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Effect of pulsed electric fields (PEFs) on the pigments extracted from spinach (Spinacia oleracea L.)
Innovative Food Science and Emerging Technologies 43 (2017) 26–34
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Effect of pulsed electric fields (PEFs) on the pigments extracted from spinach (Spinacia oleracea L.)
MARK
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: Pulsed electric fields Pigments Physicochemical properties Antioxidant activity
In order to investigate the effect of PEF on the pigments of spinach, pigments were extracted and were treated by different electric field strength at different temperatures (20, 35 and 45 °C). Results showed that with the increasing temperature, the degradation of chlorophyll content was appeared for pigments without PEF treatment. However, the concentrations of chlorophyll a, b and carotenoids were increased by the PEF treatment, especially for the higher electric field strength. Moreover, the structure of spinach pigments treated by PEF also was determined by UV–Vis, FT-IR and XRD. From the structural information, it was indicated that the effect 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 modified. In addition, the PEF treatment also significantly increased the antioxidation activity of pigments over the series of temperatures. Industrial relevance: This study provide evidences that the PEF treatment can inhibit the degradation of chlorophyll 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 oligomers chlorophyll. Therefore, the PEF treatment is an efficient food processing method, especially foods rich in pigment.
1. Introduction 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 specific health benefits, such as antioxidant activity, hematopoietic activity and anti-inflammatory properties (Ferruzzi & Blakeslee, 2007; Lanfer-Marquez, Barros, & Sinnecker, 2005; Miret, Tascioglu, van der Burg, Frenken, & Klaffke, 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 provitamin 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
⁎
Corresponding author. E-mail address: [email protected] (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.
of several degenerative processes, including cancers, cardiovascular 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 vegetables as well as their processed products. In addition, phytochemicals 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. (Özkan & Bilek, 2015; Pott, Marx, Neidhart, Mühlbauer, & Carle, 2003; Zhao et al., 2013). Therefore, it is useful to explore innovative and non-thermal technologies in processing to sterilize the food while also protect the favorable bioactivity of pigments. Pulsed electric fields (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, & Toepfl, 2013; Pina-Pérez, Rodrigo, & Martínez, 2016). PEF treatments typically involve the application of pulses of high electric field intensity (above 20 kV/cm) to liquid foods placed between two electrodes. PEF treatments are conducted at ambient or moderately high temperatures (generally < 45 °C)
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Afterwards, the spinach puree was centrifuged at 5000g for 10 min at 4 °C (TDL-5-A, Shanghai Anting Scientific Instrument Factory, China). The precipitation was collected and was extracted with 400 mL of absolute 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 filled in a 1 L volumetric flask and stored in a refrigerator at 4 °C until further PEF treatment.
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 affects the flavor and quality of the foods (Odriozola-Serrano, Aguilo-Aguayo, SolivaFortuny, & Martin-Belloso, 2013; Zeng, Han, & Zi, 2010). A number of studies have shown the effectiveness of PEF treatment in better retaining nutritious compounds, such as ascorbic acid, carotenoids and phenols, in comparison with heat treatments of different food stuffs, such as tomato and strawberry juices (Buckow et al., 2013; OdriozolaSerrano et al., 2013). Moreover, many investigations have proved that PEF can enhance the extraction of intracellular compounds, such as pigments, flavors and polysaccharides from the plant cells (Chemat et al., 2017). However, the effect of PEF treatment on the structure and physicochemical properties of compounds, especially for plant pigments rarely reported until now. Therefore, the aim of this study was to investigate the effect of PEF on the pigments of spinach including the physicochemical properties and the antioxidant activity. Furthermore, the mechanism of PEF affecting the chlorophyll and carotenoids also was explored based on the changes in the structure.
2.4. PEF treatment of pigment solution Before performing PEF treatments, bubbles in the extraction solutions were removed by a vacuum pump (SHZ-D III, Gongyi Yuhua Instrument Co., Ltd., Gongyi, China). The pH value and the conductivity 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 specific electric field intensity (0, 3.3, 6.7, 13.3, 20.0 and 26.7 kV/cm) and different temperatures (20, 35 and 45 °C). The treatment temperature was controlled using a thermostatic device (HH-4, KEJIE instrument, Jintan, China). Reaction conditions are listed in Table 1. In this study, the flow rate of the extraction solution was set at 100 mL/min. The effective 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 effective treatment time and energy input of different PEF treatments are shown in Table 1.
2. Material and methods 2.1. Materials Fresh spinach (Spinacia oleracea L.) was purchased at the local supermarket (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-picrylhydrazyl (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).
f×V S
(1)
t = Np × Wp × n
(2)
Q = E2 × t × δ
(3)
Np =
2.2. PEF treatment system The continuous PEF system was designed by the PEF team of South China University of Technology (Guangzhou, China). The structure of the device has been reported in our previous studies (Wang et al., 2016; Wang, Wang, Zeng, & Liu, 2016). The main parameters of this device were a unipolar square wave, the maximum voltage of 15 kV, maximum current of 1.77 A, frequency of 1 kHz, and pulse width of 20 μs, respectively. The electric field strength of PEF system was monitored by a two-channel digital storage oscilloscope (DST1102B, Tekway Technologies Co., Ltd., Nanjing, China). Two parallel titanium-based alloy electrodes were inserted in the treatment chamber that was made from Teflon. The distance between two the electrodes was 0.3 cm. The volume of the treatment chamber was 0.02 mL. The sample solution was transported through the silicone tube (inner diameter of 6 mm), and was introduced into the treatment chamber by a constant flow pump (323 E/D, Watson Marlow, NC, USA). The flow rate was controlled by a flow meter (LZB-4, Changzhou Shuangfa Thermal Instrument Factory, Changzhou, China). The inlet and outlet temperatures of sample solution were monitored by two K-type thermocouples (Pico technology Inc., St Neots, United Kingdom). The temperature of the sample was controlled with a cooling circulator (DLSK 3/10, Ketai Laboratory Equipment Co., Ltd., Zhengzhou, China).
where Np represents the pulse number, f represents the pulse repetition rates, V represents the volume of the chamber (mL), S represents the flow rate (mL/s), t represents the effective treatment time (μs), Wp represents the pulse width (μs), n represents the sample cycles, Q represents the energy input (J/m3) and δ represents the conductivity of the sample (S/m). 2.5. Content of pigments The concentration of pigments of treated samples was determined Table 1 The effective treatment time and energy input of different PEF treatment. Treatment method
Treatment time (min)
Effective time (ms)
Input energy (kJ/m3)
20 °C
60 60 60 60 60 60 60 60 60 60 60 60 60 60 60
0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32
2.43 9.65 38.72 87.04 154.66 2.91 11.57 46.41 104.32 185.37 1.49 5.93 23.77 53.44 94.96
35 °C
2.3. Extraction of pigments from spinach leaves In order to prevent pigments in spinach degradation, such as chlorophylls and carotenoids, each extraction step was performed under dim light and low temperature (4 ± 1 °C). 200 g of cleaned spinach leaves were milled by a mixer grinder (JYL-C022E, Joyoung, Hangzhou, China) for 15 s with deionized water at a ratio of 1:2 (w/v) in order to remove the water-soluble compounds of spinach leaves.
