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Journal of Food Science and Technology logoLink to Journal of Food Science and Technology
. 2022 Sep 1;59(12):4863–4869. doi: 10.1007/s13197-022-05574-2

Chlorine dioxide gas mediated inactivation of the biofilm cells of

Seyeon Kim 1, Sanghyun Park 1,✉
PMCID: PMC9579236  PMID: 36276550

Abstract

This study evaluated the chlorine dioxide (ClO2) gas mediated inactivation of the biofilm cells of foodborne pathogens on food contact surfaces. Biofilm cells of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes were developed on stainless steel (SS) and high density polyethylene (HDPE) coupon surfaces, and 5-day-old biofilms were treated with ClO2 gas at 60 and 90% relative humidity (RH) for up to 20 min. With an increase in gas concentration and treatment time, significant differences (p < 0.05) were observed between reduction levels under different RH conditions. Treatment with 50 ppmv of ClO2 gas (60% RH) for 20 min resulted in log reductions from 2.08 to 4.62 and 2.08 to 4.41 of the biofilm cells of three pathogens on SS and HDPE surfaces, respectively. The levels of biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes on SS and HDPE surfaces were reduced to below the detection limit (0.48 log CFU/cm2) within 15, 20, and 20 min, respectively, when exposure to 50 ppmv of ClO2 gas at 90% RH.

Keywords: Biofilm cells, E. coli (all potentially pathogenic types), Salmonella, Listeria, Non-thermal processes

Introduction

Biofilms are characterized as clusters of microorganisms embedded in a self-generated extra polymeric substances (EPS) matrix (Donlan and Costerton 2002; Wei and Ma 2013). EPS is primarily composed of extracellular polysaccharides, proteins, lipids, and biomolecular materials (Cortés et al. 2011). Biofilms can be formed on biotic or abiotic surfaces and create a dynamic environment in which microbial cells utilize available nutrients efficiently (Cortés et al. 2011). Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes are known to form biofilms in various food contact surfaces (Wang et al. 2014; Ban and Kang 2016). The biofilm-forming ability of foodborne pathogens is key for survival in adverse environments as biofilm cells are resistant to common disinfectants typically used in the food industry, as well as stressful environment including pH, UV, heat, and antimicrobials (Srey et al. 2013; Gupta et al. 2016). Also, biofilms of pathogenic bacteria on food contact materials are considered to be a continuous source of cross-contamination in the food industry as they could disintegrate and disperse during the biofilm maturation process (Cappitelli et al. 2014).

Daily cleaning processes are conducted using sanitizers to disinfect food contact surfaces in the food manufacturing plants (Chmielewski and Frank 2003). Several sanitizers have been evaluated to inactivate biofilms of pathogenic bacteria on food contact surfaces. However, treatment with individual sanitizer showed a limited effect on the inactivation of biofilm cells of pathogenic bacteria despite long exposure intervals. Treatment with ozonated water (4 ppm), quaternary ammonium compounds (200 ppm), and chlorine (100 ppm) for 1 min resulted in about 1 log reduction of 7-day-old biofilm cells of L. monocytogenes (Korany et al. 2018; Wang et al. 2018) reported treatment with 20 mg/l of acidic electrolyzed water for 25 min resulted in about 5 log reduction of the biofilm cells of Pseudomonas fluorescens. Biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes were reduced by about 1.0–1.5 log after treatment with benzalkonium chloride (50 ppm), iodophor (50 ppm), and hydrogen peroxide (1%) (Ban and Kang 2016). Several physical processing techniques, such as steam (Ban and Kang 2016), superheated steam (Ban et al. 2014), cold atmospheric plasma (Govaert et al. 2019), ultrasound (Baumann et al. 2009), ultraviolet light-C (Gora et al. 2019), and 405-nm LED (Li et al. 2018) have also been extensively utilized to inactivate biofilm cells of foodborne pathogens. However, these methods could not be applied to decontaminate pathogenic biofilm cells on inaccessible food contact surface. Therefore, it is needed to find effective method for inactivating biofilm cells of foodborne pathogens on various food contact surfaces.

