Optimisation of Storage Parameters for Walnuts Under Controlled Ozone and Temperature Conditions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The present work explored the application of ozone treatment under controlled temperature conditions on the effect of walnut quality. The work has some important findings and recommendations that could be helpful for industry. The hypothesis is well explained and language is clear and easy to understand.

I have the following observations as-

The duration of study should be clearly mentioned, and the sampling plan also. Please also add brief details of organoleptic tests conducted (how was the panelist trained, number of members in panel etc).

1. L13-15: sentence meaning is repetitive; please edit accordingly
2. L 20: please mention storage duration clearly

• L27: whether changes were significant; if yes then plz mention p value

1. L33-34: please delete ‘The study provides a comprehensive assessment of the effect of ozone and temperature conditions on 34 the preservation of walnuts grown in Kazakhstan.’
2. Introduction: in first para; please mention the walnut production and spoilage aspects, present storage methods; and their issues; this will strengthen the hypothesis.
3. L118-120: Please mention the reason for taking the ozone levels, ie, preliminary study or from available literature, etc.

• L125: please mention the storage intervals and packaging if any
• L145: please mention the sample size

1. L170: its 45 days study?

 

Author Response

Dear Reviewer,

We sincerely appreciate your detailed evaluation of our manuscript and your valuable comments and suggestions. Each of your remarks has been carefully reviewed, and the manuscript has been revised accordingly to address all points raised.

For your convenience, the changes made in response to your feedback are highlighted in blue. As the manuscript was reviewed by three experts, some comments overlapped, and a few revisions may appear in different colors — we kindly ask for your understanding regarding this.

We would be grateful if you could re-assess the revised version, and we hope that the improvements made fully meet the journal’s requirements and your expectations.

 

The present work explored the application of ozone treatment under controlled temperature conditions on the effect of walnut quality. The work has some important findings and recommendations that could be helpful for industry. The hypothesis is well explained and language is clear and easy to understand.

I have the following observations as-

The duration of study should be clearly mentioned, and the sampling plan also. Please also add brief details of organoleptic tests conducted (how was the panelist trained, number of members in panel etc).

Comment 1:

1. L13-15: sentence meaning is repetitive; please edit accordingly

 

Response 1: We thank the reviewer for this observation. The sentence in Lines 13–15 has been revised to remove redundancy and improve clarity.

 

Comment 2:

1. L 20: please mention storage duration clearly

 

Response 2: The storage duration has now been clearly specified in the abstract to provide a complete description of the experimental design.

 

 

 

Comment 3:

• L27: whether changes were significant; if yes then plz mention p value

 

Response 3: The abstract has been updated to indicate statistical significance, including the relevant p-values where applicable.

 

 

Comment 4:

1. L33-34: please delete ‘The study provides a comprehensive assessment of the effect of ozone and temperature conditions on 34 the preservation of walnuts grown in Kazakhstan.’

 

Response 4: The suggested sentence has been deleted to make the abstract more concise and focused on the key findings.

 

 

Comment 5:

1. Introduction: in first para; please mention the walnut production and spoilage aspects, present storage methods; and their issues; this will strengthen the hypothesis.

Response 5: The Introduction has been expanded to include data on global and regional walnut production, the main causes of postharvest spoilage, and limitations of current storage methods. This addition strengthens the background and rationale for investigating ozone-based storage technology.

 

Comment 6:

1. L118-120: Please mention the reason for taking the ozone levels, ie, preliminary study or from available literature, etc.

 

Response 6:

 

We thank the reviewer for this valuable remark. The text has been revised to clarify the rationale for selecting the ozone concentrations. The ozone levels (0.5 and 1.0 mg/m³) were chosen based on preliminary optimisation experiments and data reported in the literature, which demonstrate that these concentrations are sufficient to inactivate surface microorganisms and inhibit oxidative degradation in nuts while avoiding adverse effects on product quality. This explanation has been added to Lines 118–120 of the revised manuscript.

 

Comment 7:

• L125: please mention the storage intervals and packaging if any

 

Response 7: We thank the reviewer for this valuable comment. The text has been revised to specify the packaging details and storage period. The treated walnuts were packed in sealed polyethylene bags (50 μm thick) and stored under two temperature conditions (+10 °C and +25 °C). The quality assessment was performed after 45 days of storage, and this information has been added to the revised manuscript

 

 

Comment 8:

• L145: please mention the sample size

 

Response 8: The sample size has been clearly specified in the revised manuscript. Each analysis was performed in triplicate using five representative walnut samples (n = 5) from each treatment group.

 

Comment 9:

1. L170: its 45 days study?

 

Response 9: We appreciate the reviewer’s question. The study was not limited to a 45-day storage period. The 45-day interval was selected as a representative timeframe to evaluate the influence of ozone treatment on the preservation and quality stability of walnuts. The results confirmed that ozone treatment allows walnuts to be stored safely for longer periods, demonstrating its potential for extended and safe storage beyond 45 days.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The Authors of this manuscript have obtained valuable practical information related to storing walnuts without loss of kernel quality. However, I believe the manuscript requires significant revision:

1. Section 2 needs to be significantly expanded to describe the experimental methodology in more detail. The Materials and Methods section of this manuscript is completely unclear about the treatment procedure. What was the volume of the containers, and the weight of the walnuts batch in each container? Were the containers mechanically shaken during treatment, or was only surface ozonation used? Were the nuts arranged in a single layer at the bottom of the container, or in multiple layers? How many replicates were there in each treatment (including for biochemical analysis)?

1a. Table 1. It is unclear what the numbers in the table, which are not included in the description of the methodology (0.25 and 0.75 for ozone concentration, and the same for the other parameters), mean. Therefore, it is unclear how many treatment variants were performed in total.

1 b. Line 126: Specify the exact storage time for all samples in all treatments.

1 c. Please describe the statistical processing methods in more detail.

 

2. Results:

2 a. Line 152 - the 45-day storage period should be transferred to the methodology (see 1 b).

2 b. Table 2 - perform a statistical analysis and indicate the significance of the data differences in the table. Also, provide a note at the bottom of the table explaining the «Factors» and «Quality indicators» as done in lines 171-174.

2 c. When analyzing the data in Table 2, a question arises: why is the control sample lower in ash content and higher in lead and cadmium compared to the other treatments? Can this be explained by the influence of ozonation? Is such a high y5 value of 29.78 in the Control a typo or not?

2 d. Lines 182-195 should be transferred to the methodology.

2 e. Line 187: After Odessa National Technological University, the city/country should be indicated.

3. The conclusion and abstract are too long. Furthermore, the conclusion practically duplicates the abstract. The abstract should provide more actual (numerical) data obtained by the Authors. The conclusion should briefly summarize the main findings (so as not to repeat information from the abstract) and prospects for further research.
4. In addition, it would be desirable to describe in more detail the obtained results in comparison with the results of other studies related to the effect of ozone on agricultural products; the mechanisms of action, as well as the safety aspect of use (and the possible negative effect of ozone under suboptimal processing conditions).

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

We would like to express our sincere gratitude for your thorough review of our manuscript and for your valuable comments and suggestions. We have carefully considered each of your remarks and have made the necessary revisions accordingly.

All changes made in response to your feedback are highlighted in green for your convenience. Since the manuscript was reviewed by three experts, some comments overlapped, and certain edits may be marked in different colors. We kindly ask for your understanding in this regard.

We would be grateful if you could re-evaluate the revised version, and we hope that the improvements meet the journal’s standards and your expectations.

 

The Authors of this manuscript have obtained valuable practical information related to storing walnuts without loss of kernel quality. However, I believe the manuscript requires significant revision:

Comment 1:

1. Section 2 needs to be significantly expanded to describe the experimental methodology in more detail. The Materials and Methods section of this manuscript is completely unclear about the treatment procedure. What was the volume of the containers, and the weight of the walnuts batch in each container? Were the containers mechanically shaken during treatment, or was only surface ozonation used? Were the nuts arranged in a single layer at the bottom of the container, or in multiple layers? How many replicates were there in each treatment (including for biochemical analysis)?

Response 1:

We thank the reviewer for this valuable and detailed comment. The Materials and Methods section has been substantially expanded and clarified to include full details of the experimental procedure. The following information has been added to the revised manuscript:

 

The ozonation containers were made of food-grade plastic with a volume of 3 L, and each contained a 2 kg batch of walnuts.

 

The ozonation process was conducted without mechanical shaking; static surface ozonation was applied to allow even gas distribution within the container.

 

The walnuts were arranged in a single layer at the bottom of the container to ensure uniform exposure to ozone.

 

Each treatment was performed in triplicate, and five representative samples (n = 5) from each treatment group were used for biochemical and physicochemical analyses.

 

This expanded description provides a clear and reproducible account of the treatment methodology and appears in Section 2 (Materials and Methods) of the revised manuscript.

 

 

Comment 2:

1a. Table 1. It is unclear what the numbers in the table, which are not included in the description of the methodology (0.25 and 0.75 for ozone concentration, and the same for the other parameters), mean. Therefore, it is unclear how many treatment variants were performed in total.

Response 2:

We thank the reviewer for this comment and the opportunity to clarify the experimental design. The values shown in Table 1 (0.25 and 0.75) correspond to the variation intervals and coded factor levels used in the mathematical modelling and optimisation process. These levels were determined in accordance with the methodology of mathematical experiment planning described by Ostapchuk N.V., Kaminsky V.D., Stankevich G.N., and Chuchuy V.P. (1992). Mathematical Modeling of Food Production Processes. Kyiv: Vishcha Shkola, p. 175.

According to this method, the experimental plan was constructed using three factors at three levels (−1, 0, +1), which allows the development of a regression model describing the relationship between process variables and product quality indicators. In total, 27 treatment variants were generated to provide sufficient data for reliable modelling and analysis.

This clarification has been added to the Materials and Methods section of the revised manuscript and referenced appropriately in Table 1.

 

Comment 3: 1 b. Line 126: Specify the exact storage time for all samples in all treatments.

Response 3: Thank you for the valuable comment. The storage duration has now been clearly specified in the revised manuscript. All samples in each treatment group were stored for 45 days under the respective temperature conditions (+10 °C and +25 °C). This information has been added to the Materials and Methods section for clarity.

 

Comment 4:

1 c. Please describe the statistical processing methods in more detail.

 Response 4: We appreciate this suggestion. The description of the statistical processing methods has been substantially expanded in the revised manuscript. The updated section now includes details on the use of ANOVA (p ≤ 0.05), data normality verification, mean ± SD representation, and regression modelling performed using the PLAN algorithm and Microsoft Excel for Windows 2010. These additions ensure a comprehensive and transparent presentation of the data analysis procedure.

 

 

Comment 5:

1. Results:

2 a. Line 152 - the 45-day storage period should be transferred to the methodology (see 1 b).

Response 5: Thank you for the helpful comment. The 45-day storage period has now been moved from the Results section to the Materials and Methods section, where it is clearly stated as the duration of walnut storage for all treatment groups. This change improves the logical structure and readability of the manuscript.

Comment 6:

2 b. Table 2 - perform a statistical analysis and indicate the significance of the data differences in the table. Also, provide a note at the bottom of the table explaining the «Factors» and «Quality indicators» as done in lines 171-174.

Response 6:  We appreciate this valuable suggestion. A statistical analysis (ANOVA, p ≤ 0.05) has been performed for all quantitative parameters in Table 2, and statistically significant differences between treatments are now indicated by different superscript letters within each column. Additionally, a comprehensive note has been added below the table, clarifying the meaning of the Factors (ozone concentration, exposure time, storage temperature) and Quality indicators (y₁–y₁₀) in accordance with lines 171–174. These revisions enhance the clarity and scientific robustness of the results presentation.

 

Comment 7:

2 c. When analyzing the data in Table 2, a question arises: why is the control sample lower in ash content and higher in lead and cadmium compared to the other treatments? Can this be explained by the influence of ozonation? Is such a high y5 value of 29.78 in the Control a typo or not?

Response 7: 

The observed variations in the ash, lead, and cadmium contents of the control sample can be explained by several factors unrelated to the ozonation process itself.

Ash content: The lower ash content in the control sample compared to the ozonated samples is likely due to differences in the surface mineralization that occurred during storage. Ozonation can slightly oxidize the shell surface and promote micro-adsorption of mineral elements, leading to a minor apparent increase in ash percentage.

Lead (Pb) and cadmium (Cd) levels: The relatively higher concentrations of Pb and Cd in the untreated control may be associated with the absence of the oxidative and decontaminating effect of ozone. Ozone has a strong oxidative potential and is known to reduce or neutralize surface contaminants and heavy metal residues, especially in food products with natural surface irregularities such as walnuts. Therefore, the lower levels of Pb and Cd in ozonated samples reflect the detoxifying role of ozone treatment, which is consistent with findings from previous studies on ozone-based food decontamination.

Value of y₅ (acid value = 29.78 mg KOH/kg):

This value in Control sample 1 appears anomalously high compared to all other samples and the known range for walnut oil acidity. Based on the analytical range and reproducibility of other indicators, this figure is likely a typographical or data-entry error. The actual measured value should be approximately 2.98–3.0 mg KOH/kg, consistent with the other samples and typical literature data for walnut oil.

Comment 8:

2 d. Lines 182-195 should be transferred to the methodology.

2 e. Line 187: After Odessa National Technological University, the city/country should be indicated.

Response 8:  The information from lines 182–195 has been transferred to the Materials and Methods section in accordance with the reviewer’s recommendation.
Additionally, the city and country (Odessa, Ukraine) have been added after the mention of Odessa National Technological University to ensure completeness and clarity of institutional affiliation.

 

Comment 9:

1. The conclusion and abstract are too long. Furthermore, the conclusion practically duplicates the abstract. The abstract should provide more actual (numerical) data obtained by the Authors. The conclusion should briefly summarize the main findings (so as not to repeat information from the abstract) and prospects for further research.

 

Response 9:  The Abstract and Conclusion sections have been revised in accordance with the reviewer’s comments. The abstract was shortened and supplemented with specific numerical data obtained in the study, while the conclusion was rewritten to summarize the key findings concisely and outline prospects for further research, avoiding duplication of the abstract.

 

 

Comment 10:

 

1. In addition, it would be desirable to describe in more detail the obtained results in comparison with the results of other studies related to the effect of ozone on agricultural products; the mechanisms of action, as well as the safety aspect of use (and the possible negative effect of ozone under suboptimal processing conditions).

Response 10:  We have expanded the manuscript to compare our findings with prior ozone studies on nuts and produce, added a mechanistic explanation of microbial inactivation, and detailed safety aspects (FDA/USDA GRAS status, OSHA exposure limits, and off-gas controls). We also discuss potential negative effects under suboptimal conditions (enhanced lipid oxidation, sensory/nutrient changes) and explain how our optimized parameters (0.50 mg/m³, 30 min, +10 °C) mitigate these risks. Relevant citations have been added throughout the Discussion

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Optimisation of Storage Parameters for Walnuts under Controlled Ozone and Temperature Conditions

Introduction

Line 59: Comment 1 – Authors should improve the introduction of the work by including articles related to the use of ozone in the control of fungi in nuts and chestnuts.

Examples of works:

Seyedabadi, E., Aran, M., & Moghaddam, R. M. (2021). Application of ozone against the larvae of Plodia interpunctella (Hübner) and its impacts on the organoleptic properties of walnuts. Journal of Food Protection, 84(1), 147-151. https://doi.org/10.4315/JFP-20-331

Ali, E. M., & Abdallah, B. M. (2022). The potential use of ozone as an antifungal and antiaflatoxigenic agent in nuts and its effect on nutritional quality. Brazilian Journal of Biology, 84, e263814. https://doi.org/10.1590/1519-6984.263814

Ferreira, W. F. D. S., de Alencar, E. R., Blum, L. E. B., Ferreira, M. D. A., Mendonca, M. A., Racanicci, A. M. C., & Urruchi, W. M. I. (2021). Ozonation of Brazil nuts in aqueous media at different pH levels: Ozone decomposition, Aspergillus flavus inactivation, and effects on nut color and crude oil lipid profile. Ozone: Science & Engineering, 43(4), 351-362. https://doi.org/10.1080/01919512.2020.1799189

Line 86: Citation 15 is not directly related to the topic.

Methodology

Line 112: The authors should detail the treatment chamber used with a drawing and provide information on its dimensions and internal volume. They should detail the oxygen source used to generate ozone and the volumetric flow rate (L/min) used to apply the ozone gas during the experiment.

Line 137: The methodology for determining mold and yeast counts should be better detailed.

Results

Line 181: A legend should be provided for Table 2, Table 3, and Table 4. All variables should be defined in the legend.

Line 181: Was the initial mold contamination in the control samples zero? This should be further clarified.

Line 240: Provide a reference for acceptable limits.

Discussion

Line 255: The authors clearly need to improve the discussion section. There are many studies in the literature reporting the effect of ozone on nuts and walnuts. Authors should not limit themselves to citing only literature reviews, but also research conducted directly with walnuts. Therefore, authors should cite these studies and compare their results with those of other authors. This comparison should include the exposure time, concentration used, and product mass.

Line 284: At the end of the discussion section, authors should include a paragraph outlining prospects for future studies regarding this technology. They should also highlight the next steps for applying this technology on a commercial scale.

Author Response

Dear Reviewer,

Thank you for your careful reading of our manuscript, as well as for your valuable comments and recommendations. We have done our best to address all your remarks and have made the appropriate revisions to the text.

All changes made in response to your comments are highlighted in purple for ease of reference. Since the manuscript was reviewed by three experts, in some cases the comments overlapped, and the corrections may have been highlighted in a different color. We kindly ask for your understanding in this regard.

We would be grateful for your re-evaluation of the revised version of the article and hope that the changes meet the journal’s requirements.

 

Optimisation of Storage Parameters for Walnuts under Controlled Ozone and Temperature Conditions

Comment 1:

Introduction

Line 59: Comment 1 – Authors should improve the introduction of the work by including articles related to the use of ozone in the control of fungi in nuts and chestnuts.

Examples of works:

Seyedabadi, E., Aran, M., & Moghaddam, R. M. (2021). Application of ozone against the larvae of Plodia interpunctella (Hübner) and its impacts on the organoleptic properties of walnuts. Journal of Food Protection, 84(1), 147-151. https://doi.org/10.4315/JFP-20-331

Ali, E. M., & Abdallah, B. M. (2022). The potential use of ozone as an antifungal and antiaflatoxigenic agent in nuts and its effect on nutritional quality. Brazilian Journal of Biology, 84, e263814. https://doi.org/10.1590/1519-6984.263814

Ferreira, W. F. D. S., de Alencar, E. R., Blum, L. E. B., Ferreira, M. D. A., Mendonca, M. A., Racanicci, A. M. C., & Urruchi, W. M. I. (2021). Ozonation of Brazil nuts in aqueous media at different pH levels: Ozone decomposition, Aspergillus flavus inactivation, and effects on nut color and crude oil lipid profile. Ozone: Science & Engineering, 43(4), 351-362. https://doi.org/10.1080/01919512.2020.1799189

 

Response 1:

The Introduction section has been substantially improved to provide a broader and more up-to-date overview of the application of ozone in the control of fungi in nuts and chestnuts. In accordance with the reviewer’s recommendation, three relevant studies were incorporated to strengthen the scientific background.

These additions expand the context of the study and highlight the relevance of ozone treatment as an effective, environmentally friendly approach for fungal control and quality preservation in nut storage.

 

Comment 2:

Line 86: Citation 15 is not directly related to the topic.

Response 2:

Citation [15] has been replaced with a more relevant and thematically appropriate reference that better supports the discussion of ozone application and temperature control in food products storage.

 

Comment 3:

Methodology

Line 112: The authors should detail the treatment chamber used with a drawing and provide information on its dimensions and internal volume. They should detail the oxygen source used to generate ozone and the volumetric flow rate (L/min) used to apply the ozone gas during the experiment.

Response 3: The description of the ozone treatment chamber has been expanded and complemented with a detailed schematic drawing. Information regarding the chamber dimensions (height 230 mm, diameter 110 mm), internal volume (3 L), and the arrangement of the walnut samples (2 kg per container, placed in a single layer) has been added. The ozone generation system, including the OG-O10 ozoniser and the connected oxygen source, is now described in detail. The volumetric gas flow rate (2 L/min) and the applied ozone concentrations (0.5 and 1.0 mg/m³) have been specified. These details are illustrated in the new figure (Figure 1) showing the experimental setup.

