Responses of fresh-cut strawberries to ethanol vapor pretreatment

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Food and Beverage Chemistry/Biochemistry

Responses of fresh-cut strawberries to ethanol vapor pretreatment: improved quality maintenance and associated antioxidant metabolism in gene expression and enzyme activity levels Meilin Li, Xiaoan Li, Jing Li, Yue Ji, Cong Han, Peng Jin, and Yonghua Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02647 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 19, 2018

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Journal of Agricultural and Food Chemistry

1

Responses of fresh-cut strawberries to ethanol vapor

2

pretreatment: improved quality maintenance and associated

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antioxidant metabolism in gene expression and enzyme activity

4

levels

5

Meilin Li,

6

Zheng

†

† † † ‡ † Xiaoan Li, Jing Li, Yue Ji, Cong Han, Peng Jin, Yonghua

*,†

7 8

9 10

11 12

13 14

†

College of Food Science and Technology, Nanjing Agricultural University, Nanjing,

210095, People’s Republic of China ‡

College of Food

Science

and

Engineering, Qilu

University

Technology, Jinan, 250353, People’s Republic of China

*

Corresponding author. Tel.:+86 25 8439 9080; Fax: +86 25 8439 5618

E-mail address: [email protected] (Y. H. Zheng)

1

ACS Paragon Plus Environment

of


15

ABSTRACT: Strawberries were treated with different concentrations of ethanol

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vapor and then cut into four wedges and stored at 4 °C for one week. It was found that

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4 mL/kg ethanol was the optimal concentration to reduce the decrease of firmness and

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weight loss. Total phenolics content, total flavonoid and anthocyanin contents,

19

antioxidant enzyme activities and gene expression related to antioxidant were elevated

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by the ethanol treatment. Ethanol vapor also suppressed microbial growth and

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promoted free radical (hydroxyl and DPPH) scavenging capacities and four kinds of

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esters and bioactive components in strawberry wedges. Moreover, ethanol enhanced

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antioxidant enzyme activities including superoxide dismutase (SOD), catalase (CAT)

24

and ascorbate peroxidase (APX) by activating related gene expression. The results of

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our research indicate that ethanol vapor has potential application in preserving quality

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and improving antioxidant capacity of fresh-cut strawberries.

27 28 29

KEYWORDS: Ethanol vapor; fresh-cut strawberries; quality; antioxidant capacity; gene expression

30 31 32 33 34 35 36 2


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■ INTRODUCTION

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With the growing market for functional and convenient foods, ready-to-eat fruits

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and vegetables become important contributors to the produce industry with the

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advantage of high nutrition and convenience while still retaining freshness.1 Previous

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research has showed that wounding, a kind of abiotic stress, was recognized as a tool

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to enhance nutraceuticals of horticultural plants.2 Research on kinds of fruits and

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vegetables, such as carrot and pitaya indicated that cutting promoted the accumulation

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of phenolics compared with whole fruits.3, 4 However, the metabolic acceleration in

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fresh-cut fruits and vegetables caused by wounding makes it more susceptible to

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microbes. In addition, the physiological deteriorations including texture breakdown,

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brown stain, as well as the development of undesirable odor are also unavoidable to

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occur.5, 6 Therefore, interest in the preservation of minimally processed fruits and

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vegetables has prompted a growing number of researchers to investigate for more

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efficient and improved techniques.

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Strawberries are appreciated for their bioactive compounds, including

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anthocyanins, flavonoids and ascorbic acid (AsA), which protect human from chronic

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diseases. As one of the most consumed berries, they are widely used for salad, juices,

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desserts and powders processing. The scientific interest in strawberries and their

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processed products has been increasing for many years. The research of Van et al.7

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demonstrated that quartered strawberries accumulated more phenolics than whole

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fruit when stored at 2 °C for 15 days. Although chitosan and passive atmosphere were

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proven to have positive influence on retaining the quality of fresh-cut strawberries, 8, 9 3



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there remains a need for an efficient method that can extend the shelf life and retard

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quality deterioration of fresh-cut strawberries.

