Food Chemistry 126 (2011) 1693–1699
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Integrated application of nitric oxide and modified atmosphere packaging to improve quality retention of button mushroom (Agaricus bisporus) Tianjia Jiang a, Xiaolin Zheng a, Jianrong Li a, Guoxing Jing a, Luyun Cai b, Tiejin Ying b,⇑ a b
College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310035, PR China Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, PR China
a r t i c l e
i n f o
Article history: Received 7 September 2010 Received in revised form 4 November 2010 Accepted 8 December 2010 Available online 15 December 2010 Keywords: Button mushroom Nitric oxide Browning Antioxidant enzymes Quality parameters
a b s t r a c t Button mushrooms (Agaricus bisporus) were dipped for 10 min in different concentrations (0.5, 1, and 2 mM) of 2,20 -(hydroxynitrosohydrazino)-bisethanamine (DETANO), a nitric oxide donor, then packed in biorientated polypropylene (BOPP) bags, heat sealed and stored at 4 °C for 16 days (d). Mushroom weight loss, firmness, colour, percent open caps, total phenolics, ascorbic acid and H2O2 contents, superoxide anion (O 2 ) production rate and activities of polyphenol oxidase (PPO), superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) were measured. The results indicate that treatment with 1 mM DETANO maintained a high level of firmness, delayed browning and cap opening, promoted the accumulation of phenolics, ascorbic acid and reduced the increases in both O 2 production rate and H2O2 content. Furthermore, NO inhibited the activity of PPO, and increased the antioxidant enzymes activities of CAT, SOD and APX throughout storage period. Thus it was observed that application of NO in combination with modified atmosphere packaging (MAP) can extend the storage life of button mushroom up to 12 d. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Button mushroom (Agaricus bisporus) is one of the most popular mushrooms, traditionally cultivated in the world. Consumption and production of this edible mushroom have grown continuously in the past years. It is the most extensively cultivated edible mushroom, comprising 32% of the worldwide production (Chang, 1999). Mushrooms are a good source of vitamin B2, niacin, folates and many mineral elements (Mattila et al., 2001). However, button mushrooms only have a short shelf life of 3–4 d, they lose their commercial value within a few days, due to browning, water loss, senescence and microbial attack. The short shelf-life of mushroom is an impediment to the distribution and marketing of the fresh product. Thus, prolonging post-harvest storage while preserving their quality would benefit the mushroom industry as well as consumers. Nitric oxide (NO) is a highly reactive free radical gas that was initially notable as an industrial pollutant but is now known to be involved in resisting vegetative stress and senescence of horticultural products. Short-term exposure to a low concentration of NO gas or its donor compounds has been shown to extend the post-harvest life of various intact fresh fruits and vegetables (Wills, Ku, & Leshem, 2000; Wills, Soegiarto, & Bowyer, 2007; Zhu & Zhou,
⇑ Corresponding author. Tel./fax: +86 571 86971162. E-mail address: [email protected] (T. Ying). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.12.060
2007). It has been shown that they delayed ripening (Wills et al., 2000; Zhu & Zhou, 2007), inhibited ethylene biosynthesis (Eum, Kim, Choi, & Lee, 2009; Zhu, Sun, Liu, & Zhou, 2008), inhibited cut-surface browning (Pristijono, Wills, & Golding, 2006; Wills, Pristijono, & Golding, 2008), and enhanced resistance to post-harvest diseases (Zhu & Zhou, 2007). The mechanism of action of NO in delaying senescence of post-harvest horticultural produce, though not completely understood, is via the inhibition of ethylene biosynthesis (Eum et al., 2009; Zhu, Liu, & Zhou, 2006). However, adequate evidence does not exist to ascertain the mode of action of NO. Modified atmosphere packaging (MAP) has become increasingly common in recent years and has been reported to be the most economical and effective method of extending the shelf life of mushroom (Tano, Arul, Doyon, & Castaigne, 1999). Active modification is created by replacing gases in the package with a desired mixture of gases. On the other hand, passive modification happens when product is sealed in a package made with a selected film, and a desired atmosphere develops naturally as a consequence of product respiration and diffusion of gases through the film. Modified atmosphere in terms of reduced O2 and elevated CO2 can extend the post-harvest life of fruits and vegetables by reducing their respiration rate as well as production of ethylene, minimising metabolic activity, delaying enzymatic browning, retaining visual appearance (Kader, 1986). In recent studies, Xing, Wang, Feng, and Tan (2008) found that among different packaging films [polyvinyl chloride (PVC), two types of polyethylene (PE6 and PE11), polyoletin (PO),
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and biaxially oriented polypropylene (BOPP)], BOPP maintained the post-harvest appearance of the mushroom most effectively by significantly reducing the incidence of unsightly aerial hyphae on the pileal surface and restricting mushroom softening. Those samples also exhibited smaller initial decreases in soluble protein, smaller increases in reducing sugar content, and lower levels of malondialdehyde accumulation during storage. To our knowledge, there are little information about the effect of NO on button mushroom during storage. Therefore, the objective of this research was to investigate the effect of integrated application of NO and MAP employing BOPP, on quality retention of button mushroom.
mushroom colour values of L⁄ = 97, a⁄ = 2 and b⁄ = 0. The hue (H), chroma (C), and browning index (BI), which represents the purity of brown colour (Palou, Lopez-Malo, Barbosa-Canovas, Welti-Chanes, & Swanson, 1999), were calculated according to the following equations: 2
Hue ¼ tan1 ðb=aÞ Chroma ¼ ða2 þ b Þ1=2 BI ¼ ½100ðx 0:31Þ=0:172;
where x
¼ ða þ 1:75LÞ=ð5:645L þ a 3:012bÞ 2.5. Percent open caps and overall acceptability
2. Materials and methods 2.1. Mushroom treatment and storage Button mushroom used in this study were harvested from a local farm in Hangzhou, China. The mushrooms were transported to the laboratory in one hour after picking, then stored in darkness at 4 ± 1 °C and 90% relative humidity (RH). The day after, mushrooms were screened for uniform size and maturity and absence of mechanical damage. The mushrooms were dipped in 0.5, 1, and 2 mM 2,20 -(hydroxynitrosohydrazino)-bisethanamine (DETANO, the donor of NO; Sigma) aqueous solutions for 10 min at 20 °C with dip in distilled water used as a control (stand-alone MAP). The mushrooms (65 ± 5 g) were then drained in the air for another 5 min and packaged in biaxially oriented polypropylene (BOPP) bags (MAP; 35 lm thickness; size 18 20 cm; O2 permeance 38 1014 mol s1 m2 Pa1 at 23 °C; Xinlei Gongmao Co. Ltd., Zhejiang, China). The MAP was sealed with a heat sealer to create a passive modified atmosphere around the mushrooms. Samples were then stored for 16 d at 4 ± 1 °C and 90% relative humidity (RH). Twenty-five replicates were included in each treatment group, and subsequently every 4 d, five replicates from each treatment group were randomly selected and analysed for physicochemical parameters. 2.2. Package atmosphere composition O2 and CO2 concentrations in packages were evaluated by using a SCY-2A O2 and CO2 Analyser (Xinrui Instrument Co., Shanghai, China). Gas samples were taken from the bags with a 20 ml syringe. 2.3. Weight loss and firmness evaluation Weight loss was determined by weighing the whole mushroom before and after the storage period. Weight loss was expressed as the percentage of loss of weight with respect to the initial weight. A penetration test was performed on the mushroom cap using a TA.XT2i texture analyser (Stable Micro Systems, UK), using a 5 mm diameter cylindrical probe. Samples were penetrated 5 mm in depth. The speed of the probe was 2.0 mm s1 during the pretest and penetration. Force and time data were recorded with Texture Expert (Version 1.0) from Stable Micro Systems. From the force vs. time curves, firmness was defined as the maximum force. 2.4. Colour The surface colour of mushroom caps was measured with a WSC-S Colourimeter (Shanghai precision instrument Co. Ltd., Shanghai, China). To analyse the L⁄ (light/dark), a⁄ (red/green) and b⁄ (yellow/blue) values, each mushroom was measured at three equidistant points of the cap and compared to the ideal
Criteria for judging the percentage of open caps were based on the development of umbrella-like shape of the cap followed by detachment of veil. The percent open caps was determined from known number of mushrooms as:
