Phytochemical Characterization of Humulus lupulus L. Varieties Cultivated in Brazil: Agricultural Zoning for the Crop in Tropical Areas

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study provides actionable insights for both academia and industry. The data are well-supported, and the conclusions align with global sustainability goals in agriculture. I recommend acceptance pending minor revisions to address the points below.

1. Briefly addressing how the observed chemical profiles might translate to beer flavor/aroma would broaden the impact.

2. A direct comparison of key compounds (e.g., α-acids, β-myrcene) with values from temperate-grown counterparts would better contextualize Brazil’s competitiveness.

3. The Results and Discussion section is excessively lengthy and subdivided into over 30 paragraphs, making it challenging to follow. To enhance clarity and readability, it is recommended that the Discussion section be separated from the Results and organized into distinct thematic subsections with descriptive subheadings (e.g., “Bitter Acid Profiles,” “Essential Oil Composition,” and “Climate Adaptation Implications”). This restructuring would streamline the narrative, improve logical flow, and allow readers to better grasp the key contributions of the study.

Author Response

Briefly addressing how the observed chemical profiles might translate to beer flavor/aroma would broaden the impact.
Agree. It was briefly added in the conclusion section [Lines 466-467].

A direct comparison of key compounds (e.g., α-acids, β-myrcene) with values from temperate-grown counterparts would better contextualize Brazil’s competitiveness.
Agree. The section was restructured according to the suggestions. 

The Results and Discussion section is excessively lengthy and subdivided into over 30 paragraphs, making it challenging to follow. To enhance clarity and readability, it is recommended that the Discussion section be separated from the Results and organized into distinct thematic subsections with descriptive subheadings (e.g., “Bitter Acid Profiles,” “Essential Oil Composition,” and “Climate Adaptation Implications”). This restructuring would streamline the narrative, improve logical flow, and allow readers to better grasp the key contributions of the study.
Agree. The section was restructured according to the suggestions.

Reviewer 2 Report

Comments and Suggestions for Authors

The paper has shown some exciting findings regarding the conditions in Brazil because the detail experimental design and clear description of the results. The paper will be worthy for readers. I have few suggestions to improve the paper, as per below-

1. The essential oil extraction method should include a standard methodological reference.
2. In line 137, the term ‘^-1’ should be correctly formatted or spelled out.
3. The manuscript inconsistently uses two spellings for "acid": ‘α’ and ‘a’. This should be unified throughout the text.
4. The authors should include information on the number of biological and/or technical replicates used in Sections 2.6 and 2.7.
5. The discussion sections are overly detailed. The authors are advised to streamline these by focusing on directly relevant literature and concise interpretations.
6. The overall range of variation for each compound should be stated at the beginning of the Results section.
7. The authors should discuss the limitations of the study before the Conclusion section.
8. A similar study (Fortuna et al., 2023) on this species in Brazil has been published (Fortuna, G.C.; Neves, C.S.; Campos, O.P.; Gomes, J.A.O.; Silva, J.C.R.L.; Souza, A.A.; Funari, C.S.d.; Marques, M.O.M.; Bonfim, F.P.G. Hop Tropicalization: Chemical Compositions of Varieties Grown under Organic and Conventional Systems in Subtropical Conditions. Horticulturae 2023, 9, 855.) The authors should compare their findings with this study, particularly in relation to how variations in cropping systems may affect phytochemical content and what happen in their case. Because both of the studies examine phytochemical properties.
9. The manuscript lacks data or discussion on physiological or agronomic traits that might be related to the phytochemical composition. This should be addressed.
10. Moreover, there is another paper published on the same species (cited in this paper) on physiological performance and reproductive biology. Please see: Neves et al., (2024). (Neves, C. S., Aires, E. S., Campos, O. P., Fortuna, G. C., de Oliveira Gomes, J. A., Callili, D., Ono, E. O., Rodrigues, J. D., & Bonfim, F. P. G. (2024). Physiological and productive performance of hop (’Humulus lupulus L.’) varieties grown under subtropical conditions in brazil. Australian Journal of Crop Science, 18(5), 280–287.). Authors should consider this paper in the discussion section.

