Effect of allyl isothiocyanate on antioxidant enzyme activities, flavonoids and post-harvest fruit quality of blueberries (Vaccinium corymbosum L., cv. Duke)

Food Chemistry 122 (2010) 1153–1158

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Effect of allyl isothiocyanate on antioxidant enzyme activities, flavonoids and post-harvest fruit quality of blueberries (Vaccinium corymbosum L., cv. Duke) Shiow Y. Wang *, Chi-Tsun Chen Genetic Improvement of Fruit and Vegetable Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, US Department of Agriculture, Beltsville, MD 20705-2350, United States

a r t i c l e

i n f o

Article history: Received 5 January 2010 Received in revised form 22 February 2010 Accepted 24 March 2010

Keywords: Allyl isothiocyanate Antioxidant enzyme activities Flavonoids Organic acids Sugars Vaccinium corymbosum L.

a b s t r a c t The effect of allyl isothiocyanate (AITC) on antioxidant enzyme activities, flavonoid content, and fruit quality of blueberries var. Duke (Vaccinium corymbosum L.) was evaluated. Results from this study showed that AITC was effective in maintaining higher amounts of sugars and lower organic acids compared to untreated fruit during storage at 10 °C. However, AITC reduced antioxidant enzyme activities [superoxide dismutase (SOD), guaiacol peroxidase (G-POD), glutathione-peroxidase (GSH-POD), ascorbate peroxidase (AsA-POD), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDAR) and glutathione reductase (GR)] and non-enzyme components, ascorbate (AsA) and glutathione (GSH). AITC treatments also reduced the amount of phenolic acids (chlorogenic acid, myricetin 3-arabinoside, quercetin 3-galactoside, quercetin 3-arabinoside, and kaempferol 3-glucoside) and anthocyanins (delphinidin 3-galactoside, delphinidon 3-glucoside, delphinidin 3-arabinoside, petunidin 3-galactoside, petunidin 3-glucoside, petunidin 3-arabinoside, malvidin 3-galactoside, and malvidin 3-arabinoside) during storage at 10 °C. The results from this study indicate that AITC does not promote antioxidant property or scavenge constitutive reactive oxygen species (ROS), but maintain blueberry fruit quality through other mechanisms. Ó 2010 Published by Elsevier Ltd.

1. Introduction Blueberries are known to have a high content of antioxidants and high oxygen radical scavenging capacity against peroxy radicals, superoxide radicals, hydrogen peroxide, hydroxyl radicals, and singlet oxygen (Prior et al., 1998; Wang & Jiao, 2000). However, blueberries are highly perishable after harvest. Various post-harvest techniques have been shown to be beneficial in extending blueberry storage life (Connor, Luby, Hancock, Berkheimer, & Hanson, 2002; Hardenburg, Watada, & Wang, 1986; Kalt et al., 2003). One method has reported to be effective in maintaining blueberry fruit quality, by using naturally occurring compounds such as treatment with essential oils and other natural volatile (Wang, Chen, & Yin, 2010; Wang, Wang, & Chen, 2008). Allyl isothiocyanate (AITC), C4H5NS, a natural compound commonly found in the Cruciferae family (Tookey, Van Etten, & Daxenbichler, 1980), possesses a strong antimicrobial activity against Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa and other pathogenic bacteria (Inoue et al., 1983; Nishida, 1958). It has been used as a preservative compound and is considered safe for human consumption (Kermanshai et al., 2001; Masuda, Harada,

* Corresponding author. Tel.: +1 301 504 5776; fax: +1 301 504 5107. E-mail address: [email protected] (S.Y. Wang). 0308-8146/$ - see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.foodchem.2010.03.106

Inoue, Kishimoto, & Tano, 1999; Shin, Masuda, & Naohide, 2004; Troncoso, Espinoza, Sánchez-Estrada, Tiznado, & García, 2005). Our previous studies (Chanjirakul, Wang, Wang, & Siriphanich, 2006; Chanjirakul, Wang, Wang, & Siriphanich, 2007; Wang et al., 2010) have shown that AITC reduced decay of strawberries, blackberries, raspberries and blueberries. However, little is known concerning the impact of AITC treatment on fruit quality of blueberries and no information is available on the effect of AITC on antioxidant enzyme activities and the levels of individual anthocyanins, phenolic acids and antioxidant enzyme activities of blueberries. The aim of the present study was to evaluate the effect of AITC treatment on fruit quality such as sugar and organic acid content, and the changes of flavonoids in blueberries during storage at 10 °C.

