Effect of 1-methylcyclopropene on shelf life, visual quality, antioxidant enzymes and health-promoting compounds in broccoli florets

Food Chemistry 118 (2010) 774–781

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Effect of 1-methylcyclopropene on shelf life, visual quality, antioxidant enzymes and health-promoting compounds in broccoli florets Gaofeng Yuan, Bo Sun, Jing Yuan, Qiaomei Wang * Department of Horticulture, Zhejiang University, 268 Kaixuan Road, Hangzhou, Zhejiang 310029, China Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture, Hangzhou 310029, China

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Article history: Received 2 December 2008 Received in revised form 12 April 2009 Accepted 21 May 2009

Keywords: Broccoli 1-Methylcyclopropene (1-MCP) Glucosinolates Visual quality Shelf life Antioxidant enzymes

a b s t r a c t The effect of 1-methylcyclopropene (1-MCP) on quality, antioxidant enzymes and glucosinolate contents in broccoli (Brassica oleracea var. italica) florets was investigated in the present study. Broccoli florets were treated with air (control) and 2.5 ll/l 1-MCP for 6 h at 20 °C, and were then stored at 20 °C for 5 days. 1-MCP treatment markedly extended shelf life, reduced postharvest deterioration, retarded chlorophyll degradation and inhibited the increase of malondialdehyde amount and activities of polyphenol oxidase and lipoxygenase in florets. The activities of superoxide dismutase, peroxidase and catalase in florets treated with 1-MCP were higher than those in control florets. 1-MCP treatment reduced the rate of decrease of total carotenoids, ascorbic acid and glucosinolates in florets when compared to those in the control. These results indicated that 1-MCP treatment could be a good candidate for extending shelf life, maintaining visual quality and reducing loss of health-promoting compounds, particularly glucosinolates in broccoli. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Broccoli (Brassica oleracea var. italica) has been described as a vegetable with a high nutritional value, due to its significant content of vitamins, antioxidants and anticarcinogenic compounds. Glucosinolates, one of the main health-promoting secondary metabolites in broccoli are a group of sulphur- and nitrogen-containing secondary metabolites. These compounds have gained much attention in recent years because of the remarkable anticarcinogenic activity of their major hydrolysis products, isothiocyanates. Glucosinolates are chemically stable until they come into contact with the enzyme myrosinase (b-thioglucoside glucohydrolase, EC 3.2.1.147), which is stored in the cells, separately from the glucosinolates. Upon tissue damage, glucosinolates are released from plant vacuoles and are rapidly hydrolysed by myrosinase to glucose and unstable thiohydroximate-O-sulphonate intermediates, which, as dictated by chemical conditions, spontaneously rearrange to isothiocyanates, thiocyanates or nitriles. Usually, production of isothiocyanates is favoured in neutral conditions. Epidemiological studies have shown that isothiocyanates have protective effects against cancer, particularly bladder, colon and lung cancers (Cartea & Velasco, 2008). Broccoli is a crop which is increasingly popular. During the last decade, storage of broccoli florets has also been studied, as part of * Corresponding author. Tel.: +86 571 8590 9333; fax: +86 571 8742 0554. E-mail address: [email protected] (Q. Wang). 0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.05.062

the increasing demand for minimally-processed vegetables (Izumi, Watada, & Douglas, 1996). However, broccoli is a highly perishable product and its visual and organoleptic qualities greatly depend on its storage conditions. The effect of different storage methods on shelf life, visual quality and nutritional quality of broccoli has been widely investigated (Han et al., 2006; Izumi et al., 1996; Serrano, Martinez-Romero, Guillen, Castillo, & Valero, 2006; Vallejo, Tomas-Barberan, & Garcia-Viguera, 2003). Cooling and controlled atmosphere are usually recommended for the storage of broccoli florets (Izumi et al., 1996). However, cooling and controlled atmosphere facilities are not always available in developing countries such as China, where high temperatures are often encountered during postharvest storage, handling and transportation, and the marketing phase of broccoli produce (Jones, Faragher, & Winkler, 2006). Commercialisation of 1-methylcyclopropene (1-MCP) has provided a new tool to improve maintenance of horticultural product quality. 1-MCP has a non-toxic mode of action, a negligible residue and is active at very low concentrations; it has been considered non-toxic for humans and the environment (Luo, Xu, Cai, & Yan, 2007). As an ethylene action inhibitor, 1-MCP was found to be effective in overcoming the effects of ethylene in a range of perishable fruit and vegetable (Watkins, 2006). Treatment of broccoli with 1-MCP resulted in lower respiration and ethylene production, delayed degreening and an extension of broccoli storage life (Fan & Mattheis, 2000; Ku & Wills, 1999). As more attention is paid to the effect of 1-MCP treatment on shelf life and visual quality, limited