45 °C
27
3.3 kV/cm 6.7 kV/cm 13.3 kV/cm 20.0 kV/cm 26.7 kV/cm 3.3 kV/cm 6.7 kV/cm 13.3 kV/cm 20.0 kV/cm 26.7 kV/cm 3.3 kV/cm 6.7 kV/cm 13.3 kV/cm 20.0 kV/cm 26.7 kV/cm
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Table 2 The concentration of chlorophyll a, b and carotenoids from spinach following different PEF treatments (μg/mL). Sample 20 °C
35 °C
45 °C
0 kV/cm 3.3 kV/cm 6.7 kV/cm 13.3 kV/cm 20.0 kV/cm 26.7 kV/cm 0 kV/cm 3.3 kV/cm 6.7 kV/cm 13.3 kV/cm 20.0 kV/cm 26.7 kV/cm 0 kV/cm 3.3 kV/cm 6.7 kV/cm 13.3 kV/cm 20.0 kV/cm 26.7 kV/cm
Chlorophyll a
Chlorophyll b
Chlorophyll a + b
Carotenoids
11.41 12.37 12.74 13.41 13.63 14.41 11.20 11.92 12.64 13.24 13.63 14.62 10.93 11.89 11.94 12.20 12.55 13.21
4.99 5.31 5.45 5.66 5.74 6.04 4.87 5.16 5.37 5.67 5.75 6.13 4.79 5.25 5.45 5.08 5.42 5.73
16.40 17.68 18.18 19.07 19.37 20.44 16.07 17.08 18.02 18.92 19.38 20.75 15.72 17.14 17.39 17.28 17.96 18.94
2.60 2.84 2.92 3.13 3.25 3.68 2.41 2.71 2.94 3.25 3.19 3.52 2.60 2.64 2.57 2.82 2.91 2.98
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.02a 0.01b 0.02c 0.04d 0.03e 0.01f 0.04a 0.02b 0.03c 0.04d 0.03e 0.02f 0.05a 0.02b 0.03b 0.02c 0.03d 0.06e
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.01a 0.03b 0.01c 0.02d 0.01e 0.02f 0.02a 0.01b 0.02c 0.02d 0.01e 0.02f 0.04a 0.02b 0.02c 0.04d 0.04c 0.03e
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.03a 0.04b 0.03c 0.06d 0.04e 0.03f 0.06a 0.03b 0.05c 0.06d 0.04e 0.04f 0.09a 0.04b 0.05c 0.06bc 0.07d 0.09e
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.02a 0.04b 0.03c 0.01d 0.02e 0.04f 0.01a 0.02b 0.01c 0.02d 0.04e 0.02f 0.03a 0.02a 0.01a 0.03b 0.04c 0.05c
Values with different letters in the same column (a–f) are significantly different according to Tukey test (p < 0.05).
Karlsruhe, Germany), using a Cu-kα radiation source (0.154 nm wavelength) at 40 kV and 40 mA. The XRD data were collected in the 2θ 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.
by the UV–Vis spectrophotometer described by previous studies (Luengo, Condón-Abanto, Álvarez, & Raso, 2014; Wintermans & De Mots, 1965). Briefly, 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 calculated by Eqs. (4) to (7).
Chla = 13.70 × A 665 − 5.76 × A 649
(4)
Chlb = 25.8 × A 649 − 7.60 × A 665
(5)
Chla + b = 6.10 × A 665 + 20.04 × A 649
(6)
Cas =
1000 × A 470 − 2.13 × Chl a − 97.64 × Chlb 209
2.9. DPPH radical scavenging activity Antioxidant activity of pigment solutions was determined by a stable DPPH radical using the method described by Gu et al. (2009) with some modifications. A 50 mg sample was added to 5 mL ethanol and was mixed vigorously until completely dissolved. 1 mL of the 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 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).
(7)
where Chla represents the concentration of chlorophyll a (μg/mL), Chlb represents the concentration of chlorophyll b (μg/mL), Chla + b represent the total concentration of chlorophyll a and chlorophyll b (μg/ mL), Cas represent the concentration of carotenoids (μg/mL) and A665, A649 and A470 represent the absorbance at 665, 649 and 470 nm, respectively.
A − A2 ⎞ RA (%) = ⎛1 − 1 × 100 A3 ⎠ ⎝ ⎜
2.6. UV–Vis spectra of pigments
⎟
(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.
UV–Vis 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.
2.10. 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 significance testing was performed by Tukey's test and the differences were statistically significant at p < 0.05.
2.7. Fourier transform infrared (FT-IR) spectra FT-IR measurements of samples were carried out with a spectrometer (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 400–4000 cm− 1 with a resolution of 4 cm− 1, with each spectrum consisting of 32 scans.
