Review Reports - Determinants of Antibacterial Activity in Humulus lupulus Hop Extracts: Phytochemical Profiling of Prenylated Acylphloroglucinols and Efficacy Against Methicillin-Resistant Staphylococcus aureus and Multidrug-Resistant Uropathogenic Escherichia coli

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents a comparative study of five extraction protocols for hop (Humulus lupulus) derived prenylated acylphloroglucinols and evaluates their antibacterial activity. While the topic is of relevance to natural product antimicrobial research, the current version suffers from several significant methodological weaknesses that limit its scientific merit and clarity.

The authors employed UPLC and GC-MS analysis for quantification,yet they did not provide adequate method validation, including selectivity/specificity, calibration curves and limits of quantification, precision and accuracy, and extraction recovery and matrix effects.
All tested hop extracts are complex mixtures containing multiple congeners of α-acids, β-acids, and iso-α-acids, rather than purified individual compounds. The scientific rigor and reliability of the conclusions cannot be fully ascertained.
In Figure 2, the authors present chromatographic profiles with retention times and peak areas. However, it is unclear whether these peaks were identified using authentic reference standards. Since the standards are available, the authors should provide a representative chromatogram of a mixed standard solution, with clear peak assignments, and construct proper calibration curves. The current presentation does not convincingly demonstrate quantitative accuracy.
The study compares five extraction methods, but the narrative is disjointed, and the main findings are not systematically highlighted.
The authors measured MIC values against reference and clinical strains, but the link between extraction efficiency and antimicrobial effect is not rigorously explored. Since the primary goal is to identify which extraction method yields the most potent antibacterial extracts, it would be sufficient to compare the MIC values of the key individual monomers (e.g., α-acids, β-acids, and iso-α-acids) and correlate these with the respective extract compositions.
A simple comparison of MICs across extracts would directly reveal which method enriches the most active compounds. The current PCA and correlation analyses meaningless.

Author Response

We sincerely thank the Reviewer for the valuable comments, constructive suggestions, and helpful guidance. We have carefully considered each of the Reviewer’s comments and have made every effort to address them thoroughly. Accordingly, the necessary revisions have been incorporated into the manuscript. Our detailed point-by-point responses to all comments are provided in the attached file. Please see the attachment.

Reviewer comments:

The manuscript presents a comparative study of five extraction protocols for hop (Humulus lupulus) derived prenylated acylphloroglucinols and evaluates their antibacterial activity. While the topic is of relevance to natural product antimicrobial research, the current version suffers from several significant methodological weaknesses that limit its scientific merit and clarity.

Comment 1: The authors employed UPLC and GC-MS analysis for quantification, yet they did not provide adequate method validation, including selectivity/specificity, calibration curves and limits of quantification, precision and accuracy, and extraction recovery and matrix effects.

Response 1:

We thank the Reviewer for highlighting the importance of method validation. We agree that rigorous validation parameters are crucial for the reliability of quantitative results. We have revised the manuscript to include comprehensive validation data for the HPLC analysis in the Materials and Methods section.

Importantly, we clarify that we performed quantification strictly according to the well-established, standardized EBC 7.8 Method (Iso-α-, α- and β-Acids in Hop and Isomerised Hop Extracts by HPLC). By adhering to this official protocol, fundamental analytical challenges such as extraction recovery, matrix effects, and selectivity are systematically addressed according to recognized industry standards for hop extract analysis.

To directly address your concerns regarding the specific validation parameters achieved in our study, we have added the following detailed data to the manuscript:

Calibration Curves and Linearity: Calibration curves were prepared using certified EBC/ASBC reference standards (ICE-4 and ICS-I4, Labor Veritas, Zürich, Switzerland). We evaluated 6 initial concentration levels. However, to ensure strict adherence to the ICH Q2(R1) guidelines, the two lowest levels were excluded from the final regression model due to back-calculated accuracy exceeding the ±20% acceptance criterion. The remaining 4 validated concentration levels demonstrated linearity, with coefficients of determination R² ≥ 0.997. The detailed regression parameters are presented in the table below:

Compound	Standard / Wavelength	Slope	Intercept	R²
trans-Isohumulone	ICS-I4 (270 nm)	6.3324	7.363	0.99779
trans-Isoadhumulone	ICS-I4 (270 nm)	6.0211	3.728	0.99697
trans-Isocohumulone	ICS-I4 (270 nm)	3.9881	0.909	0.99670
Cohumulone	ICE-4 (314 nm)	25.1481	33.665	0.99806
n+adhumulone	ICE-4 (314 nm)	22.5608	86.660	0.99808
Colupulone	ICE-4 (314 nm)	17.5546	28.272	0.99805
n+adlupulone	ICE-4 (314 nm)	18.3054	30.444	0.99807

Limits of Detection (LOD) and Quantification (LOQ): LOD and LOQ were robustly determined based on the residual standard deviation of the calibration line and its slope, with the LOQ further verified by the back-calculated accuracy (±20% criterion). The specific ranges, which have now been added to the article, are as follows:

α-acids: LOD = 3.0–8.7 µg/mL; LOQ = 5.5–15.8 µg/mL

β-acids: LOD = 3.6–3.7 µg/mL; LOQ = 6.5–6.8 µg/mL

iso-α-acids: LOD = 1.5–6.1 µg/mL; LOQ = 2.1–8.8 µg/mL

System Stability and EBC Formula Verification: To ensure the highest accuracy and stability throughout the analysis, the calibration solution was injected both before and after each series of samples. Furthermore, as an additional step of methodological verification, the quantified amounts were cross-checked using the standard EBC external standard procedure formula:

where:

C_i = concentration of component i in the sample expressed as % (m/m).

M_cs = weight of the calibration extract in g.

C_ic = concentration of component i in the calibration extract expressed as % (m/m).

A_i = peak area of component i from the sample run (average).

M_s = weight of the sample in g.

A_ic = peak area of component i from the calibration run (average).

The concentrations obtained directly from our validated calibration curves differed only slightly from the values calculated using this official equation, confirming the robustness and high precision of our quantification approach.

We believe that the incorporation of this detailed validation information—encompassing linearity, LOQ, LOD, analytical stability, and strict adherence to official guidelines—thoroughly addresses your comments and strengthens the analytical rigor of our paper.

To address your concerns regarding the robustness of our GC-MS methodology, we have expanded Section 4.6 to fully detail the identification criteria, quantification approach, and quality controls applied.

Selectivity and Specificity. Volatile compounds were identified based on Kovàts retention indices (KI) and mass spectral comparison with the NIST 17 library. A strict similarity index (SI) cut-off of ≥ 90% was applied; only compounds meeting this threshold were retained. To eliminate matrix and solvent background interference, compounds detected in blank runs (neat cyclohexane) or in internal standard-only control runs - performed with the same syringes and vials as the sample injections, were entirely excluded from the dataset.

Sample Preparation. Approximately 100 mg of dried hop extract was accurately weighed into a 10 mL volumetric flask. A volume of 20 µL of the internal standard stock solution (2-undecanone, 50 µg/mL in cyclohexane) was added, and the flask was filled to the mark with cyclohexane. After thorough mixing, an aliquot was withdrawn, filtered through a 0.22 µm hydrophobic PTFE syringe filter, and transferred to a GC vial.

Calibration and Semi-quantification. Because this study aims at comparative volatile profiling rather than absolute quantification, individual external calibration curves and formal LOQ values were not established. Concentrations were instead estimated using an internal standard (IS) method with molecular weight (MW) correction, according to the formula:

where A denotes GC-MS peak area, MW_IS (2-undecanone) = 170.25 g/mol, and C_IS = 0.1 µg/mL (20 µL of a 50 µg/mL 2-undecanone stock solution brought to 10 mL with cyclohexane). Equal molar detector response relative to 2-undecanone was assumed for all analytes. Results are therefore semi-quantitative and are reported as molecular weight-corrected 2-undecanone equivalents [µg/mL].

Precision. All sample preparations and GC-MS injections were performed in triplicate (n = 3 independent sample preparations per extract). Results are reported as mean ± standard deviation (SD).

We trust that this clarification—covering the ≥ 90% SI threshold, blank and IS-only run exclusions, the full sample preparation procedure, the explicit MW-corrected IS quantification formula, and the triplicate preparation design—fully addresses your concerns regarding the validity and transparency of the volatile compound profiles presented.

Comment 2: All tested hop extracts are complex mixtures containing multiple congeners of α-acids, β-acids, and iso-α-acids, rather than purified individual compounds. The scientific rigor and reliability of the conclusions cannot be fully ascertained.

Response 2:

We thank the Reviewer for this important comment. We agree that the tested hop extracts are complex mixtures containing multiple congeners of α-acids, β-acids, and iso-α-acids. Therefore, the observed biological effects cannot be unequivocally attributed to a single class of compounds or to an individual congener. We have revised the manuscript accordingly and have modified the interpretation of our findings to refer to the activity of the hop extracts rather than to the activity of individual constituents.

We have also added this issue as a limitation of the study and clarified that interactions between different constituents, including possible additive or synergistic effects, cannot be excluded.

Comment 3: In Figure 2, the authors present chromatographic profiles with retention times and peak areas. However, it is unclear whether these peaks were identified using authentic reference standards. Since the standards are available, the authors should provide a representative chromatogram of a mixed standard solution, with clear peak assignments, and construct proper calibration curves. The current presentation does not convincingly demonstrate quantitative accuracy.

Response 3:

We thank the reviewer for this constructive feedback. We agree that clear peak assignment and proper demonstration of standard separation are essential.

In response to your comment, we have thoroughly revised Figure 2 to improve its overall clarity and visibility. Specifically, we have restructured the figure so that the first panel, Figure 2A, now presents a representative chromatogram of the mixed authentic standard solutions (certified EBC/ASBC standards ICS-I4 and ICE-4) with clear and explicit peak assignments for all analyzed compounds. This allows for a direct visual comparison and confirmation of the identified peaks in our samples.

Furthermore, as requested and detailed in our response to your previous comment, we have constructed proper calibration curves using these reference standards. The calibration was performed at four validated concentration levels demonstrating linearity (R² ≥ 0.997), and the comprehensive method validation parameters (including LOQ and LOD) have been added to the Materials and Methods section.

We believe that the newly updated Figure 2, combined with the comprehensive calibration and validation data now included in the manuscript, clearly and convincingly demonstrates the quantitative accuracy of our analysis.

Comment 4: The study compares five extraction methods, but the narrative is disjointed, and the main findings are not systematically highlighted.

Response 4:

We thank the Reviewer for this constructive feedback. We agree that a cohesive and well-structured narrative is essential for effectively presenting the comparison among the extraction methods and emphasizing the key findings.

In response to your suggestion, the entire manuscript has been thoroughly restructured and rewritten. We have systematically highlighted the main findings to ensure a logical and smooth progression of the narrative. Furthermore, as noted in our response to your previous comments, we have significantly condensed and streamlined the Introduction section, removing off-topic elements to maintain a strict and clear focus on the optimization and comparison of the extraction methods.

We believe these revisions have greatly improved the clarity, coherence, and impact of the study.

Comment 5: The authors measured MIC values against reference and clinical strains, but the link between extraction efficiency and antimicrobial effect is not rigorously explored. Since the primary goal is to identify which extraction method yields the most potent antibacterial extracts, it would be sufficient to compare the MIC values of the key individual monomers (e.g., α-acids, β-acids, and iso-α-acids) and correlate these with the respective extract compositions.

Response 5:

We thank the Reviewer for this valuable comment. We agree that establishing a relationship between the chemical composition of the extracts and their antimicrobial activity is important for evaluating the effectiveness of the extraction methods. To address this issue, we performed an additional correlation analysis between the concentrations of the major hop-acid fractions (α-acids, β-acids, and iso-α-acids) determined by HPLC and the MIC values obtained for the corresponding extracts.

A strong inverse correlation was observed between α-acid content and MIC values for E. coli ATCC 25922 (Spearman's ρ = −0.858, p < 0.001), S. aureus ATCC 29213 (ρ = −0.927, p < 0.001), and clinical MRSA isolates (ρ = −0.891, p < 0.001). Because all tested laboratory preparations yielded an identical MIC of 15 mg/mL for the clinical E. coli isolate, Spearman correlation analysis was not applicable for this strain, as zero variance in MIC values precludes meaningful rank-based correlation. This is clearly indicated in the revised manuscript and in Table 4. β-acid content also showed significant inverse correlations with MIC values for E. coli ATCC 25922, S. aureus ATCC 29213, and clinical MRSA isolates, whereas no significant associations were observed for iso-α-acid content.

These findings indicate that the antimicrobial activity of the extracts is associated particularly with their α-acid content. The extracts obtained by ethanol-based extraction, which generally contained higher concentrations of α-acids, exhibited lower MIC values than the extracts obtained using aqueous extraction. The highest α-acid content was observed in the supercritical CO₂ extract of Marynka (43.47% w/w), which also exhibited low MIC values against all tested bacterial groups.

However, because the tested extracts are complex mixtures containing several hop-acid fractions, these correlations should not be interpreted as evidence that α-acids alone are responsible for the observed antimicrobial activity. Rather, they suggest that α-acids may make an important contribution to the overall antibacterial activity of the extracts, while possible additive or synergistic interactions among their constituents cannot be excluded.

The correlation analysis and the corresponding interpretation have been added to the revised manuscript.

Comment 6: A simple comparison of MICs across extracts would directly reveal which method enriches the most active compounds. The current PCA and correlation analyses meaningless.

Response 6:

We thank the Reviewer for this valuable comment. We agree that direct comparison of MIC values provides the most straightforward assessment of the antibacterial potency of extracts obtained using different extraction methods. We have therefore revised the presentation of the results to emphasize the direct comparison of MIC values across extraction methods and have complemented this analysis with correlations between extract composition and antimicrobial activity.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents a new natural alternative to antibiotic-resistant bacteria. Authors have provided abundant data and systematic analysis. Here are a few suggestions that can help the authors improve the manuscript and make it have sufficient academic value to be published in this journal.

Figures 1 can be improved by applying different colors and symbols for different individual data points inside box illustrations.
Figure 2 . Stacks are vertical, making it appear low. Also, the font size of the label is too small. Increase size of chromatograms and improve readability.
Figure 3. Data points are overlapping and concentrate on specific MIC values. Distribute overlapping data and increase size of symbols.
Figure 4: Add statistical significance p – value and asterisk.
Table 1 should be spit into 2 parts HPLC results and MIC results, and MIC should be separated. At the bottom of the table write full of ND.
Why was only 1 clinical isolate used?
Apart from planktonic, should biofilm cells also be studied?
For commercial CO2, the manufacturer's extraction parameters should be mentioned to help with reproducibility of results in the future. Add SFE conditions: pressure, temperature, flow rate , and metabolomic profile, mechanistic should be supplemented.
In vivo studies and food challenge testing in real world application should be added.
GSmS was done only for cold ethnol extract not other .any specific reason?
Optimise industrial application. How cost-effective are all methods.
Introduction section needs to be improved. Don’t focus on other topics other than optimization of extraction methods. Everything is scattered.
Antibacterial activity does not always guarantee safety for human consumption. It is advisable to include cytotoxicity assessment and hemolysis test.
There are a few typos, hyphens, punctuation errors, and wrong spellings that should be thoroughly checked.

Author Response

Reviewer comments:

Comment 1: Figures 1 can be improved by applying different colors and symbols for different individual data points inside box illustrations.

Response 1:

We thank the Reviewer for this helpful suggestion, and we fully agree. Enhancing the visual distinction of the individual data points significantly improves the overall readability and interpretation of the plot.

We have revised Figure 1 according to your recommendation. Different colors and distinct symbols have now been applied to the individual data points within the box plots, making the data presentation much clearer in the updated manuscript.

Comment 2: Figure 2. Stacks are vertical, making it appear low. Also, the font size of the label is too small. Increase size of chromatograms and improve readability.

Response 2:

We thank the Reviewer for this constructive feedback. We fully agree with your observations regarding the formatting of the chromatograms.

We have adjusted Figure 2 in accordance with your suggestions. The layout of the stacks has been modified, and the overall size of the chromatograms has been increased to prevent them from appearing compressed. Furthermore, we have significantly increased the font size of the axes and general labels. Please note that the font size for the specific peak labels was increased to the maximum extent possible without causing the text to overlap and obscure the data. We believe these changes have greatly improved the readability and visual quality of the figure in the revised manuscript.

Comment 3: Figure 3. Data points are overlapping and concentrate on specific MIC values. Distribute overlapping data and increase size of symbols.

Response 3:

We thank the Reviewer for this valuable feedback. We fully agree that the overlapping data points made the figure difficult to interpret.

We attempted to modify the figure according to your suggestions by attempting to distribute the overlapping data points and increasing the symbol sizes. However, due to the highly concentrated nature of the specific MIC values, these visual adjustments were ultimately unsuccessful in improving the overall clarity of the plot.

To resolve this issue and ensure that the data is presented as clearly and unambiguously as possible, we have decided to remove Figure 3 entirely from the revised manuscript. In its place, we have introduced a new table (Table 3), which explicitly lists all the MIC values. We believe this tabular format provides much better readability and allows the readers to evaluate the exact quantitative results far more easily.

Comment 4: Figure 4: Add statistical significance p – value and asterisk.

Response 4:

We thank the Reviewer for this valuable suggestion. We agree that clearly indicating statistical significance is crucial for the proper interpretation of the results.

We have modified Figure 4 in accordance with your recommendation. The statistical significance, including the appropriate asterisks and p-values, has now been added to the graph to clearly highlight significant differences.

Additionally, to provide a more comprehensive overview of our findings, we have further expanded this figure to include the relationship between the extraction yield and the MIC. We believe these additions significantly enhance both the clarity and the scientific value of the presented data in the revised manuscript.

Comment 5: Table 1 should be split into 2 parts HPLC results and MIC results, and MIC should be separated. At the bottom of the table write full of ND.

Response 5:

We thank the Reviewer for this helpful suggestion. We agree that separating the analytical and microbiological data significantly improves the clarity and readability of the manuscript.

In accordance with your recommendation, we have split the original Table 1. The HPLC quantification results remain in Table 1, while the MIC results have been separated and are now presented in the newly created Table 3.

Furthermore, as requested, we have added clear explanatory footnotes at the bottom of both tables to define the abbreviations. To ensure absolute clarity and precision, we have differentiated between two distinct abbreviations used in our dataset:

ND (No Data): This abbreviation indicates that the specific information was not provided or could not be obtained from the extract manufacturer.
nd (not detected): This abbreviation indicates that the analytical signal from the HPLC or GC-MS analysis was below the Limit of Quantification of the method.

We believe these modifications provide a much clearer and more precise presentation of our results.

Comment 6: Why was only 1 clinical isolate used?

Response 6:

We thank the Reviewer for this comment. We would like to clarify that the study included four bacterial strains in total: two reference strains, representing Gram-positive and Gram-negative bacteria, and two clinical isolates, comprising one Gram-positive and one Gram-negative isolate. Thus, clinical isolates were included for both bacterial groups investigated.

The use of a limited number of clinical isolates was intentional, as the primary aim of the study was to compare the antibacterial activity of hop extracts obtained using different extraction methods rather than to characterize the variability of susceptibility among clinical isolates. The reference strains were used as standardized bacterial models, while the clinical isolates were included to provide an additional indication of whether the observed activity could also be detected in clinically derived strains.

We acknowledge that the inclusion of only one clinical isolate per bacterial group limits the generalizability of the findings and does not allow conclusions to be drawn regarding the variability of susceptibility among clinical isolates. We have therefore clarified this limitation in the revised manuscript and have moderated the relevant statements accordingly.

Comment 7: Apart from planktonic, should biofilm cells also be studied?

Response 7:

Thank you for this important and constructive comment. We fully agree that the assessment of biofilm-associated cells would provide an important complementary perspective, particularly because biofilm formation may substantially alter bacterial susceptibility and tolerance to antimicrobial agents compared with planktonic cells.

The present study was designed as an initial investigation focused on the antimicrobial activity of the tested extracts against planktonic bacterial cells. We would like to clarify that experiments addressing the activity of the extracts against biofilm-associated cells are currently in progress. These experiments involve a dedicated experimental design and additional parameters required for the reliable assessment of biofilm biomass, metabolic activity, and susceptibility.

We consider these studies to constitute an important subsequent stage of our research. Because the biofilm experiments are not yet completed, we believe that incorporating preliminary or incomplete data into the present manuscript would not be appropriate and could compromise the consistency and scientific rigor of the study. The results obtained from the biofilm experiments will therefore be presented in a separate, follow-up publication.

We have acknowledged this aspect as a limitation and an important direction for future research in the revised manuscript. We believe that the current study provides a necessary preliminary assessment of the activity of the extracts against planktonic cells, which will serve as a basis for the subsequent evaluation of their activity against biofilm-associated bacterial populations.

Comment 8: For commercial CO₂, the manufacturer's extraction parameters should be mentioned to help with reproducibility of results in the future. Add SFE conditions: pressure, temperature, flow rate, and metabolomic profile, mechanistic should be supplemented.

Response 8:

We thank the reviewer for raising this important point. We completely agree that precise extraction parameters are crucial for the reproducibility of results and for a deeper understanding of the extract's profile.

Unfortunately, because we utilized a commercial CO₂ hop extract, we were not provided with the specific SFE parameters, such as pressure, temperature, and flow rate. The manufacturer considers these precise extraction conditions to be proprietary information and a corporate trade secret. We only possess the official certificate of analysis issued by the manufacturer, and all the relevant data provided in that certificate has been fully incorporated into the article.

We acknowledge this as a constraint of working with commercially sourced extracts. We agree that in future experiments, performing SFE extraction in a controlled laboratory setting should be considered. This would allow for full control over the extraction parameters, providing complete data that can be directly correlated with the metabolomic profile and mechanistic studies.

To ensure full transparency, we have added a discussion regarding the lack of specific commercial extraction parameters and the necessity of future laboratory-scale SFE studies to the “Limitations” section in the revised manuscript.

Comment 9: In vivo studies and food challenge testing in real world application should be added.

Response 9:

We thank the Reviewer for this valuable suggestion. We agree that in vivo studies and challenge testing in relevant food matrices would provide important additional evidence regarding the practical applicability of hop extracts. However, the present study was designed as an in vitro comparative assessment of the antimicrobial activity of hop extracts obtained using different extraction approaches, including cold- and hot-water extraction, cold- and hot-ethanol extraction, pressurized extraction, and supercritical CO₂ extraction. The primary objective was to determine whether the extraction method affects the antimicrobial properties of the resulting hop extracts.

In vivo studies and food challenge testing were beyond the scope of the present work and could not be performed within the framework of this study. Moreover, the potential application of the investigated extracts in food matrices would require a separate evaluation, taking into account the composition of the food matrix, extract stability, sensory properties, effective concentrations, and safety. We have therefore acknowledged the lack of in vivo and food-matrix validation as a limitation of the present study and indicated that these aspects should be addressed in future research.

Comment 10: GSmS was done only for cold ethnol extract not other any specific reason?

Response 10:

GC-MS analysis was exclusively presented for the cold ethanol extract because the other extraction methods utilized in our experiments were not suitable for the preservation or recovery of the targeted volatile compounds. Specifically, the processing conditions of the other extraction methods led to the evaporation and subsequent loss of these sensitive volatiles. Furthermore, in some cases, the volatile compounds failed to dissolve adequately in the alternative solvents utilized.

As a direct consequence of these two factors (evaporation and poor solubility), no volatile compounds were detected during the GC-MS analysis of those other extracts. Therefore, to ensure the accuracy and relevance of the data presented in the manuscript, we only included the successful GC-MS profiling obtained from the cold ethanol extract, where these compounds were effectively preserved, extracted, and quantified. We have added a brief explanatory note regarding this to the text to clarify this approach for the readers.

Comment 11: Optimise industrial application. How cost-effective are all methods.

Response 11:

We thank the Reviewer for highlighting the importance of the economic feasibility of the extraction methods for potential industrial application. We agree that antimicrobial activity alone is not sufficient to determine the optimal extraction process and that extraction yield, solvent consumption, energy requirements, process time, equipment costs, solvent recovery, and scalability should also be considered.

A detailed techno-economic analysis was beyond the scope of the present study, as the experiments were performed at laboratory scale and comprehensive data required for a reliable cost analysis were not available for all extraction procedures. We have therefore avoided making quantitative claims regarding the cost-effectiveness of individual methods.

