Unconventional Material from In Vitro Plant Cell Cultures: Vitis labrusca var. Isabella Case Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comment for applsci-3767627

This manuscript investigates the feasibility of utilizing Vitis labrusca var. Isabella callus cultures as a novel source of “superfood” materials. The authors established undifferentiated cell lines and comprehensively analyzed their phenolic composition and nutritional profiles using LC-MS/MS and HPLC-DAD techniques, supplemented by antioxidant activity assays and nutritional labeling analysis. The study is well-structured and presents a relatively systematic workflow with a certain degree of novelty and practical relevance. However, there are several areas that require improvement regarding methodological clarity, data interpretation, linguistic precision, and logical organization.

1.Abstract: The first two sentences are overly long and unfocused. Consider condensing the background to one concise sentence to enhance clarity.

2.Abstract: The expressions “different polyphenol contents” and “different antioxidant activities” are too vague. It is recommended to include quantitative results or highlight key differences to emphasize the comparative value of the study.

3.Keywords: Please consider adding terms such as “sustainable food production” and “plant cell metabolism” to better reflect the broader context and scientific focus of the study.

4.Introduction: While sustainability is briefly mentioned, the manuscript does not clearly articulate the limitations of conventional agricultural production. The authors are encouraged to elaborate on specific issues such as land use, climate dependence, or inefficiencies in bioactive compound yields, to better highlight the advantages of plant cell culture technologies.

5.Introduction: In lines 81–82, a citation should be provided for the FAO and UN information.

6.Introduction: In line 101, the insertion of references 16–18 lacks contextual explanation. This paragraph should clearly define the research objective of the present study and explain how it builds upon or differs from previous work.

7.Materials and Methods: The experimental protocols are described in detail and generally ensure reproducibility. However, in the data analysis section, the authors mention “two independent experiments, each performed in duplicate.” For robust statistical analysis, at least three independent biological replicates are recommended. This would reduce random variability and increase the reliability of statistical significance.

8.Results and Discussion: When interpreting differences in total polyphenol content and antioxidant activity, the manuscript does not adequately explain why the V B5A cell line exhibits higher levels than V MSA. The authors should discuss this from the perspective of plant cell metabolism and how media composition or hormonal regulation may have influenced secondary metabolite accumulation.

Author Response

We would like to thank the Reviewer for his/her comments. The answer to Your questions/suggestions are reported below, following Your comments, point by point.

 

Reviwer 1

 

Comment for applsci-3767627

This manuscript investigates the feasibility of utilizing Vitis labrusca var. Isabella callus cultures as a novel source of “superfood” materials. The authors established undifferentiated cell lines and comprehensively analyzed their phenolic composition and nutritional profiles using LC-MS/MS and HPLC-DAD techniques, supplemented by antioxidant activity assays and nutritional labeling analysis. The study is well-structured and presents a relatively systematic workflow with a certain degree of novelty and practical relevance. However, there are several areas that require improvement regarding methodological clarity, data interpretation, linguistic precision, and logical organization.

1.Abstract: The first two sentences are overly long and unfocused. Consider condensing the background to one concise sentence to enhance clarity.

Done

 

2.Abstract: The expressions “different polyphenol contents” and “different antioxidant activities” are too vague. It is recommended to include quantitative results or highlight key differences to emphasize the comparative value of the study.

Done

 

3.Keywords: Please consider adding terms such as “sustainable food production” and “plant cell metabolism” to better reflect the broader context and scientific focus of the study.

Done

 

4.Introduction: While sustainability is briefly mentioned, the manuscript does not clearly articulate the limitations of conventional agricultural production. The authors are encouraged to elaborate on specific issues such as land use, climate dependence, or inefficiencies in bioactive compound yields, to better highlight the advantages of plant cell culture technologies.

The Reviewer is right, and following his/her suggestions, we added additional references to highlight the advantages of this technology better

 

5.Introduction: In lines 81–82, a citation should be provided for the FAO and UN information.

Sorry, we missed it; now, the two documents are cited.

 

6.Introduction: In line 101, the insertion of references 16–18 lacks contextual explanation. This paragraph should clearly define the research objective of the present study and explain how it builds upon or differs from previous work.

The Reviewer is right, the intention of the paper was not clear enough, we modified the end of the introduction according to his/her suggestion.