%Open caps ¼ Noc =Nt 100 where Nt = total number of mushrooms; Noc = number of opencaped mushrooms. The overall acceptability based on colour, texture and percent open caps was done by a panel of four judges on round table basis using four-point scale where 1 = poor, 2 = fair, 3 = good and 4 = excellent. 2.6. Analysis of functional components Quantification of the total soluble phenolic compounds was carried out using the method proposed by Singleton and Rossi (1965). Five grams of mushroom cap were homogenised with 20 ml of 80% ethanol for 24 h; the homogenised mix was then filtered through two layers of cheesecloth, and the filtered liquid was centrifuged at 10,000g for 15 min. One millilitre of the supernatant liquid was mixed with 1 ml of Folin Ciocalteu reagent and 10 ml of sodium carbonate (7%). This was topped up to 25 ml with distilled water and left to settle for 1 h. The absorbance was then read at 750 nm. A standard curve of gallic acid was used for quantification. The determination of total ascorbic acid was carried out as described by Hanson et al. (2004). On the basis of coupling 2,4-dinitrophenylhydrazine (DNPH) with the ketonic groups of dehydroascorbic acid through the oxidation of ascorbic acid by 2,6-dichlorophenolindophenol (DCPIP) to give a yellow/orange colour in acidic conditions. Superoxide anion production rate was measured by monitoring the nitrite formation from hydroxylamine in the presence of O 2 , as described by Wang and Luo (1990). Mushroom tissues (4.0 g) were homogenised with 12 ml of 50 mM potassium phosphate buffer (pH 7.8) containing 1% (w/v) polyvinylpyrrolidone at 0 °C, and then centrifuged at 5000g and 4 °C for 15 min. The obtained supernatant (1 ml) was mixed with 0.9 ml of 50 mM potassium phosphate buffer (pH 7.8) and 0.1 ml of 10 mM hydroxylamine hydrochloride, and then incubated for 30 min at 25 °C. The incubated solution (1 ml) was added to 1 ml of 17 mM 3-aminobenzenesulphonic acid and 1 ml of 7 mM 1-naphthylamine, and then further kept for 20 min at 25 °C. The absorbance was recorded at 530 nm. A standard curve with NO2 was used to calculate the O 2 production rate from the reaction equation of O 2 with hydroxylamine. The O2 production rate was expressed as nmol/min/g FW. The assay for H2O2 content was carried out by the procedure previously described by Patterson, Macrae, and Ferguson (1984). Mushroom tissues (2 g) were homogenised with 10 ml of acetone at 0 °C. After centrifugation for 15 min at 6000g at 4 °C, the supernatant phase was collected. The supernatant (1 ml) was mixed with 0.1 ml of 5% titanium sulphate and 0.2 ml ammonia, and then centrifuged for
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2.7. Measurement of enzyme activities For analysis of enzymatic activities, mushroom tissues (4.0 g) were homogenised with 12 ml of 50 mM K-phosphate buffer (pH 7.3) containing 1 mM EDTA, 2 mM DTT. After centrifugation for 15 min at 10,000g and 4 °C, the supernatant was collected and used as the crude enzyme extract for the PPO, SOD, CAT and APX assays. Protein content was determined according to the method of Bradford (1976), with bovine serum albumin used as the standard. PPO (EC 1.10.3.2) activity was measured by incubating 0.5 ml of enzyme extract to 2.5 ml of buffered substrate (100 mM sodium phosphate, pH 6.4 and 50 mM Catechol), and then monitoring the change of absorbance at 398 nm (Wang, Tian, Xu, Qin, & Yao, 2004). One unit of activity of PPO was defined as the amount of enzyme causing 0.01 absorbance increase per minute under the conditions of assay. The specific PPO activity was expressed as U/mg protein. SOD (EC 1.15.1.1) activity was assayed by its ability to inhibit photochemical reduction of nitrotetrazolium blue chloride (NBT) at 560 nm. The assays were performed at 25 °C. The reaction mixture (3 ml) contained 33 lM NBT, 10 mM L-methionine, 0.66 mM EDTANa2, and 0.0033 mM riboflavin in 0.05 M sodium phosphate buffer (pH 7.8). The reaction was started by adding riboflavin and placing the tubes containing reaction mixture under 300 lmol m1 s1 irradiance at 25 °C for 10 min. The absorbance was recorded at 560 nm. One unit of SOD enzyme activity was defined as the quantity of enzyme that reduced the absorbance reading of samples to 50% in comparison with tubes without enzymes. The specific SOD activity was expressed as U/mg protein. CAT (EC 1.11.1.6) activity was determined according to Candan and Tarhan (2003). It was assayed in reaction mixture containing 50 mM phosphate buffer (pH 7.0) 10 mM H2O2 and enzyme. One unit of CAT activity was defined as the amount of enzyme, which decomposes 1 lmol H2O2 per minute at 25 °C. The specific CAT activity was expressed as U/mg protein. APX (EC 1.11.1.11) activity was determined according to Nakano and Asada (1981). The assay depends
on the decrease in absorbance of ascorbic acid at 290 nm because of oxidation of ascorbic acid to monodehydroascorbic acid and dehydroascorbic acid. The reaction mixture contained 0.05 M Naphosphate buffer (pH 7.0), 0.5 mM ascorbate, 0.1 mM EDTA Na2, 1.2 mM H2O2, and 0.1 ml enzyme extract in a final assay volume of 1 ml. The reaction was started with the addition of hydrogen peroxide. The decrease in absorbance at 470 nm was recorded for 3 min. One unit of enzymatic activity was defined as the amount of the enzyme that caused a change of 0.01 in absorbance per minute. The specific APX activity was expressed as U/mg protein. 2.8. Statistical analysis Experiments were performed using a completely randomised design. Data were subjected to one-way analysis of variance (ANOVA). Mean separations were performed by Duncan’s multiple range test (DPS version 6.55). Differences at P < 0.05 were considered significant. 3. Results and discussion 3.1. Effect of NO and MAP integrated treatments on gas composition All treatments showed similar patterns of CO2 and O2 levels within the packaging (Fig. 1). The equilibrium-modified
3 2.5
Weight loss (%)
10 min at 6000g and 4 °C. The pellets were dissolved in 3 ml of 10% (v/v) H2SO4 and centrifuged for 10 min at 5000g. Absorbance of the supernatant phase was measured at 410 nm. H2O2 content was calculated using H2O2 as a standard and then expressed as lmol/g FW.
2 1.5 1 0.5 0 0
4
8
12
16
12
16
Storage time (days)
25
20
17 15
Firmness (N)
O2 and CO2 composition (%)
18
10
16 15 14
5
13 0 0
4
8
12
16
Storage time (days) Fig. 1. Changes in gas composition of button mushrooms packaged in stand-alone MAP (CO2; O2), DETANO (0.5 mM) + MAP (CO2j; O2h), DETANO (1 mM) + MAP (CO2N; O24), and DETANO (2 mM) + MAP (CO2; O2}) stored at 4 °C for 16 d. Each data point is the mean of three replicate samples. Vertical bars represent standard errors of means.
12 0
4
8
Storage time (days) Fig. 2. Changes in weight loss (A) and firmness (B) of button mushrooms packaged in stand-alone MAP (), DETANO (0.5 mM) + MAP (h), DETANO (1 mM) + MAP (N), and DETANO (2 mM) + MAP (j) stored at 4 °C for 16 d. Each data point is the mean of three replicate samples. Vertical bars represent standard errors of means.
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Table 1 Changes in colour of button mushrooms packaged in DETANO + MAP stored at 4 °C for 16 d.A,B
A B
Treatments
L
a
b
DE
Hue
Chroma
BI
0d Control 0.5 mM 1 mM 2 mM
91.9 ± 0.13 91.4 ± 0.06 90.9 ± 0.08 90.4 ± 0.24
a b c d
0.1 ± 0.01 1.7 ± 0.06 0.6 ± 0.12 0.3 ± 0.01
d a b c
15.6 ± 0.15 15.7 ± 0.15 14.8 ± 0.06 16.4 ± 0.09
b b c a
16.5 ± 0.23 17.1 ± 0.07 16.2 ± 0.05 17.8 ± 0.16
c b d a
89.6 ± 0.42 83.8 ± 0.16 87.7 ± 0.05 89.0 ± 0.07
a d c b
15.6 ± 0.42 15.7 ± 0.26 14.8 ± 0.21 16.4 ± 0.13
b b c a
18.0 ± 0.73 19.6 ± 0.55 17.6 ± 0.27 19.6 ± 0.63
b a b a
4d Control 0.5 mM 1 mM 2 mM
85.1 ± 0.30 86.0 ± 0.11 88.7 ± 0.15 86.6 ± 0.26
c b a b
2.5 ± 0.07 1.6 ± 0.23 1.7 ± 0.19 2.8 ± 0.04
b c c a
19.4 ± 0.64 16.3 ± 0.33 15.9 ± 0.14 18.5 ± 0.42
a c c b
23.2 ± 0.33 20.0 ± 0.41 18.3 ± 0.53 21.8 ± 0.62
a c d b
82.7 ± 0.13 84.4 ± 0.26 83.9 ± 0.20 81.4 ± 0.25
c a b d
19.6 ± 0.43 16.4 ± 0.59 16.0 ± 0.35 18.7 ± 0.66
a b b a
27.2 ± 0.44 21.7 ± 0.86 20.5 ± 0.38 25.6 ± 0.61
a c d b
8d Control 0.5 mM 1 mM 2 mM
81.8 ± 0.06 84.0 ± 0.12 86.3 ± 0.27 83.5 ± 0.15
d b a c
3.0 ± 0.36 2.8 ± 0.13 1.1 ± 0.06 2.2 ± 0.14
a b d c
19.7 ± 0.13 17.5 ± 0.26 17.0 ± 0.53 18.8 ± 0.27
a c c b
25.4 ± 0.24 22.3 ± 0.47 20.3 ± 0.11 23.5 ± 0.26
a c d b
81.3 ± 0.43 80.9 ± 0.17 86.3 ± 0.58 83.3 ± 0.63
c c a b
19.9 ± 0.46 17.7 ± 0.73 17.0 ± 0.32 18.9 ± 0.57
a c c b
29.4 ± 0.23 25.0 ± 0.46 22.1 ± 0.71 26.6 ± 0.82
a c d b
12 d Control 0.5 mM 1 mM 2 mM
78.1 ± 0.04 81.4 ± 0.06 83.8 ± 0.17 80.2 ± 0.11
d b a c
4.9 ± 0.11 4.4 ± 0.24 4.3 ± 0.17 4.6 ± 0.10
a c c b
24.3 ± 0.06 22.9 ± 0.26 22.4 ± 0.22 23.4 ± 0.28
a c c b
31.5 ± 0.47 28.4 ± 0.73 26.8 ± 0.34 29.6 ± 0.56
a c d b
78.6 ± 0.35 79.1 ± 0.73 79.1 ± 0.12 78.9 ± 0.06
b a a ab
24.8 ± 0.40 23.3 ± 0.25 22.8 ± 0.21 23.8 ± 0.27
a b c b
40.7 ± 0.33 36.0 ± 0.65 33.9 ± 0.88 37.6 ± 0.64
a c d b
16 d Control 0.5 mM 1 mM 2 mM
75.5 ± 0.28 79.1 ± 0.45 81.4 ± 0.37 78.4 ± 0.62
c b a b
5.8 ± 0.08 4.8 ± 0.12 4.8 ± 0.17 5.2 ± 0.23
a c c b
25.2 ± 0.24 25.5 ± 0.13 23.9 ± 0.06 24.3 ± 0.26
a a c b
34.0 ± 0.14 31.9 ± 0.37 29.3 ± 0.08 31.4 ± 0.28
a b d c
77.0 ± 0.21 79.3 ± 0.42 78.6 ± 0.33 77.9 ± 0.55
d a b c
25.9 ± 0.22 25.9 ± 0.42 24.4 ± 0.21 24.9 ± 0.35
a a c b
45.0 ± 0.90 42.1 ± 0.86 38.0 ± 1.01 40.8 ± 1.06
a b d c
Means of three replications ± standard deviation. Means in same column with different letters are significantly different (P < 0.05).