3. Results and discussion 3.1. The content of pigments analysis
2.8. X-ray diffraction (XRD) pattern The concentration of chlorophyll a, b and carotenoids from spinach following different PEF treatments is shown in Table 2. It can be seen that with the electric field strength of PEF was gradually increased from 0 to 26.7 kV/cm at 20 °C, the concentration of chlorophyll a was
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 diffractometer (D8 Advance, Bruker, 28
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significantly increased from 11.41 to 12.37, 12.74, 13.41, 13.63 and 14.41 μg/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 μg/mL (p < 0.05), respectively. The concentration of carotenoids was 3.68 μg/mL after 26.7 kV/cm under 20 °C, which was significantly increased by 41.5% compared to sample without PEF treatment sample of 2.60 μg/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 μg/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 field strength, and the concentration of chlorophyll a, b and carotenoids was reached to 14.62, 6.13 and 3.52 μg/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 μg/mL under the 3.3 kV/cm, and with the increase in the electric field 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 spinach leaves. However, during the various PEF treatment could inhibit the degradation process of chlorophyll a, b and carotenoids extracted from spinach leaves. This finding was consistent with previous study, which reported that the degradation of chlorophyll a and b followed a first-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) reported that increasing the electric field 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 phenomenon 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 selfaggregation 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 UV–Vis absorption spectra are at 663, 648 and 470 nm, respectively (Ilić, Milenković, Šunić, & Fallik, 2015). In the previous studies reported that the blue or red shift and the alterations in the peak shape in the UV–Vis spectra could indicate the changes of the chemical structure (Kaiser, Wang, Stepanenko, & Würthner, 2007). The UV–Vis spectral of pigments by the different PEF treatments at different temperature is shown in Fig. 1. It can be seen that the absorption of peaks did not appeared obvious red or blue shift, but the shape of peak (664 nm) with PEF treatment became sharper than untreated sample. This phenomenon indicated that PEF treatment could influence the nonconjugate structure of pigments, such as hydrogen bond and electrostatic forces, rather than the conjugate structure or carbon chain of pigments. This finding was consistent with previous studies, which reported that the PEF treatment could affect the secondary and tertiary structure of the enzymes and proteins through affecting some of weak chemical bond and intermolecular force, such as disulfide bond, hydrogen bond and hydrophobic force, et al. (Budi, Legge, Treutlein, & Yarovsky, 2005; Zhao, Tang, Lu, Chen, & Li, 2014). Moreover, from the Fig. 1, it also can be observed that with the increasing of
Fig. 1. The UV–Vis spectral of pigments by the different PEF treatments (A.20 °C, B. 35 °C, C. 45 °C).
the electric field strength, the absorption intensity of UV–Vis was gradually strengthened at 20, 35 and 45 °C. This finding indicated that the concentration of chlorophyll a, b and carotenoids was increased. This viewpoint was consistent of the results of Section 3.1 that the concentration of pigments without PEF treatment sample was decreased with an increase in temperature. Moreover, the concentrations of chlorophyll a, chlorophyll b and carotenoids by PEF treatment were higher than the sample without PEF treatment at 20, 35 and 45 °C. This finding was consistent with the previous studies, which have showed that the thermal treatments such as blanching, steaming and microwave cooking could destroy the molecules of chlorophyll a and b, thereby decreased the concentration of pigments of the food (Benlloch-Tinoco et al., 2015; Zheng et al., 2014). Meanwhile, the previous study has 29
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Fig. 3. XRD pattern of the pigments by different of PEF treatments (A. 20 °C, B. 35 °C, 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/ cm).
Fig. 2. FT-IR spectrum of the pigments by different of PEF treatments (A. 20 °C, B. 35 °C, 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/cm).
Mukherjee, & Das, 2009). The band at around 1734 cm− 1 is ascribed to 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 represents 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 field strength was increased at 20 °C (Fig. 3(A)), the characteristic absorption peaks of the chlorophyll was decreased, such as
reported that PEF treatment can increase the content of health-promoting 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 effect. 3.2.2. The functional groups characterization Fig. 2 shows the FT-IR spectrum of the pigments following various PEF treatments at different temperature. The information of IR characteristic absorption bands was based on the previous studies (Hong, Wang, Meng, Wei, & Zhao, 2002; Manna, Basu, Mitra, 30
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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 effect 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 field 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 enhanced, which showed that the out-of-plane bending vibration of carbon hydrogen bonds (–CH) was increased. Moreover, the new absorbance peak was appeared in 1317 cm− 1, which was represented the stretching vibration of carbon oxygen bonds (–CO). These phenomena indicated that the effect 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 hydrogen 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 geranylgeranyl diphosphate molecules, which contain some unsaturated bonds (Schoefs, 2002). Therefore, the π electrons of the double bonds were more susceptible to be influenced by heating and other treatments, 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 (Bermúdez-Aguirre & Barbosa-Cánovas, 2012). When the electric field 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).
Fig. 4. The DPPH radical scavenging activity of pigments solutions by different PEF treatments. Values with different letters in the same column (a–e) are significantly different 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). Furthermore, Han et al. (2012) has reported that the PEF treatment was capable of destroying the crystalline region of starches. 3.4. Antioxidant activity The DPPH radical scavenging activity of pigments following various PEF treatments at different 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 increased 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 significantly higher than the untreated sample under different temperatures (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 ascribed 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 field 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 μg/mL, respectively, compared with the untreated sample of 19.0 μg/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 μg/mL, respectively, compared with the untreated sample of 18.48 and 18.32 μg/mL. These results were in agreement with the findings of El-Sayed, Hussin, Mahmoud, and AlFredan (2013) who found that DPPH scavenging and other biological activities of Conyza extracts closely were related to its chlorophyll content. Furthermore, the concentration of total pigments at 35
3.3. The property of crystallization The XRD patterns of the pigments following various PEF treatments at different treatment temperatures are shown in Fig. 3.While there are many diffraction peaks, the four largest appeared at 2θ values of around 27.3, 29.8, 32.7 and 39.3 in Fig. 3A(a). With the increase of PEF intensity, a new diffraction peak appeared at around 2θ = 19.0. Meanwhile, the intensity of 27.3, 28.3, 29.8 and 32.7 (2θ) significantly decreased while 43.1 and 44.8 (2θ) disappeared. On the other hand, when the treatment temperature was set at 35 °C, the intensity of some peaks (2θ = 19.0, 23.9, 27.4, 41.1) increased, the intensity of other peaks (2θ = 17.6, 18 and 36.4) almost decreased and some new diffraction peaks (2θ = 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 diffraction peaks vanished in both the PEF-treated and no-PEF treated samples. These results indicated that the PEF treatment can modify the crystal structure of the compound. The crystal size of the compound became larger under the 31
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Fig. 5. The proposed mechanism of effect of PEF treatment on pigments (A: chlorophyll, B: β-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.
