Physiological and Metabolomic Analysis of Cold Plasma Treated

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Food and Beverage Chemistry/Biochemistry

Physiological and Metabolomic Analysis of Cold Plasma Treated Fresh-Cut Strawberries Meilin Li, Xiaoan Li, Cong Han, Nana Ji, Peng Jin, and Yonghua Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00656 • Publication Date (Web): 18 Mar 2019 Downloaded from http://pubs.acs.org on March 19, 2019

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Journal of Agricultural and Food Chemistry

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Physiological and Metabolomic Analysis of Cold Plasma Treated

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Fresh-Cut Strawberries

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Meilin Li, †Xiaoan Li, ‡Cong Han,§ Nana Ji,†Peng Jin, †YonghuaZheng*,†

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

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210095, PR China

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‡School of Agricultural Engineering and Food Science, Shandong University of

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Technology, Zibo 255000, PR China

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§College of Food

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Technology, Jinan, 250353,PR China

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

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

Science

and

Engineering, Qilu

1

ACS Paragon Plus Environment

University

of


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ABSTRACT: Cold plasma technology offers new opportunities to the

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decontamination and preservation of fruits and vegetables. In the present research,

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strawberries were cut into four wedges and then treated with dielectric barrier

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discharge plasma of 45kV for 1 min and stored for one week (4 °C). Metabolomic

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analysis suggested that plasma treatment improved the biosynthesis of the metabolites

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in

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phenylpropanoids”

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demonstrated that plasma treatment maintained the texture properties and inhibited

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microbial growth of fresh-cut strawberries. In addition, plasma treatment also

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promoted the accumulation of total phenolics, total flavonoid and anthocyanin by

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enhancing the critical enzyme activities and activating related gene expression in

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phenylpropanoid as well as reactive oxygen species metabolism, which contributed

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greatly to the enhancement of antioxidant capacity of strawberry wedges.Our

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investigation provided a new perspective of the effect of plasmatreatment on the

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safety and quality of strawberry wedges and suggested that cold plasma treatment

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holds promise as an emerging processing technology for improving the quality and

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antioxidant activity of postharvest fruits and vegetables.

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KEYWORDS: Cold plasma; fresh-cut strawberries; quality; antioxidantcapacity;

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“flavones

and

flavonol pathway

biosynthesis” in

fresh-cut

pathway

and

strawberries.

gene expression

2


“biosynthesis

Physiological

of

assay

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■ INTRODUCTION

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Fresh-cut fruits and vegetables are popular because of their nutritional benefits

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and convenience. In recent years, a great number of studies have shown that

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horticultural crops would respond quickly tobiotic or abiotic stresses by synthesizing

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phytoalexin. Cutting is an abiotic stress which promotes the accumulation of bioactive

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compounds in fresh produce.1,2In carrots,3dragon fruits4 and onions,5 cutting induced

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the increase of phenolic contents and enhanced the antioxidant capacity compared

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with intact fruits and vegetables. However, minimally processed products are more

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perishable and they deteriorate rapidly due to the growth of microorganisms, the

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formation of off-flavor and the acceleration of physiological breakdown. Therefore,

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some effective and innovative technologies that can be used as postharvest treatments

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for minimally processed produce have attracted much attention around the world.

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In recent years, electric discharge ionized cold plasma has emerged as an effective

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and potential processing technology in agriculture and food industry. It can be

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generated by microwaves, radio frequency, alternating or direct current. A variety of

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equipment including plasma jet, gliding arc discharge, corona discharges and

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dielectric barrier discharge (DBD) have been developed and used in food

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industry.6Cold plasma has the expediency of energy effciency, short processing times

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and operation at low temperature7and it has been employed for the microbial

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decontamination of mandarin, 8Chinese bayberries9 and cherry tomatoes,10 the

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modification of the functionality of banana starch11 and the degradation of

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agrochemical residues in bluberries12 and strawberries.13DBD plasma is a promising 3



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toolowing to its flexibility of electrode geometry and the uniformity of discharge

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ignition. It is generated by ionizing the gas mixture between two metal

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electrodeswhichare separated by dielectric barriers such as ceramic, quartz and

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plastic. The dielectric materials movewith electrodes and thwart the formation of

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sparks caused by charge movement.6It was reported that DBD plasmainhibited the

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tissue browning of fresh-cut apples,14 improved the color retention of fresh-cut

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kiwifruit15 and delayed the microbial growth of fresh-cut melon.16Fresh-cut

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strawberries are a good source of vitamins, minerals and antioxidant compounds and

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they could be sold as a fully edible and highly convenient produce.

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confirmed that there were higher levels of bioactive compounds in fresh-cut

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strawberries compared with intact fruit.19The abundant anthocyanins and flavonoids

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in strawberries have great effects on scavenging free radical in the body and

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preventing people from cardiovascular diseases, diabetes, neurodegeneration,

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inflammation and cancer.18Previous studies suggested that ethanol vapor,20 modified

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atmosphere storage21 and pulsed light22treatment had positive effects on retarding the

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quality deterioration of minimally processed strawberries. However, little information

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is available on how cold plasma influences the quality and physiological metabolism

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of fresh-cut strawberries. Therefore, the objective of present research was to study the

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effect of DBD plasma on the quality and antioxidant activity of strawberry wedges by

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physiological and metabolomic assays.

