Microbial decontamination system combining antimicrobial solution washing and atmospheric dielectric barrier discharge cold plasma treatment for preservation of mandarins

Postharvest Biology and Technology 162 (2020) 111102

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Microbial decontamination system combining antimicrobial solution washing and atmospheric dielectric barrier discharge cold plasma treatment for preservation of mandarins

T

In Hee Bang1, Eun Song Lee1, Ho Seon Lee, Sea C. Min* Department of Food Science and Technology, Seoul Women’s University, Seoul 01797, Republic of Korea

A R T I C LE I N FO

A B S T R A C T

Keywords: Mandarin Cold plasma Calcium oxide Fumaric acid Electrolyzed water Penicillium

The effect of a microbial decontamination system that integrates antimicrobial washing and in-package atmospheric dielectric barrier discharge cold plasma (ADCP) treatment on mandarin preservation was studied. A 0.2 % highly activated calcium oxide (CaO) aqueous solution and slightly acidic electrolyzed water containing 0.5 % fumaric acid (FS solution) were tested as antimicrobial washing solutions. ADCP treatment was applied to mandarins packaged in commercial polyethylene terephthalate (PET) containers at 26 or 27 kV for 1, 2, 3, or 4 min. Penicillium digitatum disease incidence on mandarins was lowest (77.1 %) after ADCP treatment at 27 kV for 2 min. P. digitatum disease incidence on untreated mandarins or those treated with ADCP after washing with CaO solution, FS solution, or CaO solution and FS solution consecutively was 97.3 %, 64.3 %, 87.1 %, or 80.0 %, respectively. ADCP treatment after washing with CaO solution (CaO-ADCP treatment) did not affect the appearance of mandarins, but altered the glossiness of the sensory attributes of mandarins (p < 0.05). CaO-ADCP treatment retarded increases in the respiration of the fruit and total soluble solid content of the flesh during storage at 4 °C, as well as the total polyphenol contents of mandarin peel at 4 °C and 25 °C. Neither ADCP treatment with nor without washing affected the pH of the flesh, ascorbic acid concentration of the flesh, antioxidant capacity of the peel, or color of the peel during storage at 4 °C and 25 °C. The results of this study demonstrated the potential of CaO-ADCP treatment for enhancing the storability of mandarins in plastic packages by inhibiting the growth of P. digitatum on fruit while minimizing changes to fruit quality during storage.

1. Introduction Satsuma mandarins are produced primarily in China, Europe, Japan, and Turkey, with a total production of 29 million tons worldwide, and are steadily purchased and eaten by consumers (Goldenberg et al., 2018). However, mandarins are susceptible to post-harvest decay, which is predominantly caused by the growth of fungi, including Penicillium spp. (Lu et al., 2018). While chlorine, chlorine dioxide, electrolyzed water, and ozone have been investigated and utilized as surface disinfection methods for fresh fruit, all of these have certain drawbacks. The use of chlorine washing has been challenged owing to public health concerns and low efficacy (Goodburn and Wallace, 2013). Although chlorine dioxide has been adopted as an alternative to chlorine, chlorine dioxide treatment requires a long exposure time (e.g., a few hours) and is prone to explosion, limiting industrial applications

(Deng et al., 2019). Furthermore, electrolyzed water washing may result in noticeable negative effects on the nutrient contents of fruit. While the use of strong acid electrolyzed water is highly effective, its application is limited due to its unstable and corrosive nature (Deng et al., 2019). When using ozone for fruit disinfection, high ozone concentrations or long treatment times are necessary to obtain effective microbial reduction. Moreover, excessive use of ozone leads to fruit quality deterioration, including discoloration and nutrient degradation (Deng et al., 2019). Therefore, it is necessary to develop new methods for disinfecting mandarins. Kim et al. (2010) reported that treatment of strawberries with a 0.5 % fumaric acid solution for 5 min inhibited the growth of strawberry yeast and fungus by 1.75 log CFU g−1. Tango et al. (2017) reported that washing with slightly acidic electrolyzed water (SAEW) inactivated Escherichia coli O157:H7 contaminated on apples and Listeria

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Corresponding author. E-mail address: [email protected] (S.C. Min). 1 The authors equally contributed to this work. https://doi.org/10.1016/j.postharvbio.2019.111102 Received 28 September 2019; Received in revised form 12 December 2019; Accepted 12 December 2019 0925-5214/ © 2019 Elsevier B.V. All rights reserved.

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current, pH, and oxidation reduction potential of the produced SAEW were 10 A, 5.4 ± 0.2, and 818–854 mV, respectively, which are the values for SAEW normally used in fresh fruit washing (Tango et al., 2017).

monocytogenes on tomatoes, reducing levels by 2.3 and 2.4 log CFU per fruit, respectively. Kang and Song (2017) reported that the treatment of kale with a 0.2 % solution of highly activated calcium oxide (CaO), obtained from pulverized shellfish shells, followed by pyrolysis at over 1,000 °C and electrolysis at over 100 kV, reduced the numbers of indigenous bacteria and E. coli O157:H7 by 1.3 and 4.0 log CFU g−1, respectively. Cold plasma treatment has been investigated as an in-package microbial disinfection method for fruit and vegetables (Deng et al., 2019). During cold plasma treatment, ultraviolet (UV) photons and electrons, as well as reactive oxygen species (ROS) and reactive nitrogen species (RNS) including ozone (O3), hydrogen peroxide (H2O2), superoxide anion (O2 %−), hydroxyl (HO2%−), singlet oxygen (1O2), hydroxyl radical (%OH), alkoxyl (RO%), peroxyl (ROO%), nitric oxide (NO%), peroxynitrite (ONOO−), nitrogen dioxide radical (%NO2), alkylperoxynitrite (ROONO), and peroxynitrous acid (OONOH), collide with the cell membranes of microorganisms to destroy chemical bonds; erode cell membranes (‘etching’); enter the cell to react with lipids, proteins, and DNA; and eventually inactivate microorganisms and inhibit their growth (Min et al., 2017b; Misra et al., 2011; Pignata et al., 2017; Sarangapani et al., 2018). One method for cold plasma discharge, atmospheric dielectric barrier discharge, is used for in-package microbial decontamination (Pignata et al., 2017). Min et al. (2017b) reported that the treatment of bulk romaine lettuce slices in polyethylene terephthalate (PET) clamshell containers with ADCP treatment at 42.6 kV for 10 min inactivated E. coli O157:H7, reducing levels by 1.1 log CFU g−1. Although cold plasma treatment has received increasing attention from the food industry for its ability to prevent potential post-processing contamination after packaging (Min et al., 2017a), a high dose of cold plasma (e.g., > 80 kV) can adversely affect the quality of fruit and vegetables (Deng et al., 2019), and the efficacy of cold plasma at low doses (e.g., < 50 kV) in terms of microbial inactivation has not been sufficiently demonstrated to replace conventional microbial decontamination methods for fruit and vegetables (Min et al., 2016). One way to improve the efficacy of cold plasma treatment at low doses, enabling a reduction in the input energy and minimizing quality deterioration, would be to reduce the microbial contamination level prior to treatment using an appropriate washing method, implementing the concept of a hurdle technology (Gómez et al., 2011). Thus, the objectives of this study were to (1) develop a microbial decontamination system combining an antimicrobial washing solution and ADCP treatment for mandarin preservation by determining the conditions under which ADCP treatment effectively reduces the Penicillium disease incidence in mandarins packaged in plastic containers and selecting an effective antimicrobial washing solution and (2) investigate the effects of the developed system on the sensory and quality properties of mandarins to evaluate the system’s potential as a method for extending the shelf life of mandarins.