Author Response

1. The essential oil extraction method should include a standard methodological reference.
It was included [Line 139].

2. In line 137, the term ‘^-1’ should be correctly formatted or spelled out.
Checked and reformulated. 

3. The manuscript inconsistently uses two spellings for "acid": ‘α’ and ‘a’. This should be unified throughout the text. Checked and reformulated. 

4. The authors should include information on the number of biological and/or technical replicates used in Sections 2.6 and 2.7.
Checked and reformulated. 

5. The discussion sections are overly detailed. The authors are advised to streamline these by focusing on directly relevant literature and concise interpretations.
Checked and restructured. 

6. The overall range of variation for each compound should be stated at the beginning of the Results section. Checked and reformulated. 

7. The authors should discuss the limitations of the study before the Conclusion section.
Checked and reformulated [Lines 450-455].

8. A similar study (Fortuna et al., 2023) on this species in Brazil has been published (Fortuna, G.C.; Neves, C.S.; Campos, O.P.; Gomes, J.A.O.; Silva, J.C.R.L.; Souza, A.A.; Funari, C.S.d.; Marques, M.O.M.; Bonfim, F.P.G. Hop Tropicalization: Chemical Compositions of Varieties Grown under Organic and Conventional Systems in Subtropical Conditions. Horticulturae 2023, 9, 855.) The authors should compare their findings with this study, particularly in relation to how variations in cropping systems may affect phytochemical content and what happen in their case. Because both of the studies examine phytochemical properties. Checked.
We have already added this paper in the discussion section [Line 238].

9. The manuscript lacks data or discussion on physiological or agronomic traits that might be related to the phytochemical composition. This should be addressed.
So far, this study has not focused on verifying the physiological traits of hops. However, this presents a valuable opportunity for future research.

10. Moreover, there is another paper published on the same species (cited in this paper) on physiological performance and reproductive biology. Please see: Neves et al., (2024). (Neves, C. S., Aires, E. S., Campos, O. P., Fortuna, G. C., de Oliveira Gomes, J. A., Callili, D., Ono, E. O., Rodrigues, J. D., & Bonfim, F. P. G. (2024). Physiological and productive performance of hop (’Humulus lupulus L.’) varieties grown under subtropical conditions in brazil. Australian Journal of Crop Science, 18(5), 280–287.). Authors should consider this paper in the discussion section. Checked. We have already cited this paper in the discussion section [Line 438].

Reviewer 3 Report

Comments and Suggestions for Authors

Please check the Instructions for Authors regarding references. All references in the list should be formatted according to the guidelines. It is not necessary to include ISSN and DOI. For websites, include the part - Available online: URL (accessed on Day Month Year).

Abstract

Lines 29-30 Please rephrase the second part of the sentence for better understanding.  For example: “…hops cultivated in Brazil may serve as an alternative to achieve the desired chemical quality.”

Materials and Methods

Lines 86-87 Please include the explanations for V, CEC, and OM under the table. Check the table formatting. Present the minerals in a row, one after the other. Check the position (cell in a table) of CaCl₂. Date and Depth should be in the first row along with the other parameters.

Lines 91-92 Could you please provide additional information for the location/name of the licensed nursery?

Line 99 Please provide adequate reference.

Lines 113-114 Please rephrase the sentence, to improve its readability.

Lines 128-130 Please add the model of the FID detector, and include the manufacturer and country or origin for all mentioned equipment.

Results

Line 172 Please restructure this sentence and include β-acids, as both are presented in Table 2.

Line 185-187 Please add a reference for this statement.

Line 193 Please include a brief explanation of what Barth Haas is in parentheses.

Lines 208-209 Check the grammar (uni-form… ).

Lines 214-219 Do you mean the “expected values” according to Barth Hass? If so, make sure to highlight it and make it easier to understand.

Line 257 In the result section, please explain and clarify why you compared the values in your study only with this company, as it is not the only one in the world. Do you have any other references?

Lines 260-262 Could you please add the discussion on how environmental conditions (e.g., fertilizers, preharvest treatments in the field) should be modified, focusing on the investigated area of Brazil in your study, and which postharvest methods might be used to improve α-acid concentration?

Lines 273-276 Please improve the style for better flow.

Lines 314-315 and 317 Please provide adequate references for those statements.

Line 355 Please introduce the abbreviation PCA for the Principal Component Analysis  at its first mention in the main text, and use only PCA thereafter.

