Potential of non-thermal N2 plasma-treated buffer (NPB) for inhibiting plant pathogenic bacteria and enhancing food storage

Journal Pre-proof Potential of non-thermal N2 plasma-treated buffer (NPB) for inhibiting plant pathogenic bacteria and enhancing food storage Hyemi Seo, Jisoo Hong, Jiseob Woo, Yoonhee Na, Won ll Choi, Daekyung Sung, Eunpyo Moon PII:

S0023-6438(20)30198-5

DOI:

https://doi.org/10.1016/j.lwt.2020.109210

Reference:

YFSTL 109210

To appear in:

LWT - Food Science and Technology

Received Date: 30 October 2019 Revised Date:

23 February 2020

Accepted Date: 24 February 2020

Please cite this article as: Seo, H., Hong, J., Woo, J., Na, Y., Choi, W.l., Sung, D., Moon, E., Potential of non-thermal N2 plasma-treated buffer (NPB) for inhibiting plant pathogenic bacteria and enhancing food storage, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/j.lwt.2020.109210. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

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19. 12. 20

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Potential of non-thermal N2 plasma-treated buffer (NPB) for inhibiting plant

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pathogenic bacteria and enhancing food storage

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Hyemi Seoa,b, Jisoo Honga,c, Jiseob Woob, Yoonhee Nab, Won ll Choib, Daekyung Sungb,* and

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Eunpyo Moona,*

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a

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World cup-ro, Yeongtong-gu, Suwon, Gyeonggi-do, 16499, Republic of Korea b

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Center for Convergence Bioceramic Materials, Korea Institute of Ceramic Engineering and Technology, 202, Osongsaengmyeong 1-ro, Osong-eup, Heungdeok-gu, Cheongju,

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Department of Biological Science, College of Natural Sciences, Ajou University, 206,

Chungbuk, 28160, Republic of Korea c

Microbial Safety Team, National Institute of Agricultural Sciences, Rural Development

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Administration, 300, Nongsaengmyeong-ro, Deokjin-gu, Jeonju, Jeonbuk, 54875, Republic

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of Korea

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* To whom the correspondence should be made

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D.K. Sung; E-mail: [email protected] / Phone: 82-43-913-1511 / Fax: 82-43-913-1597

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E.P. Moon; E-mail: [email protected] / Phone: 82-31-219-2620 / Fax: 82-31-219-1615

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Abstract

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Effective inhibition of microbial growth is essential to increase the yield and shelf life of food.

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In this study, non-thermal N2 plasma-treated buffer (NPB) was tested for its ability to

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inactivate plant pathogenic bacteria. Our results showed that NPB strongly inhibited bacterial

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growth, and 100% inhibitory activity was maintained when up to 10-fold diluted NPB was

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tested. Scavenger assay using various antioxidants against specific reactive oxygen species

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(ROS) and reactive nitrogen species (RNS) showed that certain ROS and RNS in NPB were

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responsible for its strong antimicrobial activity. NPB inhibited the growth of plant pathogens

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on raw produce and was non-toxic to animal cells at all the concentrations tested. In

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conclusion, our study showed that NPB is a safe and effective antimicrobial agent against

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plant pathogenic bacteria, and may have wider applications to enhance crop production and

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the shelf life of stored food.

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Keywords: N2 plasma-treated buffer (NPB), Pathogenic bacteria, Food storage, Antibacterial

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effect, Non-thermal atmospheric pressure plasma (NAPP)

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2

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1. Introduction

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Enhancing food storage has greatly relied on the application of effective disinfection

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processes that inactivate plant pathogenic bacteria (Amit, Uddin, Rahman, Islam, & Khan,

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2017; Moye, Woolston, & Sulakvelidze, 2018). Heat treatment is a traditional method that

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has been used for a long time to produce microbiologically safe food with an extended shelf

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life. However, disadvantages such as the loss of nutrients and a significant decrease in the

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organoleptic quality of certain foods are associated with heat sterilization (Barba, Koubaa, do

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Prado-Silva, Orlien, & Sant’Ana, 2017; Weaver et al., 2014). Non-thermal food processing

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technologies present alternative methods for food preservation and minimizing the negative

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effect on the nutritional profile of preserved food (Morris, Brody, & Wicker, 2007; Ortega-

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Rivas & Salmerón-Ochoa, 2014). Some of these technologies are based on irradiation, high

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pressure, ultraviolet (UV) light, and ozonation (Koutchma, 2008; López et al., 2019; Z. H.

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Zhang, Wang, Zeng, Han, & Brennan, 2018). Nevertheless, these approaches are costly, have

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potential risks, and require controlled and reproducible conditions, limiting their utilization

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on a larger scale (Zhang et al., 2018).

