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by
  • Yaiza Rodríguez,
  • Juan Manuel Del Fresno and
  • Antonio Morata *
  • et al.

Reviewer 1: Anonymous Reviewer 2: Anonymous Reviewer 3: Anonymous Reviewer 4: Anonymous Reviewer 5: Anonymous

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

The topic of your manuscript, entitled "Ozone as a sanitation method in winemaking: improving fermentation control in the context of climate change" is very interesting and up-to-date. The review is generally well structured and it successfully integrates climate change challenges, microbial control strategies, and emerging technologies. The comparison with other non-thermal technologies is especially valuable. However, there some things that should be clarified in order to improve manuscript quality.

First, there is no connection between the title and the manuscript topic. The only one place where is mentioned climate change and its effect on grape and wine quality is Introduction.

Second, it is very strange that according to you ozone can be used as a pipe sanitizer because ozone is strong oxidant and if there is ozone residue in the pipe after desinfection it will oxidize wine. Moreover, ozone can oxidize some of grape substances such as lipids, terpens, etc. So, it will be good to mentioned it

Third, there is no data about EU regulation aboutusing ozone in food and wine production.

Fourth, it will be good to extend the information about ozone safety and how ozone concentration in wineries should be measured.

Fifth, please, explain the idea of Figure 3. According to me, Table 2 is enough. Moreover, it is very interesting to me how you chose the values in radar diagram for different criteria.

Minor revisions

ln. 85 Please, use Italic for Latin names

ln. 236, 249, 276, 277, 287 Please, use the name of the first author or the reference, followed by  the reference number

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors:

The manuscript entitled “Ozone as a sanitation method in winemaking: improving fermentation control in the context of climate change” addresses a highly relevant topic for contemporary oenology. The progressive reduction of sulfur dioxide inputs, increasing microbial instability under warmer harvest conditions, and the need for more sustainable sanitation strategies justify a critical evaluation of ozone technologies in wineries. The review provides a useful mechanistic overview of ozone chemistry, summarizes reported antimicrobial effects, and attempts to position ozone within a broader sustainability framework.

However, several conceptual and structural aspects require clarification and strengthening to ensure scientific rigor, prevent overgeneralization, and enhance practical relevance for winery operations. The following points outline key areas for improvement.

 

1.- Clearer Separation Between Postharvest and Winery Applications

The manuscript currently merges evidence derived from table-grape storage and cold-room postharvest trials with winery-relevant sanitation practices (e.g., sorting-table sprays, fermentation management). These systems differ substantially in objectives, exposure times, environmental control (temperature, relative humidity), and microbial ecology. Presenting them as a unified evidence base risks overstating the operational feasibility of ozone under real winery conditions.

A clearer structural separation is recommended (or eliminate postharvest items):

- Section A: Ozone in grape postharvest/storage systems (cold storage, long exposure, RH control).

- Section B: Ozone in winery environments (sorting tables, equipment sanitation, fermentation management).

This distinction would prevent extrapolation beyond the experimental context and enhance technical credibility.

Differentiation of Microbial Targets:

The review would benefit from a more precise differentiation of sanitation objectives, which currently appear conflated:

- Reduction of apiculate yeasts and acetic acid bacteria at crush: with the aim of limiting early spoilage or excessive volatile acidity.

- Control of Brettanomyces bruxellensis: typically a cellar-ageing and bottling-line contamination problem rather than exclusively a grape-surface issue.

-  Influence on lactic acid bacteria (LAB): distinguishing between suppression of unwanted spoilage LAB and preservation of controlled malolactic fermentation.

In particular, Brettanomyces should be framed explicitly as a major risk in barrel ageing and bottling environments, supported by authoritative technical documents and winery-based studies rather than only grape-level experiments.

Moreover, where ozone application on grapes is discussed as a strategy to limit Brettanomyces, the manuscript should incorporate controlled winemaking studies in which inoculated grapes were treated with aqueous or gaseous ozone and fermentation outcomes were monitored. Without this, the argument remains suggestive rather than demonstrative.

2) Operationalization: Adding an “Engineering” Dimension

The manuscript effectively discusses qualitative factors influencing ozone efficacy (eg.: pH, temperature, organic matter), yet it stops short of providing actionable operational guidance. For practitioners and researchers alike, translation into applied parameters is essential.

CT-Based Guidance:

A short dedicated subsection should translate available data into CT-style considerations (concentration × time), clearly separating:

- Aqueous ozone applications

- Gaseous ozone applications

For each, the authors should explicitly address:

- Dependence on organic load (e.g., COD), turbidity, and matrix composition

- Limitations imposed by rapid ozone decay in must or wash water

- The matrix-specific nature of thresholds (e.g., statements such as rapid decay above COD ≥ 9 mg/L must be properly sourced and contextualized)

Such clarification would prevent oversimplification and enhance reproducibility.