and 45 °C without PEF treatment significantly was decreased by 2.8% and 3.7%, respectively, compared with the sample at 20 °C (19.0 μg/ mL). The result suggested that heat treatment promoted the degradation of pigments (Koca et al., 2007). In addition, the previous study indicates that the different pigment ingredients have different antioxidant capacities and that PEF treatment has different influences 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 modification of the structure of vitamin C was induced by PEF treatment. From the above discussion, it appears that the PEF treatment can
3.5. The mechanism of PEF influence in pigments The determination of pigments mainly includes chlorophyll, chlorophyll derivatives and carotenes from spinach, which had two kinds of special structure, pyrrole ring and conjugated double bond system. The possible mechanism for the effect 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 nitrogen atom derived from amino group. Then, the magnesium ion can combine with the carbonyl group (C]O) of the surrounding 32
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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, 353–377. Chutintrasri, B., & Noomhorm, A. (2007). Color degradation kinetics of pineapple puree during thermal processing. LWT-Food Science and Technology, 40(2), 300–306. Cotton, T., Loach, P., Katz, J., & Ballschmiter, K. (1978). Studies of chlorophyll-chlorophyll and chlorophyll-ligand interactions by visible absorption and infrared spectroscopy at low temperatures. Photochemistry and Photobiology, 27(6), 735–749. 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), 1–12. Gu, F., Kim, J. M., Hayat, K., Xia, S., Feng, B., & Zhang, X. (2009). Characteristics and antioxidant activity of ultrafiltrated Maillard reaction products from a casein–glucose model system. Food Chemistry, 117(1), 48–54. 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, 10–15. Han, Z., Zeng, X. A., Fu, N., Yu, S. J., Chen, X. D., & Kennedy, J. F. (2012). Effects of pulsed electric field treatments on some properties of tapioca starch. Carbohydrate Polymers, 89(4), 1012–1017. Hong, F., Wang, L., Meng, X., Wei, Z., & Zhao, G. (2002). The effect of cerium (III) on the chlorophyll formation in spinach. Biological Trace Element Research, 89(3), 263–276. Hong, J., Zeng, X. A., Buckow, R., Han, Z., & Wang, M. S. (2016). Nanostructure, morphology and functionality of cassava starch after pulsed electric fields assisted acetylation. Food Hydrocolloids, 54, 139–150. 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 hydrostatic pressure and high temperature short time. Innovative Food Science & Emerging Technologies, 18, 74–82. Ilić, Z. S., Milenković, L., Šunić, L., & Fallik, E. (2015). Effect of coloured shade-nets on plant leaf parameters and tomato fruit quality. Journal of the Science of Food and Agriculture, 95(13), 2660–2667. Kaiser, T. E., Wang, H., Stepanenko, V., & Würthner, F. (2007). Supramolecular construction of fluorescent J-aggregates based on hydrogen-bonded perylene dyes. Angewandte Chemie International Edition, 46(29), 5541–5544. Kephart, J. C. (1955). Chlorophyll derivatives—Their chemistry? Commercial preparation and uses. Economic Botany, 9(1), 3–38. Khachik, F., Beecher, G. R., & Whittaker, N. F. (1986). Separation, identification, and quantification of the major carotenoid and chlorophyll constituents in extracts of several green vegetables by liquid chromatography. Journal of Agricultural and Food Chemistry, 34(4), 603–616. Koca, N., Karadeniz, F., & Burdurlu, H. S. (2007). Effect of pH on chlorophyll degradation and colour loss in blanched green peas. Food Chemistry, 100(2), 609–615. Krasnovsky, A., & Bystrova, M. (1980). Self-assembly of chlorophyll aggregated structures. Biosystems, 12(3), 181–194. Lanfer-Marquez, U. M., Barros, R. M., & Sinnecker, P. (2005). Antioxidant activity of chlorophylls and their derivatives. Food Research International, 38(8), 885–891. Luengo, E., Condón-Abanto, S., Álvarez, I., & Raso, J. (2014). Effect of pulsed electric field treatments on permeabilization and extraction of pigments from Chlorella vulgaris. The Journal of Membrane Biology, 247(12), 1269–1277. Luo, W. B., Han, Z., Zeng, X. A., Yu, S. J., & Kennedy, J. F. (2010). Study on the degradation of chitosan by pulsed electric fields treatment. Innovative Food Science & Emerging Technologies, 11(4), 587–591. 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), 1068–1072. Miret, S., Tascioglu, S., van der Burg, M., Frenken, L., & Klaffke, W. (2009). In vitro bioavailability of iron from the heme analogue sodium iron chlorophyllin. Journal of Agricultural and Food Chemistry, 58(2), 1327–1332. Odriozola-Serrano, I., Aguilo-Aguayo, I., Soliva-Fortuny, R., & Martin-Belloso, O. (2013). Pulsed electric fields processing effects on quality and health-related constituents of plant-based foods. Trends in Food Science & Technology, 29(2), 98–107. Özkan, G., & Bilek, S. E. (2015). Enzyme-assisted extraction of stabilized chlorophyll from spinach. Food Chemistry, 176, 152–157. Parniakov, O., Barba, F. J., Grimi, N., Lebovka, N., & Vorobiev, E. (2014). Impact of pulsed electric fields and high voltage electrical discharges on extraction of highadded value compounds from papaya peels. Food Research International, 65, 337–343. Pina-Pérez, M. C., Rodrigo, D., & Martínez, A. (2016). Nonthermal inactivation of Cronobacter sakazakii in infant formula milk: A review. Critical Reviews in Food Science and Nutrition, 56(10), 1620–1629. Pott, I., Marx, M., Neidhart, S., Mühlbauer, W., & Carle, R. (2003). Quantitative determination of β-carotene stereoisomers in fresh, dried, and solar-dried mangoes (Mangifera indica L.). Journal of Agricultural and Food Chemistry, 51(16), 4527–4531. 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), 361–371. Singh, A., Ahmad, S., & Ahmad, A. (2015). Green extraction methods and environmental applications of carotenoids-a review. RSC Advances, 5(77), 62358–62393. Terefe, N. S., Buckow, R., & Versteeg, C. (2015). Quality-related enzymes in plant-based products: Effects of novel food processing technologies part 2: Pulsed electric field processing. Critical Reviews in Food Science and Nutrition, 55(1), 1–15. 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),
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 effect, 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 influencing 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 effect of thermal degradation of the pigments was inhibited, and the stability of the pigments was increased. In addition, other workers have proved that PEF treatment can affect weakly covalent bonds, non-covalent bonds (such as hydrogen bonds, disulfide bonds and hydrophobic bonds) and weak chemical forces (such as van der Waals bonding, hydrophobic and electrostatic interactions) (Terefe, Buckow, & Versteeg, 2015). 4. Conclusions The effect 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 different electric field 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 degradation of pigments, especially for the relative concentration of chlorophyll a, b and carotenoids under different treated temperatures (20, 35 and 45 °C). Based on the UV–Vis and FT-IR spectrum analysis, the PEF treatment influenced 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 structures thus increasing the stability of chlorophyll. PEF treatment also affected 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 different electric field intensities of PEF treatments was increased, especially at low temperature. Therefore, it can be concluded that the PEF acts as an efficient food processing method, providing the potential for enhancement of the biological activities and antioxidant activity of natural compounds. Acknowledgments The authors gratefully acknowledge the financial 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., Martínez-Navarrete, N., & Bohn, T. (2015). Chlorophylls and carotenoids of kiwifruit puree are affected similarly or less by microwave than by conventional heat processing and storage. Food Chemistry, 187, 254–262. Bermúdez-Aguirre, D., & Barbosa-Cánovas, 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), 383–392. Buckow, R., Ng, S., & Toepfl, S. (2013). Pulsed electric field 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), 455–467. Budi, A., Legge, F. S., Treutlein, H., & Yarovsky, I. (2005). Electric field effects on insulin chain-B conformation. The Journal of Physical Chemistry B, 109(47), 22641–22648. 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.
