Innovative Food Science and Emerging Technologies 43 (2017) 2634

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Innovative Food Science and Emerging Technologies

journal homepage: www.elsevier.com/locate/ifset

Eect of pulsed electric elds (PEFs) on the pigments extracted from spinach MARK
(Spinacia oleracea L.)
Zhi-Hong Zhang, Lang-Hong Wang, Xin-An Zeng, Zhong Han, Man-Sheng Wang
School of Food Science and Engineering, South China University of Technology, Guangzhou, Guangdong 510640, China

A R T I C L E I N F O A B S T R A C T

Keywords: In order to investigate the eect of PEF on the pigments of spinach, pigments were extracted and were treated by
Pulsed electric elds dierent electric eld strength at dierent temperatures (20, 35 and 45 C). Results showed that with the in-
Pigments creasing temperature, the degradation of chlorophyll content was appeared for pigments without PEF treatment.
Physicochemical properties However, the concentrations of chlorophyll a, b and carotenoids were increased by the PEF treatment, especially
Antioxidant activity
for the higher electric eld strength. Moreover, the structure of spinach pigments treated by PEF also was
determined by UVVis, FT-IR and XRD. From the structural information, it was indicated that the eect of PEF
treatment mainly focused on the hydrogen bonds and the pyrrole ring of chlorophyll, as well as the crystal
structure of the compound was modied. In addition, the PEF treatment also signicantly increased the anti-
oxidation activity of pigments over the series of temperatures.
Industrial relevance: This study provide evidences that the PEF treatment can inhibit the degradation of chlor-
ophyll and carotenoids, and can increase the antioxidation activity of extraction solution compared to the
thermal treatment. From the structural information, it was indicated that PEF can promote the crosslink reaction
with other chlorophyll moleculars, and formed the chlorophyll-aggregated structures, such as dimers and oli-
gomers chlorophyll. Therefore, the PEF treatment is an ecient food processing method, especially foods rich in
pigment.

1. Introduction of several degenerative processes, including cancers, cardiovascular

Chlorophyll is the major pigment in green vegetables and some
fruits. Its content may exceed 2 g/kg wet weight in some species, such
as raw spinach and kale (Khachik, Beecher, & Whittaker, 1986).
Chlorophyll and its derivatives have a chemical structure that contains
a tetrapyrrole ring, a long phytol chain and a central magnesium ion
(Han et al., 2016). Chlorophyll and its derivatives in the human diet are
associated with specic health benets, such as antioxidant activity,
hematopoietic activity and anti-inammatory properties
(Ferruzzi & Blakeslee, 2007; Lanfer-Marquez, Barros, & Sinnecker,
2005; Miret, Tascioglu, van der Burg, Frenken, & Klake, 2009).
Moreover, chlorophyll and its various derivatives are widely used in
traditional medicines and therapies in various countries and regions
(Chernomorsky & Segelman, 1988; Han et al., 2016; Kephart, 1955).
Carotenoids are also important bioactive compounds in plant food,
which have various biological functions and activities, such as provi-
tamin A activity, antioxidant capacity and enhancement of immune
system (Singh, Ahmad, & Ahmad, 2015). Some epidemiological studies
have demonstrated that eating fruits and vegetables may reduce the risk
problems and eye diseases (Huang et al., 2013). As a result, nutritionists
and epidemiologists suggest that people should increase their dietary
intake of phytochemicals through consumption of raw fruits and ve-
getables as well as their processed products. In addition, phytochem-
icals also are used as additives in food products due to their attractive
color and other physicochemical properties. However, these pigments
are unstable in nature and may degrade during the processing of food as
a result of exposure to heat, light and oxygen, etc. (zkan & Bilek, 2015;
Pott, Marx, Neidhart, Mhlbauer, & Carle, 2003; Zhao et al., 2013).
Therefore, it is useful to explore innovative and non-thermal technol-
ogies in processing to sterilize the food while also protect the favorable
bioactivity of pigments.
Pulsed electric elds (PEFs) treatment is a type of non-thermal
technique that is studied in the sterilization of liquid foods, such as fruit
and vegetable juices and milk (Buckow, Ng, & Toep, 2013; Pina-Prez,
Rodrigo, & Martnez, 2016). PEF treatments typically involve the ap-
plication of pulses of high electric eld intensity (above 20 kV/cm) to
liquid foods placed between two electrodes. PEF treatments are con-
ducted at ambient or moderately high temperatures (generally < 45 C)

Corresponding author.
E-mail address: xazeng@scut.edu.cn (X.-A. Zeng).

http://dx.doi.org/10.1016/j.ifset.2017.06.014
Received 3 January 2017; Received in revised form 18 April 2017; Accepted 27 June 2017
Available online 29 June 2017
1466-8564/ 2017 Published by Elsevier Ltd.
Z.-H. Zhang et al. Innovative Food Science and Emerging Technologies 43 (2017) 2634

