Comparative stability and efficacy of selected chlorine-based biocides against Escherichia coli in planktonic and biofilm states

Accepted Manuscript Comparative stability and efficacy of selected chlorine-based biocides against Escherichia coli in planktonic and biofilm states

Ana Meireles, Carla Ferreira, Luís Melo, Manuel Simões PII: DOI: Reference:

S0963-9969(17)30599-9 doi: 10.1016/j.foodres.2017.09.033 FRIN 6982

To appear in:

Food Research International

Received date: Revised date: Accepted date:

18 July 2017 7 September 2017 9 September 2017

Please cite this article as: Ana Meireles, Carla Ferreira, Luís Melo, Manuel Simões , Comparative stability and efficacy of selected chlorine-based biocides against Escherichia coli in planktonic and biofilm states, Food Research International (2017), doi: 10.1016/ j.foodres.2017.09.033

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ACCEPTED MANUSCRIPT Comparative stability and efficacy of selected chlorine-based biocides

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against Escherichia coli in planktonic and biofilm states

Ana Meireles, Carla Ferreira, Luís Melo, Manuel Simões*

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LEPABE, Department of Chemical Engineering, Faculty of Engineering, University of

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Porto, Rua Dr. Roberto Frias, s/n, 4200-465 Porto, Portugal

*Author to whom correspondence should be addressed: Manuel Simões ([email protected]) 1

ACCEPTED MANUSCRIPT Abstract Microbial contamination is an unavoidable problem in industrial processes. Sodium hypochlorite (SH) is the most common biocide used for industrial disinfection. However, in view of the current societal concerns on environmental and public health aspects, there is a trend to reduce the use of this biocide as it can lead to the formation

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of organochlorinated carcinogenic compounds. In this work the efficacy of SH was

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assessed against Escherichia coli in planktonic and biofilm states and compared with

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three alternative chlorine-based biocides: neutral electrolyzed oxidizing water (NEOW), chlorine dioxide (CD) and sodium dichloroisocyanurate (NaDCC). The planktonic tests

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revealed that SH had the fastest antimicrobial action, NaDCC exhibited the highest antimicrobial rate and NEOW caused the highest antimicrobial effects. Additionally,

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NEOW was the biocide that allowed the highest formation of reactive oxygen species (ROS). In biofilm control, NEOW and CD were the most efficient biocides causing 3.26

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and 3.20 log CFU.cm-2 reduction, respectively. In terms of stability for chlorine

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depletion, NEOW had the longest decay time for chlorine loss (70 days at 5 ºC) and the lowest chlorine loss rate (0.013 ppm.min-1 at 5 ºC). CD and NaDCC had equivalent

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stability. The overall results demonstrated NEOW as a good alternative to SH due to its

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higher antimicrobial effects and lower chlorine depletion over time.

Keywords: Antimicrobial action; Biofilm control; Chlorine dioxide; Neutral electrolyzed oxidizing water; Sodium dichloroisocyanurate; Sodium hypochlorite.

List of Abbreviations CD

Chlorine dioxide

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ACCEPTED MANUSCRIPT CFU

Colony forming units

DCFH-DA Dichloro-dihydro-fluorescein diacetate Extracellular polymeric substances

FC

Free chlorine

HOCl

Hypochlorous acid

MHB

Mueller–Hinton broth

MPV

Minimally processed vegetables plant

NaDCC

Sodium dichloroisocyanurate

NEOW

Neutral electrolyzed oxidizing water

O.D.

Optical density

ORP

Oxidation reduction potential

PS

Polystyrene

RMSE

Root mean square error

ROS

Reactive oxygen species

SH

Sodium hypochlorite

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SS

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EPS

Stainless steel

List of Symbols FC(t)

FC at a determined time

FC0

initial value of the FC

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ACCEPTED MANUSCRIPT Final value of the FC

kCFU

maximum antimicrobial rate (min-1)

kFC

maximum chlorine loss rate (ppm.days-1)

CFU(t)

CFU at a determined time t

CFU0

initial value of CFU

CFUf

final value of CFU

T

time (min)

λCFU

time for antimicrobial action (min)

λFC

time for chlorine loss (days)

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FCf

1. Introduction

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In the fresh vegetables industry microorganisms can contaminate the process water and get easily attached to the produce or to the equipment, forming biofilms (Yaron & Romling, 2014). Microorganisms in biofilms excrete extracellular polymeric substances

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(EPS) that protect the inner cells from stressful conditions allowing them to grow and

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survive, therefore, hampering produce disinfection (Kumar & Anand, 1998). Biofilms can harbor microorganisms able to cause human infections and/or food spoilage (Kumar & Anand, 1998; Martínez-Vaz et al., 2014). Therefore, a minimal but effective dose of biocides has to be maintained during disinfection in order to guarantee the microbiological safety of the final produce (Erickson, 2012; Ge et al., 2013). Sodium hypochlorite (SH) is the most common biocide applied to disinfect tank surfaces, the produce and water (Bott, 2011; Harms & O’Brien, 2010). SH is a very effective biocide, but its activity is reduced in the presence of organic matter (Ramos et 4

ACCEPTED MANUSCRIPT al., 2013). Furthermore, it can lead to the formation of organochlorinated compounds, some of them recognized as carcinogenic (Hahn & Weber, 2014; Van Haute et al., 2013). New chlorine-based decontamination strategies are emerging for the decontamination of fresh products and disinfection of surfaces (Meireles et al., 2016). These include neutral electrolyzed oxidizing water (NEOW), chlorine dioxide (CD) and

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sodium dichloroisocyanurate (NaDCC) (Cheng et al., 2012; Tomás-Callejas et al.,

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2012).