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radical species generated under pulsed electric fields processing. LWT-Food Science and Technology, 44(4), 1233–1235. Zhang, Z. H., Zeng, X. A., Brennan, C. S., Brennan, M., Han, Z., & Xiong, X. Y. (2015). Effects of pulsed electric fields (PEF) on vitamin C and its antioxidant properties. International Journal of Molecular Sciences, 16(10), 24159–24173. Zhang, Z. H., Han, Z., Zeng, X. A., & Wang, M. S. (2017). The preparation of Fe-glycine complexes by a novel method (pulsed electric fields). Food chemistry, 219, 468–476. Zhao, L., Wang, S., Liu, F., Dong, P., Huang, W., Xiong, L., & Liao, X. (2013). Comparing the effects of high hydrostatic pressure and thermal pasteurization combined with nisin on the quality of cucumber juice drinks. Innovative Food Science & Emerging Technologies, 17, 27–36. Zhao, W., Tang, Y., Lu, L., Chen, X., & Li, C. (2014). Review: Pulsed electric fields processing of protein-based foods. Food and Bioprocess Technology, 7(1), 114–125. Zheng, Y., Shi, J., Pan, Z., Cheng, Y., Zhang, Y., & Li, N. (2014). Effect of heat treatment, pH, sugar concentration, and metal ion addition on green color retention in homogenized puree of Thompson seedless grape. LWT-Food Science and Technology, 55(2), 595–603.
98366–98376. Wang, L. H., Wang, M. S., Zeng, X. A., & Liu, Z. W. (2016). Temperature-mediated variations in cellular membrane fatty acid composition of Staphylococcus aureus in resistance to pulsed electric fields. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1858(8), 1791–1800. 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 effect with a pulsed electric field. Journal of agricultural and food chemistry, 64(32), 6355–6363. 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), 448–453. Yin, Y., Han, Y., & Liu, J. (2007). A novel protecting method for visual green color in spinach puree treated by high intensity pulsed electric fields. Journal of Food Engineering, 79(4), 1256–1260. Zeng, X. A., Han, Z., & Zi, Z. H. (2010). Effects of pulsed electric field treatments on quality of peanut oil. Food Control, 21(5), 611–614. Zhang, S., Yang, R., Zhao, W., Liang, Q., & Zhang, Z. (2011). The first ESR observation of
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Innovative Food Science and Emerging Technologies journal homepage: www.elsevier.com/locate/ifset
Effect of pulsed electric fields (PEFs) on the pigments extracted from spinach (Spinacia oleracea L.)
MARK
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: Pulsed electric fields Pigments Physicochemical properties Antioxidant activity
In order to investigate the effect of PEF on the pigments of spinach, pigments were extracted and were treated by different electric field strength at different temperatures (20, 35 and 45 °C). Results showed that with the increasing temperature, the degradation of chlorophyll content was appeared for pigments without PEF treatment. However, the concentrations of chlorophyll a, b and carotenoids were increased by the PEF treatment, especially for the higher electric field strength. Moreover, the structure of spinach pigments treated by PEF also was determined by UV–Vis, FT-IR and XRD. From the structural information, it was indicated that the effect 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 modified. In addition, the PEF treatment also significantly increased the antioxidation activity of pigments over the series of temperatures. Industrial relevance: This study provide evidences that the PEF treatment can inhibit the degradation of chlorophyll 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 oligomers chlorophyll. Therefore, the PEF treatment is an efficient food processing method, especially foods rich in pigment.
1. Introduction 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 specific health benefits, such as antioxidant activity, hematopoietic activity and anti-inflammatory properties (Ferruzzi & Blakeslee, 2007; Lanfer-Marquez, Barros, & Sinnecker, 2005; Miret, Tascioglu, van der Burg, Frenken, & Klaffke, 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 provitamin 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
⁎
Corresponding author. E-mail address: [email protected] (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.
of several degenerative processes, including cancers, cardiovascular 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 vegetables as well as their processed products. In addition, phytochemicals 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. (Özkan & Bilek, 2015; Pott, Marx, Neidhart, Mühlbauer, & Carle, 2003; Zhao et al., 2013). Therefore, it is useful to explore innovative and non-thermal technologies in processing to sterilize the food while also protect the favorable bioactivity of pigments. Pulsed electric fields (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, & Toepfl, 2013; Pina-Pérez, Rodrigo, & Martínez, 2016). PEF treatments typically involve the application of pulses of high electric field intensity (above 20 kV/cm) to liquid foods placed between two electrodes. PEF treatments are conducted at ambient or moderately high temperatures (generally < 45 °C)
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Afterwards, the spinach puree was centrifuged at 5000g for 10 min at 4 °C (TDL-5-A, Shanghai Anting Scientific Instrument Factory, China). The precipitation was collected and was extracted with 400 mL of absolute 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 filled in a 1 L volumetric flask and stored in a refrigerator at 4 °C until further PEF treatment.