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Ethanol, the anaerobic metabolites of fruits and vegetables, is approved as a safe

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compound by the Food and Drug Administration (FDA) of US. It has not only

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fungicidal and insecticidal activity, but it is also capable of suppressing ethylene

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production, improving antioxidant capacity and retarding senescence of horticultural

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products. Studies on broccoli and grape have shown that ethanol vapor would be a

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good candidate to preserve the quality of fruits and vegetables.10, 11 Besides, ethanol

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vapor has the wide application prospect on fresh-cut fruits and vegetables. Ethanol

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vapor decreased spoilage in fresh-cut mango, inhibited the red discoloration of

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sunchoke tubers and reduced physiological metabolism activity of fresh-cut eggplant.

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12-14

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fresh-cut strawberries. Thus, the main purpose of our research was to evaluate

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changes of quality attributes and antioxidant gene expression in fresh-cut strawberries

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after pretreatment with ethanol vapor.

However, few studies were carried out on the effect of ethanol vapor treatment on

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■ MATERIALS AND METHODS

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Plant Materials and Treatments. Commercial mature strawberry fruit (Fragaria

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ananassa Duch. cv. Benihoppe) was hand-harvested from orchard located in Jiangsu,

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China and transferred to the laboratory in two hours. Intact berries of uniform size

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were selected for the experiment. Ethanol vapor treatment was carried out using the

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method of Xu et al.10 In the first experiment, strawberries were weighed (500±10 g) 4


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and placed in an air-tight plastic container (20 cm × 12 cm × 8 cm) for treatment.

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Three replicates of 4 containers of strawberries per treatment were used in this

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experiment. Containers with filter papers soaked with liquid ethanol at the bottom

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were incubated for 6 h at 20 °C to evaporate ethanol. Ethanol concentrations of 1, 2, 4

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and 8 mL/kg were calculated on the basis of the weight of the fruit in each container

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and the volume of ethanol liquid. Water was used for the control. After the treatment,

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all the containers were ventilated for 1 h. Then strawberries were sanitized in 200 µL

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L-1 NaClO solution for 2 min, and then rinsed with water. A solution of 75% ethanol

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was used to wipe the work surface and knife for sample preparation. Next, every fruit

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was cut into four wedges and packaged in 15 cm × 10 cm × 4 cm polypropylene

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containers after peduncle and calyx were removed by hand. The fresh-cut strawberries

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were stored at 4 °C in darkness with 95% relative humidity for one week and fruit

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samples were collected every other day. The most effective concentration of ethanol

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vapor to maintain the firmness and inhibited the weight loss of strawberry wedges

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was applied in the second experiment. There were 3 replicates of 6 containers of

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strawberries per treatment. Quality attributes and enzyme activities were assayed

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initially (0 h) and every other day during storage. Gene expression was determined at

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3 h (pre 3) , 6 h (pre 6) during pretreatment of ethanol vapor, then 3 h, 6 h and 12 h

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following treatment in order to measure the transient and continuous changes of genes

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expression. The experiment was carried out twice. All samples were sliced into small

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segments in liquid nitrogen and stored in ultra low temperature freezer (-80 °C)

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(DW86L388A, Haier, China) until analysis. 5



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Quality Attributes Assays. The firmness of strawberry wedges was measured

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using a TA-XT plus texture analyzer (Stable Micro Systems Ltd., UK) based on Aday,

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Caner, and Rahvalı’s15 procedure with certain modifications. The same position of

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fresh-cut strawberries was selected for each test. The trigger force of texture analyzer

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was 5 g and the pre-test speed, test speed and post-test speed was 3.0 mm/s, 1.0 mm/s

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and 5.0 mm/s, respectively. Strawberry wedges were weighed initially and then every

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other day during storage to determine the weight loss in fruit, which expressed as the

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percentage of initial weight. To evaluate total soluble solids (TSS) of strawberry

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wedges, an Abbe refractometer (14081S/N, USA) was used. Titratable acidity (TA) of

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fruit samples was measured by the method of Yang, Zheng, and Cao’s .16 PH 8.1 was

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the endpoint, and it was measured with a pH-meter (PH610, Wiggens, Germany). For

114

each replicate, ten wedges per treatment were used in firmness, TSS, TA and weight

115

loss assays, respectively.

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Microbiological Analysis. Total aerobic bacterial count (TABC) was evaluated

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according the assay of Adiani et al. 17 A sample of 25 g was cut into small pieces then

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added into a conical flask with 225 mL sterile saline. After homogenization, the

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mixture was continuously diluted in sterile saline. 1 mL dilution of each concentration

120

was used to enumerate on plate count agar (PCA) after incubating at 37 °C for 48 h.

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The results were calculated as log10CFU g−1.