atmosphere (steady state) was attained within the packaging after 4 d. Mushrooms subjected to stand-alone MAP showed slightly higher CO2 and lower O2 concentrations within the packaging than DETANO (0.5, 1, and 2 mM) + MAP, indicating that stand-alone MAP samples have a higher rate of respiration. Mushrooms treated with DETANO at higher concentrations (1 and 2 mM) showed lower CO2 level within the packaging. It was obvious that the respiration rate after 4 d of storage at 4 °C was inhibited by DETANO (0.5, 1, and 2 mM) treatment and the respiration rate decreased with increasing DETANO concentration. Similar results have also been found by Singh, Singh, and Swinny (2009) for Japanese plums (Prunus salicina Lindell). During the steady state, mushroom respiration (O2 consumption and CO2 production) was balanced by O2 and CO2 diffusion through the film: the O2 and CO2 concentrations reached values of 14% and 6% respectively in stand-alone MAP. 3.2. Effect of NO and MAP integrated treatments on weight loss and texture Mushroom weight loss is mainly caused by the water transpiration rate and CO2 loss during respiration. As shown in Fig. 2A, weight loss increased as the storage period progressed in all the treatments. Their weight losses were all below 3% during storage. However, after 8 d of storage, the weight loss was relative higher in stand-alone MAP than DETANO (0.5, 1, and 2 mM) + MAP. The maximum weight loss of 2.47% was recorded in stand-alone MAP samples after 16 d of storage, suggesting that dehydration is an important process in mushroom quality loss during post-harvest storage. This could be attributed to the fact that mushrooms are only protected by a thin epidermal structure, which does not prevent a quick superficial dehydration. DETANO (2 mM) + MAP reduced the weight loss throughout the entire storage period and recorded the minimum weight loss of 1.9% after 16 d of storage. The weight loss is also related to increased cap opening, as a result of which surface becomes more exposed to transpiration rates. As NO is known to delay the physiological processes leading to
senescence, it prevents the separation of cell walls and increases oxygen diffusion, which results in reduced respiration rate and water loss. The texture of button mushroom is often the first of many quality attributes judged by the consumer and is, therefore, extremely important in overall product acceptance. In the present study, mushrooms in stand-alone MAP gradually softened when stored at low temperature (Fig. 2B). DETANO (0.5 and 1 mM) + MAP maintained a high level of mushroom firmness within the 16 d. On the other hand, a significant reduction of firmness of mushrooms in DETANO (2 mM) + MAP treatments was found after 8 d. Similar results were obtained by Flores et al. (2008) and Zhu et al. (2008), who reported fumigation of peaches with 5 or 10 ll l1 NO and kiwifruit with 1 lM NO can retard fruit softening during storage and ripening, but a higher concentration of 15 ll l1 NO in peaches and 2 lM NO in kiwifruit enhanced softening. The results described above showed that NO could prevent the gradual decline
Table 2 Changes in percent open caps and overall acceptability of button mushrooms packaged in DETANO + MAP stored at 4 °C for 16 d.A,B Treatments
Storage time (d) 0
Percent open Control 0.5 mM 1 mM 2 mM
A
caps 0 0 0 0
4
8
12
16
14.7 ± 0.24 13.5 ± 0.13 13.3 ± 0.26 12.8 ± 0.37
a b b c
36.4 ± 0.15 31.1 ± 0.18 27.4 ± 0.42 28.5 ± 0.31
a b d c
64.3 ± 0.26 52.8 ± 0.32 48.7 ± 0.58 45.3 ± 0.41
a b c d
87.8 ± 0.73 76.7 ± 0.56 66.2 ± 0.81 64.3 ± 0.44
a b c d
Overall acceptability Control 5 3.6 ± 0.05 0.5 mM 5 3.8 ± 0.04 1 mM 5 4.0 ± 0.06 2 mM 5 4.1 ± 0.02
d c b a
2.1 ± 0.03 2.5 ± 0.04 2.9 ± 0.02 2.6 ± 0.03
d c a b
1.0 ± 0.00 1.8 ± 0.06 2.2 ± 0.05 1.6 ± 0.02
d b a c
1.0 ± 0.00 1.3 ± 0.03 1.8 ± 0.02 1.2 ± 0.01
d b a c
Means of three replications ± standard deviation. Means in same column with different letters are significantly different (P < 0.05). B
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of the mushroom toward softening. The impact of DETANO (1 mM) + MAP was apparent. This effect was also similar to the observation that NO could delay the ripening and senescence of fruit. This could be attributed to the fact that treatment with DETANO (1 mM) + MAP suppressed the increased rate of leakage, protected the cells (Zhu, Zhou, Zhu, & Guo, 2009). 3.3. Effect of NO and MAP integrated treatments on colour Table 1 shows the different values obtained after application of DETANO (0.5, 1, and 2 mM) + MAP, compared to the stand-alone MAP without any treatment. From this table, higher luminosity (L) values and lower total colour variation (DE) were observed in DETANO (0.5, 1, and 2 mM) + MAP compared to stand-alone MAP after 4 d. The L value of stand-alone MAP samples decreased sharply after the first 4 d, and it was 81.8 at the 8th day and 78.1 at the 12th day, the last value may not be considered as commercially acceptable if a L value of 80 for wholesalers was take into account (López-Briones et al., 1992). The browning index (BI) of mushrooms was higher in stand-alone MAP than DETANO (0.5, 1, and 2 mM) + MAP during the whole storage. At the end of the storage, mushrooms treated with DETANO (1 mM) + MAP browned slightly, but they also had commercial value and edibility. Compared with stand-alone MAP, DETANO (0.5, 1, and 2 mM) + MAP treatments can inhibit the browning of the button mushrooms.
3.4. Effect of NO and MAP integrated treatments on percent open caps and overall acceptability The percentage of open-cap mushrooms increased as the storage period advanced in all the treatments and was higher in stand-alone MAP samples. The percentage of open-cap mushrooms in stand-alone MAP was 87.8% after 16 d of storage (Table 2). On the other hand, the percentage of open caps mushrooms subjected to DETANO (0.5, 1, and 2 mM) + MAP was in the range of 65.3– 76.7% after 16 d. Among treatments, the minimum percentage (64.3%) was recorded in case of DETANO (2 mM) + MAP samples. The cap opening of mushrooms is related to the dryness of mushrooms as a result of water loss during storage. The increased water loss during storage causes decrease in cohesive forces of water and other hydrophilic molecules, such as proteins responsible for the intact position of the caps and veil in mushrooms. As NO reduces water loss, the cap opening of mushrooms was less in DETANO (0.5, 1, and 2 mM) + MAP samples, particularly in DETANO (1 and 2 mM) + MAP. The overall acceptability based on colour, texture and percent open caps of mushrooms decreased as the storage period advanced in all the treatments. Based on judgements made by sensory panel members, the stand-alone MAP samples were unacceptable after 12 d of storage. However, mushrooms in DETANO (0.5, 1, and 2 mM) + MAP did not exhibit these characteristics even on day
50
-1
Ascorbic acid (mg •kg )
-1
Total phenolics (mg •kg )
1000
900
800
700
600
0
4
8
12
40
30
20
10
0
16
0
4
90
1.1
80
1
70
H2O2 content (µmol g -1 FW)
1.2
0.9 0.8
0.6
30
4
8
Storage time (days)
16
12
16
50 40
0
12
60
0.7
0.5
8
Storage time (days)
•-
O2 production rate -1 -1 (nmol g FW min )
Storage time (days)
12
16
20
0
4
8
Storage time (days)
Fig. 3. Changes in total phenolics (A), ascorbic acid (B), O 2 production rate (C), and H2O2 content (D) of button mushrooms packaged in stand-alone MAP (), DETANO (0.5 mM) + MAP (h), DETANO (1 mM) + MAP (N), and DETANO (2 mM) + MAP (j) stored at 4 °C for 16 d. Each data point is the mean of three replicate samples. Vertical bars represent standard errors of means.