for short times (milliseconds) so that the energy losses due to heating of
the food matrix are negligible. For these reasons, PEF results in limited
degradation of compounds in the food and rarely aects the avor and
quality of the foods (Odriozola-Serrano, Aguilo-Aguayo, Soliva-
Fortuny, & Martin-Belloso, 2013; Zeng, Han, & Zi, 2010). A number of
studies have shown the eectiveness of PEF treatment in better re-
taining nutritious compounds, such as ascorbic acid, carotenoids and
phenols, in comparison with heat treatments of dierent food stus,
such as tomato and strawberry juices (Buckow et al., 2013; Odriozola-
Serrano et al., 2013). Moreover, many investigations have proved that
PEF can enhance the extraction of intracellular compounds, such as
pigments, avors and polysaccharides from the plant cells (Chemat
et al., 2017). However, the eect of PEF treatment on the structure and
physicochemical properties of compounds, especially for plant pig-
ments rarely reported until now.
Therefore, the aim of this study was to investigate the eect of PEF
on the pigments of spinach including the physicochemical properties
and the antioxidant activity. Furthermore, the mechanism of PEF af-
fecting the chlorophyll and carotenoids also was explored based on the
changes in the structure.
2. Material and methods
2.1. Materials
Fresh spinach (Spinacia oleracea L.) was purchased at the local su-
permarket (Guangzhou, China). Spinach leaves were washed with
deionized water and drained. Ethanol was purchased from Sinopharm
Chemical Regent Co. Ltd. (Shanghai, China). 1,1-Diphenyl-2-picrylhy-
drazyl (DPPH) was purchased from Sigma-Aldrich, Chemie GmbH (St.
Louis, MO, USA). All solvents used were of analytical grade and were
purchased from a local reagent company (Guangzhou Chemical
Reagent Factory, Guangzhou, China).
Afterwards, the spinach puree was centrifuged at 5000g for 10 min at
4 C (TDL-5-A, Shanghai Anting Scientic Instrument Factory, China).
The precipitation was collected and was extracted with 400 mL of ab-
solute ethanol for 30 min, and then the mixture was centrifuged at
5000g for 10 min. The supernatant was transformed in a glass beaker.
In order to abundant extract, the precipitation was re-extraction several
times under the same conditions until the residue became colorless. In
order to separate the proteins, the extraction solution was place in the
refrigerator at 4 C overnight and centrifuged at 5000 g for 10 min.
Finally, the purity of extraction solution of pigments was lled in a 1 L
volumetric ask and stored in a refrigerator at 4 C until further PEF
treatment.
2.4. PEF treatment of pigment solution
Before performing PEF treatments, bubbles in the extraction solu-
tions were removed by a vacuum pump (SHZ-D III, Gongyi Yuhua
Instrument Co., Ltd., Gongyi, China). The pH value and the con-
ductivity of the extraction solutions were 7.92 0.08 and
0.55 0.02 mS/cm at 20 C, respectively. In this study, 150 mL of
extraction solution was treated by specic electric eld intensity (0,
3.3, 6.7, 13.3, 20.0 and 26.7 kV/cm) and dierent temperatures (20, 35
and 45 C). The treatment temperature was controlled using a ther-
mostatic device (HH-4, KEJIE instrument, Jintan, China). Reaction
conditions are listed in Table 1. In this study, the ow rate of the ex-
traction solution was set at 100 mL/min. The eective treatment time
and the input energy of PEF treatment were calculated by Eqs. (1) to (3)
as described by previous study (Han et al., 2012). The eective treat-
ment time and energy input of dierent PEF treatments are shown in
Table 1.
fV
Np =
S (1)

t = Np Wp n (2)
2.2. PEF treatment system
Q = E2 t (3)
The continuous PEF system was designed by the PEF team of South
China University of Technology (Guangzhou, China). The structure of where Np represents the pulse number, f represents the pulse repetition
the device has been reported in our previous studies (Wang et al., 2016; rates, V represents the volume of the chamber (mL), S represents the
Wang, Wang, Zeng, & Liu, 2016). The main parameters of this device ow rate (mL/s), t represents the eective treatment time (s), Wp re-
were a unipolar square wave, the maximum voltage of 15 kV, maximum presents the pulse width (s), n represents the sample cycles, Q re-
current of 1.77 A, frequency of 1 kHz, and pulse width of 20 s, re- presents the energy input (J/m3) and represents the conductivity of
spectively. The electric eld strength of PEF system was monitored by a the sample (S/m).
two-channel digital storage oscilloscope (DST1102B, Tekway Technol-
ogies Co., Ltd., Nanjing, China). Two parallel titanium-based alloy 2.5. Content of pigments
electrodes were inserted in the treatment chamber that was made from
Teon. The distance between two the electrodes was 0.3 cm. The vo- The concentration of pigments of treated samples was determined
lume of the treatment chamber was 0.02 mL. The sample solution was
transported through the silicone tube (inner diameter of 6 mm), and Table 1
was introduced into the treatment chamber by a constant ow pump The eective treatment time and energy input of dierent PEF treatment.
(323 E/D, Watson Marlow, NC, USA). The ow rate was controlled by a
Treatment method Treatment time Eective time Input energy
ow meter (LZB-4, Changzhou Shuangfa Thermal Instrument Factory, (min) (ms) (kJ/m3)
Changzhou, China). The inlet and outlet temperatures of sample solu-
tion were monitored by two K-type thermocouples (Pico technology 20 C 3.3 kV/cm 60 0.32 2.43
6.7 kV/cm 60 0.32 9.65
Inc., St Neots, United Kingdom). The temperature of the sample was
13.3 kV/cm 60 0.32 38.72
controlled with a cooling circulator (DLSK 3/10, Ketai Laboratory 20.0 kV/cm 60 0.32 87.04
Equipment Co., Ltd., Zhengzhou, China). 26.7 kV/cm 60 0.32 154.66
35 C 3.3 kV/cm 60 0.32 2.91
2.3. Extraction of pigments from spinach leaves 6.7 kV/cm 60 0.32 11.57
13.3 kV/cm 60 0.32 46.41
20.0 kV/cm 60 0.32 104.32
In order to prevent pigments in spinach degradation, such as 26.7 kV/cm 60 0.32 185.37
chlorophylls and carotenoids, each extraction step was performed 45 C 3.3 kV/cm 60 0.32 1.49
under dim light and low temperature (4 1 C). 200 g of cleaned 6.7 kV/cm 60 0.32 5.93
13.3 kV/cm 60 0.32 23.77
spinach leaves were milled by a mixer grinder (JYL-C022E, Joyoung,
20.0 kV/cm 60 0.32 53.44
Hangzhou, China) for 15 s with deionized water at a ratio of 1:2 (w/v) 26.7 kV/cm 60 0.32 94.96
in order to remove the water-soluble compounds of spinach leaves.

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Z.-H. Zhang et al. Innovative Food Science and Emerging Technologies 43 (2017) 2634

Table 2
The concentration of chlorophyll a, b and carotenoids from spinach following dierent PEF treatments (g/mL).