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NEOW has been applied in the food industry and its in situ production is considered an environmentally friendly method as the raw materials necessary are sodium chloride

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and water (Gómez-López et al., 2017; Huang et al., 2008). The production involves an electrolysis chamber with an anode and a cathode physically separated by a membrane

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(Cheng et al., 2012; Demirci & Bialka, 2010; Deza et al., 2003). When electrodialysis occurs two separate streams are formed: one acidic (at the anode) and the other alkaline

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(cathode) (Hricova et al., 2008; Ongeng et al., 2006). NEOW is the combination of

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these two solutions (Cheng et al., 2012). Since this is a neutral solution, it can be applied on equipment surfaces and in the process water to disinfect the produce.

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Furthermore, it does not change the organoleptic properties of the produce (Rico et al., 2008). The use of NEOW minimizes the formation of by-products when compared to

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SH (Ayebah & Hung, 2005; Demirci & Bialka, 2010). Still it has limitations such as the equipment high cost and the need to be placed in a ventilated space due to the generation of Cl2 (Cheng et al., 2012). CD has FDA (Food and Drug Administration) approval (21CFR173.300, 2014), but its application is still being overviewed by the European Union (EU) regulation (Regulation number 1062/2014) (EFSA, 2015b). This biocide penetrates the cell membrane, inhibiting metabolic functions (Joshi et al., 2013). Furthermore, it is as 5

ACCEPTED MANUSCRIPT effective as SH (López-Gálvez et al., 2010), does not form harmful by-products (Rico et al., 2007b), is less corrosive than chlorine, has lower reactivity with organic matter (Ölmez & Kretzschmar, 2009), and can prevent enzymatic browning (Chen et al., 2010). The main drawbacks are its instability (Gómez-López et al., 2009), the pH dependency (Ölmez & Kretzschmar, 2009) and its degradability under sunlight

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exposure (Tomás-Callejas et al., 2012).

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Another emergent biocide is NaDCC. It is usually used in swimming pools and in

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emergency situations in drinking water systems (Clasen & Edmondson, 2006; Clasen et al., 2007). Its mode of action is similar to SH, however, when it is applied, only 50% is

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released as free available chlorine. The other 50% stay “preserved”, being used when the first 50% is depleted (Clasen & Edmondson, 2006; Jain et al., 2010). Additionally it

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is bounded to cyanuric acid that allows the containment of chlorine in a solid state (Clasen & Edmondson, 2006), and it is less susceptible to organic matter interaction

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than SH (McDonnell & Russell, 1999). It has been described as safe (it is used as tablets

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and the spilling is avoided) and stable with a shelf-life of 5 years (Clasen & Edmondson, 2006; Clasen et al., 2007). In 2004 the World Health Organization

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recommended that the daily intake of anhydrous NaDCC from treated water should be between 0 and 2 mg per kg of body weight (Clasen & Edmondson, 2006; Lantagne et

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al., 2010; WHO, 2004). The disadvantage on the use of this biocide is its relatively high cost and the possible production of trihalometanes (Lantagne et al., 2010). The aim of the present work was to compare the efficacy of four chlorine-based biocides (SH, NEOW, CD and NaDCC) on the control of Escherichia coli in both planktonic and sessile states. Pathogenic strains of E. coli are occasionally causative agents of food contamination and foodborne illness outbreaks (Almasoud et al., 2015; Warriner et al., 2009). Diverse studies already demonstrated their ability to form 6

ACCEPTED MANUSCRIPT biofilms on materials used in industrial settings (Deering et al., 2012; Meireles et al. 2015; Sharma et al., 2016). The production of reactive oxygen species (ROS) was assessed to understand the action of the selected biocides. Moreover, the chemical stability of the biocides was

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determined through over time evaluation of the levels of active chlorine.

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2.1. Microorganism and culture conditions

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2. Materials and Methods

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E. coli CECT 434 (purchased from the Spanish Type Culture Collection - CECT) was the microorganism used in this study. This strain was already used as model

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microorganism for antimicrobial tests (Abreu et al., 2014; Borges et al., 2012; Meireles et al., 2015). The microorganism was cultured overnight (15 h) in 100 mL flasks with

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25 mL of Mueller-Hinton broth (MHB, Merck, Germany), incubated at 30 ºC and 120

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rpm (CERTOMAT® BS-1, Sartorius AG, Germany), according to Meireles et al. (2015). E. coli was stored at -80 ± 2 °C in cryovials with doubly concentrated medium

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and 30% (v.v-1) glycerol (Panreac, Spain). It was subcultured in Plate Count Agar (PCA, Merck, Germany) before testing. All media were prepared according to the

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manufacturer instructions and sterilized in an autoclave (Uniclave 88, AJC, Portugal) at 121 °C for 20 min.

2.2. Chemical solutions SH 13% (w.w-1) was obtained from Acros Organics (Belgium). CD at a stock solution of 2 g.L-1 was provided by Loehrke (Germany) and NEOW was produced in an electrolysis chamber (ECAse) developed by the same company. A sterilized saline 7

ACCEPTED MANUSCRIPT solution (2.5 g.L-1 NaCl) was used to produce NEOW with 300 ppm of free chlorine content. The ECAse consumed 26 L.h-1 of brine and the overall flow was divided into 13 L.h-1 of anolyte and 13 L.h-1 of catholyte. NaDCC was acquired from Acros Organics (Belgium). 2’7’-dichlorodihydrofluorescein diacetate (DCFH-DA) ≥ 97% (Sigma Aldrich, USA) was prepared in absolute ethanol (Fisher Scientific, UK)

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(Rastogi et al., 2010).