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12, the DETANO (1 mM) + MAP samples were acceptable and in marketable condition and recorded an overall acceptability of 2.2 after 12 d of storage. These results suggest that the DETANO (0.5, 1, and 2 mM) + MAP were effective in retarding mushroom sensory deterioration. 3.5. Effect of NO and MAP integrated treatments on functional components Phenolic compounds have been reported as the major antioxidant components in mushrooms. These antioxidant compounds have been widely reported to have beneficial effects on the maintenance of health and the prevention of cancer and cardiovascular diseases. In this study, total phenolics levels declined in all treatments during the 16-d period (Fig. 3A). The lowest phenolics content was found in mushrooms from stand-alone MAP. DETANO (0.5, 1, and 2 mM) + MAP was more effective in delaying decrease of phenolics than stand-alone MAP. DETANO (1 mM) + MAP treatment best maintained the phenolic content of mushrooms. As mentioned above, this treatment also reduced browning on mushrooms. It appears that lower levels of browning correlated fairly well with phenolic content, which seems to be the limiting factor of the discoloration process. Little is known about the effects of NO treatment and storage on the retention of dietary antioxidants such as ascorbic acid (AA) in button mushroom. As shown in Fig. 3B, the content of AA
A
diminished in both stand-alone MAP and DETANO (0.5, 1, and 2 mM) + MAP treated mushrooms during storage, reaching levels about 50% lower than the initial values. However, treatment with DETANO (1 mM) + MAP induced a lower rate of decrement in AA during storage. The AA content in stand-alone MAP samples declined rapidly throughout storage, and no significant differences (P > 0.05) were observed between DETANO (0.5 mM) + MAP and stand-alone MAP mushroom. Therefore DETANO (1 mM) + MAP maintained high levels of AA, which indicated that NO treatment delayed the senescence of button mushroom during storage. Similar results have also been found by Duan et al. (2007) for longan fruit. It is known that low temperatures can reduce AA degradation in fresh-cut produce. The accumulation of CO2 within packages seems to have a negative effect on AA content of some fruits. Fruit and vegetable ripening have been described as an oxidative phenomenon which requires a turnover of reactive oxygen species (ROS), such as H2O2 and O 2 . In the present study, the increases in both O 2 production rate and H2O2 content were observed during 16 d of storage (Fig. 3C and D). The use of standalone MAP does not seem to substantially contribute to enhancement of antioxidant capacity of mushroom. On the other hand, DETANO (0.5 and 1 mM) + MAP treatments generally reduced the increase in both O 2 production rate and H2O2 content of mushroom during storage. Mushrooms treated with DETANO (1 mM) + MAP presented higher antioxidant capacity, compared with DETANO (0.5 and 2 mM) + MAP. In summary, NO treatment
B
96
2.1
64 48 32
(Units mg-1 protein)
1.8
SOD activity
PPO activity (Units mg -1 protein)
80
16 0
1.5
1.2
0.9
0.6 0
4
8
12
16
0
4
Storage time (days)
C
D
450
360
12
16
12
16
5
4
APX activity -1 (Units mg protein)
CAT activity (Units mg -1 protein)
8
Storage time (days)
270
180
90
3
2
1
0
0 0
4
8
Storage time (days)
12
16
0
4
8
Storage time (days)
Fig. 4. Changes in PPO (A), SOD (B), CAT (C), and APX (D) of button mushrooms packaged in stand-alone MAP (), DETANO (0.5 mM) + MAP (h), DETANO (1 mM) + MAP (N), and DETANO (2 mM) + MAP (j) stored at 4 °C for 16 d. Each data point is the mean of three replicate samples. Vertical bars represent standard errors of means.
T. Jiang et al. / Food Chemistry 126 (2011) 1693–1699
had a positive effect on antioxidant capacity, mainly influenced by total phenolics and ascorbic acid of button mushrooms. Similar results have also been found by Duan et al. (2007) and Flores et al. (2008) for longan fruit and peach. 3.6. Effect of NO and MAP integrated treatments on enzymes activity Enzymatic browning is a consequence of polyphenol oxidase (PPO) catalysed oxidation of phenolic substrates into quinones, which undergo further reactions to dark pigments called melanins. The major PPO enzyme responsible for browning in mushrooms appears to be tyrosinase. PPO activity increased in the stand-alone MAP samples during the first 12 d of storage then decreased (Fig. 4A). Treatment with DETANO (1 mM) + MAP significantly (P < 0.05) reduced the increase in PPO activity, compared with DETANO (0.5 and 2 mM) + MAP. NO inhibited the increase of PPO activity and suggested that NO could interact with transition metals (e.g. iron, copper and zinc) and thiol-containing enzymes. It has also been reported that NO inhibited the activity of haem-containing enzymes, such as CAT and POD, the important H2O2-scavenging enzymes during pathogen attack (Clark, Durner, Navarre, & Klessig, 2000). PPO is a complex enzyme with two copper ions in its active center. It is speculated that NO could react with the copper of PPO to form copper–nitrosyl complexes (NO–Cu–PPO), which could change the normal structure of the active site in PPO and thus reduce PPO activity. Antioxidant enzymes such as SOD, CAT and APX play a crucial role in antioxidant defense during the fruit ripening process. These enzymatic components, together with the low-molecular-weight antioxidants ascorbic acid (AA) and glutathione (GSH), ultimately scavenge H2O2 at the expense of NADPH or NADH (Foyer & Halliwell, 1976). In the present study, the DETANO (1 mM) + MAP treatment significantly (P < 0.05) delayed the decrease in the activities of SOD and CAT (Fig. 4B and C), and increase in activities of APX during storage (Fig. 4D), in agreement with the reduced ROS production (Fig. 3C and D). The results obtained seem to indicate that NO, as an antisenescent agent, affects positively the response to the oxidative stress associated with mushroom senescence. It has been observed that NO seems to have an antioxidant activity in the leaves of rice, where it delays the initiation of senescence induced by the plant-growth regulator methyl jasmonate, probably due to its capacity for removing ROS (Hung & Kao, 2004). 4. Conclusion Our research showed that the senescence inhibition of coldstored button mushroom by the DETANO (1 mM) + MAP (BOPP) treatment involved in the maintenance of tissue firmness, inhibition of browning, increase of phenolics and ascorbic acid contents, and enhancement of antioxidant ability compare with stand-alone MAP. NO treatment also reduced weight loss and delayed cap opening during the storage period. These results suggest that DETANO (1 mM) + MAP (BOPP) treatment may be a useful technique of maintaining button mushroom quality and extending their postharvest life. References Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254. Candan, N., & Tarhan, L. (2003). Relationship among chlorophyll-carotenoid content, antioxidant enzyme activities and lipid peroxidation levels by Mg2+ deficiency in the Mentha pulegium leaves. Plant Physiology and Biochemistry, 41, 35–40. Chang, S. T. (1999). World production of cultivated edible and medicinal mushrooms in 1997 with emphasis on Lentinus edodes (Berk.) Sing. in China. International Journal of Medicinal Mushrooms, 1, 291–300.
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Clark, D., Durner, J., Navarre, D. A., & Klessig, D. F. (2000). Nitric oxide inhibition of tobacco catalase and ascorbate peroxidase. Molecular Plant–Microbe Interaction, 13, 1380–1384. Duan, X. W., Su, X. G., You, Y. L., Qu, H. X., Li, Y. B., & Jiang, Y. M. (2007). Effect of nitric oxide on pericarp browning of harvested longan fruit in relation to phenolic metabolism. Food Chemistry, 104, 571–576. Eum, H. L., Kim, H. B., Choi, S. B., & Lee, S. K. (2009). Regulation of ethylene biosynthesis by nitric oxide in tomato (Solanum lycopersicum L.) fruit harvested at different ripening stages. European Food Research and Technology, 228, 331–338. Flores, F. B., Sánchez-Bel, P., Valdenegro, M., Romojaro, F., Martínez-Madrid, M. C., & Egea, M. I. (2008). Effects of a pretreatment with nitric oxide on peach (Prunus persica L.) storage at room temperature. European Food Research and Technology, 227, 1599–1611. Foyer, C. H., & Halliwell, B. (1976). The presence of glutathione and glutathione reductase in chloroplast: A proposed role in ascorbic acid metabolism. Planta, 133, 5–21. Hanson, P. M., Yang, R. Y., Wu, J., Chen, J. T., Ledesma, D., Tsou, C. S. C., et al. (2004). Variation for antioxidant activity and antioxidants in tomato. Journal of the American Society for Horticultural Science, 129, 704–711. Hung, K. T., & Kao, C. H. (2004). Nitric oxide acts as an antioxidant and delays methyl jasmonate-induced senescence of rice leaves. Journal of Plant Physiology, 161, 43–52. Kader, A. A. (1986). Biochemical and physiological basis for effects of controlled atmospheres. Food Technology, 40, 99–104. López-Briones, G. L., Varoguaux, P., Chambroy, Y., Bouquant, J., Bureau, G., & Pascat, B. (1992). Storage of common mushroom under controlled atmospheres. International Journal of Food Science and Technology, 27, 493–505. Mattila, P., Konko, K., Eurola, M., Pihlava, J. M., Astola, J., Vahteristo, L., et al. (2001). Contents of vitamins, mineral elements, and some phenolic compounds in cultivated mushrooms. Journal of Agricultural and Food Chemistry, 49, 2343–2348. Nakano, Y., & Asada, K. (1981). Hydrogen peroxide is scavenged by ascorbatespecific peroxidase in spinach chloroplasts. Plant Cell Physiology, 22, 867–880. Palou, E., Lopez-Malo, A., Barbosa-Canovas, G. V., Welti-Chanes, J., & Swanson, G. B. (1999). Polyphenoloxidase activity and colour of blanched and high hydrostatic pressure treated banana puree. Journal of Food Science, 64, 42–45. Patterson, B. D., Macrae, E. A., & Ferguson, I. B. (1984). Estimation of hydrogen peroxide in plant extracts using titanium (IV). Analytical Biochemistry, 139, 487–492. Pristijono, P., Wills, R. B. H., & Golding, J. B. (2006). Inhibition of browning on the surface of apple slices by short term exposure to nitric oxide (NO) gas. Postharvest Biology and Technology, 42, 256–259. Singh, S. P., Singh, Z., & Swinny, E. E. (2009). Postharvest nitric oxide fumigation delays fruit ripening and alleviates chilling injury during cold storage of Japanese plums (Prunus salicina Lindell). Postharvest Biology and Technology, 53, 101–108. Singleton, V. L., & Rossi, J. A. (1965). Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal Enology and Viticulture, 16, 144–158. Tano, K., Arul, J., Doyon, G., & Castaigne, F. (1999). Atmospheric composition and quality of fresh mushrooms in modified atmosphere packages as affected by storage temperature abuse. Journal of Food Science, 64, 1077–1079. Wang, A. G., & Luo, G. H. (1990). Quantitative relation between the reaction of hydroxylamine and superoxide anion radicals in plants. Plant Physiology Communications, 26, 55–57. Wang, Y. S., Tian, S. P., Xu, Y., Qin, G. Z., & Yao, H. J. (2004). Changes in the activities of pro- and anti-oxidant enzymes in peach fruit inoculated with Cryptococcus laurentii or Penicillium expansum at 0 or 20 °C. Postharvest Biology and Technology, 34, 21–28. Wills, R. B. H., Ku, V. V. V., & Leshem, Y. Y. (2000). Fumigation with nitric oxide to extend the postharvest life of strawberries. Postharvest Biology and Technology, 18, 75–79. Wills, R. B. H., Soegiarto, L., & Bowyer, M. C. (2007). Use of a solid mixture containing diethylenetriamine/nitric oxide (DETANO) to liberate nitric oxide gas in the presence of horticultural produce to extend postharvest life. Nitric oxide, 17, 44–49. Wills, R. B. H., Pristijono, P., & Golding, J. B. (2008). Browning on the surface of cut lettuce slices inhibited by short term exposure to nitric oxide (NO). Food Chemistry, 107, 1387–1392. Xing, Z. T., Wang, Y. S., Feng, Z. Y., & Tan, Q. (2008). Effect of different packaging films on postharvest quality and selected enzyme activities of hypsizygus marmoreus mushrooms. Journal of Agricultural and Food Chemistry, 56, 11838–11844. Zhu, L., Zhou, J., Zhu, S., & Guo, L. (2009). Inhibition of browning on the surface of peach slices by short-term exposure to nitric oxide and ascorbic acid. Food Chemistry, 114, 174–179. Zhu, S., Liu, M., & Zhou, J. (2006). Inhibition by nitric oxide of ethylene biosynthesis and lipoxygenase activity in peach fruit during storage. Postharvest Biology and Technology, 42, 41–48. Zhu, S., Sun, L., Liu, M., & Zhou, J. (2008). Effect of nitric oxide on reactive oxygen species and antioxidant enzymes in kiwifruit during storage. Journal of the Science of Food and Agriculture, 88, 2324–2331. Zhu, S., & Zhou, J. (2007). Effect of nitric oxide on ethylene production in strawberry fruit during storage. Food Chemistry, 100, 1517–1522.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Integrated application of nitric oxide and modified atmosphere packaging to improve quality retention of button mushroom (Agaricus bisporus) Tianjia Jiang a, Xiaolin Zheng a, Jianrong Li a, Guoxing Jing a, Luyun Cai b, Tiejin Ying b,⇑ a b
College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310035, PR China Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, PR China
a r t i c l e
i n f o
Article history: Received 7 September 2010 Received in revised form 4 November 2010 Accepted 8 December 2010 Available online 15 December 2010 Keywords: Button mushroom Nitric oxide Browning Antioxidant enzymes Quality parameters
a b s t r a c t Button mushrooms (Agaricus bisporus) were dipped for 10 min in different concentrations (0.5, 1, and 2 mM) of 2,20 -(hydroxynitrosohydrazino)-bisethanamine (DETANO), a nitric oxide donor, then packed in biorientated polypropylene (BOPP) bags, heat sealed and stored at 4 °C for 16 days (d). Mushroom weight loss, firmness, colour, percent open caps, total phenolics, ascorbic acid and H2O2 contents, superoxide anion (O 2 ) production rate and activities of polyphenol oxidase (PPO), superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) were measured. The results indicate that treatment with 1 mM DETANO maintained a high level of firmness, delayed browning and cap opening, promoted the accumulation of phenolics, ascorbic acid and reduced the increases in both O 2 production rate and H2O2 content. Furthermore, NO inhibited the activity of PPO, and increased the antioxidant enzymes activities of CAT, SOD and APX throughout storage period. Thus it was observed that application of NO in combination with modified atmosphere packaging (MAP) can extend the storage life of button mushroom up to 12 d. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Button mushroom (Agaricus bisporus) is one of the most popular mushrooms, traditionally cultivated in the world. Consumption and production of this edible mushroom have grown continuously in the past years. It is the most extensively cultivated edible mushroom, comprising 32% of the worldwide production (Chang, 1999). Mushrooms are a good source of vitamin B2, niacin, folates and many mineral elements (Mattila et al., 2001). However, button mushrooms only have a short shelf life of 3–4 d, they lose their commercial value within a few days, due to browning, water loss, senescence and microbial attack. The short shelf-life of mushroom is an impediment to the distribution and marketing of the fresh product. Thus, prolonging post-harvest storage while preserving their quality would benefit the mushroom industry as well as consumers. Nitric oxide (NO) is a highly reactive free radical gas that was initially notable as an industrial pollutant but is now known to be involved in resisting vegetative stress and senescence of horticultural products. Short-term exposure to a low concentration of NO gas or its donor compounds has been shown to extend the post-harvest life of various intact fresh fruits and vegetables (Wills, Ku, & Leshem, 2000; Wills, Soegiarto, & Bowyer, 2007; Zhu & Zhou,
⇑ Corresponding author. Tel./fax: +86 571 86971162. E-mail address: [email protected] (T. Ying). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.12.060
2007). It has been shown that they delayed ripening (Wills et al., 2000; Zhu & Zhou, 2007), inhibited ethylene biosynthesis (Eum, Kim, Choi, & Lee, 2009; Zhu, Sun, Liu, & Zhou, 2008), inhibited cut-surface browning (Pristijono, Wills, & Golding, 2006; Wills, Pristijono, & Golding, 2008), and enhanced resistance to post-harvest diseases (Zhu & Zhou, 2007). The mechanism of action of NO in delaying senescence of post-harvest horticultural produce, though not completely understood, is via the inhibition of ethylene biosynthesis (Eum et al., 2009; Zhu, Liu, & Zhou, 2006). However, adequate evidence does not exist to ascertain the mode of action of NO. Modified atmosphere packaging (MAP) has become increasingly common in recent years and has been reported to be the most economical and effective method of extending the shelf life of mushroom (Tano, Arul, Doyon, & Castaigne, 1999). Active modification is created by replacing gases in the package with a desired mixture of gases. On the other hand, passive modification happens when product is sealed in a package made with a selected film, and a desired atmosphere develops naturally as a consequence of product respiration and diffusion of gases through the film. Modified atmosphere in terms of reduced O2 and elevated CO2 can extend the post-harvest life of fruits and vegetables by reducing their respiration rate as well as production of ethylene, minimising metabolic activity, delaying enzymatic browning, retaining visual appearance (Kader, 1986). In recent studies, Xing, Wang, Feng, and Tan (2008) found that among different packaging films [polyvinyl chloride (PVC), two types of polyethylene (PE6 and PE11), polyoletin (PO),
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and biaxially oriented polypropylene (BOPP)], BOPP maintained the post-harvest appearance of the mushroom most effectively by significantly reducing the incidence of unsightly aerial hyphae on the pileal surface and restricting mushroom softening. Those samples also exhibited smaller initial decreases in soluble protein, smaller increases in reducing sugar content, and lower levels of malondialdehyde accumulation during storage. To our knowledge, there are little information about the effect of NO on button mushroom during storage. Therefore, the objective of this research was to investigate the effect of integrated application of NO and MAP employing BOPP, on quality retention of button mushroom.
