Chemical Analyses of Volatiles from Kumquat Species Grown in Greece—A Study of Antimicrobial Activity

Round 1

Reviewer 1 Report

The study titled ‘Comparative Essential Oil Analysis and Antimicrobial Activity from Kumquat Species Grown in Greece’ has been reviewed. The manuscript is well written, but the results are poor. Some information is missing in the Tables and before publication, the manuscript needs to include some more data.

This manuscript is only descriptive, except for the antimicrobial activity. More bioactivities or more information is needed to reach enough quality in the manuscript. I hardly recommend analyzing antioxidant bioactivities or comparing extraction techniques instead. Also, a deeper statistical analysis (like PCA) is necessary to relate which compound might be the responsible of the antimicrobial activity. This information is missing to enhance the manuscript interest.

You pointed that the flower showed antimicrobial activity…but can you include possible bioactivities regarding the composition of the other parts? i.e. antioxidant.

Did you try another extraction method? Maybe you could include supercritical fluid extraction (SFE) as a novel extraction methodology to improve the bioactivity. Indeed, you reference two manuscripts regarding SFE.

Some minor comments:

Line 50 at the beginning of what…? Maybe you can include sweet as a start flavor, and bitter as second or end-flavor.

Line 74 include examples of other essential oils.

Line 94 Did you check the maturity of the fruits? How?

Line 110 What is EO? I do not find the abbreviation meaning

Line 186 Could you please include CAS number, KI calculated and in the literature? Also make a full list of compounds detected instead of three different Table depending on the fruit part (peel, leaves, flower).

Line 165 Scientific names in italic. Same in Line 262, 259 please check

Author Response

The study titled ‘Comparative Essential Oil Analysis and Antimicrobial Activity from Kumquat Species Grown in Greece’ has been reviewed. The manuscript is well written, but the results are poor. Some information is missing in the Tables and before publication, the manuscript needs to include some more data. 

This manuscript is only descriptive, except for the antimicrobial activity. More bioactivities or more information is needed to reach enough quality in the manuscript. I hardly recommend analyzing antioxidant bioactivities or comparing extraction techniques instead. Also, a deeper statistical analysis (like PCA) is necessary to relate which compound might be the responsible of the antimicrobial activity. This information is missing to enhance the manuscript interest.

 You pointed that the flower showed antimicrobial activity…but can you include possible bioactivities regarding the composition of the other parts? i.e. antioxidant.

 Did you try another extraction method? Maybe you could include supercritical fluid extraction (SFE) as a novel extraction methodology to improve the bioactivity. Indeed, you reference two manuscripts regarding SFE. 

Response: Thank you for your suggestions. The scope of this study was the chemical analysis of the essential oils and the evaluation of their antimicrobial activity, as the citrus family is well known in this field. Greece even is a small country has an interesting economically important citrus species production with many different nationally recognized cultivars. The research on their chemistry is scarce.  In the framework of our studies on different citrus species cultivated in Greece (e.g. kumquats, oranges, lemons, grapefruits, tangerines etc), based to Dr Ziogas’ knowledge from the Institute of Olive Tree, Subtropical Plants and Viticulture, Hellenic Agricultural Organization and his specialization and responsibility on citrus plants from the Greek Ministry of Agriculture, we have designed their chemical analyses and antimicrobial evaluation. So, at this moment no antioxidant activities have been planned (eventhough we have a general interest), while in the close future in accordance of the analyses and results of the antimicrobial activities, we could further plan such studies

The techniques that we followed for the extraction, where selected according to the general practice and international literature where is suggested always the cold press procedure for the essential oil from the peels in order to keep low temperature and protect the volatiles from thermal stress. The best practice for the leaves and flowers accordingly was the conventional hydro-distillation

PCA has been added and a whole paragraph has been inserted in the Results. An author who did the PCA analysis has been added in the authorship.

 Some minor comments:

Line 50 at the beginning of what…? Maybe you can include sweet as a start flavor, and bitter as second or end-flavor. Response: Thank you for your comment. We made the appropriate change in the manuscript, as proposed.

Line 74 include examples of other essential oils. Response: Thank you for your comment. We could not provide the proposed example of other essential oils, since Zeng et al., 2023 (reference 14) on page 273, in section 1, wrote: “The essential oil is mild in nature, and it is considered to have the highest quality of all citrus essential oils”. Therefore we cannot imagine and add examples of essential oils, because this would be misleading since they were not clearly stated in the reference.

Line 94 Did you check the maturity of the fruits? How? Response: Thank you for your comment. It is known by the literature and by observations that unripe kumquat fruit is green-colored, whereas ripe ones (mature) are bright orange-yellow. In our case, all fruits were bright orange-yellow and we also measured the total soluble solids content, which was above 10. We did not include such quality measurement in the current manuscript, since this was not the scope of the article and we keep these data for future publication. Also, the fruits were harvested during March, 8 months after the flowering period, a period that is needed for the fruits to ripen, as it is already known.

Line 110 What is EO? I do not find the abbreviation meaning. Response: Thank you for your comment. We added the definition of EO in the manuscript (EO = essential oil).

Line 186 Could you please include CAS number, KI calculated and in the literature? Also make a full list of compounds detected instead of three different Table depending on the fruit part (peel, leaves, flower). Response: According to the comment and the global bibliography, where the CAS number is not usually included in the papers, we collected all the data from the three chemical tables (1,2,3) in a full list table (Table 1) and we added the calculated KI and these from literature.