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2.3. Time kill curves

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The time kill curves were obtained with planktonic cells. The cells were centrifuged (4000 g, 15 min) and washed one time with saline solution (8.5 g.L-1 NaCl). Afterwards,

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the cells were resuspended in NaCl 8.5 g.L-1 to obtain an optical density at 600 nm (O.D.600) of 0.2 ± 0.02 corresponding to 3.9 × 107 CFU (colony forming units).mL-1.

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These cells were centrifuged again (4000 g, 15 min) and NaCl 8.5 g.L-1 was replaced by the biocides (SH, NEOW, CD and NaDCC prepared in NaCl 8.5 g.L-1) at different

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concentrations (20, 50, 80 and 100 ppm of free chlorine). The range of concentrations chosen were based in the values usually applied in the minimally processed vegetables (MPV) industry: 70-90 ppm (Meireles et al., 2017). NaCl 8.5 g.L-1 was used as control.

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These solutions were placed in 96-well flat-bottomed polystyrene (PS) tissue culture

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plates with a lid (Orange Scientific, USA) using a total volume of 200 µL. The plates were incubated (Synergy HT, Biotek Instruments, Inc., USA) with 120 rpm agitation at 30 ⁰C. At defined interval times, samples were collected and the necessary dilutions were performed to determine CFU on PCA using the motion drop method (Reed & Reed, 1948). The Gompertz model (Gompertz, 1825) was used to determine the time kill kinetic parameters related to the over time CFU variation. This kinetic model has already been applied to characterize a biocide kinetic action by Garthright (1991). Equation 1 was applied to determine the theoretical model: 8

ACCEPTED MANUSCRIPT [kCFU ×(λCFU −t)+1]

CFU(t) = CFU0 + (CFUf − CFU0 ) × e−e

(1)

Where CFU(t), CFU0 and CFUf correspond to: the CFU at a defined time t, the initial value of the CFU (the upper asymptote curve), and the CFU final value, respectively; kCFU is the kinetic constant (log CFU.mL-1.min-1) and represents the maximum

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antimicrobial rate, λCFU is the lag time for antimicrobial action (min), t is the time (min). The kinetic parameters (kCFU and λCFU) were determined using the Solver supplement of

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Microsoft Excel 2016. The quality of the model was evaluated through the coefficient of

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determination (R2) and with RMSE (root mean square error) determination.

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2.4. Tests with biofilms

These tests were performed using a glass chemostat biofilm reactor (2 L) under

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conditions similar to Ferreira et al. (2012). A schematic representation of the biofilm reactor setup can be observed in Figure 1. The reactor was inoculated with 500 mL of E.

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coli CECT 434 from an overnight (15 h) culture (5.5 g.L-1 glucose, 2.5 g.L-1 peptone,

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1.25 g.L-1 yeast extract, 1.88 g.L-1 monopotassium phosphate and 2.6 g.L-1 sodium phosphate dibasic) with an initial number of cells of 4 × 108 CFU.mL-1. This volume

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was added to 1.5 L of NaCl 8.5 g.L-1. The feeding process began 2 hours after inoculation. To allow biofilm formation instead of planktonic growth, the reactor was

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continuously fed with 0.2 L.h-1 of a sterile solution with the same medium previously described but 100× diluted (except for monopotassium phosphate and sodium phosphate dibasic). The biofilms were formed at a dilution rate of 0.1 h-1. Fourteen slides (2.0 cm × 2.0 cm × 0.1 cm) of stainless steel (SS) AISI 316 were placed vertically in contact with the bacterial suspension for 5 days. After biofilm formation, the SS slides were transferred to closed sterile flasks where the following conditions were tested: control (NaCl 8.5 g.L-1), SH, NEOW, CD and NaDCC at 50 ppm. The concentration chosen (50 ppm) was based on the requisites of the MPV industry to reduce the concentrations 9

ACCEPTED MANUSCRIPT usually applied (Goodburn & Wallace, 2013; Rico et al., 2007a). The flasks were placed in orbital shakers at 30 ºC, for 20 min (exposure time used in the MPV industry (Meireles et al., 2015)). Afterwards, the slides were subjected to a neutralisation step by diluting the biocides to sub-inhibitory levels (Johnston et al., 2002). For that, the SS slides were inserted in 50 mL sterile tubes with 10 mL of NaCl 8.5 g.L-1. The tubes

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were subjected to vigorous (100% of maximum power) vortex (VWR, Portugal), and

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the necessary dilutions were performed to determine the variation in the number of CFU

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in PCA using the motion drop method (Merck, Germany) plates (Reed & Reed, 1948). 2.5. Reactive oxygen species diacetate

(DCFH-DA)

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Dichloro-dihydro-fluorescein

assay was

used

for

the

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determination of reactive oxygen species (ROS). In this method, DCFH-DA is oxidized by ROS resulting in a fluorescent molecule - 2’7’-dichlorofluorescein (Jambunathan, 2010). Bacteria from overnight growth were centrifuged (4000 g, 15 min) and washed

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one time with NaCl 8.5 g.L-1. Afterwards, bacteria were resuspended in NaCl 8.5 g.L-1 to obtain an O.D.600 of 0.2 ± 0.02. These cells were centrifuged again (4000 g, 15 min) and NaCl 8.5 g.L-1 was replaced by the biocides (SH, NEOW, CD and NaDCC prepared

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in NaCl 8.5 g.L-1) in the concentrations tested (20, 50, 80 and 100 ppm of free chlorine).

