Effects of cooking methods on chlorophylls, pheophytins
and colour of selected green vegetables
Nihal Turkmen, Ender Sinan Poyrazoglu, Ferda Sari & Y. Sedat Velioglu*
Faculty of Engineering, Department of Food Engineering, Ankara University, 06110 Diskapi, Ankara, Turkey
(Received 23 November 2004; Accepted in revised form 28 April 2005)
Summary
The effect of microwave and conventional cooking methods on chlorophyll pigments and
colour properties of squash, green beans, peas, leek, broccoli and spinach were studied, by
HPLC and colorimetry, respectively. In five of six vegetables, chlorophyll a was found
more heat resistant compared with chlorophyll b, except in peas. Chlorophylls in peas were
retained to the 80–90%, the highest in all vegetables evaluated. Chlorophylls were retained
to 19–100%, depending on the vegetable type and cooking method. Pheophytins increased
in all vegetables after cooking. Highest chlorophyll a and chlorophyll b losses were
observed in boiled leek while microwaved peas and boiled peas retained the most
chlorophyll a and chlorophyll b, respectively. Pheophytin a and pheophytin b formation
was highest at boiled squash and boiled green beans, which were fifty-ninefold and twentyonefold compared with fresh ones, respectively. Most of the pheophytin formations
occurred in boiled and the least in microwaved vegetables. Surface colour changed
depending on the type of vegetable and cooking method.
Keywords
Chlorophyll, colour, cooking methods, green vegetables, HPLC, pheophytin.
Introduction Chlorophyll is the principal
pigment in green plants and easily degraded
during processing
(Boekel, 1999). It has been well established that
chlorophylls are susceptible to chemical and
physical changes during processing of vegetables
(Teng & Chen, 1999). Thermally processed green
vegetables exhibit poor colour quality as compared with the fresh ones. The degree of greenness,
attributed to chlorophyll pigments, is important in
determining the final quality of these kinds of
vegetables (Nisha et al., 2004) since colour is one
of the major quality indicators for vegetable
products (Teng & Chen, 1999; Tan et al., 2000;
Tijskens et al., 2001). Colour changes in green
vegetables because of heat treatments are the
result of the conversion of chlorophyll to
pheophytin through the Mg substitution of the
*Correspondent: Fax: +90-312-317-87-11;
e-mail: [email protected]
chlorophyll by hydrogen (Canjura et al., 1991;
Lau et al., 2000; Ahmed et al., 2002). Chlorophyll
compounds fulfil certain biological functions that
are effective as long as the chlorophyll derivative
conserves the basic porphyrin ring structure. Thus,
the presence of chlorophylls and their transformation products contribute added value to the plant
(Minguez-Mosquera
et al.,
2002).
product
Recently, chlorophyll and its derivatives were also
shown to exhibit important health promoting
functions such as antimutagenic and anticarcinogenic activities, tumoricidal potencies (Yoshikawa
et al., 1996; Chernomorsky et al., 1999; HigashiOkai et al., 1999; Ma & Dolphin, 1999; Morita
et al., 2001). In the study reported by Negishi
et al. (1997), crude and purified chlorophyll
extracted from spinach inhibited the mutagenicity
of various mutagens.
Chlorophyllin, which has been intensively studied as an antimutagenic agent and widely used in
oral hygiene and hypertension showed inhibition
at an extent similar to that of natural chlorophylls
(Ma & Dolphin, 1999). According to Chernomorsky et al. (1999), food sources that yield
chlorophyll derivatives might play a significant
role in cancer prevention.
Green beans, peas, leek, broccoli and spinach are
mostly consumed green vegetables and it is a common practice that most of vegetables are cooked by a
simple boiling process before use. However,
steaming and microwave cooking are also used for
this purpose. Cooking by a simple boiling process is a
common practice for some of the green vegetables
like squash, green beans, peas, leek, broccoli and
spinach. Besides, steaming and microwave cooking
can also be used depending on the preferences.