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Major Volatile Compounds Analysis. Volatile compounds in strawberry wedges

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were analyzed following the method of Pan et al18 with minor modifications. The

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volatiles in tissue were collected by headspace solid phase micro-extraction 6


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(HS-SPME) and assayed by GC-MS (7890GC/5975MSD, Agilent, USA). SPME fiber

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was aged for half an hour at 250 °C in the GC injector. Five grams of fresh-cut

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strawberries were sealed in a 20 mL headspace vial and equilibrated in a thermostatic

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water-bath (40 °C) for 10 min. Next, aromas from fruit were extracted and

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concentrated by SPME fiber (65 µm, polydimethylsiloxane/DVB) (Supelco,

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Bellefonte, PA, USA) for 40 min and then was carried out into the GC-MS injector in

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a split-less mode directly. The GC-MS system was equipped with a fused silica

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capillary column HP-5 (0.25 µm × 0.25 µm × 30 m) and the velocity of carrier

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gas (He) was 1.0 mL/min. The mass spectrometry scanned the mass from m/z 30 to

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450 with an electron impact mode at 70 eV. The injector temperature was 230 °C and

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the temperature of chromatographic column held 40 °C for 2 min, and then raised at

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2 °C/min to 100 °C, at 5 °C/min to 250 °C, and finally kept at 250 °C for 2 min. The

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GC-MS system was controlled by Agilent 7890/5975-GC/MSD chemstation. The

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peaks were compared with the mass spectra in NIST library19 and then identified. The

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results of volatile compounds expressed as the relative content.

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Antioxidants Measurements. Total phenolic content (TPC) was quantified with

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the method of Swain and Hillis.20 For each replicate, five grams of tissue was pestled

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in 25 mL methanol and the homogenate was stored at 4 °C overnight in refrigerator

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then centrifuged for 20 min with a rotational speed of 13,000 g at 4 °C. The mixture

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was prepared to react at room temperature for 1 h, which contained 1 mL

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Folin-Ciocalteu reagent, 20 µL supernatant, 180 µL distilled water and 0.8 mL

146

Na2CO3 (7.5%, w/v). The absorbance was determined with an ultraviolet 7



147

spectrophotometer (UV 6000, METASH, Shanghai) set at 725 nm and TPC was

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expressed as milligrams of gallic acid (GAE) per gram fresh weight (FW).

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The pH differential method21 was used to analyze total anthocyanin content (TAC).

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Five mL acetone (80%) contained 0.2% formic acid was applied to extract total

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anthocyanin in two grams fresh cut strawberry tissue and the supernatant centrifuged

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for 20 min at 13,000 g. NaAC buffer and KCl buffer at pH 4.5 and 1.0 were prepared,

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respectively. Four mL buffer was mixed with 1 mL extract. The reaction conducted

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under 40 °C for 20 min. It is necessary to measure the absorbance at 510 nm and 700

155

nm in order to eliminate interference from background turbidity. The formula below

156

was used and molar extinction coefficient of pelargonidin-3-glucoside was 22400.

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Milligrams of pelargonidin-3-glucoside per gram (FW) was used to quantify TAC.

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A=(A515-A700)pH1.0-(A515-A700)pH4.5

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Aluminum chloride (AlCl3) colorimetric method22 was applied to measure the

160

content of total flavonoid (TFC) in wedges. The extract procedure to assay TAC was

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also used to measure TFC. Two mL supernatant was mixed with 2 mL of 3% AlCl3

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(m/v) and 1 mL of 30% ethanol (v/v). The homogenate remained at room temperature

163

for 20 min and detection was performed at 430 nm. Results were expressed as rutin

164

equivalents.

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AsA was assayed by using Arakawa’s23 method with modifications. Frozen tissue

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sample (2 g) was ground in 5 mL trichloroacetic acid (TCA) solution (5%, m/v) and

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centrifuged at 13,000 g for 20 min (4 °C). The reaction mixture of 0.2 mL

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supernatant, 1.8 mL of 5% TCA, 1 mL ethanol, 0.5 mL of 0.03% (m/v) ferric 8


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trichlorid (FeCl3), 1.0 mL of 0.5% (m/v) phenanthroline and 0.5 mL of 0.4% (v/v)

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phosphoric acid was incubated at water-bath (30 °C) for 1 h before reading at 534 nm.

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The AsA contents in fruit were expressed as milligrams of AsA per gram FW.