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information is available on the effect of 1-MCP treatment on physiological and biochemical responses, towards, for example, antioxidant enzymes and lipid peroxidation, as well as the alteration of nutritional quality in broccoli florets. To date, there are no reports on the effect of 1-MCP treatment on glucosinolates of products including broccoli florets. The objective of the present study was to investigate the effect of treatment of broccoli florets with 2.5 ll/l 1-MCP on shelf life, visual quality, physiological and biochemical responses and health-promoting compounds during storage at 20 °C. 2. Materials and methods 2.1. Plant material Broccoli heads (Br. oleracea var. italica cv. Lvxiong) of prime quality were harvested in the early morning from the greenhouse of Zhejiang University (Hangzhou, China), top-iced and then transported to the Postharvest Laboratory of Vegetable Institute, Zhejiang University within 10 min. Broccoli heads with a diameter ranging from 15 to 20 cm were chosen, and the inner branches (with florets having stalks of approximately 2 cm) were then cut from these heads for experimentation. The florets were surfacesterilised by washing with a solution of 50 mg/kg NaOCl for 1 min and dried using a household model spin drier for 2 min. The florets were then randomly distributed into the required number of treatment units. 2.2. 1-MCP and storage treatments Broccoli florets were treated with air (control), 0.1, 1, 2.5, 5 and 10 ll/l 1-MCP for 6 h at 20 °C. The broccoli florets were placed in plastic bags and 1-MCP was injected, then the injection pore of the bags was immediately sealed and the samples were stored for 6 h at 20 °C. After treatment, all experimental units were stored according to randomised complete block design in incubation chamber with 95% RH at 20 °C. The shelf life was then determined. The concentration of 2.5 ll/l 1-MCP was selected for further study. There were three replicates per treatment with 200 g of broccoli florets per replicate. Shelf life, visual quality and visual colour scale rating were made in replicates of 10 florets taken daily. The treated broccoli florets were used for determination of chlorophyll, antioxidant enzymes, polyphenol oxidase (PPO, EC 1.14.18.1), lipoxygenase (LOX, EC 1.13.11.12), malondialdehyde (MDA), ascorbic acid, total carotenoids and glucosinolates. 2.3. Determination of shelf life Shelf life was determined according to previous reports (Ku & Wills, 1999; Xu, Guo, Yuan, Yuan, & Wang, 2006). The time for quality to decline to 30% yellowing in florets was assigned as their shelf life. 2.4. Determination of visual colour rating scale and visual quality Visual quality was determined as previously described (Jia et al., 2009). Visual quality of florets was scored on a 1–9 scale, where 9 refers to excellent and fresh appearance, 7 to good, 5 to fair (limit of marketability), 3 to fair (useable but not saleable), and 1 to unusable. Intermediate numbers were assigned where appropriate. Colour of broccoli florets was visually rated using colour rating scales 1–5, as described by Rangkadilok et al. (2002), where 1 refers to dark green, 2 to trace yellow (10% yellow), 3 to slightly yellow (25% yellow), 4 to medium yellow (50% yellow), and 5 to completely yellow (100%).

