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Effects of radio frequency assisted blanching on polyphenol oxidase, weight loss, texture, color and microstructure of potato
Food Chemistry 248 (2018) 173–182
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Effects of radio frequency assisted blanching on polyphenol oxidase, weight loss, texture, color and microstructure of potato
T
⁎
Zhenna Zhang, Juan Wang, Xueying Zhang, Qingli Shi, Le Xin, Hongfei Fu, Yunyang Wang College of Food Science and Engineering, Northwest A&F University, Yangling, Shaanxi 712100, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Radio frequency heating Blanching Potato Polyphenol oxidase Physicochemical properties
This paper is focused on the effects of radio frequency (RF) heating on the relative activity of polyphenol oxidase (PPO), weight loss, texture, color, and microstructure of potatoes. The results showed that pure mushroom PPO was almost completely inactivated at 80 °C by RF heating. The relative activity of potato PPO reduced to less than 10% with increasing temperature (25–85 °C). Enzyme extract showed the lowest PPO relative activity at 85 °C after RF treatment, followed by the potato cuboids and mashed potato, about 0.19 ± 0.017%, 3.24 ± 0.19%, and 3.54 ± 0.04%, respectively. Circular dichroism analysis indicated that RF heating changed the secondary structure of PPO, as α-helix content decreased. Both electrode gap and temperature had significant effect (P < .05) on weight loss, color, and texture of the potato cuboids. Microstructure analysis showed the changes of potato cell and starch during RF heating.
1. Introduction Potatoes are widely cultivated all over the world, especially in many developing countries. It is commonly processed into potato flours, purees, and chips, which are favored by consumers. However, enzymatic activity in the potato can result in a series of deterioration reactions including undesirable color and texture, off-flavors, bad odor, and loss of nutrients. Polyphenol oxidase (PPO) is a copper-containing enzyme contributing to enzymatic browning of vegetables and fruits. PPO can be divided into two categories: laccase (EC 1.10.3.2) and catechol oxidase (EC 1.10.3.1). Catechol oxidase is referred to as phenolase, polyphenol oxidase, tyrosinase, catecholase or cresolase, which is clearly distinguished from laccase. Catechol oxidase can catalyze two distinct reactions: (1) the insertion of oxygen in a position ortho to an existing hydroxyl group, usually followed by oxidation of the diphenol to the corresponding quinone, which is referred to as cresolase activity; (2) the oxidation of o-diphenol with hydrogen abstraction, which is referred to as catecholase activity (Mayer & Harel, 1979). Quinones subsequently undergo nonenzymatic reactions resulting in the formation of dark-colored melanins (Duangmal & Apenten, 1999; Kuijpers et al., 2014; Tribst, Júnior, Oliveira, & Cristianini, 2016). Therefore, the inhibition of enzyme activity plays an important role in the food industry. Blanching is a crucial step that is carried out prior to food processes such as frying, drying, freezing, and storing because it can inactivate enzymes, destroy microorganisms, and eliminate air in the potato (Wang et al., 2017).
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The methods of inactivation of PPO have been widely studied. The activity of PPO can be inhibited by using chemical reagents, such as sulphites, ascorbic acid, and amino acids, which have been applied in potato (Ali, El-Gizawy, El-Bassiouny, & Saleh, 2016), lettuce (Altunkaya & Gokmen, 2008), and blueberry (Siddiq & Dolan, 2017). However, the residual chemical agents pose a threat to human health. It is necessary to develop novel technologies and to reduce the use of chemical food additives. Thermal treatment is the most common and effective technique to control enzymatic browning in the food industry. A number of studies have been explored to overcome enzymatic browning to improve the quality of products by thermal processes. The methods include hot water, hot and boiling solutions containing acids and/or salts, steam heating, microwave, infrared, ohmic heating, and radio frequency heating (Castro et al., 2008; Icier, Yildiz, & Baysal, 2006; Manzocco, Anese, & Nicoli, 2008; Mukherjee & Chattopadhyay, 2007; Sotome et al., 2009; Vishwanathan, Giwari, & Hebbar, 2013; Wang et al., 2017). Radio frequency (RF) heating utilizes electromagnetic energy at a frequency range of 3 kHz to 300 MHz and initiates volumetric heating due to frictional interaction between molecules. The advantages of RF heating include higher penetration depth and higher heating rate. Therefore, it can save processing time and improve food quality. RF heating has been studied on drying nuts, thawing meats, post-baking cookies, pasteurization, and controlling insects (Farag, Lyng, Morgan, & Cronin, 2011; Palazoglu, Coskun, Kocadagli, & Gokmen, 2012; Wang et al., 2014; Zhou & Wang, 2016). However, the study about the effect
Corresponding author. E-mail address: [email protected] (Y. Wang).
https://doi.org/10.1016/j.foodchem.2017.12.065 Received 13 August 2017; Received in revised form 15 December 2017; Accepted 18 December 2017 Available online 18 December 2017 0308-8146/ © 2017 Elsevier Ltd. All rights reserved.
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a
(1)
(2)
(3)
(4)
b
Fig. 1. Schematic of the sample in the centrifuge tube (a) and schematic of the potato sample placed in the RF heating system (b). (1) The position in the cuboid for measuring temperature and analyzing texture, (2) potato cuboid, (3) potato puree, and (4) enzyme extract (all dimensions are in mm).
(Kikani & Singh, 2015; Zhong et al., 2007), to the best of our knowledge, there is no published data so far regarding the conformation changes of enzyme during RF heating. The objectives of this study were: (1) to investigate the effects of RF heating on model enzyme (mushroom PPO) inactivation and the changes of enzyme secondary structure, (2) to analyze the effects of RF heating on the inhibition of PPO of potato, (3) to evaluate the changes of weight loss, texture, color, and microstructure of potato after RF heating.
of RF heating on the activity of enzymes in vegetables is still limited so far. It has been reported that RF heating could efficiently inactivated PPO and lipoxygenase (LOX), particularly PPO was more sensitive to an increase in the RF electric field (Manzocco et al., 2008). It showed that RF heating retained a higher sweetness and produced highly appreciated vegetable derivatives than the conventional blanching. Moreover, RF treatment can not only saves time, water, and energy, but also reduces the cost of waste treatment. Orsat, Gariépy, Raghavan, and Lyew (2001) compared the quality of vacuum-packaged carrot sticks treated with RF heating, chlorinated water dipping or hot water dipping, showing that the color and taste were better, and the vacuum of packages were greater of samples in RF treatment than that in chlorinated water or hot water. Lopez and Baganis (1971) also reported that peroxidase, polyphenolase, pectinesterase, catalase, and α-amylase were partially or totally inactivated by RF heating at 70 °C. These results indicated that RF heating has the potential in blanching fruits and vegetables. The main purpose of blanching is to inactivate enzymes which are related to the browning of food. Color, weight loss, and texture were used as indicator to evaluate the food quality during blanching. However, the changes of texture are attributed to the altering of microstructure during blanching (Araya et al., 2007). Therefore, the microstructure of food should be investigated to provide sufficient knowledge of blanching. In addition should be very interesting to add new information on the effect of RF heating to the secondary structure of proteins. The secondary structure of the protein is a stable conformation of the local peptides in the peptide chain of the protein. Enzymes are proteins which have some active sites. Heat, ultrasound, pulsed electric field, etc., can disturb the delicate balance of covalent and non-covalent interactions that maintain the native structure, which can result in a loss of enzyme activity (Baltacıoğlu, Bayındırlı, & Severcan, 2017). Nevertheless, the reasons of enzyme inactivation whether were due to changes in the active site or the changes of secondary structure need to be studied. Although some researchers have studied the changes in secondary structure of peroxidase (POD) and PPO treated by PEF
2. Materials and methods 2.1. Materials and preparation of samples The potato (Solanum tuberosum L.) numbering 15–7 was provided by the Potato Molecular Genetics and Breeding Lab of the College of Agriculture at Northwest A & F University in China and stored in a refrigerator at 4 °C. Before the experiments, the samples were taken out and equilibrated to room temperature (25 °C) overnight. Potatoes were washed, hand-peeled, and cut into cuboid with the dimension (length × width × height) of 30 × 10 × 60 mm3 having an average weight of 20 g. The mashed potato was obtained by crushing 20 g potato tubers using an electrical hand blender (Multiquick 7 MQ705, 4199, De’Longhi Braun Household Gmbh, Germany) at room temperature. The method to prepare enzyme extract was described in the section 2.3. Mushroom PPO (98% purity) as model enzyme (E.C. 1.14.18.1), catechol (≥99% purity) were purchased from Sigma (Shanghai, China) and Aladdin (Shanghai, China), respectively. Other chemicals used in this study were of analytical grade. 2.2. RF heating system RF assisted blanching treatments were performed by a 6 kW, 27.12 MHz pilot-scale free running oscillator RF system (GJG-2.1-10A174
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0.1 mg/mL. The secondary structures contents of PPO were calculated by CD spectra Deconvolution Software (Institut für Biotechnologie, Martin-Luther-Universität Halle-Witttenberg, Germany).
JY; Hebei Huashijiyuan High Frequency Equipment Co., Ltd, Hebei, China). The system included a generator and a RF applicator with two parallel rectangular electrodes. A high voltage alternating electric field was applied to a medium sandwiched between two parallel electrodes. The schematic of the RF heating system was shown in Fig. 1b. All of the experiments were repeated three times.
2.6. RF heating treatment of potato samples To evaluate the effect of RF heating on the inactivation of PPO and the quality of potato products, three types of sample matrices including potato cuboids, mashed potato, and enzyme extract were employed in this study. One potato cuboid was put in a centrifuge tube of 100 mL with a height of 72 mm. Twenty grams of mashed potato and or enzyme extract was filled in a centrifuge cube of 50 mL with a height of 50 mm. The details of sample loading were illustrated in Fig. 1a. The centrifuge tube containing potato sample was surrounded by the expanded polyethylene (EPE) foam and placed at the center in the horizontal direction and middle in the vertical direction between the top and bottom electrodes as shown in Fig. 1b (Zhang et al., 2017). The changes of sample temperature during RF heating were monitored by a fluorescence-based optical fiber temperature measurement system (HQFTS-D1F00; Xi’an Heqi Opto-Electronic Technology Co., Ltd, Xi’an, China). An optical fiber sensor was inserted in position 2 to monitor the central temperature of the potato cuboids during RF heating processing (Fig. 1a (2)). An optical fiber sensor was inserted in the mashed potato or enzyme extract to monitor the temperature. The initial temperature of potato cuboids and mashed potato were 25 °C, and enzyme extract solution of potato was 4 °C. Three electrode gaps (110, 120, and 130 mm) were tested. RF heating was terminated when the final temperature of the sample reached target temperatures (65, 70, 75, 80, and 85 °C). Then the sample was removed from the tube immediately and cooled rapidly in the ice water prior to the preparation of enzyme extract and evaluate of the quality of blanched samples.