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Non-thermal atmospheric pressure plasma (NAPP) is proposed as a promising tool for food

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preservation owing to its simplicity and ease of use (López et al., 2019; Morris et al., 2007;

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Mishra et al. 2016). Plasma, referred to as the fourth state of matter, is composed of positive

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and negative ions, electrons, excited and neutral atoms, free radicals, UV photons, and

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molecules in the ground and excited electronic states, which contribute to its antimicrobial

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activity (Adamovich et al., 2017; López et al., 2019). Many studies have explored the use of

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gas plasma directly over the food surface to obtain maximum antimicrobial efficiency

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(Adamovich et al., 2017; Misra et al., 2014). However, a few negative effects such as loss of

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color, change in surface topography due to etching, and degradation of bioactive compounds

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were reported following surface treatment with gas plasma (Thirumdas et al., 2018). To

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overcome these problems, plasma-activated water (PAW), also called plasma acid, and

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plasma-activated liquids containing mainly reactive species have been proposed as

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alternatives for food disinfection. Studies have reported the utilization of PAW for bacterial

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inactivation and efficiently controlling bacterial growth (Kim, 2018; Park et al., 2017; Shen et

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al., 2016; Thirumdas et al., 2018; Zhang et al., 2013). Ma et al. reported non-thermal PAW-

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mediated inactivation of plant pathogens on fresh produce such as strawberries (Ma et al.,

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2015), while Xu et al. demonstrated increased microbial inactivation of mushrooms (Xu, Tian,

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Ma, Liu, & Zhang, 2015). We have previously reported the development of N2 plasma-treated

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buffer (NPB) with potent antibiofilm effects against Pseudomonas syringae pv. tomato

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DC3000 (P. syringae pv. tomato DC3000), a plant pathogenic bacterium (Yang, Kim, Seo,

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Bae, & Moon, 2018). Our results demonstrated that the ability of NPB to penetrate through

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the multilayered biofilms was its most important characteristic, making it a reliable control

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agent against biofilm-forming plant pathogens. However, the potential of NPB for food

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preservation due to its antibacterial and antibiofilm effect remains unreported.

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The pathogens used in this study were plant pathogens that cause disease in plants and crops.

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Pseudomonas and Pectobacterium bacterial genera are considered the main bacteria that

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cause damage during storage. Pseudomonas marginalis is an important postharvest pathogen

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that causes soft rot in a wide variety of harvested fruits and vegetables by means of

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pectinolytic enzyme products (pectin lyase and pectate lyase). Pectobacterium carotovorum

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(formerly known as Erwinia carotovora subsp. carotovora) is a gram-negative

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phytopathogen that causes soft-rot disease, wilt, or blackleg in various crops by actively

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secreting plant cell wall-degrading enzymes.

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In this study, we report the inactivation of pathogenic bacteria by non-thermal NPB as a

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method to enhance food storage. Our results showed that the NPB effectively inhibited

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biofilm formation and had an antibacterial effect.

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2. Materials and methods

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2.1. Bacterial culture and biofilm formation

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Bacterial strains P. carotovorum and P. marginalis were used in this study. The two strains

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were cultured in Luria-Bertani (LB) media at 37 °C with shaking for 24 h. For the

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development of biofilm, the exponentially growing bacterial culture was diluted (1:100) in

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fresh LB medium containing 0.5% glucose. An aliquot containing 1 ml of diluted bacterial

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culture was added in the wells of 12-well plates with 12-mm Ø microscope cover glasses and

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incubated statically for 24 h at 37 °C. Subsequently, the medium was removed, and the

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attached cells were washed three times with sterile PBS. Next, the remaining attached cells

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were incubated for 10 min at 60 °C and stained with 0.1% (w/v) crystal violet (CV) for 15

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min at room temperature. Excessive CV stain was removed by washing cells with sterile PBS.

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Finally, ethanol: acetone (95:5, v/v, 0.5 ml) was added to each well to dissolve the CV stain,

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and the absorbance was measured at 570 nm (Ziuzina, Patil, Cullen, Boehm, & Bourkea,

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2014; Ziuzina, Patil, Cullen, Keener, & Bourke, 2014).

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2.2. NPB generation and treatment

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To generate NPB (N2 plasma buffer), 1 ml of PBS was added to the wells of the 12-well plate,

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and the plasma was generated using N2 gas with the nozzle located 1 cm above the PBS

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solution. A total of 250 µl of NPB was added to an equal volume of bacteria-containing PBS

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(108-109 CFU/ml) or directly on the biofilm for 20 min at room temperature. Finally, the

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supernatant was removed, and the pathogenic cells and the biofilm were washed twice with

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PBS and then subjected to further analyses.