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2.5. Determination of chlorophyll content Florets (0.5 g) were ground and extracted in 10 ml of 80% acetone, centrifuged at 3000 rpm for 10 min at room temperature, and then residue was removed. Total chlorophyll content was determined by reading the absorbance at 652 nm with a spectrophotometer. Total chlorophyll content was estimated as mg/g fresh weight (FW). 2.6. Enzyme assay Broccoli florets (0.5 g) were homogenised with 5 ml, 50 mM phosphate buffer (pH 7.8) in an ice-water bath, and then centrifuged at 8000 rpm for 20 min at 4 °C. The supernatant was collected and used for enzyme assay. Total superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and peroxidase (POD, EC 1.11.1.7) activities were assayed according to the method of Toivonen and Sweeney (1998). SOD activity was determined based on the ability of SOD to inhibit the reduction of nitroblue tetrazolium. The absorbance was monitored at 560 nm. One unit of SOD is the amount of extract that gives 50% inhibition of the reduction rate of NBT. CAT activity was determined by monitoring the enzyme-catalysed decomposition of H2O2 by potassium permanganate. One unit of CAT activity was defined as the amount of H2O2 (lmol) depleted per min at 240 nm. POD activity was measured based on the determination of guaiacol oxidation at 470 nm by H2O2. The change in absorbance at 470 nm was followed every 30 s by spectrophotometer. PPO activity was assayed by measuring the increase in absorbance at 420 nm with 100 mM catechol solution as substrate, according to Kumar, Mohan, and Murugan (2008). One unit of enzyme activity was defined as the amount of enzyme that caused an increase in absorbance of 0.001 min 1. LOX activity was determined according to the method of Anese and Sovrano (2006) with minor changes. The substrate solution was prepared by mixing 20 ll of linoleic acid, 48 ml of H2O, 2 ml of 0.1 N NaOH and 10 ll of Tween 20. Each test contained 0.1 ml enzymatic extracts, 0.15 ml substrate solution and 1.25 ml phosphate buffer (pH 5.7). One unit of LOX is defined as the amount of enzyme which caused an increase in absorption at 234 nm of 0.1 min 1 (3 min period). The SOD, CAT, POD, PPO and LOX activities were estimated by units/mg protein. Protein content of samples was determined by using Folin phenol reagent (Lowry, Rosebrough, Farr, & Randall, 1951). 2.7. Malondialdehyde (MDA) determination MDA was measured by 2-thiobarbituric acid reaction. One gram fresh weight of broccoli florets sample was homogenised in 5 ml of 0.1% (w/v) TCA. The homogenate was centrifuged at 12,500 rpm for 20 min. One millilitre of supernatant was precipitated with 4 ml 20% TCA containing 0.5% (w/v) 2-thiobarbituric acid. The mixture was heated in a water-bath shaker at 95 °C for 30 min and quickly cooled in an ice-bath. The absorbance was read at 532 nm after centrifugation at 3000 rpm for 10 min and the value for non-specific absorption at 600 nm was subtracted. The MDA content was calculated using its extinction coefficient of 155 mM 1 cm 1. The amount of MDA was estimated as lmol/g FW. 2.8. Total carotenoids determination Five grams of florets were ground and extracted with a mixture of acetone and petroleum ether (1:1, v/v) repeatedly using the mortar and pestle until a colourless residue was obtained. After washing several times with water, the upper phase was collected and combined as crude extract. The extracts were made up to a

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known volume with petroleum ether. Total carotenoids content was determined by reading the absorbance at 451 nm with a spectrophotometer. Total carotenoids content was estimated as mg/ 100 g FW. 2.9. Ascorbic acid determination Twenty grams of frozen broccoli florets were ground to a fine powder at liquid nitrogen temperature, extracted twice with 20 ml of 1.0% (w/v) oxalic acid and centrifuged for 20 min at 3000 rpm. The combined aqueous extract was filtered under vacuum in a volumetric flask. Each sample was filtered through a 0.45 lm cellulose acetate filter prior to injection. HPLC analysis of ascorbic acid was carried out using a Shimadzu (Tokyo, Japan) mode VP liquid chromatograph with a dual-wavelength spectrophotometer. Samples (5 ll) were separated at room temperature on a Waters Spherisorb C18 column (150  4.6 mm id; 5 lm particle size) (Milford, MA) using a solution of 0.05 M KH2PO4 at a flow rate of 1.0 ml/min. The amount of ascorbic acid was calculated from absorbance at 254 nm, using ascorbic acid as a standard. Results were expressed as mg/100 g FW. 2.10. Glucosinolate assay Freshly harvested and stored broccoli florets were frozen in liquid N2 and kept in polyethylene bags at 70 °C until freeze-drying. Freeze-dried broccoli samples were stored in sealed polyethylene bags at 4 °C until analysis. Glucosinolates were extracted and analysed as previously described with minor modifications (Wang, Grubb, & Abel, 2002; Xu et al., 2006). Freeze-dried samples (25 mg) were boiled in 1 ml water for 10 min. After recovery of the liquid, the residues were washed with water (1 ml), and the combined aqueous extract was applied to a DEAE-Sephadex A25 (40 mg) column (pyridine acetate form) (GE Healthcare, Piscataway, NJ). The column was washed three times with 20 mM pyridine acetate and twice with water. The glucosinolates were converted into their desulfo analogues by overnight treatment with 100 ll of 0.1% (1.4 units) aryl sulfatase, and the desulfoglucosinolates were eluted with 2  0.5 ml water. HPLC analysis of desulfoglucosinolates was carried out using a Shimadzu mode VP liquid chromatograph with a dual-wavelength spectrophotometer. Samples (100 ll) were separated at 30 °C on a Waters Spherisorb C18 column (150  4.6 mm id; 5 lm particle size) using acetonitrile and water at a flow rate of 1.0 ml/min. The procedure employed isocratic elution with 1.5% acetonitrile for the first 5 min; a linear gradient to 20% acetonitrile over the next 15 min, followed by isocratic elution with 20% acetonitrile for the final 10 min. Absorbance was measured at 226 nm and 280 nm. ortho-Nitrophenyl-b-D-galactopyranoside (Sigma) was used as an internal standard for HPLC analysis. Concentrations of individual glucosinolates were determined according to published response factors. The integrated area of the desulfo-4-methylsulfinylbutyl glucosinolate peak was converted to a molar amount under the assumption that this compound has a molar extinction coefficient at 226 nm equal to that of sinigrin. 2.11. Statistical analyses Statistical analysis was performed using the SPSS package program version 11.5 (SPSS Inc., Chicago, IL). Data was analysed by analysis of variance (ANOVA model one-way). Sources of variation were storage duration and treatments. The means were compared by the least significant differences (LSD) test at a significance level of 0.05. The values are reported as means with their standard deviations for all results.