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■ MATERIALS AND METHODS 4


17, 18It

was

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Plasma System and Treatments. DBD plasma system, which consisted of two

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aluminumcircular electrodes, two polypropylene barrier layers and an AC Dielectric

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Test Set (BK-130, Phenix Technologies, USA),were installed in two separate rooms.

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The polypropylene trays of 0.4mm thickness (178mm × 126 mm × 35 mm, HS-6,

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Shanghai Chuo Kagaku, China) exposed to the plasma system were sealed with

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laminated polyethylene film using a packaging machine (MAP-H360, Suzhou Senrui

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Fresh Equipment Co., Ltd, China) and placed between two polypropylene barrier

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layers. There was a distanceof 40 mm between two electrodes and the schematic of

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the DBD plasma system is presented in Fig.S1.

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Strawberries (Fragaria ananassa Duch. cv. Benihoppe) at stage of 90% colored

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based on the visual appearance of red color on the fruit surface were harvested by

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hand from a commercial farm in Jiangsu, China. Fruits of uniform size were sterilized

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in sodium hypochlorite solution (0.02%) and then rinsed in water. Next, the calyxes

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were removed. The work surface was wiped with 75% ethanol and fresh-cut

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strawberries were manually obtained by cutting every fruit into four wedges with a

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sharp blade. In the preliminary experiment, an orthogonal design was carried out to

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determine the optimal voltage and length of time of plasma treatment (data not

94

shown). In the subsequent experiment, fresh-cut strawberries were divided into two

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groups and four wedges from different strawberries were packaged in a tray, which

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was immediately exposed to 45kV of DBD plasma for 1 min (20 °C) and then stored

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at 4 °C for one week. For each replicate, there were 140 trays of strawberry wedges in

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each group. Samples were collected every other day for physiological assays. For the 5



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molecular experiment, samples were taken at 3 h, 6 h and 12 h and every other day to

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investigate the continuous and transient gene expression. Samples were collected by

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slicing into small segments in liquid nitrogen and then stored in a freezer at -80 °C

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(DW-HL398S, MELING, China) until analysis. There were three replicates in a

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complete experiment and the whole experiment was conducted twice. For the

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measurement of each parameter, each replicate of the sample was determined twice.

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Quality Parameters Measurements. The texture of fresh strawberries was

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determined following the method of Aday, Caner, and Rahvalı using a texture

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analyzer (TMS-Pro, Food Technology Corporation, USA)with a load cell of 50 N and

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the diameter ofthe cylindrical probewas 5 mm.23 Fresh-cut strawberries of ten

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replicates were compressed twice to 30% of their original height and the time interval

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was 5 s between two compression cycles. The initial trigger force was 50 N and the

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test speed was 60 mm/min. The texture parameters including firmness, springiness,

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adhesiveness, gumminess, cohesiveness and chewiness were calculated according to

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the force-time curve. Total soluble solid (TSS) was assayed with an Abbe

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refractometer (14081S/N, USA). Titratable acidity (TA) in strawberry wedges was

115

titrated with 0.05 M sodium hydroxide (NaOH) and the endpoint of titration was pH

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8.1, which was determined using a pH-meter (PH610, Wiggens, Germany).24

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Total Aerobic Bacterial Count (TABC) Assay. The method of Adiani et al was

118

used to determine TABC in strawberris.25Fresh samples (25g) were homogenized in

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225mLsterile saline and the mixture was continuously diluted with saline to different

120

concentrations. Next, 1 mL dilution was poured into plate count agar, which was 6


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incubated for 48h at 37°C.The results of TABC were expressed as log10CFU g−1.

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Vitamin C (Vc) Content Assay. Vc in frozen samples (2g) was extracted in 5

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mL trichloroacetic acid (TCA) (5%) using a chilled mortar.26Next, the homogenate

124

was centrifuged at 4 °C for 20 min with a rotational speed of 13 000 g in a centrifugal

125

machine (H1850R, XIANGYI, Hunan) and the supernatant was used in the

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determination of Vc content. The mixture contained1 mL ethanol, 1 mL

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phenanthroline (0.5%), 1.8 mL TCA, 0.5 mL phosphoric acid (0.4%), 0.05 mL ferric

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trichlorid (0.03%) and 0.2 mL supernatant and the reaction was conducted at 30 °C

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for 1 h. The readings was recorded at 534 nm using a spectrophotometer (UV 6000,

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METASH, Shanghai) and Vc content in tissue was calculated according to the

131

standard curve based on Vc and expressed as mg g-1 of fresh weight (FW).

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Antioxidant Capacity Assay. For the determination of the capacity to scavenge

133

hydroxyl radical of strawberry wedges, frozen strawberry tissue (2g) was extracted in

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5 mL ethanol (50%) and the homogenate was centrifuged for 20 min at 15000 g. An

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aliquot of 0.2 mL supernatant was reacted with the mixture of 1.5 mL salicylic acid

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(18 mM), 2.0 mL ferrous sulfate (18 mM) and 0.1 mL of 0.3%hydrogen peroxide

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(H2O2).27The reaction was conducted for 30 min at 37 °C and the readings at 510 nm

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were determined. Results were described as% ·OH inhibition.