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E. coli control (only cells) and biocides control (without cells) were also prepared in NaCl 8.5 g.L-1. Before adding the previous prepared and described solutions to a 96well flat-bottomed PS microplate (Orange Scientific, USA), a solution of DCFH-DA was added to the same 96-well PS microplate in a volume of 20 µL (5 µM as final concentration in 200 µL final volume) (Rastogi et al., 2010). Then, control (only cells), biocide solutions (20, 50, 80 and 100 ppm) and biocides (20, 50, 80 and 100 ppm) with cells were placed in the same microplates (180 µL). The fluorescence was measured (optics position on top) during 30 min with interval times of 2 min at 30 ⁰C using a 10

ACCEPTED MANUSCRIPT Synergy HT fluorescence reader (Biotek Instruments, Inc., USA). Wavelength of 485/20 and 528/20 were used for excitation and emission, respectively (Rosenkranz et al., 1992). 2.6. Chemical stability of biocide solutions

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The stability of the biocidal solutions, i.e. over time depletion of free chlorine, was evaluated with a free chlorine portable meter HI 93701 (Hanna Instruments, England) at

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5, 25 and 30 ⁰C, during 200 days. The four biocides (SH, NEOW, CD and NaDCC)

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were prepared at 100 ppm in a total volume of 100 mL for each temperature.

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The Gompertz model (Gompertz, 1825) was used to determine the kinetic parameters related to the over time variation of free chlorine levels. Equation 2 was applied to

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determine the theoretical model:

[kFC ×(λFC −𝑡)+1]

FC(t) = FC0 + (FCf − FC0 ) × e−e

(2)

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Where FC(t), FC0 and FCf correspond to: the free chlorine (FC) at a determined time,

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the initial value of the FC (the upper asymptote curve), and the FC final value, respectively; kFC is the kinetic constant (ppm.days-1) that represents the maximum

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chlorine loss rate, λFC is the time for chlorine loss (days), t is the time (days). The

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kinetic parameters (kFC and λFC) were determined using the Solver supplement of Microsoft Excel 2016. The quality of the model was evaluated through the coefficient of determination (R2) and with RMSE determination. 2.7. Statistical analysis Three independent assays were performed for each condition tested. The data were analyzed using paired samples t-test from the statistical software SPSS 24.0 (SPSS Inc., Chicago, IL, USA). Statistical calculations were based on a confidence level of ≥ 95% (P < 0.05 was considered statistically significant). 11

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3. Results and Discussion In the fresh-cut vegetables industry, equipment and water disinfection is crucial to control microbial growth and prevent biofilm formation. SH is the biocide commonly

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applied for that purposes (Meireles et al. 2016). However, its use has many drawbacks, namely pH dependency (Ramos et al., 2013), potential production of carcinogenic and

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mutagenic compounds and limited efficacy due to its reaction with organic matter

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(EFSA, 2015a; Parish et al., 2003; Ramos et al., 2013). In fact, its use is prohibited in some European countries (Belgium, Denmark, Germany and The Netherlands) (Fallik,

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2014; Ramos et al., 2013). With the aim to reduce the use of SH in disinfection

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processes, three chlorine-based biocides, NEOW, CD and NaDCC, were evaluated as alternatives.

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Time kill curves of each biocide were determined in order to evaluate their

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antimicrobial effects (Figure 2). Kinetic parameters related to antimicrobial activity were obtained from the time kill curves (Table 1): kCFU that represents the maximum antimicrobial rate, λCFU which is the time needed to exert antimicrobial action and the

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microbial elimination evaluated by the log CFU.mL-1 decrease. From the model curves

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determined, the R2 (close to 1) and RMSE values, the Gompertz model demonstrated to be suitable to describe the antimicrobial activity of the selected biocides. From the biocides tested, SH is the one requiring less time to have antimicrobial action (Fig. 2). This is reinforced by the kinetic data (Table 1) where SH had the shortest λCFU. NEOW follows SH with the shortest time, while NADCC is the biocide requiring higher time to have antimicrobial activity. Additionally, the action of CD and NaDCC was very similar (P > 0.05) as can be seen from Figure 2 and Table 1. Furthermore, by observing the kCFU values it is possible to state that among all the biocides NaDCC is the one with 12

ACCEPTED MANUSCRIPT the highest antimicrobial rates, which means that this biocide is the fastest to have an antimicrobial action. However, despite the fact that NEOW is the biocide with the lowest antimicrobial rates, 120 min after exposure to 80 and 100 ppm it caused the highest log CFU.mL-1 reductions (0.779 and 1.052 at 80 and 100 ppm, respectively). Under these conditions (120 min, 80 and 100 ppm), the log CFU.mL-1 reduction

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obtained was 0.274 and 0.591 for SH; 0.045 and 0.050 for CD; 0.021 and 0.025 for

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NaDCC, for 80 and 100 ppm, respectively. Those values are significantly different (P <

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0.05) from those obtained with NEOW. Therefore, NEOW at 80 ppm had a higher antimicrobial activity than 100 ppm of SH, as it was able to promote higher log

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CFU.mL-1 reduction (1.3 times higher). While, NaDCC was the least effective biocide in reducing E. coli CFU. Summarizing, in planktonic cultures, SH demonstrated to be

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the fastest biocide having antimicrobial action. NaDCC and CD had the highest antimicrobial rates. When NEOW was used, it was possible to achieve the highest log

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CFU.mL-1 reductions, which means that cell growth was controlled in a higher extent by

