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A short-term carbon dioxide treatment inhibits the browning of fresh-cut burdock
Postharvest Biology and Technology 110 (2015) 96–102
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
A short-term carbon dioxide treatment inhibits the browning of fresh-cut burdock Tiantian Donga,1, Jingying Shia,1, Cai-Zhong Jiangb , Yanyan Fenga , Yu Caoa , Qingguo Wanga,* a b
College of Food Science and Engineering, Shandong Agricultural University, Tai’an 271018, China Crops Pathology & Genetic Research Unit, USDA–ARS, One Shields Avenue, Davis, CA 95616, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 31 March 2015 Received in revised form 13 July 2015 Accepted 15 July 2015 Available online 31 July 2015
Fresh-cut burdock is susceptible to browning. The effect of short-term carbon dioxide (CO2) treatment on inhibiting browning of fresh-cut burdock during storage at 2–4 C was investigated. The results showed that the burdock slices treated with CO2 for 4 h, 6 h and 8 h exhibited better visual quality during 8 d storage, compared with the ones treated with air. CO2 treatment for 6 h on the fresh-cut burdock slices reduced the respiration rate, lowered the activity of PPO and PAL, and the content of total phenolic compounds. On the other hand, CO2 treatment increased the content of H2O2, enhanced the activity of CAT, POD, and SOD, maintained DPPH inhibition rate and decreased the content of MDA. The results indicate that the short-term pure CO2 treatment can extend the shelf life of fresh-cut burdock by inhibiting its browning and improving its quality. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Burdock Fresh-cut Carbon dioxide Browning
1. Introduction Fresh-cut burdock has been gradually recognized to have high potential because of the high nutritional value and biological properties (Chow et al., 1997; Duh, 1998; Chen et al., 2004). The white flesh of fresh-cut burdock root is very susceptible to browning which could limit the shelf life and decrease the nutritional value and visual quality (Tomás-Barberán and Espín, 2001). A research studied the fresh-cut burdock roots treated with 3% citric acid, 3% sodium chloride, 0.3% cystein and 3% sodium acetate to inhibit browning (Chung et al., 2012). Zhu et al. (2009) had studied the effects of the Ca(AsA)2 and 4-hexylresorcinol (4HR) on browning and quality of fresh-cut burdock. However, consumers nowadays tend to avoid the chemical additives due to potential harmful effects (Kim et al., 2014). The utilization of nontoxic, cheap, residue-free and environmentally friendly gases to retard the browning, preserve the quality and extend the shelf life of fruits and vegetables has become the main focus of consumers and researchers. CO2, as a low-cost, odorless, tasteless and colorless gas, represents a novel approach to non-thermally
* Corresponding author at: College of Food Science and Engineering, Shandong Agricultural University, Room 304, Building 9, 61 Daizong Street, Tai’an 271018, Shandong Province, China. Fax: +86 538 8249204. E-mail address: [email protected] (Q. Wang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.postharvbio.2015.07.014 0925-5214/ ã 2015 Elsevier B.V. All rights reserved.
inactivate undesirable enzymes and preserve the overall quality of fresh-cut produce. CO2 has been reported to decrease respiration rates, retard senescence, reduce or delay the overall enzymatic activity and alleviate physiological disorders (Aharoni et al., 1989; Kader et al., 1989; Day, 1994). Moreover, low respiration rate made the activity of phenylalanine ammonia lyase (PAL) decrease (Ke and Saltveit, 1989), which is the catalyst in the first step of phenylpropanoids biosynthesis (Jones, 1984), and thus affects the synthesis of phenolic compounds. Polyphenolic oxidase (PPO) is considered as a key enzyme in enzymic browning, which oxidize ortho-phenolsto quinones under the action of oxygen (Kavrayan and Aydemir, 2001; Eidhin et al., 2006). Peroxidase (POD) is another main enzyme in addition to PPO that functions in enzymatic browning (Yingsanga et al., 2008), browning mediated by which is different from PPOmediated browning related to the hydrogen peroxide (H2O2) generation with ongoing of PPO-mediated reactions (Toivonen and Brummell, 2008). Besides, catalase (CAT), as the H2O2 quenching agent had been demonstrated to form the biological defense system together with POD and superoxide dismutase (SOD) (Huang et al., 2005; Zhang and Tian, 2007) as they can eliminate free oxygen radicals (Peng et al., 2006). Membrane stability is also a major factor affecting browning rate for the compartmentalization of phenolic compounds and associated enzymes (Toivonen and Brummell, 2008) as fresh-cut processing easily disrupts cellular membranes, which causes lipid peroxidation and forms
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malondialdehyde (MDA), contributing to the deterioration and browning of the fresh-cut burdock (Duh, 1998). Low and high O2/CO2 atmospheres could reduce respiration rate and browning in minimally processed potatoes (Angós et al., 2007) and retard discoloration effectively in fresh-cut jicama (Aquinobolanos et al., 2000). High CO2 concentration alter intracellular pH and metabolic regulation, inhibits enzymes of phenolic metabolism and delays browning in lettuce tissues (Siriphanich and Kader, 1985). However, there is no report yet on the application of high concentration of CO2 for controlling browning of fresh-cut burdock. The objective of this research was to determine the possible application of CO2 as an anti-browning method for the fresh-cut burdock by evaluating its effects on the changes in visual quality and the physiological parameters. 2. Materials and methods 2.1. Raw material and sample preparation Burdock (Arctium lappa L.) purchased from Yangtze River Agriculture Co., Ltd., Weifang, Shandong Province, China, was immediately transported to the laboratory. The experiment used a batch of good quality burdock, selected with a diameter of about 3 cm and a length of 80 cm without injury and hollowness. The burdock was washed in tap water, rinsed in sodium chlorinated solution (200 mL L 1 NaClO) for 5 min to reduce the surface contamination, hand-peeled and cut diagonally into 3 mm slices. The burdock slices were immediately rinsed in 50 mL L 1 NaClO solution for 5 min, dried by draining and blotting with cheesecloth, and packed into 100 cm 120 cm polyethylene (PE) plastic bags. The air in the bag was removed by gently squeezing it. CO2 gas with purity over 99.9% (Spring Company, Taian, Shandong province, China) was passed through a wet cloth gauze for maintaining higher humidity, then through each bag from one side of the bag and out from the other side with appropriate flow rate to keep the bags fully inflated. Samples were treated for 4 h, 6 h and 8 h at 10 C. The treated slices (2.5 kg) and the control non-treated slices (2.5 kg) were packed into 40 cm 80 cm PE bags respectively and stored at 2–4 C. The samples were evaluated for quality, physiological and biochemical changes after 0, 2, 4, 6 and 8 d during the storage after CO2 treatment. For physiological and biochemical evaluations, only burdock slices treated with CO2 for 6 h were used and each sample had three replications for analysis. 2.2. Visual quality assay 2.2.1. Sensory evaluation The overall visual quality was evaluated on a 9–1 scale as described by Amodio et al. (2007). The sensory evaluation standard was as follows: 9 equaled excellent, with no defects; 7 equaled good, with minor defects; 5 equaled fair, with moderate defects; 3 equaled poor, with major defects; and 1 equaled unusable. A score of 6 was considered to be the limit of salability and the shelf-life was defined. 2.2.2. Color measurement The L* (lightness), a* (reddish–greenish) and b* (yellowish– bluish) indice of the CIELAB colorimetric system were used to evaluate the color change of the burdock samples (Abbott, 1999). The surface color change of slices was determined by the CIE L*, a*, b* scale using a colorimeter (CR-400, Minolta Co., Osaka, Japan) and calibrated on a standard white tile (L* = 97.06, a* = 0.04, b* = 2.01) before measured. Ten individual slices of each sample were measured twice (once on each side) at various time intervals.
