An Effective Sanitizer for Fresh Produce Production: In Situ Plasma-Activated Water Treatment Inactivates Pathogenic Bacteria and Maintains the Quality of Cucurbit Fruit

ABSTRACT The effect of plasma-activated water (PAW) generated with a dielectric barrier discharge diffusor (DBDD) system on microbial load and organoleptic quality of cucamelons was investigated and compared to the established sanitizer, sodium hypochlorite (NaOCl). Pathogenic serotypes of Escherichia coli, Salmonella enterica, and Listeria monocytogenes were inoculated onto the surface of cucamelons (6.5 log CFU g−1) and into the wash water (6 log CFU mL−1). PAW treatment involved 2 min in situ with water activated at 1,500 Hz and 120 V and air as the feed gas; NaOCl treatment was a wash with 100 ppm total chlorine; control treatment was a wash with tap water. PAW treatment produced a 3-log CFU g−1 reduction of pathogens on the cucamelon surface without negatively impacting quality or shelf life. NaOCl treatment reduced the pathogenic bacteria on the cucamelon surface by 3 to 4 log CFU g−1; however, this treatment also reduced fruit shelf life and quality. Both systems reduced 6-log CFU mL−1 pathogens in the wash water to below detectable limits. The critical role of superoxide anion radical (·O2−) in the antimicrobial power of DBDD-PAW was demonstrated through a Tiron scavenger assay, and chemistry modeling confirmed that ·O2− generation readily occurs in DBDD-PAW generated with the employed settings. Modeling of the physical forces produced during plasma treatment showed that bacteria likely experience strong local electric fields and polarization. We hypothesize that these physical effects synergize with reactive chemical species to produce the acute antimicrobial activity seen with the in situ PAW system. IMPORTANCE Plasma-activated water (PAW) is an emerging sanitizer in the fresh food industry, where food safety must be achieved without a thermal kill step. Here, we demonstrate PAW generated in situ to be a competitive sanitizer technology, providing a significant reduction of pathogenic and spoilage microorganisms while maintaining the quality and shelf life of the produce item. Our experimental results are supported by modeling of the plasma chemistry and applied physical forces, which show that the system can generate highly reactive ·O2− and strong electric fields that combine to produce potent antimicrobial power. In situ PAW has promise in industrial applications as it requires only low power (12 W), tap water, and air. Moreover, it does not produce toxic by-products or hazardous effluent waste, making it a sustainable solution for fresh food safety.