2. Materials and methods 2.1. Chemicals Ascorbate, chlorogenic acid, b-carotene, histidine, hydrogen peroxide (30% w/w), hydroxylamine hydrochloride, N,N-dimethylp-nitrosoaniline, xanthine, xanthine oxide ascorbate oxidase, dithiothreitol (DTT), glutathione (oxidised form), glutathione (GSH, reduced form), glutathione reductase, guaiacol, b-nicotinamide

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adenine dinucleotide (b-NADH, reduced form), b-nicotinamide adenine dinucleotide phosphate (b-NADPH, reduced form), nitro blue tetrazolium (NBT) were purchased from Sigma Chemical Co. (St. Louis, MO). EDTA (ethylenediaminetetracetic acid, disodium salt, dihydrate-Na2 EDTA
2H2O) and trichloroacetic acid were purchased from Aldrich (Milwaukee, WI). 2.2. Fruit sample handling and treatments with natural volatile compound allyl isothiocyanate (AITC) Blueberries var. Duke (Vaccinium corymbosum L.) used in this study were grown at a farm near Beltsville, Maryland, USA and were hand-harvested at a commercially mature stage, sorted to eliminate damaged, shriveled, and unripe fruit, and selected for uniform size and colour. Selected berries were randomized and used for the experiments. Fifty fruit were placed into 1 l polystyrene containers with snap-on lids. The volatile compound AITC (5 ll l 1) was spotted onto a piece of filter paper which was subsequently hung inside the plastic containers just before the lids were closed. The AITC was allowed to vaporise inside the containers spontaneously at 20 °C for 16 h. The containers were then stored at 10 °C. Each container contained 50 berries. Twenty-four containers were used for control and AITC treatment. Control samples were handled similarly, however the volatile AITC treatment was omitted. Samples were taken initially and after 3, 7, 10 and 14 days of storage for chemical analysis. The samples were then frozen in liquid nitrogen and then stored at 80 °C until assayed for sugars, organic acids, antioxidant capacity, antioxidant enzyme activities, non-enzyme components, anthocyanin and phenolic compounds. 2.3. Analysis of sugars and organic acids Triplicate samples of 4 g of blueberries were extracted twice with 15 ml of imidazole buffer (20 mM, pH 7.0) using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY). The extraction, purification, and derivatization procedures for nonstructural sugars and organic acids have been described previously (Wang, Ji, & Faust, 1987). A Hewlett–Packard 6890 gas chromatograph equipped with a flame ionisation detector and a fused silica capillary column (dimethylsilicone fluid, 12.5 m  0.2 mm) was used for separation of sugars and organic acids. Sugars and organic acids were quantified by comparing peak area with those of standards. 2.4. HPLC analysis of blueberry anthocyanins and phenolic compounds Triplicate samples of 5 g blueberries were extracted with 25 ml of 80% acetone containing 0.2% formic acid using a Polytron (Brinkmann Instruments, Inc., Westbury, NY). The homogenised samples from the acetone extracts were then centrifuged at 14,000g for 20 min at 4 °C. The supernatants were transferred to vials, stored at 80 °C, and used for HPLC analysis. Twenty millilitres from the above acetone-formic acid extracts were concentrated to 1 ml using a Buchler Evapomix (Fort Lee, NJ) in a water bath at 30 °C. The concentrated samples were dissolved in 4 ml of acidified water (3% formic acid) and then passed through a C18 Sep-Pak cartridge (Waters), which was previously activated with methanol followed by water and then 3% aqueous formic acid. Anthocyanins and other phenolics were then recovered with 2.0 ml of acidified methanol containing 3% formic acid. The methanol extract was passed though a 0.45-lm membrane filter (Millipor, MSI, Westboro, MA) and 20 ll was analysed by HPLC (Waters, Milford, MA). Samples were injected at ambient temperature (20 °C) into a reverse phase NOVA-PAK C18 column (150  3.9 mm, particle size 4 lm) with a guard column (NOVAPAK C18, 20  3.9 mm, particle size 4 lm) (Waters Corporation, MA). The mobile phase consisted of 5% aqueous formic acid (A)