mushroom colour values of L⁄ = 97, a⁄ = 2 and b⁄ = 0. The hue (H), chroma (C), and browning index (BI), which represents the purity of brown colour (Palou, Lopez-Malo, Barbosa-Canovas, Welti-Chanes, & Swanson, 1999), were calculated according to the following equations: 2
Hue ¼ tan1 ðb=aÞ Chroma ¼ ða2 þ b Þ1=2 BI ¼ ½100ðx 0:31Þ=0:172;
where x
¼ ða þ 1:75LÞ=ð5:645L þ a 3:012bÞ 2.5. Percent open caps and overall acceptability
2. Materials and methods 2.1. Mushroom treatment and storage Button mushroom used in this study were harvested from a local farm in Hangzhou, China. The mushrooms were transported to the laboratory in one hour after picking, then stored in darkness at 4 ± 1 °C and 90% relative humidity (RH). The day after, mushrooms were screened for uniform size and maturity and absence of mechanical damage. The mushrooms were dipped in 0.5, 1, and 2 mM 2,20 -(hydroxynitrosohydrazino)-bisethanamine (DETANO, the donor of NO; Sigma) aqueous solutions for 10 min at 20 °C with dip in distilled water used as a control (stand-alone MAP). The mushrooms (65 ± 5 g) were then drained in the air for another 5 min and packaged in biaxially oriented polypropylene (BOPP) bags (MAP; 35 lm thickness; size 18 20 cm; O2 permeance 38 1014 mol s1 m2 Pa1 at 23 °C; Xinlei Gongmao Co. Ltd., Zhejiang, China). The MAP was sealed with a heat sealer to create a passive modified atmosphere around the mushrooms. Samples were then stored for 16 d at 4 ± 1 °C and 90% relative humidity (RH). Twenty-five replicates were included in each treatment group, and subsequently every 4 d, five replicates from each treatment group were randomly selected and analysed for physicochemical parameters. 2.2. Package atmosphere composition O2 and CO2 concentrations in packages were evaluated by using a SCY-2A O2 and CO2 Analyser (Xinrui Instrument Co., Shanghai, China). Gas samples were taken from the bags with a 20 ml syringe. 2.3. Weight loss and firmness evaluation Weight loss was determined by weighing the whole mushroom before and after the storage period. Weight loss was expressed as the percentage of loss of weight with respect to the initial weight. A penetration test was performed on the mushroom cap using a TA.XT2i texture analyser (Stable Micro Systems, UK), using a 5 mm diameter cylindrical probe. Samples were penetrated 5 mm in depth. The speed of the probe was 2.0 mm s1 during the pretest and penetration. Force and time data were recorded with Texture Expert (Version 1.0) from Stable Micro Systems. From the force vs. time curves, firmness was defined as the maximum force. 2.4. Colour The surface colour of mushroom caps was measured with a WSC-S Colourimeter (Shanghai precision instrument Co. Ltd., Shanghai, China). To analyse the L⁄ (light/dark), a⁄ (red/green) and b⁄ (yellow/blue) values, each mushroom was measured at three equidistant points of the cap and compared to the ideal
Criteria for judging the percentage of open caps were based on the development of umbrella-like shape of the cap followed by detachment of veil. The percent open caps was determined from known number of mushrooms as:
%Open caps ¼ Noc =Nt 100 where Nt = total number of mushrooms; Noc = number of opencaped mushrooms. The overall acceptability based on colour, texture and percent open caps was done by a panel of four judges on round table basis using four-point scale where 1 = poor, 2 = fair, 3 = good and 4 = excellent. 2.6. Analysis of functional components Quantification of the total soluble phenolic compounds was carried out using the method proposed by Singleton and Rossi (1965). Five grams of mushroom cap were homogenised with 20 ml of 80% ethanol for 24 h; the homogenised mix was then filtered through two layers of cheesecloth, and the filtered liquid was centrifuged at 10,000g for 15 min. One millilitre of the supernatant liquid was mixed with 1 ml of Folin Ciocalteu reagent and 10 ml of sodium carbonate (7%). This was topped up to 25 ml with distilled water and left to settle for 1 h. The absorbance was then read at 750 nm. A standard curve of gallic acid was used for quantification. The determination of total ascorbic acid was carried out as described by Hanson et al. (2004). On the basis of coupling 2,4-dinitrophenylhydrazine (DNPH) with the ketonic groups of dehydroascorbic acid through the oxidation of ascorbic acid by 2,6-dichlorophenolindophenol (DCPIP) to give a yellow/orange colour in acidic conditions. Superoxide anion production rate was measured by monitoring the nitrite formation from hydroxylamine in the presence of O 2 , as described by Wang and Luo (1990). Mushroom tissues (4.0 g) were homogenised with 12 ml of 50 mM potassium phosphate buffer (pH 7.8) containing 1% (w/v) polyvinylpyrrolidone at 0 °C, and then centrifuged at 5000g and 4 °C for 15 min. The obtained supernatant (1 ml) was mixed with 0.9 ml of 50 mM potassium phosphate buffer (pH 7.8) and 0.1 ml of 10 mM hydroxylamine hydrochloride, and then incubated for 30 min at 25 °C. The incubated solution (1 ml) was added to 1 ml of 17 mM 3-aminobenzenesulphonic acid and 1 ml of 7 mM 1-naphthylamine, and then further kept for 20 min at 25 °C. The absorbance was recorded at 530 nm. A standard curve with NO2 was used to calculate the O 2 production rate from the reaction equation of O 2 with hydroxylamine. The O2 production rate was expressed as nmol/min/g FW. The assay for H2O2 content was carried out by the procedure previously described by Patterson, Macrae, and Ferguson (1984). Mushroom tissues (2 g) were homogenised with 10 ml of acetone at 0 °C. After centrifugation for 15 min at 6000g at 4 °C, the supernatant phase was collected. The supernatant (1 ml) was mixed with 0.1 ml of 5% titanium sulphate and 0.2 ml ammonia, and then centrifuged for
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2.7. Measurement of enzyme activities For analysis of enzymatic activities, mushroom tissues (4.0 g) were homogenised with 12 ml of 50 mM K-phosphate buffer (pH 7.3) containing 1 mM EDTA, 2 mM DTT. After centrifugation for 15 min at 10,000g and 4 °C, the supernatant was collected and used as the crude enzyme extract for the PPO, SOD, CAT and APX assays. Protein content was determined according to the method of Bradford (1976), with bovine serum albumin used as the standard. PPO (EC 1.10.3.2) activity was measured by incubating 0.5 ml of enzyme extract to 2.5 ml of buffered substrate (100 mM sodium phosphate, pH 6.4 and 50 mM Catechol), and then monitoring the change of absorbance at 398 nm (Wang, Tian, Xu, Qin, & Yao, 2004). One unit of activity of PPO was defined as the amount of enzyme causing 0.01 absorbance increase per minute under the conditions of assay. The specific PPO activity was expressed as U/mg protein. SOD (EC 1.15.1.1) activity was assayed by its ability to inhibit photochemical reduction of nitrotetrazolium blue chloride (NBT) at 560 nm. The assays were performed at 25 °C. The reaction mixture (3 ml) contained 33 lM NBT, 10 mM L-methionine, 0.66 mM EDTANa2, and 0.0033 mM riboflavin in 0.05 M sodium phosphate buffer (pH 7.8). The reaction was started by adding riboflavin and placing the tubes containing reaction mixture under 300 lmol m1 s1 irradiance at 25 °C for 10 min. The absorbance was recorded at 560 nm. One unit of SOD enzyme activity was defined as the quantity of enzyme that reduced the absorbance reading of samples to 50% in comparison with tubes without enzymes. The specific SOD activity was expressed as U/mg protein. CAT (EC 1.11.1.6) activity was determined according to Candan and Tarhan (2003). It was assayed in reaction mixture containing 50 mM phosphate buffer (pH 7.0) 10 mM H2O2 and enzyme. One unit of CAT activity was defined as the amount of enzyme, which decomposes 1 lmol H2O2 per minute at 25 °C. The specific CAT activity was expressed as U/mg protein. APX (EC 1.11.1.11) activity was determined according to Nakano and Asada (1981). The assay depends
on the decrease in absorbance of ascorbic acid at 290 nm because of oxidation of ascorbic acid to monodehydroascorbic acid and dehydroascorbic acid. The reaction mixture contained 0.05 M Naphosphate buffer (pH 7.0), 0.5 mM ascorbate, 0.1 mM EDTA Na2, 1.2 mM H2O2, and 0.1 ml enzyme extract in a final assay volume of 1 ml. The reaction was started with the addition of hydrogen peroxide. The decrease in absorbance at 470 nm was recorded for 3 min. One unit of enzymatic activity was defined as the amount of the enzyme that caused a change of 0.01 in absorbance per minute. The specific APX activity was expressed as U/mg protein. 2.8. Statistical analysis Experiments were performed using a completely randomised design. Data were subjected to one-way analysis of variance (ANOVA). Mean separations were performed by Duncan’s multiple range test (DPS version 6.55). Differences at P < 0.05 were considered significant. 3. Results and discussion 3.1. Effect of NO and MAP integrated treatments on gas composition All treatments showed similar patterns of CO2 and O2 levels within the packaging (Fig. 1). The equilibrium-modified
3 2.5
Weight loss (%)
10 min at 6000g and 4 °C. The pellets were dissolved in 3 ml of 10% (v/v) H2SO4 and centrifuged for 10 min at 5000g. Absorbance of the supernatant phase was measured at 410 nm. H2O2 content was calculated using H2O2 as a standard and then expressed as lmol/g FW.
2 1.5 1 0.5 0 0
4
8
12
16
12
16
Storage time (days)
25
20
17 15
Firmness (N)
O2 and CO2 composition (%)
18
10
16 15 14
5
13 0 0
4
8
12
16
Storage time (days) Fig. 1. Changes in gas composition of button mushrooms packaged in stand-alone MAP (CO2; O2), DETANO (0.5 mM) + MAP (CO2j; O2h), DETANO (1 mM) + MAP (CO2N; O24), and DETANO (2 mM) + MAP (CO2; O2}) stored at 4 °C for 16 d. Each data point is the mean of three replicate samples. Vertical bars represent standard errors of means.
12 0
4
8
Storage time (days) Fig. 2. Changes in weight loss (A) and firmness (B) of button mushrooms packaged in stand-alone MAP (), DETANO (0.5 mM) + MAP (h), DETANO (1 mM) + MAP (N), and DETANO (2 mM) + MAP (j) stored at 4 °C for 16 d. Each data point is the mean of three replicate samples. Vertical bars represent standard errors of means.
