Inactivation of Shiga-toxin-producing Escherichia coli, Salmonella enterica and natural microflora on tempered wheat grains by atmospheric cold plasma

Food Control 104 (2019) 231–239

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Inactivation of Shiga-toxin-producing Escherichia coli, Salmonella enterica and natural microflora on tempered wheat grains by atmospheric cold plasma

T

Emalie Thomas-Popoa,b, Aubrey Mendonçaa,b,c,∗, N.N. Misraa,c, Allison Littlea, Zifan Wana,c, Rkia Moutiqa,c, Shannon Colemana, Kevin Keenera,c a b c

Department of Food Science and Human Nutrition, Iowa State University, 536 Farmhouse Lane, Ames, IA, 50010, USA Interdepartmental Program in Microbiology, Iowa State University, Ames, IA, 50010, USA Center for Crop Utilization Research, Department of Food Science and Human Nutrition, Iowa State University, 536 Farmhouse Lane Ames, Iowa, 50011, USA

A R T I C LE I N FO

A B S T R A C T

Keywords: Cold plasma Escherichia coli Salmonella Wheat grains

Bacterial foodborne disease outbreaks implicating raw wheat products have focused much attention on decontaminating wheat grains used for manufacturing flour and other milled products. The present study investigated the effectiveness of atmospheric cold plasma (ACP) for destroying Shiga-toxin-producing Escherichia coli, Salmonella enterica and natural microflora on tempered wheat grains. Portions (10 g) of grains were each inoculated with a 5-strain mixture of E. coli or S. enterica to obtain an initial count of ∼7.0 log10 CFU/g. Inoculated or non-inoculated wheat grains were sealed in plastic bags filled with atmospheric air and exposed to ACP (44 kV) dielectric barrier discharge for 0 (control), 5, 10, 15 and 20 min. Pathogen survivors were evaluated by washing wheat grains with sterile diluent, plating the wash solution on thin agar layer (TAL) media and appropriate selective (SEL) agar, and counting bacterial colonies after 48 h of incubation (35 ᵒC). Non-inoculated grains were analyzed for yeast and molds, mesophiles, psychrotrophs, and Enterobacteriaceae. After 20 min, initial viable counts (log CFU/g) on TAL medium and SEL agar, respectively, decreased by 3.09 and 4.84 (for E. coli), and 4.40 and 4.32 (for S. enterica). A higher level of sub-lethal injury occurred in E. coli compared to S. enterica (P < 0.05). After ACP treatment for 20 min, log CFU/g reductions of mesophiles psychrotrophs, and Enterobactericeae were 0.96, 2.14 and 1.38, respectively. In contrast, yeast and molds were totally destroyed (3.29 log CFU/g reduction) after only 10 min. ACP has good potential for killing both enteric pathogens and spoilage microorganisms on tempered wheat grains destined for the manufacture of wheat flour. Further research on the baking characteristics of flour manufactured from ACP-treated wheat grains is warranted.

1. Introduction Cereal crops, including wheat grown in fields, are exposed to many sources of microbial contamination from the natural environment including air, water, windblown dust, soil, insects, and feces of birds and rodents. Microbial contamination of wheat also occurs during harvesting, handling, post-harvest drying and storage (Magan & Aldred, 2006). Microorganisms that contaminate wheat can be spread within and among batches of wheat grains by harvesting and handling equipment, and during transfer of the grains into storage silos (Bullerman & Bianchini, 2009; Nierop, 2006). The microbiological quality of wheat grains is a major factor affecting the microbial safety and quality of flour and other milled products derived from this

∗

commodity (Berghofer, Hocking, Miskelly, & Jansson, 2003; Seiler, 1986). In fact, wheat flour of poor microbiologically quality is invariably derived from wheat grains with very high microbial counts (Berghofer et al., 2003). 1.1. Implication of wheat flour in foodborne disease For wheat grains used in flour production, there is no established kill step for pathogens in the grain milling process. Consequently, wheat flour has been implicated in several foodborne disease outbreaks caused by Escherichia coli and Salmonella enterica. For example Shiga-toxinproducing Escherichia coli (STEC) were isolated from wheat flour, dry dough mix and/or ready-to-bake commercial cookie dough involved in

Corresponding author. Department of Food Science and Human Nutrition, 536 Farm House Lane, Ames, IA 50011, USA. E-mail address: [email protected] (A. Mendonça).

https://doi.org/10.1016/j.foodcont.2019.04.025 Received 13 December 2018; Received in revised form 24 April 2019; Accepted 25 April 2019 Available online 26 April 2019 0956-7135/ © 2019 Elsevier Ltd. All rights reserved.

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multistate outbreaks in the United States (CDC, 2009; CDC, 2016; Crowe et al., 2017; US FDA, 2017; Gieraltowski et al., 2017) as well as outbreaks in several Canadian provinces (BCCDC, 2017; Beach, 2017; PHAC, 2017). There were 67 reported cases of illness from consumption of an uncooked baking mixture that contained wheat flour contaminated with Salmonella Typhimurium phage type 42 (McCallum et al., 2013). Also, S. Typhimurium was incriminated in a multistate outbreak involving contaminated raw cake mix which was used as an ingredient in ice cream (Zang et al., 2007). Based on the occurrence of enteric pathogens in wheat flour, there is an urgent need for effective methods to decontaminate wheat grains destined for the production of flour.

microflora on cereal grains (Brasoveanu, Nemtanu, Surdu-Bob, Karaca, & Erper, 2015; Filatova et al., 2013; Kordas, Pusz, Czapka, & Kacprzyk, 2015; Selcuk, Oksuz, & Basaran, 2008; Zahoranova et al., 2016) and microorganisms on artificially inoculated cereal grains and seeds (Butscher, Zimmermann, Schuppler, & Rudolf von Rohr, 2016b, 2016a; Schnabel et al., 2012; Zahoranova et al., 2016). More recently Los et al. (2018b) reported on the efficacy of a high voltage (80 kV) dielectric barrier discharge (DBD) closed system for inactivating native microflora on cereal grains (barley and wheat) and two bacteria (E. coli NCTC 12900 and Bacillus atrophaeus var niger vegetative cells and endospores) and spores of Penicillium verrucosum DSM 12639 that were artificially inoculated on those grains.

1.2. Microbial decontamination of wheat grains

1.4. Tempering of wheat grains for flour production

Conventional methods for reducing pathogenic and spoilage microorganisms on cereal grains involve heating and chemical treatments. Unfortunately, those methods can negatively alter quality characteristics and functional properties of cereal products including the baking performance of flour (Guerrieri & Cerletti, 1996; Schofield, Bottomley, Timms, & Booth, 1983); therefore, novel grain decontamination methods that can prevent those problems are needed. Alternative methods such as ozone treatment (Allen, Wu, & Doan, 2003; Tiwari et al., 2010; Wu, Doan, & Cuenca, 2006), ionizing irradiation (Lorenz & Miller, 1975), pulsed ultraviolet light (Maftei, Ramos-villarroel, Nicolau, Mart, & Soliva-fortuny, 2013), microwave radiation (Reddy, Raghavan, Kushalappa, & Paulitz, 1998; Vadivambal, Jayas, & White, 2007) and ACP treatment (Butscher et al., 2016; Los, Ziuzina, & Bourke, 2018a) have been reported. While limitations of several of those methods preclude their practical application for cereal grains (Los et al., 2018a; Schmidt, Zannini, & Arendt, 2018), ACP offers the flexibility of delivering this treatment at a number of stages in grain processing (Los et al., 2018b).