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2.3. Analysis of respiration rate Burdock slices (100 g) from the CO2 treatment for 6 h were placed into LOCK plastic boxes (150 95 85 mm, Shanghai Lock trade Co., LTD) and sealed for 4 h to measure the concentration of CO2 at 0, 2, 4, 6 and 8 d during the storage to examine the respiration rate. The volume of CO2 was 1.0 L and its concentration was measured by a CO2 analyzer (PBI-940437B, PBI Dansensor, Denmark) and the respiration rate was calculated following the method of Castelló et al. (2006) and CO2 production rate expressed as mg kg 1 s 1. Three replications of each sample were conducted. 2.4. Physiological and biochemical assays Burdock slices selected randomly were collected at 0, 2, 4, 6, 8 d during storage after the 6 h CO2 treatment, frozen immediately with liquid nitrogen, ground into powder with a liquid nitrogen grinding apparatus (IKA A11 basic; IKA Werke GmbH & Co., KG, Staufen, Germany), and then stored at 80 C until measurement. 2.4.1. Determination of total phenolics The total phenolic content was measured according to the Folin–Ciocalteu procedure (Singleton and Rossi, 1965), with some modifications. Burdock powder (2.5 g) were mixed with 10 mL of 70% acetone and incubated for 2 h at 25 C. The sample was then centrifuged at 11,140 g for 10 min at 4 C, and the supernatant was used for analysis. For analysis, 0.2 mL of the supernatant was mixed with 0.5 mL of the Folin–Ciocalteu reagent. After mixing thoroughly for 3–4 min, 0.5 mL of 10% Na2CO3 solution was added into the mixture. The volume was then brought to 10 mL with water. After 1 h at room temperature, the absorbance was measured at 765 nm with a UV-spectrophotometer (TU1810, Beijing Purkinje General Instrument Co., Ltd., China). The absorbance of different gallic acid concentrations was used as the standard to determine the concentration of the total phenolic compounds of the burdock samples. 2.4.2. Assessment of PAL activity PAL activity was measured as previously described by MartínezTéllez and Lafuente (1997), with slight modifications. Burdock powder (2.5 g) were placed in a plastic test tube containing 0.25 g soluble PVPP, then mixed with 10 mL of 50 mmol L 1 borate buffer (pH 8.5). The sample was centrifuged at 11,140 g for 20 min at 4 C, and the supernatant was used for analysis. The reaction mixture in the testing tubes included 2 mL buffer (pH 8.5), 2 mL of 20 mmol L 1 phenylalanine and 0.1 mL of enzyme solution. The tubes were incubated at 40 C for 1 h. Absorbance was measured at 290 nm. One unit of PAL activity was defined as an increase of 0.01 absorbance units in one minute by the amount of the enzyme. The enzyme activity was expressed on a fresh weight basis as units kg 1. 2.4.3. Assessment of polyphenol oxidase (PPO) activity PPO activity was determined with some modifications to the procedure mentioned by Galeazzi et al. (1981). Burdock powder (2.5 g) were weighed into a plastic test tube containing 0.25 g insoluble polyvinylpolypyrrolidone (PVPP), mixed with 10 mL phosphate buffer (pH 6.8), centrifuged at 11,140 g for 20 min at 4 C, and the supernatant was used for analysis. The reaction mixture consisted of 0.1 mL of crude enzyme extract solution, 2.5 mL of 0.2 mol L 1 phosphate buffer (pH 6.8) and 0.5 mL of 20 mmol L 1 catechol solution. Enzyme activity was measured every five seconds for one minute by measuring the absorbance at 410 nm. One unit of enzyme activity was defined as the increase in absorbance by 0.01 per minute under assay conditions. The
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enzyme activity was expressed as units of activity per gram fresh weight. 2.5. Antioxidative capability assays 2.5.1. Measurement of hydrogen peroxide (H2O2) content H2O2 content was measured using the method described by Dagmar et al. (2001) with slight modification. Two grams of burdock powder were mixed with 10 mL of cooled acetone then centrifuged at 11,140 g for 10 min at 4 C. Then, 2 mL of the supernatant was mixed with 0.2 mL of 5% TiSO4 (v) solution, 0.4 mL ammonia water saturated solution to precipitate the titaniumhydro peroxide complex and the reaction mixture was centrifuged at 11,140 g for 10 min at 4 C. After removing the supernatant, the precipitate was dissolved in 5 mL of 2 mol L 1 H2SO4 and centrifuged again. The absorbance of the supernatant was read at 415 nm, using a mixture of 2 mL distilled water, 0.2 mL of 5% TiSO4 (v) solution, 0.4 mL ammonia water saturated solution and 5 mL of 2 mol L 1 H2SO4 as blank. The H2O2 content was calculated according to the standardcurve made by a known concentration of hydrogen peroxide and expressed on a fresh weight basis as mmol kg 1. 2.5.2. Measurement of catalase (CAT) activity Catalase (CAT) activity was determined following the method of Chance and Maehly (1955) with some modifications. Burdock powder (2.5 g) was weighed into a plastic test tube (50 mL) containing 0.25 g insoluble polyvinylpolypyrrolidone (PVPP), mixed with 10 mL of 0.1 mol L 1 cooled phosphate buffer (pH 7.0), centrifuged at 11,140 g for 10 min at 4 C and the supernatant was used for CAT activity analysis by monitoring the decomposition of H2O2 at 240 nm. One unit of CAT activity is defined as the amount of enzyme that produced a decrease of 0.01 absorbance units per minute. The enzyme activity was expressed on a fresh weight basis as units kg 1.
2.5.3. Assessment of superoxide dismutase (SOD) activity SOD was assayed by the nitroblue tetrazolium (NBT) methoddescribed by Dhindsa et al. (1980) with some modification. Burdock powder (2.5 g) was weighed into a plastic test tube (50 mL), mixed with 10 mL of 0.05 mol L 1 cooled phosphate buffer (PH 7.8), centrifuged at 11,140 g for 10 min at 4 C and the supernatant was used for analysis. The reaction mixture contained 0.3 mL of 130 mmol L 1 Met solution, 0.3 mL of 0.75 mmol L 1,0.3 mL of 0.1 mmol L 1 EDTA-Na2, 0.3 mL of 0.02 mmol L 1 riboflavin, 0.5 mL distilled water and 0.1 mL of the supernatant. The absorbance was measured at 560 nm. One unit of SOD was defined as the amount of enzyme required to cause a 50% inhibition of NBT reduction, under the assay conditions. The enzyme activity was expressed on a fresh weight basis as units kg 1. 2.5.4. Analysis of the peroxidase (POD) activity POD activity was assayed following the procedure described by Yang et al. (2008) with some modification. Burdock powder (2.5 g) were weighed into a plastic test tube, then homogenized with 10 mL of 0.1 mol L 1 borate buffer (pH 6.0), centrifuged at 11,140 g for 20 min at 4 C, and the supernatant was used for analysis. The reaction mixture contained 50 mL of 0.1 mol L 1 borate buffer (PH 6.0), 28 mL of guajacolum and 19 mL of 30% H2O2. For analysis, 3 mL of the reaction liquid and 200 mL of enzyme extract were added into one tube and 3 mL of the reaction liquid and 200 mL of 0.1 mol L 1 borate buffer (PH 6.0) were added into another tube as blank, and the absorbance was measured at 420 nm every 30 s for 5 min. One unit of enzyme activity was defined as the increase in absorbance by 0.01 per min under assay conditions, and enzyme activity was expressed on a fresh weight basis as units kg 1. 2.5.5. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) antioxidant assay The DPPH antioxidant assay of the fresh-cut burdock was carried out according to the method described by Kenny and O’Beirne (2010) with some modifications. Firstly, 0.2 mmol L 1 of
Fig. 1. Effects of CO2 treatment for 4 h, 6 h and 8 h on overall visual quality of fresh-cut burdock on day 4 after treatment. All the burdock slices treated with CO2 for 4 h, 6 h and 8 h and the control were stored at the same conditions with the temperature of 2–4 C and selected randomly from each group to take photographs to compare the visual quality on day 4.
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DPPH solution formulated with 95% ethanol was kept at 0–4 C in a dark place. Two grams of burdock powder were mixed with 10 mL of 95% ethanol and the sample was placed on an ultrasonic shaker for 30 min at 50 C, then centrifuged at 11,140 g for 10 min at 4 C and the supernatant was used for analysis. Then, 2 mL of the supernatant was mixed with 2 mL of the DPPH solution and incubated for 30 min. The absorbance was measured at 517 nm with the UV-spectrophotometer. The mixture of 2 mL of the DPPH solution and 2 mL of 95% ethanol solution was as the blank. Each sample had three technical replications. 2.5.6. Malondialdehyde (MDA) analysis Malondialdehyde content was determined based on the procedure described in (Hao and Liu, 2001), with slight modifications. Burdock powder (2.5 g) were weighed into a plastic test tube containing 0.25 g soluble PVPP, then mixed with 10 mL of phosphate buffer (pH 6.4), centrifuged at 11,140 g for 20 min at 4 C, and the supernatant was used for analysis. The mixture of 2.0 mL of the supernatant and 2 mL of 0.5% thiobarbituric acid (TBA, formulated with 15% trichloroacetic acid) was heated in boiling water for 18 min and then cooled rapidly, re-centrifuged at 11,140 g for 5 min. The absorbance of supernatant was separately measured at 532 nm and 600 nm. MDA concentration was calculated with the extinction coefficient and expressed on a fresh weight basis as mmol kg 1. 2.6. Experimental design and statistical analysis Three replicates of per treatment were conducted in a completely randomized design in all of the experiments, and all data analyzed and represented in the figures were the means of averages standard deviation by ANOVA with mean separation and LSD at *P < 0.05. 3. Results and discussion 3.1. Effects of short-term carbon dioxide treatment on the visual appearance of fresh-cut burdock The control slices showed browning by 2 d after cutting while the color of burdock slices CO2-treated for 4 h, 6 h, and 8 h was basically unchanged until 8 d after treatment (Fig. 1). The overall visual quality of the burdock was scored subjectively. The result shown in Fig. 2A indicated the score of control burdock slices declined to about 6 after 2 d, reaching the unacceptable commercial score, and losing the commercial value afterwards. Meanwhile the score of burdock slices treated with CO2 for 6 h was above 6 and still had commercial value by 8 d. Compared with the control, CO2 treatment could maintain the visual quality of fresh-cut burdock and extend shelf life. Changes in the color parameters L* and a* are shown in Fig. 2B and Fig. 2C. The L* value of control slices decreased during storage time while that of burdock slices treated with CO2 for 6 h was basically unchanged (Fig. 2B) and a* value of CO2treated burdock was significantly lower than that of control (*P < 0.05) (Fig. 2C), which demonstrated that CO2 treatment for 6 h prevented the browning of the burdock slices during storage as the increased in a* was related to the development of reddish colours as a consequence of the browning process (Castañer et al., 1999). 3.2. The effects of carbon dioxide treatment on the content of phenolic compounds, PAL activity, and PPO activity of fresh-cut burdock Changes in the total phenolic content and activities of PAL and PPO in CO2-treated burdock and the control showed a similar trend during the experiment period (Fig. 3). Total phenolics content of
Fig. 2. Changes of overall visual quality (A) and the values of L* (B) and a* (C) of the fresh-cut burdock treated with CO2 for 6 h and the control at different storage times. Data are the averages of three replications standard deviation. Each replicate has 10 burdock slices. Different letters in the same time indicate significant difference (*P 0.05) according to LSD test.