Sample Chlorophyll a Chlorophyll b Chlorophyll a + b Carotenoids

20 C 0 kV/cm 11.41 0.02a 4.99 0.01a 16.40 0.03a 2.60 0.02a

3.3 kV/cm 12.37 0.01b 5.31 0.03b 17.68 0.04b 2.84 0.04b
6.7 kV/cm 12.74 0.02c 5.45 0.01c 18.18 0.03c 2.92 0.03c
13.3 kV/cm 13.41 0.04d 5.66 0.02d 19.07 0.06d 3.13 0.01d
20.0 kV/cm 13.63 0.03e 5.74 0.01e 19.37 0.04e 3.25 0.02e
26.7 kV/cm 14.41 0.01f 6.04 0.02f 20.44 0.03f 3.68 0.04f
35 C 0 kV/cm 11.20 0.04a 4.87 0.02a 16.07 0.06a 2.41 0.01a
3.3 kV/cm 11.92 0.02b 5.16 0.01b 17.08 0.03b 2.71 0.02b
6.7 kV/cm 12.64 0.03c 5.37 0.02c 18.02 0.05c 2.94 0.01c
13.3 kV/cm 13.24 0.04d 5.67 0.02d 18.92 0.06d 3.25 0.02d
20.0 kV/cm 13.63 0.03e 5.75 0.01e 19.38 0.04e 3.19 0.04e
26.7 kV/cm 14.62 0.02f 6.13 0.02f 20.75 0.04f 3.52 0.02f
45 C 0 kV/cm 10.93 0.05a 4.79 0.04a 15.72 0.09a 2.60 0.03a
3.3 kV/cm 11.89 0.02b 5.25 0.02b 17.14 0.04b 2.64 0.02a
6.7 kV/cm 11.94 0.03b 5.45 0.02c 17.39 0.05c 2.57 0.01a
13.3 kV/cm 12.20 0.02c 5.08 0.04d 17.28 0.06bc 2.82 0.03b
20.0 kV/cm 12.55 0.03d 5.42 0.04c 17.96 0.07d 2.91 0.04c
26.7 kV/cm 13.21 0.06e 5.73 0.03e 18.94 0.09e 2.98 0.05c

Values with dierent letters in the same column (af) are signicantly dierent according to Tukey test (p < 0.05).

by the UVVis spectrophotometer described by previous studies
(Luengo, Condn-Abanto, lvarez, & Raso, 2014; Wintermans & De
Mots, 1965). Briey, 0.5 mL of sample solution was mixed with 4.5 mL
of deionized water. The absorbance of the chlorophyll and carotenoids
extract at 665, 649 and 470 nm was measured spectrophotometrically.
The concentration of chlorophyll and carotenoids content were calcu-
lated by Eqs. (4) to (7).
Chla = 13.70 A 665 5.76 A 649
Chlb = 25.8 A 649 7.60 A 665
Chla + b = 6.10 A 665 + 20.04 A 649
1000 A 470 2.13 Chl a 97.64 Chlb
Cas =
209
where Chla represents the concentration of chlorophyll a (g/mL), Chlb
represents the concentration of chlorophyll b (g/mL), Chla + b re-
present the total concentration of chlorophyll a and chlorophyll b (g/
mL), Cas represent the concentration of carotenoids (g/mL) and A665,
A649 and A470 represent the absorbance at 665, 649 and 470 nm, re-
spectively.
Karlsruhe, Germany), using a Cu-k radiation source (0.154 nm wave-
length) at 40 kV and 40 mA. The XRD data were collected in the 2
range from 4 to 60 at a scan rate of 5/min. The step width and step
time were set at 0.04 and 0.03 s, respectively.
2.9. DPPH radical scavenging activity
Antioxidant activity of pigment solutions was determined by a
(4)
stable DPPH radical using the method described by Gu et al. (2009)
(5) with some modications. A 50 mg sample was added to 5 mL ethanol
and was mixed vigorously until completely dissolved. 1 mL of the
(6) sample was mixed with 2 mL of 0.2 mmol/L DPPH solution in ethanol
and vortex-mixed for 5 s. After leaving the sample to stand in the dark
for 30 min, the absorbance values were determined at 517 nm using a
(7)
spectrophotometer. A blank was prepared in the same manner, except
that distilled water was used instead of the extraction solutions. For the
control, the assay was conducted in the same manner, but ethanol was
added instead of DPPH solution. The percentage of DPPH radical
scavenging activity was calculated by the following Eq. (8).
A A2
RA (%) = 1 1
100

2.6. UVVis spectra of pigments
UVVis absorption spectra of treated samples were measured as
described previously (Wang, Tao, Zhang, Li, & Gong, 2015) using a
spectrophotometer (UV-1800, SHIMADZU, Tokyo, Japan) equipped
with 1.0 cm quartz cell. 2.5 mL of each sample solution was transform
in the quartz cell and recorded over a wavelength range from 450 to
700 nm at 25 C.
2.7. Fourier transform infrared (FT-IR) spectra
FT-IR measurements of samples were carried out with a spectro-
meter (Vector33, Bruker, Germany) as described previously (Zhang,
Han, Zeng, & Wang, 2017) using KBr discs at 25 1 C.The spectra
were recorded over the range of 4004000 cm 1 with a resolution of
4 cm 1, with each spectrum consisting of 32 scans.
2.8. X-ray diraction (XRD) pattern
The crystal and structural properties of the PEF-treated pigment
samples were characterized as described previously (Han et al., 2012)
carried out with an X-ray diractometer (D8 Advance, Bruker,
A3 (8)
where RA is the DPPH radical scavenging activity, A1 is the absorbance
of the sample solution, A2 is the absorbance of the blank, and A3 is the
absorbance of the control.
2.10. Statistical analyses
All experiments were independent carried out three times, and the
results were reported as the mean standard deviation using SPSS
16.0 software (IBM, New York, NY, USA). All analyses were performed
on triplicate samples. Variance analysis and graphs were obtained by
using Origin 8.0 software (OriginLab, Massachusetts, MA, USA). The
signicance testing was performed by Tukey's test and the dierences
were statistically signicant at p < 0.05.
3. Results and discussion
3.1. The content of pigments analysis
The concentration of chlorophyll a, b and carotenoids from spinach
following dierent PEF treatments is shown in Table 2. It can be seen
that with the electric eld strength of PEF was gradually increased from
0 to 26.7 kV/cm at 20 C, the concentration of chlorophyll a was