Consequently, these cooking processes bring about a
number of changes in physical characteristics and
chemical composition of vegetables and other food
materials (Rehman et al., 2003; Zhang & Hamauzu,
2004). However, very little information is available
in the literature regarding the chlorophyll composition of green vegetables cooked by different
methods and their colour degradation in terms of
tristimulus colorimetry after cooking. Therefore,
the objectives of this study were to determine the
effects of various heat treatments on the conversion
of chlorophyll a and b to respective pheophytins
using a HPLC method that the authors have
developed and to measure colour changes directly
with respect to chlorophyll degradation in cooked
green vegetables.
Materials and methods
Plant materials Fresh broccoli, spinach, squash,
leek, peas, green beans were purchased from
several local markets in Ankara, Turkey and used
as research material.
These vegetables were selected because they are
frequently consumed vegetables, with different
parts of the plants being edible, such as seed
(peas), leaf (spinach, leek), floret (broccoli) and
fruit (squash, green beans). The vegetables (2.5 kg
of each vegetable) randomly sampled from the
shelf were washed with tap water after the removal
of inedible parts with a sharp knife. Vegetables
were dried on paper towel, sliced or cut into small
pieces and mixed well for homogeneity. To obtain a
more homogenized sample, 1200 g batches of
each vegetable were obtained and divided into
four equal portions. One portion was retained
raw, others were cooked in three different methods
in duplicate, as given below.
Heat treatment Several of the most common
cooking methods, including boiling, steaming
and microwave coo- king were applied to cook
vegetables. Cooking conditions were
determined with preliminary experiments for
each vegetable. For all cooking treatments, the
minimum level of tap water and cooking time to
reach a similar tenderness for an adequate
palatability and taste were used.
Boiling Vegetable (100 g) was added to 150 mL
of water that had just reached the boil in a
stainless steel pan and cooked for 5 min. Samples
were drained off and cooled rapidly on ice.
Microwave cooking
Vegetable (100 g) was placed in a glass dish and
6 mL (12 mL for green beans) of distilled water
was added. Dishes were covered with a cooking
bag, having several holes, and cooked in a
commercial-1000 W microwave oven (Arcelik,
MD554-Intellowave; Tuzla, Turkey). Cooking
took 1 min for squash, spinach, peas and
1.5 min for leek, broccoli and green beans. Samples were drained off and cooled rapidly on ice.
Steaming Vegetable (100 g) was placed on tray
in a steam cooker (Tefal, Clipso-clipsoval,
Model 4101; Groupe SEB, Ecully, France)
covered with a lid and steamed over boiling
water for 7.5 min under atmospheric pressure.
The samples were rapidly cooled on ice.
Dry matter determination Due to various water
content of vegetables, all calculations were
made according to dry matter
(dm) basis. For determination of the dry matter
content, 3–4 g of raw or cooked homogenized
sample (as triplicate) was dried in a convection
oven at 70 C for at least 2 days until reaching
constant weight (Teng & Chen, 1999; Nilsson
et al., 2004).
Colour measurement The CIE L*, a*, and b*
values were measured using Minolta CR-300
colorimeter having D25 optical sensor (Osaka,
Japan). The instrument was calibrated with a
standard white tile. For colour measurement
raw and cooked vegetables were ground and the
resulted purees were deliv- ered into a glass dish
and CIE L*, a*, and b* values were recorded.
The colour changes of vegetables because of
heat treatments were inter- preted by calculating
the hue angle. The hue angle was obtained by
calculating tan)1b/a (Little,
1975).
HPLC analysis
The equipment (Shimadzu Class-VP HPLC system (Shimadzu Corp., Kyoto, Japan)) consists of a
computer-controlled system with Class-VP software and SLC-10 A VP system controller. Other
accessories were a Shimadzu DGU-14A degasser,
LC-10 ADVP Shimadzu pumps, a CTO-10 ASVP
column oven and an SPD-MIOA VP photo diode
array (PDA) detector (Shimadzu Corp., Kyoto,
Japan). The chlorophyll and derivatives were
analysed by using Zorbax Eclipse XDB-C8
(150 · 4.6 mm ID, 5 lm) reversed phase column
(Agilent Technologies, Palo Alto, CA, USA). The
absorption spectrum of each pigment was determined on line using the PDA detector. The
chromatograms were recorded at 430 nm for
chlorophyll a and pheophytin b, 467 nm for
chlorophyll b and 408 nm for pheophytin a,
which are the wavelengths of their maximum
absorption. Area measurements of individual
peaks for quantitative analyses were made on
these wavelengths. A method previously applied by
Gokmen et al.
