Determination of Concentration of Prenylated Flavonoids and Analysis of Physicochemical Parameters of Beers Available on the Polish Market

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

The paper is a survey on the content of xanthohumol, isoxanthohumol, and prenylated flavonoids in 35 Polish commercially beers of different style.

However, you should have analyzed the antioxidant properties of beers and then correlate them with the content of xanthohumol, isoxanthohumol, and prenylated flavonoids.

Moreover, you should find a better way to show your results, using figures. 

Figures 1 and 2 may be inserted as supplementary material.

The text should be deeply revised for the English language.

Line 153-159: please, This paragraph is a repetition of the same information written in Materials and Methods. It shoud be removed.

 

Comments on the Quality of English Language

The text should be revised for the English language.

Line 56-57 modify the sentence, this is a suggestion:

Using this technology, XN does not isomerize, since the beer is not heated during dry-hopping [26].

Lines 59-62 modify the sentence, this is a suggestion:

Another possible way to increase the XN concentration in beer is the production of dark beers (using caramel or chocolate malts), since in previous studies there have been indications that XN can create chemical complexes with the products of the Maillard reaction.

Check the use of the English time tenses in all the text, in particular the use of the present perfect.

For example, line 30 ..have received...better received, line 33 have determined better determined

Minor revisions of the English language:

Line 22-23: improved the sentence, this is a suggestion: In the recent years many studies concerned the ways of increasing...

Author Response

Reviewer 1

However, you should have analyzed the antioxidant properties of beers and then correlate them with the content of xanthohumol, isoxanthohumol, and prenylated flavonoids.

The goal was to assess concentration of these substances, not their antioxidant activity.

 

Moreover, you should find a better way to show your results, using figures. 

Figures become unreadable with such amount of homogenous groups, but we can provide them, if necessary.

Figures 1 and 2 may be inserted as supplementary material.

They were transferred to supplementary materials.

 

The text should be deeply revised for the English language.

We have revised the English language.

Line 153-159: please, This paragraph is a repetition of the same information written in Materials and Methods. It shoud be removed.

These information is not given in the Materials and Methods section. Perhaps line numbering error?

Line 56-57 modify the sentence, this is a suggestion:

Corrected.

Lines 59-62 modify the sentence, this is a suggestion:

Corrected.

Check the use of the English time tenses in all the text, in particular the use of the present perfect.

For example, line 30 ..have received...better received, line 33 have determined better determined

Minor revisions of the English language:

Line 22-23: improved the sentence, this is a suggestion: In the recent years many studies concerned the ways of increasing...

English language was corrected throughout the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

 Concentration of prenylated flavonoids in beers available on the Polish market

 

Title:

• Specify what was done. Determination? Evaluation? Also, physicochemical analysis was performed, which showed an influence on the contents of the target analytes. It would be more interesting if statements were added to the introduction.

 

Authors and Affiliations:

• No issues found

Abstract:

• Provide a short background and maybe move the statement about the number of samples in the methodology part, wherein HPLC analysis was mentioned. Also, a short background why phenylated flavonoids are important.
• In the methodology part, please mention if these beers are locally produced and or some are imported.
• Mention that physicochemical analysis were performed as well and include it in the abstract.
• Authors should include the highlight of the physicochemical differences in the content of prenylated flavonoids.
• Also, mention the detector used.
• Provide a short conclusion or significance of the said findings.

 

Keywords:

• Add more keywords. Please include the instrumentation and prenylated flavonoids, beer, beverage, etc.