Chlorine dioxide (ClO2), which is a strong oxidizing disinfectant, has a broad antimicrobial spectrum, and its antimicrobial effect against pathogenic bacteria on produce and food contact surfaces has been evaluated (Bhagat et al. 2011; Trinetta et al. 2013; Park and Kang 2015). Gaseous ClO2 is more effective in inactivating pathogenic bacteria than aqueous ClO2 due to its superior ability of penetration (Han et al. 2001). The antimicrobial effect of gaseous ClO2 against biofilm cells of Bacillus cereus and L. monocytogenes on stainless steel surfaces has been evaluated (Vaid et al. 2010; Nam et al. 2014). These studies investigated the antimicrobial effect of ClO2 gas under conditions of high relative humidity (RH) (> 75%). However, no studies are known that have evaluated ClO2 gas mediated inactivation of the biofilm cells of foodborne pathogens relative to RH conditions, gas concentration, and treatment time. It is well known that several factors such as gas concentration, RH, treatment time, and treatment temperature could affect the antimicrobial effect of ClO2 gas, and especially the antimicrobial effect of ClO2 gas increases with increasing RH (Han et al. 2001).

The objective of this study was to evaluate ClO2 gas mediated inactivation of the biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes formed on stainless steel and high density polyethylene surfaces depending on the process conditions.

Materials and methods

Bacterial culture preparation

Three strains of E. coli O157:H7 (ATCC 35,150, ATCC 43,889, ATCC 43,890), S. Typhimurium (ATCC 19,586, ATCC 43,174, DT 104), and L. monocytogenes (ATCC 19,111, ATCC 19,114, ATCC 19,115) were obtained from the bacterial culture collection of the Department of Food Science and Biotechnology at Seoul National University (Seoul, South Korea). Each strain, maintained on frozen stocks (− 80 °C), was streaked onto tryptic soy agar (TSA; MB cell, Seoul, Korea) and incubated for 24 h at 37 °C. Each strain was cultured and incubated separately in 5 mL of tryptic soy broth (TSB; MB cell) for 24 h at 37 °C. Following incubation, bacterial cells from each culture were centrifuged at 4000× g for 20 min at 4 °C and the supernatant was decanted. Cells of each pathogen were resuspended in sterile phosphate-buffered saline (PBS, pH 7.3; LPS solution, Daejeon, Korea) and combined to produce an inoculum, corresponding to approximately 107–108 CFU/mL.

Coupon preparation

The examined food contact surfaces included high density polyethylene (HDPE; Kumjoung EPS, Ansan, Korea) and stainless steel (SS) type 304 with no. 4 finish (Ian industry, Ansan, Korea). HDPE and SS coupons (5 × 2 cm) were rinsed with distilled water, and then sterilized by autoclaving for 15 min at 121 ℃. The coupons were dried in a dry oven at 50 °C and stored at room temperature until further use.

Biofilm formation

Prepared coupons were transferred to sterile 50 mL conical tubes (SPL Life Sciences, Pocheon, Korea) which contain 30 mL of suspensions of each pathogen and incubated at 25 ℃ for 24 h for the attachment of bacterial cells. Each coupon was moved out from the cell suspension with sterile forceps and gently washed in sterile PBS for 5 s to remove unattached planktonic cells. The rinsed coupons were placed into 50 mL conical tubes which contain 30 mL of TSB and then incubated at 25 ℃ for 5 days for biofilm formation.

ClO2 gas treatment

Coupons were removed, rinsed with sterile PBS, and transferred to the gas treatment chamber (length × width × height, 84 cm × 56 cm × 54 cm). ClO2 gas was produced by gas generating system (PurgoFarm Co., Ltd., Hwasung, Korea) and transferred to the gas treatment chamber. A ClO2 gas transmitter (ATi F12, Analytical Technology, U.K.) was used to maintain steady state of ClO2 gas concentrations in the treatment chamber. Coupons were treated with 10, 30, and 50 ppmv of ClO2 gas at 22 ± 1 °C for up to 20 min. The RH of the gas treatment chamber was adjusted to 60 and 90% (± 2%) during treatment using a humidifier. RH was monitored with a thermohygrometer.