and HPLC grade acetonitrile (B). The flow rate was 1 ml/min, with a gradient profile consisting of A with the following proportions (v/ v) of B: 0–1 min, 4%, 1–10 min, 4–6% B; 10–15 min, 6% B; 15– 35 min, 6–18% B; 35–40 min, 18–20% B; 40–42 min, 20–45% B; 42–45 min, 45–100% B; 45–50 min, 100% B. The phenolic compounds in fruit extracts were identified by their retention times and UV spectra, recorded with a diode array detector and by chromatographic comparison with authentic markers (Gao, Mazza, & Mazza, 1994; Kader, Rovel, Girardin, & Metche, 1996; Wang, Chen, Sciarappa, & Wang, 2008; Zheng & Wang, 2003). The results were also confirmed by co-injection with authentic standards. Each standard was dissolved in methanol at a concentration of 1 mg/ ml, and five dilute solutions from these stock solutions were used to prepare calibration curves of each standard. Recoveries were measured by extracting the recovered amounts of pure substances added to frozen blueberries before the experiment. Three replicates from each sample were used for HPLC analyses. The transresveratrol in the purified extracts was injected into a HPLC system as described by Wang, Chen, Wang, and Chen (2007). Results of the analyses for resveratrol were expressed as lg of trans-resveratrol per gramme of fresh weight. 2.5. Antioxidant enzyme measurements 2.5.1. Glutathione-peroxidase (GSH-POD, EC 1.11.1.9), and glutathione reductase (GR, EC 1.6.4.2) Triplicate samples of fruit tissue (10 g fresh weight) were homogenised in 10 ml 0.1 M Tris–HCl buffer (pH 7.8) containing 2 mM EDTA-Na, 2 mM dithiothreitol (DTT). The homogenate was centrifuged at 20,000g for 30 min at 4 °C, and the supernatant was used for the GSH-POD and GR assays. GSH-POD activity was determined using the method of Tappel (1978) with a slight modification. The reaction mixture contained 0.1 M Tris–HCl buffer (pH 8.0), 0.4 mM EDTA, 1.0 mM NaN3, 1.0 mM H2O2, 1.0 mM glutathione (GSH), 0.15 mM NADPH, 1 unit of glutathione reductase and 100 ll enzyme extract. The total reaction volume was 1.0 ml. H2O2 was added to start the reaction. GSHPOD activity was determined by the rate of NADPH oxidation at 340 nm via a spectrophotometer (Shimadzu UV-160U, Columbia, MD). Enzyme activity was expressed as nmole of NADPH oxidised per mg of protein per min. GR activity was assayed according to Smith, Vierheller, and Thorne (1988). The activity of GR was determined by monitoring glutathione-dependent oxidation of NADPH at 340 nm. GSSG was added to start the reaction and the rate of oxidation was calculated using the extinction coefficient of NADPH (6.22 mM 1 cm 1). GR activity was expressed as nmole of NADPH oxidised per mg of protein per min. 2.5.2. Superoxide dismutase (SOD, EC 1.15.1.1) Triplicate samples of fruit tissues (10 g) were pulverised in a cold mortar and pestle with 10 ml K-phosphate buffer (0.1 M, pH 7.3) containing 1 mM EDTA, 2 mM DTT. The homogenate was strained through 4 layers of miracloth and centrifuged at 12,000g for 10 min at 4 °C. The supernatant was purified according to Wang, Jiao, and Faust (1991) before assaying the SOD enzyme activity. Total SOD activity was assayed photochemically. Dicoumarol was included in the reaction mixture to inhibit reduction by pyridine nucleotide and to obtain a completely O2 dependent reduction of NBT. One unit of SOD was defined as the amount of enzyme which produced a 50% inhibition of NBT reduction under assay conditions. Since inhibition is not linearly correlated with SOD concentration, a V/v transformation was used to obtain linearity (V = basic reaction rate without fruit extract, v = reaction rate