2.2. Inoculum preparation The microorganisms used in this study were P. digitatum (KACC 40822) isolated from Citrus unshiu Marc., which were provided by the Rural Development Administration-Genebank Information Center (Jeonju, Korea). P. digitatum was cultured in potato dextrose agar (PDA, Difco™, Becton and Dickinson, Detroit, MI, USA) supplemented with 10 % (w/v) tartaric acid (Sigma–Aldrich Co., St. Louis, MO, USA) at 23 ± 2 °C for 7 d. A suspension prepared by dispensing 10 mL of sterilized 0.1 % (w/w) Tween-80 solution to the cultured fungi was filtered with cheesecloth to remove fungal mycelium, and the filtrate was washed three times by centrifugation (GyroSpin, Gyrozen, Seoul, Korea) at 9,100 ×g for 2 min. Washed P. digitatum conidia were diluted with 0.1 % peptone water to prepare an inoculum with a concentration of ∼6 log conidia mL−1, which was enumerated using a hemocytometer (Paul Marienfeld GmbH & Co. KG, Lauda-Konigshofen, Germany). 2.3. Inoculation of microorganism P. digitatum conidium inoculation was conducted using the method described by Won et al. (2017). To disinfect the mandarin surface, 70 % (v/v) ethanol was sprayed onto the peel, which was wiped with sterilized paper towels followed by gentle rubbing and rinsing of mandarins in sterilized distilled water (one fruit in 200 mL each) for 30 s. Washed mandarins were dried in a biohazard hood (Hanbaek Co., Ltd., HB-402, Bucheon, Korea) for 1 h. Once dried, a sterilized stainless-steel needle (diameter: 0.07 mm) was used to make wounds of ∼0.1 mm depth at different spots on the surface (peel) of each fruit. Each wound was inoculated with 20 μL of a ∼6.0 log conidia mL−1 inoculum of P. digitatum. Two wounds were made per fruit. The inoculated mandarins were further dried in the biohazard hood for 1 h prior to treatment. 2.4. ADCP treatment system and conditions ADCP treatment was conducted using the system (Renosem, Bucheon, Korea) described previously by Kim et al. (2019), which generates a plasma field between the base dielectric barrier and the upper aluminum rectangular electrodes (20 × 16 cm) (AL6061; Kwanglim Co. Ltd., Hwasung, Korea). Prior to ADCP treatment, mandarins (5 fruits) were packaged in a commercial standard PET container (DL-208, width × depth × height: 180 × 180 × 60 mm, thickness: 0.368 mm, Dong-yang D&P, Daegu, Korea), which was sterilized with 70 % ethanol, rinsed with sterile distilled water, and dried in the biohazard hood prior to being filled. The container was then placed in the gap between the upper electrode and borosilicate glass (dielectric barrier: 29 × 25 cm, thickness: 0.4 cm). The distance between the two electrodes was 6.5 cm. The upper electrode and the top surface of the container were dropped to 0.5 cm to shake the container efficiently during treatment. A Bakelite (polyoxybenzylmethylenglycolanhydride) stick (diameter: 0.6 mm, length: 640 mm) was connected to the container to induce shaking back and forth at 1.5 turns/s. An AC power supply (220 V, 60 Hz) delivering a high voltage (up to 40 kV, peak-topeak voltage) was coupled to the electrodes. To determine ADCP treatment conditions to effectively inactivate P. digitatum inoculated on the mandarin surface, the fruit was subjected to an ADCP treatment voltage of 26 kV for 2 min, 3 min, or 4 min, and 27 kV for 1 min or 2 min. Voltage measurements were conducted using a high-voltage electric probe (EP-50, Pulse Electronic Engineering Co., Ltd., Noda, Japan). The surface temperature of the mandarins in the container was measured before and immediately after treatment with an infrared

2. Materials and methods 2.1. Materials The satsuma mandarins (Citrus unshiu Marc., Jeju, Korea) used in this study were harvested between April and June, 2018. Mandarins that had been transported for 1 or 2 d at 5–25 °C within 2 d of harvest were purchased and stored in a refrigerator at 4 °C until use in experiments, which were conducted within 2 d. Mandarins with physical scars or decay were removed, and intact fruit (40 ± 5 g) was selected for use in the study. CaO, supplied by Eco-Biotech Company (Hwaseong, Korea), was produced by pulverizing shellfish shells to a 1–5 mm grain size, followed by pyrolysis at 1,400–1,500 °C for 9–11 h to remove carbonic acid gas and impurities, and electrolysis at 110–130 kV for 90 min. SAEW was produced using an electrolytic water-generating device (BC-360, Cosmic Round Korea Co., Ltd., Seongnam, Korea). The 2

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2.9. Storage study

thermometer (DT 44 L, DIAS Infrared GmbH, Dresden, Germany).

Untreated mandarins, single ADCP-treated mandarins, and CaOADCP-treated mandarins (5 fruits per container) were stored at 4 °C for 0, 7, 14, 21, 28, and 35 d, and at 25 °C for 0, 3, 7, 10, and 14 d. Changes in the CO2 generated by the fruit; the total soluble solid content (SSC), pH, and ascorbic acid concentration of the mandarin flesh; and the total phenol content (TPC), antioxidant capacity, and color of the mandarin peel were observed. The temperatures inside the containers of samples stored at 4 °C and 25 °C were 4.0 ± 0.4 °C and 24.8 ± 0.1 °C, respectively, while the RHs inside the containers were 82.6 ± 1.6 % and 82.9 ± 1.6 %, respectively. Temperature and RH during storage were measured using a data logger (8829S, AZ Instrument Corp., Taichung City, Taiwan).