Bacterial enumeration

Each coupon treated with ClO2 gas was moved to sterile 50 mL conical tubes which contain 30 mL of sterile PBS and 3 g of sterile glass beads (425–600 μm; Sigma, St. Louis, MO). After vortexing for 2 min, cell suspensions were 10-fold serially diluted in peptone water (PW; MB cell). Undiluted or diluted cell suspensions (100 µL) were spread-plated onto Sorbitol MacConkey agar (SMAC; MB cell), Xylose Lysine Desoxycholate agar (XLD; Difco, Becton, Dickinson, Sparks, MD), and Oxford agar base (OAB; MB cell) with antimicrobial supplement (MB cell) for enumeration of E. coli O157:H7, S. Typhimurium, and L. monocytogenes, respectively. To lower the detection limit, 250 µL of the undiluted sample was spread onto four plates of each medium. SMAC, XLD, and OAB agar plates were incubated at 37 °C for 24–48 h. Colonies were counted after incubation and represented as log CFU/cm2.

Statistical analysis

All experiments were performed in triplicate independently, and obtained data were expressed as means ± standard deviations (SD). Analysis of variance (ANOVA) was performed using Duncan’s multiple range tests in the Statistical Analysis System (SAS Institute, Cary, NC, USA) and differences with p < 0.05 were considered significant.

Results and discussion

Tables 1, 2 and 3 shows the effects of ClO2 gas treatment against biofilm cells of three pathogens on SS surfaces. Initial biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes formed on SS coupons were 6.55–6.96, 8.34–8.55, and 6.66–7.06 log CFU/cm2, respectively. Generally, reduction levels of biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes did not significantly (p > 0.05) differ according to RH after exposure to 10 ppmv of ClO2 gas (Table 1). After treatments under 60 and 90% RH for 20 min, 1.93 and 2.49 log reductions of E. coli O157:H7 were achieved, respectively. S. Typhimurium was reduced by 1.07 and 2.57 log after 20 min treatment under 60 and 90% RH, respectively. Treatment with 10 ppmv of ClO2 gas under 60 and 90% RH for 20 min resulted in 2.02 and 2.78 log reductions of L. monocytogenes, respectively. ClO2 gas treatment with increasing concentrations at 90% RH resulted in significantly higher (p < 0.05) inactivation of biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes than gas treatment at 60% RH. After treatments with 30 ppmv of ClO2 gas under 60 and 90% RH for 20 min, 3.70 and more than 6.26 log reductions of E. coli O157:H7 were achieved, respectively (Table 2). S. Typhimurium and L. monocytogenes were reduced by 1.70 and 4.14, and 3.57 and 4.60 log after 20 min treatment under 60 and 90% RH, respectively. When exposed to 50 ppmv of ClO2 gas under 60% RH for 20 min, the biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes were reduced by 4.19, 2.08, and 4.62 log, respectively (Table 3). The level of biofilm cells of E. coli O157:H7 was reduced to below the detection limit (0.48 log CFU/cm2) within 15 min when treated with 50 ppmv of ClO2 gas at 90% RH. The 50 ppmv of ClO2 gas treatment under 90% RH for 20 min inactivated the biofilm cells of S. Typhimurium and L. monocytogenes to undetectable levels (0.48 log CFU/cm2).

Table 1.

Log reductionsa of biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes on SS coupon after treatment with 10 ppmv ClO2 gas

Bacteria Relative humidity (%) Reduction (log CFU/cm2)
1 min 5 min 10 min 15 min 20 min
E. coli O157:H7 60 0.71 ± 0.45Aab 1.17 ± 0.11ABa 1.67 ± 0.20BCa 1.73 ± 0.57BCa 1.93 ± 0.44Ca
90 0.54 ± 0.24Aa 1.51 ± 0.08Bb 1.64 ± 0.33Ba 2.28 ± 0.29Ca 2.49 ± 0.11Ca
S. Typhimurium 60 0.68 ± 0.39Aa 0.86 ± 0.25Aa 0.98 ± 0.45Aa 1.03 ± 0.36Aa 1.07 ± 0.43Aa
90 0.67 ± 0.41Aa 0.69 ± 0.40Aa 1.53 ± 0.52Ba 2.18 ± 0.20BCb 2.57 ± 0.09Cb
L. monocytogenes 60 0.69 ± 0.38Aa 1.20 ± 0.29ABa 1.26 ± 0.32ABa 1.71 ± 0.33BCa 2.02 ± 0.33Ca
90 0.83 ± 0.08Aa 1.18 ± 0.13ABa 1.43 ± 0.37ABa 1.52 ± 0.38Ba 2.78 ± 0.52Ca
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aReduction = population before treatment—population after treatment. Initial biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes on SS coupons were 6.96, 8.55, and 6.83 log CFU/cm2, respectively