This is the part where the need for disinfection of fresh produce should be most emphasized to contribute to food safety. It is recommended to present the current status of food poisoning caused by fresh produce using the latest statistics.
C2 Materials and methods (Figure 1, line 145-147) Unlike conventional PAWs, this device has a structure in which plasma is injected into water in real time. Therefore, the section where electrical discharge takes place is located very closely with water. Could this structure be a dangerous environment for workers? C3 Materials and methods (line 181-184) It is recommended to make the expression clearer. [ten × 10 mL]: Is 100 mL of bacterial suspension (200 mL for L. monocytogenes) used in total?
C4 Materials and methods (Figure 1, It is recommended to specify the position of the sample in the solution. In particular, the author explains that depending on the position of the sample in the solution, the electric field may be affected differently. In addition, the behavior of the sample may vary as bubbles enter the solution. As a result, the bactericidal effect of this device may be reduced in environments where the sample is located at the top of the solution. This means that the effect may vary depending on how many layers the sample is stacked in the solution. In addition, the effect may vary if the sample is light and floats in water. C5 Results / Discussion ( Figure 6, line 622-624) The author explained that the amount of ozone dissolved in PAW is small due to the low solubility of ozone. Ozone is the most significant gas generated by a typical DBD system, and it is thought that a significant amount of ozone will also be generated in this system. Therefore, much of the ozone that is eventually insoluble in water can be exposed above the liquid. Please give us your opinion on this part and suggest alternative measures to the possibility of ozone exposure as the author considers industrial applicability important.
C6 Discussion (Figure 8, line 596-603) Is there any way to directly observe how the electric field actually affected microbial cells? If the electric field is forming to a degree that has a profound effect on cells as the author suggests, I wonder if some cells should be killed even in an environment with tiron (·O2-scavenger) added. Perhaps this is most similar to the environment in which only electrical discharge, other than reactive species, affects. How about observing cell membrane integrity after electrical discharge with the addition of tiron?
Reviewer #5 (Comments for the Author): The manuscript by Rothwell et al. describes multidisciplinary research in the field of food science evaluating the use of plasma activated water for sanitizing cucamelons. It is the perception of this reviewer that the modelling components described in the current version would typically not be assessed or used by microbiologists. The modelling could have better served as an initial approach to optimize treatments so that a greater log reduction of pathogens was achievable. As presented the modeling is used to demonstrate mechanism. The ability of an audience of microbiologists to benefit from the modeling data is questionable. The description of the modeling activities is vague in the materials and methods section for a microbiology audience. It is unclear how the discharge volume was estimated (line 309), how the residence time was calculated (line 310), and how were the settings applied for the activated plasma were chosen. Also, how is the E/N ratio used? A number of gas products are considered in Table S1 but the text implies that only one of those is relevant in the system used. Please resolve this incongruence, is table S1 needed? Are nitrogen oxides produced in the plasma system chosen, possibly from N2 in air? It seems that the rapid solvation of the oxygen species (line 629) works against the effectiveness of the treatment as the log reduction achieved is minimal and the produce spoils in about 10 days. Can the PAW be optimized to increase the shelf-life of the produce? The modeling section should occur first in the materials and methods, so that it aligns with Figure 2 as an introductory piece. Although the protocols used for microbial counts, and texture, color and appearance evaluation may be standard, references are needed to support them in the materials and methods section. A sentence should be added in line 132 about the use of tiron as a control treatment. It seems to come out of nowhere when the reader first encounters it in line 201. Tiron inclusion is working as a control treatment and should be presented as such. Cucamelons, like other fruits and vegetables, are susceptible to equilibration with water in its surroundings (reference 32). Such dynamic needs to be considered regarding the use of solutions that were refrigerated (4C) prior to treatment application (line 170). The equilibration of fruits and vegetables with water depends on temperature. Was the temperature of the fruits during treatment measured? Were the solutions tempered? Also, was there any indication in the electron microscopy work that the pathogens had penetrated the cucamelons' flesh? The effectiveness of the sanitizer to that of the plasma treatment may not be comparable as one treatment can reduce the microbes in the 1mm outer flesh near the skin, but plasma cannot. Please elaborate on this aspect. (Mattos FR, Fasina OO, Reina LD, Fleming HP. Breidt F Jr, Damasceno GS, Passos FV. 2005. Heat transfer and microbial kinetics modeling to determine the location of microorganisms within cucumber fruit. J Food Sci 70(5):E324-E330.) The use of tap water (line 103 &139) and lactic acid (line 145): Was the mineral content of the tap water used evaluated? Would researchers be able to reproduce your findings with any tap water? Lactic acid (10% or 1.1 M) is an antimicrobial that was added to the chlorine solution for pH control. Thus, the sanitizer treatment could have been more effective than it typically is. Is lactic acid routinely used by processors in the wash waters for pH control? Is there a reason for not studying the flavor of the treated and control samples? Minor comments: Line 190: How was the probe sanitized/sterilized between treatments? Line 206: What was the definition of biological controls? Line 231, 239 and throughout the manuscript: Please use the term microbiota instead of microflora (small plants). Line 239: Assessment of the microbial load on cucamelons as a function of treatment. Line 79: ...from food sanitation are hazardous... Line 296 to 302: panelist Line 423 to 425: Please show data for microbial counts from wash waters. Line 662: Please spell out CTO Staff Comments:

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Report on Spectrum00034-23 by J.G. Rothwell et al
The submitted manuscript reports on the use of dielectric barrier discharge diffusor for decontamination of bacteria in solution and on the cucamelon surface. The author tried to design experiments considering the economic feasibility of the technology for industrial application. As an innovative approach in a demanding technological field, it results that the paper may deserves publication in the Microbiology Spectrum but after consideration of the 6 following comments.

C1
Introduction (line 64-68) This is the part where the need for disinfection of fresh produce should be most emphasized to contribute to food safety. It is recommended to present the current status of food poisoning caused by fresh produce using the latest statistics.