3. Results 3.1. Effect of 1-MCP treatment on shelf life All 1-MCP treatments greatly extended the shelf life of broccoli florets, the most significant being broccoli florets treated with 2.5 ll/l 1-MCP (Fig. 1). The shelf life of broccoli florets treated with 2.5 ll/l 1-MCP was extended to two times that of control florets (2.5 days at 20 °C). Based on the results of shelf life studies, 1MCP at a dose of 2.5 ll/l was selected, to analyse the effect of 1MCP treatment on visual and nutritional quality of broccoli florets. 3.2. Effect of 1-MCP treatment on colour, visual quality and chlorophyll degradation There was yellowing and rapid loss in visual quality occurred in control florets during storage at 20 °C. 1-MCP treatment effectively inhibited this yellowing and visual quality deterioration of broccoli florets. A significant difference in colour and visual quality between 1-MCP treatment and control was observed after 1 days storage at 20 °C (Fig. 2). Chlorophyll content decreased in both control and 1-MCP-treated broccoli florets during storage. However, chlorophyll degradation rate was significantly lower in treated broccoli florets than in control florets (Fig. 2). After storage at 20 °C for 5 days, 1-MCP-treated broccoli florets contained 67% of the initial chlorophyll content, while control florets contained only 24% of initial value. 3.3. Effect of 1-MCP treatment on SOD, CAT, POD and PPO activities SOD activity in both the control and treatment declined during the first 3 days of storage, significantly increased during the following day and then fell again at the 5th day. CAT activity in both the control and treatment showed the same trend as SOD during the first 3 days of storage, and then increased steadily to the 5th day. 1-MCP treatment significantly improved the SOD and CAT activities, which made the SOD and CAT activities always higher in the treatment than in control across the whole storage period (Fig. 3). POD activity increased in both control and 1-MCP treatment during the first day, decreased over the next 3 days and then

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The contents of ascorbic acid and total carotenoids in both control and 1-MCP treated broccoli florets declined progressively, with a higher level of both in 1-MCP treated florets than in the control after 5 days of storage at 20 °C. The loss of ascorbic acid and total carotenoids were 19.4% and 30.0%, respectively, in 1-MCP-treated florets, while losses of as much as 57.8% and 50.0% occurred, respectively, in control florets over 5 days of storage at 20 °C. The contents of ascorbic acid and total carotenoids were 2-fold and 1.3-fold higher, respectively, in 1-MCP treatment florets than in control florets on the 5th day (see Fig. 5).

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3.6. Effect of 1-MCP treatment on total and individual glucosinolates

Visual qualit y

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Days of storage at 20 C Fig. 2. Changes of colour, visual quality and chlorophyll content in broccoli florets. Florets were treated with air (control) and 2.5 ll/l 1-MCP for 6 h at 20 °C. Florets were stored at 20 °C for 5 days. Each data point is the mean of three replicate samples. Vertical bars represent standard deviation of the mean.

increased again. The activity of POD in treated broccoli florets was significantly higher than in the control florets during 5 days storage at 20 °C (Fig. 3). PPO activity in both control and treatment markedly increased during the initial 4 days of storage and then decreased at day 5. However, the florets treated with 1-MCP possessed relatively lower PPO activity in comparison with the control florets during storage (Fig. 3).