2.3. Enzyme extraction and activity assay Crude potato PPO was extracted according to the method as described by Tribst et al. (2016) with some modifications. Added 40.00 mL cold phosphate buffer (0.1 M, pH 6.5, 4 °C) in 20.00 g potato cuboid or mashed potato. The mixture was homogenized for 3 min by a digital high-speed dispersing homogenizer under an ice water bath (FJ200-S, Shanghai Suoying Instruments Co., Ltd, China). Subsequently, the homogenized samples were centrifuged at 10,000g, 4 °C for 25 min. All steps were carried out at 4 °C. The supernatant was collected as crude enzyme extract for determining the PPO activity and stored in the refrigerator at 4 °C within two hours before enzyme activity evaluation. PPO activity was assessed by using a spectrophotometer at 410 nm. The phosphate buffer (0.1 M, pH 5.5) and catechol (0.2 M) were incubated at 25 °C for 10 min prior to experiments. The reaction mixture was composed of 1.5 mL phosphate buffer and 1 mL catechol. Then 0.5 mL crude PPO extract was added to the reaction mixture. Phosphate buffer was used as the blank to replace the enzyme extract. The absorbance was measured every 30 s for a total of 2 min. One unit of enzyme activity was defined as the amount of enzyme required to increase the absorbance by 0.001 per min under the assay conditions. The relative enzyme activity (REA) was calculated according to Eq. (1)
REA = (enzyme activitytreated/enzyme activitynative) × 100%
(1)
2.7. Weight loss
2.4. RF heating of mushroom PPO solution
Weight loss of potato cuboids was analyzed to evaluate the loss of water exuded from the samples during RF heating. The cuboids were weighed prior to RF heating. Immediately after RF heating, the samples were taken out quickly and put into ice water until the temperature dropped to room temperature (25 °C). Surface water was removed using absorbent paper and weighed again. The results were presented as a fractional decrease in weight Eq. (2):
Mushroom PPO was purchased for analyzing the effect of RF heating on the enzyme activity and the changes of secondary structure. The molecular weight of PPO was 119.5 kDa with 556 amino acids. PPO solution was prepared by diluting the enzyme in 0.1 M, pH 6.5 phosphate buffer. The concentration of the PPO solution was 0.1 mg/mL. Twenty milliliter PPO solution was filled in a centrifuge cube of 50 mL with a height of 50 mm. The centrifuge tube containing PPO solution was surrounded by expanded polyethylene (EPE) foam and placed at the center in the horizontal direction and middle in the vertical direction between the top and bottom electrodes of the RF system as shown in Fig. 1b. Subsequently, PPO solution was heated under three electrode gaps (110, 120, and 130 mm) until the temperature of the PPO solution reached the final temperature (50, 60, 70, 80, and 90 °C), respectively. The changed temperature of the PPO solution was monitored by a fluorescence-based optical fiber temperature measurement system (HQFTS-D1F00; Xi’an Heqi Opto-Electronic Technology Co., Ltd, Xi’an, China) during RF heating. After the designated temperature was reached, the sample was taken out immediately and cooled rapidly in ice water preventing further inactivation of the enzyme, and stored at 4 °C in a refrigerator within two hours before enzyme activity evaluation.
Weight loss (%) = [(W0−W1)/ W0] × 100
(2)
where W0 is initial weight of the samples untreated, W1 is the weight of treated samples. 2.8. Color measurement The surface color of non-processed and treated samples was measured by a computer vision system (CVS) (Zhou & Wang, 2016) and the color was expressed as L∗ (lightness), a∗ (redness ± greenness), and b∗ (yellowness ± blueness). Generally, a computer vision system consists of the illuminant, a digital camera, an image capture board, computer hardware, and software. A standardized lighting system included two CIE source D65 lamps (18 W, Model TLD/965, Philips, Shanghai) which were placed above the sample at a 45 angle to reduce reflection and shadows, thus enhancing the image quality and assuring repeatability. The color images of samples were obtained with a Cannon EOS-600D digital camera (1800 megapixel resolution and EF-S 18–55 mm f/ 3.5–5.6 Zoom Lens) in M mode (1/400, F13, ISO800). Then the images were analyzed by Adobe Photoshop (Adobe System Inc., USA). The sample area was selected by using the rectangle marquee tool in the menu bar. Subsequently, choosing the Lab color mode, the L, a, b values of selected area were obtained in the histogram of the menu bar, which can be converted to CIE LAB (L∗, a∗, and b∗) values using the Eqs. (3)-(5) (Zhou & Wang, 2016). Total color difference (ΔE) was calculated
2.5. Circular dichroism (CD) analysis of mushroom PPO CD spectra were recorded with a Chirascan CD spectrometer (Applied Photophysics Ltd., Britain), using quartz cuvette of 1 mm optical path length at room temperature (25 ± 1 °C). CD spectra were scanned at the far UV range (260–190 nm). The band width was 1.0 nm. The CD data was expressed in terms of mean residual ellipticity (θ), in mdeg. The concentration of mushroom PPO for CD analysis was 175
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Fig. 2. Time-temperature heating profiles (a, c) and the relative activity-temperature profiles (b, d) of mushroom PPO and potato cuboids subjected to RF heating at different electrode gaps (110, 120, and 130 mm), respectively.
phosphate buffer (pH 7.2) overnight. Then, samples were washed three times with the same buffer for 10 min each time. The samples were postfixed with 1% (w/v) OsO4 in the same buffer for 2 h at room temperature, and then washed with phosphate buffer three times. Samples were dehydrated in a graded series of ethanol/water (30%, 50%, 70%, 80%, 90% and 100%, v/v) and kept in each gradient for 10 min and 100% for 2 h. The samples were embedded with an ethanol:LR white mixture in 3:1, 1:1, 1:3 (v/v) on a stirrer for 2 h, 8 h, 12 h, respectively, and then replaced the ethanol:LR white mixture with the pure LR white for another 4 h on a stirrer by twice. Subsequently, the samples were moulded in pure LR white at 55 °C for 48 h. Samples were made into semi-thin sections (1 μm) using a glass knife and ultramicrotome (Leica EM UC7, Leica Microsyetems, Germany), stained in 0.05% Toludine Blue and then were observed by the Leica DM6 B upright microscope with the magnification of 20 under bright field.
according to Eq. (6):
L∗
(3)
= L/2.5
a∗ = (240a/255)−120
(4)
b∗ = (240b/255)−120
(5)
ΔE =
(L∗−L0∗)2 + (a∗−a0∗)2 + (b∗−b0∗)2 ∗
∗
(6)
∗
where L , a , and b were the color of treated samples, while the L0∗, a0∗, and b0∗ were the color of fresh samples. 2.9. Texture assessment The texture of fresh and treated potato cuboids was determined at room temperature (25 °C) using a texture analyzer (TA.XT Plus, Stable Micro system Ltd., Britain) equipped with a 50 kg load cell. For puncture tests, the potato cuboid was punctured on the stainless steel platform with a 2 mm diameter cylinder probe, at a rate of 2 mm/s. Each sample was punctured at three different positions (position of 1, 2, and 3 in Fig. 1a). The properties of hardness, springiness, cohesiveness, fracturability, and chewiness were measured for different RF treatment samples.
2.11. Microstructure analysis Scanning electron microscopy (S-4800, Hitachi, Ltd., Japan) was employed to observe the microstructure of fresh and treated samples. Samples were cut into thin slice (5 × 5 × 3 mm3), and were fixed in 2 mL of 4% glutaraldehyde overnight. Then, samples were washed four times with phosphate buffer (0.1 M, pH 6.8) for 10 min each time. Samples were dehydrated in a graded series of ethanol (30%, 50%, 70%, 80%, and 90% (v/v)) 15 min per step, and then were further dehydrated in 100% ethanol three times (30 min each). Finally, samples were immersed in isoamyl acetate, and were vacuum dried, and then sputter coated with gold. The samples were analyzed using a scanning electron microscopy (SEM) at an accelerating voltage of 10.0 kV under high vacuum.
2.10. Parenchyma cell change analysis The Leica DM6 B upright microscope with the Leica DFC7000 T camera and LASX software (Leica Microsyetems, Wetzlar, Germany) was used to analyse the changes of potato parenchyma cell before and after RF heating. Samples were cut into the right size and fixed with 2% (w/v) formaldehyde and 2.5% (w/v) glutaraldehyde in 0.1 M 176
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Fig. 3. Far-UV CD spectra of the mushroom PPO (a) and the content of mushroom PPO secondary structures (b) before and after RF treatment.
relative activity. PPO relative activity decreased with increasing temperature. PPO activity decreased sharply from 50 to 70 °C at all of electrode gaps. PPO relative activities were 0.48 ± 0.049%, 0.25 ± 0.0088%, and 6.39 ± 0.15% at 70 °C with the electrode gaps of 110, 120, and 130 mm, respectively. This result was in agreement with a previous study founding that 90% PPO activity was inactivated when PPO solution (mushroom tyrosinase) was exposed to 6.0 kV for 5.53 min under RF treatment (Manzocco et al., 2008). Furthermore, similar results are obtained by Baltacıoğlu, Bayındırlı, Severcan, and Severcan (2015), showing that mushroom PPO was inactivated between 50 and 70 °C and 99% PPO inactivation was achieved at 70 °C subjected to thermal treatment.
2.12. Data analysis Means and standard deviations (SD) were calculated with three replicates. The data were analyzed by ANOVA and the Tukey test using the SPSS (IBM Inc., USA) statistics at the 95% confidence level. 3. Results and discussion 3.1. Inactivation of mushroom PPO by RF heating Fig. 2a shows the time-temperature profiles of mushroom PPO subjected to RF heating at different electrode gaps (110, 120, and 130 mm). The fastest heating rate was obtained when the electrode gap was 110 mm. At all of electrode gaps, the heating rates gradually decreased with increasing temperature. Fig. 2b shows the relative enzyme activity of mushroom PPO submitted to RF treatments at different electrode gaps (110, 120, and 130 mm) and final temperatures (50, 60, 70, 80, and 90 °C). Both electrode gap and temperature had significant (P < .05) effects on PPO
3.2. Circular dichroism (CD) analysis of mushroom PPO CD is an important tool for analyzing protein structure in solution because many common conformational motifs have characteristic at far UV CD spectra, which directly characterize the change of protein secondary conformation (Greenfield, 1999). 177
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Fig. 4. The relative enzyme activity of PPO in different potato matrices (a) and weight loss of potato cuboids (b) after RF heating.
