Effectiveness of Ozonation for Improving the Microbiological Safety of Fresh-Cut Parsley (Petroselinum crispum) Leaves

Round 1

Reviewer 1 Report

The manuscript outlines the importance of evaluating novel strategies for bacterial decontamination, which is a timely topic. The manuscript is well written, though the authors could provide more information on the current utility and acceptability of ozone treatment in the industry.

Some minor comments are below

Abstract

Line 20 – Is the ÷ in the microbial contamination level an error?

Introduction

Line 90-91 – Remove the first “of” in the sentence.

Materials and methods

Line 242 – remove “;” and replace with “and”

Results and Discussion

Lines 274-275 – be consistent with the use of either “to” of “-”

All the figures – the y axis of the figures can consistently read “reduction of microorganisms (%)” instead of the way figures 3 and 4 were written.

Figure 2 – re-write the ozone dose “0.5mg/l” not 0,5mg/l

Figure 4 – change “Tytul osi” on the x axis to the appropriate axis title

Lines 427-429: authors should expound that the reduction level they are talking about here is compared to water and not chlorine as the reference article they explained

Conclusion

The authors should express the limitations to the study. For instance, consumer perception on the acceptability of ozone treatment with the slight color change to parsley as observed in the study to accept the conclusion that ozonation can be scaled for industrial use.

Author Response

Our thanks to the reviewers and the subject editor for thoughtful critiques of our manuscript. We have adopted all of the suggestions, including broader literature. We think that the manuscript has been greatly improved by these revisions and we hope that you will now find it suitable for publication. Additionally, the manuscript has been improved and corrected by Dr John Speller native English speaker ([email protected]).  Changed sections have been highlight in yellow.  Our point-by-point responses to comments are detailed bellow.

Answers for Reviewers’ comments

The manuscript outlines the importance of evaluating novel strategies for bacterial decontamination, which is a timely topic. The manuscript is well written, though the authors could provide more information on the current utility and acceptability of ozone treatment in the industry.

Some minor comments are below

Abstract

• Line 20 – Is the ÷ in the microbial contamination level an error?

It has been corrected and rephrased to the following form:  „at 10⁴ to 10⁶ CFU/g for fungi and 10⁶ to 10⁸ CFU/g for bacteria”

Introduction

• Line 90-91 – Remove the first “of” in the sentence.

The sentence has been corrected.

Materials and methods

• Line 242 – remove “;” and replace with “and”

The sentence has been corrected

Results and Discussion

• Lines 274-275 – be consistent with the use of either “to” of “-”

The sentence has been corrected

• All the figures – the y axis of the figures can consistently read “reduction of microorganisms (%)” instead of the way figures 3 and 4 were written.

All y axis titles have been corrected

• Figure 2 – re-write the ozone dose “0.5mg/l” not 0,5mg/l

It has been rewritten

• Figure 4 – change “Tytul osi” on the x axis to the appropriate axis title

The x axis titles has been corrected

• Lines 427-429: authors should expound that the reduction level they are talking about here is compared to water and not chlorine as the reference article they explained

The mistake has been corrected.

 Conclusion

The authors should express the limitations to the study. For instance, consumer perception on the acceptability of ozone treatment with the slight color change to parsley as observed in the study to accept the conclusion that ozonation can be scaled for industrial use.

The whole conclusion section has been rewritten. Additional text has been added also added to Introduction: Lines 48-52 and 129-139.

Author Response File: Author Response.docx

Reviewer 2 Report

General comment:

At a broad level, the authors quantified the microbial content of parsley leaves before and after disinfecting them with various ozone treatments, which varied in ozone volume and exposure time. I believe the authors can make better use of their results.

1. I recommend that this article establish the importance of parsley in terms of food, gastronomy, and food safety. In other words, the authors should highlight the role of parsley in dishes (due to its color, flavor, and aroma) and the importance of ensuring its safety for consumers. Therefore, the results of the microbial count before and after the treatments should be addressed. The article should also include the results of color variation. The well-discussed article could end here, but it would be a plus to include some organoleptic testing, where a panel (even if non-specialized) determines if the taste and smell of parsley changed with the treatments and prior to them.