Humidity in Gaseous Applications: For gaseous ozone, the role of relative humidity should be operationalized rather than described qualitatively. The manuscript should indicate approximate RH ranges under which antimicrobial efficacy improves and explain mechanistically why dry environments may reduce effectiveness (limited surface moisture and reduced radical formation).

Mass-Transfer Constraints in Aqueous Systems:  For aqueous ozone, the review briefly mentions diffusion and transfer issues. This section should be consolidated into a practical summary of what wineries must control:

- Bubble size and diffuser design

- Contact geometry

- Mixing intensity

- Ozone generator capacity and real-time monitoring

This “engineering layer” would significantly increase the manuscript’s applied value.

3) CIP and Bottling-Line Sanitation: Strengthening the Sustainability Argument

The sustainability dimension is one of the strongest premises of the review, yet it is not fully supported with concrete winery-based evidence.

A dedicated subsection titled, for example, “Ozone in CIP and Bottling Hygiene” would improve coherence. This section should:

- Summarize microbial inactivation data from winery bottling-line or equipment sanitation studies.

- Compare ozone with conventional agents such as peracetic acid or chlorine-based systems.

- Include environmental indicators where available (water consumption, chemical residues, occupational safety considerations).

Explicit comparison with conventional CIP strategies would allow readers to evaluate ozone not only as a microbiological tool but as a process-integrated sustainability measure.

4) Barrel Sanitation: Avoiding Overgeneralization

Barrels represent one of the most challenging sanitation contexts in oenology due to wood porosity and microbial penetration depth. Ozone is frequently described as promising; however, its efficacy is highly protocol-dependent.

The manuscript should:

- Cite controlled studies evaluating microflora reduction in barrels after ozone treatment.

- Discuss reported impacts on subsequent wine composition and sensory attributes.

- Integrate systematic reviews emphasizing that protocol standardization (exposure time, concentration, gas vs aqueous phase) remains unresolved.

Without this nuance, statements on barrel sanitation risk appearing overly optimistic.

5) Comparative Section and Radar Chart (Figure 3): Improving Reproducibility

The comparative radar chart currently lacks methodological transparency. The scoring criteria and weighting appear subjective, and the evidence base supporting each axis is not explicitly mapped.

To ensure scientific robustness, the authors should either:

Replace the radar chart with a systematic, referenced comparison table including:

Defined evaluation criteria, explicit scoring rubric, citations supporting each score, indication of evidence strength, or

Retain the figure but substantially revise the caption to include:

Scoring methodology, scale anchors, weighting approach, number and type of studies informing each parameter, acknowledgment of uncertainty.

Without such clarification, the figure may be perceived as interpretative rather than evidence-based.

 

Remarks. This review addresses a timely and strategically important topic for modern winemaking. It presents a solid conceptual framework and a useful synthesis of ozone chemistry and antimicrobial mechanisms. However, to reach its full scientific potential, the manuscript requires substantial revision to:

  1. Strengthen the evidence chain around winery-relevant applications (CIP, bottling hygiene, barrels, grape sorting).
  2. Avoid overgeneralization from postharvest systems.
  3. Introduce operational and engineering clarity.
  4. Enhance transparency in comparative analyses.
  5. Refine and consolidate the reference base.
  6. Correct typographical artifacts (e.g., hard hyphenations such as “fer-mentation”).
  7. Standardize units (ppm, µL/L, mg/L) and define them clearly at first use.
  8. Ensure all figures contain standalone captions with defined abbreviations and explicit description of the evidence base.

With these improvements, the work could make a meaningful and authoritative contribution to the field of oenological sanitation and sustainable fermentation management.

Best regards.

 

Comments on the Quality of English Language

The manuscript is generally understandable and communicates its main arguments clearly. The technical vocabulary is appropriate for an academic readership in oenology and fermentation science, and the overall structure supports logical progression of ideas. 

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

 

This manuscript offers interesting perspective regarding usage of ozone in the sanitation during the production of wine.

 

Highlight in the abstract that SO2 kills unwanted microorganisms which could cause spoilage of wine and unwanted aromas, and as a sours of sulphur also promote growth of Saccharomyces yeasts which is responsible for pleasant wine aromas.

In the paragraph from lines 64 to 76 highlight that yeast species which you mentioned are used together with Saccharomyces yeasts during fermentation process.

Give more information about stability of ozone during the application in wine production and economic viability.

Did application of ozone during the wine production affected on the sensory properties of wines? How it looks like in comparison with wines produced with addition of SO2? Briefly explain it in the manuscript.

What is the safe concentration of ozone for application during the wine production? Which concentration will not cause health issues and negative effect on sensory properties of wines? Explain it in the manuscript.