The total glucosinolate content as well as the individual aliphatic (methylsulfinylalkyl: glucoiberin, glucoraphanin and progoitrin) and indole (glucobrassicin, neoglucobrassicin and 4methoxyglucobrassicin) glucosinolate contents were quantitatively determined in broccoli florets over 5 days of storage (Fig. 6). The predominant aliphatic glucosinolates in broccoli florets were 4-methylsulfinylbutyl glucosinolate (glucoraphanin), followed by 3-methylsulfinylpropyl glucosinolate (glucoiberin), while the predominant indole glucosinolates were 3-indolylmethyl glucosinolate (glucobrassicin) and 1-methoxy-3-indolymethyl glucosinolate (neoglucobrassicin). The change of the total aliphatic glucosinolates content was not significant in both control and 1-MCP treatment over the first two days storage, then a rapid decrease of total aliphatic glucosinolates content was observed in florets of control and 1-MCP treatment. However, the decrease rate in treated florets was significantly lower than in control florets. The total aliphatic glucosinolates was reduced by 71.2% after 5 days of storage at 20 °C in control florets, but only reduced by 51.3% in florets treated with 2.5 ll/l 1-MCP (Fig. 6). Amongst the individual aliphatic glucosinolates, glucoraphanin, progoitrin and glucoiberin showed similar trends to total aliphatic glucosinolates in both control and treatment during 5 days of storage at 20 °C. The loss rates of glucoraphanin, progoitrin and glucoiberin in broccoli florets treated with 1-MCP (56.4%, 23.6% and 32.4%, respectively) were much lower than those in control florets (77.7%, 48.6% and 42.7%, respectively) at the end of the 5 days storage period (Fig. 6). The total indole glucosinolates content decreased during 5 days storage at 20 °C, although lower losses of indole glucosinolates than aliphatic glucosinolates in both control and treatment were detected. The total indole glucosinolates decreased by 37.0% in the control, but only 23.7% in the treatment (Fig. 6). The decrease in total indole glucosinolates was predominantly due to the progressive degradation of glucobrassicin and neoglucobrassicin in both control and treatment. However, the decrease rate of glucobrassicin and neoglucobrassicin in the treatment (26.7% and 20.6%, respectively) was lower than in the control (41.5% and 33.2%, respectively) (Fig. 6).

3.4. Effect of 1-MCP treatment on MDA content and LOX activity

4. Discussion

The initial content of MDA was 1.54 lmol/g FW, and no significant difference in MDA content was observed between control and 1-MCP treatment during the first day of storage; subsequently a larger rise in MDA content was found in control florets than in florets treated with 2.5 ll/l 1-MCP from the second day at 20 °C (Fig. 4). The results indicated that 1-MCP treatment could inhibit the rise of MDA content. Similarly, the rise of LOX activity during storage was inhibited by the 1-MCP treatment (Fig. 4).

The effect of 1-MCP treatment on shelf life, visual quality, antioxidant enzymes and health-promoting compounds in broccoli florets was investigated in the present study. The results showed that the shelf life of broccoli florets treated with various concentrations of 1-MCP was significantly improved. This is consistent with the findings of Ku and Wills (1999), Fan and Mattheis (2000), Able, Wong, Prasad and O’Hare (2002), and Forney, Song, Fan, Hildebrand, and Jordan (2003). However, Ku and Wills (1999) found that

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Days of storage at 20 ° C Fig. 3. Changes of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and polyphenol oxidase (PPO) activities in broccoli florets. Florets were treated with air (control) and 2.5 ll/l 1-MCP for 6 h at 20 °C. Florets were stored at 20 °C for 5 days. Each data point is the mean of three replicate samples. Vertical bars represent standard deviation of the mean.

the higher the concentration of 1-MCP was (ranging from 0.02 to 50 ll/l), the greater was the storage life of broccoli florets, but also suggested that a lower concentration range (1–10 ll/l) needs to be used to avoid toxicity. Able et al. (2002) showed that a concentration of 12 ll/l 1-MCP was considered optimal for extension of the shelf life of broccoli florets. Our study showed that the extension of

shelf life was most significant in broccoli florets treated with 2.5 ll/l 1-MCP. This discrepancy could be due to different cultivars and temperatures used.