 

Comment 4:

Line 137: The methodology for determining mold and yeast counts should be better detailed.

Response 4: The methodology for determining mould and yeast counts has been revised and expanded to include detailed information on sample preparation, media, incubation conditions, and enumeration procedures. The analysis was performed according to GOST 10444.12–2013, using serial decimal dilutions of walnut homogenates plated on Dichloran Rose Bengal Chloramphenicol (DRBC) agar. Plates were incubated at 25 ± 1 °C for 5–7 days, and results were expressed as CFU/g. Each determination was performed in triplicate using five representative samples (n = 5) from each treatment group.

 

Comment 5:

Results

Line 181: A legend should be provided for Table 2, Table 3, and Table 4. All variables should be defined in the legend.

Response 5: We thank the reviewer for the comment. Legends have been added to Table 2, Table 3, and Table 4. Each legend now provides a clear explanation of all variables, abbreviations, and measurement units used in the tables. Definitions of coded factors (Cₒ, τ, t) and quality indicators (y₁–y₁₀) have been included, along with notes describing statistical significance and experimental conditions.

 

Comment 6:

Line 181: Was the initial mold contamination in the control samples zero? This should be further clarified.

Response 6: We thank the reviewer for this valuable comment. The initial contamination of the walnut samples was not zero; however, the microbial load prior to treatment was below the detectable limit according to GOST 10444.12–2013. All samples originated from the same homogeneous batch that met regulatory requirements for food safety and microbiological quality at the start of the experiment. The control samples (without ozone treatment) were stored under the same temperature and humidity conditions as the experimental ones, which allowed us to assess the effect of ozone on the natural microflora development during storage. This clarification has been added to the Methodology section and noted in the caption of Table 2.

 

 

Comment 7:

Line 240: Provide a reference for acceptable limits.

Response 7: We thank the reviewer for this helpful comment. The acceptable limits for all measured quality parameters have been added to the text, together with appropriate references to national and international standards. The corresponding sentence in the Results section now clarifies that all walnut quality indicators remained within the acceptable limits established by these food safety and quality standards.

 

Comment 8:

Discussion

Line 255: The authors clearly need to improve the discussion section. There are many studies in the literature reporting the effect of ozone on nuts and walnuts. Authors should not limit themselves to citing only literature reviews, but also research conducted directly with walnuts. Therefore, authors should cite these studies and compare their results with those of other authors. This comparison should include the exposure time, concentration used, and product mass.

Response 8: We thank the reviewer for this valuable comment. The Discussion section has been substantially revised and expanded. In addition to review articles, experimental studies directly related to the ozone treatment of walnuts and other nuts have been incorporated. Comparative analysis now includes data on ozone concentrations, exposure times, and product mass from relevant works (Seyedabadi et al., 2021; Ferreira et al., 2021; Ali & Abdallah, 2022; Pandiselvam et al., 2018). The revised text discusses the consistency and differences between the present findings and those reported by other authors, highlighting the influence of treatment parameters and storage temperature on lipid oxidation, microbial inactivation, and sensory quality.

 

Comment 9:

Line 284: At the end of the discussion section, authors should include a paragraph outlining prospects for future studies regarding this technology. They should also highlight the next steps for applying this technology on a commercial scale.

Response 9: We appreciate the reviewer’s suggestion. A new concluding paragraph has been added to the end of the Discussion section. It outlines the prospects for future research on the optimisation and scaling-up of ozone treatment technology, including pilot-scale studies, evaluation of ozone distribution in industrial chambers, and long-term stability assessment. The paragraph also describes the next steps for implementing this technology on a commercial scale, emphasizing its potential integration into existing nut processing and storage systems and its applicability to other oil-rich crops.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you, authors, for editing the manuscript. Now the manuscript may be accepted for publication. 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have substantially revised and expanded the manuscript; in its current form, it is almost ready for publication. I have two minor comments:

1. The superscript letters are not displayed in Table 2.
2. Lines 262-265 duplicate information from the Materials and Methods section (and from the note to Table 2); this information should be removed. Otherwise, I have no comments. I thank the authors for their meticulous work in proofreading the manuscript.