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NEOW although the rate of antimicrobial action was lower than for the other biocides. Further tests were performed by exposing biofilm cells to the biocides at 50 ppm for 20

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min (Fig. 3). The log CFU.cm-2 reductions were 3.26, 3.20, 2.64 and 2.46 for NEOW, CD, NaDCC and SH, respectively. NEOW and CD caused significantly higher biofilm

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CFU.cm-2 reductions than SH and NADCC (P > 0.05). Previous studies also demonstrated the high efficacy of NEOW and CD on the control of sessile cells. Arevalos-Sánchez et al. (2013) applied 70 ppm NEOW for 3 min against 4 days old Listeria monocytogenes biofilms developed on SS surfaces causing 2 log CFU.cm-2 reduction. These authors also used the same conditions (concentration and time) to test SH and obtained the same log reduction, concluding that NEOW and SH had similar effects on the control of SS-adhered L. monocytogenes. Kim et al. (2001) used 56 ppm

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ACCEPTED MANUSCRIPT NEOW to control 2 days old L. monocytogenes biofilms formed on SS surfaces. These authors observed a reduction of 9 log CFU.cm-2 after 5 min exposure. Kreske et al. (2006) used 200 ppm CD against B. cereus biofilms formed on SS coupons causing 4.42 log CFU reduction. Robbins et al. (2005) used CD at 50 000 ppm for 10 min and obtained 4.14 log CFU.chip-1 reduction of L. monocytogenes biofilms. These authors

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used higher concentrations than the one tested in this study. Therefore, the CFU

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reductions were also higher. The differences in the NEOW and CD biofilm CFU

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reduction between previous reports and the results of the present study are apparently related to the different bacterial species/strain selected for biofilm formation and

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control, as well as to the distinct process conditions (biofilm age, adhesion surface, biocide exposure time). Regarding the biofilm control action of NADCC and SH, a

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comparison of previous studies proposed a lower biofilm control action than NEOW or CD. Block (2004) used 1000 ppm NaDCC to remove Clostridium difficile and

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Bacillus atrophaeus from SS surfaces in a 10 min treatment and obtained reductions of

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1 and 1.5 log CFU, respectively. Ungurs et al. (2011) obtained similar results, 2.19 log CFU reduction of C. difficile biofilms from SS surfaces when applying 1000 ppm

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NaDCC for 20 min. In what concerns SH, Kim et al. (2016) applied 50 ppm SH for 1 min on SS surfaces and achieved only 0.34 log CFU.cm-2 reduction of L.

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monocytogenes. Rossoni and Gaylarde (2000) used SH to remove E coli biofilms from SS surfaces and were able to eliminate 1.77 log cells.cm-2 using 100 ppm SH for 10 min. The formation of ROS was assessed in order to understand the distinct antimicrobial effects of the biocides tested (Fig. 4). ROS such as singlet oxygen, hydrogen peroxide, superoxide and hydroxyl radical are normally produced during cell metabolism (Jambunathan, 2010). However, in some conditions, such as the addition of certain

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ACCEPTED MANUSCRIPT biocides, the ROS formation increases exponentially destroying cellular structure and function (Cabiscol et al., 2000; Rastogi et al., 2010). However, only biocides that have oxygen to react with the cell, producing a superoxide anion that is a precursor and propagator of oxidative chain reactions, promote ROS production (Turrens, 2003). In this study, only SH and NEOW lead to the production of ROS (Fig. 4). No relative

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fluorescence units (RFU) were detected with the use of CD and NaDCC, proposing that

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under the conditions tested those biocides do not induce detectable ROS production.

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Additionally, when comparing the RFU after SH and NEOW treatments, the maximum values obtained for each condition tested were: 68.00 (20 ppm), 510.00 (50 ppm),

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534.00 (80 ppm), 575.00 (100 ppm) for SH and 103.50 (20 ppm), 526.50 (50 ppm), 590.00 (80 ppm), 657.00 (100 ppm) for NEOW. In fact, the RFU values were always

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higher for NEOW (P < 0.05), which justifies the highest ability of this biocide to produce ROS and helps to explain the highest antimicrobial effects of NEOW (Fig. 2

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and Table 1).

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The chemical stability of the four biocides in terms of chlorine depletion rate was measured over time (Fig. 5), allowing the assessment of kinetic parameters (Table 2).

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From the model curves, the R2 (close to 1) and RMSE values, the Gompertz model demonstrated to be suitable for describing the over time biocides decay. When

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comparing the decay time for free chlorine loss, at 5 ⁰C, NEOW was the most stable biocide with a depletion time of 70 days (P < 0.05), while for NaDCC the depletion time was 25 days and for SH and CD it was 0 days. In terms of chlorine loss rate at 5 ⁰C, SH and NaDCC had the highest value (0.025 and 0.027 ppm.min-1, respectively). Heling et al. (2001) already proposed that NaDCC antimicrobial action and effectiveness is similar to SH. At 5 ⁰C, NEOW had the lowest chlorine loss rate (P < 0.05). This is an important feature in the food industry as disinfection procedures are 15

ACCEPTED MANUSCRIPT usually performed at 5 ⁰C (Parish et al., 2003) and the maintenance of a stable and effective biocide concentration is fundamental to control microbial contaminations. Temperatures of 25 and 30 °C were also studied since they are commonly used in laboratory studies to test biocides efficacy (Abreu et al., 2014; Borges et al., 2012; Kim et al., 2001; Meireles et al., 2015). At 25 °C SH demonstrated to be more stable than

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NEOW, CD and NaDCC, with a delay time for chlorine loss of 12 days, which is

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significantly higher than for all the other biocides (P < 0.05). At the same time the chlorine loss rate value for SH was the lowest (0.033 ppm.min-1). CD was the biocide

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with the highest chlorine loss rate with kFC values of 0.281 ppm.min-1 (P < 0.05),

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followed by NaDCC and NEOW that had kFC values of 0.060 and 0.053 ppm.min-1 (P > 0.05), respectively. At 30 ⁰C SH had once again a higher delay time for chlorine loss

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(20 days). However, NEOW had the smallest kFC value (0.098 ppm.min-1) and it was followed by SH (0.117 ppm.min-1), NaDCC (0.123 ppm.min-1) and CD (0.414 ppm.min). Additionally, among all the biocides, CD had the fastest chlorine loss (0 days) for all

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the temperatures tested (P < 0.05).