Comments on the Quality of English Language

Please check the style and grammar in some sentences.

Author Response

Abstract

Lines 29-30 Please rephrase the second part of the sentence for better understanding.  For example: “…hops cultivated in Brazil may serve as an alternative to achieve the desired chemical quality.”
Checked and reformulated. 

Materials and Methods

Lines 86-87 Please include the explanations for V, CEC, and OM under the table. Check the table formatting. Present the minerals in a row, one after the other. Check the position (cell in a table) of CaCl2. Date and Depth should be in the first row along with the other parameters. The explanations were included.
The table formatting was checked and updated according to the suggestions. 

Lines 91-92 Could you please provide additional information for the location/name of the licensed nursery?
Checked and included [Line101].

Line 99 Please provide adequate reference.
Checked and included [Line 111]. 

Lines 113-114 Please rephrase the sentence, to improve its readability.
Checked and rephrased.

Lines 128-130 Please add the model of the FID detector, and include the manufacturer and country or origin for all mentioned equipment.
The model of the FID detector is the same as the GC because the equipment already has the FID attached, it is not a separate equipment. The country of origin was included [Line 144]. 

Results

Line 172 Please restructure this sentence and include β-acids, as both are presented in Table 2.
Checked. The  β-acids are mentioned in the same paragraph.

Line 185-187 Please add a reference for this statement.
Checked and included.

Line 193 Please include a brief explanation of what Barth Haas is in parentheses.
Checked and included [Line 213].

Lines 208-209 Check the grammar (uni-form… ).
Checked and reformulated.

Lines 214-219 Do you mean the “expected values” according to Barth Hass? If so, make sure to highlight it and make it easier to understand.
Checked and reformulated.

Line 257 In the result section, please explain and clarify why you compared the values in your study only with this company, as it is not the only one in the world. Do you have any other references?
In this study, we use BarthHaas values as a reference, as the company is one of the oldest and largest in the world dedicated to hop trading, processing, and research. Their data are standardized and widely recognized across the brewing industry. Additionally, we compare our results with other references from the scientific literature in the discussion section.

Lines 260-262 Could you please add the discussion on how environmental conditions (e.g., fertilizers, preharvest treatments in the field) should be modified, focusing on the investigated area of Brazil in your study, and which postharvest methods might be used to improve α-acid concentration?
We acknowledge that our manuscript does not address agronomic practices for optimizing α‑acid production in Brazilian conditions. This presents a valuable opportunity for future research. However, we briefly included the postharvest method (storage) more indicated for bitter acids stability. 

Lines 273-276 Please improve the style for better flow.
Checked and modified for better flow.

Line 355 Please introduce the abbreviation PCA for the Principal Component Analysis  at its  first mention in the main text, and use only PCA thereafter.
Checked and changed.

Lines 314-315 and 317 Please provide adequate references for those statements.
Checked and provided.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript presents an important and timely contribution to the ongoing search for viable hop cultivation in subtropical areas, such as Brazil. The chemical characterization of bitter acids and essential oil components across seven Humulus lupulus L. varieties is well executed and highly relevant.

However, several points need improvement:

General structure

• All required sections are present and generally well organized.

• There is no explicit "Discussion" section—Results and discussion are merged, which may reduce clarity!!

2. Logical coherence and argumentation

• In the Results/Discussion section, the flow between data interpretation and literature comparison is sometimes interrupted or overly verbose!!

3. Methodology

• Clear description of the experimental design, climate, location, and cultivation practices.
• Analytical methods (GC-MS, UV-Vis) are well described and referenced.
• Statistical analysis using ANOVA and PCA is appropriate.

• While methods are solid, there is limited detail on the replication of chemical analyses, especially regarding the sample size per variety.
• Soil data are provided but not used to interpret results , a missed opportunity to link soil fertility with compound variation

4. Results

• Table 3 is very dense and hard to interpret.
• Please split the table into compound classes (e.g., monoterpenes, sesquiterpenes).
• Adding visual summaries (radar charts) of key compound families.
• Decimal formatting issue:
  All numeric data in tables use commas for decimal separators (e.g., "56,8%" instead of "56.8%").
  This must be corrected to periods, per English-language scientific standards and formatting norms

5. Discussion and conclusion

• The discussion integrates literature well to contextualize findings.
• Climate change and terroir effects are addressed intelligently.
• Some discussion points repeat results rather than interpreting them!!