A critical point in wheat flour production is the grain tempering process which is carried out mainly to improve the efficiency of extracting the flour (Postner & Hibbs, 2005). During tempering, water is added in specified amounts to wheat grains followed by holding of the grains in conditioning bins for about 6–18 h (Berghofer et al., 2003; Postner & Hibbs, 2005). Berghofer et al. (2003) reported that higher mesophilic aerobic counts occurred more frequently in tempered wheat. In that same study E. coli was detected in tempered wheat grains but not in those same grains prior to tempering. The addition of water and the likelihood of microbial contamination and/or growth in wheat grains during tempering justify the application of ACP for pathogen control at this critical point. Also, the major ROS formed when ACP contacts water is the hydroxyl radical (OH·) and since there is no enzyme to detoxify this radical, it is extremely toxic and lethal to microorganisms (Imlay, 2008). To our knowledge there are no published reports on the fate of enteric pathogens and natural microflora following ACP treatment of tempered wheat grains. Accordingly, the main objective of the present study was to evaluate inactivation of shiga-toxin producing E. coli and S. enterica following ACP treatment (44 kV) on artificially contaminated wheat grains moistened to simulate addition of water for grain tempering. Additional objectives were to determine: i) the extent of sublethal injury in pathogen survivors following ACP treatment and ii) the effect of ACP on the viability of naturally occurring microflora of the wheat grains.

1.3. Atmospheric cold plasma Atmospheric cold plasma refers to non-equilibrated, ionized gas (air) generated at atmospheric pressure and ambient temperature. It contains a variety of energetic species including free electrons, free radicals, positive and negative ions, neutral or excited atoms, molecules, and photons. The antimicrobial effectiveness of ACP is attributed to reactive oxygen species (ROS), reactive nitrogen species (RNS) among other chemical and bioactive radicals formed during electrical discharge (Laroussi & Leipold, 2004; Scholtz, Pazlarova, Souskova, Khun, & Julak, 2015). Apart from its antimicrobial characteristics, ACP causes very little or no thermal damage to food products (Niemira, 2012; Niemira, Boyd, & Sites, 2014); therefore, negative changes in quality characteristics and functional properties of ACP-treated cereal products can be substantially minimized. There are several reports on application of ACP using surface-only or fluidized bed approaches for destroying naturally occurring

2. Materials and methods 2.1. Atmospheric cold plasma system The ACP system (Fig. 1) used in the present study was a dielectric barrier discharge (DBD) system, consisting of two 10-cm diameter aluminum disk electrodes that were concentrically placed at a gap of 20 mm. The ACP treatment conditions including ambient temperature and relative humidity of the gaseous atmosphere (air) were recorded for each experiment. The system operated at an average wattage of 56.5 W

Fig. 1. Schematic of the experimental set-up for atmospheric cold plasma treatment of wheat grains. 232

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(saline). For each pathogen the washed cells were harvested by centrifugation as previously described and re-suspended in 30 mL of sterile saline to obtain a viable cell concentration of ∼9.0 log10 colony forming units (CFU)/mL. Dilutions (10-fold) of the five-strain cell suspensions were prepared in fresh saline and aseptically transferred to separate calibrated spray bottles. Colony counts of each diluted cell suspension were determined by serial dilution (10-fold) and surface plating on selective agar (sorbitol MacConkey agar (SMAC) or xylose lysine tergitol 4 (XLT 4) agar) and on Thin Agar Layer (TAL) medium (Kang & Fung, 2000). The TAL medium was prepared by overlaying 14 mL of molten tryptic soy agar supplemented with 0.6% yeast extract (TSAYE) (48 °C) onto solidified selective agar (SMAC or XLT 4). Bacterial colonies were counted after aerobic incubation (35 ᵒC) of TAL plates and selective agar plates for 24 and 48 h, respectively. 2.5. Preparation of non-inoculated and inoculated wheat grains Organic soft white winter wheat grains were purchased from a local grocery store in Ames, Iowa. The wheat grains were placed into a sterile stomacher bag and the contents were mixed by manually shaking the closed bag. Ten-gram portions of wheat grains were analyzed for the presence of native E. coli or S. enterica by washing grains with 0.1% (w/ v) peptone and surface plating aliquots (1.0 or 0.1-mL) of wash solution on selective agar (sorbitol MacConkey agar or xylose lysine tergitol 4 agar) and on appropriate TAL media. Bacterial colonies were counted after incubating the agar plates aerobically at 35 ᵒC for 48 h. Ten-gram portions of wheat grains were aseptically weighed and placed into separate sterile 150 mm × 15 mm petri dishes. Each Petri dish was gently agitated to form a mono-layer of wheat grains. For artificially inoculated grains, 10-g samples were each mist-inoculated with E. coli or S. enterica using a sterile calibrated spray bottle, which delivered ∼1.70 mL of cell suspension via two sprays applied to wheat grains to obtain ∼1.7 × 108 CFU per 10-g sample. All inoculations were performed in a large biohazard bag in a BSL2 laminar flow biosafety cabinet. The petri dishes containing the inoculated wheat grains were placed on an ethanol (100%) soaked paper towel on a sheet of aluminum foil. The outer sides of the petri dishes were disinfected using cotton balls soaked with ethanol (100%). The inoculated wheat grains were held at ambient temperature (22 ± 1 °C) for 6 h in the biosafety cabinet without the fan in operation. Inoculated and non-inoculated wheat grains were held separately and sprayed with sterile distilled water immediately before ACP treatment to simulate the beginning of the tempering process which conditions the grains for subsequent milling. A calibrated spray bottle was used to dispense ∼0.85 mL of sterile distilled water onto the wheat grains via application of one spray per sample (10 g).

Fig. 2. Calibration of the dial graduation (%) on variac against the measured voltage output of the step-up transformer.

and a frequency of 60 Hz. 2.2. Calibration of the variac (voltage regulator) In order to calibrate the dial on the variac (voltage regulator) against the output from the transformer, the voltage applied across the electrodes was measured using a high voltage probe (Fluke 80K-40, Fluke Corporation, Everett, WA, USA) with a maximum rating of 40 kV connected to a multimeter (Fluke 87-V, Fluke Corporation, Everett, WA, USA). The % graduation on the variac was found to vary linearly with the measured voltage output. Thus, the applied root mean square (RMS) voltage was approximated via extrapolation of the linear regression between the % dial graduation and the measured voltage. From Fig. 2 it can be observed that the measured voltages were lower than the theoretically predicted linear relationship. This was attributed to the fact that the lower electrode was at a floating potential, rather than an absolute zero (i.e. ground potential). 2.3. Bacterial strains and culture conditions Five strains of shiga-toxin-producing Escherichia coli: O121, and O157 (ATCC 35150, ATCC 43894, ATCC 43895, FRIK 125) and five serotypes of Salmonella enterica (Enteritidis-ATCC13076, Heidelberg, Typhimurium ATCC 14028, Gaminara ATCC 8324, and Oranienburg ATCC 9239) were obtained from the culture collection of the Microbial Food Safety Laboratory, Iowa State University, Ames, IA. Frozen stock cultures (−80 ᵒC) in brain heart infusion (BHI) broth (Difco; Becton Dickinson, Sparks, MD) with added 10% (v/v) glycerol were thawed under cold running water and activated in tryptic soy broth supplemented with 0.6% yeast extract (TSBYE; pH 7.2; Difco; Becton Dickinson) at 35 ᵒC. Working cultures were held at 4 ᵒC until used in experiments. Two consecutive 24 h transfers of each working culture in TSBYE (35 ᵒC) were performed before preparing the cells for inoculation of wheat grains for each experiment.