2. With the identification and quantification results of oils and a thorough analysis, another article could be written to address whether the profile of essential oils was modified before and after the treatments. However, this article should focus on the valorization of parsley leaves (and not on culinary aspects).

My previous recommendations are based on the fact that I believe there is no strong link to quantify the oils, it is not discussed in the justification, and the introduction is not focused on it.

Specific comments:

Title

The title could be improved after reading the comments.

Abstract

El abstract could be improved after reading the comments.

Keywords

The keywords are adequate.

1. Introduction

Lines 44-103: The introduction contains the necessary information; however, the order can be improved. For example, the sentence "The most common pathogens contaminating plant material are Salmonella spp., Bacillus cereus, Staphylococcus aureus, Clostridium perifingens, and Escherichia coli [6-8]" should be placed after "...including opportunistic pathogens, and food spoilage microorganisms [3,4,5]."

Lines 104-115: The objective can be summarized. In lines 104-115, the justification and objective of the study are addressed. However, it would be appropriate to address the justification of the study after line 103 (explaining the choice of spice type), linking it with the previous paragraph, and then briefly stating the objective.

2. Materials and Methods

Lines 120-123. 1) The wording is not the best; it should start by saying something like: "Fresh parsley leaves were obtained...". 2) It is not clear if there were 4 samples of how many grams or of how many leaves. 3) How was it established that this sampling was sufficient or adequate? Was any experimental design conducted?

Lines 125-140. The statistical analysis is so important that it should have a separate section to define it.

Lines 142-184. 1) It is necessary to explain the methods of preliminary identification, whether any kit was used, and provide references. 2) More emphasis should be placed on the operational values of MALDI-TOF MS and less on its interpretation. 3) The last paragraph does not align with "microorganism identification" as indicated in the title. The effectiveness should be described in a separate section.

Lines 185-197. The paragraph is not understandable. The terminology A, B, a, b... is confusing, given that Figure 1 also has sections A and B. It is important to improve the wording.

Figure 1. It is not clear if Figure 1A represents a process flow diagram or a block diagram with names. If it is the former, it is necessary to use appropriate nomenclature and process flow diagrams.

Equation 1. Use the equation function.

Lines 223-234. It is not clear why the oil was extracted.

3. Results and discussion

Table 1. 1) The way the mean and standard deviation are expressed is incorrect. The correct format should be: 8.83x10^4 ± 2.95x10^4. 2) The table footnote should indicate the sample size and the expression of the standard deviation. 3) The statistical analysis should be indicated (columns or rows) and expressed with letters in all assays.

Table 1, Figure 2, and Figure 3. The included results are appropriate, but their presentation can be improved. For example, for better visualization, Figures or Tables can be included showing before and after microbial content, statistically analyzing the reduction, and including a discussion on it.

Figure 3. There are spelling errors.

Figures 2-4. If the axes already have the label "%," it should not be included in the numerical intervals.

Table 3. The same comments as for Table 1 apply here.

Table 4. The data cannot be properly discussed without statistical analysis.

I will limit my review to the discussion since I believe that numerical comparison and statistical analyses of the microorganism concentration before and after would allow for a better discussion of the results.

4. Conclusion

Conclusion is adequate, considering that it can be improved after the proposed corrections mentioned earlier.

5. References

The number of references is adequate, 50% of them are from over 5 years ago.

Author Response

Our thanks to the reviewers and the subject editor for thoughtful critiques of our manuscript. We have adopted all of the suggestions, including broader literature. We think that the manuscript has been greatly improved by these revisions and we hope that you will now find it suitable for publication. Additionally, the manuscript has been improved and corrected by Dr John Speller native English speaker ([email protected]).  Changed sections have been highlight in yellow.  Our point-by-point responses to comments are detailed bellow.

Answers for Reviewers’ comments

General comment:

At a broad level, the authors quantified the microbial content of parsley leaves before and after disinfecting them with various ozone treatments, which varied in ozone volume and exposure time. I believe the authors can make better use of their results.