In conclusion, from the planktonic tests SH demonstrated to be the fastest biocide and

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NaDCC exhibited the highest antimicrobial rate but with a reduced efficacy. CD and

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NaDCC had similar antimicrobial activity. NEOW caused the highest antimicrobial action. The biofilm tests showed that NEOW and CD were the biocides allowing higher CFU reduction (3.26 and 3.20 log CFU.cm-2 for NEOW and CD, respectively). The ROS determination proposed that antimicrobial actions of NEOW and SH were related to ROS formation. Furthermore, in the food industry, the fresh produce is typically disinfected at temperatures around 5 ⁰ C, thus the biocide stability (maintenance of a constant active concentration) at this temperature is important. NEOW had the longest decay time for chlorine loss (70 days) and the smallest chlorine loss rate (0.013 16

ACCEPTED MANUSCRIPT ppm.min-1). Consequently, the overall results propose that SH can be replaced by a greener alternative (NEOW) that is more effective in microbial growth control and has lower over time chlorine decay when used at 5 ºC. In addition and according to the literature (Demirci & Bialka, 2010), no organochlorinated by-products from the use of

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NEOW are produced.

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Acknowledgments

POCI-01-0145-FEDER-006939

(Laboratory for

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(i)

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This work was the result of the project:

Process

Engineering,

Environment, Biotechnology and Energy – UID/EQU/00511/2013) funded the

European

COMPETE2020

Regional -

Development

MA

by

Programa

Fund

Operacional

(ERDF),

through

Competitividade

e

D

Internacionalização (POCI) and by national funds, through FCT - Fundação

(ii)

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para a Ciência e a Tecnologia. European project SusClean (Contract number FP7-KBBE-2011-5, ref.

(iii)

FCT

(iv)

AC

CE

287514).

(Fundação

para

a

Ciência

e

a

Tecnologia)

PhD

grant

SFRH/BD/52624/2014. POMACEA project (INNO INDIGO Partnership Programme)

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ACCEPTED MANUSCRIPT Goodburn, C., & Wallace, C. A. (2013). The microbiological efficacy of decontamination methodologies for fresh produce: A review. Food Control, 32(2), 418427. Hahn, K., & Weber, J. A. (2014). Bleach. In P. Wexler (Ed.), Encyclopedia of Toxicology (3rd ed., pp. 519-521). Oxford: Academic Press.

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Hricova, D., Stephan, R., & Zweifel, C. (2008). Electrolyzed water and its application in the food industry. Journal of Food Protection, 71(9), 1934-1947.

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Huang, Y.-R., Hung, Y.-C., Hsu, S.-Y., Huang, Y.-W., & Hwang, D.-F. (2008). Application of electrolyzed water in the food industry. Food Control, 19(4), 329-345.

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Jain, S., Sahanoon, O. K., Blanton, E., Schmitz, A., Wannemuehler, K. A., Hoekstra, R.

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M., & Quick, R. E. (2010). Sodium dichloroisocyanurate tablets for routine treatment of household drinking water in periurban Ghana: a randomized controlled trial. The

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(ROS), lipid peroxidation, and electrolyte leakage in plants. Methods in Molecular Biology, 639, 292-298. Johnston, M. D., Lambert, R. J., Hanlon, G. W., & Denyer, S. P. (2002). A rapid method for assessing the suitability of quenching agents for individual biocides as well as combinations. Journal of Applied Microbiology, 92(4), 784-789.

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ACCEPTED MANUSCRIPT Joshi, K., Mahendran, R., Alagusundaram, K., Norton, T., & Tiwari, B. K. (2013). Novel disinfectants for fresh produce. Trends in Food Science & Technology, 34(1), 5461. Kim, C., Hung, Y.-C., Brackett, R. E., & Frank, J. F. (2001). Inactivation of Listeria monocytogenes biofilms by electrolyzed oxidizing water. Journal of Food Processing

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and Preservation, 25(2), 91-100.

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Kim, M., Park, S. Y., & Ha, S.-D. (2016). Synergistic effect of a combination of

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ultraviolet–C irradiation and sodium hypochlorite to reduce Listeria monocytogenes biofilms on stainless steel and eggshell surfaces. Food Control, 70, 103-109.

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Kreske, A. C., Ryu, J. H., Pettigrew, C. A., & Beuchat, L. R. (2006). Lethality of chlorine, chlorine dioxide, and a commercial produce sanitizer to Bacillus cereus and

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Pseudomonas in a liquid detergent, on stainless steel, and in biofilm. Journal of Food Protection, 69(11), 2621-2634.