2.6. Treatment of wheat grains with atmospheric cold plasma Petri dishes containing inoculated or non-inoculated wheat grains were placed in 20 cm × 20 cm high barrier polypropylene Cryovac bags (B2630; Cryovac Sealed Air Corp., Duncan, SC, USA) filled with atmospheric air at atmospheric pressure. The relative humidity of the air was recorded for each experiment. Bags with petri dishes containing the samples of grains were heat sealed and placed between the aluminum electrodes (separated by dielectric barriers) connected to the transformer. Samples were exposed to ACP at 44 KV for 0 (control) to 20 min in 5 min increments (direct HVACP discharge) in atmospheric air and at atmospheric pressure. Control and ACP-treated samples were subjected to post-treatment storage conditions of ambient temperature (22 ± 1 °C) for 24 h before they were analyzed for survivors. The experiment was replicated four times.

2.4. Preparation of inocula Equal volumes (6 mL per culture) of each of the five working cultures of E. coli or S. enterica were combined in a sterile 50 mL centrifuge tube. Cells were harvested by centrifugation (10,000×g, 10 min, 4 ᵒC) using a Sorvall Super T21 centrifuge (American Laboratory Trading, Inc., East Lyme, CT) and washed once in sterile 0.85% (w/v) NaCl

2.7. Optical emission spectroscopy of plasma The light emission from plasma carries information about the 233

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2.10. Determination of sub-lethal injury in Escherichia coli and Salmonella enterica

excited species in the plasma discharge. To identify the excited species, optical emission spectroscopy (OES) was carried out using a computer controlled, custom-built Ocean Optics spectrometer (Ocean Optics, Inc., Florida, USA) with 0.2 nm/pixel resolution over the wavelength window of 200–800 nm of the electromagnetic spectrum. Spectral acquisition was carried out using a MATLAB® (The Mathworks, MA, USA) computer code developed in-house, that uses the instrument control toolbox. The light from the plasma was captured using a 5 mm diameter collimating lens directed towards a solarized optical fiber with a core diameter of 1000 μm. The length from the collimating lenses to the edge of the sample containment box was 7 cm. The integration time for spectral recording was set to 5 s and an average of 10 spectra was recorded, thereby maximizing the signal to noise ratio. Each spectrum was corrected for dark current and the background noise via subtraction, and the averaged spectra were reported. Data were collected for both empty petri plates, as well as petri plates with tempered wheat. A time resolved emission spectroscopy was also performed for the entire duration of treatment. The major peaks from the time-series OES data were detected using a MATLAB® (The Mathworks, MA, USA) computer code developed in-house and the intensities plotted as a function of time. The characteristic molecular and atomic transitions associated with the spectral bands and lines were identified using published reports and NIST (National Institute of Standards and Technology) database.

Based on bacterial colonies on selective (SMAC or XLT 4 agar) and TAL agar or each time of exposure to ACP, survivor curves were prepared and the reduction in viability of each pathogen was determined. That reduction was expressed as the logarithm of the reduction factor (RF) which is the ratio of the CFU/g of non-treated (control) sample to the CFU/g of the ACP-treated sample as expressed by the following equation (Wuytack et al., 2003): log RF = log [CFU/g before ACP treatment ÷ CFU/g after ACP treatment] For each type of agar medium the logarithm of the RF was used to determine reduction in pathogen viability after each exposure time to ACP. The log of RF for XLT-4 or SMAC was plotted on the y-axis against the log of RF for TAL agar on the x-axis. Linear regression lines were fitted through the data points and sub-lethal injury was determined by comparing the slopes and y-intercepts of the regression lines to 1 and 0 respectively (Wuytack et al., 2003). 2.11. Statistical analysis Four replications of the experiments were performed. Mean numbers of survivors were analyzed using JMP Pro statistical software version 14 (SAS Institute, Inc., Cary NC). Significant differences (p < 0.05) in the survival of the pathogens was tested as a function of time, for both pathogen, and exposure time to ACP using analysis of variance. The linear regression model and D-values were prepared for each treatment using JMP Pro statistical software version 14. Tests were carried out at a 5% significance level.

2.8. Enumeration of surviving pathogens and background microflora Bags were aseptically cut open and samples of wheat grains were aseptically transferred to sterile stomacher bags. To each 10 g sample, 90 mL of 0.1% (w/v) peptone (diluent) were added. The mixtures of grains and diluent were vigorously shaken manually for about 20 s. Ten-fold serial dilutions of the diluent were prepared in 0.1% (w/v) peptone and aliquots were surface plated (in duplicate) on selective agar (SMAC or XLT 4 agar) and on Thin Agar Layer (TAL) recovery media to enumerate non-injured and sub-lethally injured pathogens. Bacterial colonies were counted after aerobic incubation of agar at 35 ᵒC for 24 h (TAL plates) or 48 h (selective agar plates). For non-inoculated wheat grains colonies of aerobic mesophilic and psychrotrophic bacteria were enumerated by surface plating samples on plate count agar (PCA) and incubating inoculated agar plates at 35 °C for 48 h (mesophiles) and 7 °C for 7 days (psychrotrophs). Enterobacteriaceae were enumerated by pour plating samples in TSAYE, holding the agar plates for ambient temperature for one hour, overlaying the plates with violet red bile (VRB) agar (Hartman, Hartman, & Lanz, 1975) and incubation at 35 °C for 24 h. Yeast and molds were enumerated by surface plating samples on Dichloran Rose Bengal Chloramphenicol Agar (DRBC) and incubation at 25 °C for 5 days.

3. Results 3.1. Inactivation of pathogens on wheat grains The effectiveness of direct ACP treatment (44 kV) against shiga toxin producing E. coli and S. enterica on artificially contaminated wheat grains is shown in Fig. 3. Initial viable counts of E. coli were 7.32 and 7.15 log CFU/g on TAL medium and XLT-4 agar, respectively. Viable counts of the pathogen declined with increased exposure to ACP irrespective of the recovery agar medium used. After 20 min, E. coli

A

Survivors Log CFU/g

TAL

2.9. Calculation of D-values for inactivation of Escherichia coli and Salmonella enterica To describe the ACP led inactivation of E. coli and Salmonella as a function of time, a classical first order inactivation model was used:

dN (t ) = −k . N (t ) dt

8 7 6 5 4 3 2 1 0

a

ab abc abc

bcd cd

cd

d

d

e

0

where N(t) is the number of survivors after plasma treatment (cfu/g), t is the time (min) and k is the inactivation rate (1/min). The D-value (Decimal value, min) is the time required to reduce an original concentration of micro-organisms by 90%. The D value is related to the kvalue by the equation D = log 10/ k . Alternatively, if the survival curve is plotted on a semi-logarithmic scale, the D-value is determined as the time for a one log10 reduction of the micro-organism.

