hl
c
on
te
nt
/ (μ
g/
g
FW
)
Ch1 b
Ch1 a
Total Ch1
220 B
200
160
100
80
40
20
0
60
140
120
180
70 60 50 40 30 20 10 0
Moisture content/%
C
hl
c
on
te
nt
/ (μ
g/
g
FW
) Ch1 b
Ch1 a
Total Ch1
Fig.2 Changes in chlorophyll contents at 50 ℃ (A) and 25 ℃(B)
※基础研究 食品科学 2015, Vol.36, No.01 23
2.2 Chl derivatives and their related enzyme activities
As shown in Table 1, the pigments in the peel of
GP-DOV were separated and identified according to the
comparison of the retention time and spectrum with the
standard Chl pigments and their derivatives. The resulting
Chl derivatives in GP-DOV were much more diverse than
those in GP-IOV. Changes in enzyme activity and Chl-related
derivatives throughout the drying process were illustrated in
Fig.3 and Fig.4, respectively.
Table 1 Chromatographic and spectroscopic characteristics of
chlorophylls and their derivatives in GP-DOV
Peak No. Compound tR/min Kc’ α λmax/nm (on-line) λmax/nm (reported)
1 Chlorophyllide b 2.68 0.69 2.68 (1,2)e 469, 605, 652 468, 608, 653[24]
2 Chlorophyllide a 7.18 1.84 2.68 (1,2)e 433, 621, 667 432, 616, 664[15]
3 Pyrochlorophyllide a 10.91 2.80 1.52 (2,3)e 434, 617, 667 432, 616, 664[15]
4 C132-OH pheophorbide a 13.04 3.34 1.20 (3,4)e 411, 507, 538, 609, 666 506, 534, 608, 666[16]
5
Pheophorbide a 13.75 3.53 1.05 (4,5)e 411, 507, 537, 610, 666 506, 534, 608, 666[16]
Pheophorbide a’ 15.09 3.87 1.10 (5, 5’)e 411, 507, 537, 610, 666 506, 534, 608, 666[16]
6 Pyropheophorbide a 16.26 4.17 1.08 (5’,6)e 412, 510, 539, 611, 667
7 C132-OH chlorophyll b 28.87 7.40 1.78 (6,7)e 462, 598, 648 600, 650[16]
8
Chlorophyll b 31.34 8.04 1.09 (7,8)e 464, 601, 650 462, 600, 648[25]
Chlorophyll b’ 32.32 8.29 1.03 (8, 8’)e 464, 601, 650 462, 600, 648[25]
9 C132-OH chlorophyll a 32.79 8.41 1.01 (8’,9)e 430, 534, 578, 618, 664 534, 590, 616, 666[16]
10
Chlorophyll a 34.99 8.97 1.07 (9,10)e 432, 497, 619, 664 430, 618, 664[25]
Chlorophyll a’ 35.97 9.22 1.03 (10, 10’)e 432, 497, 619, 664 430, 618, 664[25]
11 C151-OH-lactone pheophytin a 39.48 10.12 1.10 (10’,11)e 410, 504, 532, 609, 667 531, 614, 670[16]
12 C132-OH pheophytin a 39.97 10.25 1.01 (11,12)e 410, 504, 534, 609, 667 506, 534, 608, 666[16]
13
Pheophytin a 41.29 10.59 1.03 (12,13)e 408, 505, 536, 608, 666 408, 506, 536, 608, 666[25]
Pheophytin a 41.87 10.74 1.01 (13, 13’)e 408, 505, 536, 608, 666 408, 506, 536, 610, 666[25]
14 Pyropheophytin a 43.86 11.25 1.05 (13’,14)e 413, 621, 666 410, 508, 538, 610, 666[25]
Note: tR=tr-t0, where tr is the retention time of pigment peak and t0 is the
retention time of unrestrained component; Kc’ and α represent capacity factor
and separation factor respectively; λmax. Maximum absorbance wavelength; e.