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Kumar, C. G., & Anand, S. K. (1998). Significance of microbial biofilms in food

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industry: a review. International Journal of Food Microbiology, 42(1–2), 9-27. Lantagne, D. S., Cardinali, F., & Blount, B. C. (2010). Disinfection by-product

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formation and mitigation strategies in point-of-use chlorination with sodium dichloroisocyanurate in Tanzania. The American Journal of Tropical Medicine and

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Hygiene, 83(1), 135-143.

López-Gálvez, F., Allende, A., Truchado, P., Martínez-Sánchez, A., Tudela, J. A., Selma, M. V., & Gil, M. I. (2010). Suitability of aqueous chlorine dioxide versus sodium hypochlorite as an effective sanitizer for preserving quality of fresh-cut lettuce while avoiding by-product formation. Postharvest Biology and Technology, 55(1), 5360.

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ACCEPTED MANUSCRIPT Martínez-Vaz, B. M., Fink, R. C., Diez-Gonzalez, F., & Sadowsky, M. J. (2014). Enteric pathogen-plant interactions: molecular connections leading to colonization and growth and implications for food safety. Microbes and Environments, 29(2), 123-135. McDonnell, G., & Russell, A. D. (1999). Antiseptics and disinfectants: Activity, action, and resistance. Clinical Microbiology Reviews, 12(1), 147-179.

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Meireles, A., Fulgêncio, R., Machado, I., Mergulhão, F., Melo, L., & Simões, M.

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(2017). Characterization of the heterotrophic bacteria from a minimally processed

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vegetables plant. LWT - Food Science and Technology, 85(B), 293-300. Meireles, A., Giaouris, E., & Simões, M. (2016). Alternative disinfection methods to

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chlorine for use in the fresh-cut industry. Food Research International, 82, 71-85. Meireles, A., Machado, I., Fulgêncio, R., Mergulhão, F., Melo, L., & Simões, M.

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(2015). Efficacy of antimicrobial combinations to reduce the use of sodium hypochlorite in the control of planktonic and sessile Escherichia coli. Biochemical

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Engineering Journal, 104, 115-122.

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Ölmez, H., & Kretzschmar, U. (2009). Potential alternative disinfection methods for organic fresh-cut industry for minimizing water consumption and environmental

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impact. LWT - Food Science and Technology, 42(3), 686-693. Ongeng, D., Devlieghere, F., Debevere, J., Coosemans, J., & Ryckeboer, J. (2006). The

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efficacy of electrolysed oxidising water for inactivating spoilage microorganisms in process water and on minimally processed vegetables. International Journal of Food Microbiology, 109(3), 187-197. Parish, M. E., Beuchat, L. R., Suslow, T. V., Harris, L. J., Garrett, E. H., Farber, J. N., & Busta, F. F. (2003). Methods to reduce/eliminate pathogens from fresh and fresh-cut produce. Comprehensive Reviews in Food Science and Food Safety, 2, 161-173.

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ACCEPTED MANUSCRIPT Ramos, B., Miller, F. A., Brandão, T. R. S., Teixeira, P., & Silva, C. L. M. (2013). Fresh fruits and vegetables - An overview on applied methodologies to improve its quality and safety. Innovative Food Science and Emerging Technologies, 20, 1-15. Rastogi, R. P., Singh, S. P., Hader, D. P., & Sinha, R. P. (2010). Detection of reactive oxygen species (ROS) by the oxidant-sensing probe 2',7'-dichlorodihydrofluorescein

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Biophysical Research Communications, 397(3), 603-607.

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diacetate in the cyanobacterium Anabaena variabilis PCC 7937. Biochemical and

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measuring the quality of fresh-cut fruit and vegetables: a review. Trends in Food

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Emerging Technologies, 9(1), 37-48. Robbins, J. B., Fisher, C. W., Moltz, A. G., & Martin, S. E. (2005). Elimination of Listeria monocytogenes biofilms by ozone, chlorine, and hydrogen peroxide. Journal of Food Protection, 68(3), 494-498. Rosenkranz, A. R., Schmaldienst, S., Stuhlmeier, K. M., Chen, W., Knapp, W., & Zlabinger, G. J. (1992). A microplate assay for the detection of oxidative products using 2',7'-dichlorofluorescin-diacetate. Journal of Immunological Methods, 156(1), 39-45.

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ACCEPTED MANUSCRIPT Rossoni, E. M. M., & Gaylarde, C. C. (2000). Comparison of sodium hypochlorite and peracetic acid as sanitising agents for stainless steel food processing surfaces using epifluorescence microscopy. International Journal of Food Microbiology, 61(1), 81-85. Sharma, G., Sharma, S., Sharma, P., Chandola, D., Dang, S., Gupta, S., & Gabrani, R. (2016). Escherichia coli biofilm: development and therapeutic strategies. Journal of

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Applied Microbiology, 121(2), 309-319.

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Tomás-Callejas, A., López-Gálvez, F., Sbodio, A., Artés, F., Artés-Hernández, F., &

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Suslow, T. V. (2012). Chlorine dioxide and chlorine effectiveness to prevent Escherichia coli O157:H7 and Salmonella cross-contamination on fresh-cut red chard.

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Food Control, 23(2), 325-332.

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Turrens, J. F. (2003). Mitochondrial formation of reactive oxygen species. The Journal

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(2011). The effectiveness of sodium dichloroisocyanurate treatments against

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Clostridium difficile spores contaminating stainless steel. American Journal of Infection Control, 39(3), 199-205.

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Van Haute, S., Sampers, I., Holvoet, K., & Uyttendaele, M. (2013). Physicochemical quality and chemical safety of chlorine as a reconditioning agent and wash water

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disinfectant for fresh-cut lettuce washing. Applied and Environmental Microbiology, 79(9), 2850-2861.