SMAC

5 10 15 Exposure me (minutes)

20

Fig. 3A. Escherichia coli survivors following HVACP (44 KV) treatment on artificially contaminated wheat grains. Bacterial counts are averages from four replicate experiments. Error bars represent standard error (SE) of the mean. For each exposure time, bars that do not share the same letter are significantly different (p < 0.05). TAL is thin agar layer medium and SMAC is Sorbitol MacKonkey agar. 234

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Table 2 Sub-lethal injury (expressed by linear regression parametersa) in shiga toxin producing Escherichia coli and Salmonella enterica survivors following HVACP (44 kV) treatment on artificially contaminated wheat grains.

B

TAL Survivors (Log CFU/g)

7

a a

6

a

a

a

XLT-4

a b

5

b

4

Pathogen

Slope

Intercept

R2

Escherichia coli Salmonella enterica

0.977 ± 0.43 0.970 ± 0.12

0.701 ± 0.44b −0.086 ± 0.30

0.414 ± 0.244 0.975 ± 0.019

a

3

c

2

Values are averages ± standard deviations from four replications of the experiment. b Intercept significantly different from 0 (P < 0.05).

c

1

3.3. Inactivation of natural microflora on wheat grains

0 0

5 10 15 Exposure me (minutes)

20

Populations (log CFU/g) of yeast and molds, Enterobacteriaceae, aerobic mesophiles and aerobic psychrotrophs on wheat grains after ACP treatment are shown in Table 3. The highest initial numbers of viable microorganisms were observed for mesophiles and pyschrotrophs (∼5.02 log CFU/g) compared to Enterobacteriaceae (4.60 log CFU/g) and yeast and molds (3.29 log CFU/g). After a 10 min exposure of the wheat grains toACP, yeast and molds were not detected (< 1.0 log CFU/g) indicating a greater than 2.29 log CFU/g reduction in viable counts for that group. After 10–20 min of ACP treatment, log CFU/g reductions in viable counts ranged from 1.03 to 2.5 (Enterobacteriaceae), 0.63 to 1.97 (mesophiles) and 0.59 to 2.14 (psychrotrophs). Of all those microbial groups evaluated, the yeast and molds exhibited the highest sensitivity to ACP after 10 or 20 min of treatment (Table 3). The relative sensitivities of natural microflora to ACP (44 kV) treatment for the maximum exposure time (20 min) in decreasing order are: yeast and molds > aerobic psychrotrophs > Enterobacteriaceae > aerobic mesophiles (Fig. 4).

Fig. 3B. Salmonella enterica survivors following HVACP (44 KV) treatment on artificially contaminated wheat grains. Bacterial counts are averages from four replicate experiments. Error bars represent standard error (SE) of the mean. For each exposure time, bars that do not share the same letter are significantly different (p < 0.05). TAL is thin agar layer medium and XLT-4 is xylose lysine tergitol agar.

survivors on TAL medium and SMAC agar decreased by 3.09 and 4.84 log CFU/g, respectively (Fig. 3a). A similar trend was observed for S. enterica on wheat grains where initial viable counts (log CFU/g) of the pathogen decreased by 4.40 and 4.32 log CFU/g on TAL medium and XLT-4 agar, respectively. For each pathogen, numbers of survivors on appropriate selective agar (SMAC or XLT-4) were consistently lower compared to survivors on TAL medium. While there was a significant difference in E. coli survivors on the two media (TAL and SMAC) following 20 min of ACP (P < 0.05), S. enterica survivors on TAL medium and XLT-4 agar were not significantly different (P > 0.05) (see Fig. 3B). The resistance to ACP expressed as decimal reduction times (D-values in minutes) for E. coli and S. enterica on TAL medium and SMAC or XLT-4 agar are shown in Table 1. No statistically significant differences in D-values of the two pathogens (P > 0.05) were observed based on colony counts on TAL medium or on selective agar media (SMAC or XLT-4).

3.4. Optical emission spectroscopy The emission spectrum for the air plasma within the first 50 s of the discharge is shown in Fig. 5 (both, with and without tempered wheat samples in the package). The spectrum presented here agrees with that observed in previous studies with ACP (Misra, Keener, Bourke, & Cullen, 2015; Misra et al., 2014). From the emission spectrum, the presence of many intense peaks in the wavelength range of 315–405 nm were recorded. Specifically, these were identified as molecular transitions from nitrogen second positive system, N2(CeB) and first negative system, N2+ (B-X). The band heads of the N2(C3Πu→B3Πg) second positive system were recorded around 314.6 nm, 335.9 nm, 356.7 nm, 379.2 nm, 398.5 nm and 404.9 nm, while the spectral emission of the nitrogen mono-positive ion + + N2 (B2 ∑u → X 2 ∑g ) was recorded at 393 nm and 425.5 nm with relatively low intensities. These intense peaks indicate the electronic transition of molecular nitrogen resulting from collision reactions. The hydroxyl molecular peak, A2 (Σ+ → X 2Π) was recorded at a very low intensity at 296.6 nm. The relatively low intensity of the hydroxyl peak is due to the low degree of ionization, as would be expected for a low temperature plasma source. An oxygen molecular band at 746.8 nm was recorded, which is suspected to have originated from the vibrations of O2 (b1 Σg+ → X3 Σg−)(0,0) magnetic dipole transition. Several emissions lines from atomic oxygen transitions were recorded at 708.6 nm, 736.7 nm, 772.5 nm, and 776.9 nm, albeit at relatively very low intensities. Thus, it can be concluded that the ACP equipment employed was a source of ROS and RNS. The time-resolved spectrum of the discharge in presence of the wheat samples is presented in Fig. 6. Overall, the intensity of emission peaks of nitrogen transitions increased up to ∼10 min of treatment, followed by a decrease until the 15th min, and a subsequent rise until the end of the treatment duration. This type of variation is a direct outcome of the complex plasma chemical kinetics within the closed volume of the petri-dish. Thus, the chemical kinetics and the

3.2. Sub-lethal injury in pathogen survivors on wheat grains Table 2 displays sub-lethal injury (expressed by linear regression parameters) in E. coli and S. enterica survivors caused by ACP treatment of those pathogens on artificially inoculated wheat grains. In this regard there was a significantly higher level of sub-lethal injury in E. coli survivors compared to that in S. enterica survivors based on the intercept parameter (P < 0.05). Table 1 Pathogen resistance (aD-value) to high voltage atmosphere cold plasma treatment of wheat grains. The D-values are based on slopes of survivor curves from bacterial colony counts (log CFU/g) on TAL medium and on sorbitol MacKonkey agar or xylose lysine tergitol agar. Pathogen Escherichia coli Salmonella enterica