 

Introduction:

• The introduction is substantial but was written in one block, which makes it difficult to read. Please divide the introduction into three parts.
• The introduction is well written, and enough studies were included to back up the research question and the research gap.
• Authors are encouraged to add sentences that indicate the extent of research done in the context of quantification of prenylated flavonoids or similar compounds to establish the significance of this study further.
• The aims and objectives are too short, and authors are requested to provide context on what has been done on the study, what comparisons were performed, etc.
• Question: Please justify why prenylated and xanthohumol, and isoxanthohumol were chosen as target analytes amongst all components available.
•  

Methodology:

• 1.1. Research material. It would be better if the revised intro to Beer Samples.
• 1.1. Research material. Please provide context on storage conditions, exposure to light, temperature control, etc. Also, please indicate whether samples are all produced locally in Poland or some were imported but are available in Poland.
• Ensure that in all analyses, the number of replications should be mentioned.
• In all parts of the methodology, use prepositions and conjunctions rather than enclosing the experimental conditions in parentheses. Also, please improve the statement of the methodology.
• Section 2.2.2. Please clarify this: which was constructed from twelve before mentioned Strata C18-columns
• Section 2.2.2. Please provide more details on Strata C18-E. Is it a syringe type? What is the height and diameter?
• Section 2.2.2. Solvent systems can be expressed as a paragraph.
• Section 2.2.2. This section would require more detail and clarity. The paper is submitted to Beverages and not to a chromatography journal; therefore, authors are encouraged to ensure that the method included here is described clearly and can easily be understood by readers who are not so familiar with chromatography.
• Section 2.2.3. Express as parentheses or tabulate the chromatographic program.
• Section 2.2.4. Please provide pore size and brand of filter paper.
• Section 2.2.5. Authors are required to describe the methodology in detail in such a way that it can be reproduced by other researchers.
• Section 2.2.5. Analysis of the beer's basic physicochemical parameters. Please justify that ethyl alcohol content, extract content, beer density, wort extract content, and beer final attenuation can really be measured properly by the Anton Paar DMA 4500M oscillating densitometer. Do you have other modules, or did you use the instrument as is? Also, were there standards used? Please provide more context.
• Section 3. Data analysis. Some beer styles are only represented by 2 samples. Please justify if whether the said number is enough to generate a sound statistical analysis.

 

Results:

• Please explain why % alcohol content as per packaging was not considered in the study.
• Overall, tables and figures are prepared properly and can easily be comprehended since it contain legends or descriptions.
• Results. The intro paragraph is a repetition of what had been mentioned about the beer samples in the methodology. Please come up or provide more important information. Probably, alcohol content, etc.
• Figures 1 and 2. Authors are encouraged to include the chromatogram of the standards either as superimposed or as a separate image to the right of that of the beer samples.
• Statistical analysis was performed, but the authors failed to describe and interpret the results in such a way that the significance and p-values are mentioned side by side with the results. Please revise.
• Section 2. Physicochemical parameters of the beers available on the Polish market. Please provide context on why each physicochemical characteristic were tested. What are they for?
•  

 

Discussion:

• Be careful in discussing melanoidins are other Maillard reaction products since these were not measured in the study.
• Discuss the importance of wort and hops in beer making and why different manufactures use different approaches or methods in applying it.
• Unfortunately the discussion never mentioned the implications of the contents of IXN and XN in terms of its health benefits, safety, taste, and consumer preference. Please reflect and look into your introduction and ensure that your research questions were answered.

 

Conclusion:

• It looks like a repetition of the abstract. Please revise this. Mention what was analysed, what are the major findings (without the inclusion of data or values) and then, indicate why the study;s findings are important.

References:

• Majority of the references are old (10 years and above) please update.

 

Comments on the Quality of English Language

Comments on Grammar and Formatting:

• Avoid starting a sentence with a number. Spell it out into words.
• Ensure that long paragraphs are divided accordingly to ensure readability.
• Double-check and ensure that concentrations adhere to the correct format of MDPI.
• Subsection titles should have a consistent format. (italicized vs normal)
• Please revise your work and ensure that grammar is correct, appropriate conjunctions and linking words are used to ensure cohesiveness of the work.

Author Response

Reviewer 2

Title:

• Specify what was done. Determination? Evaluation? Also, physicochemical analysis was performed, which showed an influence on the contents of the target analytes. It would be more interesting if statements were added to the introduction.

Title was corrected

 

Abstract:

• Provide a short background and maybe move the statement about the number of samples in the methodology part, wherein HPLC analysis was mentioned. Also, a short background why phenylated flavonoids are important.