(2002) was used with some minor modifications
indicated as italicized in parentheses. Several
mobile phases and elution profiles were evaluated
and the best separation was observed with
following conditions: the mobile phase (A) was
made up of distilled water and mobile phase (B)
was 100% methanol. The HPLC gradient programme was 0–5 min, 90% B; 5–10 min, 90–94%
B; 10–50 min, 94% B, then 10 min post run at
the initial conditions (90% solvent B isocratic for
5 min followed by a 90–95% linear gradient for
5 min with solvent B and holding with 95%
solvent B for 5 min, and finally followed by a 95–
90% linear gradient with solvent A for 5 min;
total run time was 20 min). The flow rate was
1 mL min)1 (0.75 mL min)1) and the injection
volume was 20 lL. The column temperature was
maintained at 35 C (room temperature). The
spectrum of each reference standard was recorded
and stored in the HPLC spectrum library. Chromatographic peaks in the samples were identified
by comparing the retention time and spectrum of
an unknown compound with HPLC library data
of standards, and by cochromatography with
added standards.
Extraction of chlorophylls and pheophytins
Extraction of chlorophyll and derivatives was
performed at low temperatures and reduced
illumination in order to minimize photodegradation of pigments. Raw and processed vegetables
were homogenized in a blender (Moulinex,
France) for 2 min. Duplicate samples of homogenate (c. 1.5–2 g) was blended with c. 0.4 g
Na2CO3 and a few milligrams MgSO4 to avoid
pheophytin formation with a mortar and pestle.
Cold acetone (8 mL for squash, green beans,
peas and leek, 8.5 mL for broccoli and 13 mL
for spinach) was added to this mixture in small
volumes and acetone extracts were collected in a
polyethylene (PE) centrifuge tube, until the
residue became colourless. The pooled extract
was centrifuged at 6200 g for 5 min. The supernatant was then filtered through a 0.45 lm
membrane filter for HPLC analysis. All extracts
were stored at )20 C until used although they
were not kept more than a few hours.
Chemicals and reagents Chlorophyll a and b
standards were purchased from Sigma Co. (St
Louis, MO, USA). Because of unavailability of
commercial pheophytins a and b, they were
obtained by pheophytinization of chlo- rophylls
adding 1 N HCl (Schwartz & Lorenzo,
1991; Van Breemen et al., 1991; Bohn & Walczyk,
2004), followed by 1 N NaOH for neutralization.
Chemicals were used in this work were either
HPLC or analytical grade, from Merck (Darmstadt, Germany), unless noted.
Statistical analysis All data were recorded as mean
values ± SE and analysed by SPSS for
Windows (version 10.1, Chicago, IL, USA). Oneway analysis of variance
(anova) and Duncan’s multiple range test were
carried out to test any significant differences
between raw and cooked vegetables.
Results and discussion
Chlorophylls and derivatives The concentrations of
chlorophyll a and b, pheo- phytin a and b and
their retentions in fresh and cooked vegetables
are presented in Table 1. As
expected, differences in chlorophyll a and b concentrations between fresh and cooked vegetables
were found to be statistically significant (P < 0.05).
More chlorophyll a was extracted than chlorophyll
b in all fresh samples and was more labile than
chlorophyll b owing to its greater susceptibility to
pheophytinization during heating as it has been
already reported in earlier studies (López-Ayerra
et al., 1998; Tan et al., 2000; Tenorio et al., 2004).
As an example, the HPLC chromatogram of
chlorophylls and their derivatives obtained for
fresh and microwaved broccoli is shown in Fig. 1.