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The activity to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical of

140

fresh-cut strawberries was assayedwith procedure of Brand-Williams et al.28Frozen

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samples (2g) were ground in 5 mL of methanol and then the homogenate was

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centrifuged. The supernatant (0.15 mL) was added to 3.85 mL DPPH solution (0.12 7



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M).After incubation at 25°C for 25min,the readings were determined at 525nmand

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results were expressed as % DPPH inhibition.

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MetabolomicAnalysis. Frozen samples were ground into powder in grinding

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miller (A11, IKA, Germany) and the powder (80 mg) were mixed with 20 uL

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chlorohenylalanine (0.3 mg mL-1) and 1 mL methanol (70%) and then extracted by

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ultrasonic at 4 °C for 30 min. After balanced for 20 min at -20 °C, the mixture was

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centrifuged at 13000 g for 15 min. Next, 5 µL supernatant was passed through a

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membrane filter of 0.22 µm(NYLON66, Jinlong, England) and injected to the ultra

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high performance liquid chromatography (ACQUITY UPLC, Waters, USA)-mass

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spectrometry (AB Triple TOF 5600, AB Sciex, USA) (UPLC-MS) system. The

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metabolites in strawberry wedges were separated by the UPLC system equipped with

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a C18 phase column (2.1 × 100mm, 1.7 µm, ACQUITY UPLC BEH C18, USA).

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The temperature of the column was 45 °C. Formic acid (FA) solution (0.1%)prepared

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with water and acetonitrile was the mobile phase A and B, respectively. The gradient

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procedure was as follows: 0/95, 2/80, 4/75, 9/40, 14/0, 18/0, 18.1/95, 19.5/95(min/%

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mobile phase B), and the flow rate of the mobile phase was 0.4 mL/min.

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The mass spectrogram was acquired by scanning the mass from 70 to 1000 in

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positive (pos) and negative (neg) ion mode using a MS system equipped with an ESI

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source. The MS parameters were as follows: nebulizer gas 40, auxiliary gas 40,

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curtain gas 35, ion source temperature 550 °C, ion spray voltage 5500 V (pos) and

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4500 V (neg), collision energy 10 eV (pos) and -10 eV (neg), declustering potential

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100 (pos) and -100 (neg), interface heater temperature 550 °C (pos) and 600 °C 8


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(neg).There are six replicates in metabolomic analysis and the mixture of six

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replicates was defined as quality control (QC) sample.

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Total Phenolics Content (TPC) Assay. To determine TPC in fresh-cut

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strawberries, the procedure described by Swainand Hillis was used.29Frozen samples

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(2 g) were ground in 5 mL methanol and then the homogenate was centrifuged. The

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mixture containing 0.8mL sodium carbonate, 1mL Folin-Ciocalteu, 180μL distilled

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water and 20μLextraction was incubated for 2hat 25 °C. The spectrophotometric

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readings at 725nm were measured and TPC was described as mg g-1 of FW, which

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based ongallic acidequivalents.

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Total Flavonoid Content (TFC) Assay. TFC in frozen samples (2 g) was

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ground in 5 mL acetone (80%) contained 0.2% FA and the homogenate was

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centrifuged.30Thesupernatant(1 mL) was reacted with 1 mL aluminum chloride (3%)

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and 0.5 mL ethanol (30%) for 20 min at ambient temperature. The readings were

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determined at 430nm and TFC was described as mg g-1 of FW, which based on rutin

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equivalents.

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Anthocyanin Content (AC) Assay. For the assay of AC in fresh-cut strawberries,

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the procedure described by Cheng and Breen was used.31The extract to determine

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TFC was also applied to determine AC. Sodium acetate buffer at pH 4.5 (2 mL) and

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potassium chloride buffer at pH 1.0 (2 mL) were mixed with the extract(0.5 mL),

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respectively. After incubation at 40°C for 20min, spectrophotometric readings at 700

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nm and 510 nm of the mixture were recorded. AC in strawberries was expressed as

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mg g-1 of FW on the basis of pelargonidin-3-glucoside equivalents. 9



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Phenylpropanoid Metabolism Related Enzymes Assays. The method of Zucker

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was used to measure phenylalanine ammoniumlyase (PAL) activity.32The frozen

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samples (2 g) was ground in sodium borate buffer at pH 8.8(0.1 M, 5 mL) containing

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β-mercaptoethanol (5mM) and ethylenediaminetetraaceticacid (EDTA, 2mM). Next,

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the homogenate was centrifuged for 30 min at 10000 g. The mixture of 0.3 mL

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supernatant, 3.7 mL extracting solution and 1.0 mL L-phenylalanine was incubated at

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37°C for 1 h. The mixture without L-phenylalanine, which was replaced by distilled

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water, was defined as control. The amount of enzymes that caused a variation of 0.1

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in absorbance at 290 nm per second was defined as one unit of PAL. The results were

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described as U mg-1based on protein content.The protein content was calculated based

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on a standard curve of bovine serum albumin following Bradford’s procedure.33

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For the assay of cinnamate-4-hydroxylase (C4H) activity, two grams of frozen

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samples was ground in 5 mL Tris-HCl buffer(pH 8.9, 0.5 M) that contained Vc (5

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mM), leupeptin (10µM), PMSF (1 mM), β-mercaptoethanol (15 mM), magnesium

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chloride (4 mM) and glycerol (10%).34Next, the homogenate was centrifuged and 0.15

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mL supernatant was added to 2.85 mL Tris-HCl buffer at pH 8.9 (0.5 M) that

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contained nicotinamide adenine dinucleotide phosphate disodium salt (2µM),

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trans-cinnamic acid (2µM) and glucose-6-phosphate disodium salt (5µM)and the

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reaction conducted at 25°C for 30 min. One unit of C4H activity was defined as the

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amount of enzymes that caused a variation of 0.1 in spectrophotometric readings at

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340 nm per second and the results were expressed as U mg-1based on protein content.