bValues followed by the same uppercase letters within rows are not significantly different (p > 0.05). Values followed by the same lowercase letters within columns are not significantly different (p > 0.05)

Table 2.

Log reductionsa of biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes on SS coupon after treatment with 30 ppmv ClO2 gas

Bacteria Relative humidity (%) Reduction (log CFU/cm2)
1 min 5 min 10 min 15 min 20 min
E. coli O157:H7 60 0.91 ± 0.37Aab 1.78 ± 0.24Ba 2.41 ± 0.16Ca 2.69 ± 0.44Ca 3.70 ± 0.20Da
90 1.17 ± 0.61Aa 2.15 ± 0.18Ba 2.88 ± 0.15Ba 4.03 ± 0.51Cb > 6.26Db
S. Typhimurium 60 0.87 ± 0.43Aa 0.99 ± 0.52Aa 1.36 ± 0.46Aa 1.57 ± 0.40Aa 1.70 ± 0.39Aa
90 0.50 ± 0.12Aa 1.15 ± 0.46Ba 2.54 ± 0.15Cb 3.44 ± 0.41Db 4.14 ± 0.38Eb
L. monocytogenes 60 1.38 ± 0.06Aa 1.56 ± 0.21Aa 2.64 ± 0.31Ba 2.88 ± 0.45Ba 3.57 ± 0.22Ca
90 0.98 ± 0.33Aa 2.40 ± 0.28Bb 3.00 ± 0.24Ba 3.70 ± 0.22Cb 4.60 ± 0.57Db
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aReduction = population before treatment—population after treatment. Initial biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes on SS coupons were 6.74, 8.34, and 6.66 log CFU/cm2, respectively

bValues followed by the same uppercase letters within rows are not significantly different (p > 0.05). Values followed by the same lowercase letters within columns are not significantly different (p > 0.05)

Table 3.

Log reductionsa of biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes on SS coupon after treatment with 50 ppmv ClO2 gas

Bacteria Relative humidity (%) Reduction (log CFU/cm2)
1 min 5 min 10 min 15 min 20 min
E. coli O157:H7 60 1.34 ± 0.54Aab 1.81 ± 0.39Aa 2.89 ± 0.34Ba 3.83 ± 0.39Ca 4.19 ± 0.25Ca
90 0.80 ± 0.27Aa 3.79 ± 0.41Bb 4.38 ± 0.56Bb > 6.07Cb > 6.07Cb
S. Typhimurium 60 1.01 ± 0.47Aa 0.98 ± 0.44Aa 1.63 ± 0.47ABa 1.83 ± 0.50ABa 2.08 ± 0.37Ba
90 0.71 ± 0.31Aa 2.36 ± 0.29Bb 4.86 ± 0.56Cb 5.71 ± 0.16Db > 7.87Eb
L. monocytogenes 60 1.66 ± 0.32Aa 2.44 ± 0.31Ba 3.11 ± 0.19Ca 3.71 ± 0.37Da 4.62 ± 0.33Ea
90 1.54 ± 0.12Aa 2.47 ± 0.39Ba 3.42 ± 0.47Ca 4.63 ± 0.43Db > 6.58Eb
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aReduction = population before treatment—population after treatment. Initial biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes on SS coupons were 6.55, 8.35, and 7.06 log CFU/cm2, respectively

bValues followed by the same uppercase letters within rows are not significantly different (p > 0.05). Values followed by the same lowercase letters within columns are not significantly different (p > 0.05)