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 affects the flavor and quality of the foods (Odriozola-Serrano, Aguilo-Aguayo, SolivaFortuny, & Martin-Belloso, 2013; Zeng, Han, & Zi, 2010). A number of studies have shown the effectiveness of PEF treatment in better retaining nutritious compounds, such as ascorbic acid, carotenoids and phenols, in comparison with heat treatments of different food stuffs, such as tomato and strawberry juices (Buckow et al., 2013; OdriozolaSerrano et al., 2013). Moreover, many investigations have proved that PEF can enhance the extraction of intracellular compounds, such as pigments, flavors and polysaccharides from the plant cells (Chemat et al., 2017). However, the effect of PEF treatment on the structure and physicochemical properties of compounds, especially for plant pigments rarely reported until now. Therefore, the aim of this study was to investigate the effect of PEF on the pigments of spinach including the physicochemical properties and the antioxidant activity. Furthermore, the mechanism of PEF affecting the chlorophyll and carotenoids also was explored based on the changes in the structure.
2.4. PEF treatment of pigment solution Before performing PEF treatments, bubbles in the extraction solutions were removed by a vacuum pump (SHZ-D III, Gongyi Yuhua Instrument Co., Ltd., Gongyi, China). The pH value and the conductivity 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 specific electric field intensity (0, 3.3, 6.7, 13.3, 20.0 and 26.7 kV/cm) and different temperatures (20, 35 and 45 °C). The treatment temperature was controlled using a thermostatic device (HH-4, KEJIE instrument, Jintan, China). Reaction conditions are listed in Table 1. In this study, the flow rate of the extraction solution was set at 100 mL/min. The effective 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 effective treatment time and energy input of different PEF treatments are shown in Table 1.
2. Material and methods 2.1. Materials Fresh spinach (Spinacia oleracea L.) was purchased at the local supermarket (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-picrylhydrazyl (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).
f×V S
(1)
t = Np × Wp × n
(2)
Q = E2 × t × δ
(3)
Np =
2.2. PEF treatment system The continuous PEF system was designed by the PEF team of South China University of Technology (Guangzhou, China). The structure of the device has been reported in our previous studies (Wang et al., 2016; Wang, Wang, Zeng, & Liu, 2016). The main parameters of this device were a unipolar square wave, the maximum voltage of 15 kV, maximum current of 1.77 A, frequency of 1 kHz, and pulse width of 20 μs, respectively. The electric field strength of PEF system was monitored by a two-channel digital storage oscilloscope (DST1102B, Tekway Technologies Co., Ltd., Nanjing, China). Two parallel titanium-based alloy electrodes were inserted in the treatment chamber that was made from Teflon. The distance between two the electrodes was 0.3 cm. The volume of the treatment chamber was 0.02 mL. The sample solution was transported through the silicone tube (inner diameter of 6 mm), and was introduced into the treatment chamber by a constant flow pump (323 E/D, Watson Marlow, NC, USA). The flow rate was controlled by a flow meter (LZB-4, Changzhou Shuangfa Thermal Instrument Factory, Changzhou, China). The inlet and outlet temperatures of sample solution were monitored by two K-type thermocouples (Pico technology Inc., St Neots, United Kingdom). The temperature of the sample was controlled with a cooling circulator (DLSK 3/10, Ketai Laboratory Equipment Co., Ltd., Zhengzhou, China).
where Np represents the pulse number, f represents the pulse repetition rates, V represents the volume of the chamber (mL), S represents the flow rate (mL/s), t represents the effective treatment time (μs), Wp represents the pulse width (μs), n represents the sample cycles, Q represents the energy input (J/m3) and δ represents the conductivity of the sample (S/m). 2.5. Content of pigments The concentration of pigments of treated samples was determined Table 1 The effective treatment time and energy input of different PEF treatment. Treatment method
Treatment time (min)
Effective time (ms)
Input energy (kJ/m3)
20 °C
60 60 60 60 60 60 60 60 60 60 60 60 60 60 60
0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32 0.32
2.43 9.65 38.72 87.04 154.66 2.91 11.57 46.41 104.32 185.37 1.49 5.93 23.77 53.44 94.96
35 °C
2.3. Extraction of pigments from spinach leaves In order to prevent pigments in spinach degradation, such as chlorophylls and carotenoids, each extraction step was performed under dim light and low temperature (4 ± 1 °C). 200 g of cleaned spinach leaves were milled by a mixer grinder (JYL-C022E, Joyoung, Hangzhou, China) for 15 s with deionized water at a ratio of 1:2 (w/v) in order to remove the water-soluble compounds of spinach leaves.
45 °C
27
3.3 kV/cm 6.7 kV/cm 13.3 kV/cm 20.0 kV/cm 26.7 kV/cm 3.3 kV/cm 6.7 kV/cm 13.3 kV/cm 20.0 kV/cm 26.7 kV/cm 3.3 kV/cm 6.7 kV/cm 13.3 kV/cm 20.0 kV/cm 26.7 kV/cm
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Table 2 The concentration of chlorophyll a, b and carotenoids from spinach following different PEF treatments (μg/mL). Sample 20 °C
35 °C
45 °C
0 kV/cm 3.3 kV/cm 6.7 kV/cm 13.3 kV/cm 20.0 kV/cm 26.7 kV/cm 0 kV/cm 3.3 kV/cm 6.7 kV/cm 13.3 kV/cm 20.0 kV/cm 26.7 kV/cm 0 kV/cm 3.3 kV/cm 6.7 kV/cm 13.3 kV/cm 20.0 kV/cm 26.7 kV/cm
Chlorophyll a
Chlorophyll b
Chlorophyll a + b
Carotenoids
11.41 12.37 12.74 13.41 13.63 14.41 11.20 11.92 12.64 13.24 13.63 14.62 10.93 11.89 11.94 12.20 12.55 13.21
4.99 5.31 5.45 5.66 5.74 6.04 4.87 5.16 5.37 5.67 5.75 6.13 4.79 5.25 5.45 5.08 5.42 5.73
16.40 17.68 18.18 19.07 19.37 20.44 16.07 17.08 18.02 18.92 19.38 20.75 15.72 17.14 17.39 17.28 17.96 18.94
2.60 2.84 2.92 3.13 3.25 3.68 2.41 2.71 2.94 3.25 3.19 3.52 2.60 2.64 2.57 2.82 2.91 2.98
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.02a 0.01b 0.02c 0.04d 0.03e 0.01f 0.04a 0.02b 0.03c 0.04d 0.03e 0.02f 0.05a 0.02b 0.03b 0.02c 0.03d 0.06e
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.01a 0.03b 0.01c 0.02d 0.01e 0.02f 0.02a 0.01b 0.02c 0.02d 0.01e 0.02f 0.04a 0.02b 0.02c 0.04d 0.04c 0.03e
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.03a 0.04b 0.03c 0.06d 0.04e 0.03f 0.06a 0.03b 0.05c 0.06d 0.04e 0.04f 0.09a 0.04b 0.05c 0.06bc 0.07d 0.09e
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.02a 0.04b 0.03c 0.01d 0.02e 0.04f 0.01a 0.02b 0.01c 0.02d 0.04e 0.02f 0.03a 0.02a 0.01a 0.03b 0.04c 0.05c
Values with different letters in the same column (a–f) are significantly different according to Tukey test (p < 0.05).