Number of beers analysed is mentioned, therefore it seems obvious, that all of the beers were analysed? Number of replicates of the HPLC analysis doesn’t seem necessary in the abstract.

• In the methodology part, please mention if these beers are locally produced and or some are imported.

This information was implemented

• Mention that physicochemical analysis were performed as well and include it in the abstract.

This information was added.

• Authors should include the highlight of the physicochemical differences in the content of prenylated flavonoids.

They were not universal, therefore it is hard to add shortened highlights to the abstract.

• Also, mention the detector used.

Added.

 

Keywords:

• Add more keywords. Please include the instrumentation and prenylated flavonoids, beer, beverage, etc.

Some more keywords were introduced

 

Introduction:

• The introduction is substantial but was written in one block, which makes it difficult to read. Please divide the introduction into three parts.

It was divided, hopefully it looks better now.

• Authors are encouraged to add sentences that indicate the extent of research done in the context of quantification of prenylated flavonoids or similar compounds to establish the significance of this study further.

Information that the xanthohumol and isoxanthohumol were analysed was added to the introduction.

• The aims and objectives are too short, and authors are requested to provide context on what has been done on the study, what comparisons were performed, etc.

We have expanded slightly the aims of the study.

• Question: Please justify why prenylated and xanthohumol, and isoxanthohumol were chosen as target analytes amongst all components available.

We concentrated mainly on the xanthohumol, as it was most interesting compound for as, and additionally expanded the study to determine amount of isoxanthohumol, to see, whether small/large concentration of xanthohumol are closely connected with small/large concentration of isoxanthohumol, because, logically, there should be some connection between these two.

Methodology:

• 1.1. Research material. It would be better if the revised intro to Beer Samples.

We left the ‘research material’, but added ‘(beer samples)’. We hope its all right.

• 1.1. Research material. Please provide context on storage conditions, exposure to light, temperature control, etc. Also, please indicate whether samples are all produced locally in Poland or some were imported but are available in Poland.

Information was added.

• Ensure that in all analyses, the number of replications should be mentioned.
• In all parts of the methodology, use prepositions and conjunctions rather than enclosing the experimental conditions in parentheses. Also, please improve the statement of the methodology.

It was provided and included throughout the manuscript.

 

• Section 2.2.2. Please clarify this: which was constructed from twelve before mentioned Strata C18-columns

SPE system was constructed from 12 Strata C18 columns, manifold and pump, as it is clearly stated in the sentence.

• Section 2.2.2. Please provide more details on Strata C18-E. Is it a syringe type? What is the height and diameter?

Circa 80 mm height and 11mm internal diameter. Information was given in the manuscript.

• Section 2.2.2. Solvent systems can be expressed as a paragraph.
• Section 2.2.2. This section would require more detail and clarity. The paper is submitted to Beverages and not to a chromatography journal; therefore, authors are encouraged to ensure that the method included here is described clearly and can easily be understood by readers who are not so familiar with chromatography.

We believe that we described this in detail. If it were chromatography journal, we would’ve just written down that extraction was done with four solvents, gave the information about the solvent and given the extraction time. This is adequate to perform the extraction for somebody with basic knowledge about SPE systems. Our description is very detailed.

• Section 2.2.4. Please provide pore size and brand of filter paper.

Model and brand was added.

• Section 2.2.5. Authors are required to describe the methodology in detail in such a way that it can be reproduced by other researchers.

It can be. Anton Paar DMA 4500M allows analyses of all these parameters with a click of a button. Nothing more needs to be added.

• Section 2.2.5. Analysis of the beer's basic physicochemical parameters. Please justify that ethyl alcohol content, extract content, beer density, wort extract content, and beer final attenuation can really be measured properly by the Anton Paar DMA 4500M oscillating densitometer. Do you have other modules, or did you use the instrument as is? Also, were there standards used? Please provide more context.