28
Z.-H. Zhang et al. Innovative Food Science and Emerging Technologies 43 (2017) 2634

signicantly increased from 11.41 to 12.37, 12.74, 13.41, 13.63 and

14.41 g/mL (p < 0.05), respectively. Moreover, the concentration of
chlorophyll b was increased from 4.99 to 5.31, 5.45, 5.66, 5.74 and
6.04 g/mL (p < 0.05), respectively. The concentration of carotenoids
was 3.68 g/mL after 26.7 kV/cm under 20 C, which was signicantly
increased by 41.5% compared to sample without PEF treatment sample
of 2.60 g/mL (p < 0.05). When the treatment temperature was 35 C,
the concentration of chlorophyll a, b and carotenoids were increased by
0.72, 0.29 and 0.3 g/mL under the 3.3 kV/cm (p < 0.05), compared
to the sample without PEF treatment. Moreover, the concentration of
these pigments were increased with increasing electric eld strength,
and the concentration of chlorophyll a, b and carotenoids was reached
to 14.62, 6.13 and 3.52 g/mL under 26.7 kV/cm. When the treatment
temperature was 45 C, the concentration of chlorophyll a and b were
increased by 0.98, 0.46 and 0.04 g/mL under the 3.3 kV/cm, and with
the increase in the electric eld strength, the concentration of pigments
was gradually increased. In addition, for the sample without PEF
treatment, with the treatment temperature raised, the concentration of
chlorophyll a, b and carotenoids were decreased. These results showed
that the thermal treatment could destroy pigments extracted from spi-
nach leaves. However, during the various PEF treatment could inhibit
the degradation process of chlorophyll a, b and carotenoids extracted
from spinach leaves. This nding was consistent with previous study,
which reported that the degradation of chlorophyll a and b followed a
rst-order kinetic model by thermal treatment, and the degradation
rate was increased with temperature raised (Koca,
Karadeniz, & Burdurlu, 2007). Moreover, Yin, Han, and Liu (2007) re-
ported that increasing the electric eld strength from 20 to 100 kV/cm
could increase the stability of the green color of spinach puree during
the PEF treatment with acetate zinc. Their study explained this phe-
nomenon could be due to the as following reasons: 1. the PEF could
inactivate microorganisms and enzymes; 2. Addition of acetate zinc as
the stabilizer could increase the stabilization of green color because
zinc ions could replace magnesium ions derived from the pyrrole ring of
chlorophyll molecules during the PEF treatment. In the present study,
the reason for PEF stabilizing pigments may be that PEF change the
microenvironment of the solution and subsequently promote the
crosslink reaction with other chlorophyll moleculars, forming the self-
aggregation of chlorophyll, which leading to an increase in the stability
of chlorophyll molecules. Moreover, the pervious study reported that
the chlorophyll a dimers and oligomers were formed in both in vitro
and vivo (Cotton, Loach, Katz, & Ballschmiter, 1978).

3.2. The structural characteristics of pigments

3.2.1. The conjugated structure characterization

The characteristic absorption peak of chlorophyll a, chlorophyll b
and carotenoids in UVVis absorption spectra are at 663, 648 and
470 nm, respectively (Ili, Milenkovi, uni, & Fallik, 2015). In the
previous studies reported that the blue or red shift and the alterations in Fig. 1. The UVVis spectral of pigments by the dierent PEF treatments (A.20 C, B.
the peak shape in the UVVis spectra could indicate the changes of the 35 C, C. 45 C).
chemical structure (Kaiser, Wang, Stepanenko, & Wrthner, 2007). The
UVVis spectral of pigments by the dierent PEF treatments at dierent
the electric eld strength, the absorption intensity of UVVis was gra-
temperature is shown in Fig. 1. It can be seen that the absorption of
dually strengthened at 20, 35 and 45 C. This nding indicated that the
peaks did not appeared obvious red or blue shift, but the shape of peak
concentration of chlorophyll a, b and carotenoids was increased. This
(664 nm) with PEF treatment became sharper than untreated sample.
viewpoint was consistent of the results of Section 3.1 that the con-
This phenomenon indicated that PEF treatment could inuence the
centration of pigments without PEF treatment sample was decreased
nonconjugate structure of pigments, such as hydrogen bond and elec-
with an increase in temperature. Moreover, the concentrations of
trostatic forces, rather than the conjugate structure or carbon chain of
chlorophyll a, chlorophyll b and carotenoids by PEF treatment were
pigments. This nding was consistent with previous studies, which re-
higher than the sample without PEF treatment at 20, 35 and 45 C. This
ported that the PEF treatment could aect the secondary and tertiary
nding was consistent with the previous studies, which have showed
structure of the enzymes and proteins through aecting some of weak
that the thermal treatments such as blanching, steaming and microwave
chemical bond and intermolecular force, such as disulde bond, hy-
cooking could destroy the molecules of chlorophyll a and b, thereby
drogen bond and hydrophobic force, et al. (Budi, Legge,
decreased the concentration of pigments of the food (Benlloch-Tinoco
Treutlein, & Yarovsky, 2005; Zhao, Tang, Lu, Chen, & Li, 2014). More-
et al., 2015; Zheng et al., 2014). Meanwhile, the previous study has
over, from the Fig. 1, it also can be observed that with the increasing of

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Z.-H. Zhang et al. Innovative Food Science and Emerging Technologies 43 (2017) 2634

Fig. 3. XRD pattern of the pigments by dierent of PEF treatments (A. 20 C, B. 35 C, C.

Fig. 2. FT-IR spectrum of the pigments by dierent of PEF treatments (A. 20 C, B. 35 C,
45 C; a. 0 kV/cm, b. 3.3 kV/cm, c. 6.7 kV/cm, d. 13.3 kV/cm, e. 20.0 kV/cm, f. 26.7 kV/
C. 45 C; a. 0 kV/cm, b. 3.3 kV/cm, c. 6.7 kV/cm, d. 13.3 kV/cm, e. 20.0 kV/cm, f. cm).
26.7 kV/cm).