• Limitations of the study are not acknowledged (For example: small-scale trial, only one location, one season)
• Conclusion is descriptive rather than analytical. Please suggest concrete next steps or implications for farmers and brewers.

6. Tables and figures

• Please provide high-resolution versions of PCA and HCA figures, with clear axes, units, and legends.
• Hierarchical clusters are formed, but the dendrogram lacks clear labeling of axes, linkage method, and sample replication. Include distance scale and group coloring (Figure 1).
• PCA shows only 51.04% cumulative variance, which is relatively low for robust separation (Figure 2).
• Please using additional components or a 3D PCA plot (PC1 vs PC2 vs PC3)!!
• Please clarify the justification for interpreting PCA with low explanatory power !!

7. References

 Minor inconsistencies in formatting (e.g., DOI placement, hyphenation of journal names).

Please ensure all references are cross-checked for accuracy and consistency with journal style!!

8. Language and style

• Some long and complex sentences reduce clarity — prefer shorter, active-voice constructions.
•  “isomers could serve as markers for hops grown in Brazil.”  Shoule be: "as potential markers"
• "the bitter potential of hops is a key quality criterion" should be more scientific: "a primary indicator of brewing quality"

9. Originality

• Please emphasize the practical implications for Brazilian hop producers and local brewers more explicitly.
• Please add a paragraph comparing cost-effectiveness or yield potential vs traditional hop regions.

Questions for the Authors

• How many replicates per variety were analyzed chemically (essential oils and acids)!?
• Were the samples taken from different plants or the same plant? Please clarify the biological vs technical replication!!
• Did you control or monitor any microclimatic variations in the plots that might affect compound synthesis!?
• Given the low percentage of explained variance, do you believe the PCA biplot allows for confident varietal differentiation? If so, could you elaborate?
• Was the essential oil yield (e.g., mL/100 g) measured and compared among varieties? It would strengthen your conclusions if yield volume were discussed alongside composition.
• You provided soil data (Table 1) but did not correlate it with any phytochemical profiles. Did you consider doing a correlation analysis between soil nutrients and compound accumulation?

Author Response

General structure

All required sections are present and generally well organized.

There is no explicit "Discussion" section—Results and discussion are merged, which may reduce clarity!!
The authors considered it most appropriate to divide the Results and Discussion section into specific topics. We hope this has contributed to improving the clarity and organization of the manuscript. 

2. Logical coherence and argumentation

In the Results/Discussion section, the flow between data interpretation and literature comparison is sometimes interrupted or overly verbose!!
We have made changes in the sections to improve fluidity. We hope it is suitable. 

3. Methodology

Clear description of the experimental design, climate, location, and cultivation practices.

Analytical methods (GC-MS, UV-Vis) are well described and referenced.

Statistical analysis using ANOVA and PCA is appropriate.

While methods are solid, there is limited detail on the replication of chemical analyses, especially regarding the sample size per variety.
The sample size is described in section 2.2 (Experimental design). 

Soil data are provided but not used to interpret results, a missed opportunity to link soil fertility with compound variation.
So far, this study has not focused on verifying the physiological or agronomic traits of hops. The soil analysis has been made to provide area characterization and indicate the necessary fertilization. However, this presents a valuable opportunity for future research.

Results

 Table 3 is very dense and hard to interpret.

Please split the table into compound classes (e.g., monoterpenes, sesquiterpenes).
The compounds were listed in a single block because the table follows the retention‑index sequence; organizing the data in chromatographic order provides a clear, standardized format that readers can interpret at a glance.

Adding visual summaries (radar charts) of key compound families.
We appreciate the suggestion, but we believe the current figures sufficiently support our points.

Decimal formatting issue:
All numeric data in tables use commas for decimal separators (e.g., "56,8%" instead of "56.8%"). This must be corrected to periods, per English-language scientific standards and formatting norms.
Checked and changed.

Discussion and conclusion

The discussion integrates literature well to contextualize findings.

Climate change and terroir effects are addressed intelligently.

Some discussion points repeat results rather than interpreting them!!
Checked and reformulated. 

Limitations of the study are not acknowledged (For example: small-scale trial, only one location, one season).
Checked and reformulated [Lines 450-455]. 