1. I recommend that this article establish the importance of parsley in terms of food, gastronomy, and food safety. In other words, the authors should highlight the role of parsley in dishes (due to its color, flavor, and aroma) and the importance of ensuring its safety for consumers. Therefore, the results of the microbial count before and after the treatments should be addressed.

Additional information has been added to Indrodiction Lines 55-74.

The article should also include the results of color variation. The well-discussed article could end here, but it would be a plus to include some organoleptic testing, where a panel (even if non-specialized) determines if the taste and smell of parsley changed with the treatments and prior to them.

The authors are aware of the importance of changes in sensory characteristics, which is why they undertook preliminary research related to monitoring changes in color and essential oil profiles. These studies will be continued, and a full senoric assessment may be prepared in a separate elaboration.

2. With the identification and quantification results of oils and a thorough analysis, another article could be written to address whether the profile of essential oils was modified before and after the treatments. However, this article should focus on the valorization of parsley leaves (and not on culinary aspects).

My previous recommendations are based on the fact that I believe there is no strong link to quantify the oils, it is not discussed in the justification, and the introduction is not focused on it.

Specific comments:

Title

The title could be improved after reading the comments.

The title has been improved.

Abstract

El abstract could be improved after reading the comments.

Abstract has been rewritten.

1. Introduction

Lines 44-103: The introduction contains the necessary information; however, the order can be improved. For example, the sentence "The most common pathogens contaminating plant material are Salmonella spp., Bacillus cereus, Staphylococcus aureus, Clostridium perifingens, and Escherichia coli [6-8]" should be placed after "...including opportunistic pathogens, and food spoilage microorganisms [3,4,5]."

The indicated part of the text has been corrected.

Lines 104-115: The Lines 104-115: The objective can be summarized. In lines 104-115, the justification and objective of the study are addressed. However, it would be appropriate to address the justification of the study after line 103 (explaining the choice of spice type), linking it with the previous paragraph,

the previous paragraph, and then briefly stating the objective.

The choice of spice type has been additionelly expained - Lines 58-68.The section considering the objective and aim of the study has been rewitten – Lines 129-139. The objective has been briefly stated - Lines 141-145.

2. Materials and Methods

Lines 120-123. 1) The wording is not the best; it should start by saying something like: "Fresh parsley leaves were obtained...". 2) It is not clear if there were 4 samples of how many grams or of how many leaves. 3) How was it established that this sampling was sufficient or adequate? Was any experimental design conducted?

The indicated part of the text has been corrected.

Lines 125-140. The statistical analysis is so important that it should have a separate section to define it.

The separate chapter Statistical analysis was added to the manuscript - Lines 294-299

Lines 142-184. 1) It is necessary to explain the methods of preliminary identification, whether any kit was used, and provide references. 3) The last paragraph does not align with "microorganism identification" as indicated in the title. The effectiveness should be described in a separate section.

Lines 142-184. 1) Reviewer noted „It is necessary to explain the methods of preliminary identification, whether any kit was used, and provide references”.

We would like to explain to the Reviewer that we performed the initial identification of microorganisms isolated from parsley. For bacteria, we performed a microscopic cell morphology assessment including sporulation, Gram-staining, as well as a biochemical test: Bactident oxidase, Bactident catalase, Bactident L-alanine aminopeptidase which are commercially available (Merck, Darmstadt, Germany). These are standard microbiological procedures that, in accordance with good laboratory practice and literature recommendations, should start the bacterial identification process (Baron, 1996; Franco-Duarte et al., 2019).

According Reviewer's comment we have made changes in the text. Lines 171-175.

Lines 142-184. 2) More emphasis should be placed on the operational values of MALDI-TOF MS and less on its interpretation

According Reviewer comment we have rewrote MALDI-TOF MS identification description drawing more attention on the operational values and less on results  interpretation. Lines 175-193.

Lines 142-184. 3). The last paragraph does not align with "microorganism identification" as indicated in the title. The effectiveness should be described in a separate section.