TAL

SMAC or XLT-4 j,x

7.00 ± 2.31 4.73 ± 0.67j,y

4.58 ± 1.35k,x 5.01 ± 0.71k,y

Same first alphabets across the columns indicate no statistical significance (p > 0.05). Same second alphabets across the rows indicate no statistical significance (p > 0.05). a Value for each decimal reduction time (minutes) represents an average (standard deviation) of four replicate experiments. 235

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Table 3 Populations (*log CFU/g) of yeast and molds, Enterobacteriaceae, aerobic mesophiles and psychrotrophs on wheat grains after high voltage cold plasma treatment at 44 kV. Exposure time (minutes) Natural Microflora Group Yeasts and Molds Enterobacteriaceae Aerobic Plate Count Psychrotrophs

0 *3.29 ± 0.17a 4.60 ± 0.25a 5.02 ± 0.52a 5.02 ± 0.46a

5 1.76 4.15 4.69 4.52

± ± ± ±

0.90b 0.44a 0.36a 0.35a

10 NDc 3.57 ± 1.11a 4.39 ± 0.48a 4.43 ± 0.40a

15 NDc 2.10 ± 1.90b 3.05 ± 1.51b 4.20 ± 0.44a

20 NDc 3.42 ± 0.16a,b 4.06 ± 0.35a,b 2.88 ± 1.41b

∗

Each value for viable counts is the average ± standard deviation of four replicate experiments. Means that do not share the same letter within a row are significantly different (p < 0.05). ND: no colonies detected at lowest dilution used; detection limit = 10 CFU g−1.

a,b,c

Log CFU/g reduc on

4. Discussion

4 3.5 3 2.5 2 1.5 1 0.5 0

PSY

APC

Enterics

Y&M

4.1. ACP inactivation on pathogens on wheat grains

a a,b

In the present study, substantial reductions of five-strain mixtures of viable shiga-toxin-producing E. coli (3.09 log CFU/g) or S. enterica (4.40 log CFU/g) on artificially contaminated wheat grains were attained by direct treatment with ACP at 44 kV for 20 min using a dielectric barrier discharge (DBD) system with atmospheric air as the plasma source and a 24-h retention time. Recently, Los et al. (2018b) treated artificially inoculated barley grains with ACP generated using 80 kV with a DBD system. They reported complete inactivation (3.5 CFU g−1 log CFU/g reduction) of E. coli NCTC 12900 on the barley grains after direct ACP treatment in atmospheric air for 20 min and a 24 h retention time before performing microbial analysis on the barley grains. These previously reported results as well as those of the present study indicate the effectiveness of ACP for destroying enteric bacteria on cereal grains. The rate of inactivation of each pathogen by ACP was expressed as the decimal reduction time (D-value) to indicate pathogen resistance to ACP. Our observation of no significant differences in D-values between E. coli and S. enterica (P > 0.05) suggests that the rate of inactivation by direct ACP (44 kV) treatment was similar for both pathogens on wheat grains. Similar reductions in populations of E. coli and S. enterica were observed after application of cold plasma generated in a gliding arc with various flow rates (10–40 L/min for 1, 2, or 3 min) to the surface of artificially inoculated whole apples. Critzer, Kelly-

b,c

c,d c,d

d d d

10

Exposure me (minutes)

20

Fig. 4. Relative sensitivities (Log CFU/g reduction) of natural microflora groups on wheat grains following treatment with ACP (44 kV) for 10 and 20 min..

corresponding rise and fall in reactive species formed is likely responsible for the variations in inactivation of Enterobacteriaceae and mesophiles at the 15th and 20th minute of treatment in our study (Table 3).

Fig. 5. Time integrated optical emission spectrum from the ACP with peaks dominated by emission from N2 second positive system. The specific vibrational transitions (v'→v'') are also denoted. Inset shows the low intensity emissions from atomic transitions of oxygen. 236

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Fig. 6. Intensity of major N2 second positive system peaks obtained from time-resolved optical emission spectroscopy.

crucial to accurately evaluate the antimicrobial efficacy of this emerging non-thermal technology. Populations of microorganisms that are exposed to a food preservation method contains three physiologically different groups of cells. Those are: i) the non-injured group which can grow equally well on selective and non-selective culture media; ii) the sub-lethally injured group which can grow in a non-selective media but not in selective media; and iii) the lethally-injured (dead) group which are incapable of growth under culture conditions (Ray, 1979). The dilemma in trying to detect sub-lethally injured pathogens in agricultural products such as cereal grains containing numerous background microflora is that nonselective culture media allow overgrowth by the background microflora, while selective media inhibit resuscitation and growth of the sublethally injured target organisms. This situation precludes accurate evaluation of the actual number of pathogens that survive antimicrobial treatments. We circumvented that problem by use of the thin agar layer (TAL) method (Kang & Fung, 2000). This TAL method facilitated the recovery of sub-lethally injured E. coli and S. enterica from ACP-treated wheat grains without interference from indigenous microflora. Also, the TAL method precluded any prior application of physical or chemical sterilizing treatments to the wheat grains to kill background microflora. Such sterilizing treatments could also alter the surface characteristics and overall structural integrity of the wheat grains. Our evaluation of sub-lethal injury in each pathogen involved plotting the logarithm of the viability reductions calculated for the selective agar medium (SMAC or XLT-4) on the y axis against the logarithm of viability reduction on TAL medium on the x axis and fitting linear regression lines through the data points. The extent to which the ACP caused sub-lethal injury was determined from the slopes and y intercepts of the regression lines (Wuytack et al., 2003). A slope of 1.0 and intercept of 0 indicate no sub-lethal injury because the same viability reduction occurs on the selective culture medium as well as on the TAL medium. When a slope is significantly > 1.0 or an intercept is significantly > 0, there is sub-lethal injury due to the higher viability reduction noted on selective medium than on the TAL medium. Additionally, it can be assumed that a greater deviation of the slope from 1.0 or a greater deviation of the intercept from 0 indicates a larger extent of sub-lethal injury. In the present study the intercept of the regression line for viability reduction in E. coli was significantly greater than 0 in contrast to that for viability reduction in S. enterica (Table 2). This observation suggests that, compared to S. enterica, a larger extent of sub-lethal injury occurred among E. coli survivors of the ACP