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The extract procedure to determine C4H activity was also used in the 10


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measurement of 4-coumarate coenzyme A ligase (4CL) activity.35 The mixture

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contained 2 mL magnesium chloride (5 mM), 0.5 mL extract, 0.05 mL coenzyme A

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(0.04 mM),0.5 mL adenosine triphosphate (5 mM)and 0.5 mL p-cumaric acid (0.6

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mM) and it was incubated at 40 °C for 10 min. One unit of 4CL was defined as the

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amount of enzyme that caused a variation of 0.1 in absorbance at 333 nm per second

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and 4CL activity was described as U mg-1based on protein content.

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Reactive Oxygen Species (ROS) Assays. For the determination of superoxide

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radicals (O2·-), two grams of frozen sample were ground in 0.1 M phosphate buffer

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solution (PBS)(pH 7.8,5 mL) and the homogenate was centrifuged.36An aliquot of 0.5

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mL extract reacted with0.5 mL hydroxylamine hydrochloride (1mM) for 1hat ambient

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temperature and after that 0.5 mL p-aminophenylsulfonic (17 mM) and 0.5 mL

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α-naphthylamine (7 mM) were added to the reaction system. After incubation for

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another 20 min, the readings at 530 nm were recorded and the results were expressed

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as nMg-1min-1FW.

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For the determination of H2O2 content, frozen samples (2 g) were ground in 5 mL

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of acetone and then the homogenate was centrifuged. Next, the supernatant (1 mL)

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was reacted with 0.1 mL titanium tetrachloride (10%) and 0.2 mL ammonium

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hydroxide.37 The precipitate was washed by acetone for four times and finally

227

dissolved insulfuric acid(3 mL,2 M). The readings at 412 nm were recorded and H2O2

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content was described as μmol g-1FW.

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Antioxidant Enzymes Activities Assays. For the measurement of superoxide

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dismutase (SOD) activity, the extract procedure to determine O2·- was also used in the 11



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extraction of SOD.38 The reaction system consisted of 0.89 mL PBS at pH 7.8 (50

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mM), crude enzyme extract (0.01 mL), 0.15 mL methionine (130 mM), 0.15 mL

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riboﬂavin (20 μM), 0.15 mL EDTA-Na2 (100μM) and 0.15 mL nitro-blue-tetrazolium

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(750 μM) was reacted under 4000 lx ﬂuorescent light for 20 min. One unit of SOD

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activity was equivalent tothe amount of enzyme that caused a 50% of NBT

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photochemical reduction per second at 560 nm. The results were described as U

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mg-1on the basis of protein content.

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To determine ascorbate peroxidase (APX) activity, frozen samples (2 g) were

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extracted by 5 mL PBS at pH 7.0 (0.1 M) containing Vc (1 mM), EDTA (0.1 mM)

240

and polyvinyl pyrrolidone (PVP, 1%).39Next, the homogenate was centrifuged for 30

241

min at 13000 g. The supernatantwas reacted with the mixture of 0.1 mL Vc (9 mM),

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2.8 mL PBS (pH 7.0, 0.1 M) and 0.1 mL H2O2 (30%) at ambient temperature.

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Readings at 240 nm were monitored and one unit of APX activity was equivalent to

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the quantity of enzyme that caused a change of 0.01 in absorbance per minutes. APX

245

activity was described as U mg-1based on protein content.

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The extract to determine APX activity was also applicable in the measurement of

247

catalase (CAT) activity.40 The absorbance of the reaction system consisting of 2.5 mL

248

PBS (pH 7.0,0.1 M), 0.2 mL H2O2(0.75%) and 0.3 mL extract was monitoredat 240

249

nm. One unit of CAT activity was equivalent tothe amount of enzyme that caused a

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variation of 0.1 in absorbance per second and it was described as U mg-1based on

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protein content.

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Gene Expression Assays. The isolation of total RNA in frozen tissue was carried 12


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out with cetyltrimethylammonium bromide (CTAB) method according to Chang et

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al.41A gel image system (Tanon 2500, Tanon Science and Technology Co., Ltd,

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China) and an ultramicrospectrophotometer (NanoDrop 2000, Thermo Scientific,

256

USA) were applied to detect the quality and concentration of total RNA, respectively.

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The amplifications were performed following the manufacturer’s instructions of

258

PrimeScriptTMRT Master Mix Kit(RR036B, TaKaRa, Japan) and SYBR Premix Ex

259

TaqTM Kit (RR420B, TaKaRa, Japan). The specific primers of genes were listed in

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Table S1. The PCR procedure was conducted on a QuantStudioTM 5 Flex Real Time

261

PCR System (Applied Biosystems, Foster city, CA, USA). The results was calculated

262

using 2-△△CT method.