C2
Materials and methods ( Figure 1, line 145-147) Unlike conventional PAWs, this device has a structure in which plasma is injected into water in real time. Therefore, the section where electrical discharge takes place is located very closely with water. Could this structure be a dangerous environment for workers?

C3
Materials and methods (line 181-184) It is recommended to make the expression clearer. [ten × 10 mL]: Is 100 mL of bacterial suspension (200 mL for L. monocytogenes) used in total?

C4
Materials and methods ( Figure 1, line 188-189) It is recommended to specify the position of the sample in the solution. In particular, the author explains that depending on the position of the sample in the solution, the electric field may be affected differently. In addition, the behavior of the sample may vary as bubbles enter the solution. As a result, the bactericidal effect of this device may be reduced in environments where the sample is located at the top of the solution. This means that the effect may vary depending on how many layers the sample is stacked in the solution. In addition, the effect may vary if the sample is light and floats in water.

C5
Results / Discussion ( Figure 6, line 622-624) The author explained that the amount of ozone dissolved in PAW is small due to the low solubility of ozone. Ozone is the most significant gas generated by a typical DBD system, and it is thought that a significant amount of ozone will also be generated in this system. Therefore, much of the ozone that is eventually insoluble in water can be exposed above the liquid. Please give us your opinion on this part and suggest alternative measures to the possibility of ozone exposure as the author considers industrial applicability important.

C6
Discussion (Figure 8, line 596-603) Is there any way to directly observe how the electric field actually affected microbial cells? If the electric field is forming to a degree that has a profound effect on cells as the author suggests, I wonder if some cells should be killed even in an environment with tiron (·O2scavenger) added. Perhaps this is most similar to the environment in which only electrical discharge, other than reactive species, affects. How about observing cell membrane integrity after electrical discharge with the addition of tiron?
End of report.

Response to reviewers (Rothwell et al, Spectrum00034-23)
We thank both reviewers for their detailed and helpful comments, which we address below. We believe these changes have greatly improved our manuscript and hope that it is now acceptable for publica on in mSpectrum.

Reviewer #4:
The submi ed manuscript reports on the use of dielectric barrier discharge diffusor for decontamina on of bacteria in solu on and on the cucamelon surface. The author tried to design experiments considering the economic feasibility of the technology for industrial applica on. As an innova ve approach in a demanding technological field, it results that the paper may deserves publica on in the Microbiology Spectrum but a er considera on of the 6 following comments. C1 Introduc on (line 64-68) This is the part where the need for disinfec on of fresh produce should be most emphasized to contribute to food safety. It is recommended to present the current status of food poisoning caused by fresh produce using the latest sta s cs.
 The first paragraph of the introduc on has been reworked to be er emphasise the importance of sani zers for fresh produce safety and we have added sta s cs on foodborne disease outbreaks due to fresh produce (lines 64-70). Unfortunately, we were not able to find a more recent paper that specifically reported on fresh produce than Li et al 2018.
C2 Materials and methods (Figure 1, line 145-147) Unlike conven onal PAWs, this device has a structure in which plasma is injected into water in real me. Therefore, the sec on where electrical discharge takes place is located very closely with water. Could this structure be a dangerous environment for workers?
 We agree that this will be an important considera on for future studies involving scale up and applica on of the technology and have added a sentence at the end of the paper (line 591-592) describing this as a possible limita on, along with the build-up of ozone men oned below in C5.