3.3. Effect of RF heating on the activity of potato PPO
Fig. 3a shows the CD spectra of mushroom PPO before and after RF treatment at the electrode gap of 120 mm with different final temperatures (50, 60, 70, 80, and 90 °C). The CD spectra of mushroom PPO changed after RF heating as compared with the controlled sample. The two negative peaks at 208 and 222 nm weakened, indicating the decrease of α-helix content of mushroom PPO. Fig. 3b summarizes the content of secondary structures of mushroom PPO before and after RF treatment. The content of α-helix decreased after RF heating while the antiparallel, parallel, β-turn, and random coil content increased, showing that RF treatment changed the conformation of protein. Similar phenomenon was reported by Luo, Zhang, Wang, Chen, and Guan (2009), founding that pulsed electric field treatment reduced the content of α-helix and increased the content of β-sheet of the PPO. Liu et al. (2009) also reported that high pressure microfluidization treatment caused a loss of α-helix content and the β-sheet, β-turn and random coil content changed irregularly of the mushroom PPO.
Fig. 2c shows the time-temperature profiles of potato cuboids during RF heating at different electrode gaps (110, 120, and 130 mm). Similar to the mushroom PPO tests, the graph showed that the heating rates under all electrode gaps gradually decreased as temperature increased. The heating rate was fastest at the electrode gap of 110 mm followed by 120 and 130 mm. Fig. 2d shows the relative enzyme activity of PPO of potato cuboids subjected to RF heating at three electrode gaps (110, 120, and 130 mm) and five final temperatures (65, 70, 75, 80, and 85 °C). The results showed that both electrode gap and temperature had significant effect (P < .05) on the activity of PPO. Under the same final temperature, potato cuboids showed the lowest relative activity of PPO at the electrode gap of 120 mm compared to 110 mm and 130 mm. According to previously study (Zhang et al., 2017), the best RF heating uniformity 178
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a
(Untreated)
(75 °C)
(65 °C)
(70 °C)
(80 °C)
(85 °C)
(65 °C)
(70 °C)
(80 °C)
(85 °C)
b
(Untreated)
(75 °C)
Fig. 5. Upright micrographs (a) and SEM micrographs (b) of untreated sample and samples subjected to RF heating at fixed electrode gap of 120 mm.
extract) at five final temperatures (65, 70, 75, 80, and 85 °C) and fixed electrode gap of 120 mm. There were significant differences (P < .05) among potato cuboids, mashed potato, and enzyme extract. The relative activity of PPO in the enzyme extract was lower than that in potato cuboids and mashed potato after RF heating to 75 °C. There was 4.18 ± 0.077%, 12.96 ± 0.39%, and 24.47 ± 0.063% of residual PPO activity in enzyme extract, potato cuboids, and mashed potato, respectively. It was found that PPO was more sensitive in tuber extract than that in cube or puree after HPP treatments in Peruvian carrot (Tribst et al., 2016). The relative activity of PPO was higher in mashed potato than that in cuboids. This phenomenon may be due to the damage of the potato physical structure, therefore facilitating the access of the enzyme to the substrate and the formation of the enzyme-substrate complex which was more stable to the RF heating. And this may also be due to the release of isoenzymes, cofactors and other food constituents. On the other hand, temperature had a significant effect (P < .05) on the PPO activity, the relative activity decreased with increasing temperature at all matrices. The relative activity of PPO was 28.89 ± 1.89%, 8.58 ± 0.25%, 4.19 ± 0.077%, 3.69 ± 0.89% and 0.19 ± 0.017% in enzyme extract submitted to RF heating at 65, 70,
was obtained at the electrode gap of 120 mm, thus resulting in the higher inactivation of PPO. PPO relative activity decreased rapidly as temperature increasing. The trend of PPO inactivation was in agreement with that described in section 3.1. Moreover, PPO relative activity was only 3.24 ± 0.19% at 85 °C under the electrode gap of 120 mm. However, there was significant variation among different blanching methods for inactivating PPO according to the reported references. Terefe, Yang, Knoerzer, Buckow, and Versteeg (2010) reported that only 28% inactivation of strawberry PPO after 30 min treatment at 100 °C. Castro et al. (2008) investigated the effects of thermal blanching on green pepper and red pepper, founding that both green pepper and red pepper PPO had more than 50% activity subjected to thermal treatment at 80 °C with 2 min. Wang et al. (2017) explored that the residual activity of red bell pepper PPO was 9.80% blanched at high microwave power (900 W) for 100 s. These studies indicated that the stability of PPO depended on source, variety, physicochemical components and also explained that RF heating had a great potential on inactivating PPO for blanching of vegetables and fruits. Fig. 4a shows the effect of RF heating on the relative PPO activity of different potato matrices (potato cuboids, mashed potato, and enzyme 179
Food Chemistry 248 (2018) 173–182 0.91bcd 0.64e 1.40ef 0.32ef 0.38d ± ± ± ± ±
3.4. Effect of RF heating on the weight loss Fig. 4b shows the effect of RF heating on the weight loss of potato cuboids at different electrode gaps (110, 120, and 130 mm) and temperatures (65, 70, 75, 80, and 85 °C). It was clear that all of the treated samples had different levels of weight loss. There were no significant differences (P > .05) on weight loss at different electrode gaps, whereas final temperature had significant effect (P < .05) on weight loss of the sample. The weight loss increased with increasing final temperature. Maximum weight loss was reached of 7.69 ± 1.08% at the temperature of 85 °C. This phenomenon was attributed to cellular damage leading to nutrients outflow, and water evaporation increasing with temperature increasing during blanching process. Moreover, RF heating may destroy the microstructure of the sample leading to weight loss. The results of scanning electron microscopy (Fig. 5) verified that the microstructure of the sample was gradually destroyed with increasing temperature. When comparing with other blanching methods, RF heating showed a slight higher values of weight loss as compared to steam blanching and PEF treatments (Ignat, Manzocco, Brunton, Nicoli, & Lyng, 2015; Mukherjee & Chattopadhyay, 2007).
0.89bc 35.61bc 12.64bc 0.54cd 0.83e
0.99 0.95 0.96 0.99 1.02
± ± ± ± ± ± ± ± ± ± 689.88 680.09 686.51 644.29 588.18
0.01ab 0.04bc 0.02abc 0.01ab 0.00a
0.11 0.12 0.14 0.16 0.19
± ± ± ± ±
0.00ef 0.00def 0.00cdef 0.01bcd 0.01ab
89.27 73.32 72.21 72.02 84.67
0.03cd 2.95ab 1.83cd 0.55efg 0.01g ± ± ± ± ± 0.98 0.98 0.98 0.99 0.98 2.24b 0.94cd 37.96bc 0.01bc 0.54de
0.29bc 0.96bc 0.08b 0.34bc 0.24b
± ± ± ± ± ± ± ± ± ± 704.90 645.10 696.72 677.09 615.68
0.01ab 0.01ab 0.01ab 0.00ab 0.01ab
0.12 0.14 0.15 0.15 0.21
± ± ± ± ±
0.01cdef 0.02cdef 0.01bcde 0.01bcde 0.00a
87.03 94.75 85.73 70.58 65.35
0.09bcd 0.31abc 1.14fg 0.30efg 0.23fg ± ± ± ± ±
96.73 ± 4.64a
88.93 91.84 67.06 69.72 66.38 0.00cdef 0.02cdef 0.00cdef 0.00bc 0.02ab ± ± ± ± ±
0.11 ± 0.00f
0.14 0.15 0.13 0.16 0.19 0.02c 0.02ab 0.01abc 0.01ab 0.00ab 0.91 0.98 0.96 0.99 0.99 ± ± ± ± ± 682.31 682.39 701.10 695.09 716.26
± ± ± ± ±
0.99 ± 0.00ab 772.22 ± 10.66a
Springiness Fracturability (g)
75, 80, and 85 °C, respectively.
0.29b 1.38g 0.86i 0.27k 0.64m ± ± ± ± ±
The changes of color and total color difference (ΔE) of samples before and after RF heating are presented in table 1. Both the electrode gap and temperature had a significant effect (P < .05) on the value of L∗, a∗, b∗, and ΔE except that temperature had no significant effect on b∗ value. The value of L∗ and b∗ decreased with increasing temperature. This phenomenon was probably due to cell collapse and liquid release leading to a lower reflectance. Similar studies were reported in Peruvian carrot and cocoyam, showing that high pressure processing induced a decrease in both L∗ and b∗ values (Tribst et al., 2016). However, a∗ value showed a lightly increase with increasing temperature. In addition, the value of ΔE ranged from 3.35 to 11.81 indicating a very noticeable color change (ΔE > 3.0) (Adekunte, Tiwari, Cullen, Scannell, & O'Donnell, 2010). The increasing value of ΔE might be on account of Maillard reaction, non-enzymatic browning, and the change of heat-sensitive components (Deylami, Rahman, Tan, Bakar, & Olusegun, 2016).
Different letters in the same column indicate a significant differences (P < .05) among fresh and blanched samples using RF heating.