2.5. Determination of mandarin washing method CaO washing solution (‘CaO solution’) was prepared by dissolving CaO in distilled water (0.2 %, w/w) by stirring for 1 h at 23 ± 2 °C. Fumaric acid (Sigma–Aldrich Co.) was mixed with SAEW (0.5 %, w/w) to prepare fumaric acid-containing SAEW washing solution (‘FS solution’). The concentrations of CaO and fumaric acid in the solutions were selected based on those that are reportedly effective for microbial decontamination (Kang and Song, 2017; Ngnitcho et al., 2017; Tango et al., 2017). During CaO solution or FS solution washing, 5 mandarins that had either been inoculated or not inoculated with P. digitatum were placed in stomacher bags (710 mL, Whirl-Pak® Write-On Bags, Nasco Co., Fort Atkinson, WI, USA) with the solution (200 mL) and completely immersed for 3 min for treatment. ‘CaO-FS washing’ was conducted by immersing mandarins in CaO solution (200 mL) for 3 min, followed by another immersion in FS solution (200 mL) for 3 min. After washing, mandarins were dried in the biohazard hood for 1 h. Five dried mandarins were packaged in each PET container and subjected to ADCP treatment at 27 kV for 2 min. CaO solution washing followed by ADCP treatment, FS solution washing followed by ADCP treatment, and CaOFS washing followed by ADCP treatment are hereafter referred to as ‘CaO-ADCP’ treatment, ‘FS-ADCP’ treatment, and ‘CaO-FS-ADCP’ treatment, respectively.

2.9.1. Determination of CO2 generation To assess CO2 generation, the concentration of CO2 in a jar (1 L) containing mandarins was measured following the method of Choi et al. (2015). Five mandarins (∼200 g) were placed in the glass container (1 L) and sealed. The glass container lid was affixed with a rubber septum (5 mm, Precision Seal1 rubber septa, Sigma–Aldrich Co.). After 1 h of incubation at 23 ± 2 °C, the needle of a gas analyzer was inserted into the septum on the container lid, and the CO2 concentration inside the glass container was measured by the gas analyzer (Check Point 2, PBI Dansensor, Ringsted, Denmark). The volume collected from the jar headspace for respiration analysis was 9 cm3. 2.9.2. Determination of SSC and pH of flesh Peel and fiber were separated from fruit flesh manually. The separated mandarin flesh was blended with a home processor (HR3556, Philips, Koninklijke Philips N.V., Netherlands) for 1 min and filtered through 4 layers of cheesecloth to obtain juice. The SSC and pH of the obtained juice were determined using a digital refractometer (PAL-1, ATAGO, Tokyo, Japan) and a pH meter (pH/mV/Temp meter Model PL500, Ezodo, Taiwan), respectively.

2.6. Determination of P. digitatum disease incidence P. digitatum-inoculated mandarins were subjected to ADCP, CaOADCP, FS-ADCP, or CaO-FS-ADCP treatment and then moved to a desiccator with a relative humidity (RH) maintained at 80–85 % using potassium chloride (Samchun Pure Chemical Co., Pyeongtaek, Korea) at 23 ± 2 °C. Fungal growth was observed after incubation for at least 4 d. The growth of P. digitatum on the fruit was confirmed when wounds (inoculation sites) showed visible mycelial growth, i.e., green conidia or white hyphae were observed in the area around the inoculation site (Won and Min, 2018). Disease incidence (%) was calculated by dividing the number of infected sites by the total number of inoculation sites and multiplying the result by 100. Thirty fruits per treatment were used for determining P. digitatum disease incidence.

2.8. Sensory evaluation

2.9.3. Determination of ascorbic acid concentration High-performance liquid chromatography (HPLC, Agilent 110 series, Agilent Technologies, Santa Clara, CA, USA) was used to determine the ascorbic acid concentration according to the method by Won et al. (2017). Juice obtained as described above was centrifuged (Supra 22 K, Hanil Science Industrial, Incheon, Korea) at 4 °C and 10,000 ×g for 20 min, and the supernatant was filtered with a syringe filter (Dismic®-25CP, cellulose acetate, pore size: 0.45 μm, Advantec MFS, Inc., Dublin, CA, USA) to prepare analytic samples. The mobile phase solution for HPLC was 2 % (v/v) acetic acid/acetonitrile (95:5 v/ v), which flowed at 0.8 mL/min. The column was a Symmetry® C18 (5 μm, 4.6 mm × 250 mm I.D., Waters Co., Milford, MA, USA), which was maintained at 23 ± 2 °C during analysis. An analytic sample of 20 μL was injected, and ascorbic acid was detected with a diode array detector (DAD, UV–vis detector, G1315B, Agilent Technologies) at a 254-nm wavelength. The ascorbic acid concentration was expressed as vitamin C equivalent antioxidant capacity (VCE g L−1). Standard ascorbic acid was purchased from Sigma–Aldrich Co.

Sensory characteristics including appearance and glossiness were evaluated without consumption in non-stored and unpeeled mandarins from untreated, ADCP-treated, and CaO-ADCP-treated samples. For sensory evaluation, 32 female panelists aged 20–29 at Seoul Women’s University (Seoul, Korea) participated. The panelists were initially screened for their consumption frequencies of mandarin. Panelists consuming mandarin more than once a week during the fruiting season (November through January) were enrolled. Fruit was coded using random three-digit numbers. Sensory characteristics were evaluated based on a 9-point scale (1: extreme dislike, 5: normal, 9: extreme like), where higher numbers reflect a higher preference for the attribute.

2.9.4. Determination of TPC and antioxidant capacity TPC and antioxidant capacity were determined in mandarin peels, which were freeze-dried (PVTFD 100R, Il-shin Bio Base Co., Ltd, Dongducheon, Korea) for 72 h. Freeze-dried fruit peel was pulverized by blending with a home processor. The peel powder (1 g) was mixed with 95 % methyl alcohol (9 g, Sigma–Aldrich Co.) by stirring at 100 rpm for 24 h at 23 ± 2 °C using an orbital shaker (JSOS-500, JS Research Inc., Gongju, Korea). The extract was filtered with Whatman No. 1 filter paper (Whatman, Maidstone, UK) to prepare the peel extract. For TPC measurement, peel extract (80 μL) was reacted with Folin-

2.7. Determination of surface morphology The surface morphology of mandarin peels was observed following the method of Won et al. (2017). Mandarin peel before and after treatment was cut to 5 × 5 mm, dried in the biohazard hood for 1 h, adhered to aluminum stubs, sputter-coated with platinum (HR 208 sputter coating device, Cressington, Warford, UK), and viewed with field-emission scanning electron microscopy (FE-SEM, S-4700; Hitachi, Tokyo, Japan) at 5 kV under 1000× magnification.

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Ciocalteu's phenol reagent (20 μL) at 23 ± 2 °C for 3 min in the dark, followed by the addition of 2 % (w/v) Na2CO3 aqueous solution (100 μL) and reaction for 30 min at 23 ± 2 °C according to the method by Schlesier et al. (2002). Subsequently, the absorbance of the mixture was measured at 750 nm. The TPCs of fruit were calculated from a standard curve obtained with gallic acid and expressed as gallic acid equivalents (GAE) in mg mL−1. Antioxidant capacity was evaluated by quantifying the 1,1-diphenyl-2-picrylhydrazyl (DPPH)-scavenging activity following the method of Blois (1958), which has been adopted in many studies for determining the antioxidant capacity of Citrus spp. peels (Safdar et al., 2017; Rahman et al., 2018). DPPH solution was prepared by dissolving 0.32 mM DPPH radical (Sigma–Aldrich Co.) in 95 % methanol (Samchun Pure Chemical Co., Pyeongtaek, Korea). After 30 min of reaction with 100 μL of sample and 100 μL of DPPH solution in the dark at 23 ± 2 °C, absorbance was measured at 517 nm. A mixture of equal volumes of the DPPH solution and methanol was used as the blank (Klimczak et al., 2007). The antioxidant capacity was expressed as the percent decline in the absorbance after 1 min, relative to that of the blank, corresponding to the percentage of DPPH that was scavenged. Antioxidant capacity was expressed as ascorbic acid equivalent (AAE) antioxidant capacity (Kim et al., 2002).