Karlsruhe, Germany), using a Cu-kα radiation source (0.154 nm wavelength) at 40 kV and 40 mA. The XRD data were collected in the 2θ 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.
by the UV–Vis spectrophotometer described by previous studies (Luengo, Condón-Abanto, Álvarez, & Raso, 2014; Wintermans & De Mots, 1965). Briefly, 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 calculated by Eqs. (4) to (7).
Chla = 13.70 × A 665 − 5.76 × A 649
(4)
Chlb = 25.8 × A 649 − 7.60 × A 665
(5)
Chla + b = 6.10 × A 665 + 20.04 × A 649
(6)
Cas =
1000 × A 470 − 2.13 × Chl a − 97.64 × Chlb 209
2.9. DPPH radical scavenging activity Antioxidant activity of pigment solutions was determined by a stable DPPH radical using the method described by Gu et al. (2009) with some modifications. A 50 mg sample was added to 5 mL ethanol and was mixed vigorously until completely dissolved. 1 mL of the 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 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).
(7)
where Chla represents the concentration of chlorophyll a (μg/mL), Chlb represents the concentration of chlorophyll b (μg/mL), Chla + b represent the total concentration of chlorophyll a and chlorophyll b (μg/ mL), Cas represent the concentration of carotenoids (μg/mL) and A665, A649 and A470 represent the absorbance at 665, 649 and 470 nm, respectively.
A − A2 ⎞ RA (%) = ⎛1 − 1 × 100 A3 ⎠ ⎝ ⎜
2.6. UV–Vis spectra of pigments
⎟
(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.
UV–Vis 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.
2.10. 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 significance testing was performed by Tukey's test and the differences were statistically significant at p < 0.05.
2.7. Fourier transform infrared (FT-IR) spectra FT-IR measurements of samples were carried out with a spectrometer (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 400–4000 cm− 1 with a resolution of 4 cm− 1, with each spectrum consisting of 32 scans.
3. Results and discussion 3.1. The content of pigments analysis
2.8. X-ray diffraction (XRD) pattern The concentration of chlorophyll a, b and carotenoids from spinach following different PEF treatments is shown in Table 2. It can be seen that with the electric field strength of PEF was gradually increased from 0 to 26.7 kV/cm at 20 °C, the concentration of chlorophyll a was
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 diffractometer (D8 Advance, Bruker, 28
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significantly increased from 11.41 to 12.37, 12.74, 13.41, 13.63 and 14.41 μg/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 μg/mL (p < 0.05), respectively. The concentration of carotenoids was 3.68 μg/mL after 26.7 kV/cm under 20 °C, which was significantly increased by 41.5% compared to sample without PEF treatment sample of 2.60 μg/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 μg/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 field strength, and the concentration of chlorophyll a, b and carotenoids was reached to 14.62, 6.13 and 3.52 μg/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 μg/mL under the 3.3 kV/cm, and with the increase in the electric field 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 spinach leaves. However, during the various PEF treatment could inhibit the degradation process of chlorophyll a, b and carotenoids extracted from spinach leaves. This finding was consistent with previous study, which reported that the degradation of chlorophyll a and b followed a first-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) reported that increasing the electric field 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 phenomenon 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 selfaggregation 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 UV–Vis absorption spectra are at 663, 648 and 470 nm, respectively (Ilić, Milenković, Šunić, & Fallik, 2015). In the previous studies reported that the blue or red shift and the alterations in the peak shape in the UV–Vis spectra could indicate the changes of the chemical structure (Kaiser, Wang, Stepanenko, & Würthner, 2007). The UV–Vis spectral of pigments by the different PEF treatments at different temperature is shown in Fig. 1. It can be seen that the absorption of peaks did not appeared obvious red or blue shift, but the shape of peak (664 nm) with PEF treatment became sharper than untreated sample. This phenomenon indicated that PEF treatment could influence the nonconjugate structure of pigments, such as hydrogen bond and electrostatic forces, rather than the conjugate structure or carbon chain of pigments. This finding was consistent with previous studies, which reported that the PEF treatment could affect the secondary and tertiary structure of the enzymes and proteins through affecting some of weak chemical bond and intermolecular force, such as disulfide bond, hydrogen bond and hydrophobic force, et al. (Budi, Legge, Treutlein, & Yarovsky, 2005; Zhao, Tang, Lu, Chen, & Li, 2014). Moreover, from the Fig. 1, it also can be observed that with the increasing of
Fig. 1. The UV–Vis spectral of pigments by the different PEF treatments (A.20 °C, B. 35 °C, C. 45 °C).
the electric field strength, the absorption intensity of UV–Vis was gradually strengthened at 20, 35 and 45 °C. This finding indicated that the concentration of chlorophyll a, b and carotenoids was increased. This viewpoint was consistent of the results of Section 3.1 that the concentration of pigments without PEF treatment sample was decreased with an increase in temperature. Moreover, the concentrations of chlorophyll a, chlorophyll b and carotenoids by PEF treatment were higher than the sample without PEF treatment at 20, 35 and 45 °C. This finding was consistent with the previous studies, which have showed that the thermal treatments such as blanching, steaming and microwave cooking could destroy the molecules of chlorophyll a and b, thereby decreased the concentration of pigments of the food (Benlloch-Tinoco et al., 2015; Zheng et al., 2014). Meanwhile, the previous study has 29
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Fig. 3. XRD pattern of the pigments by different of PEF treatments (A. 20 °C, B. 35 °C, 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/ cm).