both groups decreased consistently and the content of CO2-treated burdock was significantly (*P < 0.05) lower than that of nontreated burdock from 2 to 6 d (Fig. 3A). Phenolic compounds reacted with PPO to form brown polymers during processing or storage, resulting in a decrease in phenolic content and quality of fresh-cut fruits and vegetables (Tomás-Barberán and Espín, 2001). Compared with the control, PAL activity in CO2-treated burdock was also significantly (*P < 0.05) lower from 2 to 6 d (Fig. 3B). PAL is the catalyst for the first step of the phenylpropanoid biosynthesis, which subsequently form into a wide variety of phenolic compounds (Jones, 1984). These compounds are the root cause of browning and loss of aesthetic quality of fresh-cut fruits and vegetables (Tomás-Barberán and Espín, 2001). PPO is one of the key enzymes in enzymatic browning (TomásBarberán and Espín, 2001). PPO activity of burdock slices treated with CO2 was significantly lower than that of the control (*P < 0.05) during the storage period (Fig. 3C), suggesting that CO2 treatment inhibits enzymatic browning of the fresh-cut burdock slices by lowering the activity of PPO. Same results had been obtained in previous studies (Bi et al., 2011; Gui et al., 2007). Before 2 d of storage, the increased phenolics stimulated PPO activity and PPO activity increased. Then, the increased of PPO activity and the reaction of PPO and phenolic substrates made the decreased of phenolics compounds (Fig. 3A and C). Enzyme’s activity is
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Fig. 4. The effects of CO2 treatment on respiration rate of fresh-cut burdock. Data are the averages of three replications standard deviation. Different letters indicate significant difference (*P 0.05) according to LSD test.
respiration rate, decrease the PAL activity and then reduce the synthesis of the phenols. 3.4. The effects of carbon dioxide treatment on H2O2 content, CAT, POD and SOD activity of fresh-cut burdock
Fig. 3. The effects of CO2 treatment on the content of phenolics (A) and the activities of PAL (B) and PPO (C) of fresh-cut burdock slices. Data are the averages of three replications standard deviation. Different letters in the same time indicate significant difference (*P 0.05) according to LSD test.
influenced by many chemical and physical factors such as protons, metals, inorganic ions, PH, temperatures, and enzyme activators, inhibitors and stabilizers (Purich, 2010). The mechanism of lowering PPO activity by CO2 treatment in fresh-cut burdock needs further study. 3.3. The effects of carbon dioxide treatment on respiration rate of fresh-cut burdock Compared with the control, the respiration rate of burdock treated with CO2 for 6 h was significantly (*P < 0.05) lower during the storage time (Fig. 4), suggesting that CO2 treatment could inhibit the respiration rate of fresh-cut burdock. The fresh-cut processing may elevate the respiration rate (Jacobo-Velázquez et al., 2011), resulting in aggravation of browning process (Cefola et al., 2012). Besides, high respiration rate led to the increase of PAL activity (Ke and Saltveit, 1989), thereby promoting the production of the phenolic compounds used as substrates by PPO to produce brown substances (Tomás-Barberán, 2000). So, CO2 treatment decreased the respiration rate of fresh-cut burdock and thus inhibited PAL activity (Fig. 3B), thereby reducing the production of the phenolic compounds (Fig. 3A) which was consistent with the findings of Tomás-Barberán (2000) that high CO2 concentration could inhibit the activity of PAL and the synthesis of phenolic compounds. Therefore, our results suggested that CO2 treatment inhibits browning of fresh-cut burdock as it can retard the
The content of H2O2 and activity of CAT, POD and SOD are shown in Fig. 5. The content of H2O2 of the fresh-cut burdock treated with CO2 was significantly (*P < 0.05) higher than that of the control (Fig. 5A). The higher H2O2 content of burdock slices treated with CO2 in the present result may be related to the mechanism that high CO2 as an abiotic stress stimulates the biological defense signaling and response, which is consistent with previous reports that elevated CO2 could induce alterations in plant metabolism and accumulation of H2O2, which possibly plays a role as a signaling molecule (AbdElgawad et al., 2015; Veal et al., 2007; Neill et al., 2002) to activate plant defense mechanisms against environmental stresses (Cingoz et al., 2014). H2O2 is one of the most important reactive oxygen species and its relative stability is necessary for avoiding the oxidative burst (Bradley et al., 1992). Besides, H2O2 could regulate antioxidative enzymes such as CAT, POD and SOD to response to oxidative stress (Veal et al., 2007; Cingoz et al., 2014). The CO2 treatment increased H2O2 content and thus significantly increased CAT (*P < 0.05), POD (*P < 0.05) and SOD (*P < 0.05) activity of burdock slices compared to the control (Fig. 5B–D). 3.5. The effects of carbon dioxide treatment on DPPH inhibition of fresh-cut burdock The scavenging of DPPH radical is the basis of antioxidant assay because of its stability (Kordali et al., 2005). The DPPH inhibition in CO2-treated and non-treated burdock had a continuous decline after cutting, but the inhibition of the control fell significantly faster (*P < 0.05) than that of treated burdock (Fig. 6). The results suggest that CO2 treatment could protect the antioxidative capability of fresh-cut burdock to retard the browning caused by oxidation, which might be related to the increase of H2O2 content by enhancing the defense system. And the increase of POD, CAT and SOD activity might also make a contribution to the maintenance of the DPPH inhibition as they formed the defence systerm against the reactive oxygen species in organisms (Zhang and Tian, 2007; Cingoz et al., 2014) and thus prevent or alleviate the oxidative browning of fresh-cut burdock.
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Fig. 5. The effects of CO2 treatment on the H2O2 content (A), CAT activity (B), POD activity (C) and SOD activity (D) of the fresh-cut burdocks during storage time. Data are the averages of three replications standard deviation. Different letters in the same time indicate significant difference (*P 0.05) according to LSD test.
Fig. 6. Effect of the CO2 treatment for 6 h on antioxidative capability measured by DPPH inactivation of the fresh-cut burdocks during storage time. Data are the averages of three replications standard deviation. Different letters in the same time indicate significant difference (*P 0.05) according to LSD test.
3.6. The effects of carbon dioxide treatment on MDA content of freshcut burdock Lipid peroxidation is supposed to be the consequence of toxic metabolites that cause intracellular membrane disruption (Dianzani et al., 2008). MDA is the end product of lipid peroxidation, and it provides a useful way to assess the lipid peroxidation in biological materials (Draper and Hadley, 1990). The MDA content in the fresh-cut burdock treated with CO2 for 6 h was significantly (*P <0 .05) lower than that of the control during the whole storage (Fig. 7). The result indicated that CO2 treatment could weaken the process of lipid peroxidation and thus inhibit the browning and quality deterioration by strengthening the antioxidative capability.
Fig. 7. Effect of the CO2 treatment for 6 h on the content of MDA. Data are the averages of three replications standard deviation. Different letters in the same time indicate significant difference (*P 0.05) according to LSD test.
4. Conclusion CO2 treatment inhibits browning of fresh-cut burdock. The shelf life of fresh-cut burdock treated with CO2 for 6 h was extended to 8 d at 2–4 C compared to 2 d for non-treated control. The results showed that CO2 treatment maintained the quality of fresh-cut burdock, reduced its respiration rate, lowered the activity of PPO and PAL and phenolic compounds content, increased the content of H2O2 and the activity of CAT, POD and SOD, kept DPPH inhibition rate, and decreased MDA content. Acknowledgement This work was partially supported by Shandong Provincial Research Center for Engineering and Technology of Food Safety of Fruits and Vegetables, Jinan, China.