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Statistical Analysis.The principle component analysis (PCA) and the hierarchical

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clustering heatmap assay of the metabolomic data was conducted using SIMCA-P

265

(version 14.1) and R statistical software (version 3.4.2), respectively. Data was

266

analyzed by one-way analysis of variance (ANOVA) and the mean separation was

267

performed using Duncan’s multiple range test in SAS (Version 9.1).Data was shown

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as the mean ± standard error (SE) and differences at p< 0.05 were considered to be

269

significant.

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■RESULTS

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Quality Attributesof Fresh-Cut Strawberries. Changes of texture, TSS and TA

273

are presented in Fig.1. Firmness, cohesiveness, springiness, chewiness and

274

gumminess of wedges decreased with storage timewhile adhesiveness presented an 13



275

increasing trend during storage period. Plasma treatment significantly (p< 0.05)

276

suppressed the increase of adhesiveness and delayed the decrease of firmness as well

277

as gumminess of fresh-cut strawberries significantly (p< 0.05), but the differences in

278

springiness and cohesiveness between two groups were insignificant (p>0.05). TSS

279

as well as TA showed a steady decline with storage duration and plasma treatment

280

significantly (p< 0.05) retarded the decrease of TA in wedges during the first 3 days.

281

TSS in treated strawberries were always higher than control throughout the storage

282

time but no significant differences (p>0.05) were determined

283

TABC, Vc Content and Antioxidant Capacity of Fresh-Cut Strawberries.

284

The microbial growth in fresh-cut strawberries was significantly (p< 0.05) suppressed

285

by plasma treatment (Fig.2A). At day 7, TABC was only 3.98 log CFU g-1 in treated

286

strawberries while that in control reached 5.39 log CFU g-1. Changes of Vc content in

287

fresh-cut strawberries is shown in Fig.2B. Vc content presented a downward trend

288

during storage period and it maintained at a higher level in wedges treated with

289

plasma compared with that in control. Significant (p< 0.05) difference was detected at

290

day 1 and day 5. Fig.2C and D present antioxidant capacity of strawberry wedges.The

291

activity to scavenge hydroxyl radical in treated strawberries increased until day 3 and

292

decreased slightly subsequently, while a gradualincrease was observed in control.

293

However, it was always significantly (p< 0.05) higher in samplestreated with plasma

294

than control from day 1 to day 5. The activity to scavenge DPPH radical in strawberry

295

wedges showed an increase from day 0 to day 3 and then decreased with storage time.

296

There was an increase of 17.85% in treated wedges at day 3 in comparison with day 0 14


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while the increase in control was only 5.64%.

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MetabolomicAnalysis of Fresh-Cut Strawberries. The data matrix of all

299

samples in LC-MS was analyzed by PCA. As shown in Fig.S2A, data points were

300

clearly distinguishable among four groups of samples and the data points of three QC

301

samples gathered in the center of the statistic model. There were 28.3% and 17.6%

302

variables of the first principal component (PC1) and the second principal component

303

(PC2), respectively. Fig.S2B shows the top ten enriched KEGG pathways in samples

304

treated with plasma compared with control and the significance levels of these

305

pathways.The first six pathways were at an extremely significant level (p< 0.01),

306

including “Flavone and flavonol biosynthesis”, “Biosynthesis of plant hormones”,

307

“Carbon fixation in photosynthetic organisms”, “Alanine aspartate and glutamate

308

metabolism”, “Aminoacyl-tRNA biosynthesis” and “Nitrogen metabolism”. The

309

remaining four pathways including “Biosynthesis of alkaloids derived from ornithine,

310

lysine and nicotinic acid”, “Biosynthesis of alkaloids derived from shikimate

311

pathway”, “Biosynthesis of phenylpropanoids” and “Zeatin biosynthesis” were at a

312

significant level (p< 0.05).

313

The hierarchical clustering heatmap analysis of themetabolitesin the top ten

314

pathways identified by LC-MS was shown in Fig.3.There were 57 common

315

differential metabolites in the three groups of samples and the heatmap was

316

constructed using their peak areas. Some of the metabolites are involved in two or

317

more metabolic pathways, such as apigenin, kaempferol and L-aspartic acid. In

318

comparison with control, plasma treatment mainly promoted the accumulation of 15



319

some metabolites in “Flavone and flavonol biosynthesis” pathway, “Biosynthesis of

320

phenylpropanoids” pathway and “Zeatin biosynthesis” pathway, the biosynthesis of

321

two kinds of plant hormones (brassinolide and cis-zeatin) and some metabolites

322

derived from phenolic compoundsnot mapped onto the specific metabolic pathway

323

were also enhanced by plasma treatment.

324

Antioxidants of Fresh-Cut Strawberries. Changes of the major antioxidants

325

involved in the metabolism of phenylpropanoids as well as flavones are presented in

326

Fig.4. TPC in treated wedges increased until the third day of storage and then

327

followedbya decrease, while that in control experienced a steady but slow increasing

328

trend. Plasma treatment improved TPC in fresh-cut strawberries during the first 5

329

days.

330

AC in strawberry wedges showed a similar pattern compared with that of TPC.

331

Plasma treatment significantly (p< 0.05) enhanced AC in strawberries from day 1 to

332

day 3 and AC in control and treated wedges were 0.5550 mg g-1 and 0.6911 mg g-1 at

333

day 3, respectively. From then on, AC in treated wedges began to decrease and it was

334

significantly less (p< 0.05) than control at day 7.