C3 Materials and methods (line 181-184)
It is recommended to make the expression clearer. [ten × 10 mL]: Is 100 mL of bacterial suspension (200 mL for L. monocytogenes) used in total?
 The text has been amended as follows: "Each cucamelon was then spot-inoculated with ten x 10 L (100 L total) of the E. coli or S. enterica inoculum at a final concentra on of 1 × 10 9 colony forming units (CFU) mL -1 . Cucamelons were spot-inoculated with twenty × 10 L spots of (200 L total) of L. monocytogenes at 8 × 10 8 CFU mL -1 , as this species has a lower adhesion to cucumber (33)." (Lines 180-184).
C4 Materials and methods (Figure 1, line 188-189) It is recommended to specify the posi on of the sample in the solu on. In par cular, the author explains that depending on the posi on of the sample in the solu on, the electric field may be affected differently. In addi on, the behavior of the sample may vary as bubbles enter the solu on. As a result, the bactericidal effect of this device may be reduced in environments where the sample is located at the top of the solu on. This means that the effect may vary depending on how many layers the sample is stacked in the solu on. In addi on, the effect may vary if the sample is light and floats in water.
 We agree with the reviewer comment that the posi on of the sample in rela on to the plasma electrodes is an important considera on in this study and for in situ PAW studies in general. Movement of the cucamelons in the wash solu on is dynamic due to the bubbling of the plasma reactor. In our modelling study we addressed this by examining two cucamelon posi ons, one with the cucamelon next to the reactor (Figure 7) and one with it towards the top of the water ( Figure S3). The bacterial cells posi oned on the cucamelon near the water surface would be expected to experience the lowest levels of electrical forces in the system, which we calculated to be approximately 0.55 kV/cm, which is around a quarter of the strength of the maximum electric field experienced when cucamelon was next to the reactor (2 kV/cm). This is explained in the text from lines 466-470: "These demonstrate that a high local electric field of over 2.0 kV/cm can be formed inside the bacterial cell under these condi ons. This is strongly dependent on the rela ve posi on of individual bacterial cells; when the cucamelon is located at the top of the water the maximum local electric field experienced by the bacteria is much lower at 0.24 to 0.55 kV/cm". We have modified the discussion to specify that "For future scale-up of this technology, reactor design and the posi on of the electrodes in the wash systems must be considered to guarantee effec ve an microbial power and thereby maximize food safety" (lines 593-5). We believe this is a sufficient caveat for the scope of this study.
C5 Results / Discussion ( Figure 6, line 622-624) The author explained that the amount of ozone dissolved in PAW is small due to the low solubility of ozone. Ozone is the most significant gas generated by a typical DBD system, and it is thought that a significant amount of ozone will also be generated in this system. Therefore, much of the ozone that is eventually insoluble in water can be exposed above the liquid. Please give us your opinion on this part and suggest alterna ve measures to the possibility of ozone exposure as the author considers industrial applicability important.
 We agree with the reviewer that the produc on of ozone and other poten ally toxic gasses from the PAW systems will be an important safety considera on for upscale and applica ons of the technologies in future studies, and ven la on and fume extrac on systems will likely need to be employed. We have noted this safety considera on alongside considera ons of electrical hazards at the end of the discussion (lines 591-593).
C6 Discussion (Figure 8, line 596-603) Is there any way to directly observe how the electric field actually affected microbial cells? If the electric field is forming to a degree that has a profound effect on cells as the author suggests, I wonder if some cells should be killed even in an environment with ron (·O2-scavenger) added. Perhaps this is most similar to the environment in which only electrical discharge, other than reac ve species, affects. How about observing cell membrane integrity a er electrical discharge with the addi on of ron?  The effects of the pulsed electric field on cells have previously been explored by another study (Mentheour R, Machala Z. Coupled An bacterial Effects of Plasma-Ac vated Water and Pulsed Electric Field. Fron ers in Physics. 2022;10.) which was men oned in our study on lines 131-133. While we agree with the reviewer that this would be an interes ng area of future research, we believe that this is outside of the scope of the current study.