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References Abbott, J.A., 1999. Quality measurement of fruits and vegetables. Postharvest Biol. Technol. 15, 207–225. AbdElgawad, H., Farfan-Vignolo, E.R., Vos de, D., Asard, H., 2015. Elevated CO2 mitigates drought and temperature-induced oxidative stress differently in grasses and legumes. Plant Sci. 231, 1–10. Aharoni, N., Reuveni, A., Dvir, O., 1989. Modified atmospheres in film packages delay senescence and decay of fresh herbs. Int. Symp. Postharvest Handing Fruit Veg. 258, 255–262. Amodio, M.L., Cabezas-Serrano, A.B., Rinaldi, R., Colelli, G., 2007. Implementation of rating scales for visual quality evaluation of various vegetable crops. Produce quality rating scales and color charts. Postharvest Hortic. Series. No. 23. Angós, I., Vírseda, P., Fernández, T., 2007. Control of respiration and color modification on minimally processed potatoes by means of low and high O2/CO2 atmospheres. Postharvest Biol. Technol. 48, 422–430. Aquino-bolanos, E.N., Cantwell, M.I., Peiser, G., Mercado-silva, E., 2000. Changes in the quality of fresh-cut jicama in relation to storage temperatures and controlled atmospheres. J. Food Sci. 65, 1238–1243. Bi, X.F., Wu, J.H., Zhang, Y., Xu, Z.H., Liao, X.J., 2011. High pressure carbon dioxide treatment for fresh-cut carrot slices. Innovative Food Sci. Emerg. Technol. 12, 298–304. Bradley, D.J., Kjellbom, P., Lamb, C.J., 1992. Elicitor-induced and wound-induce doxidative cross-linking of a proline-rich plant-cell wall protein-a novel, rapid defense response. Cell 70, 21–30. Castelló, M.L., Fito, P.J., Chiralt, A., 2006. Effect of osmotic dehydration and vacuum impregnation on respiration rate of cut strawberries. LWT—Food Sci. Technol. 39, 1171–1179. Castañer, M., María, I.G., Ruíz, M.V., Artés, F., 1999. Browning susceptibility of minimally processed baby and romaine lettuces. Eur. Food Res. Technol. 209, 52–56. Cefola, M., D'Antuono, I., Pace, B., Calabrese, N., Carito, A.V., Cardinali, L.A., 2012. Biochemical relationships and browning index for accessing the storage suitability of artichoke genotypes. Food Res. Int. 48, 397–403. Chance, B., Maehly, A., 1955. Assay of catalases and peroxidases. Methods Enzymol. 2, 764–775. Chen, F.A., Wu, A.B., Chen, C.Y., 2004. The influence of different treatments on the free radical scavenging activity of burdock and variations of its active components. Food Chem. 86, 479–484. Chow, L.W., Wang, S.J., Duh, P.D., 1997. Antibacterial activity of burdock. Food Sci. 24, 195–202. Chung, H.S., Moon, K.D., Seong, J.H., 2012. Effects of processing temperature and browning inhibitor on quality ptoperties of fresh-cut burdock roots. Korean J. Food Preserv. 19, 31–36. Cingoz, G.S., Verma, S.K., Gurel, E., 2014. Hydrogen peroxide-induced antioxidant activities and cardiotonic glycoside accumulation in callus cultures of endemic Digitalis species. Plant Physiol. Biochem. 82, 89–92. Dagmar, P., Sairam, R.K., Srivastava, G.C., Singh, D.V., 2001. Oxidative stress and antioxidant activity as the basis of senescence in maize leaves. Plant Sci. 161, 765–771. Day, B.P.F., 1994. Modified atmosphere packaging and active packaging of fruits and vegetables. In: Ahvenainen, R., Mattila-Sandholm, T., Ohlsson, T. (Eds.), Minimal Processing of Foods. VTT Symposium series 142, Majvik, pp. 14–15. Dhindsa, R.S., Dhindsa, P.P., Thorpe, T.A., 1980. Leaf senescence correlated with increased levels of membrane permeability and lipid-peroxidationand decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 32, 93–101. Dianzani, M.U., Barrera, G., Alvarez, S., Evelson, P., Boveris, A., 2008. Pathology and physiology of lipid peroxidation and its carbonyl products. Free Radical Pathophysiol. 19–38. Draper, H.H., Hadley, M., 1990. Malondialdehyde determination as index of lipid perqxidation. Methods Enzymol. 186, 421–423. Duh, P.D., 1998. Antioxidant activity of burdock (Arctium lappa Linné): its scavenging effect on free-radical and active oxygen. J. Am. Oil Chem. Soc. 75, 455–461. Eidhin, D.M., Murphy, E., O’beirne, D., 2006. Polyphenoloxidase from apple (malus domestica Borkh. Cv Bramley’s Seedling): purification strategies and characterization. J. Food Sci. 71, C51–C58. Galeazzi, M.A.M., Sagarbieri, V., Constantidines, S.M., 1981. Isolation, purification and physicochemical characterization of polyphenoloxidase (PPO) from a dwarf variety of banana (Musa cavendishii L.). J. Food Sci. 46, 150–155.
Gui, F.Q., Wu, J.H., Chen, F., Liao, X.J., Hu, X.S., Zhang, Z.H., Wang, Z.F., 2007. Inactivation of polyphenol oxidases in cloudy apple juice exposed to supercritical carbon dioxide. Food Chem. 100, 1678–1685. Hao, J.J., Liu, Y.J., 2001. The Technology of Plant Physiology Experiment. Liaoning Science and Technology Press, Shenyang, China. Huang, Y.H., Hua, H., et al., 2005. The research on the extraction factor of catalase in pork liver. Lett. Biotechnol. 16, 40–42. Jones, H.D., 1984. Phenylalanine ammonia-lyase: regulation of its induction, and its role in plant development. Phytochemistry 23, 1349–1359. Jacobo-Velázquez, D.A., Martínez-Hernández, G.B., Rodríguez, S., Del, C., Cao, C.M., Cisneros-Zevallos, L., 2011. Plants as biofactories: physiological role ofreactive oxygen species on the accumulation of phenolic antioxidants in carrot tissue under wounding and hyperoxia stress. J. Agric. Food Chem. 59, 6583–6593. Kader, A.A., Zagory, D., Kerbal, E.L., Wang, C.Y., 1989. Modified atmosphere packaging of fruits and vegetables. Critical. Revi. Food Sci. Nutr. 28, 1–30. Kavrayan, D., Aydemir, T., 2001. Partial purification and characterization of polyphenoloxidase from peppermint (Mentha piperita). Food Chem. 74, 147– 154. Ke, D., Saltveit, M.E., 1989. Carbon dioxide-induced brown stain development as related to phenolic metabolism in iceberg lettuce. J. Am. Soc. Hortic. Sci. 114, 789–794. Kenny, O., O'Beirne, D., 2010. Antioxidant phytochemicals in fresh-cut carrot disks as affected by peeling method. Postharvest Biol. Technol. 58, 247–253. Kim, D.H., Kim, H.B., Chung, H.S., Moon, K.D., 2014. Browning control of fresh-cut lettuce by phytoncide treatment. Food Chem. 159, 188–192. Kordali, S., Cakir, A., Mavi, A., Kilic, H., Yildirim, A., 2005. Screening of chemical composition and antifungal and antioxidant activities of the essential oils from three Turkish Artemisia species. J. Agric. Food Chem. 53, 1408–1416. Martínez-Téllez, M.A., Lafuente, M.T., 1997. Effect of high temperature conditioning on ethylene, phenylalanine ammonia-lyase, peroxidase and polyphenol oxidase activities in flavedo of chilled ‘Fortune’ mandarin fruit. J. Plant Physiol. 150, 674– 678. Neill, S., Desikan, R., Hancock, J., 2002. Hydrogen peroxide signalling. Curr. Opin. Plant Biol. 5, 388–395. Peng, Y., Li, Y., Yang, G.X., He, G.Y., 2006. Effects of aluminum stress on the activities of SOD, POD, CAT and the contents of MDA in the seedlings of different wheat cultivars. J. Biotechnol. 3, 38–42 (in Chinese). Purich, D.L., 2010. Factors influencing enzyme activity. In: Purich, D.L. (Ed.), Enzyme Kinetics: Catalysis & Control: A Reference of Theory and Best-practice Methods. Elsevier, pp. 379–484. Singleton, V.L., Rossi, J.A., 1965. Colourimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16, 144– 158. Siriphanich, J., Kader, A., 1985. Effects of CO2 on total phenolics,phenylalanine ammonia lyase, and polyphenol oxidasein lettuce tissue. J. Am. Soc. Hortic. Sci. 110, 249–253. Toivonen, P.M.A., Brummell, D.A., 2008. Biochemical bases of appearance and texture changes in fresh-cut fruit and vegetables. Postharvest Biol. Technol. 48, 1–14. Tomás-Barberán, F.A., 2000. Strategies for reducing phenolic-related quality loss in fresh-cut lettuce. In: improving postharvest technologies of fruits. Vegetables and Ornamentals.V. 1. IRR Conference, Murcia, Spain, pp. 374–380. Tomás-Barberán, F.A., Espín, J.C., 2001. Phenolic compounds and related enzymes as determinants of quality in fruits and vegetables. J. Sci. Food Agric. 81, 853–876. Veal, E.A., Day, A.M., Morgan, B.A., 2007. Hydrogen peroxide sensing and signalling. Mol. Cell. 26, 1–14. Yang, Z., Zheng, Y., Cao, S., 2008. Effect of high oxygen atmosphere storage on quality, antioxidant enzymes, and DPPH-radical scavenging activity of Chinese bayberry fruit. J. Agric. Food Chem. 57, 176–181. Yingsanga, P., Srilaong, V., Kanlayanarat, S., Noichinda, S., McGlasson, W.B., 2008. Relationship between browning and related enzymes (PAL, PPO and POD) in rambutan fruit (Nephelium lappaceum Linn.) cvs. Rongrien and See-Chompoo. Postharvest Biol. Technol. 50, 164–168. Zhang, K.S., Tian, H.L., 2007. Research on the function of peroxidase. Food Sci. Technol. 1, 8–11. Zhu, Y.Z., Wang, J., Wang, D.Y., Xu, W.M., Wang, Z.J., Cao, J.M., 2009. Effects of Ca (AsA)2 on browning and quality of fresh-cut burdock (Arctium lappa L.). Jiangsu J. Agric. Sci. 25, 655–659.