774.03 645.12 601.57 486.95 418.62 0.73b 0.37b 2.19b 1.31b 1.16b ± ± ± ± ± 3.93 4.14 6.05 3.35 5.11 0.68ab 0.36ab 2.95ab 1.32ab 1.15ab 35.89 35.64 34.75 37.57 34.89 0.27ab 0.57ab 0.07ab 0.56ab 0.42ab 130
± ± ± ± ± 68.56 67.03 65.15 65.23 65.99 65 70 75 80 85
0.42abcd 0.05abc 0.81bcd 0.67bcd 0.26abcd
−5.25 −5.46 −4.10 −5.51 −5.61
± ± ± ± ±
± ± ± ± ±
2.22c 1.20e 4.07f 0.79k 1.27m ± ± ± ± ± 744.15 690.01 651.92 490.19 416.44 0.50b 0.33b 0.36b 0.47b 0.89b ± ± ± ± ± 3.80 4.47 5.20 5.07 4.13 0.80ab 0.25ab 1.40ab 0.93ab 1.05ab 36.50 35.38 36.60 35.76 36.12 0.35ab 0.15ab 1.67ab 0.46ab 0.62b 120
± ± ± ± ± 66.31 66.68 64.19 64.68 65.83 65 70 75 80 85
0.72abcd 0.63abcd 0.51cd 0.55bcd 0.32abcd
−4.44 −5.34 −4.37 −5.15 −6.21
± ± ± ± ±
± ± ± ± ±
0.00b 0.47d 1.09h 0.35k 1.00L ± ± ± ± ± 771.35 701.25 618.17 497.52 431.87
824.19 ± 2.09a –
4.09 ± 0.94b 5.12 ± 0.62b 7.59 ± 4.79ab 11.81 ± 1.06a 6.76 ± 1.03b 1.12ab 1.17ab 4.41ab 4.12b 0.72ab ± ± ± ± ±
39.68 ± 1.13a
35.84 35.11 34.13 32.55 34.10 0.68ab 0.99ab 1.39ab 0.91a 0.53ab ± ± ± ± ±
−5.74 ± 0.53b
−4.68 −4.45 −3.96 −3.35 −5.26 0.35abc 1.14abcd 3.28d 1.49e 1.10cd ± ± ± ± ±
67.72 ± 0.14ab
67.25 66.52 63.55 59.34 64.02
25
Hardness (g) L
*
65 70 75 80 85
Control
110
Temperature (°C)
Color
a
*
b
*
ΔE
Texture
3.5. Effect of RF heating on the color
Electrode gap (mm)
Table 1 Color and texture values (mean ± SD) of potato cuboids subjected to RF heating at three electrode gaps (110, 120, and 130 mm) and five temperatures (65, 70, 75, 80, and 85 °C).
Cohesiveness
Chewiness
Z. Zhang et al.
3.6. Effect of RF heating on the texture Table 1 shows the texture of fresh and treated potato cuboids at different treatment conditions. Both electrode gap and temperature had significant effects (P < .05) on hardness, fracturability, and chewiness. Under the same electrode gap, hardness of RF treated samples decreased with increasing temperature. This phenomenon was resulted from water loss which directly reduced turgor pressure (Rocculi, Romani, Galindo, & Rosa, 2009). Furthermore, the higher content of starch granules exerted a greater swelling pressure, consequently resulted in a softer texture during blanching processing (Liu & Scanlon, 2007). Under all electrode gaps, the change of hardness in texture was less than 20% as compared to control sample at temperature of 65 and 70 °C. This trend is consistent with that obtained by Abu-Ghannam and Crowley (2006). However, the texture degraded rapidly when temperature was above 80 °C. The results showed that RF heating had significant effect on the texture of potato cuboids. 3.7. Effect of RF heating on the microstructure Upright microscope showed the morphology of potato parenchyma cell before and after RF heating in Fig. 5a. The photographs revealed the apparent differences among samples after different RF heating treatment. In the untreated sample, parenchyma cells showed the 180
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regular arrangement with integrated cell wall. Some different sized starch granules were observed in parenchyma cell. The parenchyma cells remained intact after RF heating with the final temperature of 65 and 70 °C. However, the cells become shrunken and some starch began to gelatinization when the samples were subjected to RF treatment with the final temperature of 75 °C. The starch granules were completely gelatinized when the RF heating reached 80 °C. Some cells were filled with the starchy matrix filled in entire cell, others presented no contents in the cell at 85 °C. This phenomenon indicated that some cells retain cell wall and some cell wall disintegrated. Similar results were observed by Bordoloi, Kaur, and Singh (2012), founding Moonlight potato kept cell wall after cooking while the cell walls of Nadine and Red Rascal potato were destroyed during cooking. Microstructure photographs of fresh and treated potato cuboids subjected to RF heating at fixed electrode gap of 120 mm with different final temperatures are listed in Fig. 5b. It was observed that the cell walls and starch grains were clearly presented in micrographs. In the fresh sample, the cells arranged neatly with certain regularity, and the middle lamella connected closely with cell wall. This phenomenon was consistent with that observed by microscope. Cell wall is composed of pectin together with other polysaccharides which glue neighboring cells and cement them firmly in the middle lamella (Bolwell, 1993). In case of 65 and 70 °C, the cell walls folded companying with wall separation. Above 75 °C, some cell wall disrupted and cell collapsed. The reason is probably that the pectin in the middle lamella was leached away leading to cell wall breakdown and the tissue softened markedly. Similar change of microstructure was observed in the blanching of carrot by hot water (Ando et al., 2016). The starch gelatinized significantly and aggravated with increasing temperature. Al-Khusaibi and Niranjan (2012) reported that hot water blanching had a significant destructive effect on the structure, while there were no noticeably effect on the cellular architecture and starch granules of potato under the highpressure treatment. Changes in microstructure further affected color, texture, and weight of potatoes. Therefore, it’s very important to explore the changes in microstructure of vegetables and fruits during RF heating.
texture of whole processed new potatoes. Journal of Food Engineering, 74(3), 335–344. Adekunte, A. O., Tiwari, B. K., Cullen, P. J., Scannell, A. G. M., & O'Donnell, C. P. (2010). Effect of sonication on colour, ascorbic acid and yeast inactivation in tomato juice. Food Chemistry, 122(3), 500–507. Al-Khusaibi, M. K., & Niranjan, K. (2012). The impact of blanching and high-pressure pretreatments on oil uptake of fried potato slices. Food and Bioprocess Technology, 5(6), 2392–2400. Ali, H. M., El-Gizawy, A. M., El-Bassiouny, R. E., & Saleh, M. A. (2016). The role of various amino acids in enzymatic browning process in potato tubers, and identifying the browning products. Food Chemistry, 192, 879–885. Altunkaya, A., & Gokmen, V. (2008). Effect of various inhibitors on enzymatic browning, antioxidant activity and total phenol content of fresh lettuce (Lactuca sativa). Food Chemistry, 107(3), 1173–1179. Ando, Y., Maeda, Y., Mizutani, K., Wakatsuki, N., Hagiwara, S., & Nabetani, H. (2016). Impact of blanching and freeze-thaw pretreatment on drying rate of carrot roots in relation to changes in cell membrane function and cell wall structure. LWT – Food Science and Technology, 71, 40–46. Araya, X. I. T., Hendrickx, M., Verlinden, B. E., Buggenhout, S. V., Smale, N. J., Stewart, C., & Mawson, A. J. (2007). Understanding texture changes of high pressure processed fresh carrots: A microstructural and biochemical approach. Journal of Food Engineering, 80(3), 873–884. Baltacıoğlu, H., Bayındırlı, A., & Severcan, F. (2017). Secondary structure and conformational change of mushroom polyphenol oxidase during thermosonication treatment by using FTIR spectroscopy. Food Chemistry, 214, 507–514. Baltacıoğlu, H., Bayındırlı, A., Severcan, M., & Severcan, F. (2015). Effect of thermal treatment on secondary structure and conformational change of mushroom polyphenol oxidase (PPO) as food quality related enzyme: A FTIR study. Food Chemistry, 187, 263–269. Bolwell, G. P. (1993). Dynamic aspects of the plant extracellular matrix. International Review of Cytology, 146, 261–324. Bordoloi, A., Kaur, L., & Singh, J. (2012). Parenchyma cell microstructure and textural characteristics of raw and cooked potatoes. Food Chemistry, 133(4), 1092–1100. Castro, S. M., Saraiva, J. A., Lopes-Da-Silva, J. A., Delgadillo, I., Loey, A. V., Smout, C., & Hendrickx, M. (2008). Effect of thermal blanching and of high pressure treatments on sweet green and red bell pepper fruits (Capsicum annuum L.). Food Chemistry, 107(4), 1436–1449. Deylami, M. Z., Rahman, R. A., Tan, C. P., Bakar, J., & Olusegun, L. (2016). Effect of blanching on enzyme activity, color changes, anthocyanin stability and extractability of mangosteen pericarp: A kinetic study. Journal of Food Engineering, 178, 12–19. Duangmal, K., & Apenten, R. K. O. (1999). A comparative study of polyphenoloxidases from taro (Colocasia esculenta) and potato (Solanum tuberosum var. Romano). Food Chemistry, 64(3), 351–359. Farag, K. W., Lyng, J. G., Morgan, D. J., & Cronin, D. A. (2011). A Comparison of conventional and radio frequency thawing of beef meats: Effects on product temperature distribution. Food and Bioprocess Technology, 4(7), 1128–1136. Greenfield, N. J. (1999). Applications of circular dichroism in protein and peptide analysis. Trends in Analytical Chemistry, 18, 236–244. Icier, F., Yildiz, H., & Baysal, T. (2006). Peroxidase inactivation and colour changes during ohmic blanching of pea puree. Journal of Food Engineering, 74(3), 424–429. Ignat, A., Manzocco, L., Brunton, N. P., Nicoli, M. C., & Lyng, J. G. (2015). The effect of pulsed electric field pre-treatments prior to deep-fat frying on quality aspects of potato fries. Innovative Food Science & Emerging Technologies, 29, 65–69. Kikani, B. A., & Singh, S. P. (2015). Enzyme stability, thermodynamics and secondary structures of α-amylase as probed by the CD spectroscopy. International Journal of Biological Macromolecules, 81, 450–460. Kuijpers, T. F. M., Herk, T. V., Vincken, J. P., Janssen, R. H., Narh, D. L., Berkel, W. J. H. V., & Gruppen, H. (2014). Potato and mushroom polyphenol oxidase activities are differently modulated by natural plant extracts. Journal of Agricultural and Food Chemistry, 62(1), 214–221. Liu, E. Z., & Scanlon, M. G. (2007). Modeling the effect of blanching conditions on the texture of potato strips. Journal of Food Engineering, 81(2), 292–297. Liu, W., Liu, J., Liu, C., Zhong, Y., Liu, W., & Wan, J. (2009). Activation and conformational changes of mushroom polyphenoloxidase by high pressure microfluidization treatment. Innovative Food Science & Emerging Technologies, 10(2), 142–147. Lopez, A., & Baganis, N. A. (1971). Effect of radio-frequency energy at 60 MHz on food enzyme activity. Journal of Food Science, 36(6), 911–914. Luo, W., Zhang, R. B., Wang, L. M., Chen, J., & Guan, Z. C. (2009). Conformation changes of polyphenol oxidase and lipoxygenase induced by PEF treatment. Journal of Applied Electrochemistry, 40(2), 295–301. Manzocco, L., Anese, M., & Nicoli, M. C. (2008). Radiofrequency inactivation of oxidative food enzymes in model systems and apple derivatives. Food Research International, 41(10), 1044–1049. Mayer, A. M., & Harel, E. (1979). Polyphenol oxidases in plants. Phytochemistry, 18(2), 193–215. Mukherjee, S., & Chattopadhyay, P. K. (2007). Whirling bed blanching of potato cubes and its effects on product quality. Journal of Food Engineering, 78(1), 52–60. Orsat, V., Gariépy, Y., Raghavan, G. S. V., & Lyew, D. (2001). Radio-frequency treatment for ready-to-eat fresh carrots. Food Research International, 34(6), 527–536. Palazoglu, T. K., Coskun, Y., Kocadagli, T., & Gokmen, V. (2012). Effect of radio frequency postdrying of partially baked cookies on acrylamide content, texture, and color of the final product. Journal of Food Science, 77(5), E113–117. Rocculi, P., Romani, S., Galindo, F. G., & Rosa, M. D. (2009). Effect of minimal processing on physiology and quality of fresh-cut potatoes: a review. Global Science Books, 18–30. Siddiq, M., & Dolan, K. D. (2017). Characterization of polyphenol oxidase from blueberry (Vaccinium corymbosum L.). Food Chemistry, 218, 216–220.