Table 1 Effects of atmospheric dielectric barrier discharge cold plasma on P. digitatum disease incidence in mandarins. Samples

Treatment (conditions)

Disease incidence (%)

Untreated Treated

– 26 kV-2 min 26 kV-3 min 26 kV-4 min 27 kV-1 min 27 kV-2 min

97.3 93.3 92.0 88.0 85.0 77.1

± ± ± ± ± ±

5.9a 8.2a 8.4ab 6.4ab 4.8b 4.5c

-: not available. Data represent the mean and standard deviation (n = 30). Means followed by different letters are significantly different (p < 0.05).

to 19.8 ± 1.0 °C; this is only a 2.4 °C increase, indicating that the inhibition of P. digitatum growth occurred non-thermally by cold plasma. Reactive species produced during ADCP treatment oxidize fungal cell walls, causing the cytoplasm to flow out; oxidizes the glycoproteins of the cell (Liang et al., 2012; Gander, 1974); and penetrates the cell to directly react with biomaterials, including DNA, to inhibit fungal growth (Park et al., 2003). A similar reduction in the disease incidence of P. digitatum on mandarins to that observed after treatment at 27 kV for 2 min has been reported with a carnauba wax coating incorporating grapefruit seed extract (Choi et al., 2019). The coating decreased disease incidence by 26 %, with a further reduction exhibited during storage at 25 °C.

2.9.5. Determination of color The color of mandarin peel was measured using a colorimeter (Minolta Chroma Meter CR-400, Minolta Camera Co., Osaka, Japan) with Illuminate D65, a 10° standard observer, and the CIELAB scale [CIE L* (lightness), a* (redness), and b* (yellowness)], which was calibrated using a white tile (Minolta calibration plate No.14233126, Y = 87.4, x = 0.3174, and y = 0.3353). Four sides of untreated, single ADCPtreated, and CaO-ADCP-treated mandarins were measured. Both L* and hue angle (0° = red hue and 90° = yellow hue), calculated by tan−1(CIE b*/CIE a*) (Siddiq et al., 2011), were used to determine the color.

3.2. Determination of the mandarin washing method based on the efficacy of P. digitatum disease incidence reduction The P. digitatum disease incidences of untreated, CaO-ADCP-treated, FS-ADCP-treated, and CaO-FS-ADCP-treated mandarins were 97.3 ± 6.0 %, 64.3 ± 11.3 %, 87.1 ± 11.1 %, and 80.0 ± 5.8 %, respectively (Fig. 1). CaO-ADCP and CaO-FS-ADCP treatments significantly decreased the growth of P. digitatum (p < 0.05). In particular, CaO-ADCP treatment was the most effective at inhibiting P. digitatum growth (p < 0.05) (Fig. 1). Therefore, CaO solution only was selected as

2.10. Statistical analysis P. digitatum disease incidence experiments were performed in duplicate, with each replicate including 30 fruits from each sample group. The storage study was repeated 4 times. For each replication, the numbers of measurements for CO2 generation, SSC, pH, ascorbic acid concentration, TPC, antioxidant capacity, and color were 3, 5, 3, 2, 3, 2, and 20, respectively, for each sample group. For each replication, 5 fruit were used per treatment to measure CO2 generation and color, while 3 fruit were used to determine SSC, pH, ascorbic acid concentration, TPC, and antioxidant capacity. Differences in the average values were analyzed using one-way analysis of variance using SPSS (Ver. 24, SPSS Inc., Chicago., IL, USA), and Tukey’ s multiple range test was conducted in cases showing a significant difference. 3. Results and discussion 3.1. Determination of ADCP treatment conditions based on the efficacy of P. digitatum disease incidence reduction Treatment using voltages under 26 kV did not inactivate P. digitatum, while over 2 min of treatment at 27 kV led to exterior damage after treatment, such as bruising or browning of the mandarin surface (data not shown). Treatment at 27 kV for 2 min significantly decreased the disease incidence of P. digitatum compared to that without treatment, without exterior damage to the mandarin (Table 1); thus, this was determined to be the optimum treatment condition. The disease incidence decreased with increases in treatment voltage and time, as could be observed in the treatments performed for 2 min and those at 27 kV, respectively. ADCP treatment at 27 kV for 2 min only minimally changed the surface temperature of the mandarin, from 17.4 ± 1.0 °C

Fig. 1. Effects of washing treatments followed by atmospheric dielectric barrier discharge cold plasma (ADCP) treatment at 27 kV for 2 min on the inhibition of Penicillium digitatum on the mandarin peel. Each point represents a mean value of 30 measurements. Error bars denote standard deviations. ADCP-treated: samples treated with ADCP without washing, CaO-ADCP-treated: samples treated with ADCP after washing with 0.2 % highly activated calcium oxide (CaO) solution, FS-ADCP-treated: samples treated with ADCP after washing with 0.5 % fumaric acid in slightly acidic electrolyzed water (FS) solution, CaOFS-ADCP-treated: samples treated with ADCP after washing with CaO solution and then FS solution. Different letters indicate significant differences among groups (p < 0.05). 4

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enhanced through engineering advances (Min et al., 2016), including the development of cold plasma treatment systems that deliver more energy to mandarins. The natural, non-thermal, and in-package nature of the current intervention technology warrants such engineering development. It should be noted that P. digitatum growth did not occur on any inoculated mandarins following storage at 4 °C for 35 days (data not shown). This could have been because of the low temperature. Unlike other Penicillium spp., P. digitatum has difficulty growing at temperatures below 10 °C (Jhalegar et al., 2014). Thus, the effects of ADCP and CaO-ADCP treatments on P. digitatum growth during storage at 4 °C could not be investigated in this study.