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
A short-term carbon dioxide treatment inhibits the browning of fresh-cut burdock Tiantian Donga,1, Jingying Shia,1, Cai-Zhong Jiangb , Yanyan Fenga , Yu Caoa , Qingguo Wanga,* a b
College of Food Science and Engineering, Shandong Agricultural University, Tai’an 271018, China Crops Pathology & Genetic Research Unit, USDA–ARS, One Shields Avenue, Davis, CA 95616, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 31 March 2015 Received in revised form 13 July 2015 Accepted 15 July 2015 Available online 31 July 2015
Fresh-cut burdock is susceptible to browning. The effect of short-term carbon dioxide (CO2) treatment on inhibiting browning of fresh-cut burdock during storage at 2–4 C was investigated. The results showed that the burdock slices treated with CO2 for 4 h, 6 h and 8 h exhibited better visual quality during 8 d storage, compared with the ones treated with air. CO2 treatment for 6 h on the fresh-cut burdock slices reduced the respiration rate, lowered the activity of PPO and PAL, and the content of total phenolic compounds. On the other hand, CO2 treatment increased the content of H2O2, enhanced the activity of CAT, POD, and SOD, maintained DPPH inhibition rate and decreased the content of MDA. The results indicate that the short-term pure CO2 treatment can extend the shelf life of fresh-cut burdock by inhibiting its browning and improving its quality. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Burdock Fresh-cut Carbon dioxide Browning
1. Introduction Fresh-cut burdock has been gradually recognized to have high potential because of the high nutritional value and biological properties (Chow et al., 1997; Duh, 1998; Chen et al., 2004). The white flesh of fresh-cut burdock root is very susceptible to browning which could limit the shelf life and decrease the nutritional value and visual quality (Tomás-Barberán and Espín, 2001). A research studied the fresh-cut burdock roots treated with 3% citric acid, 3% sodium chloride, 0.3% cystein and 3% sodium acetate to inhibit browning (Chung et al., 2012). Zhu et al. (2009) had studied the effects of the Ca(AsA)2 and 4-hexylresorcinol (4HR) on browning and quality of fresh-cut burdock. However, consumers nowadays tend to avoid the chemical additives due to potential harmful effects (Kim et al., 2014). The utilization of nontoxic, cheap, residue-free and environmentally friendly gases to retard the browning, preserve the quality and extend the shelf life of fruits and vegetables has become the main focus of consumers and researchers. CO2, as a low-cost, odorless, tasteless and colorless gas, represents a novel approach to non-thermally
* Corresponding author at: College of Food Science and Engineering, Shandong Agricultural University, Room 304, Building 9, 61 Daizong Street, Tai’an 271018, Shandong Province, China. Fax: +86 538 8249204. E-mail address: [email protected] (Q. Wang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.postharvbio.2015.07.014 0925-5214/ ã 2015 Elsevier B.V. All rights reserved.
inactivate undesirable enzymes and preserve the overall quality of fresh-cut produce. CO2 has been reported to decrease respiration rates, retard senescence, reduce or delay the overall enzymatic activity and alleviate physiological disorders (Aharoni et al., 1989; Kader et al., 1989; Day, 1994). Moreover, low respiration rate made the activity of phenylalanine ammonia lyase (PAL) decrease (Ke and Saltveit, 1989), which is the catalyst in the first step of phenylpropanoids biosynthesis (Jones, 1984), and thus affects the synthesis of phenolic compounds. Polyphenolic oxidase (PPO) is considered as a key enzyme in enzymic browning, which oxidize ortho-phenolsto quinones under the action of oxygen (Kavrayan and Aydemir, 2001; Eidhin et al., 2006). Peroxidase (POD) is another main enzyme in addition to PPO that functions in enzymatic browning (Yingsanga et al., 2008), browning mediated by which is different from PPOmediated browning related to the hydrogen peroxide (H2O2) generation with ongoing of PPO-mediated reactions (Toivonen and Brummell, 2008). Besides, catalase (CAT), as the H2O2 quenching agent had been demonstrated to form the biological defense system together with POD and superoxide dismutase (SOD) (Huang et al., 2005; Zhang and Tian, 2007) as they can eliminate free oxygen radicals (Peng et al., 2006). Membrane stability is also a major factor affecting browning rate for the compartmentalization of phenolic compounds and associated enzymes (Toivonen and Brummell, 2008) as fresh-cut processing easily disrupts cellular membranes, which causes lipid peroxidation and forms
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malondialdehyde (MDA), contributing to the deterioration and browning of the fresh-cut burdock (Duh, 1998). Low and high O2/CO2 atmospheres could reduce respiration rate and browning in minimally processed potatoes (Angós et al., 2007) and retard discoloration effectively in fresh-cut jicama (Aquinobolanos et al., 2000). High CO2 concentration alter intracellular pH and metabolic regulation, inhibits enzymes of phenolic metabolism and delays browning in lettuce tissues (Siriphanich and Kader, 1985). However, there is no report yet on the application of high concentration of CO2 for controlling browning of fresh-cut burdock. The objective of this research was to determine the possible application of CO2 as an anti-browning method for the fresh-cut burdock by evaluating its effects on the changes in visual quality and the physiological parameters. 2. Materials and methods 2.1. Raw material and sample preparation Burdock (Arctium lappa L.) purchased from Yangtze River Agriculture Co., Ltd., Weifang, Shandong Province, China, was immediately transported to the laboratory. The experiment used a batch of good quality burdock, selected with a diameter of about 3 cm and a length of 80 cm without injury and hollowness. The burdock was washed in tap water, rinsed in sodium chlorinated solution (200 mL L 1 NaClO) for 5 min to reduce the surface contamination, hand-peeled and cut diagonally into 3 mm slices. The burdock slices were immediately rinsed in 50 mL L 1 NaClO solution for 5 min, dried by draining and blotting with cheesecloth, and packed into 100 cm 120 cm polyethylene (PE) plastic bags. The air in the bag was removed by gently squeezing it. CO2 gas with purity over 99.9% (Spring Company, Taian, Shandong province, China) was passed through a wet cloth gauze for maintaining higher humidity, then through each bag from one side of the bag and out from the other side with appropriate flow rate to keep the bags fully inflated. Samples were treated for 4 h, 6 h and 8 h at 10 C. The treated slices (2.5 kg) and the control non-treated slices (2.5 kg) were packed into 40 cm 80 cm PE bags respectively and stored at 2–4 C. The samples were evaluated for quality, physiological and biochemical changes after 0, 2, 4, 6 and 8 d during the storage after CO2 treatment. For physiological and biochemical evaluations, only burdock slices treated with CO2 for 6 h were used and each sample had three replications for analysis. 2.2. Visual quality assay 2.2.1. Sensory evaluation The overall visual quality was evaluated on a 9–1 scale as described by Amodio et al. (2007). The sensory evaluation standard was as follows: 9 equaled excellent, with no defects; 7 equaled good, with minor defects; 5 equaled fair, with moderate defects; 3 equaled poor, with major defects; and 1 equaled unusable. A score of 6 was considered to be the limit of salability and the shelf-life was defined. 2.2.2. Color measurement The L* (lightness), a* (reddish–greenish) and b* (yellowish– bluish) indice of the CIELAB colorimetric system were used to evaluate the color change of the burdock samples (Abbott, 1999). The surface color change of slices was determined by the CIE L*, a*, b* scale using a colorimeter (CR-400, Minolta Co., Osaka, Japan) and calibrated on a standard white tile (L* = 97.06, a* = 0.04, b* = 2.01) before measured. Ten individual slices of each sample were measured twice (once on each side) at various time intervals.