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Antioxidant capacity measurement. Antioxidant capacity of strawberry wedges

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was determined by the method of Brand-Williams et al.24 with some modifications.

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The extraction used to evaluated TPC was also applicable to determine the capacity of

175

scavenging DPPH radical. The mixture of 3.9 ml of 0.12 mM DPPH solution prepared

176

with methanol and 0.1 ml extracts reacted at 25 °C for 30 min in the dark. Methanol

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was used to replace the extraction as the control. Spectrophotometric readings at 525

178

nm were registered and % DPPH inhibition was used to express the antioxidant

179

capacity. The following equation was applied when calculated:

180 181

% DPPH inhibition =1- [(the absorbance of the sample - the absorbance of sample mixed with 3.9 mL methanol) / the absorbance of the control] × 100 %

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The Hydroxyl radical (·OH) scavenging capacity was conducted using a

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previously published procedure with slight modifications. 25 Two grams of fruit tissue

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were homogenized in 5 mL of 50% (v/v) ethanol and the mixture was centrifuged at

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13,000 g for 25 min (4 °C). The supernatant (0.15 mL) was mixed with 2.0 mL of 18

186

mM ferrous sulfate (FeSO4) and 1.5 mL of 18 mM salicylic acid (SA, prepared with

187

ethanol), after that 0.1 mL hydrogen peroxide (H2O2) (0.3%, v/v) was added to started

188

the reaction. Readings at 510 nm was recorded after incubation at 37 °C for 30 min.

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The following formula was used to calculate the ·OH scavenging capacity:

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% ·OH inhibition =1- [(the absorbance of the sample - the absorbance of the reaction 9



191

mixture without FeSO4) / the absorbance of the control] ×100 % ·

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O2·- Production and H2O2 Content Measurements. Fresh tissue samples (2 g)

193

were homogenated in 5 mL phosphate buffer at pH7.8 (0.1 M) and centrifuged at

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13,000 g for 20 min (4 °C) to evaluated superoxide radicals (O2·-) production

195

according to Elstner and Heupel.26 The extracts (1 mL) were added to 1 mM

196

hydroxylamine hydrochloride (1 mL) and stored at ordinary temperature for 1 h. Next,

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7 mM α-naphthylamine (1 mL) and 17 mM p-aminophenylsulfonic acid (1 mL) were

198

added into the incubated solution, and then reacted for 20 min at ordinary temperature

199

and we measured its absorbance at 530 nm. The results were expressed as nMg-1min-1

200

FW, based on a standard curve.

201

The method described by Patterson, MacRae, and Ferguson27 was used to

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measure H2O2 content with minor modifications. Fresh tissue (2 g) was mixed with 5

203

mL acetone then centrifuged at 13,000 g for 20 min (4 °C), and then 0.1 mL HCl

204

containing ten percent TiCl4 and 0.2 mL ammonium hydroxide (NH3·H2O) were

205

added into 1 mL extracts. Next, the mixture was centrifuged for 10 min at 11,000 g

206

(4 °C). Acetone was applied three times to remove the pigment in the sediment. The

207

sediment was dissolved with 3 mL of 2 M sulfuric acid (H2SO4) solution and the

208

absorbance at 412 nm was recorded. We used µmol g-1·FW to express the content of

209

H2O2 in strawberries.

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Antioxidant Enzymes Measurements. Superoxide dismutase (SOD) activity was

211

determined according to Rao, Paliyath, and Ormrod’s.28 The same extracts prepared

212

for the O2·- production were also used for SOD activity assay. The reaction medium 10


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contained 0.02 mL extract, 0.3 mL of 100 µM EDTA-Na2, 0.3 mL of 750 µM

214

nitro-blue-tetrazolium (NBT) , 1.78 mL of 50 mM sodium phosphate buffer (PBS) at

215

pH 7.8, 0.3 mL of 20 µM riboﬂavin and 0.3 mL of 130 mM methionine. The mixture

216

was placed under 4000 lx ﬂuorescent lamp and the reaction started when the light was

217

turned on. The detection wavelength of spectrophotometer was 560 nm. A 50%

218

inhibition of NBT photochemical reduction per minutes was defined as one unit of

219

SOD activity and described as U mg-1 protein.