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2.3. ROS measurement

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Peroxide solution (H2O2 in Tris buffer, Clarity Western ECL Substrate, Bio-Rad, Hercules,

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CA, USA) and luminol/enhancer solution (acridan solution in dioxane and ethanol, Clarity

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Western ECL Substrate, Bio-Rad) were mixed in a 40:1 ratio. A total of 50 µl of NPB was

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mixed with equal volumes of PBS and the peroxide-luminol/enhancer mixture in a 96-well

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plate. The plate was incubated in the dark for 5 min at room temperature (25 °C), and the

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luminous intensity indicating ROS generation was measured using a Chemi-Doc analyzer

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(Infinity Gel Documentation, Vilber, France).

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2.4. Scavenger assay using antioxidants

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The following specific antioxidants were used in this study: L-histidine (for singlet oxygen),

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mannitol, glutathione (for hydroxyl radical, OH•), NAC (for O free radical), sodium pyruvate

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(for hydrogen peroxide, H2O2), Tiron (for superoxide anion, •O2–), Trolox (for peroxyl radical,

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ROO•), carboxy-PTIO (for nitric oxide, NO), vitamin E (for lipid-soluble peroxyl radical),

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and uric acid (for peroxynitrite anion, ONOO–). The antioxidants were freshly prepared and

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filtered through a 0.22-µm filter before use. For the scavenger assay, P. carotovorum was

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treated with the following freshly prepared antioxidant solution at a working concentration

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(Franco, Panayiotidis, & Cidlowski, 2007): L-histidine (50 mM), mannitol (50 mM),

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glutathione (50 mM), NAC (20 µM), sodium pyruvate (10 mM), Tiron (10 µM), Trolox (100

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µM), carboxy-PTIO (100 µM), vitamin E (100 µM), and uric acid (100 mM). Subsequently,

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NPB was added at an equal volume, and the mixture was incubated for 20 min before making

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serial dilutions. Next, 100-µl aliquots of diluted cultures were spread on Luria agar (LA)

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plates and incubated overnight. Finally, the percentage of viable cells was calculated by

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counting the number of colonies.

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2.5. LIVE/DEAD bacterial viability assay

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The biofilm was grown on 12-mm Ø microscope cover glasses as described before and

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covered with 300 µl of SYTO9/ PI solution (LIVE/DEAD BacLight Bacterial Viability

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Assay Kit, Invitrogen Co., Carlsbad, CA, USA), and incubated at RT for 15 min in the dark.

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The biofilm-containing coverslips were transferred onto a glass slide and observed under a

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fluorescence microscope (Zeiss Axioscope 2, Carl Zeiss, Germany) equipped with a GFP and

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rhodamine filter (X600). SYTO9/PI staining is used to determine the killing efficiency of a

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substance with an unknown number of dead cells. A standard curve was generated, and the

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ratio of SYTO9 (green) to PI (red) fluorescence (G/R ratio) was used to calculate the

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percentage of live/dead cells.

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2.6. NPB antipathogenic assay of fresh produce

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The competent cells of P. carotovorum were mixed with 3 µl of GFP plasmid DNA on ice

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and transformed by electroporation. This was followed by quick addition of 1 ml of LB

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medium and 40 min of incubation to rescue cells from shock. An aliquot of 100 µl of diluted

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cells was spread on ampicillin-containing LA plates and incubated overnight at 28°C.

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Surfaces of sliced white radishes and potatoes were smeared with 100 µl of NPB, and a 100-

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µl aliquot of diluted GFP-tagged bacterial culture was spread on top. The treated vegetables

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were placed in sterilized containers and cultured at 30°C and 85% humidity for 16 h. The

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bacteria-infected areas of sliced vegetables were processed with VISIRAYS (ATTO, Tokyo,

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Japan), an LED illuminator fitted with an SCF-515 filter.

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2.7. Water-soluble tetrazolium salt (WST-1) assay to measure cell viability/cytotoxicity

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The cytotoxic effects of NPB on the mouse embryonic line NIH-3T3 were evaluated by

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WST-1 assay. A suspension of 104 cells/ml was serially diluted, and 100 µl of each dilution

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was added in the wells of a 96-well microplate and incubated in a CO2 incubator for 48 h.

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Next, 10 µl of WST-1 reagent was added to each well and incubated in a CO2 incubator for 4

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h. Finally, the absorbance was measured at 450 nm using a microplate reader. DMSO was

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used as the vehicle control.