Thank Rewiever for this comment. We completely agree with Reviewer. Therefore we have made a separate subsection for the last paragraph and titled it „Selection of microorganisms”. We have consequently changed the numbering of subsequent subsections in the methodology.

Lines 185-197. The paragraph is not understandable. The terminology A, B, a, b... is confusing, given that Figure 1 also has sections A and B. It is important to improve the wording.

Sections A and B on Figure 1 has been changed to I and II.

Figure 1. It is not clear if Figure 1A represents a process flow diagram or a block diagram with names. If it is the former, it is necessary to use appropriate nomenclature and process flow diagrams.

The nomenclature has been changed.

Equation 1. Use the equation function.

The equation functionhas been used.

Lines 223-234. It is not clear why the oil was extracted.

 Additional justification has been incorporated in the tekst . Lines 530-536.

3. Results and discussion

Table 1. 1) The way the mean and standard deviation are expressed is incorrect. The correct format should be: 8.83x10^4 ± 2.95x10^4. 2) The table footnote should indicate the sample size and the expression of the standard deviation. 3) The statistical analysis should be indicated (columns or rows) and expressed with letters in all assays.

In the literature there are different ways of expressing the number of microorganisms. Most often the number is presented in the format e.g. 8.83×10⁴ cfu/ml . We removed marking: M-mean, SD- standard deviation, we used partially the format suggested by the reviewer and changed in all tables to the format: 8.83×10⁴ ± 2.95×10⁴.

 We changed tables footnotes as follow: mean ± standard deviation, sample number N=3

1. Statistical analysis was done according reviewer suggestion for the microbial contamination results as well for the reduction of microorganisms number and the optical parameters (Table 1, Table 3, supplementary Tables: S1, S2, S3), all tables footnotes were changed, the statistically different samples are marked with different letters within the same column (a,b,c) or row (A,B,C) (Tukey’s post hoc test at a significance level of 0.05)

Table 1, Figure 2, and Figure 3. The included results are appropriate, but their presentation can be improved. For example, for better visualization, Figures or Tables can be included showing before and after microbial content, statistically analyzing the reduction, and including a discussion on it.

Table 3. The same comments as for Table 1 apply here.

Presentation results was improved, in the supplementary Tables S1-S3 the number of microorganisms before and after ozonation in different time are marked. Statistical analysis of the microorganisms reduction for each measurement was performed, thanks to which standard deviations were added to the reduction numbers in Figures: Fig.2, Fig.3, Fig.4, moreover in the supplementary Tables S1-S3 and the Tukey statistical test was performed for these results, which allowed to illustrate the differences between the results. In addition, fragments of the text describing the results obtained have been changed according to statistical analysis.

Figure 3. There are spelling errors.

Spelling errors have been corrected.

Figures 2-4. If the axes already have the label "%," it should not be included in the numerical intervals.

It was correted.

Table 4. The data cannot be properly discussed without statistical analysis.

I will limit my review to the discussion since I believe that numerical comparison and statistical analyses of the microorganism concentration before and after would allow for a better discussion of the results.

Statistical analysis was included in the description and discussion of the results.

4. Conclusion

Conclusion is adequate, considering that it can be improved after the proposed corrections mentioned earlier.

The whole conclusion section has been rewritten. Additional

5. References

The number of references is adequate, 50% of them are from over 5 years ago.

The list of references has been extended including publications from the last five years: 1, 13, 27, 42, 47, 48, 50.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The manuscript improved; however, I consider that it lacks scientific quality to support its results.

General comment:

I thank the authors for their responses. Indeed, the manuscript has improved considerably; however, I believe that there are some important points that cannot be ignored, since they are requirements in all scientific research.

1. Figure 1I is not a process flow diagram, to be one you need to use the equipment and nomenclature figures.

2. The abstract has the necessary information; however, the organization of ideas is important: context, objective, important methodology and relevant results (perspectives may exist).