Similar inactivation patterns were observed after the biofilm cells of three pathogens on HDPE surfaces were exposed to ClO2 gas (Tables 4, 5 and 6). Initial biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes formed on HDPE coupons were 6.80–6.95, 7.85–7.90, and 7.06–7.08 log CFU/cm2, respectively. After treatments with 10 ppmv of ClO2 gas under 60 and 90% RH for 20 min, 1.85 and 2.70, 1.25 and 2.51, and 2.09 and 2.26 log reductions of E. coli O157:H7, S. Typhimurium, and L. Monocytogenes were achieved, respectively (Table 4). After treatments with 30 ppmv of ClO2 gas under 60 and 90% RH for 20 min, 2.47 and 4.55, 1.26 and 4.34, and 3.13 and 4.13 log reductions of E. coli O157:H7, S. Typhimurium, and L. monocytogenes were occurred, respectively (Table 5). When exposed to 50 ppmv of ClO2 gas under 60% RH for 20 min, the biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes were reduced by 4.14, 2.08, and 4.41 log, respectively (Table 6). The 50 ppmv of ClO2 gas treatment under 90% RH inactivated the biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes to undetectable levels (0.48 log CFU/cm2) within 15, 20, and 20 min, respectively.

Table 4.

Log reductionsa of biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes on HDPE coupon after treatment with 10 ppmv ClO2 gas

Bacteria Relative humidity (%) Reduction (log CFU/cm2)
1 min 5 min 10 min 15 min 20 min
E. coli O157:H7 60 0.27 ± 0.43Aab 0.69 ± 0.38ABa 1.33 ± 0.39BCa 1.73 ± 0.54Ca 1.85 ± 0.43Ca
90 0.46 ± 0.20Aa 1.16 ± 0.23Ba 1.25 ± 0.20Ba 1.64 ± 0.37Ba 2.70 ± 0.28Cb
S. Typhimurium 60 0.27 ± 0.09Aa 0.58 ± 0.41Aa 1.07 ± 0.17Ba 1.14 ± 0.23Ba 1.25 ± 0.12Ba
90 0.29 ± 0.38Aa 0.98 ± 0.40ABa 1.13 ± 0.40Ba 2.18 ± 0.29Cb 2.51 ± 0.47Cb
L. monocytogenes 60 1.20 ± 0.25Aa 1.62 ± 0.25Ba 1.91 ± 0.22BCa 1.98 ± 0.07Ca 2.09 ± 0.10Ca
90 0.87 ± 0.55Aa 1.69 ± 0.41Ba 2.07 ± 0.33Ba 2.21 ± 0.19Ba 2.26 ± 0.19Ba
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aReduction = population before treatment—population after treatment. Initial biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes on HDPE coupons were 6.80, 7.85, and 7.08 log CFU/cm2, respectively

bValues followed by the same uppercase letters within rows are not significantly different (p > 0.05). Values followed by the same lowercase letters within columns are not significantly different (p > 0.05)

Table 5.

Log reductionsa of biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes on HDPE coupon after treatment with 30 ppmv ClO2 gas

Bacteria Relative humidity (%) Reduction (log CFU/cm2)
1 min 5 min 10 min 15 min 20 min
E. coli O157:H7 60 0.55 ± 0.39Aab 0.92 ± 0.48ABa 1.32 ± 0.44ABa 1.78 ± 0.40BCa 2.47 ± 0.59Ca
90 1.08 ± 0.41Aa 2.14 ± 0.15Bb 3.31 ± 0.49Cb 3.63 ± 0.20Cb 4.55 ± 0.23Db
S. Typhimurium 60 0.54 ± 0.22Aa 0.63 ± 0.28Aa 0.83 ± 0.11ABa 0.94 ± 0.27ABa 1.26 ± 0.31Ba
90 0.49 ± 0.28Aa 1.54 ± 0.20Bb 2.03 ± 0.35Bb 3.69 ± 0.51Cb 4.34 ± 0.35Db
L. monocytogenes 60 1.41 ± 0.31Aa 2.12 ± 0.48Ba 2.32 ± 0.31Ba 2.49 ± 0.38Ba 3.13 ± 0.23Ca
90 1.71 ± 0.40Aa 2.55 ± 0.52Ba 3.03 ± 0.18BCb 3.40 ± 0.25Cb 4.13 ± 0.30Db
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a Reduction = population before treatment—population after treatment. Initial biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes on HDPE coupons were 6.95, 7.88, and 7.07 log CFU/cm2, respectively

b Values followed by the same uppercase letters within rows are not significantly different (p > 0.05). Values followed by the same lowercase letters within columns are not significantly different (p > 0.05)

Table 6.