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with extract). Linear correlation gave the equation: SOD units/ ml = (0.459 V/v 0.032)  dilution factor. 2.5.3. Ascorbate peroxidase (AsA-POD, EC 1.11.1.11), guaiacol peroxidase (G-POD, EC 1.11.1.7), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and monodehydroascorbate reductase (MDAR, EC 1.6.5.4) assay Triplicate fruit tissue (10 g) was pulverised in a cold mortar and pestle with 10 ml K-phosphate buffer (0.1 M, pH 7.3) containing 1 mM EDTA, 2 mM DTT. The homogenate was centrifuged at 12,000g for 10 min at 4 °C. The supernatant was used for the AsA-POD, G-POD, DHAR and MDAR assays. AsA-POD activity was assayed according to the method of Amako, Chen, and Asada (1994). H2O2 was added to start the reaction. Enzyme activity was expressed as nmole of ascorbate oxidised per mg of protein per min. The G-POD assay mixture contained 0.1 M phosphate buffer (pH 6.1), 4 mM guaiacol as donor, 3 mM H2O2 as substrate and 1.0 ml crude enzyme extract. The total reaction volume was 3.0 ml. The rate of change in absorbance at 420 nm was measured, and the level of enzyme activity was expressed as the difference in absorbance (OD) per mg protein, per min. DHAR activity was assayed by measuring the rate of NADPH oxidation at 340 nm (Shigeoka, Nakano, & Kitaoka, 1980). The reaction mixture contained 50 mM potassium phosphate (pH 6.1), 0.2 mM NADPH, 2.5 mM dehydroascorbate, 2.5 mM glutathione, 0.6 unit glutathione reductase (GR; from spinach, EC 1.6.4.2) and 0.1 ml of diluted crude enzyme extract (2 ml was diluted with 2 ml 50 mM potassium phosphate, pH 6.1). The reaction was started by adding dehydroascorbate. Enzyme activity was expressed as nmole of NADPH oxidised per mg of protein per min. MDAR activity was assayed by measuring the rate of NADH oxidation at 340 nm (Nakagawara & Sagisaka, 1984). The reaction mixture contained 50 mM K-phosphate buffer (pH 7.3), 0.2 mM NADH, 1.0 mM ascorbate, 1.0 unit of ascorbate oxidase and 0.1 ml of 50 mM K-phosphate buffer (pH 7.3) diluted crude enzyme extract (2–1 dilution) in a total volume of 1.0 ml. The reaction was started by adding ascorbate oxidase (from Cucurbita, EC 1.10.3.3). Enzyme activity was expressed as nmole of NADH oxidised per mg of protein per min. 2.5.4. Determination of ascorbate (AsA) and glutathione (GSH) For measurement of AsA, fruit samples of 4 g were homogenised with a cold mortar and pestle using 8 ml ice-cooled 5% trichloroacetic acid (TCA). The homogenate was filtered through four layers of miracloth and centrifuged at 16,000g for 10 min at 4 °C. The supernatant was used for the AsA assays. AsA was deter-

mined using the methods of Arakawa, Tsutsumi, Sanceda, Kurata, and Inagaki (1981). A standard curve in the range 0–10 lmol AsA was used. For measurement of GSH, triplicate samples of blueberry fruit of 4 g were homogenised in 8.0 ml ice-cold, degassed 7.57 mM sodium ascorbate solution with chilled mortar and pestle under N2 at 0 °C. The homogenate was filtered through four layers of miracloth and centrifuged at 30,000g for 15 min at 0 °C. The supernatant was deproteined in glass test tubes by incubation in a water bath at 100 °C for 3 min and then centrifuged at 15,000g for 15 min at 0 °C. The supernatants were used for the GSH assay. GSH was assayed using the method described by Castillo and Greppin (1988). 2.6. Protein determination Protein was determined according to Bradford (1976) using bovine serum albumin (BSA) as a standard. 2.7. Statistical analysis Data presented were the means ± S.D. values. All data were subjected to analysis of variance (ANOVA) using the NCSS Statistical Analysis System (Statistical Analysis and Graphics, Kaysville, UT, USA) (NCSS, 2007). Values of sugars (fructose, glucose, and sucrose), organic acids (citric and malic acid), and individual flavonoids were evaluated by the Duncan’s test. Differences at p 6 0.05 were considered significant. 3. Results Fructose and glucose are major sugars and citric acid is the main organic acid in blueberries (Table 1). During storage at 10 °C, fructose and glucose in control samples started to decrease after 3 or 7 days. However, a steady increase of fructose and glucose in the AITC treated samples was observed during first 10 days of storage then declined after 10 days. Malic acid and citric acid continued to decrease in blueberries during storage. The decreases of malic and citric acid were accentuated by AITC treatment. As a result, AITCtreated blueberry fruit had higher sugar contents and lower acid levels than the control fruit during storage at 10 °C (Table 1). Blueberries contain high amounts of phenolic acids (chlorogenic acid, myricetin 3-arabinoside, quercetin 3-glucoside, quercetin 3-galactoside, and kaempferol 3-glucoside) and anthocyanins (delphinidin 3-galactoside, delphinidon 3-glucoside, delphinidin 3-arabinoside, petunidin 3-galactoside, petunidin 3-glucoside,

Table 1 Effect of allyl isothiocyanate (AITC) treatment on sugar and organic acid content in ‘Duke’ Blueberries.a Treatment

Storage (days)