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ACCEPTED MANUSCRIPT Yaron, S., & Romling, U. (2014). Biofilm formation by enteric pathogens and its role in

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plant colonization and persistence. Microbial Biotechnology, 7(6), 496-516.

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ACCEPTED MANUSCRIPT Figure captions Figure 1 - Schematic representation of the experimental set-up of the agitated reactor used to form the E. coli biofilms. (a) air filter; (b) agitated reactor; (c) SS coupons; (d) air pump; (e) magnetic bar; (f) power supply; (g) magnetic stirrer; (h) peristaltic pump;

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(i) waste container; (j) feed container. Figure 2 – Time kill curves of SH (a), NEOW (b), CD (c) and NaDCC (d) at four

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different concentrations (20, 50, 80 and 100 ppm). The lines represent the Gompertz

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models for the experimental data. To facilitate the observation, not all the points measured are represented. SH – sodium hypochlorite; NEOW – neutral electrolyzed

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oxidizing water; CD – chlorine dioxide; NaDCC - sodium dichloroisocyanurate; □ 20

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ppm; ◊ 50 ppm; Δ 80 ppm; ○ 100 ppm; — 20 ppm model; — 50 ppm model; — 80 ppm model; — 100 ppm model.

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Figure 3 – Log CFU.cm-2 achieved after the application of the four biocides at 50 ppm.

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SH – sodium hypochlorite; NEOW – neutral electrolyzed oxidizing water; CD – chlorine dioxide; NaDCC - sodium dichloroisocyanurate.

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Figure 4 – Relative fluorescence units (RFU) for SH (a), NEOW (b), CD (c) and NaDCC (d) at four different concentrations (20, 50, 80 and 100 ppm) and the control (cells with DCFH-DA). SH – sodium hypochlorite; NEOW – neutral electrolyzed oxidizing water; CD – chlorine dioxide; NaDCC - sodium dichloroisocyanurate; × Control; ■ 20 ppm; ♦ 50 ppm; ▲ 80 ppm; ● 100 ppm. Figure 5 – Chemical stability of SH (a), NEOW (b), CD (c) and NaDCC (d) at three different temperatures (5, 25 and 30 ⁰ C). The lines represent the Gompertz models for

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ACCEPTED MANUSCRIPT the experimental data. SH – sodium hypochlorite; NEOW – neutral electrolyzed oxidizing water; CD – chlorine dioxide; NaDCC - sodium dichloroisocyanurate; ■ 5 °C;

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♦ 25 °C; ▲ 30 °C; — 5 °C model; — 25 °C model; — 30 °C model.

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ACCEPTED MANUSCRIPT List of Tables Table 1 – Kinetic parameters of the Gompertz model for the experimental data of the time kill curves for the selected biocides (SH, NEOW, CD and NaDCC) using four different concentrations (20, 50, 80 and 100 ppm)

CD

-1

R2

RMSE (log CFU.mL-1)

(ppm)

(log CFU.mL .min )

(min)

20

0.027

13.077

50

0.034

2.471

80

0.033

3.316

100

0.030

7.508

0.988

0.200

20

0.032

15.315

0.980

0.024

50

0.030

22.854

0.991

0.101

80

0.031

25.075

0.985

0.316

100

0.038

9.573

0.979

0.517

20

0.025

34.683

0.916

0.190

50

0.042

46.473

0.980

0.008

0.046

58.887

0.954

0.012

0.041

47.405

0.971

0.011

0.041

48.075

0.982

0.015

0.051

59.778

0.911

0.037

0.056

58.282

0.898

0.034

0.061

72.069

0.935

0.025

80 100 20

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NaDCC

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50 80

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0.932

0.030

0.988

0.082

0.988

0.093

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NEOW

λCFU

kCFU

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Concentration

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Biocide

kCFU is the kinetic constant (log CFU.mL-1.min-1) that represents the maximum antimicrobial

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rate, λCFU is the time for antimicrobial action (min), R2 is the coefficient of determination

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obtained with the Gompertz model and RMSE is the root mean square error (log CFU.mL-1).

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ACCEPTED MANUSCRIPT Table 2 – Kinetic parameters of the Gompertz model for the experimental data of the chemical stability evaluation for the selected biocides (SH, NEOW, CD and NaDCC) at

Temperature

kFC (ppm.days-

λFC

(⁰C)

1

)

(days)

5

0.025

0.000

0.886

15.077

25

0.033

11.829

0.976

20.531

30

0.117

20.219

0.986

5

0.013

70.342

0.935

8.548

25

0.053

3.606

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19.484

0.991

16.746

30

0.098

6.337

0.998

6.308

5

0.020

0.000

0.957

19.597

25

0.281

0.000

0.998

6.094

30

0.414

0.000

0.874

34.849

5

0.027

24.785

0.955

21.891

25

0.060

0.000

0.988

14.408

0.000

0.990

12.953

CD

NaDCC

30

0.123

R2

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SH

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Biocide

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three different temperatures (5, 25 and 30 ⁰ C) RMSE (ppm)

kFC is the kinetic constant (ppm.days ) that represents the maximum chlorine loss rate, λFC is the -1

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time for chlorine loss (days), R2 is the coefficient of determination obtained with the Gompertz

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model and RMSE is the root mean square error (ppm).

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ACCEPTED MANUSCRIPT Highlights Sodium hypochlorite had the fastest antimicrobial action



NEOW was the most effective biocide in reducing bacterial counts



NEOW and CD were the most efficient in biofilm removal



NEOW was the most effective and stable biocide

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