335

TFC in tissue increased with storage period and the accumulation of it was

336

promoted by plasma treatment. The significant (p< 0.05) difference was detected at

337

day 3 and day 5.At the last day of storage, there was an increase of 43.58% and

338

31.39% in TFC of treated wedges and control compared with day 0, respectively.

339

Activities of PAL, C4H and 4CL and Relevant Gene Expression. Fig.5A, B

340

and C show changes of the activities of PAL, C4H and 4CL with storage duration, 16


Page 16 of 41

Page 17 of 41


341

respectively. These three enzyme activities in strawberries without any treatment

342

presented a consistently increasing trend. The activity of PAL in wedges treated with

343

plasma increased sharply during the first three days and followed by a gradual

344

decrease in the subsequent time. It was always higher in plasma treated strawberries

345

compared with control throughout the whole storage. A similar tendency was also

346

found in the activity of 4CL. The activity of C4H was enhanced significantly (p
0.05) was observed between two

349

groups in the activities of C4H and 4CL from day 5 to day 7.

350

The changes of the expression levels of FaPAL, FaC4H and Fa4CL are shown in

351

Fig.5D, E and F.The relative expression levels of FaPAL and FaC4H increasedduring

352

the early storage timeand afterthat a descending trend was presented.The

353

transcriptional peak of FaPAL and FaC4H appeared at day 3 and 12 h, respectively.

354

Plasma treatment elevated the transcription level of these two genes,but the

355

expression level of FaC4H in treated wedges was lower than that in control from day

356

3 to day 7. Plasma treatment also induced the earlier peaking of Fa4CLcompared with

357

control and the expression level of Fa4CL was always higher in treated samples than

358

in control during the first 3 days of storage.

359

ROS Contents of Fresh-Cut Strawberries. As shown in Fig. S3A, the production

360

of O2·- increased with storage period and plasma treatment promoted its enhancement.

361

The difference between two groups was significant (p< 0.05) from day 3 to day 7.

362

H2O2 content exhibited a slow but steady increasing trend in control with the storage 17



363

duration (Fig. S3B). Plasma treatment significantly (p< 0.05) accelerated the

364

accumulation of H2O2 in strawberry wedges at earlier storage but no significant

365

(p>0.05) difference between the two groups was detected at the last day of storage.

366

Activities of SOD, APX and CAT and Relevant Gene Expression. As shown in

367

Fig.6A, SOD activity showed a sharp increase intreated strawberries at day 1 and then

368

a decrease trend was presented. It was significantly (p< 0.05) lower than that in

369

control in the last two days. APX activity in wedges treated with plasma reached the

370

maximum value at day 5 and then decreased slightly (Fig.6B). It was always higher in

371

plasma treated samples than in control samples throughout the whole storage duration.

372

Plasma treatment also promoted the continuous increase of CAT activity and there

373

was an increase of 72.41% and 110.34% in control and treated wedges from day 0

374

today 7, respectively (Fig.6C). Fig.6D, E, F and G show the changes of relative

375

expression levels of genes that encoded the antioxidant enzymes. Plasma treatment

376

not only induced the earlier peaking of FaCu/ZnSOD as well as FaMnSOD but also

377

improved the relative expression levels of FaAPX and FaCAT. The transcriptional

378

peak of FaAPX and FaCAT in fresh-cut strawberries treated with plasma appeared at

379

day 3 and day 1, respectively, and significant (p < 0.05) difference between two

380

groups was detected from 12 h to day 3.

381 382

■DISCUSSION

383

Dielectric barrier discharge plasma is an innovative processing technology that

384

has great potential in retarding the quality deterioration and inhibiting the microbial 18


Page 18 of 41

Page 19 of 41


385

growth of fruits and vegetables. In our experiment, the changes of texture, TSS, TA,

386

Vc and TABC were determined to illustrate the impacts of DBD plasma on the quality

387

and safety of strawberry wedges. Texture profile analysis (TPA) was one of the

388

pivotal methods to characterize textural properties of postharvest fruits and vegetables

389

and it provides comprehensive information about the elasticity as well as plasticity of

390

foods.42Firmness refers to the stability of cell wall and it decreased with storage time

391

in fresh-cut strawberries. Loss in firmness is probably caused by the cell debonding,

392

cell rupture and hydrolysis of cell wall components.23 Our results demonstrated that

393

plasma treatment maintained the firmness as well as springiness of strawberry wedges

394

and this result revealed the positive effect of plasma on the intercellular adhesion and

395

the structural integrity of the cell wall. Besides, plasma treatment also reduced the

396

increase of adhesiveness, which could be explained by the inhibition of the

397

depolymerization and solubilization of pectin in cell wall.43Likewise, the hydrolysis

398

of pectin as well as starch led to the decrease of turgor pressure and the increase of

399

osmotic pressure during storage, and thus the primary cell wall and middle lamella

400

were disintegrated.42,

401

and chewiness indicating that more force and energy were required to masticate the

402

strawberries until swallowing. Strawberries with TSS more than 7.0% and TA less

403

than 0.8% are generally regarded as acceptable for consumers.44Our research

404

indicated that plasma treatment had no adverse impact on TSS as well as TA of

405

fresh-cut strawberries. Vc was an important nutrient component in strawberries.