Reviewer #5:
The manuscript by Rothwell et al. describes mul disciplinary research in the field of food science evalua ng the use of plasma ac vated water for sani zing cucamelons. It is the percep on of this reviewer that the modelling components described in the current version would typically not be assessed or used by microbiologists. The modelling could have be er served as an ini al approach to op mize treatments so that a greater log reduc on of pathogens was achievable. As presented the modeling is used to demonstrate mechanism. The ability of an audience of microbiologists to benefit from the modeling data is ques onable.
 We understand the concern regarding the applicability of the modelling components described in our study to microbiologists. However, we believe that the modelling approach has significant value for both microbiologists and PAW researchers. Op misa on of PAW parameters to achieve the highest an microbial ac vity was completed in our previous study (Rothwell et al. "The an microbial efficacy of plasma-ac vated water against Listeria and E. coli is modulated by reactor design and water composi on." Journal of Applied Microbiology 132.4 (2022): 2490-2500). The modelling in the current study built on this and on our new SEM work to be er understand how the highly effec ve an microbial capacity of this system was generated and to advance knowledge in this emerging and mul disciplinary field. While plasma modelling is not a typical component of a microbiology research paper, we feel that presen ng a comprehensive study that combines experimental data with modelling can provide a deeper understanding of the mechanisms at play, and that this approach can facilitate cross-disciplinary collabora on and spark new research ques ons that can drive innova on in the field.
The descrip on of the modeling ac vi es is vague in the materials and methods sec on for a microbiology audience. It is unclear how the discharge volume was es mated (line 309), how the residence me was calculated (line 310), and how were the se ngs applied for the ac vated plasma were chosen.
 To address the discharge volume and residence me comment, addi onal informa on has been added to the text: "The discharge volume was es mated to be 1.1 cm 3 based on the reactor's dimensions, which included an outer radius of 0.4 cm, an inner radius of 0.3 cm, and a height of 5 cm. By dividing the discharge volume by the gas flow rate, the residence me of gas species within the discharge volume was determined to be 0.067 seconds at the gas flow rate of 1 SLM" (Lines 306-310).  To address the comment on how the se ngs for the plasma ac va on were chosen, further clarifica ons have been made in the text as follows: "PAW was generated with a DBDD probe (PlasmaLeap Technologies) and power was supplied from a Leap100 micropulse generator (PlasmaLeap Technologies). The power supply se ngs used were based on the findings of our previous study (30). These se ngs included a frequency of 1500 Hz, a voltage of 120 volts, a duty cycle of 100 microseconds, and a resonance frequency of 60 kHz. Compressed air at a flow rate of 1 standard litre per minute (SLM) was used as the processing gas." (Lines 148-153).
Also, how is the E/N ra o used?
 The E/N ra o is an important parameter used in all of the plasma modelling in the current study. It represents the ra o of the electric field strength (E) to the gas density (N) in the plasma. The E/N ra o provides insights into the energy levels and behaviour of electrons, making it a crucial factor in plasma modelling.  The following text has been amended in the methods to be er explain how the E/N ra o was used: "The reduced electric field (E/N) is calculated by dividing the electric field strength (E) by the density of neutral gas molecules (N) in the plasma. The E/N ra o is related to the energy distribu on of electrons in the plasma, and it is an important parameter for plasma modelling. However, plasma exhibits highly transient and non-uniform characteris cs, as depicted in Figure S1. Therefore, an averaged E/N value that accounts for these spa al and temporal varia ons was es mated using the QV Lissajous plot technique (39). The es mated E/N was approximately 30 Td as shown in Table S1 and so E/N values of 30, 40 and 50 Townsends (Td) were used for the ini al plasma chemistry simula on. The influence of these parameters on the produc on of ·O2-and other important gas products listed in Table 2 was inves gated. As the gas composi on at 30 Td was closest to that determined empirically in our previous study (30) we used 30 Td as the assumed E/N value used for subsequent plasma simula ons." (Line 314-325)  The following addi onal table has been included in the supplementary figures to help address this reviewer comment: Table S1. Es mated characteris c capacitance of the given plasma system and calculated reduced electric field (E/N) following Wagner et al. (39)  A number of gas products are considered in Table S1 but the text implies that only one of those is relevant in the system used. Please resolve this incongruence, is table S1 needed? Are nitrogen oxides produced in the plasma system chosen, possibly from N2 in air?
 The species listed in what is now Table S2 are the total species included in the model. In Fig 6( Table S2 are from reac ons in the N 2 /O 2 (air) plasma system, therefore the nitrogen oxides are from the reac on of the air molecules in the plasma discharge.
