Ethanol and Methyl Jasmonate Fumigation Impact on Quality, Antioxidant Capacity, and Phytochemical Content of Broccoli Florets during Storage^†

Round 1

Reviewer 1 Report

The authors conducted on broccoli to examine the impact of post-harvest treatments 15 involving abiotic stress to delay the process of senescence. The article is well writte, but there are a few minor issues that need revising.

1. All units of measurement in the manuscript are missing a point in the middle.

2. All the figures are a little too small and can be merged properly, for example, Figures 1 and 2 can be merged without the need to separate.

3. No mathematical statistical analysis seems to have been done in the figure, and the difference between treatment and treatment is not reflected.

 

 

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Author Response

Dear reviewer,

 

We appreciate the time you have taken to read this manuscript. Your comments are very much welcomed as they are pertinent, in particular as regards the statistical analysis of the data. Note that we have included LSD values in all the figures displaying differences between the treatments applied to the florets for a better understanding of the results as you have suggested. We have also included a dot between the units as you also pointed out. Finally, the size of the figures can be modified by the publisher, as they are of sufficient quality for this to be done. Nevertheless, we decided to continue with the arrangement of figures 1 and 2 (separately), as they show very different aspects of the experiment. Figure 1 is basically the determination of the dose, and Figure 2 is a consequence of the first one.

All suggested modifications, as well as additional ones, can be found in the attached file. We look forward to meeting your expectations, and please accept our best regards.

 

Author Response File: Author Response.docx

Reviewer 2 Report

I cannot properly review manuscript entitled ‘Ethanol and methyl jasmonate fumigation impact on quality, antioxidant capacity and phytochemical content of broccoli florets during storage’ due to the fact that storage conditions are not specified nor properly reported. Also spectrophotometric methods used (chlorophyll content, ORAC, TPC, etc.) are not provided with details allowing me to conclude on which day of treatment / storage they were conducted. All those details should be put in materials and methods section. E.g. Table 2 and 3 report that content of the compounds were averaged (0, 7 and 14 days) and table 1 (which is placed in the manuscript after T2 and T3) reports that values were averaged in a different way (0, 7, 14, 21 days). What is more in section 2.1.3. it can be concluded that storage period lasted for 30 days, however measurements from this section were carried out only for 21 days (I think it should include period 21-30 as well?). Since the manuscript investigates number of parameters during the storage period it have to be precisely revised and clarified.

Methodology of kaempferol quantification is not provided.

Why periods of fumigation for ethanol  and MeJA are different? 30-120min vs 45-180min according to the abstract and 0-540 min vs 0-720 min according to the 2.2. Figure 1 presents color differences for ethanol and MeJA fumigations. In case of ethanol there are 8 measuring points and in case of MeJA 5 points. Why it was decided like that?

 

 

Author Response

Dear reviewer,

 

We thank you for taking the time to read this manuscript. Your comments are most welcome as they are pertinent to our work. We have tried to answer each of your questions in the manuscript and will try to detail the way in which we made these corrections.

 

1. I cannot properly review manuscript entitled ‘Ethanol and methyl jasmonate fumigation impact on quality, antioxidant capacity and phytochemical content of broccoli florets during storage’ due to the fact that storage conditions are not specified nor properly reported.

A new section (2.1.3) has been included in the text to clarify this observation.

2. Also spectrophotometric methods used (chlorophyll content, ORAC, TPC, etc.) are not provided with details allowing me to conclude on which day of treatment / storage they were conducted. All those details should be put in materials and methods section. E.g. Table 2 and 3 report that content of the compounds were averaged (0, 7 and 14 days) and table 1 (which is placed in the manuscript after T2 and T3) reports that values were averaged in a different way (0, 7, 14, 21 days).

A new paragraph in section 2.1.5 has been written to provide additional details on this methods, sections 2.1.3 and 2.1.4 were also modified for a better understanding on how treatments were performed.

3. What is more in section 2.1.3. it can be concluded that storage period lasted for 30 days, however measurements from this section were carried out only for 21 days (I think it should include period 21-30 as well?). Since the manuscript investigates number of parameters during the storage period it have to be precisely revised and clarified.

This was an error since the maximum storage time was 21 days. However, the analysis of glucosinolates was limited to 14 days due to budget constraints.

 

4. Methodology of kaempferol quantification is not provided.

This compound was analyzed together with HCA, following the same methodology, using sinigrin as the internal standard.

5. Why periods of fumigation for ethanol  and MeJA are different? 30-120min vs 45-180min according to the abstract and 0-540 min vs 0-720 min according to the 2.2. Figure 1 presents color differences for ethanol and MeJA fumigations.

The methodology involved utilizing a diverse array of treatments to determine the hormetic dose first, followed by evaluating the effects of three treatments, comprising of the hormetic dose, a control, and a dose four times greater, on the quality parameters and phytochemical compounds of florets over the course of storage.

6. In case of ethanol there are 8 measuring points and in case of MeJA 5 points. Why it was decided like that?

The dose range varies because in the case of ethanol two breakpoints were detected and therefore more measurements were needed to ensure that the concentration effect remained constant. For methyl jasmonate, only one breakpoint was observed, and the increase in floret yellowing behaved in a reciprocal manner as the concentration of this growth regulator was increased.

We look forward to meeting your expectations, and please accept our best regards.