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2.8. Statistical analysis

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The data were obtained from three independent experiments and analyzed using analysis of

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variance (ANOVA). The statistical analysis was performed using SPSS 22.0; results with a p-

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value <0.05 were considered statistically significant.

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3. Results and discussion

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3.1. Detection of ROS in NPB and the scavenging effect of various antioxidants

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ROS are biologically active molecules that contribute to bacterial death (Franco et al., 2007).

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We examined the possibility of ROS as the mediator of NPB inhibitory activity against plant

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pathogens. Our results from the Chemi-Doc analyzer showed that ten times higher ROS were

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generated in NPB compared to untreated PBS (Fig. 2A) and could have contributed to the

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observed antibacterial activity. To confirm the role of reactive species as the active mediator

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of the antibacterial effect of NPB, we performed ROS and RNS scavenging assays using

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specific antioxidants. RNS included nitric oxide (NO), which is relatively unreactive, and its 8

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derivative peroxynitrite (ONOO−), a powerful oxidant that can damage many biological

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molecules. ROS and RNS are attractive for their ability to kill pathogenic microorganisms.

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As shown in Fig. 2B, NAC, glutathione, L-histidine, and sodium pyruvate protected P.

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carotovorum 10057 from NPB based on the scavenger assay, while mannitol, uric acid,

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Trolox, and Tiron remained ineffective. Based on the target specificity of glutathione (a

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scavenger of hydroxyl radical), L-histidine (a scavenger of singlet oxygen), and sodium

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pyruvate (a scavenger of hydrogen peroxide), our results clearly indicated that hydroxyl

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radical (OH•), singlet oxygen (O2), and hydrogen peroxide (H2O2) were the major active

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species responsible for microbial inhibition of P. carotovorum 10057 by NPB. Nitric oxide

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(NO), peroxynitrite anion (ONOO–), peroxyl radical (ROO•), and superoxide anion (•O2–) did

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not have any significant effect.

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3.2. Inhibitory effect and efficacy of NPB against pathogenic bacteria under various

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conditions.

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Earlier studies have shown that irrespective of the devices and conditions used for the plasma

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generation, NAPP exhibited the highest antimicrobial activity when N2 was used. Further, the

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overall antimicrobial efficacy of NPB depends on the N2 plasma exposure time, the inhibitory

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effects of NPB were evaluated following varying treatment duration. Our results were

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consistent with the earlier findings and showed that the plasma, resulting from N2 as the gas

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source, showed effective inhibition of the plant pathogens, and the maximum inhibitory

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activity was observed following a 5-min exposure to plasma (Fig. 3A). Using bacteria

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responsible for soft-rot, we showed that a 5-min exposure to NPB had a strong antibacterial

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effect, while a shorter treatment (1 min) did not show any significant inactivation. In contrast,

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a 3-min exposure of P. marginalis to NPB showed a minimal antibacterial effect. The

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inhibitory effects of NPB were evaluated on a liquid bacterial culture (105-109 CFU/ml)

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responsible for soft-rot, and the living cells were counted and visualized (Fig. 3B). Our

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results revealed that the NPB treatment caused strong bacterial inactivation at all four

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bacterial dilutions. NPB resulted in complete inactivation of P. marginalis and P.

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carotovorum 10057, especially at the initial concentration of 109 CFU/ml. Most importantly,

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NPB showed an antimicrobial effect even at the 10-fold dilution used for the assay (Fig. 3C).

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No inhibitory effect of 100-fold diluted NPB on both soft-rot bacteria was observed. Since

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the antimicrobial efficiency depends on the NPB concentration, and our results showed that

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10-fold diluted NPB could retain its maximum antibacterial activity, further studies are

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warranted to standardize the minimum effective concentration for NPB that could be used for

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effective antibacterial function for wider use in agriculture and food storage.

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3.3. LIVE/DEAD bacterial viability assay

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We analyzed the NPB-mediated antibacterial effects against P. carotovorum 10057 using

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fluorescence microscopy (Fig 4) and the LIVE/DEAD staining kit. Pectobacterium is well

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known as a biofilm-forming plant pathogenic bacterium, and effective inhibition of its

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biofilm formation is considered as one of the possible ways to reduce its pathogenicity

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(Brelles-Mariño, 2012; Xiong, Du, Lu, Cao, & Pan, 2011). We evaluated the inhibitory

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activity of NPB against biofilm generation by P. carotovorum 10057. Fluorescence images

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clearly showed strong inhibition of biofilm formation of P. carotovorum 10057 by NPB.