Sugar Fructose

Organic acid Glucose

Sucrose

Citric

Malic

23.5 ± 0.2 22.5 ± 0.2 25.9 ± 0.1 21.5 ± 0.4 26.0 ± 0.3 16.7 ± 0.2 26.9 ± 0.1 13.6 ± 0.3 24.3 ± 0.2 *

0.17 ± 0.3 0.17 ± 0.4 0.25 ± 0.5 0.11 ± 0.5 0.14 ± 0.3 0.13 ± 0.4 0.16 ± 0.3 0.11 ± 0.2 0.18 ± 0.3 ns

3.2 ± 0.08 3.1 ± 0.14 2.7 ± 0.15 2.8 ± 0.13 2.4 ± 0.06 2.6 ± 0.03 2.3 ± 0.01 2.4 ± 0.14 2.2 ± 0.07 *

0.065 ± 0.005 0.061 ± 0.001 0.045 ± 0.003 0.042 ± 0.002 0.031 ± 0.006 0.038 ± 0.004 0.024 ± 0.002 0.029 ± 0.003 0.022 ± 0.001 ns

mg/g fresh weight Initial Control AITC Control AITC Control AITC Control AITC Significanceb Treatment a b

0 3 3 7 7 10 10 14 14

20.1 ± 0.2 20.1 ± 0.2 22.1 ± 0.5 18.2 ± 0.4 24.1 ± 0.3 15.3 ± 0.4 25.1 ± 0.9 14.2 ± 0.2 19.8 ± 0.3 *

Data expressed as mean ± SEM (n = 3). *, ns, Significant or non-significant, respectively, at p 6 0.05.

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Table 2 Effect of allyl isothiocyanate (AITC) treatment on chlorogenic acid, resveratrol, myricetin 3-arabinoside, quercetin 3-galactoside, quercetin 3-glucoside, quercetin 3-arabinoside, kaempferol 3-glucoside, and kaempferol derivative in ‘Duke’ blueberries.a

a b c d e f g

Treatment

Storage (days)

Chlorogenic acidb

Resveratrolc

Myricetin 3arabinosided

Quercetin 3galactosidee

Quercetin3glucosidee

Quercetin 3arabinosidee

Kaempferol derivativef

Kaempferol 3glucosidef

Initial Control AITC Control AITC Control AITC Control AITC Significanceg Treatment

0 3 3 7 7 10 10 14 14

59.6 ± 0.2 69.2 ± 3.2 62.4 ± 3.0 74.2 ± 1.2 65.5 ± 0.2 63.8 ± 2.6 55.3 ± 1.6 69.0 ± 0.4 53.3 ± 0.9 *

3.1 ± 0.2 3.3 ± 0.2 3.4 ± 0.0 3.5 ± 0.4 3.3 ± 0.3 4.0 ± 0.4 3.1 ± 0.2 3.3 ± 0.2 3.1 ± 0.1 ns

4.6 ± 0.9 5.4 ± 0.1 4.7 ± 0.5 6.9 ± 1.1 5.4 ± 0.9 9.5 ± 1.6 5.4 ± 0.0 8.8 ± 0.4 5.9 ± 0.1 *

33.1 ± 0.8 44.2 ± 2.4 39.1 ± 1.6 47.1 ± 1.4 38.4 ± 0.6 57.5 ± 3.3 40.6 ± 0.1 50.0 ± 3.4 43.0 ± 2.7 *

16.5 ± 0.5 17.8 ± 1.6 12.6 ± 1.0 17.9 ± 0.2 16.8 ± 6.3 7.6 ± 2.4 8.6 ± 2.2 9.9 ± 3.0 9.8 ± 1.7 ns

7.4 ± 0.2 8.3 ± 1.1 7.8 ± 0.3 9.6 ± 0.4 8.8 ± 1.8 8.4 ± 0.3 7.9 ± 1.8 8.1 ± 2.5 7.5 ± 0.2 *

1.2 ± 0.2 1.1 ± 0.3 1.0 ± 0.1 1.5 ± 0.2 1.3 ± 0.1 1.1 ± 0.2 0.9 ± 0.2 1.0 ± 0.1 1.1 ± 0.0 ns

5.7 ± 0.2 6.3 ± 0.0 5.9 ± 0.5 6.9 ± 0.1 6.0 ± 0.3 7.5 ± 0.6 5.7 ± 0.4 6.2 ± 0.1 5.8 ± 0.0 *

Data expressed as mean ± SEM (n = 3). Data expressed as microgrammes of chlorgenic acid equivalents per gramme of fresh weight. Data expressed as microgrammes of trans-resveratrol equivalents per gramme of fresh weight. Data expressed as microgrammes of myricetin equivalents per gramme of fresh weight. Data expressed as microgrammes of quercetin equivalents per gramme of fresh weight. Data expressed as microgrammes of kaempferol equivalents per gramme of fresh weight. *, ns, Significant or non-significant, respectively, at p 6 0.05.