Mukherjee, & Das, 2009). The band at around 1734 cm 1 is ascribed to

reported that PEF treatment can increase the content of health-pro-
moting compounds compared to the heating treatment, such as vitamin
and anthocyanins (Odriozola-Serrano et al., 2013). In the present study,
it also was revealed that the PEF treatment can inhibit the degradation
of pigments, and 26.7 kV/cm presented the highest inhibition eect.
3.2.2. The functional groups characterization
Fig. 2 shows the FT-IR spectrum of the pigments following various
PEF treatments at dierent temperature. The information of IR char-
acteristic absorption bands was based on the previous studies (Hong,
Wang, Meng, Wei, & Zhao, 2002; Manna, Basu, Mitra,
the stretching vibration of keto group (C]O) between C13. The band
at around 1260 cm 1 is assigned to the skeletal vibration of the carbon
bond (CeC). The band of 934 cm 1 observed in this spectrum re-
presents the out-of-plane bending vibration of cis-carbon hydrogen
bonds (CH). The band at around 826 cm 1 is due to the out-of-plane
bending vibration of the amino group (NeH) in the porphyrin rings.
The band of 800 and 765 cm 1 is observed in this spectrum represents
the out-of-plane bending vibration of carbon hydrogen bonds (CH). As
the electric eld strength was increased at 20 C (Fig. 3(A)), the char-
acteristic absorption peaks of the chlorophyll was decreased, such as