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Similar to the antibacterial effects seen on live bacteria, NPB had a strong inhibitory effect on

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biofilm formation. Our results showed that following treatment with NPB, the number of

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dead cells increased by more than 50% compared to the untreated cells (Fig. 4B). In light of

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the difficulty reported in inhibition of biofilm-forming bacteria due to the protection imparted

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by the biofilm (Traba, Chen, & Liang, 2013; Wei & Ma, 2013), the ability of NPB to inhibit P. 10

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carotovorum is promising. Thus, NPB could be an effective and valuable agent to prolong the

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shelf life of stored food.

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3.4. Pathogenicity test of NPB on fresh produce

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Labeling of bacterial pathogens with fluorescent protein enables researchers to trace bacterial

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pathogen movement and location in host cells. We evaluated the antibacterial effects of NPB

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on vegetable slices to confirm the bactericidal effects of NPB (Fig. 5). Using the fluorescent

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labelled P. carotovorum (GFP-P. carotovorum 10057), our results demonstrated that NPB

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exerted a strong antibacterial effect on the soft-rot-causing bacteria. Further, the potatoes and

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white radishes pre-treated with NPB showed no changes in their original color, aroma,

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hardness, and thickness, and no damage to the tissue was observed. These results suggested

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that NPB treatment could be useful for decontamination of fresh food surfaces without

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causing any adverse effects on the organoleptic properties.

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3.5. Cell viability/cytotoxicity

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The cytotoxicity of NPB was assessed using mouse embryonic cell line NIH-3T3 to verify

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the biocompatibility of NPB for various disinfection applications. As shown in Fig. 6, >80%

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cell viability was observed with NPB concentrations ranging from 1/8 to 1/2, implying that

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NPB, with favorable biocompatibility, could be suitable for disinfection in agriculture and

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food.

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4. Conclusion

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Pathogenic bacteria cause substantial damage to crops and food. Here, we report NPB-

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mediated inhibition of pathogenic bacteria. NPB showed a strong antibacterial effect on the

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viability of pathogens P. marginalis and P. carotovorum 10057 in stored food as well as their

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biofilm-forming activity. Measurement of ROS following treatment with ROS-specific

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scavengers showed that the ROS (H2O2, singlet oxygen, and NO) of NPB were the clear

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mediators of its antimicrobial effects. Furthermore, NPB pretreatment significantly reduced

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the infection of the fresh produce by pathogenic bacteria, while keeping the structure and

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texture of the produce intact. NPB was observed to be non-cytotoxic to animal cells. These

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properties indicate NPB as a reliable antimicrobial agent for producing microbially safe food.

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These findings have practical implications for the use of NPB in food storage and improved

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crop production.

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Funding

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This study was supported by a research grant from the National Research Foundation of

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Korea (NRF), funded by the Ministry of Science, ICT, & Future Planning (NRF-

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2012M3A9B2052872). Financial support was provided by a grant from the Korea Institute of

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Ceramic Engineering and Technology (KICET).

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Declarations of interest

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The authors declare that they have no known competing financial interests or personal

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relationships that could have appeared to influence the work reported in this paper.

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Figure legends

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Figure 1. A schematic diagram depicting the experimental design of generation of N2 plasma-

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treated buffer (NPB) using a micro-plasma jet, generation of GFP-P. carotovorum 10057 as

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plant pathogenic bacterium, and inhibition of bacterial growth by NPB.

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Figure 2. (a) Detection of ROS in NPB and (b) the scavenging effect of various antioxidants

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on antimicrobial activity of NPB.

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Figure 3. Inhibition efficacy of NPB against P. marginalis and P. carotovorum 10057 as per

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(a) N2 plasma exposure time, (b) initial cell concentration, and (c) dilution factor of NPB.

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Figure 4. Fluorescence microscopy images using LIVE/DEAD bacterial viability assay,

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showing the inhibitory effect of NPB on (a) free-living bacteria and (b) biofilm of P.

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carotovorum 10057. Scale bars = 20 µm.

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Figure 5. A fluorescence image showing the growth of GFP-P. carotovorum 10057 (a) after

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NPB treatment and (b) after PBS treatment on the cut surface of potatoes and white radishes.

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Figure 6. Evaluation of NPB cytotoxicity using WST-1 assay and mouse embryonic cell line

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NIH-3T3. NPB remained non-cytotoxic to animal cells at all tested dilutions.

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Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Highlights •

N2 plasma treated buffer (NPB) shows strong inhibition of pathogenic bacteria.

•

Specific ROS in NPB are responsible for the pathogen-inhibition activity.

•

NPB has no significant cytotoxic effect on animal cell lines.

•

NPB holds potential for enhancing the shelf-life of stored food.

Declaration of interests ■ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: