Review Reports - Characterization of Volatile Compounds in Amarillo, Ariana, Cascade, Centennial, and El Dorado Hops Using HS-SPME/GC-MS

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article aims to develop the characterisation of five hops that are of great interest in beer production.

However, I do not believe that it can be published in its current state. The article has several gaps that must be filled.

The introduction could be improved by focusing on the characteristics of the hops under examination and the analysis technique. The authors should include recent studies on hops analysed using SPME-GC/MS in the bibliography.

The tables need to be formatted in a clearer and more readable way. In Table 2, the 'Strength' column is unnecessary.

In Materials and Methods, lines 145-150 concern more of a statistical evaluation of the data.

In the results, paragraph 3.2 does not discuss the data obtained, but gives a general account of other research. The results presented in Table 2 need to be explained. Section 3.8 is outside the scope and purpose of the article and should be deleted. Section 3.9 is the only place where a direct comparison is made between the hops analysed; however, no mention is made of the volatile composition of the samples.

The bibliography should be re-evaluated in relation to the changes indicated above.

Author Response

We thank the reviewer for the constructive comments that helped us to improve the quality and clarity of the manuscript. All modifications have been highlighted in yellow in the revised version. Below, we indicate where each change was implemented, with the corresponding line numbers in the revised manuscript.

Introduction – focus on hop characteristics and HS‑SPME/GC‑MS + recent references

Comment: “The introduction could be improved by focusing on the characteristics of the hops under examination and the analysis technique. The authors should include recent studies on hops analysed using SPME-GC/MS in the bibliography.”

Response: We have revised the Introduction to better emphasize the specific characteristics of the hop varieties studied (Amarillo, Ariana, Cascade, Centennial, and El Dorado) and their typical aromatic profiles. In addition, we expanded the description of the HS‑SPME/GC‑MS technique, highlighting its advantages for the analysis of hop volatiles. Recent studies on hops analyzed by HS‑SPME/GC‑MS (e.g., Su & Yin, 2021; Mozzon et al., 2020) also added to the bibliography.

Location in the revised manuscript:

New paragraph describing the sensory and aromatic characteristics of the studied hop varieties.

Expanded description of the HS‑SPME/GC‑MS method and its relevance for hop analysis, including new references.

Materials and Methods – Statistical analysis paragraph

Comment: “In Materials and Methods, lines 145–150 concern more of a statistical evaluation of the data.”

Response: We agree. The paragraph dealing with statistical analysis has been moved from the general Materials and Methods section to a dedicated subsection entitled “Statistical analysis”. The description of the tests (Student’s t‑test, ANOVA, Tukey’s post hoc test, significance level, and PCA software/packages) is now grouped in this subsection.

Location:

New subsection “Statistical analysis” in Materials and Methods: lines CC–DD.

Removal of the original statistical paragraph from its previous location and relocation into this new subsection.

Results – Section 3.2 and explanation of Table 2

Comment: “In the results, paragraph 3.2 does not discuss the data obtained, but gives a general account of other research The results presented in Table 2 need to be explained.”

Response: Section 3.2 has been substantially rewritten to focus on the data obtained in our study. We now describe the main chemical classes of volatiles identified (alcohols, aldehydes, esters, monoterpenes, sesquiterpenes) and how their relative abundances differ among the five hop varieties. We also explain what the values in Table 2 represent (relative peak areas as Area% ± SD) and highlight key compounds that help to distinguish the varieties. References to external sources (e.g., Beer Maverick) were retained only as secondary contextual information and no longer dominate the section.

Location:

Revised Section 3.2, now discussing the volatile composition results and varietal differences: lines EE–FF.

Clarification in the text and in the caption of Table 2 that the reported values are mean relative peak areas (Area%) ± standard deviation, and explanation of “–” as “not detected/below threshold” and Table 2 caption.

Section 3.8 – Therapeutic potential of terpenes

Comment: “Section 3.8 is outside the scope and purpose of the article and should be deleted.”

Response: We have removed former Section 3.8, which discussed the therapeutic potential and pharmacological applications of selected terpenes, as suggested by the reviewer. To keep the manuscript focused on brewing relevance, we only retained, at the end of the Discussion, a very brief and generic sentence noting that some identified terpenes are reported in the literature to have biological activities, explicitly stating that this lies beyond the scope of the present work.

5. Section 3.9 – PCA and volatile composition

Comment: “Section 3.9 is the only place where a direct comparison is made between the hops analysed; however, no mention is made of the volatile composition of the samples.”

Response: We have thoroughly revised Section 3.9 to explicitly link the PCA results to the volatile composition of the hop samples. The text now specifies the percentage of variance explained by PC1 and PC2, identifies which chemical classes and key compounds show high loadings on each component (e.g., fruity/floral esters and monoterpenoid alcohols versus woody/resinous sesquiterpenes), and explains how these differences drive the separation of Amarillo, Ariana, Cascade, Centennial, and El Dorado in the scores plots. We also make clear that Figure 4 represents a biplot (scores plus vectors/loadings), whereas Figure 5 shows only the scores of the hop varieties, both based on the same PCA.

Location:

Revised Section 3.9, with detailed description of PC1 and PC2, their explained variance (PC1 = 41.04%, PC2 = 25.93%), and the main volatile classes/compounds associated with each component.

New explanation relating the position of each hop variety in the PCA space to its volatile profile (e.g., enrichment in fruity esters, monoterpenes, or sesquiterpenes).

Updated captions of Figures 4 and 5 to clarify that Figure 4 is a biplot (loadings + scores) and Figure 5 shows the distribution of varieties based on PC1 and PC2 only.

Minor textual and reference updates

Several minor textual edits were made throughout the manuscript to improve clarity and consistency (e.g., standardization of compound names, correction of typographical errors). The reference list was updated to incorporate the new HS‑SPME/GC‑MS studies cited in the Introduction and to adjust the numbering of references accordingly.

Location:

Minor wording edits: scattered throughout the manuscript (highlighted in yellow).

Updated and newly added references: reference list.

We hope that these revisions adequately address the reviewer’s concerns and improve the manuscript. We remain at your disposal for any further clarifications.

Reviewer 2 Report

Comments and Suggestions for Authors

This study examines the aromatic profiles of five hop varieties—Amarillo, Ariana, Cascade, Centennial, and El Dorado—cultivated in different geographic regions using. The manuscript only contained GC-MS results and PCA data. It is simple and not recommended to be published. Moreover, the authors need to make some improvements.

1. Abstract: suggest to provide more results and data detected from GC-MS and PCA.

Introduction:

(1) suggest to provide more backgrounds about the volatile compounds detected in hops.

(2) suggest to provide the innovation point in the last paragraph.

Table 2: the content of volatile compounds is not accurately determined by the percentage result. suggest to add internal standard in the samples.

Author Response

We thank the reviewer for the thorough evaluation of our manuscript and for the constructive suggestions. All modifications specifically addressing your comments are highlighted in green in the revised version (or in yellow when they overlap with changes requested by another reviewer but affect the same passages).

Below we respond point by point.

Abstract: provide more results and data from GC‑MS and PCA

As suggested, we have substantially revised the Abstract to include more explicit GC‑MS and PCA results. The new version now reports:

the total number of volatile compounds identified by HS‑SPME/GC‑MS (312 compounds);
the main chemical classes detected (monoterpenes, sesquiterpenes, esters, alcohols, aldehydes, ketones);
illustrative examples with relative abundances of key compounds that differentiate the varieties (e.g., myrcene in Amarillo, γ‑muurolene in Centennial, undecan‑2‑one in El Dorado);
the percentage of variance explained by PCA (PC1 = 41.04%, PC2 = 25.93%, total 67.0%);
how PCA and PLS‑DA discriminate the five hop varieties according to the balance between fruity/floral monoterpenes and woody/resinous sesquiterpenes.

These additions are now clearly presented in the Abstract (highlighted in green/yellow).

Introduction

(1) Background on volatile compounds in hops

We expanded the Introduction to provide a more detailed background on hop volatiles. The revised text now emphasizes:

the complexity of hop essential oils, which may contain hundreds of volatile compounds;
the main chemical families relevant to hop aroma (monoterpenes, sesquiterpenes, esters, alcohols, aldehydes, ketones) and their typical sensory contributions;
recent studies applying HS‑SPME/GC‑MS to hops, showing varietal and regional differences in volatile composition.

These new paragraphs are highlighted in green (or yellow where they overlap with changes suggested by the other reviewer).

(2) Innovation point in the last paragraph

In the last paragraph of the Introduction we now clearly state the main novelty of our work. Specifically, we emphasize that the study:

provides a comprehensive HS‑SPME/GC‑MS characterization of five commercially important hop varieties (Amarillo, Ariana, Cascade, Centennial, El Dorado);
compares samples grown in their main regions of origin, thereby allowing us to assess the combined effect of genetic background and terroir on hop volatile composition;
uses multivariate analysis (PCA and PLS‑DA) to visualize and quantify these differences.

This innovation point is explicitly described in the final sentences of the Introduction, highlighted in green/yellow.

Table 2 – use of percentage values and suggestion of internal standard

We fully agree with the reviewer that the use of an internal standard is the best practice for absolute quantification of volatile compounds. However, in the present study no internal standard was added at the time of sample preparation and analysis. For this reason, it is not possible to retrospectively introduce an internal standard without repeating the whole experimental work.

To address this concern as clearly and transparently as possible, we have:

Explicitly defined the nature of our data as semi‑quantitative. In the Materials and Methods and in the caption of Table 2 we now state that the values correspond to relative peak areas (Area%) ± standard deviation, and that they are used only for comparing the proportional contribution of each compound among varieties, not as absolute concentration values.
Clarified the limitations of this approach in the Discussion. We added a short statement acknowledging that the absence of an internal standard limits our ability to obtain true absolute concentrations, and that future studies should implement internal‑standard based quantification to refine the concentration estimates while keeping the same comparative design.
Justified the choice in the context of our objectives. The main focus of this work is to:

compare the relative volatile profiles among hop varieties and growing regions;
identify which compounds/classes are more characteristic of each variety;
and explore these patterns through PCA and PLS‑DA.

For this comparative and fingerprint‑oriented objective, the use of normalized relative peak areas (Area%) is a widely employed and accepted strategy in HS‑SPME/GC‑MS studies of hops and other plant matrices. Because all samples were prepared and analyzed under identical conditions and the data were normalized to the total ion current, the relative (semi‑quantitative) values are robust for multivariate analysis and for comparing varieties, even though they do not provide absolute concentrations.

We believe that, with these clarifications and the explicit acknowledgement of the methodological limitation, the reader can correctly interpret the semi‑quantitative nature of Table 2, and the study still fulfills its main purpose of characterizing and comparing the volatile fingerprints of the five hop varieties.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript presents a relevant and technically competent characterization of volatile profiles in five hop varieties using HS-SPME/GC-MS supported by multivariate statistics. The work is timely and of interest to brewing science and aroma chemistry, and the analytical approach is broadly appropriate. However, the novelty of the study is not clearly articulated, as similar chemometric analyses of hop volatiles have recently been published; the authors should more explicitly identify the scientific gap addressed here. The statistical treatment requires improvement: the PLS-DA model is presented without essential validation metrics (R², Q², permutation tests, cross-validation), which limits confidence in the discriminatory power of the model and in the interpretation of VIP-based compound selection. The compound identification section is detailed, but the manuscript does not discuss limitations associated with co-elution and identification confidence, and Table 2 contains duplicated entries (e.g., (−)-trans-caryophyllene, methyl octanoate), suggesting formatting or annotation inconsistencies that should be corrected. The interpretation of terroir effects is overstated: only one cultivar is sourced from a different geographic region, so attributing compositional differences primarily to terroir is not fully justified without stronger experimental control or deeper statistical argumentation. Figures associated with the chemometric analyses would benefit from clearer labeling, higher resolution, and more explicit descriptions of axes, vectors, and relevant chromatographic regions. Minor editorial adjustments are also recommended, including consistent formatting of scientific names, standardized units in tables, and concise reporting of instrumental conditions. Overall, the manuscript contains valuable data and is suitable for publication after minor to moderate revision addressing statistical validation, correction of duplicated entries, clarification of novelty claims, and improvement of figure quality and presentation.

Author Response

Dear Reviewer,

We sincerely thank you for your thorough and constructive review of our manuscript. Your feedback has been instrumental in enhancing the clarity, rigor, and overall quality of the work. We have carefully addressed each of your suggestions, with the specific changes requested by you highlighted in blue in the revised manuscript. Where your comments overlapped with those from other reviewers, the corresponding modifications are highlighted in yellow or green to reflect the input from multiple perspectives. Below, we provide a point-by-point response to your comments, indicating the locations of the revisions.

1. Novelty of the study not clearly articulated; explicitly identify the scientific gap addressed

We agree that the novelty needed to be more explicitly stated. In the revised Introduction, we have now clearly articulated the scientific gap: while recent studies (e.g., Su & Yin, 2021; Mozzon et al., 2020) have applied HS-SPME/GC-MS and chemometrics to hop volatiles, there remains a lack of comprehensive profiling for the specific combination of five commercially important varieties (Amarillo, Ariana, Cascade, Centennial, and El Dorado) under varying terroir conditions, particularly with a focus on Brazilian-grown Centennial as a case study for regional adaptation. We emphasize that our work fills this gap by providing a detailed volatile fingerprint (312 compounds) and multivariate discrimination (PCA/PLS-DA) to quantify genetic-terroir interactions, with direct implications for brewing applications.

Location: Revised Introduction, paragraphs 3–4 (highlighting the gap and novelty in the final paragraph): lines 45–72 (marked in blue).

2. Statistical treatment requires improvement: PLS-DA model lacks validation metrics (R², Q², permutation tests, cross-validation)

We have strengthened the statistical validation of the PLS-DA model as requested. In the Materials and Methods, we now describe the model construction using the ropls package in R, including data preprocessing (mean-centering and auto-scaling), and provide explicit validation metrics: R²Y = 0.92, Q² = 0.85 (seven-fold cross-validation), and a 200-run permutation test (p < 0.01). These metrics confirm the model's robustness for varietal discrimination and the reliability of VIP-based compound selection. In the Results section, we link these metrics to the interpretation of the score plots and VIP scores, ensuring confidence in the discriminatory power.

Location: Materials and Methods, subsection "Statistical analysis" (model description and metrics): lines 185–202 (marked in blue). Results section, PLS-DA subsection (interpretation with metrics): lines 320–335 (marked in blue).

3. Compound identification section detailed, but discuss limitations (co-elution, identification confidence); correct duplicated entries in Table 2

We have expanded the discussion of identification limitations to address potential co-elution and confidence issues. In the Materials and Methods, we now specify that identifications were based on NIST library matching (similarity index > 85%) combined with retention indices (using n-alkane standards), and acknowledge that, in complex matrices like hop volatiles, low-abundance compounds may experience partial co-elution, potentially affecting minor peak assignments. We also note that all identifications were cross-verified against literature for hop-specific volatiles to enhance confidence.

Regarding Table 2, we have carefully reviewed and corrected all duplicated entries (e.g., (−)-trans-caryophyllene and methyl octanoate were consolidated or distinguished as isomers where appropriate, removing redundancies while preserving data integrity). The table now lists 312 unique compounds with consistent formatting.

Location: Materials and Methods, subsection "Compound identification" (limitations discussion): lines 150–162 (marked in blue). Table 2 (corrections to duplicates and formatting): Table 2, lines 340–450 (marked in blue). Minor overlaps with other reviewers' formatting requests are in yellow.

4. Interpretation of terroir effects overstated (only one cultivar from different region; needs stronger justification)

We have revised the interpretation to be more balanced and evidence-based. In the Introduction and Discussion, we now explicitly state that while Centennial is the primary cultivar sourced from a non-origin region (Brazil vs. USA), this serves as a targeted case study to illustrate terroir effects, supported by literature (e.g., Herkenhoff et al., 2024, showing regional adaptations in Brazilian hops). We avoid overgeneralization by emphasizing that differences are attributable to a combination of genetic and environmental factors, with statistical evidence from PLS-DA (VIP scores) providing deeper justification. We also added a caveat that broader terroir assessment would require multi-site replications, which we suggest for future work.

Location: Introduction (revised terroir context): lines 60–68 (marked in blue). Discussion (balanced interpretation and justification): lines 520–540 (marked in blue). Some phrasing overlaps with other reviewers' suggestions on scope and are in green.

5. Figures for chemometric analyses: clearer labeling, higher resolution, explicit descriptions of axes, vectors, and chromatographic regions

We have improved all relevant figures as suggested. Figure 4 (PCA biplot) and Figure 5 (PCA scores) now include higher-resolution images with explicit axis labels (e.g., "PC1 (41.04% variance)" and "PC2 (25.93% variance)"), vector abbreviations for key compounds (e.g., MeHpEst for methyl heptanoate), and legends explaining scores, loadings, and clusters. For PLS-DA figures (e.g., Figure 1), we added descriptions of chromatographic regions (e.g., 15–25 min for sesquiterpenes) and VIP thresholds. All figures have been redrawn for clarity and submitted in high-resolution format (300 dpi).

Location: Figure captions and revisions: Figure 1 caption, lines 310–315; Figure 4 caption, lines 550–558; Figure 5 caption, lines 560–565 (all marked in blue). Image files updated in the submission package.

6. Minor editorial adjustments: consistent formatting of scientific names, standardized units in tables, concise reporting of instrumental conditions

We have implemented these editorial improvements throughout. Scientific names (e.g., Humulus lupulus L.) are now consistently italicized and formatted. Units in tables (e.g., Area% for volatiles, % w/w for acids) are standardized, and instrumental conditions in Methods (e.g., GC-MS parameters) have been condensed for conciseness while retaining all essential details. A full proofread ensured uniformity.

Location: Scattered throughout (e.g., scientific names in Introduction: lines 10–80; units in Table 2: lines 340–450; instrumental conditions in Methods: lines 120–140) – marked in yellow/green where overlapping with other reviewers, or blue for unique instances.

We believe these revisions fully address your concerns and elevate the manuscript to a publication-ready state. We are grateful for your expertise and remain available for any further clarifications.

Sincerely,

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

cThe authors have improved the manuscript according to the suggestions provided. The manuscript now clearly illustrates the good results obtained and is considered suitable for publication in its current form.

Author Response

Dear Author,

Thank you for the additional opportunity to revise the manuscript. I confirm that the requested changes have now been addressed. Table 2 has been substantially revised and currently includes 155 compounds only, restricted to those of greater relevance and relative abundance. A single nomenclature system has been adopted throughout the main table, prioritizing commonly used traditional names rather than detailed IUPAC denominations. All IUPAC names, together with the full list of identified compounds and their chemical structures, are now provided exclusively in the Supplementary Information, as requested.

Figures 1 and 2 have also been revised to follow a consistent graphical standard. Both figures now use a transparent layout with a white background, uniform font type, and harmonized font size for axes, legends, and labels, ensuring readability and visual coherence. While the analytical comparisons shown in Figures 1 and 2 are inherently different and the software used does not allow identical panel layouts, the overall graphical style and visual language have been standardized across both figures to the maximum extent possible.

With these revisions, I believe the manuscript now fully complies with the editorial requirements and is suitable for acceptance.

Kind regards,

Reviewer 2 Report

Comments and Suggestions for Authors

accept in present form

Author Response

Dear Author,

With these revisions, I believe the manuscript now fully complies with the editorial requirements and is suitable for acceptance.

Kind regards,