Log reductionsa of biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes on HDPE coupon after treatment with 50 ppmv ClO2 gas

Bacteria Relative humidity (%) Reduction (log CFU/cm2)
1 min 5 min 10 min 15 min 20 min
E. coli O157:H7 60 0.72 ± 0.42Aab 2.06 ± 0.53Ba 2.64 ± 0.28Ba 3.38 ± 0.39Ca 4.14 ± 0.40Da
90 1.09 ± 0.32Aa 2.11 ± 0.29Ba 3.52 ± 0.31Cb > 6.41Db > 6.41Db
S. Typhimurium 60 0.66 ± 0.04Aa 1.18 ± 0.42ABa 1.38 ± 0.37Ba 1.63 ± 0.34BCa 2.08 ± 0.19Ca
90 0.86 ± 0.07Ab 2.77 ± 0.57Bb 3.96 ± 0.56Cb 4.53 ± 0.16Cb > 7.42Db
L. monocytogenes 60 1.93 ± 0.53Aa 2.60 ± 0.44ABa 3.20 ± 0.44BCa 3.92 ± 0.50CDa 4.41 ± 0.37Da
90 1.84 ± 0.43Aa 2.88 ± 0.37Ba 3.26 ± 0.56Ba 4.21 ± 0.34Ca > 6.58Db
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aReduction = population before treatment—population after treatment. Initial biofilm cells of E. coli O157:H7, S. Typhimurium, and L. monocytogenes on HDPE coupons were 6.89, 7.90, and 7.06 log CFU/cm2, respectively

bValues followed by the same uppercase letters within rows are not significantly different (p > 0.05). Values followed by the same lowercase letters within columns are not significantly different (p > 0.05)

In this study, ClO2 gas represented a significant antimicrobial effect against biofilm cells of pathogenic bacteria on SS and HDPE surfaces. When treated with 50 ppmv of ClO2 gas under conditions of 90% RH, biofilm cells of three pathogens were reduced to below the detection limit (0.48 log CFU/cm2) within 20 min. Good efficacy of ClO2 gas against biofilm cells of pathogenic bacteria on food contact surfaces has been reported by other studies. Vegetative cells and spores of B. cereus in the biofilm were completely inactivated within 6 h when exposed to ClO2 gas (peak concentration: 115.3 ± 5.0 ppm) (Nam et al. 2014). Biofilm cells of L. monocytogenes on SS coupon were reduced by 3.21 log after treatment with 10 min of 0.3 mg/l ClO2 gas (Vaid et al. 2010). The U.S. Food and Drug Administration (FDA) Food Code defines sanitization as the application of cumulative heat or chemicals on cleaned food-contact surfaces that is sufficient to yield a reduction of 5 logs of representative disease microorganisms of public health importance (U. S. FDA 2017). The results of this study indicate that ClO2 gas treatment for the decontamination of biofilm cells of foodborne pathogens on food contact surfaces could meet the FDA standards.

As ClO2 gas has high solubility in water, the antimicrobial effect of ClO2 gas increases with increasing RH (Linton et al. 2006). Therefore, several studies have been evaluated the antimicrobial effect of ClO2 gas under high RH conditions (> 80%) (Vandekinderen et al. 2009; Bhagat et al. 2011). The antimicrobial effect of ClO2 gas against biofilm cells of pathogens on food contact surfaces also has been evaluated under high RH conditions (> 75%) (Vaid et al. 2010; Nam et al. 2014). However, little information is known about the ClO2 gas mediated inactivation of the biofilm cells of foodborne pathogens relative to RH conditions. In this study, significant differences (p < 0.05) were observed between the inactivation levels of biofilm cells of three pathogens under different RH conditions as gas concentration and treatment time increased. When exposed to ClO2 gas, populations of E. coli O157:H7, S. Typhimurium, and L. monocytogenes on spinach leaves and tomatoes were reduced more under conditions of 90% RH than under 50 and 70% RH (Park and Kang 2015; Park et al. 2018). These results are in agreement with the results of this study. This result may be attributed to the differences in the solubility of ClO2 gas at various RH levels. Park et al. (2018) reported that ClO2 residue on tomato surfaces significantly (p < 0.05) increased with increasing RH. The authors also observed a close association between reduction in the amount of pathogens and ClO2 gas residue on tomato surfaces.