Numbers in parentheses stand for the two neighboring peaks.
0.45 A
0.40
0.30
0.20
0.15
0.10
0.05
0.25
0.35
70 60 50 40 30 20 10 0
Moisture content/%
C
hl
as
e
ac
tiv
ity
/(U
/m
g
pr
o)
50 ć,10 h
25 ć,108 h
4.0 B
3.0
2.0
1.5
1.0
0.5
2.5
3.5
70 60 50 40 30 20 10 0
Moisture content/%
M
IC
S
ac
tiv
ity
/ (U
/ m
g
pr
o)
50 ć,10 h
25 ć,108 h
0.18 C
0.14
0.10
0.08
0.06
0.04
0.02
0.12
0.16
70 60 50 40 30 20 10 0
Moisture content/%
Ph
eid
as
e a
cti
vi
ty
/(U
/m
g p
ro
)
50 ć,10 h
25 ć,108 h
8 D
6
4
3
2
1
5
7
70 60 50 40 30 20 10 0
Moisture content/%
Ch
1-
PO
D
ac
tiv
ity
/(U
/m
g p
ro
)
50 ć,10 h
25 ć,108 h
Fig.3 Changes in enzyme activities involved in Chl degradation and in
Mg-dechalating activity in GP-DOV
20 A
14
8
6
2
0
10
18
16
12
4ˉ2
70 60 50 40 30 20 10 0
Moisture content/%
C
hl
id
e
a
co
nt
en
t/(
μg
/g
F
W
)
50 ć,10 h
25 ć,108 h
30 B
28
22
20
16
14
24
26
18
12
70 60 50 40 30 20 10 0
Moisture content/%
Ph
ei
de
a
c
on
te
nt
/ (μ
g/
g
FW
)
50 ć,10 h
25 ć,108 h
30 C
25
20
10
5
15
0
70 60 50 40 30 20 10 0
Moisture content/%
Py
roP
he
ide
a
co
nte
nt/
(μ
g/g
FW
)
50 ć,10 h
25 ć,108 h
6.0 D
5.0
5.5
4.0
3.0
1.5
4.5
2.5
2.0
3.5
1.0
70 60 50 40 30 20 10 0
Moisture content/%
Py
roC
hli
de
a
co
nte
nt/
(μ
g/ g
FW
)
50 ć,10 h
25 ć,108 h
24 2015, Vol.36, No.01 食品科学 ※基础研究
2.8 E
2.0
2.4
1.2
0.4
1.6
0.8
0.0
70 60 50 40 30 20 10 0
Moisture content/%
Py
ro
Ph
y a
co
nt
en
t/(
μg
/g
FW
)
50 ć,10 h
25 ć,108 h
16 F
12
14
8
4
10
6
2
70 60 50 40 30 20 10 0
Moisture content/%
Ph
y
a
co
nt
en
t/(
μg
/g
F
W
)
50 ć,10 h
25 ć,108 h
7 G
6
4
2
5
3
1
70 60 50 40 30 20 10 0
Moisture content/%
C1
32 -
OH
Ph
eid
e a
co
nte
nt/
(μg
/ g
FW
)
50 ć,10 h
25 ć,108 h
8
7
H
6
4
2
1
5
3
0
70 60 50 40 30 20 10 0
Moisture content/%
C1
32 -
OH
C
h1
b
co
nte
nt/
(μg
/ g
FW
)
50 ć,10 h
25 ć,108 h
4
3
I
2
1
0
70 60 50 40 30 20 10 0
Moisture content/%
C1
32 -
OH
C
hl
a c
on
ten
t/ (μ
g/ g
FW
)
50 ć,10 h
25 ć,108 h
3.6
3.2
2.4
1.6
1.2
0.4
2.8
J
2.0
0.8
0.0
70 60 50 40 30 20 10 0
Moisture content/%
C1
32 -
OH
Ph
y a
co
nte
nt/
(μg
/g
FW
) 50 ć,10 h
25 ć,108 h
6.0
5.5
3.5
2.5
2.0
1.0
5.0
4.5
4.0
K
3.0
1.5
0.5
70 60 50 40 30 20 10 0
Moisture content/%
C1
51 -
OH
-lac
ton
e P
hy
a c
ont
ent
/(μg
/g F
W) 50 ć,10 h
25 ć,108 h
Fig.4 Changes in chlorophyll derivatives in GP-DOV
2.2.1 Changes in Chlase, MCS, and their associated Chl
derivatives
As shown in Fig. 4A, Chlase showed similar patterns in
both drying modes, which corresponded with other findings
that reported a reducing activity when leaf senescence was
initiated[18,21]. This activity change was parallel with the
changes in Chlide-a, the product of Chlase reaction, which
occupied the majority (18.48 μg/g FW) of Chl derivatives at
the beginning of drying but declined (<3 μg/g FW) in dried
GPs under both drying conditions.