30
Z.-H. Zhang et al.
the band at 1734, 800 and 767 cm 1, which showed the vibration of
carbonyl and methyl group of chlorophyll was limited. The reason for
this phenomenon may be the formations the steric eect that arised
from the structure of chlorophyll aggregation by PEF treatment, such as
dimers and oligomers chlorophyll. From Fig. 2(B), it can be seen that
the band at 1317 cm 1 almost disappeared, and the band at 826 cm 1
was enhanced with increase the electric eld strength at 35 C, which
revealed that the in-plane bending vibration of the hydroxyl group
(OH) was decreased, and the out-of-plane bending vibration of the
amino group (NeH) was enhanced, respectively. The reason for this
phenomenon was that the high intensity of PEF could promote the
formation of chlorophyll aggregations, and the interaction between the
magnesium ion and the amino group (NeH) in the porphyrin rings was
reduce during the formation process of chlorophyll aggregations. When
the temperature was increased to 45 C (Fig. 2(C)), it can be seen that
the intensity of IR absorbance at 767, 839 and 534 cm 1 was en-
hanced, which showed that the out-of-plane bending vibration of
carbon hydrogen bonds (CH) was increased. Moreover, the new ab-
sorbance peak was appeared in 1317 cm 1, which was represented the
stretching vibration of carbon oxygen bonds (CO). These phenomena
indicated that the eect of PEF treatment mainly focused on the carbon
hydrogen bonds and pyrrole ring of chlorophyll, and promoted the
formation of chlorophyll-aggregated structures. With the increase in
temperature, the intensity of vibration derived from the carbon hy-
drogen bonds was increased, which proved that heating treatment can
destroy the structure of chlorophyll with thermal treatment.
As for the carotenoids molecule, its basic structure is a symmetrical
tetraterpene skeleton formed by tail-to-tail connection of two ger-
anylgeranyl diphosphate molecules, which contain some unsaturated
bonds (Schoefs, 2002). Therefore, the electrons of the double bonds
were more susceptible to be inuenced by heating and other treat-
ments, and it was proved in previous study that the heating treatment
could cause the carotenoids isomerization (Chutintrasri & Noomhorm,
2007). From the Fig. 2, it can be seen that when the temperature was
increased from 20 to 45 C, the band at 935 cm 1 was decreased, and
the band at 767 cm 1 was increased. These phenomena indicated that
the bending vibration of carbon hydrogen bonds led to isomerization
and high temperature could promote the intensity, which can be seen
from the Fig. 2(B) and (C). This result was consistent with the previous
study (Bermdez-Aguirre & Barbosa-Cnovas, 2012). When the electric
eld strength was increased, the degree of carbon hydrogen bonds
isomerization was promoted. In related previous work, researchers
have reported that PEF can cause the conformation of vitamin C to
change from the enol to the ketone form (Zhang et al., 2015b), as well
as creating alterations in starch and chitosan molecular structures
(Hong, Zeng, Buckow, Han, & Wang, 2016; Luo, Han, Zeng,
Yu, & Kennedy, 2010).
3.3. The property of crystallization
The XRD patterns of the pigments following various PEF treatments
at dierent treatment temperatures are shown in Fig. 3.While there are
many diraction peaks, the four largest appeared at 2 values of around
27.3, 29.8, 32.7 and 39.3 in Fig. 3A(a). With the increase of PEF in-
tensity, a new diraction peak appeared at around 2 = 19.0. Mean-
while, the intensity of 27.3, 28.3, 29.8 and 32.7 (2) signicantly de-
creased while 43.1 and 44.8 (2) disappeared. On the other hand, when
the treatment temperature was set at 35 C, the intensity of some peaks
(2 = 19.0, 23.9, 27.4, 41.1) increased, the intensity of other peaks
(2 = 17.6, 18 and 36.4) almost decreased and some new diraction
peaks (2 = 31.2, 38.6 and 43) appeared after the PEF treatment (20.0,
26.7 kV/cm) compared to the untreated sample. When the treatment
temperature increased to 45 C, the major diraction peaks vanished in
both the PEF-treated and no-PEF treated samples. These results in-
dicated that the PEF treatment can modify the crystal structure of the
compound. The crystal size of the compound became larger under the
31
Innovative Food Science and Emerging Technologies 43 (2017) 2634
Fig. 4. The DPPH radical scavenging activity of pigments solutions by dierent PEF
treatments. Values with dierent letters in the same column (ae) are signicantly dif-
ferent according to Tukey test (p < 0.05).
PEF treatment that may be a result of the chlorophyll-aggregated
structures generated by the PEF treatment. Moreover, with the increase
in temperature, the pigments may have transitioned to a non-crystalline
state following the PEF treatment (forming chlorophyll-aggregated
structures) that hinders the formation of crystalline forms due to the
intermolecular interactions (Krasnovsky & Bystrova, 1980). Further-
more, Han et al. (2012) has reported that the PEF treatment was cap-
able of destroying the crystalline region of starches.
3.4. Antioxidant activity
The DPPH radical scavenging activity of pigments following various
PEF treatments at dierent treatment temperature is shown in Fig. 4. At
20 C, The DPPH radical scavenging activities of samples subjected to
3.3, 6.7, 13.3, 20.0 and 26.7 kV/cm of PEF treatment were 50.65%,
60.90%, 54.26%, 55.44% and 54.59%, respectively, which was in-
creased by 3.72%, 13.97%, 7.33%, 8.51% and 7.66%, respectively,
compared with untreated sample of 46.93%. When the treatment
temperature was increased to 35 C, DPPH radical scavenging activity
of samples treated by PEF was increased by 13.89%, 15.25%, 13.44%,
14.87% and 14.55%, respectively, compared with untreated sample of
20.87%. When the temperature further was increased to 45 C, DPPH
radical scavenging activity of the sample was 17.8%, 31.89%, 32.10%,
35.07%, 34.15% and 24.11%, respectively, while the intensity of
sample treated by PEF was 0 to 26.7 kV/cm. These results showed that
DPPH radical scavenging activity of PEF-treated samples was sig-
nicantly higher than the untreated sample under dierent tempera-
tures (p < 0.05), and DPPH radical scavenging activity of samples
with PEF treatment at lower temperature exhibited higher activity than
the sample at high temperatures. The reasons for the phenomena of
increased DPPH radical scavenging activity by PEF treatment were as-
cribed to two aspects: 1) the degradation process of reducing state
pigments were inhibited, which implied increasing the concentration of
reducing state pigments by various PEF treatments. From Table 2, it can
be seen that with the increase in the electric eld strength from 0 to
26.7 kV/cm at 20 C, the concentration of total pigments (chlorophyll a
+ chlorophyll b + carotenoids) increased by 1.85, 2.10, 3.2 and
1.52 g/mL, respectively, compared with the untreated sample of
19.0 g/mL. Moreover, with the increase in temperature to 35 and
45 C following 26.7 kV/cm of PEF treatment, the concentration of total
pigments increased by 5.79 and 3.60 g/mL, respectively, compared
with the untreated sample of 18.48 and 18.32 g/mL. These results
were in agreement with the ndings of El-Sayed, Hussin, Mahmoud,
and AlFredan (2013) who found that DPPH scavenging and other bio-
logical activities of Conyza extracts closely were related to its chlor-
ophyll content. Furthermore, the concentration of total pigments at 35
Z.-H. Zhang et al.
and 45 C without PEF treatment signicantly was decreased by 2.8%
and 3.7%, respectively, compared with the sample at 20 C (19.0 g/