 

7.Materials and Methods: The experimental protocols are described in detail and generally ensure reproducibility. However, in the data analysis section, the authors mention “two independent experiments, each performed in duplicate.” For robust statistical analysis, at least three independent biological replicates are recommended. This would reduce random variability and increase the reliability of statistical significance.

We understand the Reviewer’s concern about the reliability of the statistical significance of the chemical experiments. The data presented in this work are the final result of analyses that we have been performed over a three-year period. As reported in the paper, the starting material (the Isabella leaves) was collected in 2022, and after only a few months, we obtained two stabilised cell lines. Thereafter, the cells were observed weekly, and the chemical analyses presented in this work were repeated at regular intervals to confirm material stability. The nutritional label analysis is the only exception, as it has been performed by an Italian company authorised for food nutritional labelling. The preliminary chemical analyses (total phenol content, DPPH discolouration, and total stilbenoid content), based on a well-defined extraction protocol, gave reliable and reproducible results. Based on this, for the data presented in this paper we decided to do two independent experiments, for which, each one was performed in duplicate, for a better measure of reproducibility and to reduce the impact of potential outliers or systematic errors; the replicates, which estimate the precision, and the independent experiments, which give more confidence that our results are not due to a one-off fluke, were extremely overlapping (standard deviations comprise between ±5%). In addition, we also decided to reduce the number of experiments based on the main goal of our paper, which is sustainability; thus, we minimised resource consumption (time, materials, and energy) on the grounds of solid and reliable data.

 

8.Results and Discussion: When interpreting differences in total polyphenol content and antioxidant activity, the manuscript does not adequately explain why the V B5A cell line exhibits higher levels than V MSA. The authors should discuss this from the perspective of plant cell metabolism and how media composition or hormonal regulation may have influenced secondary metabolite accumulation.

We thank the Reviewer for his/her suggestion. We tried to improve the discussion about the results obtained for the total phenol content and antioxidant activity; however, it is challenging to provide an unequivocal and definitive explanation for our results. As reported in the manuscript, the different biosynthetic capability of the two cell lines may only be attributed to the basal medium composition, as the carbon source is qualitatively and quantitatively the same, as well as the hormonal balance, pH value, and explant type. Moreover, we contextualised our results with other cell culture based papers, highlighting that the optimal conditions for a specific culture are the result of systematic trials and experimentations.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The article entitled “Unconventional material from in vitro plant cell cultures: Isabella case study” is good idea to explore the potential of plants in foods. The article is good; however, suggestions and recommendations are below which needed to be addressed before it goes to further stage.

Major comments:

1. The introduction is very lengthy with very old litrature with important stuff missing, such as why this research is needed. Whats the objectives of this research? And what's the hypothesis and the recent studies about this topic?
2. For the section 2.4, the number of sample n=2, is very less, for scientific research at least have 3 replication.
3. Revise the 2.4.2 section and write it in scientific way.
4. Lines 233-235, related to material section not results. Also write year.
5. There is only results (section 3) where is discussion?
6. The article needs complete revision, with logic and specially to compere the results and then justify it. The current version is not suitable.

 

Minor comments:

1. The title is ok but I suggest to the scientific name of this variety instead of the common English name.
2. Revise the sentence line 34-35.
3. Line 39-43. The sentences are too long, I suggest revise the long sentence into simple sentences that’s will easy for reader.
4. Line 42-43. Why does the author interrelate the wine to super foods..as super foods are good according to Refrences 4, but in case of wine it could be harm for health as well.
5. Lines 49, 50, 52 and son on…please use V. labrusca after appear it first.
6. Line 59, I don’t know whats mean of thanks in this sentence. If its species name use scientific name.
7. Line 108 “ Isabella were harvested at different period” which period please mention.
8. Table 1 arrangement is not correct. I cant understand the current version of table 1. Revise it please.
9. Line 135, check wether there is space between Pteri dishes or not.
10. Line 161, use full name of LC-MS/MS and HPLC-DAD at first time.
11. Line 205, write the unit same as in the figure.
12. Line 199-200, use first the nutrient name and then abbreviation, although the full form of N is mentioned many time, please check it and revise it with abbreviation, same for other nutrients.
13. Line 157; line 222, the SD with full and abbrevation form have been used. Revise it.
14. Write more detail for figure 1 caption. And write A and B instead of left and right.
15. Line 295 42^nd should be in uppercase.
16. Lines 319-321. Repetition of the same introduction. And why no reference for this sentence?
17. Figure 3, I think you should remove antioxidant activity words because its huge word not for specific indicator as you did here (DPPH).
18. Add conclusion.

 

 

Comments on the Quality of English Language

The quality of English is poor. need to improve it. 

Author Response

We would like to thank the Reviewer for his/her comments. The answer to Your questions/suggestions are reported below, following Your comments, point by point.

 

 

Reviewer 2

 

The article entitled “Unconventional material from in vitro plant cell cultures: Isabella case study” is good idea to explore the potential of plants in foods. The article is good; however, suggestions and recommendations are below which needed to be addressed before it goes to further stage.

 

Major comments:

1. The introduction is very lengthy with very old litrature with important stuff missing, such as why this research is needed. Whats the objectives of this research? And what's the hypothesis and the recent studies about this topic?

 

The articles cited in the introduction were published between 2011 and 2025, with most of them published after 2020; we do not think that the literature was as old as the Reviewer suggested. In any case, according to the Reviewer’s suggestion, we shortened non-essential parts and added some recent literature on this topic. Moreover, we better highlighted the advantages of this technique and the aim of the work

 

 

1. For the section 2.4, the number of sample n=2, is very less, for scientific research at least have 3 replication.

 

We are sorry for the lack of clarity. We reported in each chemical method paragraph the sentence “the samples were analysed in two independent experiments, each performed in duplicate”.

We understand the Reviewer’s concern about the reliability of the statistical significance of the chemical experiments. The data presented in this work are the final analyses of a material that we have been following over a three-year period. As reported in the paper, the starting material (the Isabella leaves) was collected in 2022, and after only a few months we obtained two stabilised cell lines. The cells were then observed weekly, and the chemical analyses presented in this work were repeated at regular intervals to confirm material stability. The nutritional label analysis is the only exception, as it has been performed by an Italian company authorised for food nutritional labelling. The preliminary chemical analyses (total phenol content, DPPH discolouration, and total stilbenoid content), based on a well-defined extraction protocol, gave reliable and reproducible results. Based on this, for the data presented in this paper we decided to do two independent experiments, for which, each one was performed in duplicate, for a better measure of reproducibility and to reduce the impact of potential outliers or systematic errors; the replicates, which estimate the precision, and the independent experiments, which give more confidence that our results are not due to a one-off fluke, were extremely overlapping (standard deviations comprise between ±5%). In addition, we also decided to reduce the number of experiments based on the main goal of our paper, which is sustainability; thus, we minimised the resource consumption (time, materials, and energy) on the grounds of solid and reliable data.

 

1. Revise the 2.4.2 section and write it in scientific way.

 

Done

 

1. Lines 233-235, related to material section not results. Also write year.

 

The Reviewer is right; we moved these sentences to the “Material and method” section. Additionally, we included the year in the “result” section.

 

1. There is only results (section 3) where is discussion?

 

The Reviewer is right, we apologise for missing “discussion” in the section title, now the section is appropriately named “results and discussion”

 

1. The article needs complete revision, with logic and specially to compere the results and then justify it. The current version is not suitable.

We thanks the Reviewer for all his/her suggestions; we improved the manuscript taking care about most of them.

 

Minor comments:

1. The title is ok but I suggest to the scientific name of this variety instead of the common English name.

 

We added the complete botanical name of the plant.

 

1. Revise the sentence line 34-35.

 

Done

 

1. Line 39-43. The sentences are too long, I suggest revise the long sentence into simple sentences that’s will easy for reader.

 

The Reviewer is right, we shortened the sentences.

 

1. Line 42-43. Why does the author interrelate the wine to super foods..as super foods are good according to Refrences 4, but in case of wine it could be harm for health as well.

 

We actually did not correlate superfoods with wine. In our paper, we mention Vitis vinifera because it is the most renowned Vitis species in the world, and the major usage of this grape is in wine production. In recent years, Vitis labrusca is gaining attention, too, not only for the wine production in some countries, but also as a source of important bioactive molecules, the same fate that happened for V. vinifera

 

1. Lines 49, 50, 52 and son on…please use V. labrusca after appear it first.

 

Done

 

1. Line 59, I don’t know whats mean of thanks in this sentence. If its species name use scientific name.

 

We modified some sentences in the paragraphs, and now it is better explained the use of the term “Isabella”, which is the common name of the V. labrusca variety that we used

 

1. Line 108 “ Isabella were harvested at different period” which period please mention.

 

We added in brackets to look at the Table 1, where the year and the months of harvesting are specified.

 

1. Table 1 arrangement is not correct. I cant understand the current version of table 1. Revise it please.

 

Revised

 

1. Line 135, check wether there is space between Pteri dishes or not.

 

We are sorry but we did not fully understand the Reviewer’s question

 

 

1. Line 161, use full name of LC-MS/MS and HPLC-DAD at first time.

 

Done

 

1. Line 205, write the unit same as in the figure.

 

Done

 

1. Line 199-200, use first the nutrient name and then abbreviation, although the full form of N is mentioned many time, please check it and revise it with abbreviation, same for other nutrients.

 

Done

 

 

1. Line 157; line 222, the SD with full and abbrevation form have been used. Revise it.

 

Done

 

1. Write more detail for figure 1 caption. And write A and B instead of left and right.

 

Done

 

1. Line 295 42^nd should be in uppercase.

 

Done

 

1. Lines 319-321. Repetition of the same introduction. And why no reference for this sentence?

 

We changed some sentences in the Introduction and added the required references.

 

1. Figure 3, I think you should remove antioxidant activity words because its huge word not for specific indicator as you did here (DPPH).

 

We agree with the Reviewer that the title “antioxidant activity” could be considered deviant and not in the context; however, in material and method section, results and discussion section, and also in Figure 3 capture we specified the usage of the DPPH method to determining the antioxidant activity. Several spectrophotometric methods are currently used to determine antioxidant activity. Among these assays, the DPPH assay is one of the most widely used methods, largely due to its simplicity (e.g., https://doi.org/10.2116/analsci.18P014). Moreover, it is one of the most used methods for determining the antioxidant activity of plant cell culture extracts too (e.g https://doi.org/10.1038/s41598-024-83096-x;  https://doi.org/10.1007/s11240-025-03090-7).

 

1. Add conclusion.

 

We thank the Reviewer for the suggestion, and we added the conclusion to better summarise the results and especially the context of the research.

 

 

 

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

The research  combines plant cell culture technology with the production of functional food materials. After a careful review, it was found that there were some major problems. 

The number of samples in the experiments is too small. The document says that "all experiments were done using eight samples in each dish" and the results are based on "two independent experiments, each done twice." The small number of samples used makes it hard to be sure that the results are reliable. This is particularly true for analyses like total phenolic content (TPC) and antioxidant activity, where small changes in the data could make the results seem less important than they really are. Also, there is not enough information about why the culture media (MSA and B5A) were chosen. The manuscript mentions studies on V. vinifera to support the media mix, but doesn't talk about the differences in how V. vinifera and V. labrusca var. Isabella grow. There is no good reason why media formulations should not be optimised specifically for V. labrusca, and this lack of rigour in the experimental design is problematic.

There are big differences in the secondary metabolites between the two cell lines (V MSA and V B5A). Seven stilbenoid derivatives were found in V B5A, compared to three in V MSA. There were also big differences in the total amount. These differences are only thought to be caused by the "media influence" and no deeper investigation has been done. The document does not look at how different parts of the media (like 2,4-D, kinetin and NAA) control the pathways that make stilbenoids, and it doesn't have any information on how important genes are expressed or how enzymes are active. This basic analysis doesn't allow us to easily understand the differences we can see.

The claim that "plant cell culture technology is a sustainable tool for added-value food production" lacks empirical support. The manuscript fails to compare the economic feasibility, energy consumption, or scalability of cell culture versus traditional cultivation methods. Without such data, the assertion of "viability" remains unsubstantiated .

Given the current shortcomings, the manuscript does not meet the standards of Applied Sciences. A rejection is therefore recommended.

Author Response

We would like to thank the Reviewer for his/her comments. The answer to Your questions/suggestions are reported below, following Your comments, point by point.

 

 

Reviewer 3

 

The research  combines plant cell culture technology with the production of functional food materials. After a careful review, it was found that there were some major problems. 