Wintenberg, South, and Golden (2007) reported reductions (∼3.0 log CFU) in initial populations of E. coli on whole apples or S. enterica on cantaloupe using an atmospheric glow discharge plasma for 2 min. No clear pattern of sensitivity was observed for E. coli and S. enterica isolates on artificially inoculated raw almond kernels exposed to a cold plasma jet for 10 or 20 s (Niemira, 2012). In contrast, Ziuzina et al. (2014) demonstrated that S. enterica was more sensitive than E. coli to indirect HVACP (70 kV) treatment of cherry tomatoes using a DBD system with air as the carrier gas at atmospheric pressure and a 24 h post-treatment holding time at ambient temperature. Differences in those previously stated results are likely due to several factors including, but not limited to the type of plasma source, treatment parameters, working gas, the humidity level, surface characteristics of the food product, bacterial strains and the physiological state of pathogens tested (Bourke, Ziuzina, Han, Cullen, & Gilmore, 2017). Variations in cold plasma systems, process parameters, food product characteristics and types of target microorganisms make it challenging to meaningfully compare reported antimicrobial efficacies of ACP. 4.2. Sub-lethal injury in pathogen survivors on wheat grains In the context of microbial food safety, evaluations of sub-lethal injury in foodborne pathogens after their exposure to physical or chemical antimicrobial treatments are important for two main reasons. Firstly, the occurrence of sub-lethally injured pathogens in foods following application of a food preservation method can erroneously overestimate the antimicrobial efficacy of that method. This is particularly true if selective culture media are used to enumerate pathogen survivors. Secondly, food preservation technologies that cause sub-lethal injury offer opportunities for their combined use with other hurdles to prevent resuscitation and subsequent growth of sub-lethally injured pathogens to ultimately cause death of those pathogens. The consistently lower numbers of pathogen survivors on selective agar media (SMAC and XLT-4) compared to TAL media (Fig. 2a and b) indicated the occurrence of E. coli and S. enterica survivors that were sub-lethally injured by ACP. The greater extent of ACP inactivation of E. coli (4.84 log CFU/g reduction; SMAC agar) compared to its inactivation (3.09 log CFU/g reduction; TAL medium) after 20 min of treatment is indicative of the substantial numbers of E. coli survivors that could be overlooked when a selective culture medium is used to evaluate numbers of survivors. Therefore, appropriate methods to facilitate resuscitation and growth of pathogens sub-lethally injured by ACP are 237

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the antimicrobial efficacy of plasma through direct chemical interactions (Moisan et al., 2001) and generation of toxic oxygen derivatives including the hydroxyl radical (Imlay, 2008). In fact, an earlier study has shown that an increase in humidity of air improves the efficacy of atmospheric plasma in inactivating bacterial spores (Patil et al., 2014). Therefore, the application of ACP at the grain tempering step, where water is added to wheat grains, can be an effective strategy for eliminating enteric pathogens on wheat grains that will be subsequently milled to produce flour. While the present study represents a laboratory scale method for efficient ACP inactivation of pathogens on wheat grains, there are likely to be challenges in commercial application of this technology. A major challenge is the decontamination of bulk quantities of wheat grains without compromising plasma uniformity. In this respect, there is a crucial need for innovative engineering ideas in design of large-scale plasma application systems.

treatment. 4.3. Inactivation of background microflora on wheat grains Apart from pathogenic microbes various groups of spoilage microorganisms can be found on the end products of milled wheat such as wheat bran and flour. Those micoorganisms are yeast and molds, and psychrotrophic, mesophilic and thermophilic bacteria (Richter, Dorneanu, Eskridge, & Rao, 1993; Berghofer et al., 2003; Manthey, Wolf-Hall, Yalla, Viajayakumar, & Carlson, 2004; Sperber, 2007; Eglezos, 2010). In the present study, we report significant reductions (P < 0.05) in viable populations of aerobic psychrotrophs and yeast and molds on wheat grains after ACP treatment for 20 min (Table 3) with yeast and molds being the most sensitive of all the microbial groups evaluated (Fig. 3). Our findings are consistent with those of previous reports on cold plasma inactivation of bacteria and fungi (yeast and molds) on wheat grains (Zahoranova et al., 2015) and maize kernels (Dasan, Boyaci, & Mutlu, 2017). The relatively high sensitivity of yeast and molds to cold plasma treatments was highlighted in those reports. The use of raw wheat flour as an ingredient for refrigerated cookie dough, pastry for making pies, and pasta products can contribute substantial amounts of spoilage microorganisms to those products. Those organisms can significantly reduce the microbial shelf-life of the refrigerated ready-to-cook or ready-to-bake products by accelerating spoilage. Therefore, substantial reduction of psychrotrophs and yeast and molds in wheat grains as demonstrated in the present study is of major significance because of the important role that these groups play in the microbial spoilage of refrigerated non-heat-treated wheat-based products.

5. Conclusions The ACP at 44 kV for 20 min in a contained DBD system was effective in reducing populations of microorganisms on the surface of soft white winter wheat grains. The ACP treatment of tempered wheat grains can inactivate shiga toxin producing E. coli and S. enterica; however, substantial sub-lethal injury can occur among pathogen survivors. In this regard, the TAL method facilitates resuscitation and growth of sub-lethally injured E. coli and S. enterica that were not detected by selective agar media. The naturally occurring fungi (yeast and molds) and psychrotrophic bacteria are most sensitive to ACP treatment of wheat grains. Therefore, flour manufactured from ACP-treated wheat grains can be of high microbial quality and contribute to improved microbial shelf-life of refrigerated non-heat-treated wheat-based products. In summary, ACP can be a promising non-thermal treatment for killing both enteric pathogens and spoilage microorganisms on tempered wheat grains destined for the manufacture of wheat flour.

4.4. Optical emission spectroscopy The chemistry of humid air plasma under atmospheric conditions is widely recognized to be quite complex, involving thousands of reactions and dozens of species; c.f. Gordillo-Vázquez, 2008. The optical emission spectroscopy of the plasma discharge helps to unravel the origin of the molecular and atomic transitions of the gas species. Cold plasma generated from atmospheric air is an excellent source of reactive species including excited atoms and molecules, free radicals, electrons, positive and negative ions, ultra violet photons and stable conversion products such as ozone (Stoffels, Sakiyama, & Graves, 2008). The species formed from commonly used sources of cold plasma include ROS such as the oxygen atom (O), singlet oxygen (1O2), superoxide anion (O2−), and ozone (O3), and RNS such as excited nitrogen [N2 (A)], the nitrogen atom (N), and nitric oxide (NO·). Also, the presence of humidity can result in production of other toxic oxygen derivatives including the hydroxyl radical (·OH), hydroxide anion (OH−), and hydrogen peroxide (H2O2) (Scholtz et al., 2015). The intensities of the spectral peaks in presence of wheat grains in the package was lower as compared to the corresponding peaks when the package was empty, which was likely due to the interaction of the excited molecules and atoms with the wheat grains. In the present study moisture (∼7.83% on a wet weight basis) was added to the wheat grains just prior to ACP treatment to simulate moisture conditions generally used for tempering wheat grains. The reactive gas species generated in air plasma and their solubilization into aqueous films on food surfaces (in this instance, wheat grains), resulting in antimicrobial products is well-documented in literature (Misra & Jo, 2017; Misra et al., 2018). Further, it may be noted that the spectral emission in the UV region is very limited, indicating that the antimicrobial action is primarily due to the ROS and RNS. Considering that the 44 kV applied in the present study is not at the higher end of voltages (80–100 kV) typically used for ACP treatments, the destruction of E. coli and S. enterica (3.09 and 4.40 log CFU/g reductions, respectively) was substantial. In this respect, the presence of moisture could have potentiated