In this study, the biofilm cells of S. Typhimurium were more resistant to ClO2 gas than those of E. coli O157:H7 and L. monocytogenes under 60% RH condition. The EPS matrix and biofilm architecture played important role in the resistance of biofilm cells to disinfectant (Xue et al. 2012; Govaert et al. 2018) reported that the 1-day-old biofilm of L. monocytogenes which appeared to be more denser than that of S. Typhimurium was more resistant to H2O2 treatment. In the present study, initial biofilm cells of S. Typhimurium were higher than those of E. coli O157:H7 and L. monocytogenes. However, the amount of EPS matrix such as carbohydrates or protein was not measured to explain different degree of biofilm resistance. Kim et al. (2020) reported that the resistance of biofilm of Staphylococcus aureus to thermal or non-thermal treatments was related to the amount of secreted exopolysaccharide. Further evaluation exploring the differences in resistance by comparing the amount of EPS produced by each foodborne pathogen is required.

Park and Kang (2017) revealed that the hydrophobicity of food contact surfaces influences the antimicrobial effect of gaseous ClO2. They discerned that differences in the inactivation levels with respect to hydrophobicity may be attributed to differences in hydration levels of each sample surface. In this study, no significant (p > 0.05) differences were observed in the inactivation levels of biofilm cells between those formed on SS and HDPE coupons. Here, wet biofilms of foodborne pathogens were treated with ClO2 gas. Thus, it seems that the hydrophobicity of SS and HDPE coupon surfaces had a low effect on the inactivation levels of biofilm cells of pathogenic bacteria on these surfaces.

Conclusion

ClO2 gas treatment represents a significant antimicrobial effect against biofilm cells of pathogenic bacteria on SS and HDPE surfaces. Significant differences (p < 0.05) were found between inactivation levels under different RH conditions as ClO2 gas concentration and treatment time increased. Based on the results of this study, ClO2 gas can be used in the food industry as a disinfectant to inactivate biofilm cells of pathogens on pipe lines, conveyor lines, or food processing equipment. Also, it is needed to evaluate the quantities of ClO2 residues on food contact surfaces after ClO2 gas treatment and its harmful effect such as corrosion. The results of this study could be useful for predicting the inactivation patterns of biofilm cells of pathogenic bacteria on food contact surfaces by ClO2 gas for practical application.

Acknowledgements

All bacterial strains used in this study were kindly provided by Dong-Hyun Kang (Department of Food Science and Biotechnology at Seoul National University). This research was financed by the National Research Foundation of Korea (NRF) and Korea Forest Service.

Abbreviations

SS

Stainless steel

HDPE

High density polyethylene

RH

Relative humidity

EPS

Extra polymeric substances

TSA

Tryptic soy agar

TSB

Tryptic soy broth

PBS

Phosphate-buffered saline

PW

Peptone water

SMAC

Sorbitol MacConkey agar

XLD

Xylose Lysine Desoxycholate agar

OAB

Oxford agar base

Author contributions

All authors contributed to this manuscript as; S-YK: Investigation, Writing—original draft. S-HP: Conceptualization, Writing—review & editing, Resources, Supervision, Funding acquisition.

Funding

This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korea government(MSIT) (NRF-2020R1F1A1048617). This study was carried out with the support of 'R&D Program for Forest Science Technology (Project No. 2021332C10-2123A01)' provided by Korea Forest Service (Korea Forestry Promotion Institute).

Data availability

The authors confirm that the data supporting the findings of this study are available within the article.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article.

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