4. Conclusions The results showed that RF heating can efficiently inactivate mushroom PPO. CD analysis indicated that the changes of secondary structures companied with PPO inactivation. RF heating had significant effects on activity of PPO, texture, color, weight loss, microstructure of potatoes. The relative activity of potato PPO decreased with increasing temperature at all electrode gaps. Furthermore, different potato matrices had significant effects (P < .05) on the PPO relative activity. The enzyme extract showed the lowest PPO relative activity, followed by the potato cuboids and mashed potato. Microstructure analysis well explained the changes of color, texture, and weight of potatoes subjected to RF heating. This study provides the valuable information to understand the effect of RF heating on preventing browning of vegetables and shows the potential of this technology for blanching vegetables and fruits. Acknowledgement We thank Professor Zhenyu Li (College of Food Science and Engineering, Northwest A&F University, China) for his helpful advice and assistance with the English language in the manuscript submitted for publication. This study was supported by the general program (Grant No. 31371854) of National Natural Science Foundation of China, the Key Research Project of Shaanxi Province (2017ZDXM-SF-104). References Abu-Ghannam, N., & Crowley, H. (2006). The effect of low temperature blanching on the
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red bell pepper (Capsicum annuum L.). LWT – Food Science and Technology, 77, 337–347. Wang, Y., Zhang, L., Johnson, J., Gao, M., Tang, J., Powers, J. R., & Wang, S. (2014). Developing hot air-assisted radio frequency drying for in-shell macadamia nuts. Food and Bioprocess Technology, 7(1), 278–288. Zhang, Z., Guo, C., Gao, T., Fu, H., Chen, Q., & Wang, Y. (2017). Pilot-scale radio frequency blanching of potato cuboids: Heating uniformity. Journal of the Science of Food & Agriculture. Zhong, K., Wu, J., Wang, Z., Chen, F., Liao, X., Hu, X., & Zhang, Z. (2007). Inactivation kinetics and secondary structural change of PEF-treated POD and PPO. Food Chemistry, 100(1), 115–123. Zhou, L., & Wang, S. (2016). Verification of radio frequency heating uniformity and Sitophilus oryzae control in rough, brown, and milled rice. Journal of Stored Products Research, 65, 40–47.
Sotome, I., Takenaka, M., Koseki, S., Ogasawara, Y., Nadachi, Y., Okadome, H., & Isobe, S. (2009). Blanching of potato with superheated steam and hot water spray. LWT – Food Science and Technology, 42(6), 1035–1040. Terefe, N. S., Yang, Y. H., Knoerzer, K., Buckow, R., & Versteeg, C. (2010). High pressure and thermal inactivation kinetics of polyphenol oxidase and peroxidase in strawberry puree. Innovative Food Science & Emerging Technologies, 11(1), 52–60. Tribst, A. A. L., Júnior, B. R.d. C., Oliveira, M. M. D., & Cristianini, M. (2016). High pressure processing of cocoyam, Peruvian carrot and sweet potato: Effect on oxidative enzymes and impact in the tuber color. Innovative Food Science & Emerging Technologies, 34, 302–309. Vishwanathan, K. H., Giwari, G. K., & Hebbar, H. U. (2013). Infrared assisted dryblanching and hybrid drying of carrot. Food and Bioproducts Processing, 91(2), 89–94. Wang, J., Yang, X. H., Mujumdar, A. S., Wang, D., Zhao, J. H., Fang, X. M., ... Xiao, H. W. (2017). Effects of various blanching methods on weight loss, enzymes inactivation, phytochemical contents, antioxidant capacity, ultrastructure and drying kinetics of
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Effects of radio frequency assisted blanching on polyphenol oxidase, weight loss, texture, color and microstructure of potato
T
⁎
Zhenna Zhang, Juan Wang, Xueying Zhang, Qingli Shi, Le Xin, Hongfei Fu, Yunyang Wang College of Food Science and Engineering, Northwest A&F University, Yangling, Shaanxi 712100, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Radio frequency heating Blanching Potato Polyphenol oxidase Physicochemical properties
This paper is focused on the effects of radio frequency (RF) heating on the relative activity of polyphenol oxidase (PPO), weight loss, texture, color, and microstructure of potatoes. The results showed that pure mushroom PPO was almost completely inactivated at 80 °C by RF heating. The relative activity of potato PPO reduced to less than 10% with increasing temperature (25–85 °C). Enzyme extract showed the lowest PPO relative activity at 85 °C after RF treatment, followed by the potato cuboids and mashed potato, about 0.19 ± 0.017%, 3.24 ± 0.19%, and 3.54 ± 0.04%, respectively. Circular dichroism analysis indicated that RF heating changed the secondary structure of PPO, as α-helix content decreased. Both electrode gap and temperature had significant effect (P < .05) on weight loss, color, and texture of the potato cuboids. Microstructure analysis showed the changes of potato cell and starch during RF heating.
1. Introduction Potatoes are widely cultivated all over the world, especially in many developing countries. It is commonly processed into potato flours, purees, and chips, which are favored by consumers. However, enzymatic activity in the potato can result in a series of deterioration reactions including undesirable color and texture, off-flavors, bad odor, and loss of nutrients. Polyphenol oxidase (PPO) is a copper-containing enzyme contributing to enzymatic browning of vegetables and fruits. PPO can be divided into two categories: laccase (EC 1.10.3.2) and catechol oxidase (EC 1.10.3.1). Catechol oxidase is referred to as phenolase, polyphenol oxidase, tyrosinase, catecholase or cresolase, which is clearly distinguished from laccase. Catechol oxidase can catalyze two distinct reactions: (1) the insertion of oxygen in a position ortho to an existing hydroxyl group, usually followed by oxidation of the diphenol to the corresponding quinone, which is referred to as cresolase activity; (2) the oxidation of o-diphenol with hydrogen abstraction, which is referred to as catecholase activity (Mayer & Harel, 1979). Quinones subsequently undergo nonenzymatic reactions resulting in the formation of dark-colored melanins (Duangmal & Apenten, 1999; Kuijpers et al., 2014; Tribst, Júnior, Oliveira, & Cristianini, 2016). Therefore, the inhibition of enzyme activity plays an important role in the food industry. Blanching is a crucial step that is carried out prior to food processes such as frying, drying, freezing, and storing because it can inactivate enzymes, destroy microorganisms, and eliminate air in the potato (Wang et al., 2017).
⁎
The methods of inactivation of PPO have been widely studied. The activity of PPO can be inhibited by using chemical reagents, such as sulphites, ascorbic acid, and amino acids, which have been applied in potato (Ali, El-Gizawy, El-Bassiouny, & Saleh, 2016), lettuce (Altunkaya & Gokmen, 2008), and blueberry (Siddiq & Dolan, 2017). However, the residual chemical agents pose a threat to human health. It is necessary to develop novel technologies and to reduce the use of chemical food additives. Thermal treatment is the most common and effective technique to control enzymatic browning in the food industry. A number of studies have been explored to overcome enzymatic browning to improve the quality of products by thermal processes. The methods include hot water, hot and boiling solutions containing acids and/or salts, steam heating, microwave, infrared, ohmic heating, and radio frequency heating (Castro et al., 2008; Icier, Yildiz, & Baysal, 2006; Manzocco, Anese, & Nicoli, 2008; Mukherjee & Chattopadhyay, 2007; Sotome et al., 2009; Vishwanathan, Giwari, & Hebbar, 2013; Wang et al., 2017). Radio frequency (RF) heating utilizes electromagnetic energy at a frequency range of 3 kHz to 300 MHz and initiates volumetric heating due to frictional interaction between molecules. The advantages of RF heating include higher penetration depth and higher heating rate. Therefore, it can save processing time and improve food quality. RF heating has been studied on drying nuts, thawing meats, post-baking cookies, pasteurization, and controlling insects (Farag, Lyng, Morgan, & Cronin, 2011; Palazoglu, Coskun, Kocadagli, & Gokmen, 2012; Wang et al., 2014; Zhou & Wang, 2016). However, the study about the effect
Corresponding author. E-mail address: [email protected] (Y. Wang).
https://doi.org/10.1016/j.foodchem.2017.12.065 Received 13 August 2017; Received in revised form 15 December 2017; Accepted 18 December 2017 Available online 18 December 2017 0308-8146/ © 2017 Elsevier Ltd. All rights reserved.
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a
(1)
(2)
(3)
(4)
b
Fig. 1. Schematic of the sample in the centrifuge tube (a) and schematic of the potato sample placed in the RF heating system (b). (1) The position in the cuboid for measuring temperature and analyzing texture, (2) potato cuboid, (3) potato puree, and (4) enzyme extract (all dimensions are in mm).