The number of samples in the experiments is too small. The document says that "all experiments were done using eight samples in each dish" and the results are based on "two independent experiments, each done twice." The small number of samples used makes it hard to be sure that the results are reliable. This is particularly true for analyses like total phenolic content (TPC) and antioxidant activity, where small changes in the data could make the results seem less important than they really are. Also, there is not enough information about why the culture media (MSA and B5A) were chosen. The manuscript mentions studies on V. vinifera to support the media mix, but doesn't talk about the differences in how V. vinifera and V. labrusca var. Isabella grow. There is no good reason why media formulations should not be optimised specifically for V. labrusca, and this lack of rigour in the experimental design is problematic.

 

The document says “All the experiments were performed using eight explants per dish”, referring to the number of explants per dish for establishing the undifferentiated in vitro cultures of V. labrusca var. Isabella. Regarding the chemical results, we understand the Reviewer’s concern about the reliability of the statistical significance of the chemical experiments. The data presented in this work are the final analyses of a material that we have been following over a three-year period. As reported in the paper, the starting material (the Isabella leaves) was collected in 2022, and after only a few months we obtained two stabilised cell lines. The cells were then observed weekly, and the chemical analyses presented in this work were repeated at regular intervals to confirm material stability. The nutritional label analysis is the only exception, as it has been performed by an Italian company authorised for food nutritional labelling. The preliminary chemical analyses (total phenol content, DPPH discolouration, and total stilbenoid content), based on a well-defined extraction protocol, gave reliable and reproducible results. Based on this, for the data presented in this paper we decided to do two independent experiments, for which, each one was performed in duplicate, for a better measure of reproducibility and to reduce the impact of potential outliers or systematic errors; the replicates, which estimate the precision, and the independent experiments, which give you more confidence that your results are not due to a one-off fluke, were extremely overlapping (standard deviations comprise between ±5%). In addition, we also decided to reduce the number of experiments based on the main goal of our paper, sustainability; thus, we minimised the resource consumption (time, materials, and energy) on the grounds of solid and reliable data.

Regarding the media choice, in the revised paper, the explanation (in the Results and Discussion) has been implemented to help the reader better understand the common passages in the medium selection.

 

 

There are big differences in the secondary metabolites between the two cell lines (V MSA and V B5A). Seven stilbenoid derivatives were found in V B5A, compared to three in V MSA. There were also big differences in the total amount. These differences are only thought to be caused by the "media influence" and no deeper investigation has been done. The document does not look at how different parts of the media (like 2,4-D, kinetin and NAA) control the pathways that make stilbenoids, and it doesn't have any information on how important genes are expressed or how enzymes are active. This basic analysis doesn't allow us to easily understand the differences we can see.

 

We thank the Reviewer for his/her concern because thanks to them we implemented the manuscript discussion about the possible/expected results in establishing productive plant cell cultures . We improved the discussion about the results obtained for the phenol content, especially for the stilbenoid class; however, it is challenging to provide an unequivocal and definitive explanation for our results. As reported in the manuscript, the different biosynthetic capability of the two cell lines may only be attributed to the basal medium composition, as the carbon source is qualitatively and quantitatively the same, as well as the hormonal balance, pH value, and explant type. Moreover we contestualised our results with other cell culture based papers, evaluating the enzyme that direct catalyse the trans-resveratrol formation, and the strategies to highlighting stilbenoid production.. We compared our results to some in which grapevine cultures were used, confirming how the results are not only genera, but species specific, and that the optimal conditions for a specific culture are the result of systematic trials and experimentations.

 

 

The claim that "plant cell culture technology is a sustainable tool for added-value food production" lacks empirical support. The manuscript fails to compare the economic feasibility, energy consumption, or scalability of cell culture versus traditional cultivation methods. Without such data, the assertion of "viability" remains unsubstantiated .

 

The literature on plant cell culture-based food is still in its infancy, few works are published on this topic (You can find the literature in the manuscript), and, until now, only two performed a comprehensive environmental impact assessment on plant cell culture systems. This paper studied the Isabella culture establishment, and the chemical investigation of them, also considering the obtained biomasses as an alternative source of food, considering the challenges we are facing. At this point, the economic feasibility evaluation versus the traditional farming products is relatively difficult; further studies are needed to confirm these hypotheses after technical process optimisation of the Isabella in vitro-derived materials, such as other culture condition, the passage on the liquid cultivation, followed by a bench-bioreactor phase, for finally optimizing the scaling up at the industrial level. The aim of this paper was to establish Isabella cell cultures, that, as far as we now, as not yet been obtained, in the light of this plant great potential. We successfully established them, obtaining two cell lines which are promising in several sectors. The biosynthetic potential of the obtained material, along with the advantages of this technology free from any type of pollutant and microbial contamination, with unlimited availability, planned and adaptable according to demand, and with guaranteed titre, can be a good basement for further studies on these calli.

 

 

 Given the current shortcomings, the manuscript does not meet the standards of Applied Sciences. A rejection is therefore recommended.

 

We improved our manuscript based on Your’s and the other two Reviewers’ comments, improving the introduction, aim of the work, discussion and possible outcomings.

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been substantially improved and can be accepted for publication.

Author Response

We would like to thank the Reviewer for the approval

Reviewer 2 Report

Comments and Suggestions for Authors

the revised version is fine, but revision by native english speaker for better presentation and fluency. 

Author Response

We would like to thank the Reviewer for appreciating the improvement of the manuscript. We have further improved the English

Reviewer 3 Report

Comments and Suggestions for Authors

I still hold my concerns. This article explores the potential of using plant cell culture technology to produce sustainable food materials from the grape variety Isabella, including establishing undifferentiated cell lines, evaluating secondary metabolites and nutritional properties. The revised version failed to adequately address the core issues of the original draft. Overall, the research design lacks rigor, the data volume is small, and the innovation is insufficient. Therefore, I have decided to reject the manuscript. Although the revised version has improved, the following key issues have not been fully addressed:
The sample size is only two cell lines, lacking biological replicates and control groups, resulting in insufficient reliability of the results.

The statistical methods (such as ANOVA) were applied improperly, without correcting for multiple comparisons, and the effect size was not reported.

The essence of the research is a technical application (PCC in grapes), and there are already numerous related literature, with limited theoretical contributions.

The quality of the figures is poor (such as low resolution, unclear labels), affecting the interpretation of the data.

Author Response

We thank Reviewer #3 for the time and effort dedicated to evaluating our Manuscript; we entirely respect his/her observations. However, we deem that some of the fundamental aspects of our study may have been misunderstood, and we would like to respectfully bring the following clarification to the attention of Reviewer #3.  

“The sample size is only two cell lines, lacking biological replicates and control groups, resulting in insufficient reliability of the results. The statistical methods (such as ANOVA) were applied improperly, without correcting for multiple comparisons, and the effect size was not reported.”

Our Manuscript focused on the establishment of in vitro cultures from a Vitis labrusca variety (the Isabella). Under specific experimental conditions, i.e. different in basal medium composition, we obtained two distinct cell lines. Due to the nature of the research, which centres on cell line establishment and characterisation (e.g., metabolite content and nutritional value), the use of biological replicates and control groups, as well as statistical analysis that are typically applied in cell culture stimulation or biological studies, is not applicable in this context. Indeed, our study is more aligned with phytochemical analyses using in vitro materials, rather than classical cell biology experiments.

“The essence of the research is a technical application (PCC in grapes), and there are already numerous related literature, with limited theoretical contributions”.

As already reported in the Manuscript, several works have been published related to in vitro plant cell cultures, but to the best of our knowledge, no studies have been published using the Isabella plant as starting material for in vitro cell cultures. We do not understand how a work can be considered “with limited theoretical contribution” just because other studies about the in vitro cell culture establishment from other plants have been published. Behind the original cell lines we characterised, in our study, we propose them as a food alternative, which is an emerging field. The use of plant cell cultures as food supplies is still an unexplored area with enormous scientific and daily-life applications, as we stated in the manuscript.

“The quality of the figures is poor (such as low resolution, unclear labels), affecting the interpretation of the data”.

We would like to clarify that the resolution and clarity of a figure, its caption, and a table were improved in the revised version of the manuscript, also in response to Reviewer #2’s recommendations. Reviewer #2 approved the revised materials. All figures and tables were prepared using the same software and formatting guidelines we have successfully applied in previous manuscripts accepted by MDPI journals, too. At this stage, we are unsure how further improvements could be made without more specific guidance on what aspects remain problematic. Of course, we are open to implementing any concrete suggestions the editorial team might have.

We would like to make clear that we respect the decisions of Reviewer #3. Our intention in sharing the above points is simply to clarify the context and rationale of our study. We hope this will provide a clearer understanding of our scientific approach in the Manuscript.