3. The introduction has the necessary information; however, it must also be organized: from the general to the specific. For example, lines 47-53 are well organized, then decontamination techniques can be addressed (lines 82-137), then all about parsley (lines 54-81) and finally the objective. It is important that the authors consider diminishing the information on parsley and highlight only what is important.

4. Table 1. In the statistical analyzes the assignment of letters is from highest to lowest, that is, a is assigned to the highest value. On the other hand, reviewing the standard deviations, you can realize that they are very high. It is to be expected for biological models, however, for such models, the way to report the data is with logarithmic or percentage adjustments. In addition to this, Tukey is not an appropriate method for data analysis in biological models. On the other hand, this table has made me question myself, what is the basis for analyzing the microbiological content between seasons and between microorganisms?

5. Table 3. Same comments as table 1.

6. Table 4. The authors express in the analysis (Lines 535-537) that there were no significant changes, however, the table does not show a statistical analysis.

Author Response

All authors would like to thank the reviewer for further valuable comments and for consideration of publication of the manucript.

General comment:

I thank the authors for their responses. Indeed, the manuscript has improved considerably; however, I believe that there are some important points that cannot be ignored, since they are requirements in all scientific research.

1. Figure 1I is not a process flow diagram, to be one you need to use the equipment and nomenclature figures

Figure 1I has been corrected using nomenclature figures. Additional information about the equipment has been added to the figure caption.

2. The abstract has the necessary information; however, the organization of ideas is important: context, objective, important methodology and relevant results (perspectives may exist).

The organization of ideas has been changed. Additional information has been added.

3. The introduction has the necessary information; however, it must also be organized: from the general to the specific. For example, lines 47-53 are well organized, then decontamination techniques can be addressed (lines 82-137), then all about parsley (lines 54-81) and finally the objective. It is important that the authors consider diminishing the information on parsley and highlight only what is important.

The organization of introduction and references sections has been changed. The information on parsley has been shortened.

4. Table 1. In the statistical analyzes the assignment of letters is from highest to lowest, that is, a is assigned to the highest value. On the other hand, reviewing the standard deviations, you can realize that they are very high. It is to be expected for biological models, however, for such models, the way to report the data is with logarithmic or percentage adjustments. In addition to this, Tukey is not an appropriate method for data analysis in biological models. On the other hand, this table has made me question myself, what is the basis for analyzing the microbiological content between seasons and between microorganisms?
5. Table 3. Same comments as table 1.

The results obtained from microbiological analyzes are always subjected to a preliminary Grubbs analysis, also in this case we performed such analysis. Indeed, in microbiological studies, standard deviations are high, also in the present studies. We have used Tukey's test many times for our microbiological analyses, also for disinfection effect , always with good results (this is evidenced by our publications in journals MOLECULES, DOI: 10.3390/molecules28083560. INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH, DOI: 10.3390/ijerph20021551, JOURNAL OF ENVIRONMENTAL MANAGEMENT, DOI: 10.1016/j.jenvman.2021.114257, INTERNATIONAL BIODETERIORATION AND BIODEGRADATION, DOI: 10.1016/j.ibiod.2017.01.021; DOI: 10.1016/j.ibiod.2017.02.011 and many other). We are not aware of microbiological tests of food products, plant materials and disinfected materials in which percentage corrections are used. Statistical analyzes with percentage corrections were used in air microbial analysis and correction tables are provided by sampler manufacturers. In our opinion, the statistical/percentage correction will not change the substantive assessment and will not affect the quality of this study. We have used the comparative analysis between groups of microorganisms and seasons of the year as suggested by the reviewer in the first round of improvement (quote: Table 1. The statistical analysis should be indicated (columns / rows) and expressed with letters in all assays). The earlier version was only a comparison between the seasons. This reviewer's suggestion seemed reasonable and answered questions about the influence of the season and microorganisms groups dominated in the tested parsley samples. 

6. Table 4. The authors express in the analysis (Lines 535-537) that there were no significant changes, however, the table does not show a statistical analysis

Thank you for this remark, we corrected the description according to the table, description “significant qualitative” was removed from the text.

Round 3

Reviewer 2 Report

The manuscript improved