DMA 4500M allows for the analyses of the alcohol content with the error up to just 0.1% v/v. Wort extract content and attenuation is automatically calculated on the basis of extract content and alcohol content of the measured beverage.

• Section 3. Data analysis. Some beer styles are only represented by 2 samples. Please justify if whether the said number is enough to generate a sound statistical analysis.

 This is why we didn’t do statistical analysis of the beer styles, but the beers individually.

• Please explain why % alcohol content as per packaging was not considered in the study.

Because the goal was not to determine differences between given alcohol content and measured alcohol content. Also, Polish legislation allows error of 0.5% of abv for beers under 6.5% abv and error of 1% for beers over 6.6%. We don’t see the reason to indicate alcohol content per packaging, as it has nothing to do with the goal of the study.

• Results. The intro paragraph is a repetition of what had been mentioned about the beer samples in the methodology. Please come up or provide more important information. Probably, alcohol content, etc.

We have shortened the into paragraph not to repeat information.

• Figures 1 and 2. Authors are encouraged to include the chromatogram of the standards either as superimposed or as a separate image to the right of that of the beer samples.

We were asked to transfer the chromatograms to Supplementary Data.

Section 2. Physicochemical parameters of the beers available on the Polish market. Please provide context on why each physicochemical characteristic were tested. What are they for?

At the beginning, we strongly believed that beers with higher starting extract content and higher alcohol concentration will prove to be the beers with higher xanthohumol content. It is not so and the correlation do not indicate anything like that. Therefore, we decided to leave the table as is, so each reader could see the same results.

 

Discussion:

• Be careful in discussing melanoidins are other Maillard reaction products since these were not measured in the study.

Our discussion about melanoidins is hypothetical, however, it is proven that darker beers, obviously, contain much higher concentration of Maillard products than light beers.

• Discuss the importance of wort and hops in beer making and why different manufactures use different approaches or methods in applying it.

We have added a section discussing this aspects.

 

• Unfortunately the discussion never mentioned the implications of the contents of IXN and XN in terms of its health benefits, safety, taste, and consumer preference. Please reflect and look into your introduction and ensure that your research questions were answered.

We cannot conclude anything about the safety and taste, as the study didn’t aim to analyse that. However, we have added a paragraph at the end of the discussion about XN content in beers.

• It looks like a repetition of the abstract. Please revise this. Mention what was analysed, what are the major findings (without the inclusion of data or values) and then, indicate why the study;s findings are important.

Conclusion was rewritten.

• Majority of the references are old (10 years and above) please update.

We added some new references. We also often cited 10-year or older references, because newer articles stated have given the same information, while citing the older references (cited by us). We believe that citing the original work should be more important than citing ‘newer’ manuscript.

• Avoid starting a sentence with a number. Spell it out into words.

Corrected.

• Ensure that long paragraphs are divided accordingly to ensure readability.

We decreased number of long sentences in the manuscript, we hope it reads better now.

• Double-check and ensure that concentrations adhere to the correct format of MDPI.

 The main discrepancy is our use of mL and uL than typical metric units, but from what we see, the MDPI articles still use this units, despite encouraging metric SI units. If that will be problem for the editorial team, we will switch them to centimetre and milimeter cubed.

Subsection titles should have a consistent format. (italicized vs normal)

The template states, that main point (1, 2 or three) should be boldened, the first level of subsection (4.1) should be italicized, while lower levels of subsections (4.1.1.; 3.1.1.4) should be normal.

• Please revise your work and ensure that grammar is correct, appropriate conjunctions and linking words are used to ensure cohesiveness of the work.

We have corrected the language throughout the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

In the article entitled “Concentration of prenylated flavonoids in beers available on 2 the Polish market” the authors have carried out an analysis of the content of xanthohumol and isoxanthohumol, components with beneficial health properties, in 35 beers of different styles from Poland.

The article is well written and structured, explaining the different materials and methods used, and the results obtained. It is an interesting research, the results of which can be published, with only a few minor changes required:

Line 20: It sounds repetitive to use “produced” twice in the same sentence.