Do you need any specific facility and installation for application of ozone? Explain it in the manuscript.

Lines 229 to 245 Highlight that presence of Botrytis cinerea in grapes significantly decreased content of phenolic compound in wines and antioxidant properties. Kindly consider to cite Fermentation 2023, 9(7), 695.

Line 241 Which reference supports this. Insert reference.

Highlight that ozone is unselected and that can affect on the cells which cause controlled fermentation too.

How ozone affect on the wine during the ageing and maturation?  What is optimal dose which did not affect on the sensory properties? Briefly explain in the manuscript.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

A very interesting article about the application of ozone in the viticulture and enology sector.

I suggest some minor modifications:

Lines 236, 246, 255, 276, 277, 287: add author

Line 332:
"This section presents a comparative analysis of several emerging technologies: ozone, pulsed light, high hydrostatic pressure, ultrasound, and pulsed electric fields."

Add abbreviations to this sentence, for example: Pulsed light (PL), high hydrostatic pressure (HHP), etc... Because they use the abbreviations later without mentioning them beforehand.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 5 Report

Comments and Suggestions for Authors

This work presents valuable information and versatile applications regarding wine safety. The manuscript focuses on the use of ozone technology as an effective, sustainable, and environmentally friendly alternative to address the impact of climate change on viticulture. 

Abstract 

Lines 12-16: The abstract discusses the use of ozone for grape microbiota disinfection and highlights non-saccharomycetic yeasts for their adaptability and aroma development. However, it overlooks lactic acid bacteria (LAB), which are affected by sulfite and ozone and are essential to the winemaking process. Including a mention of LAB would enhance the abstract.

Lines 180-185: The disinfectant effect varies depending on the microorganism´s structure and physiology, particularly the thick peptidoglycan layer in Gram-positive bacteria. LAB relevant to oenology, and some harmful ones, also have an S-layer. 

It would be useful to comment on the ozone sensitivity of the S-layer at this point.

Lines 189-193: The text mentioned studies on grapes that have been treated with ozone technologies, noting the percentage reductions in acetic acid bacteria and mold population. However, it does not provide any data on LAB. In grapes, LAB are present in low quantities. What would be the degree of sensitivity to ozone treatment?

Table 1 provides valuable information. Adding LAB data to understand the impact of ozone on the full range of microorganisms involved in the winemaking process.

Lines 278-282: Mentioned that short-term ozone treatment reduces tartaric acid, citric acid, and glycerol, also producing the occurrence of precipitates in wine. These effects are more evident with prolonged exposure to ozone. This item requires further explanation for clarity. 

Comments on the Quality of English Language

The manuscript is clear and easy to read. I suggest minor improvements:

Line 34: Remove the word "this" or split it into two sentences.

Line 67: Add a comma in front of the word "which"

Line 150: Replace the word "This" with "these" 

Line 175: Replace the word "cause" with "causes"

Line 198: Remove the comma after "such as"

 

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for taking into account my remarks! I still continue thinking about that the usage of ozone as sanitizing agent in pipes is not good idea. You are right that ozone is unstable and will decompose to oxygen but what will happen if this oxygen is in contact with wine?

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors:

The revised version shows clear improvement compared with the initial submission, and you have made a genuine effort to address the main reviewer comments. The topic is relevant and the manuscript compiles useful information on ozone applications in enological contexts.

Nevertheless, a few aspects could still be strengthened to improve scientific rigor and practical usefulness:

  • Although Table 1 now clarifies methodological contexts, the manuscript still occasionally merges postharvest cold-storage experiments with winery sanitation applications. These systems differ substantially in exposure time, environmental conditions, and microbial ecology. A clearer distinction in the discussion would prevent potential overgeneralization.

  • The review discusses several factors affecting ozone efficacy (pH, temperature, organic matter), but the manuscript would benefit from a more operational perspective.For example, briefly summarizing:  

    • typical concentration ranges used in studies

    • approximate exposure times

    • differences between aqueous and gaseous ozone systems

    would increase the practical value of the review for researchers and winemakers.

  • Barrel sanitation discussion:  The additional reference included improves this section. However, it would be useful to emphasize that barrel sanitation remains challenging due to wood porosity and microbial penetration depth, and that ozone efficacy depends strongly on treatment protocols. This nuance would prevent overly optimistic interpretations.

The manuscript now presents a useful and informative review of ozone applications in winemaking, with clear relevance for sustainable winery practices. After the minor revisions, the manuscript is close to publishable, although minor improvements in clarity and methodological transparency would further strengthen it.

Regards.

Author Response

Please see the attachment 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Authors have given answers on comments and suggestions which improved manuscript significanlty. 

Author Response

We thank the reviewer for their positive assessment and for the time dedicated to reviewing our manuscript and we are pleased to hear that our revisions have significantly improved the manuscript.