The sharp decrease in Chlide-a may also be attributed
to the consistent level of MCS activity (Fig. 3B), reflecting
its non-enzymatic origin and non-inducement in aging[3,5].
As shown in Fig.4B, the high drying temperature did not
affect MCS activity, and the stable MCS continuously
dechelated magnesium from Chlide-a to yield Pheide-a,
which substantially accumulated in both drying modes,
culminating at 26.91 μg/g FW within 3 h for fast drying
and at 19.04 μg/g FW within 24 h for slow drying.
Pheide-a was diminished in the following drying but was
left at high levels in the dried GPs.
2.2.2 Changes in Phedase and its associated Chl derivatives
Although it remained almost constant in the first 3 h of
drying at 50 ℃, Phedase (Fig. 3C) that facilitated the removal
of carbomethoxy showed a downward trend in the drying
process.
However, the formation of carbomethoxy-free Chl
pigments was diverse. The majority of pyrochlorophyll pigments
belonged to PyroPhiede-a (Fig. 4C). In the 25 ℃  and 50 ℃
drying modes, its content increased dramatically from less than
3 μg/g FW to approximately 20 μg/g FW, which consisted most
of the Chl pigments in the dried GP-DOV. As shown in Fig. 4D,
PyroChlide-a showed an upward trend in the drying process.
The content of PyroPhy-a (Fig. 4E), which was not detected in
GP-IOV drying fluctuated in a trace amount throughout the
drying process[1]. The appearance of carbomethoxy-free Chl may
be attributed to the enzyme reactions.
※基础研究 食品科学 2015, Vol.36, No.01 25
Several studies reported that PyroPhiede-a can be
induced by Pheidase from Pheide-a. Moreover, other
decarbomethoxy Chl derivatives (PyroChlide-a and
PyroPhy-a) can be catalyzed in a similar way by Pheidase[9].
Our previous study also confirmed the finding that Pheidase
is involved in decarbomethoxy reactions[1]. The destruction
of oil vacuoles increased the accessibility of enzymes
and substrates. This structural change benefited Pheidase
reactions, which are also helpful for pyrochlorophyll
accumulation. Pheidase had a relatively high activity before
3 h in fast drying or 24 h in slow drying. This finding may
clarify the increase in pyrochlorophyll pigment content. As
shown in Fig. 4F, Phy-a had an upward trend in the drying
process. This phenomenon may be influenced by the release
of organic acid in the fruits after oil vacuole destruction;
hence, the acidic environment activated the formation of
Phy-a[26-27].
Chl degradation was initiated when fresh GPs were
picked from the trees. A previous study demonstrated that the
PAO pathway plays a crucial role in the decomposition of Chl
pigments in GP-IOV. However, the Chl degradation pathways
were intervened after oil vacuoles were damaged. Chl-a and
Chl-b only constituted a very small part of the pigments in
the final dried GP-DOV (25 ℃), and the majority of pigments
focused on Pheide-a (18%) and PyroPheide-a (27%), both of
which presented a brown color.