Line 165 Scientific names in italic. Same in Line 262, 259 please check. Response: Thank you for your comment. Line 165, has no scientific name to write in italic. In the uploaded pdf file all the scientific names were in italics. The MDPI software changed them to normal. All of them have been reformatted to the whole body of the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

The study is on interesting topic, but there are some questions to be answered:

1. In M&M there is no information about collection, preparation, and distillation of flowers samples;

2. Why the information for analyses and antimicrobial activity of Nagami flower essential oil is missing? If this oil was not produced this cannot be defined as comparative study and in such case the information presented in title, abstract and conclusions is speculative and misleading;

3. There are many typing errors - for example the names of the plants and microorganisms must be formatted in italic in entire text.

Author Response

The study is on interesting topic, but there are some questions to be answered:

1. In M&M there is no information about collection, preparation, and distillation of flowers samples; Response: Thank you for your comment. We made the appropriate change in the manuscript, as proposed to provide information regarding the collection of flowers. The preparation and distillation of flowers is indicated in line 116 and line 120, respectively.

2. Why the information for analyses and antimicrobial activity of Nagami flower essential oil is missing? If this oil was not produced this cannot be defined as comparative study and in such case the information presented in title, abstract and conclusions is speculative and misleading; Response: The yield of the essential oil of the Nagami flower from the distillation was not the required for chemical analysis and biological testing, as these flowers are very small and it is very difficult to collect enough amount. According to your suggestion, we changed the title and the appropriate parts of the manuscript.

3. There are many typing errors - for example the names of the plants and microorganisms must be formatted in italic in entire text.Response: Thank you for your comment. We made the appropriate changes in the manuscript. There was a different format from the platform that converted the format from italics to normal.

Author Response File: Author Response.pdf

Reviewer 3 Report

Generally, the authors present an interesting study. However, there are some problems in this paper and it may be considered for publication with some correction. Please find my specific comments below:

1.      Line 47, what is the meaning of ‘tn’?

2.      Line 77, what is the meaning of ‘(93,73%)’?

3.      Line 96-97, the information of flowers was lacking. Please add the harvest year of the samples.

4.      Line 109-112, why not use hydro-distillation to prepare the peel EO? The cold pressed peel EO may contain figment, flavonoid, and pesticide residue, which may interfere the bioactivity test.

5.      Line 131-132, ‘and firmed via a comparison of Kovats retention indices (KIs) with literature data’, I would suggest the authors list the retention index values of each compound in Table 1 and Table 2 to show the chemical composition assignment was correct. The information of which one was terpene or alcohol was common sense; it not necessary to use a column to list this information in the table.

6.      Line 370-371, the Latin name of the citrus should be written in italic.

Author Response

Generally, the authors present an interesting study. However, there are some problems in this paper and it may be considered for publication with some correction. Please find my specific comments below:

1. Line 47, what is the meaning of ‘tn’? Response: Thank you for your comment. We made the appropriate change in the manuscript and changed ‘tn’ with ‘tonnes’
2. Line 77, what is the meaning of ‘(93,73%)’? Response: Thank you for your comment. We made the appropriate changes in the manuscript. We deleted the number so as not to confuse the reader. The presentation of a number value in the introduction was not necessary and we thank the reviewer for this.
3. Line 96-97, the information of flowers was lacking. Please add the harvest year of the samples. Response: Thank you for your comment. We made the appropriate changes in the manuscript. We added the year of sample harvesting.
4. Line 109-112, why not use hydro-distillation to prepare the peel EO? The cold pressed peel EO may contain figment, flavonoid, and pesticide residue, which may interfere the bioactivity test. Response: The techniques that we followed for the extraction, where selected according to our experience in this family, As referred in the answer to the 1^st Reviewer

[Thank you for your suggestions. The scope of this study was the chemical analysis of the essential oils and the evaluation of their antimicrobial activity, as the citrus family is well known in this field. Greece even is a small country has an interesting economically important citrus species production with many different nationally recognized cultivars. The research on their chemistry is scarce.  In the framework of our studies on different citrus species cultivated in Greece (e.g. kumquats, oranges, lemons, grapefruits, tangerines etc), based to Dr Ziogas’ knowledge from the Institute of Olive Tree, Subtropical Plants and Viticulture, Hellenic Agricultural Organization and his specialization and responsibility on citrus plants from the Greek Ministry of Agriculture, we have designed their chemical analyses and antimicrobial evaluation. So, at this moment no antioxidant activities have been planned (even though we have a general interest), while in the close future in accordance of the analyses and results of the antimicrobial activities we could further plan such studies]

5. Line 131-132, ‘and firmed via a comparison of Kovats retention indices (KIs) with literature data’, I would suggest the authors list the retention index values of each compound in Table 1 and Table 2 to show the chemical composition assignment was correct. The information of which one was terpene or alcohol was common sense; it not necessary to use a column to list this information in the table. Response: According to the comment of the Reviewer 1 we collected all the data from the three chemical tables (1,2,3) in a full list table (Table 1) and we added the calculated KI and these from literature. The chemical classification of the compounds accordingly has been omitted.
6. Line 370-371, the Latin name of the citrus should be written in italic. Response: Thank you for your comment. All scientific names are written in italics. During the upload procedure, the MDPI software changed them to normal. All of them have been reformatted to the whole body of the manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Authors did modified the changes I suggested.

I believe now the manuscript is ready to publish in Horticulturae journal. 

But I recommend to include more than one bioactivities in further experiments. Then, the chemical characterization might be better correlated to the bioactivities. 

Author Response

Author Response File: Author Response.pdf

Reviewer 2 Report

The revised version of the paper now is clearly presented and well organized. 

Author Response

The revised version of the paper now is clearly presented and well organized.

Answer: We thank the reviewer for the approval.

Author Response File: Author Response.pdf