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Fig. 4. Change of malondialdehyde (MDA) content and lipoxygenase (LOX) activity in broccoli florets. Florets were treated with air (control) and 2.5 ll/l 1-MCP for 6 h at 20 °C. Florets were stored at 20 °C for 5 days. Each data point is the mean of three replicate samples. Vertical bars represent standard deviation of the mean.

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The green colour of broccoli is an important commercial quality index. Degreening of broccoli after harvest occurs rapidly during storage at 20 °C (Wang, 1977). Treatment of broccoli with 1-MCP resulted in delayed loss of green colour and delayed onset of yellowing (Fan & Mattheis, 2000; Gong & Mattheis, 2003; Ku & Wills, 1999). Our study supported the evidence that treatment of broccoli florets with 2.5 ll/l 1-MCP inhibited chlorophyll degradation, yellowing and quality deterioration. Senescence is considered to be associated with the defence system, including antioxidant enzymes and antioxidants. Antioxidant enzymes (SOD, CAT and POD) are considered to be important in the oxy-radical detoxification process in plant tissues (Han et al., 2006;

Mittler, 2002). Toivonen and Sweeney (1998) reported that antioxidant protection offered by SOD and POD is important for the retention of green colour in broccoli flower buds, and the increases in SOD, POD and CAT were likely responses to the increases in oxygen radical production in broccoli, which could subsequently lead to yellowing. Postharvest treatment could change the activity of antioxidant enzymes in vegetables and fruits. Ethanol vapour treatment leads to higher activities of SOD, POD and CAT in broccoli florets during storage, as compared with the control (Han et al., 2006). 1-MCP treatment has also been shown to increase the activities of antioxidant enzymes in mango (Singh & Dwivedi, 2008) and Yali pear (Fu, Cao, Li, Lin, & Jiang, 2007). In our study,

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it was found that SOD, CAT and POD activities were consistently higher in the 1-MCP treatment than in the control during 5 days storage at 20 °C. It was suggested that the increases in the activities of SOD, CAT and POD were generally a consequence of the system ability to delay senescence (Toivonen & Sweeney, 1998). From the results of our study, it can be hypothesised that 1-MCP might delay the senescence of broccoli florets by means of regulating the antioxidant enzymes system. The specific mechanism of 1-MCP in delaying the senescence of broccoli florets and its relationship with the antioxidant enzymes still remain to be further investigated. PPO represents a marker compound for the extent of oxidation and is responsible for browning in fresh-cut fruits and vegetables. 1-MCP treatment has been shown to reduce browning, which was associated with reduced PPO activity in avocados (Watkins, 2006). It is suggested in the present study that 1-MCP treatment could reduced the browning of broccoli florets by reduction of PPO activity. The thiobarbituric acid-reactive substance MDA is the product of membrane peroxidation and has been used as a direct indicator of membrane injury. According to Zhuang, Hildebrand, and Barth (1995, 1997), postharvest senescence of broccoli is correlated with lipid peroxidation (MDA content), leading to cell-membrane disintegration. LOX are regarded to be responsible for membrane degradation because they catalyse the peroxidation of polyunsaturated fatty acids, producing hydroperoxy fatty acids (Macri, Braidot, Petrusa, & Vianello, 1994). It was found in our study that 1-MCP treatment could inhibit the increase of lipid peroxidation (MDA content) and LOX activity in broccoli florets, compared with control florets, during postharvest storage. Vitamin C is one of the most important nutritional components in broccoli, as well as in many other horticultural crops, and has many biological activities in the human body. The concentration of vitamin C/ascorbic acid (the predominant form of vitamin C) in broccoli generally decreased during storage and the loss rate during storage could be lowered by postharvest handling (Serrano et al., 2006; Vallejo et al., 2003). In our study, the control florets lost more than half of their initial ascorbic acid after 5 days of storage, while these losses were minimised in those florets treated with 1-MCP. The present study showed that 1-MCP treatment resulted in significantly better retention of ascorbic acid during postharvest storage. Carotenoids have been extensively studied for their potential protection against numerous cancers. However, limited information is available on the effect of postharvest handling on carotenoids in broccoli. In the present work, total carotenoids in both control and treated florets decreased during 5 days of storage. However, treatment of broccoli florets with 2.5 ll/l 1-MCP significantly lowered the magnitude of total carotenoids loss during postharvest storage. Glucosinolates are among the most important health-promoting natural products in broccoli florets. They are known to contribute to the anticarcinogenic activity of broccoli. Several hydrolysis products of methylsulfinyl aliphatic glucosinolates, such as glucoraphanin, in broccoli are considered to reduce the risk of cancers. Sulforaphane (derived from glucoraphanin) is the most potent, naturally-occurring inducer of Phase 2 enzymes that detoxify carcinogens (Brown et al., 2002). The same is true for breakdown products of indole glucosinolates, notably glucobrassicin (Weng, Tsai, Kulp, & Chen, 2008). However, higher losses of glucoraphanin (or sulforaphane) and other glucosinolates have been reported after harvesting (Cieslik, Leszczynska, Filipiak-Florkiewicz, Sikora, & Pisulewski, 2007; Jia et al., 2009; Rangkadilok et al., 2002; Vallejo et al., 2003). In fact, control florets lost over half of their initial glucosinolates after 5 days storage at 20 °C (Fig. 6). Broccoli florets suffer a series of stresses after harvest (washing/pre-cutting) and during storage, which might trigger complex metabolism of glucosinolates. Cutting of broccoli heads to broccoli florets brings myrosinase in contact with glucosinolates, which might lead to a high