Line 83: I find it unnecessary to cite three previous articles in this case, since you are only talking about the reagents used. It does not matter whether they were already available or newly purchased; it is only necessary to cite the brand and indicate that they are HPLC grade.

Line 239: Could you indicate which varieties have the highest xantohumol content?

Author Response

Reviewer 3

Line 20: It sounds repetitive to use “produced” twice in the same sentence.

Corrected.

Line 83: I find it unnecessary to cite three previous articles in this case, since you are only talking about the reagents used. It does not matter whether they were already available or newly purchased; it is only necessary to cite the brand and indicate that they are HPLC grade.

We didn’t exactly bought the standards – we created them and analysed them ourselves. The methods used are described in these references.

Line 239: Could you indicate which varieties have the highest xantohumol content?

They are mostly traditional German bittering varieties, such as Magnum, Taurus, Perle, with the notable exception of English Brewer’s Gold, which is a parent hop for varieties such as Centennial and other famous ‘new age’ hops.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

the paper has been modified, but it needs more corrections.

Title: please, change the title, if you use “Determination” it is it is superfluous to use “of concentration”,

Determination of prenylated flavonoids and analysis of physicochemical parameters of in beers available on the Polish market

The paper needs to be reorganized, please read the Instructions for Authors and use the template. This should be the structure of the article:

• Introduction 
• Materials and Methods
• Results
• Discussion
• Conclusions

A question: Is the method for analyzing the different compounds so precise that it can determine their concentration to three decimal places? Please, check this for the prenylated flavonoids.

Line 104-110: this paragraph should be modified.

Here is a suggestion:

In this study the analyses of 35 commercially beers available in the Polish market are reported. In particular, the content of the physicochemical parameters and of the prenylated flavonoid (XN and IXN).

Comments on the Quality of English Language

The text needs some revisions of the English language.

 Check the use of the term "present" in all the text..

Line  36: substitute the term present: The main prenylated flavonoids, originating from hops and found in beer, are ... 

Line 372: improve the sentence

However, it can be converted by the human gut microbiota into a powerful phytoestrogen,

Line 384: Usually, in beer brewing technology, the bitterness of the hops should be balanced by the sweetness of the sugars contained in the beer;

Line 375-376: revise these lines, the name of the compound is written in 2 lines.

Line 410-417:  This paragraph should be modified.

The samples analyzed were 35 commercially available beers sold in the Polish market. Their styles were different: 8 American Indian Pale Ale style (AIPA), 9 Pale Ale style (PALE), 3 Indian Pale Ale style (IPA); 2 Stout style (ST), 3 Porter style (POR), 4 Lager style (LAG), 4 Pilsener style (PIL) and 2 Witbier style (WIT).

Line 437: 6 mL is better than six mL

Line 449, 452, 456, 457,  471: 10 µL is better than ten µL

Line 459: 5 mL of Solvent  is better than five

Line 474:  for instead of over

 Line 479: All Each of the beers

Line 482: 200 mL is better than Two hundred mL

Author Response

Title: please, change the title, if you use “Determination” it is it is superfluous to use “of concentration”,

Corrected.

The paper needs to be reorganized, please read the Instructions for Authors and use the template. This should be the structure of the article:

• Introduction 
• Materials and Methods
• Results
• Discussion
• Conclusions

This structure is acceptable, we will leave it for now. If the editorial team decides to change it, we will do it, if the manuscript is accepted.

A question: Is the method for analyzing the different compounds so precise that it can determine their concentration to three decimal places? Please, check this for the prenylated flavonoids.

Yes, we always used three decimal places for that method.

Line 104-110: this paragraph should be modified.

Here is a suggestion:

In this study the analyses of 35 commercially beers available in the Polish market are reported. In particular, the content of the physicochemical parameters and of the prenylated flavonoid (XN and IXN).

Thank you, modified.

 

All of the English corrections were introduced. We decided to use the words, instead of numbers, as the other reviewers suggested that. In the first version we used 2 for two, 5 for five and so on, but were asked to modify it, unfortunately. We also believe that in most of MDPI journals, if the number is small (i.e. 1-10) they preffer to use word than number.