mL). The result suggested that heat treatment promoted the degrada-
tion of pigments (Koca et al., 2007). In addition, the previous study
indicates that the dierent pigment ingredients have dierent anti-
oxidant capacities and that PEF treatment has dierent inuences on
pigments compounds. These results were consistent with previous study
(Lanfer-Marquez et al., 2005). 2) The structure of pigments was
changed by various PEF treatments. The change in structure following
PEF treatment was evident in the XRD and FT-IR spectra described
above. The previous study reported that there is a relationship between
the antioxidant property of chlorophyll and the chlorophyll structure
(Ferruzzi & Blakeslee, 2007). Moreover, Zhang et al. (2015) reported
that PEF could improve the antioxidant activity of vitamin C due to the
modication of the structure of vitamin C was induced by PEF treat-
ment.
From the above discussion, it appears that the PEF treatment can
32
Innovative Food Science and Emerging Technologies 43 (2017) 2634
Fig. 5. The proposed mechanism of eect of PEF treatment on
pigments (A: chlorophyll, B: -carotene).
inhibit the degradation of pigments and change the structure of those
pigments that leads to the enhancement of the DPPH radical scavenging
activity of pigments over a series of temperatures.
3.5. The mechanism of PEF inuence in pigments
The determination of pigments mainly includes chlorophyll, chlor-
ophyll derivatives and carotenes from spinach, which had two kinds of
special structure, pyrrole ring and conjugated double bond system. The
possible mechanism for the eect of PEF treatment on the structure of
the pyrrole ring is shown in Fig. 5A. Hydrogen peroxide or free radicals
generated by PEF treatment (Parniakov, Barba, Grimi,
Lebovka, & Vorobiev, 2014; Zhang, Yang, Zhao, Liang, & Zhang, 2011)
attack the pyrrole ring (II and IV) of chlorophyll molecules leading to
the dissociation of interaction force between magnesium ion and ni-
trogen atom derived from amino group. Then, the magnesium ion can
combine with the carbonyl group (C]O) of the surrounding
Z.-H. Zhang et al.
chlorophyll molecules (pyrrole ring IV) and generate the intermolecular
force in the ethanol due to their relatively high electronegativity. This
reaction process may increase the molecular weight of the pigments by
forming chlorophyll-aggregated structures, such as dimers or oligomers
chlorophyll (Krasnovsky & Bystrova, 1980). This special molecule
structure could increase the steric eect, and then protects the pyrrole
ring of the chlorophyll molecule. Furthermore, as for the conjugated
double bond system, it may be due to the hydrogen peroxide or free
radicals generated by PEF treatment inuencing the vibration of these
unsaturated bonds leading to the change of conformation (from cis to
trans) which is shown in Fig. 5B. Due to the structural changes by PEF
treatment was increased, the eect of thermal degradation of the pig-
ments was inhibited, and the stability of the pigments was increased. In
addition, other workers have proved that PEF treatment can aect
weakly covalent bonds, non-covalent bonds (such as hydrogen bonds,
disulde bonds and hydrophobic bonds) and weak chemical forces
(such as van der Waals bonding, hydrophobic and electrostatic inter-
actions) (Terefe, Buckow, & Versteeg, 2015).
4. Conclusions
The eect of PEF on the pigments of spinach was investigated in this
study. The pigments from fresh green spinach were extracted using
ethanol, and then treated by dierent electric eld intensity of PEF at a
series of temperature conditions. The physicochemical properties and
antioxidant activity of the treated pigment were also determined.
Results showed that PEF treatment to some extent inhibited the de-
gradation of pigments, especially for the relative concentration of
chlorophyll a, b and carotenoids under dierent treated temperatures
(20, 35 and 45 C). Based on the UVVis and FT-IR spectrum analysis,
the PEF treatment inuenced the structure of pigments mainly related
to the chemical bonds between the pyrrole ring and central magnesium
ions of chlorophyll that could form the chlorophyll aggregated struc-
tures thus increasing the stability of chlorophyll. PEF treatment also
aected the unsaturated bond of carotenoids, which could have led to
the change of conformation from cis to trans. Furthermore, compared to
the thermal treatment, the antioxidant property of sample solution
treated by the dierent electric eld intensities of PEF treatments was
increased, especially at low temperature. Therefore, it can be concluded
that the PEF acts as an ecient food processing method, providing the
potential for enhancement of the biological activities and antioxidant
activity of natural compounds.
Acknowledgments
The authors gratefully acknowledge the nancial support provided
by the National Natural Science Foundation of China (Grant no.
21576099, 21376094 and 31301559), China Postdoctoral Science
Foundation (2017M612672) and the Fundamental Research Funds for
the central Universities (2017MS067) as well as the S & T projects of
Guangdong province (Grant no. 2015A030312001, 2013B051000010
and 2013B020203001).
References
Benlloch-Tinoco, M., Kaulmann, A., Corte-Real, J., Rodrigo, D., Martnez-Navarrete, N., &
Bohn, T. (2015). Chlorophylls and carotenoids of kiwifruit puree are aected simi-
larly or less by microwave than by conventional heat processing and storage. Food
Chemistry, 187, 254262.
Bermdez-Aguirre, D., & Barbosa-Cnovas, G. V. (2012). Inactivation of Saccharomyces
cerevisiae in pineapple, grape and cranberry juices under pulsed and continuous
thermo-sonication treatments. Journal of Food Engineering, 108(3), 383392.
Buckow, R., Ng, S., & Toep, S. (2013). Pulsed electric eld processing of orange juice: A
review on microbial, enzymatic, nutritional, and sensory quality and stability.
Comprehensive Reviews in Food Science and Food Safety, 12(5), 455467.
Budi, A., Legge, F. S., Treutlein, H., & Yarovsky, I. (2005). Electric eld eects on insulin
chain-B conformation. The Journal of Physical Chemistry B, 109(47), 2264122648.
Chernomorsky, S., & Segelman, A. (1988). Biological activities of chlorophyll derivatives.
New Jersey Medicine: The Journal of the Medical Society of New Jersey, 85(8), 669.
33
Innovative Food Science and Emerging Technologies 43 (2017) 2634
Chemat, F., Rombaut, N., Meullemiestre, A., Turk, M., Perino, S., Fabiano-Tixier, A. S., &
Abert-Vian, M. (2017). Review of Green Food Processing techniques. Preservation,
transformation, and extraction. Innovative Food Science & Emerging Technologies, 41,
353377.
Chutintrasri, B., & Noomhorm, A. (2007). Color degradation kinetics of pineapple puree
during thermal processing. LWT-Food Science and Technology, 40(2), 300306.
Cotton, T., Loach, P., Katz, J., & Ballschmiter, K. (1978). Studies of chlorophyll-chlor-
ophyll and chlorophyll-ligand interactions by visible absorption and infrared spec-
troscopy at low temperatures. Photochemistry and Photobiology, 27(6), 735749.
El-Sayed, W. M., Hussin, W. A., Mahmoud, A. A., & AlFredan, M. A. (2013). The Conyza
triloba extracts with high chlorophyll content and free radical scavenging activity had
anticancer activity in cell lines. BioMed Research International, 2013.
Ferruzzi, M. G., & Blakeslee, J. (2007). Digestion, absorption, and cancer preventative
activity of dietary chlorophyll derivatives. Nutrition Research, 27(1), 112.
Gu, F., Kim, J. M., Hayat, K., Xia, S., Feng, B., & Zhang, X. (2009). Characteristics and
antioxidant activity of ultraltrated Maillard reaction products from a caseinglucose