Table 3 Effect of allyl isothiocyanate (AITC) treatment on delphinidin 3-galactoside, delphinidon 3-glucoside, delphinidin 3-arabinoside, petunidin 3-galactoside, petunidin 3-glucoside, petunidin 3-arabinoside, malvidin 3-galactoside, and malvidin 3-arabinoside in ‘Duke’ blueberries.a,b

a b c

Treatment

Storage (days) time

Delphinidin 3galactoside

Delphinidin 3-glucoside

Delphinidin 3arabinoside

Petunidin 3galactoside

Petunidin 3glucoside

Petunidin 3arabinoside

Malvidin 3galactoside

Malvidin 3arabinoside

Initial Control AITC Control AITC Control AITC Control AITC Significancec Treatment

0 3 3 7 7 10 10 14 14

114 ± 3.9 119 ± 8.8 83.7 ± 1.8 127 ± 8.1 90.3 ± 5.4 107 ± 1.2 66.4 ± 8.4 100 ± 6.2 68.0 ± 1.2 *

41.1 ± 0.9 47.8 ± 3.1 41.6 ± 0.1 47.5 ± 6.4 34.8 ± 0.3 45.5 ± 5.2 29.6 ± 1.6 44.9 ± 1.1 29.1 ± 5.0 *

46.1 ± 11.3 58.0 ± 8.5 51.6 ± 0.2 66.6 ± 2.7 45.0 ± 8.3 68.3 ± 5.6 36.6 ± 2.5 58.5 ± 2.0 41.1 ± 1.7 *

118 ± 14.7 137 ± 8.1 126 ± 15.4 161 ± 11.6 121 ± 16.0 148 ± 12.7 89.6 ± 10.9 146 ± 9.2 113 ± 8.0 *

43.7 ± 15.7 84.6 ± 1.9 80.9 ± 2.8 89.5 ± 17.4 79.7 ± 7.2 69.4 ± 1.8 33.1 ± 8.3 59.2 ± 3.5 47.8 ± 3.4 *

56.5 ± 17.5 77.1 ± 0.1 73.1 ± 2.1 86.4 ± 1.9 74.2 ± 7.8 70.9 ± 3.9 46.2 ± 0.2 70.3 ± 3.8 58.5 ± 6.4 *

431 ± 19.7 468 ± 11.2 455 ± 15.7 496 ± 15.5 445 ± 11.3 504 ± 13.4 350 ± 0.8 469 ± 14.7 381 ± 16.4 *

210 ± 1.3 244 ± 4.1 232 ± 8.7 262 ± 14.7 227 ± 3.1 265 ± 9.6 178 ± 3.7 234 ± 7.7 196 ± 6.8 *

Data expressed as mean ± SEM (n = 3). Data expressed as microgrammes of cyanidin 3-glucoside equivalents per gramme of fresh weight. *, Significant at p 6 0.05.

Table 4 Effect of allyl isothiocyanate (AITC) treatment on activities of antioxidant enzymes [superoxide dismutase (SOD; U/mg protein), guaiacol peroxidase (G-POD; DA/mg protein min), glutathione-peroxidase (GSH-POD; nmol/mg protein min), ascorbate peroxidase (AsA-POD; nmol/mg protein min), monodehydroascorbate reductase (MDAR; nmol/mg protein min), dehydroascorbate reductase (DHAR; nmol/mg protein min), glutathione reductase (GR; nmol/mg protein min)] and non-enzyme antioxidants [ascorbic acid (AsA; lmol/g fwt) and reduced glutathione (GSH; nmol/g fwt)] in ‘Duke’ Blueberries.a

a b

Treatment

Storage (days)

SOD

G-POD

GSH-POD

AsA-POD

MDAR

DHAR

GR

GSH

AsA

Initial Control AITC Control AITC Control AITC Control AITC Significanceb Treatment

0 3 3 7 7 10 10 14 14

0.89 ± 0.05 1.01 ± 0.06 0.78 ± 0.03 1.18 ± 0.07 0.72 ± 0.02 1.02 ± 0.04 0.54 ± 0.02 0.83 ± 0.01 0.46 ± 0.02 *