the washing solution for the integrated treatment and was used for washing mandarins when preparing fruit for the subsequent sensory and storage analyses. The disease incidence of CaO-ADCP-treated mandarins was significantly lower than that of ADCP only-treated mandarins, indicating that CaO washing was effective in inhibiting the growth of P. digitatum. Washing with CaO solution may remove P. digitatum from the mandarin surface due to the high pH (pH: 12.3 ± 0.1) of the solution (Sawai and Yoshikawa, 2004). Additionally, CaO may directly inactivate P. digitatum. The calcium cation derived from CaO destabilizes the microbial cell membrane by ion exchange and the formation of transmembrane pores and modifies the function of the membrane by altering the concentrations of sodium and potassium ions in the cell (Sawai and Yoshikawa, 2004; Xing et al., 2013). Similar to this study, Tango et al. (2017) used an FS solution in SAEW for washing apples and tomatoes inoculated with E. coli O157:H7 or L. monocytogenes for 3 min. This reduced E. coli O157:H7 and L. monocytogenes on apples by 3.1 and 3.0 log CFU fruit−1, respectively, and on tomatoes by 3.8 and 3.6 log CFU fruit−1, respectively. However, in this study, FS solution did not effectively inhibit the growth of P. digitatum on mandarins. This could be due to the fact that the pH of the FS solution was ∼4.4, which is within the optimum growth range of P. digitatum (pH 3–6) (Gross and Robbins, 2000) and insufficiently low for P. digitatum inhibition. The inactivation effect of CaO-FS-ADCP treatment was similar to that of ADCP treatment (p > 0.05), which could reflect the neutralization of the CaO solution by the FS solution. The pH of the mandarin surface immediately after CaO-FS washing was actually 5.0 ± 0.1. Using SEM, the surfaces of untreated, ADCP-treated, and CaOADCP-treated mandarin peels were observed (Fig. 2). ADCP-treated and CaO-ADCP-treated mandarin peels did not exhibit any structural differences, but a partial lamination layer was observed on the CaO-ADCPtreated peel (Fig. 2C), which may have been due to calcium deposit. Therefore, SEM images support the hypothesis that calcium residues on the mandarin surface contributed to inhibiting the growth of P. digitatum. Moreover, the peel surfaces of untreated and ADCP-treated mandarins did not exhibit any structural differences, implying that the conditions for ADCP treatment used in this study did not cause etching or changes in roughness of the mandarin peel surface that may be induced by cold plasma treatment (Song et al., 2016). Alternatively, the mandarins used in the study may have possessed sturdy flavedo cells, as susceptibility to injury is known to depend on maturity and cultivar (Montesinos-Herrero et al., 2009). The efficacy of CaO-ADCP treatment at inhibiting the growth of P. digitatum obtained in this study may not be considered substantial. Nonetheless, the current study, performed with a first-generation prototype for cold plasma treatment, provides a foundation for further research on the development of cold plasma-combined microbial decontamination systems. The efficacy of microbial inhibition could be

3.3. Sensory evaluation Preferences for the appearance of untreated, ADCP-treated, and CaO-ADCP-treated mandarins were 5.3 ± 1.3, 5.4 ± 1.4, and 5.2 ± 1.7, respectively, indicating no significant difference according to treatment method (p > 0.05). However, the glossiness of the CaOADCP-treated mandarins was 4.6 ± 1.2, indicating a lower preference (p < 0.05) compared to that for untreated (6.0 ± 1.3) and single ADCP-treated mandarins (5.6 ± 1.4). The lower preferences for the glossiness of the CaO-ADCP-treated mandarin may reflect that the CaO deposited on the mandarin peel surface (Fig. 2) negatively affected its glossiness.

3.4. Storage tests 3.4.1. CO2 generation CO2 concentrations in the headspaces of plastic containers, which are indicative of the respiration rate of the fruit in the containers, were analyzed as CO2 generation (Won and Min, 2018). Immediately after treatment (day 0), CO2 generation by untreated, single ADCP-treated, and CaO-ADCP-treated mandarins did not exhibit any significant differences (p > 0.05). Furthermore, during storage at 4 °C and 25 °C, CO2 generation by ADCP-treated mandarins did not differ from that of untreated mandarins, suggesting that ADCP treatment did not alter the respiration rate of mandarins. When produce undergoes physiological stress due to damage, its respiration rate increases (Rico et al., 2007). Therefore, ADCP treatment at the conditions used in this study appeared not to induce noticeable physiological stress in the mandarins. Comparison of the CO2 generation of untreated or ADCP-treated mandarins with that of CaO-ADCP-treated mandarins at 4 °C storage demonstrated that CaO-ADCP treatment delayed the increase in CO2 generation. Calcium adsorbed on the mandarin surface following CaO washing (Fig. 2) might reduce the respiratory rate of mandarins stored at 4 °C. However, this effect was not sufficient to inhibit active respiration, as observed at 25 °C.

Fig. 2. Scanning electron microscopy (SEM) images of the peel surfaces of mandarins not washed with an antimicrobial solution and untreated with atmospheric dielectric barrier discharge cold plasma (ADCP) (A), not washed with an antimicrobial solution but treated with ADCP at 27 kV for 2 min (B), and treated with ADCP at 27 kV for 2 min after washing with a 0.2 % highly activated calcium oxide solution (C). 5

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Table 2 Effects of atmospheric dielectric barrier discharge cold plasma (ADCP) treatment at 27 kV for 2 min, with and without washing with 0.2 % highly activated calcium oxide solution, on the total soluble solid content (%) and pH of mandarin flesh during storage at 4 °C and 25 °C. Storage temperature (˚C)

4

25

Storage time (d) 0 7 14 21 28 35 0 3 7 10 14

Total soluble solid contents (%)

pH

Untreated

ADCP- treated

CaO-ADCP- treated

12.3 ± 0.1Ca 12.3 ± 0.1BCa 12.4 ± 0.1Ba 12.7 ± 0.2 Aa 12.7 ± 0.1 Aa 12.8 ± 0.1 Aa 12.3 ± 0.1Ca 12.5 ± 0.1ABa 12.4 ± 0.1ABCa 12.4 ± 0.3BCb 12.6 ± 0.4Ab

12.3 ± 0.1Ca 12.3 ± 0.1BCa 12.4 ± 0.1BCa 12.4 ± 0.1Bb 12.7 ± 0.1Aa 12.8 ± 0.1Aa 12.3 ± 0.1Da 12.4 ± 0.1BCa 12.5 ± 0.1Ba 12.4 ± 0.1CDb 13.0 ± 0.1Aa

12.3 ± 0.1Aa 12.2 ± 0.3Aa 12.4 ± 0.1Aa 12.5 ± 0.1Ab 12.5 ± 0.1Ab 12.5 ± 0.1Ab 12.3 ± 0.1Ba 12.2 ± 0.1Bb 12.6 ± 0.1Aa 12.6 ± 0.4Aa 12.6 ± 0.1Ab

Untreated 3.5 ± 0.1 Aa 3.5 ± 0.1 Aa 3.5 ± 0.1 Aa 3.6 ± 0.1 Aa 3.5 ± 0.1 Aa 3.5 ± 0.1 Aa 3.5 ± 0.1Ca 3.6 ± 0.1BCa 3.6 ± 0.0Ba 3.7 ± 0.1Aa 3.7 ± 0.1Aa