Conclusion is descriptive rather than analytical. Please suggest concrete next steps or implications for farmers and brewers.
Checked and reformulated. 

Tables and figures 

Please provide high-resolution versions of PCA and HCA figures, with clear axes, units, and legends. Hierarchical clusters are formed, but the dendrogram lacks clear labeling of axes, linkage method, and sample replication. Include distance scale and group coloring (Figure 1).
We supplied files with higher quality. However, some crowding of points is unavoidable and reflects the intrinsic data structure rather than image resolution.

Please using additional components or a 3D PCA plot (PC1 vs PC2 vs PC3)!!
We retained the 2D biplot rather than presenting a 3D PCA because the first two principal components already capture a substantial share of the variance (60.15 %). 

Please clarify the justification for interpreting PCA with low explanatory power !!
After incorporating essential‑oil yield into the multivariate matrix, the explanatory power of the ordination improved: PC 1 now accounts for 37.61 % of the total variance and PC 2 for 22.54 %. These higher values confirm that yield is an informative variable and provides a more robust basis for interpreting the relationships among samples.

References

 Minor inconsistencies in formatting (e.g., DOI placement, hyphenation of journal names). Please ensure all references are cross-checked for accuracy and consistency with journal style!!
Checked and reformulated. 

Language and style

Some long and complex sentences reduce clarity — prefer shorter, active-voice constructions.

• •  “isomers could serve as markers for hops grown in Brazil.”  Shoule be: "as potential markers" Checked and reformulated. 
  • "the bitter potential of hops is a key quality criterion" should be more scientific: "a primary indicator of brewing quality"
    Checked and reformulated. 

Originality

Please emphasize the practical implications for Brazilian hop producers and local brewers more explicitly.
It was added in the conclusion section. Regarding local producers, the practical implications involve supporting the selection of hop varieties with phytochemical profiles of interest, particularly those compounds responsible for aroma and bitterness, adapted to tropical climate conditions. For the brewing industry, the practical implications include the adoption of varieties with phytochemical profiles similar to those of temperate-climate varieties, enabling the production of traditional beers. Additionally, the choice of varieties with distinct profiles could result in a unique terroir, contributing to the development of a distinctive national beverage.

Please add a paragraph comparing cost-effectiveness or yield potential vs traditional hop regions.
Talking about the value of hops in relation to acids, saying that the theory for Brazilian hops may not yet be so competitive, but it may be attractive in techniques in which the aim is to add aroma and not necessarily the bitterness coming from acids.

Questions for the Authors

How many replicates per variety were analyzed chemically (essential oils and acids)!? Were the samples taken from different plants or the same plant? Please clarify the biological vs technical replication!!
Both analyses have been made on triplicates. The samples were taken from two different plants. The experimental design is described in Section 2.2. 

Did you control or monitor any microclimatic variations in the plots that might affect compound synthesis!?
We addressed potential microclimatic effects through a randomized‑block design that used two experimental units per variety, all within a single field exhibiting uniform edaphoclimatic conditions, identical irrigation scheduling, and standardized cultural practices. This layout minimizes external influences—such as soil heterogeneity or localized moisture differences—thereby isolating genotype‑driven variation in compound synthesis.

Given the low percentage of explained variance, do you believe the PCA biplot allows for confident varietal differentiation? If so, could you elaborate?
To improve varietal resolution, we added essential‑oil yield, as suggested, to the data matrix. In the revised PCA, PC 1 and PC 2 now explain 37.61 % and 22.54 % of the variance, respectively, yielding a combined 60.15 %. This increase strengthens the separation among varieties, particularly by distinguishing high‑yielding, α‑acid–rich cultivars (e.g., Comet). Consequently, the updated biplot offers a more reliable basis for varietal differentiation while maintaining visual clarity.

Was the essential oil yield (e.g., mL/100 g) measured and compared among varieties? It would strengthen your conclusions if yield volume were discussed alongside composition.
A new table was included for essential oil yield data (Table 2). 

You provided soil data (Table 1) but did not correlate it with any phytochemical profiles. Did you consider doing a correlation analysis between soil nutrients and compound accumulation?
Soil analysis was carried out with the aim of characterizing the experimental area, in addition to providing the basis for correct soil treatment, from the necessary correction to the calculation of fertilization.