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Table 1 Changes in colour of button mushrooms packaged in DETANO + MAP stored at 4 °C for 16 d.A,B
A B
Treatments
L
a
b
DE
Hue
Chroma
BI
0d Control 0.5 mM 1 mM 2 mM
91.9 ± 0.13 91.4 ± 0.06 90.9 ± 0.08 90.4 ± 0.24
a b c d
0.1 ± 0.01 1.7 ± 0.06 0.6 ± 0.12 0.3 ± 0.01
d a b c
15.6 ± 0.15 15.7 ± 0.15 14.8 ± 0.06 16.4 ± 0.09
b b c a
16.5 ± 0.23 17.1 ± 0.07 16.2 ± 0.05 17.8 ± 0.16
c b d a
89.6 ± 0.42 83.8 ± 0.16 87.7 ± 0.05 89.0 ± 0.07
a d c b
15.6 ± 0.42 15.7 ± 0.26 14.8 ± 0.21 16.4 ± 0.13
b b c a
18.0 ± 0.73 19.6 ± 0.55 17.6 ± 0.27 19.6 ± 0.63
b a b a
4d Control 0.5 mM 1 mM 2 mM
85.1 ± 0.30 86.0 ± 0.11 88.7 ± 0.15 86.6 ± 0.26
c b a b
2.5 ± 0.07 1.6 ± 0.23 1.7 ± 0.19 2.8 ± 0.04
b c c a
19.4 ± 0.64 16.3 ± 0.33 15.9 ± 0.14 18.5 ± 0.42
a c c b
23.2 ± 0.33 20.0 ± 0.41 18.3 ± 0.53 21.8 ± 0.62
a c d b
82.7 ± 0.13 84.4 ± 0.26 83.9 ± 0.20 81.4 ± 0.25
c a b d
19.6 ± 0.43 16.4 ± 0.59 16.0 ± 0.35 18.7 ± 0.66
a b b a
27.2 ± 0.44 21.7 ± 0.86 20.5 ± 0.38 25.6 ± 0.61
a c d b
8d Control 0.5 mM 1 mM 2 mM
81.8 ± 0.06 84.0 ± 0.12 86.3 ± 0.27 83.5 ± 0.15
d b a c
3.0 ± 0.36 2.8 ± 0.13 1.1 ± 0.06 2.2 ± 0.14
a b d c
19.7 ± 0.13 17.5 ± 0.26 17.0 ± 0.53 18.8 ± 0.27
a c c b
25.4 ± 0.24 22.3 ± 0.47 20.3 ± 0.11 23.5 ± 0.26
a c d b
81.3 ± 0.43 80.9 ± 0.17 86.3 ± 0.58 83.3 ± 0.63
c c a b
19.9 ± 0.46 17.7 ± 0.73 17.0 ± 0.32 18.9 ± 0.57
a c c b
29.4 ± 0.23 25.0 ± 0.46 22.1 ± 0.71 26.6 ± 0.82
a c d b
12 d Control 0.5 mM 1 mM 2 mM
78.1 ± 0.04 81.4 ± 0.06 83.8 ± 0.17 80.2 ± 0.11
d b a c
4.9 ± 0.11 4.4 ± 0.24 4.3 ± 0.17 4.6 ± 0.10
a c c b
24.3 ± 0.06 22.9 ± 0.26 22.4 ± 0.22 23.4 ± 0.28
a c c b
31.5 ± 0.47 28.4 ± 0.73 26.8 ± 0.34 29.6 ± 0.56
a c d b
78.6 ± 0.35 79.1 ± 0.73 79.1 ± 0.12 78.9 ± 0.06
b a a ab
24.8 ± 0.40 23.3 ± 0.25 22.8 ± 0.21 23.8 ± 0.27
a b c b
40.7 ± 0.33 36.0 ± 0.65 33.9 ± 0.88 37.6 ± 0.64
a c d b
16 d Control 0.5 mM 1 mM 2 mM
75.5 ± 0.28 79.1 ± 0.45 81.4 ± 0.37 78.4 ± 0.62
c b a b
5.8 ± 0.08 4.8 ± 0.12 4.8 ± 0.17 5.2 ± 0.23
a c c b
25.2 ± 0.24 25.5 ± 0.13 23.9 ± 0.06 24.3 ± 0.26
a a c b
34.0 ± 0.14 31.9 ± 0.37 29.3 ± 0.08 31.4 ± 0.28
a b d c
77.0 ± 0.21 79.3 ± 0.42 78.6 ± 0.33 77.9 ± 0.55
d a b c
25.9 ± 0.22 25.9 ± 0.42 24.4 ± 0.21 24.9 ± 0.35
a a c b
45.0 ± 0.90 42.1 ± 0.86 38.0 ± 1.01 40.8 ± 1.06
a b d c
Means of three replications ± standard deviation. Means in same column with different letters are significantly different (P < 0.05).
atmosphere (steady state) was attained within the packaging after 4 d. Mushrooms subjected to stand-alone MAP showed slightly higher CO2 and lower O2 concentrations within the packaging than DETANO (0.5, 1, and 2 mM) + MAP, indicating that stand-alone MAP samples have a higher rate of respiration. Mushrooms treated with DETANO at higher concentrations (1 and 2 mM) showed lower CO2 level within the packaging. It was obvious that the respiration rate after 4 d of storage at 4 °C was inhibited by DETANO (0.5, 1, and 2 mM) treatment and the respiration rate decreased with increasing DETANO concentration. Similar results have also been found by Singh, Singh, and Swinny (2009) for Japanese plums (Prunus salicina Lindell). During the steady state, mushroom respiration (O2 consumption and CO2 production) was balanced by O2 and CO2 diffusion through the film: the O2 and CO2 concentrations reached values of 14% and 6% respectively in stand-alone MAP. 3.2. Effect of NO and MAP integrated treatments on weight loss and texture Mushroom weight loss is mainly caused by the water transpiration rate and CO2 loss during respiration. As shown in Fig. 2A, weight loss increased as the storage period progressed in all the treatments. Their weight losses were all below 3% during storage. However, after 8 d of storage, the weight loss was relative higher in stand-alone MAP than DETANO (0.5, 1, and 2 mM) + MAP. The maximum weight loss of 2.47% was recorded in stand-alone MAP samples after 16 d of storage, suggesting that dehydration is an important process in mushroom quality loss during post-harvest storage. This could be attributed to the fact that mushrooms are only protected by a thin epidermal structure, which does not prevent a quick superficial dehydration. DETANO (2 mM) + MAP reduced the weight loss throughout the entire storage period and recorded the minimum weight loss of 1.9% after 16 d of storage. The weight loss is also related to increased cap opening, as a result of which surface becomes more exposed to transpiration rates. As NO is known to delay the physiological processes leading to
senescence, it prevents the separation of cell walls and increases oxygen diffusion, which results in reduced respiration rate and water loss. The texture of button mushroom is often the first of many quality attributes judged by the consumer and is, therefore, extremely important in overall product acceptance. In the present study, mushrooms in stand-alone MAP gradually softened when stored at low temperature (Fig. 2B). DETANO (0.5 and 1 mM) + MAP maintained a high level of mushroom firmness within the 16 d. On the other hand, a significant reduction of firmness of mushrooms in DETANO (2 mM) + MAP treatments was found after 8 d. Similar results were obtained by Flores et al. (2008) and Zhu et al. (2008), who reported fumigation of peaches with 5 or 10 ll l1 NO and kiwifruit with 1 lM NO can retard fruit softening during storage and ripening, but a higher concentration of 15 ll l1 NO in peaches and 2 lM NO in kiwifruit enhanced softening. The results described above showed that NO could prevent the gradual decline
Table 2 Changes in percent open caps and overall acceptability of button mushrooms packaged in DETANO + MAP stored at 4 °C for 16 d.A,B Treatments
Storage time (d) 0
Percent open Control 0.5 mM 1 mM 2 mM
A
caps 0 0 0 0
4
8
12
16
14.7 ± 0.24 13.5 ± 0.13 13.3 ± 0.26 12.8 ± 0.37
a b b c
36.4 ± 0.15 31.1 ± 0.18 27.4 ± 0.42 28.5 ± 0.31
a b d c
64.3 ± 0.26 52.8 ± 0.32 48.7 ± 0.58 45.3 ± 0.41
a b c d
87.8 ± 0.73 76.7 ± 0.56 66.2 ± 0.81 64.3 ± 0.44
a b c d
Overall acceptability Control 5 3.6 ± 0.05 0.5 mM 5 3.8 ± 0.04 1 mM 5 4.0 ± 0.06 2 mM 5 4.1 ± 0.02
d c b a
2.1 ± 0.03 2.5 ± 0.04 2.9 ± 0.02 2.6 ± 0.03
d c a b
1.0 ± 0.00 1.8 ± 0.06 2.2 ± 0.05 1.6 ± 0.02
d b a c
1.0 ± 0.00 1.3 ± 0.03 1.8 ± 0.02 1.2 ± 0.01
d b a c
Means of three replications ± standard deviation. Means in same column with different letters are significantly different (P < 0.05). B
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of the mushroom toward softening. The impact of DETANO (1 mM) + MAP was apparent. This effect was also similar to the observation that NO could delay the ripening and senescence of fruit. This could be attributed to the fact that treatment with DETANO (1 mM) + MAP suppressed the increased rate of leakage, protected the cells (Zhu, Zhou, Zhu, & Guo, 2009). 3.3. Effect of NO and MAP integrated treatments on colour Table 1 shows the different values obtained after application of DETANO (0.5, 1, and 2 mM) + MAP, compared to the stand-alone MAP without any treatment. From this table, higher luminosity (L) values and lower total colour variation (DE) were observed in DETANO (0.5, 1, and 2 mM) + MAP compared to stand-alone MAP after 4 d. The L value of stand-alone MAP samples decreased sharply after the first 4 d, and it was 81.8 at the 8th day and 78.1 at the 12th day, the last value may not be considered as commercially acceptable if a L value of 80 for wholesalers was take into account (López-Briones et al., 1992). The browning index (BI) of mushrooms was higher in stand-alone MAP than DETANO (0.5, 1, and 2 mM) + MAP during the whole storage. At the end of the storage, mushrooms treated with DETANO (1 mM) + MAP browned slightly, but they also had commercial value and edibility. Compared with stand-alone MAP, DETANO (0.5, 1, and 2 mM) + MAP treatments can inhibit the browning of the button mushrooms.