Concentrations of chlorophyll a and b varied in
significant amounts between the raw vegetables
analysed. Spinach had the highest chlorophyll
(chlorophyll a + b) content, followed by broccoli,
Table 1 Effects of cooking methods on chlorophyll and pheophytin contents (mg g)1 dm; mean ± SE) of vegetables and their
retention (%)
Chlorophyll a
Vegetable and
cooking method
Squash
Fresh
Boiling
Steaming
Microwaving
Green Beans
Fresh
Boiling
Steaming
Microwaving
Peas
Fresh
Boiling
Steaming
Microwaving
Leek
Fresh
Boiling
Steaming
Microwaving
Broccoli
Fresh
Boiling
Steaming
Microwaving
Spinach
Fresh
Boiling
Steaming
Microwaving
Content
Chlorophyll b
%
Content
Pheophytin b
%
Content
%
Content
%
100
67
57
49
0.01 ± 0.00a
0.59 ± 0.04c
0.32 ± 0.00b
0.25 ± 0.00b
100
5900
3200
2500
0.03 ± 0.00a
0.17 ± 0.01c
0.14 ± 0.01bc
0.09 ± 0.02ab
100
567
467
300
1.05 ± 0.02b
0.48 ± 0.03a
0.42 ± 0.03a
0.38 ± 0.02a
100
46
40
36
0.61
0.41
0.35
0.30
1.11 ± 0.00c
0.51 ± 0.01a
0.49 ± 0.02a
0.65 ± 0.01b
100
46
44
59
0.74 ± 0.00c
0.53 ± 0.01b
0.47 ± 0.01a
0.46 ± 0.00a
100
72
64
62
0.07 ± 0.01a
1.29 ± 0.02d
1.05 ± 0.08c
0.60 ± 0.01b
100
1843
1500
857
0.01 ± 0.00a
0.21 ± 0.01d
0.19 ± 0.00c
0.13 ± 0.00b
100
2100
1900
1300
0.80 ± 0.01b
0.73 ± 0.01a
0.78 ± 0.01ab
0.80 ± 0.02b
100
91
98
100
0.51 ± 0.03b
0.44 ± 0.02ab
0.42 ± 0.01a
0.41 ± 0.01a
100
86
82
80
0.03 ± 0.00a
0.22 ± 0.02c
0.26 ± 0.00d
0.10 ± 0.00b
100
733
867
333
0.02 ± 0.01a
0.21 ± 0.00b
0.25 ± 0.04b
0.21 ± 0.01b
100
1050
1250
1050
1.11 ± 0.02c
0.21 ± 0.00a
0.24 ± 0.01a
0.35 ± 0.01b
100
19
22
32
0.69 ± 0.02c
0.27 ± 0.01a
0.29 ± 0.03ab
0.36 ± 0.02b
100
39
42
52
0.09 ± 0.00a
0.98 ± 0.01b
0.96 ± 0.01b
0.94 ± 0.04b
100
1089
1067
1044
0.01 ± 0.00a
0.13 ± 0.00b
0.12 ± 0.01b
0.10 ± 0.01b
100
1300
1200
1000
4.39 ± 0.27c
2.37 ± 0.04b
1.54 ± 0.01a
2.54 ± 0.02b
100
54
35
58
2.55 ± 0.13c
1.77 ± 0.04b
1.26 ± 0.01a
1.56 ± 0.03b
100
69
49
61
0.10 ± 0.03a
2.41 ± 0.07c
2.08 ± 0.21c
1.15 ± 0.09b
100
2410
2080
1150
0.07 ± 0.00a
0.69 ± 0.02c
0.49 ± 0.00b
0.47 ± 0.00b
100
575
408
392
± 2.00b
± 0.09a
± 0.27a
± 0.15a
100
64
63
63
0.79b
0.24a
0.01a
0.12a
100
70
68
63
1.64
8.10
7.83
4.56
100
494
477
278
0.39 ± 0.17a
5.27 ± 1.59b
3.09 ± 0.02ab
1.73 ± 0.12a
100
1351
792
444
24.39
15.51
15.44
15.47
14.7
10.26
9.94
9.27
± 0.00c
± 0.02b
± 0.04ab
± 0.01a
Pheophytin a
±
±
±
±
±
±
±
±
0.27a
0.27b
0.21ab
0.85ab
Different letters in same columns indicates the difference between two means is statistically significant (P < 0.05) for each
treatment.