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2.3. Analysis of respiration rate Burdock slices (100 g) from the CO2 treatment for 6 h were placed into LOCK plastic boxes (150 95 85 mm, Shanghai Lock trade Co., LTD) and sealed for 4 h to measure the concentration of CO2 at 0, 2, 4, 6 and 8 d during the storage to examine the respiration rate. The volume of CO2 was 1.0 L and its concentration was measured by a CO2 analyzer (PBI-940437B, PBI Dansensor, Denmark) and the respiration rate was calculated following the method of Castelló et al. (2006) and CO2 production rate expressed as mg kg 1 s 1. Three replications of each sample were conducted. 2.4. Physiological and biochemical assays Burdock slices selected randomly were collected at 0, 2, 4, 6, 8 d during storage after the 6 h CO2 treatment, frozen immediately with liquid nitrogen, ground into powder with a liquid nitrogen grinding apparatus (IKA A11 basic; IKA Werke GmbH & Co., KG, Staufen, Germany), and then stored at 80 C until measurement. 2.4.1. Determination of total phenolics The total phenolic content was measured according to the Folin–Ciocalteu procedure (Singleton and Rossi, 1965), with some modifications. Burdock powder (2.5 g) were mixed with 10 mL of 70% acetone and incubated for 2 h at 25 C. The sample was then centrifuged at 11,140 g for 10 min at 4 C, and the supernatant was used for analysis. For analysis, 0.2 mL of the supernatant was mixed with 0.5 mL of the Folin–Ciocalteu reagent. After mixing thoroughly for 3–4 min, 0.5 mL of 10% Na2CO3 solution was added into the mixture. The volume was then brought to 10 mL with water. After 1 h at room temperature, the absorbance was measured at 765 nm with a UV-spectrophotometer (TU1810, Beijing Purkinje General Instrument Co., Ltd., China). The absorbance of different gallic acid concentrations was used as the standard to determine the concentration of the total phenolic compounds of the burdock samples. 2.4.2. Assessment of PAL activity PAL activity was measured as previously described by MartínezTéllez and Lafuente (1997), with slight modifications. Burdock powder (2.5 g) were placed in a plastic test tube containing 0.25 g soluble PVPP, then mixed with 10 mL of 50 mmol L 1 borate buffer (pH 8.5). The sample was centrifuged at 11,140 g for 20 min at 4 C, and the supernatant was used for analysis. The reaction mixture in the testing tubes included 2 mL buffer (pH 8.5), 2 mL of 20 mmol L 1 phenylalanine and 0.1 mL of enzyme solution. The tubes were incubated at 40 C for 1 h. Absorbance was measured at 290 nm. One unit of PAL activity was defined as an increase of 0.01 absorbance units in one minute by the amount of the enzyme. The enzyme activity was expressed on a fresh weight basis as units kg 1. 2.4.3. Assessment of polyphenol oxidase (PPO) activity PPO activity was determined with some modifications to the procedure mentioned by Galeazzi et al. (1981). Burdock powder (2.5 g) were weighed into a plastic test tube containing 0.25 g insoluble polyvinylpolypyrrolidone (PVPP), mixed with 10 mL phosphate buffer (pH 6.8), centrifuged at 11,140 g for 20 min at 4 C, and the supernatant was used for analysis. The reaction mixture consisted of 0.1 mL of crude enzyme extract solution, 2.5 mL of 0.2 mol L 1 phosphate buffer (pH 6.8) and 0.5 mL of 20 mmol L 1 catechol solution. Enzyme activity was measured every five seconds for one minute by measuring the absorbance at 410 nm. One unit of enzyme activity was defined as the increase in absorbance by 0.01 per minute under assay conditions. The
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enzyme activity was expressed as units of activity per gram fresh weight. 2.5. Antioxidative capability assays 2.5.1. Measurement of hydrogen peroxide (H2O2) content H2O2 content was measured using the method described by Dagmar et al. (2001) with slight modification. Two grams of burdock powder were mixed with 10 mL of cooled acetone then centrifuged at 11,140 g for 10 min at 4 C. Then, 2 mL of the supernatant was mixed with 0.2 mL of 5% TiSO4 (v) solution, 0.4 mL ammonia water saturated solution to precipitate the titaniumhydro peroxide complex and the reaction mixture was centrifuged at 11,140 g for 10 min at 4 C. After removing the supernatant, the precipitate was dissolved in 5 mL of 2 mol L 1 H2SO4 and centrifuged again. The absorbance of the supernatant was read at 415 nm, using a mixture of 2 mL distilled water, 0.2 mL of 5% TiSO4 (v) solution, 0.4 mL ammonia water saturated solution and 5 mL of 2 mol L 1 H2SO4 as blank. The H2O2 content was calculated according to the standardcurve made by a known concentration of hydrogen peroxide and expressed on a fresh weight basis as mmol kg 1. 2.5.2. Measurement of catalase (CAT) activity Catalase (CAT) activity was determined following the method of Chance and Maehly (1955) with some modifications. Burdock powder (2.5 g) was weighed into a plastic test tube (50 mL) containing 0.25 g insoluble polyvinylpolypyrrolidone (PVPP), mixed with 10 mL of 0.1 mol L 1 cooled phosphate buffer (pH 7.0), centrifuged at 11,140 g for 10 min at 4 C and the supernatant was used for CAT activity analysis by monitoring the decomposition of H2O2 at 240 nm. One unit of CAT activity is defined as the amount of enzyme that produced a decrease of 0.01 absorbance units per minute. The enzyme activity was expressed on a fresh weight basis as units kg 1.
2.5.3. Assessment of superoxide dismutase (SOD) activity SOD was assayed by the nitroblue tetrazolium (NBT) methoddescribed by Dhindsa et al. (1980) with some modification. Burdock powder (2.5 g) was weighed into a plastic test tube (50 mL), mixed with 10 mL of 0.05 mol L 1 cooled phosphate buffer (PH 7.8), centrifuged at 11,140 g for 10 min at 4 C and the supernatant was used for analysis. The reaction mixture contained 0.3 mL of 130 mmol L 1 Met solution, 0.3 mL of 0.75 mmol L 1,0.3 mL of 0.1 mmol L 1 EDTA-Na2, 0.3 mL of 0.02 mmol L 1 riboflavin, 0.5 mL distilled water and 0.1 mL of the supernatant. The absorbance was measured at 560 nm. One unit of SOD was defined as the amount of enzyme required to cause a 50% inhibition of NBT reduction, under the assay conditions. The enzyme activity was expressed on a fresh weight basis as units kg 1. 2.5.4. Analysis of the peroxidase (POD) activity POD activity was assayed following the procedure described by Yang et al. (2008) with some modification. Burdock powder (2.5 g) were weighed into a plastic test tube, then homogenized with 10 mL of 0.1 mol L 1 borate buffer (pH 6.0), centrifuged at 11,140 g for 20 min at 4 C, and the supernatant was used for analysis. The reaction mixture contained 50 mL of 0.1 mol L 1 borate buffer (PH 6.0), 28 mL of guajacolum and 19 mL of 30% H2O2. For analysis, 3 mL of the reaction liquid and 200 mL of enzyme extract were added into one tube and 3 mL of the reaction liquid and 200 mL of 0.1 mol L 1 borate buffer (PH 6.0) were added into another tube as blank, and the absorbance was measured at 420 nm every 30 s for 5 min. One unit of enzyme activity was defined as the increase in absorbance by 0.01 per min under assay conditions, and enzyme activity was expressed on a fresh weight basis as units kg 1. 2.5.5. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) antioxidant assay The DPPH antioxidant assay of the fresh-cut burdock was carried out according to the method described by Kenny and O’Beirne (2010) with some modifications. Firstly, 0.2 mmol L 1 of
Fig. 1. Effects of CO2 treatment for 4 h, 6 h and 8 h on overall visual quality of fresh-cut burdock on day 4 after treatment. All the burdock slices treated with CO2 for 4 h, 6 h and 8 h and the control were stored at the same conditions with the temperature of 2–4 C and selected randomly from each group to take photographs to compare the visual quality on day 4.
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DPPH solution formulated with 95% ethanol was kept at 0–4 C in a dark place. Two grams of burdock powder were mixed with 10 mL of 95% ethanol and the sample was placed on an ultrasonic shaker for 30 min at 50 C, then centrifuged at 11,140 g for 10 min at 4 C and the supernatant was used for analysis. Then, 2 mL of the supernatant was mixed with 2 mL of the DPPH solution and incubated for 30 min. The absorbance was measured at 517 nm with the UV-spectrophotometer. The mixture of 2 mL of the DPPH solution and 2 mL of 95% ethanol solution was as the blank. Each sample had three technical replications. 2.5.6. Malondialdehyde (MDA) analysis Malondialdehyde content was determined based on the procedure described in (Hao and Liu, 2001), with slight modifications. Burdock powder (2.5 g) were weighed into a plastic test tube containing 0.25 g soluble PVPP, then mixed with 10 mL of phosphate buffer (pH 6.4), centrifuged at 11,140 g for 20 min at 4 C, and the supernatant was used for analysis. The mixture of 2.0 mL of the supernatant and 2 mL of 0.5% thiobarbituric acid (TBA, formulated with 15% trichloroacetic acid) was heated in boiling water for 18 min and then cooled rapidly, re-centrifuged at 11,140 g for 5 min. The absorbance of supernatant was separately measured at 532 nm and 600 nm. MDA concentration was calculated with the extinction coefficient and expressed on a fresh weight basis as mmol kg 1. 2.6. Experimental design and statistical analysis Three replicates of per treatment were conducted in a completely randomized design in all of the experiments, and all data analyzed and represented in the figures were the means of averages standard deviation by ANOVA with mean separation and LSD at *P < 0.05. 3. Results and discussion 3.1. Effects of short-term carbon dioxide treatment on the visual appearance of fresh-cut burdock The control slices showed browning by 2 d after cutting while the color of burdock slices CO2-treated for 4 h, 6 h, and 8 h was basically unchanged until 8 d after treatment (Fig. 1). The overall visual quality of the burdock was scored subjectively. The result shown in Fig. 2A indicated the score of control burdock slices declined to about 6 after 2 d, reaching the unacceptable commercial score, and losing the commercial value afterwards. Meanwhile the score of burdock slices treated with CO2 for 6 h was above 6 and still had commercial value by 8 d. Compared with the control, CO2 treatment could maintain the visual quality of fresh-cut burdock and extend shelf life. Changes in the color parameters L* and a* are shown in Fig. 2B and Fig. 2C. The L* value of control slices decreased during storage time while that of burdock slices treated with CO2 for 6 h was basically unchanged (Fig. 2B) and a* value of CO2treated burdock was significantly lower than that of control (*P < 0.05) (Fig. 2C), which demonstrated that CO2 treatment for 6 h prevented the browning of the burdock slices during storage as the increased in a* was related to the development of reddish colours as a consequence of the browning process (Castañer et al., 1999). 3.2. The effects of carbon dioxide treatment on the content of phenolic compounds, PAL activity, and PPO activity of fresh-cut burdock Changes in the total phenolic content and activities of PAL and PPO in CO2-treated burdock and the control showed a similar trend during the experiment period (Fig. 3). Total phenolics content of
Fig. 2. Changes of overall visual quality (A) and the values of L* (B) and a* (C) of the fresh-cut burdock treated with CO2 for 6 h and the control at different storage times. Data are the averages of three replications standard deviation. Each replicate has 10 burdock slices. Different letters in the same time indicate significant difference (*P 0.05) according to LSD test.