ADCP- treated

CaO-ADCP- treated

3.5 ± 0.1 Aa 3.6 ± 0.1 Aa 3.6 ± 0.1 Aa 3.6 ± 0.1 Aa 3.5 ± 0.1 Aa 3.5 ± 0.1 Aa 3.5 ± 0.1Ca 3.6 ± 0.1Ba 3.7 ± 0.1Ba 3.8 ± 0.1Aa 3.8 ± 0.0Aa

3.6 ± 0.1 Aa 3.6 ± 0.1 Aa 3.5 ± 0.1 Aa 3.5 ± 0.1 Aa 3.5 ± 0.1 Aa 3.5 ± 0.1 Aa 3.6 ± 0.1Ca 3.6 ± 0.1BCa 3.6 ± 0.1Ba 3.8 ± 0.1Aa 3.7 ± 0.1Aa

ADCP-treated: mandarin samples treated with ADCP without washing with an antimicrobial solution. CaO-ADCP-treated: samples treated with ADCP after washing with a 0.2 % highly activated calcium oxide aqueous solution. Data represent the mean and standard deviation (n = 8). Means followed by the same lowercase letter within a row are not significantly different between treatments in the same property, while those followed by the same uppercase letter within a column are not significantly different between samples stored at each temperature (p < 0.05).

25 °C, no significant differences among samples were observed on the same storage day (p > 0.05). The increase in fruit pH during 25 °C storage may be due to fruit respiration, which leads to metabolism that decreases the organic acid in the fruit (Dávila-Aviña et al., 2011; Yang et al., 2006). An increase in the mandarin reparation rate at 25 °C storage compared to that at 4 °C storage (Fig. 3) may be the reason for the increase in mandarin pH at 25 °C. The results of this study show that the ADCP and CaO-ADCP treatments did not affect the pH of the mandarin flesh. Storage at 4 °C and 25 °C after microwave-powered cold plasma treatment (900 W, 10 min) using nitrogen gas was also shown not to affect the pH of mandarins (Won et al., 2017).

3.4.2. SSC and pH Increases in the SSC with increasing storage time were observed in untreated and ADCP-treated mandarins at all storage temperatures (Table 2). However, SSC stayed constant in CaO-ADCP-treated mandarins stored at 4 °C (p > 0.05), and this was significantly lower on the last day of storage (day 35) than that of untreated and ADCP-treated mandarins (p < 0.05). Furthermore, CaO-ADCP-treated mandarins at 25 °C maintained a constant SSC from day 7 (p > 0.05). Respiration and transpiration of fruit promote metabolic activity in mandarins, leading to water loss and resulting in an increase in the sugar content of fruit (Hong et al., 2007). The CaO-ADCP treatment, which delayed mandarin respiration at 4 °C (Fig. 3), could have effectively maintained the SSC at 4 °C. Moreover, since ADCP treatment did not affect the CO2 generation of mandarins, regardless of storage temperature, this treatment might not have affected SSC. The effects of the ADCP and CaO-ADCP treatments on the pH of mandarin flesh during storage are summarized in Table 2. During 4 °C storage, pH values for all samples were within the 3.5–3.6 range, and they were not significantly different from one another (p > 0.05). Although there was a trend for the pH to increase in all samples stored at

3.4.3. Ascorbic acid concentration Ascorbic acid concentrations in all samples were maintained at similar levels at 4 °C were not generally different by treatment method and storage time (Table 3). In contrast, all samples showed a decrease in ascorbic acid concentrations at 25 °C, although the concentrations did not generally differ by treatment method on the same day of sampling (Table 3). Ascorbic acid in mandarins is reported to be degraded by ascorbate oxidase during storage at room temperature (about 25 °C) (Toǧrul and Arslan, 2004; Yahia et al., 2001). ADCP treatment (80 kV) of strawberries also led to a decrease in ascorbic acid concentration, which was explained by oxidation of ascorbic acid by oxidizing reactive species, including ozone, produced during cold plasma treatment (Misra et al., 2015). However, the mandarins used in this study have thicker peels than many other fruit and vegetables, and this could protect the flesh from oxidization by penetrating reactive species produced by cold plasma, which has a low penetration depth (≤ 10 nm) (Morent et al., 2011). 3.4.4. TPC and antioxidant capacity The effects of ADCP and CaO-ADCP treatments on the TPC and antioxidant capacity were evaluated only in the peel, assuming that the treatments would not noticeably affect the properties of the flesh due to the thick peels of mandarins, based on our analysis of ascorbic acid. Regardless of the storage temperature and treatment, TPC increased during storage (Table 3). Immature citrus ripens during storage, increasing the content of the flavonoid hesperidin (Kalt, 2005). The mandarins used in this study may not have been fully mature, which could have led to an increase in TPC during storage. The TPC values for ADCP-treated and CaO-ADCP-treated mandarins were generally lower than those of untreated mandarins at 4 °C and 25 °C (p < 0.05, Table 3), reflecting an inhibition of the increase in TPC

Fig. 3. Effect of atmospheric dielectric barrier discharge cold plasma (ADCP) treatment at 27 kV for 2 min, with and without washing with 0.2 % highly activated calcium oxide solution (CaO), on the CO2 concentration (%) of mandarin fruit during storage at 4 °C and 25 °C. Each point represents a mean value of 12 measurements. Error bars denote standard deviations. ADCPtreated: samples treated with ADCP without washing treatment, CaO-ADCPtreated: samples treated with ADCP after washing with 0.2 % highly activated calcium oxide (CaO) solution. 6

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Table 3 Effects of atmospheric dielectric barrier discharge cold plasma treatment (ADCP) treatment at 27 kV for 2 min, with and without washing with a 0.2 % highly activated calcium oxide solution, on the ascorbic acid concentration of mandarin flesh and the total polyphenol content and antioxidant capacity of mandarin peel during storage at 4 °C and 25 °C. Storage temperature (°C)

4

25

Storage time (d)

0 7 14 21 28 35 0 3 7 10 14

Ascorbic acid (g L−1)

Total phenol contents (GAE mg L−1)

Antioxidant capacity (AAE mg L−1)

Untreated

ADCP-treated

CaO-ADCPtreated

Untreated

ADCP-treated

CaO-ADCP-treated

Untreated

ADCP-treated

CaO-ADCP-treated

2.2 ± 0.2ABa 2.0 ± 0.1Ab 2.4 ± 0.1ABa 2.3 ± 0.2ABa 2.5 ± 0.1Ba 2.4 ± 0.3ABa 2.2 ± 0.2Aa 1.7 ± 0.1Ba 1.3 ± 0.1Ca 1.3 ± 0.1Ca 1.3 ± 0.1Ca

2.0 ± 0.1Ba 2.4 ± 0.1Aa 2.4 ± 0.1Aa 2.2 ± 0.1ABa 2.2 ± 0.2ABb 2.4 ± 0.1Aa 2.0 ± 0.1Aa 1.4 ± 0.1Bb 1.3 ± 0.1Ba 1.3 ± 0.1Ba 1.4 ± 0.1Ba