(Kikani & Singh, 2015; Zhong et al., 2007), to the best of our knowledge, there is no published data so far regarding the conformation changes of enzyme during RF heating. The objectives of this study were: (1) to investigate the effects of RF heating on model enzyme (mushroom PPO) inactivation and the changes of enzyme secondary structure, (2) to analyze the effects of RF heating on the inhibition of PPO of potato, (3) to evaluate the changes of weight loss, texture, color, and microstructure of potato after RF heating.
of RF heating on the activity of enzymes in vegetables is still limited so far. It has been reported that RF heating could efficiently inactivated PPO and lipoxygenase (LOX), particularly PPO was more sensitive to an increase in the RF electric field (Manzocco et al., 2008). It showed that RF heating retained a higher sweetness and produced highly appreciated vegetable derivatives than the conventional blanching. Moreover, RF treatment can not only saves time, water, and energy, but also reduces the cost of waste treatment. Orsat, Gariépy, Raghavan, and Lyew (2001) compared the quality of vacuum-packaged carrot sticks treated with RF heating, chlorinated water dipping or hot water dipping, showing that the color and taste were better, and the vacuum of packages were greater of samples in RF treatment than that in chlorinated water or hot water. Lopez and Baganis (1971) also reported that peroxidase, polyphenolase, pectinesterase, catalase, and α-amylase were partially or totally inactivated by RF heating at 70 °C. These results indicated that RF heating has the potential in blanching fruits and vegetables. The main purpose of blanching is to inactivate enzymes which are related to the browning of food. Color, weight loss, and texture were used as indicator to evaluate the food quality during blanching. However, the changes of texture are attributed to the altering of microstructure during blanching (Araya et al., 2007). Therefore, the microstructure of food should be investigated to provide sufficient knowledge of blanching. In addition should be very interesting to add new information on the effect of RF heating to the secondary structure of proteins. The secondary structure of the protein is a stable conformation of the local peptides in the peptide chain of the protein. Enzymes are proteins which have some active sites. Heat, ultrasound, pulsed electric field, etc., can disturb the delicate balance of covalent and non-covalent interactions that maintain the native structure, which can result in a loss of enzyme activity (Baltacıoğlu, Bayındırlı, & Severcan, 2017). Nevertheless, the reasons of enzyme inactivation whether were due to changes in the active site or the changes of secondary structure need to be studied. Although some researchers have studied the changes in secondary structure of peroxidase (POD) and PPO treated by PEF
2. Materials and methods 2.1. Materials and preparation of samples The potato (Solanum tuberosum L.) numbering 15–7 was provided by the Potato Molecular Genetics and Breeding Lab of the College of Agriculture at Northwest A & F University in China and stored in a refrigerator at 4 °C. Before the experiments, the samples were taken out and equilibrated to room temperature (25 °C) overnight. Potatoes were washed, hand-peeled, and cut into cuboid with the dimension (length × width × height) of 30 × 10 × 60 mm3 having an average weight of 20 g. The mashed potato was obtained by crushing 20 g potato tubers using an electrical hand blender (Multiquick 7 MQ705, 4199, De’Longhi Braun Household Gmbh, Germany) at room temperature. The method to prepare enzyme extract was described in the section 2.3. Mushroom PPO (98% purity) as model enzyme (E.C. 1.14.18.1), catechol (≥99% purity) were purchased from Sigma (Shanghai, China) and Aladdin (Shanghai, China), respectively. Other chemicals used in this study were of analytical grade. 2.2. RF heating system RF assisted blanching treatments were performed by a 6 kW, 27.12 MHz pilot-scale free running oscillator RF system (GJG-2.1-10A174
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0.1 mg/mL. The secondary structures contents of PPO were calculated by CD spectra Deconvolution Software (Institut für Biotechnologie, Martin-Luther-Universität Halle-Witttenberg, Germany).
JY; Hebei Huashijiyuan High Frequency Equipment Co., Ltd, Hebei, China). The system included a generator and a RF applicator with two parallel rectangular electrodes. A high voltage alternating electric field was applied to a medium sandwiched between two parallel electrodes. The schematic of the RF heating system was shown in Fig. 1b. All of the experiments were repeated three times.
2.6. RF heating treatment of potato samples To evaluate the effect of RF heating on the inactivation of PPO and the quality of potato products, three types of sample matrices including potato cuboids, mashed potato, and enzyme extract were employed in this study. One potato cuboid was put in a centrifuge tube of 100 mL with a height of 72 mm. Twenty grams of mashed potato and or enzyme extract was filled in a centrifuge cube of 50 mL with a height of 50 mm. The details of sample loading were illustrated in Fig. 1a. The centrifuge tube containing potato sample was surrounded by the expanded polyethylene (EPE) foam and placed at the center in the horizontal direction and middle in the vertical direction between the top and bottom electrodes as shown in Fig. 1b (Zhang et al., 2017). The changes of sample temperature during RF heating were monitored by a fluorescence-based optical fiber temperature measurement system (HQFTS-D1F00; Xi’an Heqi Opto-Electronic Technology Co., Ltd, Xi’an, China). An optical fiber sensor was inserted in position 2 to monitor the central temperature of the potato cuboids during RF heating processing (Fig. 1a (2)). An optical fiber sensor was inserted in the mashed potato or enzyme extract to monitor the temperature. The initial temperature of potato cuboids and mashed potato were 25 °C, and enzyme extract solution of potato was 4 °C. Three electrode gaps (110, 120, and 130 mm) were tested. RF heating was terminated when the final temperature of the sample reached target temperatures (65, 70, 75, 80, and 85 °C). Then the sample was removed from the tube immediately and cooled rapidly in the ice water prior to the preparation of enzyme extract and evaluate of the quality of blanched samples.
2.3. Enzyme extraction and activity assay Crude potato PPO was extracted according to the method as described by Tribst et al. (2016) with some modifications. Added 40.00 mL cold phosphate buffer (0.1 M, pH 6.5, 4 °C) in 20.00 g potato cuboid or mashed potato. The mixture was homogenized for 3 min by a digital high-speed dispersing homogenizer under an ice water bath (FJ200-S, Shanghai Suoying Instruments Co., Ltd, China). Subsequently, the homogenized samples were centrifuged at 10,000g, 4 °C for 25 min. All steps were carried out at 4 °C. The supernatant was collected as crude enzyme extract for determining the PPO activity and stored in the refrigerator at 4 °C within two hours before enzyme activity evaluation. PPO activity was assessed by using a spectrophotometer at 410 nm. The phosphate buffer (0.1 M, pH 5.5) and catechol (0.2 M) were incubated at 25 °C for 10 min prior to experiments. The reaction mixture was composed of 1.5 mL phosphate buffer and 1 mL catechol. Then 0.5 mL crude PPO extract was added to the reaction mixture. Phosphate buffer was used as the blank to replace the enzyme extract. The absorbance was measured every 30 s for a total of 2 min. One unit of enzyme activity was defined as the amount of enzyme required to increase the absorbance by 0.001 per min under the assay conditions. The relative enzyme activity (REA) was calculated according to Eq. (1)
REA = (enzyme activitytreated/enzyme activitynative) × 100%
(1)
2.7. Weight loss
2.4. RF heating of mushroom PPO solution
Weight loss of potato cuboids was analyzed to evaluate the loss of water exuded from the samples during RF heating. The cuboids were weighed prior to RF heating. Immediately after RF heating, the samples were taken out quickly and put into ice water until the temperature dropped to room temperature (25 °C). Surface water was removed using absorbent paper and weighed again. The results were presented as a fractional decrease in weight Eq. (2):
Mushroom PPO was purchased for analyzing the effect of RF heating on the enzyme activity and the changes of secondary structure. The molecular weight of PPO was 119.5 kDa with 556 amino acids. PPO solution was prepared by diluting the enzyme in 0.1 M, pH 6.5 phosphate buffer. The concentration of the PPO solution was 0.1 mg/mL. Twenty milliliter PPO solution was filled in a centrifuge cube of 50 mL with a height of 50 mm. The centrifuge tube containing PPO solution was surrounded by expanded polyethylene (EPE) foam and placed at the center in the horizontal direction and middle in the vertical direction between the top and bottom electrodes of the RF system as shown in Fig. 1b. Subsequently, PPO solution was heated under three electrode gaps (110, 120, and 130 mm) until the temperature of the PPO solution reached the final temperature (50, 60, 70, 80, and 90 °C), respectively. The changed temperature of the PPO solution was monitored by a fluorescence-based optical fiber temperature measurement system (HQFTS-D1F00; Xi’an Heqi Opto-Electronic Technology Co., Ltd, Xi’an, China) during RF heating. After the designated temperature was reached, the sample was taken out immediately and cooled rapidly in ice water preventing further inactivation of the enzyme, and stored at 4 °C in a refrigerator within two hours before enzyme activity evaluation.
Weight loss (%) = [(W0−W1)/ W0] × 100
(2)
where W0 is initial weight of the samples untreated, W1 is the weight of treated samples. 2.8. Color measurement The surface color of non-processed and treated samples was measured by a computer vision system (CVS) (Zhou & Wang, 2016) and the color was expressed as L∗ (lightness), a∗ (redness ± greenness), and b∗ (yellowness ± blueness). Generally, a computer vision system consists of the illuminant, a digital camera, an image capture board, computer hardware, and software. A standardized lighting system included two CIE source D65 lamps (18 W, Model TLD/965, Philips, Shanghai) which were placed above the sample at a 45 angle to reduce reflection and shadows, thus enhancing the image quality and assuring repeatability. The color images of samples were obtained with a Cannon EOS-600D digital camera (1800 megapixel resolution and EF-S 18–55 mm f/ 3.5–5.6 Zoom Lens) in M mode (1/400, F13, ISO800). Then the images were analyzed by Adobe Photoshop (Adobe System Inc., USA). The sample area was selected by using the rectangle marquee tool in the menu bar. Subsequently, choosing the Lab color mode, the L, a, b values of selected area were obtained in the histogram of the menu bar, which can be converted to CIE LAB (L∗, a∗, and b∗) values using the Eqs. (3)-(5) (Zhou & Wang, 2016). Total color difference (ΔE) was calculated
2.5. Circular dichroism (CD) analysis of mushroom PPO CD spectra were recorded with a Chirascan CD spectrometer (Applied Photophysics Ltd., Britain), using quartz cuvette of 1 mm optical path length at room temperature (25 ± 1 °C). CD spectra were scanned at the far UV range (260–190 nm). The band width was 1.0 nm. The CD data was expressed in terms of mean residual ellipticity (θ), in mdeg. The concentration of mushroom PPO for CD analysis was 175
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Fig. 2. Time-temperature heating profiles (a, c) and the relative activity-temperature profiles (b, d) of mushroom PPO and potato cuboids subjected to RF heating at different electrode gaps (110, 120, and 130 mm), respectively.