degree of glucosinolate hydrolysis. On the other hand, it might also induce the biosynthesis of glucosinolates during 1-MCP treatment and storage. The glucosinolate level of stored broccoli florets is a reflection of two opposing mechanisms, hydrolysis of glucosinolates by myrosinase and induction of glucosinolate biosynthesis by an unknown mechanism. The elevated level of some individual glucosinolates (progoitrin, glucoraphanin, and 4-methoxyglucobrassicin) in broccoli florets during the first day of storage under control or 1-MCP treatment was observed in our study (Fig. 6). The predominance of the induction of biosynthesis of these glucosinolates might be the cause of the increase of the content under these cases during storage, though the specific mechanism involved remains to be further elucidated. Postharvest handing is required to maintain glucosinolate content in broccoli florets and low temperature and high relative humidity are effective conditions (Jia et al., 2009; Jones et al., 2006; Rodrigues & Rosa, 1999). The present study showed that 1MCP treatment leads to better retention of glucosinolates, in comparison with the control, during storage at 20 °C, which suggested that 1-MCP treatment is also an effective method to maintain glucosinolate content. To the best of our knowledge, this is the first report on the effect of 1-MCP treatment on the glucosinolate content of foods. The mechanism by which broccoli florets treated with 2.5 ll/l 1-MCP inhibited the decrease rate of glucosinolate content compared with the control is not clear. However, previous studies and the data from the current study could provide possible explanations. Firstly, as mentioned above, 1-MCP treatment inhibited the increase of MDA as compared with the control. MDA is the product of membrane peroxidation, and could damage the structure and integrity of membrane during the senescence of broccoli florets. Thus, it can be hypothesised that 1-MCP could inhibit probable loss of membrane integrity and mixing of glucosinolates with myrosinase, leading to less glucosinolates hydrolysis. Furthermore, the biosynthesis of secondary metabolites, such as glucosinolates, is thought to be part of the plant defensive mechanism, and ethylene seems to play a role in this process (Mikkelsen et al., 2003). On the other hand, 1-MCP is well-known as an ethylene inhibitor and ethylene production was decreased by 1-MCP treatment. It is possible that the blockage of ethylene action by 1-MCP treatment favoured the biosynthesis of glucosinolates or inhibited some ethylene-related degradative pathways. Further studies are warranted to provide a clearer understanding of the effect of 1-MCP treatment on glucosinolate content. It was reported that aliphatic glucosinolates were generally more stable than indole glucosinolates during storage and processing of Brassica vegetables (Jia et al., 2009; Rungapamestry, Duncan, Fuller, & Ratcliffe, 2007). Boiling of these vegetables leads to inactivation of myrosinase and partial decomposition of thermolabile glucosinolates, especially indole glucosinolates (Cieslik et al., 2007). The observation that indole glucosinolates were preserved better than aliphatic glucosinolates under 1-MCP treatment in the current study suggests that 1-MCP treatment might have different effects on the stability of myrosinase and individual glucosinolates metabolites, though the mechanism involved remains to be further investigated. Previous studies have found that glucoraphanin, the predominant glucosinolate in broccoli, dramatically decreased at room temperature (Rodrigues & Rosa, 1999). Our current study showed similar results. On the other hand, 1-MCP treatment significantly inhibited the loss rate of glucoraphanin and glucobrassicin. In this regard, it is likely that broccoli florets treated with 1-MCP will be preferable for human consumption than control broccoli florets. As high temperatures are often encountered during the handling, transportation and retail marketing of broccoli, they have become the main cause of postharvest loss of broccoli, especially in developing countries. The use of 1-MCP can reduce the require-