model system. Food Chemistry, 117(1), 4854.
Han, J. H., Jo, Y. G., Kim, J. C., Lee, J.-B., Kim, Y. C., Kang, H., & Hwang, I.-W. (2016).
Contrast agent free detection of bowel perforation using chlorophyll derivatives from
food plants. Chemical Physics Letters, 643, 1015.
Han, Z., Zeng, X. A., Fu, N., Yu, S. J., Chen, X. D., & Kennedy, J. F. (2012). Eects of
pulsed electric eld treatments on some properties of tapioca starch. Carbohydrate
Polymers, 89(4), 10121017.
Hong, F., Wang, L., Meng, X., Wei, Z., & Zhao, G. (2002). The eect of cerium (III) on the
chlorophyll formation in spinach. Biological Trace Element Research, 89(3), 263276.
Hong, J., Zeng, X. A., Buckow, R., Han, Z., & Wang, M. S. (2016). Nanostructure, mor-
phology and functionality of cassava starch after pulsed electric elds assisted acet-
ylation. Food Hydrocolloids, 54, 139150.
Huang, W., Bi, X., Zhang, X., Liao, X., Hu, X., & Wu, J. (2013). Comparative study of
enzymes, phenolics, carotenoids and color of apricot nectars treated by high hydro-
static pressure and high temperature short time. Innovative Food Science & Emerging
Technologies, 18, 7482.
Ili, Z. S., Milenkovi, L., uni, L., & Fallik, E. (2015). Eect of coloured shade-nets on
plant leaf parameters and tomato fruit quality. Journal of the Science of Food and
Agriculture, 95(13), 26602667.
Kaiser, T. E., Wang, H., Stepanenko, V., & Wrthner, F. (2007). Supramolecular con-
struction of uorescent J-aggregates based on hydrogen-bonded perylene dyes.
Angewandte Chemie International Edition, 46(29), 55415544.
Kephart, J. C. (1955). Chlorophyll derivativesTheir chemistry? Commercial preparation
and uses. Economic Botany, 9(1), 338.
Khachik, F., Beecher, G. R., & Whittaker, N. F. (1986). Separation, identication, and
quantication of the major carotenoid and chlorophyll constituents in extracts of
several green vegetables by liquid chromatography. Journal of Agricultural and Food
Chemistry, 34(4), 603616.
Koca, N., Karadeniz, F., & Burdurlu, H. S. (2007). Eect of pH on chlorophyll degradation
and colour loss in blanched green peas. Food Chemistry, 100(2), 609615.
Krasnovsky, A., & Bystrova, M. (1980). Self-assembly of chlorophyll aggregated struc-
tures. Biosystems, 12(3), 181194.
Lanfer-Marquez, U. M., Barros, R. M., & Sinnecker, P. (2005). Antioxidant activity of
chlorophylls and their derivatives. Food Research International, 38(8), 885891.
Luengo, E., Condn-Abanto, S., lvarez, I., & Raso, J. (2014). Eect of pulsed electric eld
treatments on permeabilization and extraction of pigments from Chlorella vulgaris.
The Journal of Membrane Biology, 247(12), 12691277.
Luo, W. B., Han, Z., Zeng, X. A., Yu, S. J., & Kennedy, J. F. (2010). Study on the de-
gradation of chitosan by pulsed electric elds treatment. Innovative Food
Science & Emerging Technologies, 11(4), 587591.
Manna, J. S., Basu, S., Mitra, M., Mukherjee, S., & Das, G. C. (2009). Study on the
biostability of chlorophyll a entrapped in silica gel nanomatrix. Journal of Materials
Science: Materials in Electronics, 20(11), 10681072.
Miret, S., Tascioglu, S., van der Burg, M., Frenken, L., & Klake, W. (2009). In vitro
bioavailability of iron from the heme analogue sodium iron chlorophyllin. Journal of
Agricultural and Food Chemistry, 58(2), 13271332.
Odriozola-Serrano, I., Aguilo-Aguayo, I., Soliva-Fortuny, R., & Martin-Belloso, O. (2013).
Pulsed electric elds processing eects on quality and health-related constituents of
plant-based foods. Trends in Food Science & Technology, 29(2), 98107.
zkan, G., & Bilek, S. E. (2015). Enzyme-assisted extraction of stabilized chlorophyll from
spinach. Food Chemistry, 176, 152157.
Parniakov, O., Barba, F. J., Grimi, N., Lebovka, N., & Vorobiev, E. (2014). Impact of
pulsed electric elds and high voltage electrical discharges on extraction of high-
added value compounds from papaya peels. Food Research International, 65, 337343.
Pina-Prez, M. C., Rodrigo, D., & Martnez, A. (2016). Nonthermal inactivation of
Cronobacter sakazakii in infant formula milk: A review. Critical Reviews in Food Science
and Nutrition, 56(10), 16201629.
Pott, I., Marx, M., Neidhart, S., Mhlbauer, W., & Carle, R. (2003). Quantitative de-
termination of -carotene stereoisomers in fresh, dried, and solar-dried mangoes
(Mangifera indica L.). Journal of Agricultural and Food Chemistry, 51(16), 45274531.
Schoefs, B.t. (2002). Chlorophyll and carotenoid analysis in food products. Properties of
the pigments and methods of analysis. Trends in Food Science & Technology, 13(11),
361371.
Singh, A., Ahmad, S., & Ahmad, A. (2015). Green extraction methods and environmental
applications of carotenoids-a review. RSC Advances, 5(77), 6235862393.
Terefe, N. S., Buckow, R., & Versteeg, C. (2015). Quality-related enzymes in plant-based
products: Eects of novel food processing technologies part 2: Pulsed electric eld
processing. Critical Reviews in Food Science and Nutrition, 55(1), 115.
Wang, L., Tao, M., Zhang, G., Li, S., & Gong, D. (2015). Partial intercalative binding of the
food colorant erythrosine to herring sperm DNA. RSC Advances, 5(119),
Z.-H. Zhang et al.
9836698376.
Wang, L. H., Wang, M. S., Zeng, X. A., & Liu, Z. W. (2016). Temperature-mediated var-
iations in cellular membrane fatty acid composition of Staphylococcus aureus in re-
sistance to pulsed electric elds. Biochimica et Biophysica Acta (BBA)-Biomembranes,
1858(8), 17911800.
Wang, L. H., Wang, M. S., Zeng, X. A., Zhang, Z. H., Gong, D. M., & Huang, Y. B. (2016).
Membrane destruction and DNA binding of Staphylococcus aureus cells induced by
carvacrol and its combined eect with a pulsed electric eld. Journal of agricultural
and food chemistry, 64(32), 63556363.
Wintermans, J., & De Mots, A. (1965). Spectrophotometric characteristics of chlorophylls
a and b and their phenophytins in ethanol. Biochimica et Biophysica Acta (BBA)-
Biophysics including Photosynthesis, 109(2), 448453.
Yin, Y., Han, Y., & Liu, J. (2007). A novel protecting method for visual green color in
spinach puree treated by high intensity pulsed electric elds. Journal of Food
Engineering, 79(4), 12561260.
Zeng, X. A., Han, Z., & Zi, Z. H. (2010). Eects of pulsed electric eld treatments on
quality of peanut oil. Food Control, 21(5), 611614.
Zhang, S., Yang, R., Zhao, W., Liang, Q., & Zhang, Z. (2011). The rst ESR observation of
34
Innovative Food Science and Emerging Technologies 43 (2017) 2634
radical species generated under pulsed electric elds processing. LWT-Food Science
and Technology, 44(4), 12331235.
Zhang, Z. H., Zeng, X. A., Brennan, C. S., Brennan, M., Han, Z., & Xiong, X. Y. (2015).
Eects of pulsed electric elds (PEF) on vitamin C and its antioxidant properties.
International Journal of Molecular Sciences, 16(10), 2415924173.
Zhang, Z. H., Han, Z., Zeng, X. A., & Wang, M. S. (2017). The preparation of Fe-glycine
complexes by a novel method (pulsed electric elds). Food chemistry, 219, 468476.
Zhao, L., Wang, S., Liu, F., Dong, P., Huang, W., Xiong, L., & Liao, X. (2013). Comparing
the eects of high hydrostatic pressure and thermal pasteurization combined with
nisin on the quality of cucumber juice drinks. Innovative Food Science & Emerging
Technologies, 17, 2736.
Zhao, W., Tang, Y., Lu, L., Chen, X., & Li, C. (2014). Review: Pulsed electric elds pro-
cessing of protein-based foods. Food and Bioprocess Technology, 7(1), 114125.
Zheng, Y., Shi, J., Pan, Z., Cheng, Y., Zhang, Y., & Li, N. (2014). Eect of heat treatment,
pH, sugar concentration, and metal ion addition on green color retention in homo-
genized puree of Thompson seedless grape. LWT-Food Science and Technology, 55(2),
595603.