220

The activity of ascorbate peroxidase (APX) was determined by previous method

221

described by Nakano and Asada.29 Two grams of sample were mixed with 5 mL of 0.1

222

M PBS (pH7.0) which contained 1% PVP, 1 mM ascorbic acid and 0.1 mM EDTA,

223

and then homogenized. After centrifuged for 30 min at 13,000 g (4 °C), 0.1 mL

224

supernatant, 0.1 mL of 9 mM ascorbic acid and 2.8 mL of 0.1 M PBS were pipette

225

into a test tube. An aliquot of 0.1 mL of thirty percent H2O2 was added to start the

226

reaction. A change of 0.01 in absorbance at 240 nm per minutes was defined as one

227

unit of APX activity and described as U mg-1 protein.

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The extract procedure of APX was also applied to evaluate catalase (CAT) activity

229

while the buffer was 0.1 M PBS (pH 7.0). According to Steel,30 the reaction mixture

230

containing 0.2 mL extract, 2.6 mL of 0.5 M PBS (pH 7.0) and 0.2 mL of 0.75% (v/v)

231

H2O2. Spectrophotometric readings at 240 nm were decreased with the disappearance

232

of H2O2, and they were recorded every 30 seconds for 5 minutes. A decrease of 0.01

233

in absorbance per minutes was defined as one unit of CAT activity and described as U

234

mg-1 protein. 11



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The procedure described by Bradford31 was used to determine the protein content in extracts above, which based on bovine serum albumin. Antioxidant Enzymes Related Gene Expression Analysis. Total RNA was

238

isolated from each sample using a modified cetyltrimethylammonium bromide (CTAB)

239

method designed for samples rich in polysaccharides and polyphenols. The

240

concentration

241

ultramicrospectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, USA) and

242

gel electrophoresis, respectively. A RT-PCR Kit (RR036A, TaKaRa, Japan) was used

243

to synthesize cDNA. A total volume of 20 µL reaction containing 10 µL of the SYBR

244

Green PCR Master Mix (RR420A, TaKaRa, Japan), 0.4 µL primers, 2 µL of the

245

synthesized cDNA, 6.8 µL of RNase-free water and 0.4 µL of ROX Reference Dye Ⅱ

246

(RR420A, TaKaRa, Japan) was used to perform amplifications and synthesized cDNA

247

was replaced by RNAase-free water in the negative control to detect the

248

contamination. The gene-specific primers were designed using NCBI Primer-Blast

249

Tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and listed in Supplementary

250

Table S1. The Actin gene from strawberries (Fragaria ananassa) was chosen as a

251

reference gene. qRT-PCR was carried out on a QuantStudioTM 6 Flex Real Time PCR

252

System (Applied Biosystems, Foster city, CA, USA) and 2-△△CT method was used to

253

analyze the relative expression level of genes.

and

quality

of

total

RNA

was

analyzed

with

254

Statistical Analysis. A completely randomized design was arranged in the

255

experiments. Three replicates were used and a complete experiment was carried out

256

for two times. Data in this research was represented as the mean ± standard error (SE) 12


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of three replicates. Statistical analysis was carried out by a one-way analysis of

258

variance (ANOVA) from SAS (Version 9.1) and mean separations were performed

259

using Duncan’s multiple range test. Differences were judged to be significant when p

260

< 0.05.

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■RESULTS

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Firmness, Weight loss, TSS and TA Contents of Fresh-cut Strawberries. Fig.1

264

showes the changes in fruit firmness, weight loss, TSS and TA in wedges treated with

265

different concentrations of ethanol vapor. Strawberry wedges held in an ethanol-free

266

environment for one week experienced significant reduction in firmness (p < 0.05),

267

but the ethanol vapor treatment inhibited the softening in fresh-cut fruit. The higher

268

the concentration (up to 4 mL/kg), the greater the reduction. Concentrations higher

269

than 4 mL/kg did not have any additional beneficial effect. The effect of ethanol

270

vapor on weight loss of wedges varied with treatment concentrations and 4 mL/kg

271

was also found to be the most effective in reducing the weight loss of strawberries. A

272

higher concentration (8 mL/kg) aggravated the weight loss at 7 d of storage. Both

273

TSS and TA content had no significant difference (p ＞ 0.05) after ethanol

274

pretreatment and a slight decline trend was shown in all strawberry wedges.

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TABC of Fresh-cut Strawberries. Ethanol vapor pretreatment significantly (p

Responses of fresh-cut strawberries to ethanol vapor pretreatment

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