Author Response File: Author Response.docx

Reviewer 3 Report

Revised Introduction section based on the structure below:

1st paragraph: Problem statement

2nd paragraph: Current ongoing solution

3rd paragraph: Proposed solution in this work.

4th paragraph: Summarized the current research novelty and objective of this work.

·        Problem statement of your introduction is not strong, need to discuss more about it.

Results:

Toward the end of this section, it is recommended to include previous related research work and has to be critically discussed.

 

How about the other nutritional components of broccoli like protein, sugar, Mg, Ca, etc. Does ethanol and methyl jasmonate fumigation maintain o decrease their concentration? Can you talk something about?   

 

Conclusion

·        Highlight the main finding of the study.

 

·        Please include the limitations and what can be done for future studies.

 

References: Please improved as some of them were before 2010.

 

Author Response

Dear reviewer,

 

We appreciate your willingness and time to review this manuscript, which we have modified based on your comments and those of other reviewers.  As you can see in the attached file, the introduction and conclusions were modified according to the proposed structure, which we appreciate, as it makes the comprehension of the text easier. Nevertheless, we believe that the references used are pertinent in spite of their antiquity and that they have been used to the best of our ability in an adequate manner for the context of the subject matter.

In response to your inquiry about how these treatments may impact minerals and nutrients, such as sugars, it is probable that they have an effect. With regards to simple sugars, it is plausible that they are utilized in the production of the secondary metabolites mentioned in this manuscript, as well as other metabolites that were not specifically examined. Additionally, based on the observed respiration values, the initial peak in florets subsequent to gasification by any of these elements suggests this to be the case. Furthermore, the yellowing produced by high doses of MeJA is evidence of chlorophyll degradation, and subsequent detachment of the magenesium atom in the pyrrole ring of chlorophyll. However, we do not know how the magnesium or other minerals such as calcium you mention might be utilized subsequently. This would definitely be a good object of study as a possible continuation of this research, to which other aspects should be incorporated, particularly the sensory ones mentioned in the conclusion of the study.

 

We look forward to meeting your expectations, and please accept our best regards.

 

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Thank you for considering my remarks. I am mostly satisfied with the results. However, the information about when analysis (2.1.5 and 2.1.6) were done is still missing. E.g. in section 2.1.4. it is written: 'Color measurement was carried out after every 24 h for a period of 21 d [32]'. I suggest to put similar details to mentioned two sections. It is important especially that Authors provided information that some of the analysis were conducted for 14 or 21 days. All those details should be put in materials and methods section, as I mentioned before. 

Introduction points out that kaempferol and quercetin are the most abundant flavonoids in broccoli. Kaempferol was detected in simillar amounts to a chlorogenic acid. So if quercitin could be found in a comparable amounts to those of kaempferol, why it was decided not to inlcude it in the results? LC-MS coupled with a DAD is a tool where you can investigate numerous compounds. Can you include in your answers short commentary about it?

Table 1 is still placed after T2 and T3.

Author Response

Dear reviewer,

Thank you again for taking the time to read our responses carefully. All your comments are still very pertinent and certainly benefit the manuscript. We have tried to address them as follows: 

1. Thank you for considering my remarks. I am mostly satisfied with the results. However, the information about when analysis (2.1.5 and 2.1.6) were done is still missing. E.g. in section 2.1.4. it is written: 'Color measurement was carried out after every 24 h for a period of 21 d [32]'. I suggest to put similar details to mentioned two sections. It is important especially that Authors provided information that some of the analysis were conducted for 14 or 21 days. All those details should be put in materials and methods section, as I mentioned before.

We have added these details in the materials and methods section, as well as modifying certain sections. In fact, the example you mention did not correspond to the appropriate section. You will now find that it corresponds to lines 172-174, where the color measurement was indeed done on a daily basis for the determination of the hormetic dose.

The paragraph concerning the color measurement of the specified dosages currently reads as follows (lines 194-195):

“The color was assessed at uniform intervals of 7 days over a period of 21 days, specifically on days 0, 7, 14, and 21”

The sampling information for chemical analysis (lines 226--227) now reads as follows:

“All measurements were performed every 7 d in triplicate on three broccoli samples for 21 days”.

The information related to secondary metabolites (GLS, HCA, etc.) can now be reported (lines 242-243) as follows:

“The measurements were carried out in triplicate on three broccoli samples on days 0, 3, 7, and 14”

 

2. Introduction points out that kaempferol and quercetin are the most abundant flavonoids in broccoli. Kaempferol was detected in simillar amounts to a chlorogenic acid. So if quercitin could be found in a comparable amounts to those of kaempferol, why it was decided not to inlcude it in the results? LC-MS coupled with a DAD is a tool where you can investigate numerous compounds. Can you include in your answers short commentary about it?

Actually, we did not detect it, and it could be for several reasons, but for the moment we can only suggest two hypotheses:

1. The cultivar utilized may produce quercetin in very low concentrations, and this could be further influenced by the growing season.
2. We have previously speculated that the synthesis of secondary metabolites may be influenced by the reductive state of cells caused by oxidative stress from different types of pressures. For instance, under UV-B radiation, the production of compounds with a reducing character may be preferred over other compounds with a lower reducing capacity. As such, indole GLS have been found in significantly higher concentrations compared to phenolic acids and other flavonoids.

 

3. Table 1 is still placed after T2 and T3.

The table numbers have been changed, and the corresponding sections in the text where they are referenced have also been updated. 

We are eager to fulfill your expectations and send our warmest regards.

Author Response File: Author Response.docx