3.4. Effect of NO and MAP integrated treatments on percent open caps and overall acceptability The percentage of open-cap mushrooms increased as the storage period advanced in all the treatments and was higher in stand-alone MAP samples. The percentage of open-cap mushrooms in stand-alone MAP was 87.8% after 16 d of storage (Table 2). On the other hand, the percentage of open caps mushrooms subjected to DETANO (0.5, 1, and 2 mM) + MAP was in the range of 65.3– 76.7% after 16 d. Among treatments, the minimum percentage (64.3%) was recorded in case of DETANO (2 mM) + MAP samples. The cap opening of mushrooms is related to the dryness of mushrooms as a result of water loss during storage. The increased water loss during storage causes decrease in cohesive forces of water and other hydrophilic molecules, such as proteins responsible for the intact position of the caps and veil in mushrooms. As NO reduces water loss, the cap opening of mushrooms was less in DETANO (0.5, 1, and 2 mM) + MAP samples, particularly in DETANO (1 and 2 mM) + MAP. The overall acceptability based on colour, texture and percent open caps of mushrooms decreased as the storage period advanced in all the treatments. Based on judgements made by sensory panel members, the stand-alone MAP samples were unacceptable after 12 d of storage. However, mushrooms in DETANO (0.5, 1, and 2 mM) + MAP did not exhibit these characteristics even on day
50
-1
Ascorbic acid (mg •kg )
-1
Total phenolics (mg •kg )
1000
900
800
700
600
0
4
8
12
40
30
20
10
0
16
0
4
90
1.1
80
1
70
H2O2 content (µmol g -1 FW)
1.2
0.9 0.8
0.6
30
4
8
Storage time (days)
16
12
16
50 40
0
12
60
0.7
0.5
8
Storage time (days)
•-
O2 production rate -1 -1 (nmol g FW min )
Storage time (days)
12
16
20
0
4
8
Storage time (days)
Fig. 3. Changes in total phenolics (A), ascorbic acid (B), O 2 production rate (C), and H2O2 content (D) of button mushrooms packaged in stand-alone MAP (), DETANO (0.5 mM) + MAP (h), DETANO (1 mM) + MAP (N), and DETANO (2 mM) + MAP (j) stored at 4 °C for 16 d. Each data point is the mean of three replicate samples. Vertical bars represent standard errors of means.
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12, the DETANO (1 mM) + MAP samples were acceptable and in marketable condition and recorded an overall acceptability of 2.2 after 12 d of storage. These results suggest that the DETANO (0.5, 1, and 2 mM) + MAP were effective in retarding mushroom sensory deterioration. 3.5. Effect of NO and MAP integrated treatments on functional components Phenolic compounds have been reported as the major antioxidant components in mushrooms. These antioxidant compounds have been widely reported to have beneficial effects on the maintenance of health and the prevention of cancer and cardiovascular diseases. In this study, total phenolics levels declined in all treatments during the 16-d period (Fig. 3A). The lowest phenolics content was found in mushrooms from stand-alone MAP. DETANO (0.5, 1, and 2 mM) + MAP was more effective in delaying decrease of phenolics than stand-alone MAP. DETANO (1 mM) + MAP treatment best maintained the phenolic content of mushrooms. As mentioned above, this treatment also reduced browning on mushrooms. It appears that lower levels of browning correlated fairly well with phenolic content, which seems to be the limiting factor of the discoloration process. Little is known about the effects of NO treatment and storage on the retention of dietary antioxidants such as ascorbic acid (AA) in button mushroom. As shown in Fig. 3B, the content of AA
A
diminished in both stand-alone MAP and DETANO (0.5, 1, and 2 mM) + MAP treated mushrooms during storage, reaching levels about 50% lower than the initial values. However, treatment with DETANO (1 mM) + MAP induced a lower rate of decrement in AA during storage. The AA content in stand-alone MAP samples declined rapidly throughout storage, and no significant differences (P > 0.05) were observed between DETANO (0.5 mM) + MAP and stand-alone MAP mushroom. Therefore DETANO (1 mM) + MAP maintained high levels of AA, which indicated that NO treatment delayed the senescence of button mushroom during storage. Similar results have also been found by Duan et al. (2007) for longan fruit. It is known that low temperatures can reduce AA degradation in fresh-cut produce. The accumulation of CO2 within packages seems to have a negative effect on AA content of some fruits. Fruit and vegetable ripening have been described as an oxidative phenomenon which requires a turnover of reactive oxygen species (ROS), such as H2O2 and O 2 . In the present study, the increases in both O 2 production rate and H2O2 content were observed during 16 d of storage (Fig. 3C and D). The use of standalone MAP does not seem to substantially contribute to enhancement of antioxidant capacity of mushroom. On the other hand, DETANO (0.5 and 1 mM) + MAP treatments generally reduced the increase in both O 2 production rate and H2O2 content of mushroom during storage. Mushrooms treated with DETANO (1 mM) + MAP presented higher antioxidant capacity, compared with DETANO (0.5 and 2 mM) + MAP. In summary, NO treatment
B
96
2.1
64 48 32
(Units mg-1 protein)
1.8
SOD activity
PPO activity (Units mg -1 protein)
80
16 0
1.5
1.2
0.9
0.6 0
4
8
12
16
0
4
Storage time (days)
C
D
450
360
12
16
12
16
5
4
APX activity -1 (Units mg protein)
CAT activity (Units mg -1 protein)
8
Storage time (days)
270
180
90
3
2
1
0
0 0
4
8
Storage time (days)
12
16
0
4
8
Storage time (days)
Fig. 4. Changes in PPO (A), SOD (B), CAT (C), and APX (D) of button mushrooms packaged in stand-alone MAP (), DETANO (0.5 mM) + MAP (h), DETANO (1 mM) + MAP (N), and DETANO (2 mM) + MAP (j) stored at 4 °C for 16 d. Each data point is the mean of three replicate samples. Vertical bars represent standard errors of means.
T. Jiang et al. / Food Chemistry 126 (2011) 1693–1699
had a positive effect on antioxidant capacity, mainly influenced by total phenolics and ascorbic acid of button mushrooms. Similar results have also been found by Duan et al. (2007) and Flores et al. (2008) for longan fruit and peach. 3.6. Effect of NO and MAP integrated treatments on enzymes activity Enzymatic browning is a consequence of polyphenol oxidase (PPO) catalysed oxidation of phenolic substrates into quinones, which undergo further reactions to dark pigments called melanins. The major PPO enzyme responsible for browning in mushrooms appears to be tyrosinase. PPO activity increased in the stand-alone MAP samples during the first 12 d of storage then decreased (Fig. 4A). Treatment with DETANO (1 mM) + MAP significantly (P < 0.05) reduced the increase in PPO activity, compared with DETANO (0.5 and 2 mM) + MAP. NO inhibited the increase of PPO activity and suggested that NO could interact with transition metals (e.g. iron, copper and zinc) and thiol-containing enzymes. It has also been reported that NO inhibited the activity of haem-containing enzymes, such as CAT and POD, the important H2O2-scavenging enzymes during pathogen attack (Clark, Durner, Navarre, & Klessig, 2000). PPO is a complex enzyme with two copper ions in its active center. It is speculated that NO could react with the copper of PPO to form copper–nitrosyl complexes (NO–Cu–PPO), which could change the normal structure of the active site in PPO and thus reduce PPO activity. Antioxidant enzymes such as SOD, CAT and APX play a crucial role in antioxidant defense during the fruit ripening process. These enzymatic components, together with the low-molecular-weight antioxidants ascorbic acid (AA) and glutathione (GSH), ultimately scavenge H2O2 at the expense of NADPH or NADH (Foyer & Halliwell, 1976). In the present study, the DETANO (1 mM) + MAP treatment significantly (P < 0.05) delayed the decrease in the activities of SOD and CAT (Fig. 4B and C), and increase in activities of APX during storage (Fig. 4D), in agreement with the reduced ROS production (Fig. 3C and D). The results obtained seem to indicate that NO, as an antisenescent agent, affects positively the response to the oxidative stress associated with mushroom senescence. It has been observed that NO seems to have an antioxidant activity in the leaves of rice, where it delays the initiation of senescence induced by the plant-growth regulator methyl jasmonate, probably due to its capacity for removing ROS (Hung & Kao, 2004). 4. Conclusion Our research showed that the senescence inhibition of coldstored button mushroom by the DETANO (1 mM) + MAP (BOPP) treatment involved in the maintenance of tissue firmness, inhibition of browning, increase of phenolics and ascorbic acid contents, and enhancement of antioxidant ability compare with stand-alone MAP. NO treatment also reduced weight loss and delayed cap opening during the storage period. These results suggest that DETANO (1 mM) + MAP (BOPP) treatment may be a useful technique of maintaining button mushroom quality and extending their postharvest life. References Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254. Candan, N., & Tarhan, L. (2003). Relationship among chlorophyll-carotenoid content, antioxidant enzyme activities and lipid peroxidation levels by Mg2+ deficiency in the Mentha pulegium leaves. Plant Physiology and Biochemistry, 41, 35–40. Chang, S. T. (1999). World production of cultivated edible and medicinal mushrooms in 1997 with emphasis on Lentinus edodes (Berk.) Sing. in China. International Journal of Medicinal Mushrooms, 1, 291–300.
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