0.10 ± 0.01 0.11 ± 0.02 0.08 ± 0.01 0.29 ± 0.03 0.06 ± 0.01 0.09 ± 0.02 0.05 ± 0.01 0.07 ± 0.02 0.04 ± 0.01 ns

744 ± 12.3 812 ± 12.5 737 ± 7.9 848 ± 12.6 678 ± 10.5 812 ± 9.6 623 ± 7.4 715 ± 8.8 586 ± 9.7 *

16.8 ± 0.9 18.6 ± 0.8 17.2 ± 0.6 19.5 ± 0.5 14.9 ± 1.1 16.1 ± 0.3 12.9 ± 0.6 14.3 ± 0.4 10.6 ± 0.3 *

132 ± 4.5 141 ± 6.2 126 ± 4.7 149 ± 5.1 108 ± 2.3 116 ± 2.6 102 ± 4.5 96 ± 2.4 74 ± 0.2 *

686 ± 9.8 699 ± 9.2 679 ± 6.3 784 ± 7.1 567 ± 8.2 658 ± 6.2 434 ± 5.1 517 ± 5.8 393 ± 4.2 *

221 ± 5.3 259 ± 6.2 182 ± 4.6 285 ± 5.2 162 ± 3.1 207 ± 4.5 116 ± 3.2 159 ± 5.2 101 ± 2.7 *

34.5 ± 1.2 38.2 ± 0.3 32.4 ± 0.8 45.3 ± 1.2 30.6 ± 0.6 38.9 ± 0.2 25.7 ± 0.3 21.7 ± 0.5 18.3 ± 0.7 *

31.3 ± 0.5 33.5 ± 0.6 29.2 ± 0.4 35.8 ± 1.1 25.6 ± 0.7 28.3 ± 0.8 20.8 ± 0.6 18.9 ± 0.4 11.7 ± 0.5 *

Data expressed as mean ± SEM (n = 3). *, ns, Significant or non-significant, respectively, at p 6 0.05.

petunidin 3-arabinoside, malvidin 3-galactoside, and malvidin 3arabinoside) and non-enzyme components, ASA and GSH (Tables 2–4). During storage at 10 °C, chlorogenic acid, quercetin 3-arabinoside, quercetin 3-glucoside, delphinidin 3-galactoside, delphinidinn 3-glucoside, petunidin 3-galactoside, petunidin 3-glucoside, and petunidin 3-arabinoside in control fruit samples increased

for 7 days and resveratrol, myricetin 3-arabinoside, quercetin 3galactoside, kaempferol 3-glucoside, delphinidin 3-arabinoside, malvidin 3-galactoside, and malvidin 3-arabinoside in control samples also increased for 10 days of storage at 10 °C, then declined. However, AITC treatments suppressed all phenolics and anthocyanins content in blueberries during storage. As a

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consequence, AITC-treated blueberry fruit had lower flavonoid levels than the control fruit during storage at 10 °C (Tables 2 and 3). Blueberries had high antioxidant enzyme activities and non-enzyme components (Table 4). Various oxygen scavenging enzymes activities such as GSH-POD, GR, SOD, AsA-POD, G-POD, DHAR, and MDAR and non-enzyme components, ASA and GSH in blueberries were detected (Table 4). These antioxidant enzyme activities and non-enzyme components, ASA and GSH in control samples increased for 7 days of storage at 10 °C, then decreased. AITC treatment reduced all antioxidant enzyme activities and non-enzyme components, ASA and GSH in blueberries during storage period (Table 4).