phosphate buffer (pH 7.2) overnight. Then, samples were washed three times with the same buffer for 10 min each time. The samples were postfixed with 1% (w/v) OsO4 in the same buffer for 2 h at room temperature, and then washed with phosphate buffer three times. Samples were dehydrated in a graded series of ethanol/water (30%, 50%, 70%, 80%, 90% and 100%, v/v) and kept in each gradient for 10 min and 100% for 2 h. The samples were embedded with an ethanol:LR white mixture in 3:1, 1:1, 1:3 (v/v) on a stirrer for 2 h, 8 h, 12 h, respectively, and then replaced the ethanol:LR white mixture with the pure LR white for another 4 h on a stirrer by twice. Subsequently, the samples were moulded in pure LR white at 55 °C for 48 h. Samples were made into semi-thin sections (1 μm) using a glass knife and ultramicrotome (Leica EM UC7, Leica Microsyetems, Germany), stained in 0.05% Toludine Blue and then were observed by the Leica DM6 B upright microscope with the magnification of 20 under bright field.
according to Eq. (6):
L∗
(3)
= L/2.5
a∗ = (240a/255)−120
(4)
b∗ = (240b/255)−120
(5)
ΔE =
(L∗−L0∗)2 + (a∗−a0∗)2 + (b∗−b0∗)2 ∗
∗
(6)
∗
where L , a , and b were the color of treated samples, while the L0∗, a0∗, and b0∗ were the color of fresh samples. 2.9. Texture assessment The texture of fresh and treated potato cuboids was determined at room temperature (25 °C) using a texture analyzer (TA.XT Plus, Stable Micro system Ltd., Britain) equipped with a 50 kg load cell. For puncture tests, the potato cuboid was punctured on the stainless steel platform with a 2 mm diameter cylinder probe, at a rate of 2 mm/s. Each sample was punctured at three different positions (position of 1, 2, and 3 in Fig. 1a). The properties of hardness, springiness, cohesiveness, fracturability, and chewiness were measured for different RF treatment samples.
2.11. Microstructure analysis Scanning electron microscopy (S-4800, Hitachi, Ltd., Japan) was employed to observe the microstructure of fresh and treated samples. Samples were cut into thin slice (5 × 5 × 3 mm3), and were fixed in 2 mL of 4% glutaraldehyde overnight. Then, samples were washed four times with phosphate buffer (0.1 M, pH 6.8) for 10 min each time. Samples were dehydrated in a graded series of ethanol (30%, 50%, 70%, 80%, and 90% (v/v)) 15 min per step, and then were further dehydrated in 100% ethanol three times (30 min each). Finally, samples were immersed in isoamyl acetate, and were vacuum dried, and then sputter coated with gold. The samples were analyzed using a scanning electron microscopy (SEM) at an accelerating voltage of 10.0 kV under high vacuum.
2.10. Parenchyma cell change analysis The Leica DM6 B upright microscope with the Leica DFC7000 T camera and LASX software (Leica Microsyetems, Wetzlar, Germany) was used to analyse the changes of potato parenchyma cell before and after RF heating. Samples were cut into the right size and fixed with 2% (w/v) formaldehyde and 2.5% (w/v) glutaraldehyde in 0.1 M 176
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Fig. 3. Far-UV CD spectra of the mushroom PPO (a) and the content of mushroom PPO secondary structures (b) before and after RF treatment.
relative activity. PPO relative activity decreased with increasing temperature. PPO activity decreased sharply from 50 to 70 °C at all of electrode gaps. PPO relative activities were 0.48 ± 0.049%, 0.25 ± 0.0088%, and 6.39 ± 0.15% at 70 °C with the electrode gaps of 110, 120, and 130 mm, respectively. This result was in agreement with a previous study founding that 90% PPO activity was inactivated when PPO solution (mushroom tyrosinase) was exposed to 6.0 kV for 5.53 min under RF treatment (Manzocco et al., 2008). Furthermore, similar results are obtained by Baltacıoğlu, Bayındırlı, Severcan, and Severcan (2015), showing that mushroom PPO was inactivated between 50 and 70 °C and 99% PPO inactivation was achieved at 70 °C subjected to thermal treatment.
2.12. Data analysis Means and standard deviations (SD) were calculated with three replicates. The data were analyzed by ANOVA and the Tukey test using the SPSS (IBM Inc., USA) statistics at the 95% confidence level. 3. Results and discussion 3.1. Inactivation of mushroom PPO by RF heating Fig. 2a shows the time-temperature profiles of mushroom PPO subjected to RF heating at different electrode gaps (110, 120, and 130 mm). The fastest heating rate was obtained when the electrode gap was 110 mm. At all of electrode gaps, the heating rates gradually decreased with increasing temperature. Fig. 2b shows the relative enzyme activity of mushroom PPO submitted to RF treatments at different electrode gaps (110, 120, and 130 mm) and final temperatures (50, 60, 70, 80, and 90 °C). Both electrode gap and temperature had significant (P < .05) effects on PPO
3.2. Circular dichroism (CD) analysis of mushroom PPO CD is an important tool for analyzing protein structure in solution because many common conformational motifs have characteristic at far UV CD spectra, which directly characterize the change of protein secondary conformation (Greenfield, 1999). 177
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Fig. 4. The relative enzyme activity of PPO in different potato matrices (a) and weight loss of potato cuboids (b) after RF heating.
3.3. Effect of RF heating on the activity of potato PPO
Fig. 3a shows the CD spectra of mushroom PPO before and after RF treatment at the electrode gap of 120 mm with different final temperatures (50, 60, 70, 80, and 90 °C). The CD spectra of mushroom PPO changed after RF heating as compared with the controlled sample. The two negative peaks at 208 and 222 nm weakened, indicating the decrease of α-helix content of mushroom PPO. Fig. 3b summarizes the content of secondary structures of mushroom PPO before and after RF treatment. The content of α-helix decreased after RF heating while the antiparallel, parallel, β-turn, and random coil content increased, showing that RF treatment changed the conformation of protein. Similar phenomenon was reported by Luo, Zhang, Wang, Chen, and Guan (2009), founding that pulsed electric field treatment reduced the content of α-helix and increased the content of β-sheet of the PPO. Liu et al. (2009) also reported that high pressure microfluidization treatment caused a loss of α-helix content and the β-sheet, β-turn and random coil content changed irregularly of the mushroom PPO.
Fig. 2c shows the time-temperature profiles of potato cuboids during RF heating at different electrode gaps (110, 120, and 130 mm). Similar to the mushroom PPO tests, the graph showed that the heating rates under all electrode gaps gradually decreased as temperature increased. The heating rate was fastest at the electrode gap of 110 mm followed by 120 and 130 mm. Fig. 2d shows the relative enzyme activity of PPO of potato cuboids subjected to RF heating at three electrode gaps (110, 120, and 130 mm) and five final temperatures (65, 70, 75, 80, and 85 °C). The results showed that both electrode gap and temperature had significant effect (P < .05) on the activity of PPO. Under the same final temperature, potato cuboids showed the lowest relative activity of PPO at the electrode gap of 120 mm compared to 110 mm and 130 mm. According to previously study (Zhang et al., 2017), the best RF heating uniformity 178
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a
(Untreated)
(75 °C)
(65 °C)
(70 °C)
(80 °C)
(85 °C)
(65 °C)
(70 °C)
(80 °C)
(85 °C)
b
(Untreated)
(75 °C)
Fig. 5. Upright micrographs (a) and SEM micrographs (b) of untreated sample and samples subjected to RF heating at fixed electrode gap of 120 mm.
extract) at five final temperatures (65, 70, 75, 80, and 85 °C) and fixed electrode gap of 120 mm. There were significant differences (P < .05) among potato cuboids, mashed potato, and enzyme extract. The relative activity of PPO in the enzyme extract was lower than that in potato cuboids and mashed potato after RF heating to 75 °C. There was 4.18 ± 0.077%, 12.96 ± 0.39%, and 24.47 ± 0.063% of residual PPO activity in enzyme extract, potato cuboids, and mashed potato, respectively. It was found that PPO was more sensitive in tuber extract than that in cube or puree after HPP treatments in Peruvian carrot (Tribst et al., 2016). The relative activity of PPO was higher in mashed potato than that in cuboids. This phenomenon may be due to the damage of the potato physical structure, therefore facilitating the access of the enzyme to the substrate and the formation of the enzyme-substrate complex which was more stable to the RF heating. And this may also be due to the release of isoenzymes, cofactors and other food constituents. On the other hand, temperature had a significant effect (P < .05) on the PPO activity, the relative activity decreased with increasing temperature at all matrices. The relative activity of PPO was 28.89 ± 1.89%, 8.58 ± 0.25%, 4.19 ± 0.077%, 3.69 ± 0.89% and 0.19 ± 0.017% in enzyme extract submitted to RF heating at 65, 70,
was obtained at the electrode gap of 120 mm, thus resulting in the higher inactivation of PPO. PPO relative activity decreased rapidly as temperature increasing. The trend of PPO inactivation was in agreement with that described in section 3.1. Moreover, PPO relative activity was only 3.24 ± 0.19% at 85 °C under the electrode gap of 120 mm. However, there was significant variation among different blanching methods for inactivating PPO according to the reported references. Terefe, Yang, Knoerzer, Buckow, and Versteeg (2010) reported that only 28% inactivation of strawberry PPO after 30 min treatment at 100 °C. Castro et al. (2008) investigated the effects of thermal blanching on green pepper and red pepper, founding that both green pepper and red pepper PPO had more than 50% activity subjected to thermal treatment at 80 °C with 2 min. Wang et al. (2017) explored that the residual activity of red bell pepper PPO was 9.80% blanched at high microwave power (900 W) for 100 s. These studies indicated that the stability of PPO depended on source, variety, physicochemical components and also explained that RF heating had a great potential on inactivating PPO for blanching of vegetables and fruits. Fig. 4a shows the effect of RF heating on the relative PPO activity of different potato matrices (potato cuboids, mashed potato, and enzyme 179
Food Chemistry 248 (2018) 173–182 0.91bcd 0.64e 1.40ef 0.32ef 0.38d ± ± ± ± ±
3.4. Effect of RF heating on the weight loss Fig. 4b shows the effect of RF heating on the weight loss of potato cuboids at different electrode gaps (110, 120, and 130 mm) and temperatures (65, 70, 75, 80, and 85 °C). It was clear that all of the treated samples had different levels of weight loss. There were no significant differences (P > .05) on weight loss at different electrode gaps, whereas final temperature had significant effect (P < .05) on weight loss of the sample. The weight loss increased with increasing final temperature. Maximum weight loss was reached of 7.69 ± 1.08% at the temperature of 85 °C. This phenomenon was attributed to cellular damage leading to nutrients outflow, and water evaporation increasing with temperature increasing during blanching process. Moreover, RF heating may destroy the microstructure of the sample leading to weight loss. The results of scanning electron microscopy (Fig. 5) verified that the microstructure of the sample was gradually destroyed with increasing temperature. When comparing with other blanching methods, RF heating showed a slight higher values of weight loss as compared to steam blanching and PEF treatments (Ignat, Manzocco, Brunton, Nicoli, & Lyng, 2015; Mukherjee & Chattopadhyay, 2007).