 

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have indeed made progress, particularly in formatting, paragraphing, keyword expansion, abstract completeness, and section organization, and these revisions appear to have been undertaken in good faith. Nevertheless, a number of substantive scientific concerns remain only partially resolved. In particular:

    Key methodological comments (e.g., reproducibility and justification of physicochemical measurements using the Anton Paar DMA 4500M, and clarity of the SPE procedure for a non-specialist audience) were not meet transparent, evidence-based clarification.
    Requests aimed at strengthening interpretive depth—such as contextualizing physicochemical parameters, articulating the broader relevance of xanthohumol and isoxanthohumol, and engaging more meaningfully with recent literature—were only minimally addressed.

My recommendation is Major Revision, with a clear directive that the next version must:

    Provide method descriptions that are genuinely reproducible and accessible to the journal’s readership.
    Justify instrumental measurements and calculations with appropriate technical and bibliographic support.
    Strengthen the scientific framing and discussion, particularly regarding the relevance of prenylated flavonoids and physicochemical parameters.
    Engage more robustly with recent literature to position the study within the current state of the field.
    Adopt a more precise, objective, and collegial scientific tone throughout.

If these core issues are not adequately resolved in the next round, rejection would then be appropriate.

Comments on the Quality of English Language

Comments on Grammar and Formatting:

• Avoid starting a sentence with a number. Spell it out into words.
• Ensure that long paragraphs are divided accordingly to ensure readability.
• Double-check and ensure that concentrations adhere to the correct format of MDPI.
• Subsection titles should have a consistent format. (italicized vs normal)
• Please revise your work and ensure that grammar is correct, appropriate conjunctions and linking words are used to ensure cohesiveness of the work.

Author Response

Key methodological comments (e.g., reproducibility and justification of physicochemical measurements using the Anton Paar DMA 4500M, and clarity of the SPE procedure for a non-specialist audience) were not meet transparent, evidence-based clarification.

    Requests aimed at strengthening interpretive depth—such as contextualizing physicochemical parameters, articulating the broader relevance of xanthohumol and isoxanthohumol, and engaging more meaningfully with recent literature—were only minimally addressed.

    Provide method descriptions that are genuinely reproducible and accessible to the journal’s readership.
    Justify instrumental measurements and calculations with appropriate technical and bibliographic support.

Anton Paar DMA 4500 equipped with alcolyzer is adequate for:

• MEBAK 9.6.3 (B-590.10.181) method
• EBC 9.2.6 Method
• BCOJ Method 4, 8, 9, 12, and 14
• ASBC Beer-4G method
• OENO 390/2010 method

We believe that if five international, large committees dealing with alcoholic beverages on scale millions-times larger than our study describe a method to be adequate, then it really is adequate. The assumed error of the method is in the range of 0.1% v/v, lower than in analysis using pycnometer (which is the standard).

If that’s not enough, method used in that particular equipment has been analysed and confirmed over 20 years ago to be adequate:

Schropp, P., Bruder, T., & Forstner, A. (2002). Beurteilung des NIR-Verfahrens zur Messung des Alkoholgehalts und weiterer damit verbundener Parameter in Bier (Alcolyzer Beer). BrewingScience, 55(11/12), 212-216.

If that is not suitable, then we believe there is no option to prove that our analysis is made properly. The description of the method used is not really necessary – equipment is turned on, tube is inserted into the beverage (which was degassed and filtered, as already described) and the ‘start’ button is pressed. That’s it. We don’t see that this kind of description is necessary for a scientific journal.

As for the XN and IXN – we have given proper description of the method and provided reference for the methodology. We have added R² for XN and IXN concentration

    Strengthen the scientific framing and discussion, particularly regarding the relevance of prenylated flavonoids and physicochemical parameters.
    Engage more robustly with recent literature to position the study within the current state of the field.
    Adopt a more precise, objective, and collegial scientific tone throughout.

We have improved our discussion section and added newer literature to the manuscript.