406

Plasma retarded the reduction of Vc content but the difference was insignificant

43The

treated wedges maintained higher levels of gumminess

19



Page 20 of 41

407

(p>0.05) between two groups at day 7. The bactericidal effect of plasma has been

408

reported in many fruits, such as fresh-cut melon and fresh-cut pitaya.16,

409

documented that cold plasma suppressed the microbial growth by inducing ROS

410

production and accelerating the accumulation of intracellular charged particles, which

411

was closely related to the apoptosis and electrostatic disruption of microorganism.6 In

412

our results, TABC in DBD plasma treated wedges was only 3.98 log CFU g-1 and it

413

was much less than the threshold (6 log CFU g-1) in the specifications proposed by

414

Regulation E.C. (NO. 2073/2005). Therefore, the results from our investigation

415

suggested that DBD plasma at 45 kV for 1 min not only retained the texture but also

416

inhibited the microbial growth of fresh-cut strawberries without any adverse influence

417

on TSS, TA and Vc content.

45It

was

418

Fresh-cut strawberries are rich in antioxidant components, which act as a critical

419

role in scavenging the free radicals in the body and thus providing beneficial health

420

effect.18In our experiments, the activities to scavenge hydroxyl as well as DPPH

421

radicals were assayed to evaluate the antioxidant capacity of fruit. Results indicated

422

that plasma treatment enhanced the antioxidant activity of strawberries and the peak

423

value was detected at day 3. Therefore, the samples at day 3 were collected and the

424

metabolomic analysis was conducted to identify the specific metabolites in tissue. The

425

dimensions of the original variable in data matrix were reduced in PCA. The

426

confidence interval (Hotelling’s T2) and the cumulative variable of the principal

427

component in PCA model were 95% and 53.60%, respectively, which demonstrated

428

the model was reliable and a good distinction was made among three groups of 20


Page 21 of 41


429

samples.46Next, we selected the top ten pathways that were sensitive to the treatment

430

and found that 57 common different metabolites were identified by LC-MS.

431

Excitingly, the biosynthesis of flavones and flavonol, phenylpropanoid, and some

432

other metabolites derived from phenolic compounds was significantly (p< 0.05)

433

activated by plasma and these metabolites were strongly associated with the

434

enhancement of antioxidant capacity in strawberry wedges.In addition, plasma

435

treatment also improved the content of brassinolide. This kind of plant hormone has

436

the ability to induce plant tolerance to biotic or abiotic stresses via modulation of

437

ROS, which act as a signal molecular in the accumulation of phenolics.47, 48It is worth

438

mentioning that some metabolites in the clustering analysis were involved in more

439

than one pathway because of the overlap and the interaction among certain pathways

440

in the metabolic network. Deriving from the metabolomic results in our experiments,

441

plasma treatment increased the contents of certain metabolites in the pathways that

442

made a great contribution to the biosynthesis of antioxidative compounds and thus

443

enhanced the antioxidant capacity of fresh-cut strawberries. Similar results were also

444

reported in mandarin peel and cashew apple juice.8,49

445

In order to further explore how DBD plasma influence the metabolic pathway

446

related to the synthesis of antioxidants in strawberry wedges, the physiological

447

analysis on phenylpropanoid metabolism was conducted. Phenolic compounds,

448

including phenolic acids and anthocyanin, are the major antioxidant constituents in

449

strawberry wedges and their synthesesare catalyzed by PAL, C4H and 4CL, which are

450

the first three crucial enzymes involved in phenylpropanoid metabolism. In our 21



Page 22 of 41

451

results, plasma treatment improved the activities of these three enzymes by elevating

452

the gene expression levels of FaPAL, FaC4H and Fa4CL, and thus increased the

453

accumulation of antioxidants. The positive effect of plasma in promoting the contents

454

of TPC, TFC and AC was also reported in blueberries and pomegranate juice.12,

455

50These

456

theimprovementof the antioxidant capacity of strawberries. Giampieri et al

457

overexpressed the anthocyanidin synthase gene in strawberries and the antioxidant

458

activity of fruit was also increased.51ROS are regarded as an important signal in

459

mediating the stress response in plants.48In the studies on carrot and pitaya, the

460

production of ROS induced by wounding played a vital role in the synthesis of

461

phenolic compounds.3,4Interestingly, higher levels of ROS contents were also detected

462

in treated wedges in our research, which suggested that the enhancement of TPC in

463

treated strawberries would be closely connect with the increase of ROS induced by

464

plasma. SOD, APX and CAT are three antioxidant enzymes that regulated the

465

contents of ROS. In plants, SOD catalyzeO2·- while APX and CAT convert

466

carbon dioxide and harmless water to prevent oxidative damage caused by excessive

467

H2O2.The present results demonstrated that plasma treatment promoted SOD activity

468

on the first day but inhibited its activity in the subsequent time of storage. This could

469

be the reason why O2 ·- production was significantly (p< 0.05) higher than control in

470

strawberries treated with plasma from day 3 to day 7.The changes of the gene

471

expression of FaMnSOD and FaCu/ZnSOD during storage were in accordance with

472

the trend of relevant enzymes, but the transcriptional peaks of these two genes were

results indicated that the up-regulation of relevant genes was related with

22


H2O2 to

Page 23 of 41


473

both detected before the first day of storage, which indicated that the enhancement of

474

enzyme activities lagged behind the up-regulation of genes. By the same token, the

475

elevation of the transcription levels of FaAPX and FaCAT was preparing for the

476

improvement of APX and CAT activities in wedges treated with plasma, respectively.