0.89bc 35.61bc 12.64bc 0.54cd 0.83e
0.99 0.95 0.96 0.99 1.02
± ± ± ± ± ± ± ± ± ± 689.88 680.09 686.51 644.29 588.18
0.01ab 0.04bc 0.02abc 0.01ab 0.00a
0.11 0.12 0.14 0.16 0.19
± ± ± ± ±
0.00ef 0.00def 0.00cdef 0.01bcd 0.01ab
89.27 73.32 72.21 72.02 84.67
0.03cd 2.95ab 1.83cd 0.55efg 0.01g ± ± ± ± ± 0.98 0.98 0.98 0.99 0.98 2.24b 0.94cd 37.96bc 0.01bc 0.54de
0.29bc 0.96bc 0.08b 0.34bc 0.24b
± ± ± ± ± ± ± ± ± ± 704.90 645.10 696.72 677.09 615.68
0.01ab 0.01ab 0.01ab 0.00ab 0.01ab
0.12 0.14 0.15 0.15 0.21
± ± ± ± ±
0.01cdef 0.02cdef 0.01bcde 0.01bcde 0.00a
87.03 94.75 85.73 70.58 65.35
0.09bcd 0.31abc 1.14fg 0.30efg 0.23fg ± ± ± ± ±
96.73 ± 4.64a
88.93 91.84 67.06 69.72 66.38 0.00cdef 0.02cdef 0.00cdef 0.00bc 0.02ab ± ± ± ± ±
0.11 ± 0.00f
0.14 0.15 0.13 0.16 0.19 0.02c 0.02ab 0.01abc 0.01ab 0.00ab 0.91 0.98 0.96 0.99 0.99 ± ± ± ± ± 682.31 682.39 701.10 695.09 716.26
± ± ± ± ±
0.99 ± 0.00ab 772.22 ± 10.66a
Springiness Fracturability (g)
75, 80, and 85 °C, respectively.
0.29b 1.38g 0.86i 0.27k 0.64m ± ± ± ± ±
The changes of color and total color difference (ΔE) of samples before and after RF heating are presented in table 1. Both the electrode gap and temperature had a significant effect (P < .05) on the value of L∗, a∗, b∗, and ΔE except that temperature had no significant effect on b∗ value. The value of L∗ and b∗ decreased with increasing temperature. This phenomenon was probably due to cell collapse and liquid release leading to a lower reflectance. Similar studies were reported in Peruvian carrot and cocoyam, showing that high pressure processing induced a decrease in both L∗ and b∗ values (Tribst et al., 2016). However, a∗ value showed a lightly increase with increasing temperature. In addition, the value of ΔE ranged from 3.35 to 11.81 indicating a very noticeable color change (ΔE > 3.0) (Adekunte, Tiwari, Cullen, Scannell, & O'Donnell, 2010). The increasing value of ΔE might be on account of Maillard reaction, non-enzymatic browning, and the change of heat-sensitive components (Deylami, Rahman, Tan, Bakar, & Olusegun, 2016).
Different letters in the same column indicate a significant differences (P < .05) among fresh and blanched samples using RF heating.
774.03 645.12 601.57 486.95 418.62 0.73b 0.37b 2.19b 1.31b 1.16b ± ± ± ± ± 3.93 4.14 6.05 3.35 5.11 0.68ab 0.36ab 2.95ab 1.32ab 1.15ab 35.89 35.64 34.75 37.57 34.89 0.27ab 0.57ab 0.07ab 0.56ab 0.42ab 130
± ± ± ± ± 68.56 67.03 65.15 65.23 65.99 65 70 75 80 85
0.42abcd 0.05abc 0.81bcd 0.67bcd 0.26abcd
−5.25 −5.46 −4.10 −5.51 −5.61
± ± ± ± ±
± ± ± ± ±
2.22c 1.20e 4.07f 0.79k 1.27m ± ± ± ± ± 744.15 690.01 651.92 490.19 416.44 0.50b 0.33b 0.36b 0.47b 0.89b ± ± ± ± ± 3.80 4.47 5.20 5.07 4.13 0.80ab 0.25ab 1.40ab 0.93ab 1.05ab 36.50 35.38 36.60 35.76 36.12 0.35ab 0.15ab 1.67ab 0.46ab 0.62b 120
± ± ± ± ± 66.31 66.68 64.19 64.68 65.83 65 70 75 80 85
0.72abcd 0.63abcd 0.51cd 0.55bcd 0.32abcd
−4.44 −5.34 −4.37 −5.15 −6.21
± ± ± ± ±
± ± ± ± ±
0.00b 0.47d 1.09h 0.35k 1.00L ± ± ± ± ± 771.35 701.25 618.17 497.52 431.87
824.19 ± 2.09a –
4.09 ± 0.94b 5.12 ± 0.62b 7.59 ± 4.79ab 11.81 ± 1.06a 6.76 ± 1.03b 1.12ab 1.17ab 4.41ab 4.12b 0.72ab ± ± ± ± ±
39.68 ± 1.13a
35.84 35.11 34.13 32.55 34.10 0.68ab 0.99ab 1.39ab 0.91a 0.53ab ± ± ± ± ±
−5.74 ± 0.53b
−4.68 −4.45 −3.96 −3.35 −5.26 0.35abc 1.14abcd 3.28d 1.49e 1.10cd ± ± ± ± ±
67.72 ± 0.14ab
67.25 66.52 63.55 59.34 64.02
25
Hardness (g) L
*
65 70 75 80 85
Control
110
Temperature (°C)
Color
a
*
b
*
ΔE
Texture
3.5. Effect of RF heating on the color
Electrode gap (mm)
Table 1 Color and texture values (mean ± SD) of potato cuboids subjected to RF heating at three electrode gaps (110, 120, and 130 mm) and five temperatures (65, 70, 75, 80, and 85 °C).
Cohesiveness
Chewiness
Z. Zhang et al.
3.6. Effect of RF heating on the texture Table 1 shows the texture of fresh and treated potato cuboids at different treatment conditions. Both electrode gap and temperature had significant effects (P < .05) on hardness, fracturability, and chewiness. Under the same electrode gap, hardness of RF treated samples decreased with increasing temperature. This phenomenon was resulted from water loss which directly reduced turgor pressure (Rocculi, Romani, Galindo, & Rosa, 2009). Furthermore, the higher content of starch granules exerted a greater swelling pressure, consequently resulted in a softer texture during blanching processing (Liu & Scanlon, 2007). Under all electrode gaps, the change of hardness in texture was less than 20% as compared to control sample at temperature of 65 and 70 °C. This trend is consistent with that obtained by Abu-Ghannam and Crowley (2006). However, the texture degraded rapidly when temperature was above 80 °C. The results showed that RF heating had significant effect on the texture of potato cuboids. 3.7. Effect of RF heating on the microstructure Upright microscope showed the morphology of potato parenchyma cell before and after RF heating in Fig. 5a. The photographs revealed the apparent differences among samples after different RF heating treatment. In the untreated sample, parenchyma cells showed the 180
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regular arrangement with integrated cell wall. Some different sized starch granules were observed in parenchyma cell. The parenchyma cells remained intact after RF heating with the final temperature of 65 and 70 °C. However, the cells become shrunken and some starch began to gelatinization when the samples were subjected to RF treatment with the final temperature of 75 °C. The starch granules were completely gelatinized when the RF heating reached 80 °C. Some cells were filled with the starchy matrix filled in entire cell, others presented no contents in the cell at 85 °C. This phenomenon indicated that some cells retain cell wall and some cell wall disintegrated. Similar results were observed by Bordoloi, Kaur, and Singh (2012), founding Moonlight potato kept cell wall after cooking while the cell walls of Nadine and Red Rascal potato were destroyed during cooking. Microstructure photographs of fresh and treated potato cuboids subjected to RF heating at fixed electrode gap of 120 mm with different final temperatures are listed in Fig. 5b. It was observed that the cell walls and starch grains were clearly presented in micrographs. In the fresh sample, the cells arranged neatly with certain regularity, and the middle lamella connected closely with cell wall. This phenomenon was consistent with that observed by microscope. Cell wall is composed of pectin together with other polysaccharides which glue neighboring cells and cement them firmly in the middle lamella (Bolwell, 1993). In case of 65 and 70 °C, the cell walls folded companying with wall separation. Above 75 °C, some cell wall disrupted and cell collapsed. The reason is probably that the pectin in the middle lamella was leached away leading to cell wall breakdown and the tissue softened markedly. Similar change of microstructure was observed in the blanching of carrot by hot water (Ando et al., 2016). The starch gelatinized significantly and aggravated with increasing temperature. Al-Khusaibi and Niranjan (2012) reported that hot water blanching had a significant destructive effect on the structure, while there were no noticeably effect on the cellular architecture and starch granules of potato under the highpressure treatment. Changes in microstructure further affected color, texture, and weight of potatoes. Therefore, it’s very important to explore the changes in microstructure of vegetables and fruits during RF heating.
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4. Conclusions The results showed that RF heating can efficiently inactivate mushroom PPO. CD analysis indicated that the changes of secondary structures companied with PPO inactivation. RF heating had significant effects on activity of PPO, texture, color, weight loss, microstructure of potatoes. The relative activity of potato PPO decreased with increasing temperature at all electrode gaps. Furthermore, different potato matrices had significant effects (P < .05) on the PPO relative activity. The enzyme extract showed the lowest PPO relative activity, followed by the potato cuboids and mashed potato. Microstructure analysis well explained the changes of color, texture, and weight of potatoes subjected to RF heating. This study provides the valuable information to understand the effect of RF heating on preventing browning of vegetables and shows the potential of this technology for blanching vegetables and fruits. Acknowledgement We thank Professor Zhenyu Li (College of Food Science and Engineering, Northwest A&F University, China) for his helpful advice and assistance with the English language in the manuscript submitted for publication. This study was supported by the general program (Grant No. 31371854) of National Natural Science Foundation of China, the Key Research Project of Shaanxi Province (2017ZDXM-SF-104). References Abu-Ghannam, N., & Crowley, H. (2006). The effect of low temperature blanching on the
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