2.0 ± 0.1Aa 2.4 ± 0.3Aab 2.2 ± 0.2Aa 2.3 ± 0.3Aa 2.3 ± 0.1Ab 2.4 ± 0.2 Aa 2.0 ± 0.1Aa 1.3 ± 0.1Bb 1.4 ± 0.1Ba 1.4 ± 0.1Ba 1.4 ± 0.1Ba

35.8 ± 2.6 Da 51.3 ± 4.7Ca 52.1 ± 4.3BCa 58.7 ± 0.6ABa 63.6 ± 1.5Aa 63.8 ± 3.4Aa 35.8 ± 2.6 Ca 54.8 ± 1.8Ba 56.3 ± 3.9ABa 58.3 ± 2.1ABa 60.2 ± 2.7Aa

36.3 ± 0.6Da 47.3 ± 1.5Cab 50.5 ± 1.3BCa 51.3 ± 1.2BCb 52.8 ± 3.2ABb 56.3 ± 1.5Ab 36.3 ± 0.6Da 45.2 ± 3.0Cb 48.7 ± 1.5BCb 51.7 ± 2.3Bb 59.2 ± 2.3Aa

36.4 ± 2.1Ca 40.7 ± 1.5BCb 49.0 ± 1.0ABa 47.0 ± 7.0ABb 49.7 ± 5.1ABb 52.8 ± 1.5Ab 36.4 ± 2.1Ba 36.2 ± 7.3Bc 43.0 ± 2.6ABb 47.7 ± 0.6Ab 48.7 ± 0.6Ab

225.0 ± 13.2Aa 218.3 ± 16.7Aa 232.0 ± 8.7Aa 238.8 ± 13.3Aa 230.0 ± 12.4Aa 238.5 ± 15.8Aa 225.0 ± 13.2Aa 237.8 ± 24.8Aa 222.0 ± 14.2Aa 239.3 ± 13.8Aa 223.0 ± 14.4Aa

221.7 ± 21.1Aa 227.8 ± 24.3Aa 232.8 ± 5.9Aa 225.3 ± 24.8Aa 245.0 ± 22.5Aa 219.0 ± 15.6Aa 221.7 ± 21.1Aa 218.2 ± 19.1Aa 234.3 ± 16.0Aa 238.3 ± 18.3Aa 213.0 ± 4.8Aa

213.5 ± 10.3Aa 229.8 ± 13.3Aa 216.5 ± 2.1Aa 211.0 ± 14.1Aa 230.7 ± 24.6Aa 253.5 ± 6.4Aa 213.5 ± 10.3Aa 238.5 ± 26.2Aa 235.8 ± 18.3Aa 226.8 ± 7.7Aa 237.5 ± 7.8Aa

ADCP-treated: mandarin samples treated with ADCP without washing with an antimicrobial solution. CaO-ADCP-treated: samples treated with ADCP after washing with a 0.2 % highly activated calcium oxide aqueous solution. Data represent the mean and standard deviation (n = 8). Means followed by the same lowercase letter within a row are not significantly different between treatments in the same property, while those followed by the same uppercase letter within a column are not significantly different between samples stored at each temperature (p < 0.05).

general, a decrease in mandarin lightness during storage can be explained by a loss of moisture due to an increased respiratory rate (Hong et al., 2007). Therefore, the lightness measurement results indirectly reflect the fact that the ADCP and CaO-ADCP treatments did not increase the mandarin respiration rate, as previously demonstrated experimentally (Fig. 3). The hue angle values of the mandarin samples were 74–81° during storage and did not significantly differ by treatment method at either temperature (p > 0.05). Hue angle values smaller than 90° indicate yellow color (Nasrin et al., 2018). Thus, the results show that the yellowness of mandarins was not affected by either treatment. The hue angle, however, generally decreased with storage time at both temperatures. The reason for the decrease in yellowness during storage could be the degradation of carotenoids and chlorophyll in the mandarin peel (Plaza et al., 2004). A decrease in the hue angle of mandarins during storage has been previously reported (Barry and Wyk, 2006).

by those treatments. Furthermore, at 25 °C, the TPC of CaO-ADCPtreated fruit was even lower than that of ADCP-treated fruit on day 14 (the last day of storage) (p < 0.05, Table 3), indicating the superior efficacy of CaO-ADCP treatment at inhibiting the TPC increase. A delay in the rate of the TPC increase in mandarin peel by ADCP and CaO solution washing could be caused by the nitrogen oxides produced during ADCP treatment and the calcium from the CaO solution, both of which penetrate cells in the peel, inhibiting the activity of phenylalanine ammonia-lyase (PAL), which is involved in the biosynthesis of phenolic compounds (Lichanporn and Techavuthiporn, 2013; Misra et al., 2016; Tomás-Barberán et al., 1997). Furthermore, due to the fact that calcium deposition on the surface can reduce the cell membrane permeability, thereby reducing oxygen transportation (Ferguson, 1984), calcium fortification by CaO solution washing is potentially beneficial for retarding the disassembly of the cell wall structure and thus extending the shelf life of mandarins by decreasing the activity of enzymes such as pectin methylesterase and polygalacturonase that require oxygen (Deytieux-Belleau et al., 2008). The antioxidant capacities of the mandarin peels did not significantly differ among treatment groups and remained constant, regardless of storage temperature (p > 0.05). Although ADCP and CaOADCP treatments inhibited the TPC increase in mandarin peels during storage, they did not affect the antioxidant capacity of mandarins. In fact, the correlations between the TPCs and antioxidant capacities of mandarins at both 4 °C and 25 °C were not strong (p > 0.05), with R2 values of 0.0 and 0.1 for the correlations at 4 °C and 25 °C, respectively. Bioactive compounds that affect the antioxidant capacity of the mandarin peel include vitamins, phenols, flavonoids, carotenoids, limonoids, and coumarins (Goldenberg et al., 2018; Zhang et al., 2011). The results of this study suggest that ADCP and CaO-ADCP treatments do not affect the overall concentrations of such antioxidant compounds.