Acknowledgments This work is supported by the Iowa Agriculture and Home Economics Experiment Station Project No. IOW03902, sponsored by Hatch Act and State of Iowa funds. References Allen, B., Wu, J., & Doan, H. (2003). Inactivation of fungi associated with barley grain by gaseous ozone. Journal of Environmental Science and Health. Part. B, Pesticides, Food Contaminants, and Agricultural Wastes, 38(5), 617–630. BC Centre for Disease Control (BCCDC) (2017). BCCDC advises British Columbians about a new outbreak of E. coli O121 associated with flour. May 19, 2017. Retrieved from http://www.bccdc.ca/about/news-stories/news-releases/2017/bccdc-advisesbritish-columbians-about-a-new-outbreak-of-e-coli-o121-associated-with-flour. Beach, C. (2017). Rogers flour sold by Costco in Canada linked to new outbreak. Food Safety News. 20 May. Retrieved from http://www.foodsafetynews.com/2017/05/ rogers-flour-sold-bycostco-in-canada-linked-to-new-outbreak/#.WSWPK-vytyz. Berghofer, L. K., Hocking, A. D., Miskelly, D., & Jansson, E. (2003). Microbiology of wheat and flour milling in Australia. International Journal of Food Microbiology, 85, 137–149. Bourke, P., Ziuzina, D., Han, L., Cullen, P. J., & Gilmore, B. F. (2017). Microbiological interactions with cold plasma. Journal of Applied Microbiology, 123, 308–324. Brasoveanu, M., Nemtanu, M. R., Surdu-Bob, C., Karaca, G., & Erper, I. (2015). Effect of glow discharge plasma on germination and fungal load of some cereal seeds. Romanian Reports in Physics, 67(2), 617–624. Bullerman, L. B., & Bianchini, A. (2009). Food safety issues and the microbiology of cereals and cereal products. In N. Heredia, I. Wesley, & S. Garcia (Eds.). Microbiologically safe foods (pp. 315–335). New York, U.S.A.: John Wiley & Sons. Butscher, D., Van Loon, H., Waskow, A., Rudolf von Rohr, P., & Schuppler, M. (2016a). Plasma inactivation of microorganisms on sprout seeds in a dielectric barrier discharge. International Journal of Food Microbiology, 238, 222–232. Butscher, D., Zimmermann, D., Schuppler, M., & Rudolf von Rohr, P. (2016b). Plasma inactivation of bacterial endospores on wheat grains and polymeric model substrates in a dielectric barrier discharge. Food Control, 60, 636–645. Centers for Disease Control and Prevention (CDC) (2009). Multistate outbreak of E. coli O157:H7 infections linked to eating raw refrigerated, prepackaged cookie dough. Updated August 7, 2009 (final web update). Retrieved from https://www.cdc.gov/

238

Food Control 104 (2019) 231–239

E. Thomas-Popo, et al.

Science and Technology, 3, 125–142. Niemira, B. A., Boyd, G., & Sites, J. (2014). Cold plasma rapid decontamination of food contact surfaces contaminated with Salmonella biofilms. Journal of Food Science, 79(5) M917– 22. Nierop, S. Van (2006). The impact of microorganisms on barley and malt quality - a review. Journal of the American Society of Brewing Chemists, 64(2), 69–78. Patil, S., Moiseev, T., Misra, N. N., Cullen, P. J., Mosnier, J. P., Keener, K. M., et al. (2014). Influence of high voltage atmospheric cold plasma process parameters and role of relative humidity on inactivation of Bacillus atrophaeus spores inside a sealed package. Journal Of Hospital Infection, 88(3), 162–169. Postner, E. S., & Hibbs, A. N. (2005). Wheat flour milling (2nd ed.). New York, NY: Springer. Public Health Agency of Canada (PHAC) (2017). Public Health notice - outbreak of E. coli infections linked to various flours and flour products. June 2, 2017 – Final update. Retrieved from http://www.phac-aspc.gc.ca/phn-asp/2017/outbreak-ecoli-eng.php. Ray, B. (1979). Methods to detect stressed microorganisms. Journal of Food Protection, 42, 346–355. Reddy, M. V. B., Raghavan, G. S. V., Kushalappa, A. C., & Paulitz, T. C. (1998). Effect of microwave treatment on quality of wheat seeds infected with Fusarium graminearum. Journal of Agricultural Engineering Research, 71(2), 113–117. Richter, K. S., Dorneanu, E., Eskridge, K. M., & Rao, C. (1993). Microbiological quality of flours. Cereal Foods World, 38, 367–369. Schmidt, M., Zannini, E., & Arendt, E. K. (2018). Recent advances in physical post-harvest treatments for shelf-life extension of cereal crops. Foods, 7, 45. https://doi.org/10. 3390/foods7040045. Schnabel, U., Niquet, R., Krohmann, U., Polak, M., Schl€uter, O., Weltmann, K.-D., et al. (2012). Decontamination of microbiologically contaminated seeds by microwave driven discharge processed gas. Journal of Agriculture Science and Applications, 1, 99–105. Schofield, J. D., Bottomley, R. C., Timms, M. F., & Booth, M. R. (1983). The effect of heat on wheat gluten and the involvement of sulphydryl-disulphide interchange reactions. Journal of Cereal Science, 1(4), 241–253. Scholtz, V., Pazlarova, J., Souskova, H., Khun, J., & Julak, J. (2015). Nonthermal plasma a tool for decontamination and disinfection. Biotechnology Advances, 33(6), 1108–1119. Seiler, D. A. (1986). The microbial content of wheat and flour. In B. Flannigan (Ed.). Proceedings of the spring meeting of the Biodeterioration Society on spoilage and mycotoxins of cereals and other stored products (pp. 35–40). Slough, UK: C.A.B. International. Selcuk, M., Oksuz, L., & Basaran, P. (2008). Decontamination of grains and legumes infected with Aspergillus spp. and Penicillum spp. by cold plasma treatment. Bioresource Technology, 99(11), 5104–5109. Sperber, W. H. (2007). Role of microbiological guidelines in the production and commercial use of milled cereal grains: A practical approach for the 21st century. Journal of Food Protection, 70 1041–53. Stoffels, E., Sakiyama, Y., & Graves, D. B. (2008). Cold atmospheric plasma: Charged species and their interactions with cells and tissues. IEEE Transactions on Plasma Science, 36, 1441–1457. Tiwari, B. K., Brennan, C. S., Curran, T., Gallagher, E., Cullen, P. J., & O' Donnell, C. P. (2010). Application of ozone in grain processing. Journal of Cereal Science, 51(3), 248–255. U.S. Food and Drug Administration (US FDA) (2017). Final summary: FDA investigation of multistate outbreak of shiga toxin-producing E. coli infections linked to flour. Retrieved from https://www.fda.gov/Food/RecallsOutbreaksEmergencies/Outbreaks/ ucm587435.htm. Vadivambal, R., Jayas, D. S., & White, N. D. G. (2007). Wheat disinfestation using microwave energy. Journal of Stored Products Research, 43(4), 508–514. Wu, J., Doan, H., & Cuenca, M. A. (2006). Investigation of gaseous ozone as an anti-fungal fumigant for stored wheat. Journal of Chemical Technology and Biotechnology, 81(May), 1288–1293. Wuytack, E. Y., Phuong, L. D., Aertsen, A., Reyns, K. M., Marquenie, D., De Ketelaere, B., et al. (2003). Comparison of sublethal injury induced in Salmonella enterica serovar Typhimurium by heat and by different nonthermal treatments. Journal of Food Protection, 66(1), 31–37. Zahoranova, A., Henselova, M., Hudecova, D., Kalinakova, B., Kovacik, D., Medvecka, V., et al. (2015). Effect of cold atmospheric pressure plasma on the wheat seedlings vigor and on the inactivation of microorganisms on the seeds surface. Plasma Chemistry and Plasma Processing, 36(2), 397–414. Zahoranova, A., Henselova, M., Hudecova, D., Kalinakova, B., Kovacik, D., Medvecka, V., et al. (2016). Effect of cold atmospheric pressure plasma on the wheat seedlings vigor and on the inactivation of microorganisms on the seeds surface. Plasma Chemistry and Plasma Processing, 36(2), 397–414. Zhang, G., Ma, L., Patel, N., Swaminathan, B., Wedel, S., & Doyle, M. P. (2007). Isolation of Salmonella Typhimurium from outbreak-associated cake mix. Journal of Food Protection, 70, 997–1001. Ziuzina, D., Patil, S., Cullen, P., Keener, K., & Bourke, P. (2014b). Atmospheric cold plasma inactivation of Escherichia coli, Salmonella enterica serovar Typhimurium and Listeria monocytogenes inoculated on fresh produce. Food Microbiology, 42, 109–116.