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ment for low temperature while still extending the shelf life, and reducing the loss of visual and nutritional quality. It has tremendous potential for maintaining broccoli florets quality during storage at relatively higher temperature. In conclusion, 2.5 ll/l 1-MCP treatment significantly extended the shelf life, reduced the postharvest deterioration, retarded the chlorophyll degradation and inhibited the increase of MDA content and the activities of PPO and LOX of broccoli florets. The activities of SDO, POD and CAT in florets treated with 1-MCP were higher than those in control florets. 1-MCP treatment reduced the losses of total carotenoids, ascorbic acid and total aliphatic, total indole and individual glucosinolates in broccoli florets when compared to those in the control. These results indicated that 1-MCP treatment could be a good candidate for extending shelf life, maintaining the visual quality and reducing the loss of health-promoting compounds, particularly chemopreventive glucosinolates in broccoli florets stored at 20 °C. Acknowledgements The authors thank Dr. Duo Li for critical reading of the manuscript. This work was supported by National High-tech R&D Program of China (863 program 2008AA10Z111), China Postdoctoral Science Foundation Funded Project (20080441259), National Natural Science Foundation of China (30320974) and Fok Ying Tong Education Foundation (104034). References Able, A. J., Wong, L. S., Prasad, A., & O’Hare, T. J. (2002). 1-MCP is more effective on a floral brassica (Brassica oleracea var. italica L.) than a leafy brassica (Brassica rapa var. chinensis). Postharvest Biology and Technology, 26(2), 147–155. Anese, M., & Sovrano, M. (2006). Kinetics of thermal inactivation of tomato lipoxygenase. Food Chemistry, 95, 131–137. Brown, A. F., Yousef, G. G., Jeffrey, E. H., Klein, B. P., Wallig, M. A., Kushad, M. M., et al. (2002). Glucosinolate profiles in broccoli: Variation in levels and implications in breeding for cancer chemoprotection. Journal of the American Society for Horticultural Science, 127(5), 807–813. Cartea, M. E., & Velasco, P. (2008). Glucosinolates in Brassica foods: Bioavailability in food and significance for human health. Phytochemistry Reviews, 7, 213–229. Cieslik, E., Leszczynska, T., Filipiak-Florkiewicz, A., Sikora, E., & Pisulewski, P. M. (2007). Effects of some technological processes on glucosinolate contents in cruciferous vegetables. Food Chemistry, 105(3), 976–981. Fan, X. T., & Mattheis, J. P. (2000). Yellowing of broccoli in storage is reduced by 1methylcyclopropene. Hortscience, 35(5), 885–887. Forney, C. F., Song, J., Fan, L. H., Hildebrand, P. D., & Jordan, M. A. (2003). Ozone and 1-methylcyclopropene alter the postharvest quality of broccoli. Journal of the American Society for Horticultural Science, 128(3), 403–408. Fu, L., Cao, J., Li, Q., Lin, L., & Jiang, W. (2007). Effect of 1-methylcyclopropene on fruit quality and physiological disorders in Yali pear (Pyrus bretschneideri Rehd.) during storage. Food Science and Technology International, 13(1), 49–54. Gong, Y. P., & Mattheis, J. P. (2003). Effect of ethylene and 1-methylcyclopropene on chlorophyll catabolism of broccoli florets. Plant Growth Regulation, 40(1), 33–38. Han, J. H., Tao, W. Y., Hao, H. K., Zhang, B. L., Jiang, W. B., Niu, T. G., et al. (2006). Physiology and quality responses of fresh-cut broccoli florets pretreated with ethanol vapor. Journal of Food Science, 71(5), S385–S389. Izumi, H., Watada, A. E., & Douglas, W. (1996). Optimum O2 or CO2 atmospheres for storing broccoli florets at various temperatures. Journal of the American Society for Horticultural Science, 121, 127–131.

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