1: 467 nm, 8 nm
BrT1E BrT1E. dat
2: 430 nm, 8 nm
BrT1E BrT1E.dat
45
3: 408 nm, 8 nm
BrT1E BrT1E.dat
2
120
120
100
(a)
1
100
80
80
60
60
40
40
20
20
0
3
0
120
5
10
1: 467 nm, 8 nm
BrT2EM BrT2EM.dat
15
20
2: 430 nm, 8 nm
BrT2EM BrT2EM.dat
25
30
Minutes
467 nm
430 nm
408 nm
4
35
40
45
Fresh
Boiled
35
Steamed
Microwaved
30
25
0
20
50
3: 408 nm, 8 nm
BrT2EM BrT2EM.dat
120
(b)
100
40
100
80
80
60
60
40
40
20
20
15
10
5
Squash G.beans
Peas
Leek
Broccoli Spinach
Figure 2 Effects of cooking methods on chlorophyll a + b
467 nm
430 nm
0
0
content.
0
408 nm
0
5
10
15
20
25
30
35
40
45
50
Minutes
Figure 1 HPLC chromatogram of chlorophylls and their
derivatives of fresh (a) and microwaved (b) broccoli*.
*1 – Chlorophyll b; 2 – Chlorophyll a; 3 – Pheophytin b;
4 – Pheophytin b.
green beans, leek, squash and peas. Few data on the
chlorophyll content of these vegetables by chromatographic method are found in the literature, but
some can be compared with those obtained in this
study (Table 1). Bohn & Walczyk (2004) reported
that the highest chlorophyll content was found in
spinach followed by lettuce, endive and iceberg
lettuce. Chlorophyll a and b content in fresh spinach
reported by Teng & Chen (1999) were 14.1 and
6.23 mg g)1 dry weight, respectively, which were
much lower than our results. For green beans, CruzGarcı́a et al. (1997) reported the values of 3.66–
10.50 mg (100 g))1 fresh weight (fw) for chlorophyll a, which agrees with the concentration of
11.37 mg (100 g))1 fw from this study and
0.2–0.46 mg (100 g))1 fw for chlorophyll b, much
less than our findings [7.59 mg (100 g))1 fw]. The
discrepancy with literature data may be because of
differences in varieties used and/or analytical methods applied. Cooking resulted in a loss of
chlorophyll a and b to various extents depending
not only on the type of method involved but also
the type of vegetable
(Fig. 2).While chlorophyll a loss ranged from 9 to
81% it was 20 to 61% in chlorophyll b (Table 1).
This result confirmed the findings of Teng & Chen
(1999) who observed that rate constant of
chlorophyll a degradation was higher than that
of chlorophyll b in heated spinach leaves. The
reduction in chlorophyll a and b contents is
attributed to degradation of both chlorophylls
into their major derivatives pheophytin a and b,
respectively (Schwartz & Elbe, 1983; Mangos &
Berger, 1997; Boekel, 1999). Pheophytin a and b
were found in the highest amount by boiling
followed by steaming and microwave cooking in
nearly all vegetables after cooking treatments
(Fig. 3). The difference can be result of variation
in the cooking conditions (e.g. time and medium)
and also lower levels of organic acids liberated
from matrix of plant tissue treated with microwave
cooking (Cruz-Garcı́a et al., 1997; Teng & Chen,
16
14
Fresh
Boiled
12
Steamed
Microwaved
10
8
6
4
2
0
Squash G.beans
Peas
Leek
Broccoli Spinach
Figure 3 Effects of cooking methods on pheophytin a + b
content.
1999). It was reported that steamed cooking
resulted in the highest formation of pheophytin
in spinach leaves (Teng & Chen, 1999) and in
green beans (Cruz-Garcı́a et al., 1997). These
differences may be attributed to the differences in
varieties used and in the cooking methods. Additionally, the level of pheophytin a was greater than
that of pheophytin b in all cooked vegetables
regardless of cooking method (Table 1), which is
in agreement with the results previously reported
(Schwartz & Elbe, 1983; Khachik et al., 1986;
Schwartz & Lorenzo, 1991; López-Ayerra et al.,
1998).