both groups decreased consistently and the content of CO2-treated burdock was significantly (*P < 0.05) lower than that of nontreated burdock from 2 to 6 d (Fig. 3A). Phenolic compounds reacted with PPO to form brown polymers during processing or storage, resulting in a decrease in phenolic content and quality of fresh-cut fruits and vegetables (Tomás-Barberán and Espín, 2001). Compared with the control, PAL activity in CO2-treated burdock was also significantly (*P < 0.05) lower from 2 to 6 d (Fig. 3B). PAL is the catalyst for the first step of the phenylpropanoid biosynthesis, which subsequently form into a wide variety of phenolic compounds (Jones, 1984). These compounds are the root cause of browning and loss of aesthetic quality of fresh-cut fruits and vegetables (Tomás-Barberán and Espín, 2001). PPO is one of the key enzymes in enzymatic browning (TomásBarberán and Espín, 2001). PPO activity of burdock slices treated with CO2 was significantly lower than that of the control (*P < 0.05) during the storage period (Fig. 3C), suggesting that CO2 treatment inhibits enzymatic browning of the fresh-cut burdock slices by lowering the activity of PPO. Same results had been obtained in previous studies (Bi et al., 2011; Gui et al., 2007). Before 2 d of storage, the increased phenolics stimulated PPO activity and PPO activity increased. Then, the increased of PPO activity and the reaction of PPO and phenolic substrates made the decreased of phenolics compounds (Fig. 3A and C). Enzyme’s activity is
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Fig. 4. The effects of CO2 treatment on respiration rate of fresh-cut burdock. Data are the averages of three replications standard deviation. Different letters indicate significant difference (*P 0.05) according to LSD test.
respiration rate, decrease the PAL activity and then reduce the synthesis of the phenols. 3.4. The effects of carbon dioxide treatment on H2O2 content, CAT, POD and SOD activity of fresh-cut burdock
Fig. 3. The effects of CO2 treatment on the content of phenolics (A) and the activities of PAL (B) and PPO (C) of fresh-cut burdock slices. Data are the averages of three replications standard deviation. Different letters in the same time indicate significant difference (*P 0.05) according to LSD test.
influenced by many chemical and physical factors such as protons, metals, inorganic ions, PH, temperatures, and enzyme activators, inhibitors and stabilizers (Purich, 2010). The mechanism of lowering PPO activity by CO2 treatment in fresh-cut burdock needs further study. 3.3. The effects of carbon dioxide treatment on respiration rate of fresh-cut burdock Compared with the control, the respiration rate of burdock treated with CO2 for 6 h was significantly (*P < 0.05) lower during the storage time (Fig. 4), suggesting that CO2 treatment could inhibit the respiration rate of fresh-cut burdock. The fresh-cut processing may elevate the respiration rate (Jacobo-Velázquez et al., 2011), resulting in aggravation of browning process (Cefola et al., 2012). Besides, high respiration rate led to the increase of PAL activity (Ke and Saltveit, 1989), thereby promoting the production of the phenolic compounds used as substrates by PPO to produce brown substances (Tomás-Barberán, 2000). So, CO2 treatment decreased the respiration rate of fresh-cut burdock and thus inhibited PAL activity (Fig. 3B), thereby reducing the production of the phenolic compounds (Fig. 3A) which was consistent with the findings of Tomás-Barberán (2000) that high CO2 concentration could inhibit the activity of PAL and the synthesis of phenolic compounds. Therefore, our results suggested that CO2 treatment inhibits browning of fresh-cut burdock as it can retard the
The content of H2O2 and activity of CAT, POD and SOD are shown in Fig. 5. The content of H2O2 of the fresh-cut burdock treated with CO2 was significantly (*P < 0.05) higher than that of the control (Fig. 5A). The higher H2O2 content of burdock slices treated with CO2 in the present result may be related to the mechanism that high CO2 as an abiotic stress stimulates the biological defense signaling and response, which is consistent with previous reports that elevated CO2 could induce alterations in plant metabolism and accumulation of H2O2, which possibly plays a role as a signaling molecule (AbdElgawad et al., 2015; Veal et al., 2007; Neill et al., 2002) to activate plant defense mechanisms against environmental stresses (Cingoz et al., 2014). H2O2 is one of the most important reactive oxygen species and its relative stability is necessary for avoiding the oxidative burst (Bradley et al., 1992). Besides, H2O2 could regulate antioxidative enzymes such as CAT, POD and SOD to response to oxidative stress (Veal et al., 2007; Cingoz et al., 2014). The CO2 treatment increased H2O2 content and thus significantly increased CAT (*P < 0.05), POD (*P < 0.05) and SOD (*P < 0.05) activity of burdock slices compared to the control (Fig. 5B–D). 3.5. The effects of carbon dioxide treatment on DPPH inhibition of fresh-cut burdock The scavenging of DPPH radical is the basis of antioxidant assay because of its stability (Kordali et al., 2005). The DPPH inhibition in CO2-treated and non-treated burdock had a continuous decline after cutting, but the inhibition of the control fell significantly faster (*P < 0.05) than that of treated burdock (Fig. 6). The results suggest that CO2 treatment could protect the antioxidative capability of fresh-cut burdock to retard the browning caused by oxidation, which might be related to the increase of H2O2 content by enhancing the defense system. And the increase of POD, CAT and SOD activity might also make a contribution to the maintenance of the DPPH inhibition as they formed the defence systerm against the reactive oxygen species in organisms (Zhang and Tian, 2007; Cingoz et al., 2014) and thus prevent or alleviate the oxidative browning of fresh-cut burdock.
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Fig. 5. The effects of CO2 treatment on the H2O2 content (A), CAT activity (B), POD activity (C) and SOD activity (D) of the fresh-cut burdocks during storage time. Data are the averages of three replications standard deviation. Different letters in the same time indicate significant difference (*P 0.05) according to LSD test.
Fig. 6. Effect of the CO2 treatment for 6 h on antioxidative capability measured by DPPH inactivation of the fresh-cut burdocks during storage time. Data are the averages of three replications standard deviation. Different letters in the same time indicate significant difference (*P 0.05) according to LSD test.
3.6. The effects of carbon dioxide treatment on MDA content of freshcut burdock Lipid peroxidation is supposed to be the consequence of toxic metabolites that cause intracellular membrane disruption (Dianzani et al., 2008). MDA is the end product of lipid peroxidation, and it provides a useful way to assess the lipid peroxidation in biological materials (Draper and Hadley, 1990). The MDA content in the fresh-cut burdock treated with CO2 for 6 h was significantly (*P <0 .05) lower than that of the control during the whole storage (Fig. 7). The result indicated that CO2 treatment could weaken the process of lipid peroxidation and thus inhibit the browning and quality deterioration by strengthening the antioxidative capability.
Fig. 7. Effect of the CO2 treatment for 6 h on the content of MDA. Data are the averages of three replications standard deviation. Different letters in the same time indicate significant difference (*P 0.05) according to LSD test.
4. Conclusion CO2 treatment inhibits browning of fresh-cut burdock. The shelf life of fresh-cut burdock treated with CO2 for 6 h was extended to 8 d at 2–4 C compared to 2 d for non-treated control. The results showed that CO2 treatment maintained the quality of fresh-cut burdock, reduced its respiration rate, lowered the activity of PPO and PAL and phenolic compounds content, increased the content of H2O2 and the activity of CAT, POD and SOD, kept DPPH inhibition rate, and decreased MDA content. Acknowledgement This work was partially supported by Shandong Provincial Research Center for Engineering and Technology of Food Safety of Fruits and Vegetables, Jinan, China.