ecoli/2009/0807.html. Centers for Disease Control and Prevention (CDC) (2016). Multistate outbreak of Shiga toxin producing Escherichia coli infections linked to flour (final update). Retrieved from https://www.cdc.gov/ecoli/2016/o121-06-16/. Critzer, F. A., Kelly-Wintenberg, K., South, S. L., & Golden, D. A. (2007). Atmospheric plasma inactivation of foodborne pathogens on fresh produce surfaces. Journal of Food Protection, 70, 2290–2296. Crowe, S. J., Bottichio, L., Shade, L. N., Whitney, B. M., Corral, N., Melius, B., et al. (2017). Shiga toxin–producing E. coli infections associated with flour. New England Journal of Medicine, 377, 2036–2043. Dasan, B. G., Boyaci, I. H., & Mutlu, M. (2017). Nonthermal plasma treatment of Aspergillus spp. spores on hazelnuts in an atmospheric pressure fluidized bed plasma. Journal of Food Engineering, 196, 139–149. Eglezos, S. (2010). Microbiological quality of wheat grain and flour from two mills in Queensland, Australia. Journal of Food Protection, 73, 1533–1536. Filatova, I., Azharonok, V., Lushkevich, V., Zhukovsky, A., Gadzhieva, G., & Spasi, K. (2013). Plasma seeds treatment as a promising technique for seed germination improvement. Paper presented at international conference on phenomena in ionized gases, Granada, Spain. Gieraltowski, L., Schwensohn, C., Meyer, S., Eikmeier, D., Medus, C., Sorenson, A., et al. (2017). Notes from the field: Multistate outbreak of Escherichia coli O157:H7 infections linked to dough mix – United States, 2016. Morbidity and Mortality Weekly Report, 66(3), 88–89. Gordillo-Vázquez, F. J. (2008). Air plasma kinetics under the influence of sprites. Journal of Physics D: Applied Physics, 41(23), 234016. Guerrieri, N., Alberti, E., Lavelli, V., & Cerletti, P. (1996). Use of spectroscopic and fluorescence techniques to assess heat-induced molecular modifications of gluten. Cereal Chemistry, 73(3), 368–374. Hartman, P. A., Hartman, P. S., & Lanz, W. W. (1975). Violet red bile 2 agar for stressed coliforms. Applied Microbiology, 29, 537. Imlay, J. A. (2008). Cellular defenses against superoxide and hydrogen peroxide. Annual Review of Biochemistry, 77, 755–776. Kang, D. H., & Fung, D. Y. C. (2000). Application of thin agar layer method for recovery of injured Salmonella Typhimurium. International Journal of Food Microbiology, 54, 127–132. Kordas, L., Pusz, W., Czapka, T., & Kacprzyk, R. (2015). The effect of low-temperature plasma on fungus colonization of winter wheat grain and seed quality. Polish Journal of Environmental Studies, 24(1), 433–438. Laroussi, M., & Leipold, F. (2004). Evaluation of the roles of reactive species, heat, and UV radiation in the inactivation of bacterial cells by air plasmas at atmospheric pressure. International Journal of Mass Spectrometry, 233(1–3), 81–86. Lorenz, K., & Miller, B. S. (1975). Irradiation of cereal grains and cereal grain products. CRC Critical Reviews in Food Science & Nutrition, 6(4), 317–382. Los, A., Ziuzina, D., Akkermans, S., Boehm, D., Cullen, P. J., Van Impe, J., et al. (2018b). Improving microbiological safety and quality characteristics of wheat and barley by high voltage atmospheric cold plasma closed processing. Food Research International, 106, 509–521. Los, A., Ziuzina, D., & Bourke, P. (2018a). Current and future technologies for microbiological decontamination of cereal grains. Journal of Food Science, 83(6), 1484–1493. Maftei, N.,A., Ramos-villarroel, A. Y., Nicolau, A. I., Mart, O., & Soliva-fortuny, R. (2013). Pulsed light inactivation of naturally occurring moulds on wheat grain. Journal of the Science of Food and Agriculture, 94, 721–726. Magan, N., & Aldred, D. (2006). Managing microbial spoilage in cereals and baking products. In C. de Blackburn (Ed.). Food spoilage microorganisms (pp. 194–212). Cambridge, UK: Woodhead Publishing Ltd. Manthey, F. A., Wolf-Hall, C. E., Yalla, S., Viajayakumar, C., & Carlson, D. (2004). Microbial loads, mycotoxins, and quality of durum wheat from the 2001 harvest of the North Plains region of the United States. Journal of Food Protection, 67, 772–780. McCallum, L., Paine, S., Sexton, K., Dufour, M., Dyet, K., Wilson, M., et al. (2013). An outbreak of Salmonella Typhimurium phage type 42 associated with the consumption of raw flour. Foodborne Pathogens and Disease, 10, 159–164. Misra, N. N., & Jo, C. (2017). Applications of cold plasma technology for microbiological safety in meat industry. Trends in Food Science & Technology, 64, 74–86. Misra, N. N., Keener, K. M., Bourke, P., & Cullen, P. J. (2015). Generation of in-package cold plasma and efficacy assessment using methylene blue. Plasma Chemistry and Plasma Processing, 35(6), 1043–1056. Misra, N. N., Martynenko, A., Chemat, F., Paniwnyk, L., Barba, F. J., & Jambrak, A. R. (2018). Thermodynamics, transport phenomena, and electrochemistry of external field-assisted nonthermal food technologies. Critical Reviews in Food Science and Nutrition, 58(11), 1832–1863. Misra, N. N., Sullivan, C., Pankaj, S. K., Alvarez-Jubete, L., Cama, R., Jacoby, F., et al. (2014). Enhancement of oil spreadability of biscuit surface by nonthermal barrier discharge plasma. Innovative Food Science & Emerging Technologies, 26, 456–461. Moisan, M., Barbeau, J., Moreau, S., Pelletier, J., Tabrizian, M., & Yahia, L. (2001). Low temperature sterilization using gas plasmas: A review of the experiments and an analysis of the inactivation mechanisms. International Journal of Pharmacy, 226, 1–21. Niemira, B. A. (2012). Cold plasma decontamination of foods. Annual Review of Food

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