135
Fresh
Boiled
Steamed
130
Microwaved
125
120
115
110
105
100
Squash G.beans
Peas
Leek
Broccoli Spinach
Colour changes
The changes in chlorophyll pigments during cooking were also monitored by hue angle values of
six vegetables studied. Heating had different
effects on hue angle values of all vegetables
depending on the type of vegetable and the
cooking method involved (Table 2). Leek and
squash showed a significant decrease (P < 0.05)
in the hue value compared with fresh ones with all
cooking methods, which means decrease in the
intensity of greenness (Maharaj & Sankat, 1996;
Lau et al., 2000). On the other hand, hue values of
peas and spinach increased after all treatments
(P < 0.05) and that of green beans and broccoli
remained unchanged with only microwave cooking. Ramesh (2000) reported that conventionally
cooked peas were brighter owing to surface
reflectance and depth of light penetration into
the tissue, which partially agrees with our results.
From Fig. 4 it is clear that all microwaved cooked
vegetables have the highest hue angle value in
comparison with the other cooked vegetables. This
might be because of the formation of other
Figure 4 Effects of cooking methods on hue angle.
chlorophyll derivatives like chlorophyllides more
than pheophytins since chlorophyllides do not
bring about changes in chromophore properties
and colour of their precursors (Minguez-Mosquera et al., 2002). In addition, the use of shortest
cooking time in microwave cooking might cause
this difference.
In this study, the reduction in hue angle value
for each vegetable could not be attributed to a loss
of chlorophylls or an increase in pheophytins in
comparison with previous studies (Maharaj &
Sankat, 1996; Lau et al., 2000; Ahmed et al., 2002)
based on changes in green colour because of time
and temperature treatments. This might be related
to other chlorophyll derivatives not considered in
this study. According to a previous study by
(Tijskens et al., 2001), the increase in colour
intensity of green beans and broccoli at the early
stage of blanching was attributed to either conversion of non or less-coloured precursor of green
Table 2 Effects of cooking
Hue angle
Vegetable and
cooking method
Fresh
Boiling
Steaming
Microwaving
Squash
Green beans
Peas
Leek
Broccoli
Spinach
120 ± 0.17b
123 ± 0.55b
120 ± 0.28a
126 ± 0.07c
123 ± 0.00b
129 ± 0.00a
115 ± 0.02a
117 ± 0.02a
123 ± 0.06b
113 ± 0.37a
120 ± 0.72a
131 ± 1.02ab
114 ± 0.61a
116 ± 0.53a
123 ± 0.36b
114 ± 0.63a
118 ± 0.37a
131 ± 0.65ab
116 ± 1.24a
123 ± 0.38b
124 ± 0.05c
116 ± 0.24b
124 ± 1.25b
132 ± 0.51b
Different letters in same rows indicates the difference between two means is
statistically significant (P < 0.05) for each treatment.
methods on hue angle of vegetables
(mg g)1 dm ± SE)
colour to more visible green colour or a decrease
in opacity by replacement of intercellular air with
blanching water and cell juice released by cell
membrane deterioration. The same mechanisms
could be a possible explanation for the result
obtained from this study, although the reason for
increase in greenness during cooking still needs
further research.
Conclusions As a result, chlorophyll a and b
content of these six vegetables were reduced
(P < 0.05) in various extents to their
derivatives, pheophytin a and b, depending on the
type of vegetable and cooking method. Together
with this modification of qualitative and
quantitative composition of chlorophylls of
vegetables, heat treatments also caused changes
in their hue angle values. Whilst colour changes
were detrimental for leek and squash because of
a loss of greenness, the colours of peas and
spinach unexpectedly improved after cooking.
From a nutritional point of view, although
cooking caused a loss of chlorophylls which are
known to have health effects such as
antimutagenic and anticarcinogenic effects, we
can conclude that nutritional properties of vegetables might have been maintained since
chlorophyll degradation products pheophytin a
and pheophytin b also have the similar health
effects.
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