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References Abbott, J.A., 1999. Quality measurement of fruits and vegetables. Postharvest Biol. Technol. 15, 207–225. AbdElgawad, H., Farfan-Vignolo, E.R., Vos de, D., Asard, H., 2015. Elevated CO2 mitigates drought and temperature-induced oxidative stress differently in grasses and legumes. Plant Sci. 231, 1–10. Aharoni, N., Reuveni, A., Dvir, O., 1989. Modified atmospheres in film packages delay senescence and decay of fresh herbs. Int. Symp. Postharvest Handing Fruit Veg. 258, 255–262. Amodio, M.L., Cabezas-Serrano, A.B., Rinaldi, R., Colelli, G., 2007. Implementation of rating scales for visual quality evaluation of various vegetable crops. Produce quality rating scales and color charts. Postharvest Hortic. Series. No. 23. Angós, I., Vírseda, P., Fernández, T., 2007. Control of respiration and color modification on minimally processed potatoes by means of low and high O2/CO2 atmospheres. Postharvest Biol. Technol. 48, 422–430. Aquino-bolanos, E.N., Cantwell, M.I., Peiser, G., Mercado-silva, E., 2000. Changes in the quality of fresh-cut jicama in relation to storage temperatures and controlled atmospheres. J. Food Sci. 65, 1238–1243. Bi, X.F., Wu, J.H., Zhang, Y., Xu, Z.H., Liao, X.J., 2011. High pressure carbon dioxide treatment for fresh-cut carrot slices. Innovative Food Sci. Emerg. Technol. 12, 298–304. Bradley, D.J., Kjellbom, P., Lamb, C.J., 1992. Elicitor-induced and wound-induce doxidative cross-linking of a proline-rich plant-cell wall protein-a novel, rapid defense response. Cell 70, 21–30. Castelló, M.L., Fito, P.J., Chiralt, A., 2006. Effect of osmotic dehydration and vacuum impregnation on respiration rate of cut strawberries. LWT—Food Sci. Technol. 39, 1171–1179. Castañer, M., María, I.G., Ruíz, M.V., Artés, F., 1999. Browning susceptibility of minimally processed baby and romaine lettuces. Eur. Food Res. Technol. 209, 52–56. Cefola, M., D'Antuono, I., Pace, B., Calabrese, N., Carito, A.V., Cardinali, L.A., 2012. Biochemical relationships and browning index for accessing the storage suitability of artichoke genotypes. Food Res. Int. 48, 397–403. Chance, B., Maehly, A., 1955. Assay of catalases and peroxidases. Methods Enzymol. 2, 764–775. Chen, F.A., Wu, A.B., Chen, C.Y., 2004. The influence of different treatments on the free radical scavenging activity of burdock and variations of its active components. Food Chem. 86, 479–484. Chow, L.W., Wang, S.J., Duh, P.D., 1997. Antibacterial activity of burdock. Food Sci. 24, 195–202. Chung, H.S., Moon, K.D., Seong, J.H., 2012. Effects of processing temperature and browning inhibitor on quality ptoperties of fresh-cut burdock roots. Korean J. Food Preserv. 19, 31–36. Cingoz, G.S., Verma, S.K., Gurel, E., 2014. Hydrogen peroxide-induced antioxidant activities and cardiotonic glycoside accumulation in callus cultures of endemic Digitalis species. Plant Physiol. Biochem. 82, 89–92. Dagmar, P., Sairam, R.K., Srivastava, G.C., Singh, D.V., 2001. Oxidative stress and antioxidant activity as the basis of senescence in maize leaves. Plant Sci. 161, 765–771. Day, B.P.F., 1994. Modified atmosphere packaging and active packaging of fruits and vegetables. In: Ahvenainen, R., Mattila-Sandholm, T., Ohlsson, T. (Eds.), Minimal Processing of Foods. VTT Symposium series 142, Majvik, pp. 14–15. Dhindsa, R.S., Dhindsa, P.P., Thorpe, T.A., 1980. Leaf senescence correlated with increased levels of membrane permeability and lipid-peroxidationand decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 32, 93–101. Dianzani, M.U., Barrera, G., Alvarez, S., Evelson, P., Boveris, A., 2008. Pathology and physiology of lipid peroxidation and its carbonyl products. Free Radical Pathophysiol. 19–38. Draper, H.H., Hadley, M., 1990. Malondialdehyde determination as index of lipid perqxidation. Methods Enzymol. 186, 421–423. Duh, P.D., 1998. Antioxidant activity of burdock (Arctium lappa Linné): its scavenging effect on free-radical and active oxygen. J. Am. Oil Chem. Soc. 75, 455–461. Eidhin, D.M., Murphy, E., O’beirne, D., 2006. Polyphenoloxidase from apple (malus domestica Borkh. Cv Bramley’s Seedling): purification strategies and characterization. J. Food Sci. 71, C51–C58. Galeazzi, M.A.M., Sagarbieri, V., Constantidines, S.M., 1981. Isolation, purification and physicochemical characterization of polyphenoloxidase (PPO) from a dwarf variety of banana (Musa cavendishii L.). J. Food Sci. 46, 150–155.
Gui, F.Q., Wu, J.H., Chen, F., Liao, X.J., Hu, X.S., Zhang, Z.H., Wang, Z.F., 2007. Inactivation of polyphenol oxidases in cloudy apple juice exposed to supercritical carbon dioxide. Food Chem. 100, 1678–1685. Hao, J.J., Liu, Y.J., 2001. The Technology of Plant Physiology Experiment. Liaoning Science and Technology Press, Shenyang, China. Huang, Y.H., Hua, H., et al., 2005. The research on the extraction factor of catalase in pork liver. Lett. Biotechnol. 16, 40–42. Jones, H.D., 1984. Phenylalanine ammonia-lyase: regulation of its induction, and its role in plant development. Phytochemistry 23, 1349–1359. Jacobo-Velázquez, D.A., Martínez-Hernández, G.B., Rodríguez, S., Del, C., Cao, C.M., Cisneros-Zevallos, L., 2011. Plants as biofactories: physiological role ofreactive oxygen species on the accumulation of phenolic antioxidants in carrot tissue under wounding and hyperoxia stress. J. Agric. Food Chem. 59, 6583–6593. Kader, A.A., Zagory, D., Kerbal, E.L., Wang, C.Y., 1989. Modified atmosphere packaging of fruits and vegetables. Critical. Revi. Food Sci. Nutr. 28, 1–30. Kavrayan, D., Aydemir, T., 2001. Partial purification and characterization of polyphenoloxidase from peppermint (Mentha piperita). Food Chem. 74, 147– 154. Ke, D., Saltveit, M.E., 1989. Carbon dioxide-induced brown stain development as related to phenolic metabolism in iceberg lettuce. J. Am. Soc. Hortic. Sci. 114, 789–794. Kenny, O., O'Beirne, D., 2010. Antioxidant phytochemicals in fresh-cut carrot disks as affected by peeling method. Postharvest Biol. Technol. 58, 247–253. Kim, D.H., Kim, H.B., Chung, H.S., Moon, K.D., 2014. Browning control of fresh-cut lettuce by phytoncide treatment. Food Chem. 159, 188–192. Kordali, S., Cakir, A., Mavi, A., Kilic, H., Yildirim, A., 2005. Screening of chemical composition and antifungal and antioxidant activities of the essential oils from three Turkish Artemisia species. J. Agric. Food Chem. 53, 1408–1416. Martínez-Téllez, M.A., Lafuente, M.T., 1997. Effect of high temperature conditioning on ethylene, phenylalanine ammonia-lyase, peroxidase and polyphenol oxidase activities in flavedo of chilled ‘Fortune’ mandarin fruit. J. Plant Physiol. 150, 674– 678. Neill, S., Desikan, R., Hancock, J., 2002. Hydrogen peroxide signalling. Curr. Opin. Plant Biol. 5, 388–395. Peng, Y., Li, Y., Yang, G.X., He, G.Y., 2006. Effects of aluminum stress on the activities of SOD, POD, CAT and the contents of MDA in the seedlings of different wheat cultivars. J. Biotechnol. 3, 38–42 (in Chinese). Purich, D.L., 2010. Factors influencing enzyme activity. In: Purich, D.L. (Ed.), Enzyme Kinetics: Catalysis & Control: A Reference of Theory and Best-practice Methods. Elsevier, pp. 379–484. Singleton, V.L., Rossi, J.A., 1965. Colourimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16, 144– 158. Siriphanich, J., Kader, A., 1985. Effects of CO2 on total phenolics,phenylalanine ammonia lyase, and polyphenol oxidasein lettuce tissue. J. Am. Soc. Hortic. Sci. 110, 249–253. Toivonen, P.M.A., Brummell, D.A., 2008. Biochemical bases of appearance and texture changes in fresh-cut fruit and vegetables. Postharvest Biol. Technol. 48, 1–14. Tomás-Barberán, F.A., 2000. Strategies for reducing phenolic-related quality loss in fresh-cut lettuce. In: improving postharvest technologies of fruits. Vegetables and Ornamentals.V. 1. IRR Conference, Murcia, Spain, pp. 374–380. Tomás-Barberán, F.A., Espín, J.C., 2001. Phenolic compounds and related enzymes as determinants of quality in fruits and vegetables. J. Sci. Food Agric. 81, 853–876. Veal, E.A., Day, A.M., Morgan, B.A., 2007. Hydrogen peroxide sensing and signalling. Mol. Cell. 26, 1–14. Yang, Z., Zheng, Y., Cao, S., 2008. Effect of high oxygen atmosphere storage on quality, antioxidant enzymes, and DPPH-radical scavenging activity of Chinese bayberry fruit. J. Agric. Food Chem. 57, 176–181. Yingsanga, P., Srilaong, V., Kanlayanarat, S., Noichinda, S., McGlasson, W.B., 2008. Relationship between browning and related enzymes (PAL, PPO and POD) in rambutan fruit (Nephelium lappaceum Linn.) cvs. Rongrien and See-Chompoo. Postharvest Biol. Technol. 50, 164–168. Zhang, K.S., Tian, H.L., 2007. Research on the function of peroxidase. Food Sci. Technol. 1, 8–11. Zhu, Y.Z., Wang, J., Wang, D.Y., Xu, W.M., Wang, Z.J., Cao, J.M., 2009. Effects of Ca (AsA)2 on browning and quality of fresh-cut burdock (Arctium lappa L.). Jiangsu J. Agric. Sci. 25, 655–659.