Both Pheidase and Chlase represented a downward
trend. However, the most significant loss (72% of total Chl)
was achieved within 24 h when GP-DOV was dried at 25 ℃,
during which the two enzymes were detected at relatively
high levels. The PAO degrading pathway seemed to be
stagnant at the point of Pheide-a when oil vacuoles were
destroyed. The accumulation of Pheide-a suggested a possible
restriction in the conversion of Pheide-a to FCCs. In addition,
the activated conversion of Pheide-a to PyroPheide-a also
convinced this fact.
2.2.3 Changes in Chl-POD and associated Chl derivatives
As shown in Fig. 3D, the activity of Chl-POD in
GP-DOV varied greatly in the different drying patterns. In
fast drying, its activity was restrained and lowered gradually
but showed a reverse “V” trend when dried at 25 ℃, peaking
at 6.26 U/mg pro, which outweighed its highest activity
(4.06 U/mg pro) in GP-IOV under the same drying condition.
This phenomenon may be attributed to the adaptation of
plants to the variations in environmental condition. Tissue
damage or senescence can increase Chl-POD activity, which
is very common in the post-harvest storage of fruits[18].
The following decrease in Chl-POD in slow drying may be
attributed to fierce dehydration. The significant enhancement
in Chl-POD activity during the 25 ℃ drying process also
yielded complicated products that included different kinds of
C132-OH-oxidized Chl pigments.
As shown in Fig. 4I, C132-OH Chl-a, the confirmed
Chl catabolite of the peroxidase degrading pathway, also
showed a reverse “V” trend in the two drying modes. This
result disagreed with other findings that boosted Chl-POD
activity could not prevent the continuous decrease of in vivo
C132-OH Chl-a[10-13]. The initial increase in C132-OH Chl-a
may be attributed to the strong Chl-POD activity and cell
structural changes.
C132-OH Pheide-a (Fig. 4G), which was not detected
in the fresh GPs, increased greatly in the two drying modes.
C132-OH Chl-b (Fig.4H), which was absent in fast drying
or GP-IOV drying, started to increase after 12 h of slow
drying. C132-OH Phy-a (Fig. 4J), which was supposed to be
converted from Phy-a, also showed an increasing trend in
both drying modes[28]. The Chl-POD acquired from GP could
also accept Pheide-a and Chl-b as substrates to generate
C132-OH Pheide-a and C132-OH Chl-b, respectively.
The oil vacuole damage helped to prompt these oxidizing
reactions by increasing the accessibility of Chl-POD and
Chls. This conclusion can be verified by the sharp increase in
C132-OH Chl catabolites in the beginning of GP-DOV slow
drying. C151-OH-lactone Phy-a (Fig. 4K), which was not
in the regular Chl degrading route, also increased in the
25 ℃ drying process. The formation of C151-OH-lactone
Chl probably began with C132-OH-lactone Chl, which was
also catalyzed by perioxidase[16]. Thus, the detected C132-
OH Chl pigments with various forms resulted from the active
catabolism of Chl-POD induced by the destruction of oil
vacuoles.
3 Conclusions
GP underwent a rapid and sharp degradation of Chl
when the oil vacuoles were destroyed. Fast drying appeared
to be ineffective in retaining Chl. Chl catabolites that
emerged in GP-DOV drying were more complicated and
diverse than those in GP-IOV drying. The destruction of oil
vacuoles facilitated several enzyme reactions but inhibited the
reactions after the formation of Pheide-a in the PAO pathway.
Therefore, the accumulation of Pheide-a and another branch
26 2015, Vol.36, No.01 食品科学 ※基础研究
conversion of Pheide-a to PyroPheide-a by Pheidase were
highly induced in this procedure. PyroPheide-a accounted for
the largest amount of Chl pigments in the dried GP-DOV.
The higher Chl-POD activity was induced and accompanied
by the accumulation of C132-OH-oxidized Chl in slow drying
when oil vacuoles were damaged.
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