4. Conclusions In this study, we developed a microbial decontamination system integrating antimicrobial washing with CaO solution and a postpackaging intervention treatment using cold plasma (ADCP). CaOADCP treatment more effectively inhibited P. digitatum growth on mandarins than FS-ADCP treatment or CaO-FS-ADCP treatment, and thus, CaO solution washing was determined to be the most appropriate washing method to be integrated with ADCP treatment. CaO-ADCPtreated mandarins showed lower preference scores in terms of glossiness than untreated mandarins, which could be due to the presence of adsorbed calcium oxide on the mandarin surface. ADCP treatment generally did not alter the appearance or respiration rate of mandarins; the SSC, pH, or ascorbic acid concentration of the flesh; or the antioxidant capacity or color of the mandarin peel at 4 or 25 °C. The lack of an effect of ADCP treatment on mandarin respiration likely led to the lack of any effect on SSC or peel color. Furthermore, CaO-ADCP treatment did not affect pH, ascorbic acid concentration, antioxidant capacity, or color, while inhibiting a TPC increase, during storage at 4 or 25 °C and effectively delaying increases in mandarin respiration and SSC at 4 °C. This suggests that CaO adsorbed on the mandarin surface inhibited an increase in the respiration rate, which in turn maintained a constant SSC. The results of this study suggest that a microbial decontamination system integrating CaO washing and ADCP treatment does not negatively affect the quality properties of mandarin, while

3.4.5. Color The individual color parameters of untreated, ADCP-treated, and CaO-ADCP-treated mandarins were not significantly different on each day of storage at 4 °C or 25 °C (p > 0.05, Table 4), indicating that neither ADCP nor CaO-ADCP treatment affected mandarin color, either immediately after treatment or during storage. The potential bleaching effect of ADCP treatment due to the presence of ozone in the package (Misra et al., 2014) was not observed in the current study. The lightness (L*) of all samples decreased with increasing storage time, regardless of the storage temperature or treatment (p < 0.05). In 7

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Table 4 Effects of atmospheric dielectric barrier discharge cold plasma (ADCP) treatment at 27 kV for 2 min, with and without washing with a 0.2 % highly activated calcium oxide solution, on the color of whole mandarins during storage at 4 °C and 25 °C. Storage temperature (°C)

4

25

Storage time (d)

L*

0 7 14 21 28 35 0 3 7 10 14

72.7 71.4 70.3 69.9 70.1 69.9 72.7 71.9 70.7 70.0 69.8

Hue angle

Untreated ± ± ± ± ± ± ± ± ± ± ±

1.3Aa 0.8Ba 0.8Ca 1.0Ca 0.8Ca 1.0Ca 1.3Aa 1.2Aa 1.5Ba 1.9BCa 1.5Ca

ADCP- treated 72.2 70.9 70.3 70.2 70.3 70.4 72.2 71.8 70.1 69.7 69.7

± ± ± ± ± ± ± ± ± ± ±

1.1Aa 1.6Ba 1.8Ba 1.8Ba 1.3Ba 1.6Ba 1.1Aa 1.1Aa 1.9Ba 1.8Ba 1.4Ba

CaO-ADCP- treated 72.6 70.7 70.3 70.2 70.3 70.1 72.6 72.2 70.6 70.3 69.8

± ± ± ± ± ± ± ± ± ± ±

1.1Aa 1.7Ba 1.3Ba 1.3Ba 1.3Ba 1.3Ba 1.1Aa 1.0Aa 1.0Ba 1.3Ba 1.7Ba

Untreated 80.9 79.7 77.8 78.3 78.1 77.0 80.9 77.5 76.5 75.3 75.8

± ± ± ± ± ± ± ± ± ± ±

4.7Aa 5.7ABa 4.4BCa 4.9BCa 3.5BCa 3.3Ca 4.7Aa 3.8Ba 5.2BCa 5.2BCa 4.8Ca

ADCP- treated 81.0 79.3 77.7 77.8 78.4 78.1 81.0 77.2 76.8 75.8 75.3

± ± ± ± ± ± ± ± ± ± ±

4.9Aa 4.3ABa 3.5Ba 4.3Ba 3.8Ba 3.9Ba 4.9Aa 3.3Ba 4.5BCa 3.3BCa 3.9Ca

CaO-ADCP- treated 80.9 79.4 77.1 77.1 78.9 78.0 80.9 76.9 76.5 74.9 74.3

± ± ± ± ± ± ± ± ± ± ±

4.5Aa 4.7ABa 3.6Ca 4.5Ca 4.1BCa 4.2BCa 4.5Aa 3.3Ba 3.7BCa 2.9CDa 3.2Da

ADCP-treated: mandarin samples treated with ADCP without washing with an antimicrobial solution. CaO-ADCP-treated: samples treated with ADCP after washing with a 0.2 % highly activated calcium oxide aqueous solution. Data represent the mean and standard deviation (n = 20). Means followed by the same lowercase letter within a row are not significantly different between treatments in the same property, while those followed by the same uppercase letter within a column are not significantly different between samples stored at each temperature (p < 0.05).

effectively inhibiting the growth of P. digitatum. Thus, the system shows potential as a technology to enhance mandarin preservation.

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CRediT authorship contribution statement In Hee Bang: Methodology, Investigation, Formal analysis, Writing - original draft. Eun Song Lee: Investigation, Formal analysis, Writing review & editing. Ho Seon Lee: Writing - review & editing, Formal analysis. Sea C. Min: Conceptualization, Funding acquisition, Methodology, Writing - review & editing, Supervision. Declaration of Competing Interest The authors declare no competing financial interests. Acknowledgements This research was supported by the collaborative R&BD program (2019) of Agency for Korea National Food Cluster (AnFC) and by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through High Value-added Food Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (318026-03). References Barry, G.H., Wyk, A.A., 2006. Low-temperature cold shock may induce rind colour development of ‘Nules Clementine’ mandarin (Citrus reticulata Blanco) fruit. Postharvest Biol. Technol. 40, 82–88. https://doi.org/10.1016/j.postharvbio. Blois, M.S., 1958. Antioxidant determinations by the use of a stable free radical. Nature 181, 1199–1200. https://doi.org/10.1038/1811199a0. Choi, D.S., Park, S.H., Choi, S.R., Kim, J.S., Chun, H.H., 2015. The combined effects of ultraviolet-C irradiation and modified atmosphere packaging for inactivating Salmonella enterica serovar Typhimurium and extending the shelf life of cherry tomatoes during cold storage. Food Packaging Shelf. 3, 19–30. https://doi.org/10. 1016/j.fpsl.2014.10.005. Choi, H.Y., Bang, I.H., Kang, J.H., Min, S.C., 2019. Development of a microbial decontamination system combining washing with highly activated calcium oxide solution and antimicrobial coating for improvement of mandarin storability. J. Food Sci. 84, 2190–2198. https://doi.org/10.1111/1750-3841.14719. Deng, L.Z., Mujumdar, A.S., Pan, Z., Vidyarthi, S.K., Xu, J., Zielinska, M., Xiao, H.W., 2019. Emerging chemical and physical disinfection technologies of fruits and vegetables: a comprehensive review. Crit. Rev. Food Sci. Nutr. 1–28. https://doi.org/10. 1080/10408398.2019.1649633. Deytieux-Belleau, C., Vallet, A., Donèche, B., Geny, L., 2008. Pectin methylesterase and polygalacturonase in the developing grape skin. Plant Physiol. Biochem. 46, 638–646. https://doi.org/10.1016/j.plaphy.2008.04.008. Dávila-Aviña, J.E.D.J., Villa-Rodríguez, J., Cruz-Valenzuela, R., Rodríguez-Armenta, M., Espino-Díaz, M., Ayala-Zavala, J.F., Olivas-Orozco, G.I., Heredia, B., GonzálezAguilar, G., 2011. Effect of edible coagings, storage time and maturity stage on

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