477

Thus, it could be concluded that plasma treatment increased the synthesis of

478

antioxidants in fresh-cut strawberries by promoting the enzyme activities and related

479

gene expression in phenylpropanoid metabolism, which was probably induced by the

480

activation of ROS metabolism.

481

According to the obtained results, DBD plasma at 45 kV for 1 min retained the

482

textural properties and suppressed the microbial growth of quarter strawberries during

483

one week of storage period at 4 °C. It also promoted the accumulation of some

484

metabolites involved in the synthesis of flavones and flavonol, as well as

485

phenylpropanoids in strawberry wedges, which contributed greatly to the

486

enhancement of TPC, TFC and AC. Besides, plasma treatment induced the

487

heightening of phenylpropanoids as well as ROS metabolism, including the

488

improvement of enzyme activities and the activation of related gene expression, and

489

thus enhanced the antioxidant activity of fruit. Our study showed clearly the positive

490

effect of DBD plasma on the quality retention and antioxidant capacity enhancement

491

of strawberry wedges through physiological and metabolomic analysis. This emerging

492

non-thermal technology is useful for the strawberry industry and may be helpful for

493

other

fruits

and

vegetables

and

warrants

23


further

investigations.


494

■ACKNOWLEDGEMENTS

495

This study was financially supported by the National Natural Science Foundation

496

of China (No. 31471632)and Natural Science Foundation of Shandong Province

497

(ZR2018PC032).

498

We are grateful toProf. Jianhao. Zhang of the National Center of Meat Quality &

499

Safety Control, Nanjing Agricultural University, for providing the equipment and

500

technical guidance. We also thank Dr. Chien Y. Wang ofU.S. Department of

501

Agriculture, for the critical review of the manuscript.

24


Page 24 of 41

Page 25 of 41


503

Supporting Information

504

Table S1. Gene-specific primers used for qRT-PCR experiments.

505

Fig. S1. The schematic diagram of DBD plasma system.

506

Fig. S2. Score scatter plots of principle component analysis (PCA) of the primary

507

metabolites derived from LC-MS data (A) and effect of plasma treatment on the

508

significance levels of the top 10 enriched KEGG pathways in fresh-cut strawberries.

509

Fig. S3. Effect of plasma treatment on O2 · - production (A)and H2O2content (B) of

510

fresh-cut strawberries stored at 4 °C for one week.

511 512

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■Legends to Figures

666

Figure 1.Effect of plasma treatment on firmness (A), adhesiveness(B), springiness

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(C), cohesiveness (D), gumminess (E), chewiness (F), total soluble solids (TSS) (G)

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and titratable acidity (TA) (H) of fresh-cut strawberries stored at 4°C for one week.

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Data represent the means of three replicates and their standard errors.Asterisk

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(*)indicates significant difference at p< 0.05 between two groups.

671

Figure 2. Effect of plasma treatment on the total aerobic bacterial count (TABC) (A),

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Vc content (B), hydroxyl radical scavenging activity (C) and DPPH radical

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scavenging activity (D) of fresh-cut strawberries stored at 4°C for one week. Data

674

represent the means of three replicates and their standard errors.Asterisk (*)indicates

675

significant difference at p< 0.05 between two groups.

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Figure 3.The hierarchical clustering heatmap of the metabolites in the top ten

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pathways identified by LC-MS. Rows represent metabolites and columns represent

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the samples. Triangle (▲) indicates the metabolites involved in two pathways and

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circle (●) indicates the metabolites involved in three or more pathways.

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Figure 4. Effect of plasma treatment on total phenolics content (TPC) (A), 33



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anocythanin content(AC)(B) and total flavonoid content (TFC)(C) of fresh-cut

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strawberries stored at 4 °C for one week. Data represent the means of three replicates

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and their standard errors.Asterisk (*) indicates significant difference at p< 0.05

684

between two groups.

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Figure 5.Effect of plasma treatment on the activities of phenylalanine

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ammoniumlyase (PAL)(A), cinnamate-4-hydroxylase (C4H) (B) and 4-coumarate

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coenzyme A ligase (4CL) (C) and the gene expression levels of FaPAL (D), FaC4H

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(E) and Fa4CL (F) of fresh-cut strawberries stored at 4 °C for one week. Data

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represent the means of three replicates and their standard errors.Asterisk (*) and

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different letters indicate significant difference at p< 0.05 between two groups.

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Figure 6.Effect of plasma treatment on theactivities of superoxide dismutase

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(SOD)(A), ascorbate peroxidase (APX) (B) and catalase (CAT) (C) and the gene

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expression levels of FaCu/ZnSOD (D), FaMnSOD(E), FaAPX (F) and FaCAT(G) of

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fresh-cut strawberries stored at 4 °C for one week. Data represent the means of three

695

replicates and their standard errors.Asterisk (*) and different letters indicate

696

significant difference at p< 0.05 between two groups.

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Figure1

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Figure2

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Figure3

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Figure4

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Figure5

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Figure6

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■TABLE OF CONTENTS GRAPH

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Physiological and Metabolomic Analysis of Cold Plasma Treated

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