4. Discussion Fungal and bacterial decay is one of the major causes of rapid post-harvest deterioration of fresh produces. The most widely used method to limit fungal decay is the application of fungicidal substances. However, due to public concerns on health risk and environmental contamination, alternative methods are needed. Investigations on the beneficial use of naturally occurring or GRAS (generally recognised as safe) substances and sustainable nonchemical techniques for minimising our dependency on potentially hazardous chemicals to reduce decay and post-harvest losses of fruits and vegetables have gained growing interest. Several naturally occurring essential oils have been reported to have antimicrobial properties and have shown promise in reducing post-harvest diseases and disorders in horticultural crops (Dorman & Deans, 2000; Serrano, Martínez-Romero, Castillo, Guillén, & Valero, 2005; Wang et al., 2008; Wang et al., 2010). Our previous report (Wang et al., 2010) has shown that AITC, a naturally occurring compound, was effective in reducing fungal decay and maintaining quality of blueberry fruit. In the present study, we have shown that blueberries contain high amount of chlorogenic acid, myricetin 3-arabinoside, quercetin 3-galactoside, quercetin 3-arabinoside, and kaempferol 3-glucoside, delphinidin 3-galactoside, delphinidon 3-glucoside, delphinidin 3-arabinoside, petunidin 3-galactoside, petunidin 3-glucoside, petunidin 3-arabinoside, malvidin 3-galactoside, and malvidin 3arabinoside and non-enzyme components, ASA and GSH. These antioxidants are capable of performing a number of functions including free radical scavengers, peroxide decomposers, singlet and triplet oxygen quenchers, enzyme inhibitors, and synergists (Larson, 1988). Therefore, antioxidants can delay or prevent the oxidation of lipids or other molecules by inhibiting the initiation or propagation of oxidising chain reactions. Different antioxidants have a wide range of capacities to scavenge various reactive oxygen species. Individual flavonoids as mentioned above in the untreated blueberries increased for 7 or 10 days during storage at 10 °C. Increases in anthocyanin and phenolic content during storage also have been reported for lowbush blueberries (Kalt, Forney, Martin, & Prior, 1999), rabbiteye blueberries (Basiouny & Chen, 1988) and cranberries (Wang & Stretch, 2001). Kalt et al. (1999) reported that storage of fresh small fruits (strawberries, raspberries, highbush blueberries and lowbush blueberries), at temperatures higher than 0 °C increased antioxidant capacity, anthocyanins, and total phenolic content. Apparently, blueberry fruit continues to synthesise anthocyanins and other phenolics after harvest in our study, since our storage temperature was 10 °C. The decrease in titratable acidity and organic acids during storage might have provided carbon skeletons for the synthesis of phenolics (Mazza & Miniati, 1993). Phenolics and anthocyanins were both strongly correlated to antioxidant capacity. In bivariate regression of anthocyanin and antioxidant capacity, R2 value equaled 0.90, and the correlation (R2) for total phenolics and antioxidant capacity was 0.83 (Kalt

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et al., 1999). Connor et al. (2002) reported that antioxidant activity, total phenolic, and anthocyanin content in blueberry were strongly correlated with each other (r = 0.87–0.99) and their increases during cold storage were variety-dependent. Another major part of plant antioxidants system in berry fruits is the involvement of antioxidant enzymes. The antioxidant enzyme defense system consists of hundreds of different substances and mechanisms and it is possible that antioxidant enzymes can prevent cellular and tissue damage. Blueberries also had high GSH-POD, GR, SOD, AsA-POD, G-POD, DHAR, and MDAR enzyme activity. The antioxidant enzymes in control blueberry fruit samples further increased for 7 days storage at 10 °C then decrease. The increase or decrease of antioxidant enzyme activity was coincided to the increase or decrease of the level of individual anthocyanin and phenolic compounds in blueberries (Tables 2–4). A positive correlation was found between antioxidants activity and activities of antioxidant enzymes (Jiao & Wang, 2000). It has been shown that treatments with other naturally occurring compound jasmonate (Chanjirakul et al., 2006; Chanjirakul et al., 2007; Wang & Zheng, 2005; Wang, Bowman, & Ding, 2007) or essential oils (thymol, menthol, eugenol carvacrol, anethole, and perillaldehyde) (Wang, Wang, Yin, Parry, & Yu, 2007; Wang et al., 2008) not only reduced fruit decay during storage but also exhibited the capability to increase antioxidant activity, phenolic, and individual flavonoid compounds. High antioxidant activity could enhance free radical scavenging capacity and increase the resistance of tissues to oxidative damage against microbial invasion and reduce the spoilage of fruit. Blueberry fruit treated with AITC maintained higher sugars and better quality than the untreated fruit (Table 1). However, our results indicate that AITC does not promote antioxidant property or scavenging of constitutive reactive oxygen species (ROS). Therefore, AITC must have reduced decay and maintained fruit quality through a different mechanism. It is possible that AITC may paradoxically generate additional amounts of ROS to inhibit the growth and proliferation of microbial cells, thereby reducing decay in fruit tissue (Wang et al., 2010). Lower concentrations of AITC or combination of AITC with other low level of antioxidants enhancing natural products (e.g. methyl jasmonate) or essential oils (e.g. thymol, menthol, eugenol, carvacrol, anethole, and perillaldehyde) need to be evaluated for their antimicrobial power and for minimising any adverse effect on fruit composition. The impact of these treatments on sensory quality of fruit also warrants further research.

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