Fig. 2. FT-IR spectrum of the pigments by different of PEF treatments (A. 20 °C, B. 35 °C, 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/cm).
Mukherjee, & Das, 2009). The band at around 1734 cm− 1 is ascribed to 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 represents 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 field strength was increased at 20 °C (Fig. 3(A)), the characteristic absorption peaks of the chlorophyll was decreased, such as
reported that PEF treatment can increase the content of health-promoting 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 effect. 3.2.2. The functional groups characterization Fig. 2 shows the FT-IR spectrum of the pigments following various PEF treatments at different temperature. The information of IR characteristic absorption bands was based on the previous studies (Hong, Wang, Meng, Wei, & Zhao, 2002; Manna, Basu, Mitra, 30
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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 effect 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 field 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 enhanced, which showed that the out-of-plane bending vibration of carbon hydrogen bonds (–CH) was increased. Moreover, the new absorbance peak was appeared in 1317 cm− 1, which was represented the stretching vibration of carbon oxygen bonds (–CO). These phenomena indicated that the effect 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 hydrogen 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 geranylgeranyl diphosphate molecules, which contain some unsaturated bonds (Schoefs, 2002). Therefore, the π electrons of the double bonds were more susceptible to be influenced by heating and other treatments, 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 (Bermúdez-Aguirre & Barbosa-Cánovas, 2012). When the electric field 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).
Fig. 4. The DPPH radical scavenging activity of pigments solutions by different PEF treatments. Values with different letters in the same column (a–e) are significantly different 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). Furthermore, Han et al. (2012) has reported that the PEF treatment was capable of destroying the crystalline region of starches. 3.4. Antioxidant activity The DPPH radical scavenging activity of pigments following various PEF treatments at different 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 increased 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 significantly higher than the untreated sample under different temperatures (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 ascribed 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 field 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 μg/mL, respectively, compared with the untreated sample of 19.0 μg/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 μg/mL, respectively, compared with the untreated sample of 18.48 and 18.32 μg/mL. These results were in agreement with the findings of El-Sayed, Hussin, Mahmoud, and AlFredan (2013) who found that DPPH scavenging and other biological activities of Conyza extracts closely were related to its chlorophyll content. Furthermore, the concentration of total pigments at 35
3.3. The property of crystallization The XRD patterns of the pigments following various PEF treatments at different treatment temperatures are shown in Fig. 3.While there are many diffraction peaks, the four largest appeared at 2θ values of around 27.3, 29.8, 32.7 and 39.3 in Fig. 3A(a). With the increase of PEF intensity, a new diffraction peak appeared at around 2θ = 19.0. Meanwhile, the intensity of 27.3, 28.3, 29.8 and 32.7 (2θ) significantly decreased while 43.1 and 44.8 (2θ) disappeared. On the other hand, when the treatment temperature was set at 35 °C, the intensity of some peaks (2θ = 19.0, 23.9, 27.4, 41.1) increased, the intensity of other peaks (2θ = 17.6, 18 and 36.4) almost decreased and some new diffraction peaks (2θ = 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 diffraction peaks vanished in both the PEF-treated and no-PEF treated samples. These results indicated that the PEF treatment can modify the crystal structure of the compound. The crystal size of the compound became larger under the 31
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Fig. 5. The proposed mechanism of effect of PEF treatment on pigments (A: chlorophyll, B: β-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.
and 45 °C without PEF treatment significantly was decreased by 2.8% and 3.7%, respectively, compared with the sample at 20 °C (19.0 μg/ mL). The result suggested that heat treatment promoted the degradation of pigments (Koca et al., 2007). In addition, the previous study indicates that the different pigment ingredients have different antioxidant capacities and that PEF treatment has different influences 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 modification of the structure of vitamin C was induced by PEF treatment. From the above discussion, it appears that the PEF treatment can
3.5. The mechanism of PEF influence in pigments The determination of pigments mainly includes chlorophyll, chlorophyll derivatives and carotenes from spinach, which had two kinds of special structure, pyrrole ring and conjugated double bond system. The possible mechanism for the effect 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 nitrogen atom derived from amino group. Then, the magnesium ion can combine with the carbonyl group (C]O) of the surrounding 32
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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 effect, 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 influencing 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 effect of thermal degradation of the pigments was inhibited, and the stability of the pigments was increased. In addition, other workers have proved that PEF treatment can affect weakly covalent bonds, non-covalent bonds (such as hydrogen bonds, disulfide bonds and hydrophobic bonds) and weak chemical forces (such as van der Waals bonding, hydrophobic and electrostatic interactions) (Terefe, Buckow, & Versteeg, 2015). 4. Conclusions The effect 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 different electric field 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 degradation of pigments, especially for the relative concentration of chlorophyll a, b and carotenoids under different treated temperatures (20, 35 and 45 °C). Based on the UV–Vis and FT-IR spectrum analysis, the PEF treatment influenced 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 structures thus increasing the stability of chlorophyll. PEF treatment also affected 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 different electric field intensities of PEF treatments was increased, especially at low temperature. Therefore, it can be concluded that the PEF acts as an efficient food processing method, providing the potential for enhancement of the biological activities and antioxidant activity of natural compounds. Acknowledgments The authors gratefully acknowledge the financial 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., Martínez-Navarrete, N., & Bohn, T. (2015). Chlorophylls and carotenoids of kiwifruit puree are affected similarly or less by microwave than by conventional heat processing and storage. Food Chemistry, 187, 254–262. Bermúdez-Aguirre, D., & Barbosa-Cánovas, 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), 383–392. Buckow, R., Ng, S., & Toepfl, S. (2013). Pulsed electric field 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), 455–467. Budi, A., Legge, F. S., Treutlein, H., & Yarovsky, I. (2005). Electric field effects on insulin chain-B conformation. The Journal of Physical Chemistry B, 109(47), 22641–22648. 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.
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