It seems that the rapid solva on of the oxygen species (line 629) works against the effec veness of the treatment as the log reduc on achieved is minimal and the produce spoils in about 10 days. Can the PAW be op mized to increase the shelf-life of the produce?
 We respec ully disagree with the comment that log reduc on is minimal and the produce spoils in about 10 days. As demonstrated in Figure 3, PAW treatment was highly effec ve, reducing the counts of pathogenic bacteria on the cucamelon surface by 3 log CFU g -1 , and completely elimina ng pathogens from the wash water. It demonstrated comparable an microbial efficacy to that of the established sani ser NaOCl, while using a rapid 2-minute wash me. With regard to shelf-life, as noted in the methods a quality scores of 3 and lower indicates that the product is no longer within acceptable specifica ons for consump on, and Figure 5 E demonstrates that the quality of the cucamelons treated with PAW remained above 3 throughout the 28 day storage trial. We have modified the text from lines 413-417 to make this clearer.  It is possible that PAW could be further op mised but there will always be a trade-off between an microbial efficacy and the quality of delicate fresh produce.
The modeling sec on should occur first in the materials and methods, so that it aligns with Figure 2 as an introductory piece.
 The structure of the manuscript begins with the an microbial capacity of PAW and its effect on produce quality, which is followed by a more in-depth analysis on the mode of ac on through modelling. We feel this order is most appropriate for a microbiology journal. Please also note that modelling is itself a result and was developed based on the results of the SEM and an microbial work in order to be er understand how PAW affects bacterial cells.
Although the protocols used for microbial counts, and texture, color and appearance evalua on may be standard, references are needed to support them in the materials and methods sec on.
 As cucamelons are not a commonly studied fresh produce item, the evalua on methods for texture, color and appearance were developed by our group for this study, and no prior references exist.
A sentence should be added in line 132 about the use of ron as a control treatment. It seems to come out of nowhere when the reader first encounters it in line 201. Tiron inclusion is working as a control treatment and should be presented as such.
 To address this comment, addi onal text has been added to the introduc on as follows: "… assays using the ·O2-scavenger ron demonstrated that ·O2-was essen al for the an microbial ac vity of this system." (Line 121) Cucamelons, like other fruits and vegetables, are suscep ble to equilibra on with water in its surroundings (reference 32). Such dynamic needs to be considered regarding the use of solu ons that were refrigerated (4C) prior to treatment applica on (line 170). The equilibra on of fruits and vegetables with water depends on temperature. Was the temperature of the fruits during treatment measured? Were the solu ons tempered?
 In our methods we specify that the "All treatments were made using autoclaved tap water cooled to 4 °C in a final volume of 200 mL" (line 143). We have clarified on line 177 that the cucamelons "were stored at 9 °C with 85% humidity" prior to the experiment.
Also, was there any indica on in the electron microscopy work that the pathogens had penetrated the cucamelons' flesh? The effec veness of the sani zer to that of the plasma treatment may not be comparable as one treatment can reduce the microbes in the 1mm outer flesh near the skin, but plasma cannot. Please elaborate on this aspect.  As specified in the introduc on of the ar cle, ("Sani zers are cri cal for reducing the risk of cross contamina on by pathogens that may have been transferred into the wash solu on", Line 72) the cri cal role of sani sers is to prevent cross-contamina on by killing pathogens in the wash water. In this study, we demonstrate the capacity for PAW to reduce pathogens to below detec ble limits in the wash water using a fresh produce model. While assessment of the penetra on of pathogens into the cucamelon surface is an interes ng concept for future studies, we believe that this is outside the scope of the current paper where the focus was to determine the efficacy of PAW with reference to the established sani ser NaOCl.
The use of tap water (line 103 &139) and lac c acid (line 145): Was the mineral content of the tap water used evaluated? Would researchers be able to reproduce your findings with any tap water? Lac c acid (10% or 1.1 M) is an an microbial that was added to the chlorine solu on for pH control. Thus, the sani zer treatment could have been more effec ve than it typically is. Is lac c acid rou nely used by processors in the wash waters for pH control?
 To address the comment on the tap water used in this study, addi onal informa on has been added to line 143-144 of the text: "The chemical analysis of the Sydney tap water used in this study has been published previously (30)."  Producers must use food safe acid to acidify their sani ser solu ons in produc on, therefore the use of lac c acid was deemed appropriate in this study. A small number of drops of the 10% lac c acid (<1mL) were added to the solu on to reduce the pH, which is insignificant compared to the concentrated chlorine solu on that provides the an microbial ac on. Acidifica on of chlorine solu ons is standard prac ce in food safety sani ser work.
Is there a reason for not studying the flavor of the treated and control samples?