Based on the present results, ethanol-based extraction appears particularly promising because it provided extracts with consistently high antimicrobial activity and substantially higher concentrations of hop bitter acids than aqueous and steam-assisted extraction. From an industrial perspective, ethanol also offers the advantage of being recoverable and recyclable, although solvent recovery would contribute to the overall process cost. Aqueous and steam-assisted methods may have advantages in terms of solvent simplicity and safety but produced extracts with lower concentrations of the major bitter acids and generally weaker antimicrobial activity, which may reduce their efficiency when a highly concentrated antimicrobial extract is required. SFE produced a highly active extract; however, its industrial implementation involves specialized high-pressure equipment and process parameters that were not available for the commercial extract used in this study. Because only one supercritical CO₂ extract was evaluated, further comparative studies are required before its economic advantages can be assessed.

We have consequently revised the manuscript to emphasize that the most industrially suitable extraction method cannot be selected solely on the basis of MIC values. A future process-scale techno-economic assessment integrating extraction yield, antimicrobial potency, chemical composition, energy and solvent requirements, solvent recovery, equipment investment, and scalability will be necessary to identify the most economically viable process.

Comment 12: Introduction section needs to be improved. Don’t focus on other topics other than optimization of extraction methods. Everything is scattered.

Response 12:

We thank the Reviewer for this highly constructivResponsese feedback. We agree that the original Introduction lacked structural coherence and contained information that detracted from the main objective of the study.

In accordance with your suggestion, we have thoroughly revised and streamlined the Introduction section. We have carefully removed the scattered, off-topic paragraphs and reorganized the text to maintain a strict and clear focus solely on the optimization of extraction methods and the direct context of our research. We believe these modifications have significantly improved the logical flow, clarity, and overall scientific rigor of the Introduction.

Comment 13: Antibacterial activity does not always guarantee safety for human consumption. It is advisable to include cytotoxicity assessment and hemolysis test.

Response 13:

We thank the Reviewer for this important and constructive comment. We fully agree that antibacterial activity alone does not establish the safety of hop extracts for potential food applications, and that cytotoxicity and hemolysis are essential components of a preliminary in vitro safety assessment.

Cytotoxicity of Humulus lupulus preparations has previously been investigated in mammalian cell models. For example, Klimek et al. reported a CC₅₀ of 155.70 ± 4.23 μg/mL for a supercritical CO₂ extract obtained from the ‘Marynka’ hop variety toward normal human dermal fibroblasts, whereas purified xanthohumol showed substantially higher cytotoxicity under the same experimental conditions [1]. Cytotoxicity of hydroalcoholic hop extracts has also been evaluated in mammalian cell lines, with effects depending on extract concentration [2]. In addition, recent research has specifically investigated the hemolytic effects of hop-derived xanthohumols toward human erythrocytes and demonstrated that the hemolytic response depends on both compound concentration and chemical structure [3].

We acknowledge, however, that these published findings cannot be directly extrapolated to the extracts investigated in the present study, because cytotoxicity and hemolytic activity may vary significantly according to the extraction method, chemical composition, and concentration of individual constituents. Since cytotoxicity and hemolysis assays were not included in our original experimental design, we have deliberately avoided making any claims regarding the safety of the investigated extracts for human consumption.

To fully address your valuable recommendation, we have explicitly added this point to the “Limitations” section in the revised manuscript. Furthermore, we would like to note that our planned future research already incorporates these exact safety evaluations. Cytotoxicity and hemolysis tests will be conducted prior to the application and testing of these extracts in any food matrix models.

References:

[1] Klimek K, Tyśkiewicz K, Miazga-Karska M, Dębczak A, Rój E, Ginalska G. Bioactive Compounds Obtained from Polish "Marynka" Hop Variety Using Efficient Two-Step Supercritical Fluid Extraction and Comparison of Their Antibacterial, Cytotoxic, and Anti-Proliferative Activities In Vitro. Molecules. 2021 Apr 19;26(8):2366. doi: 10.3390/molecules26082366.

[2] Di Sotto A, Checconi P, Celestino I, Locatelli M, Carissimi S, De Angelis M, Rossi V, Limongi D, Toniolo C, Martinoli L, Di Giacomo S, Palamara AT, Nencioni L. Antiviral and Antioxidant Activity of a Hydroalcoholic Extract from Humulus lupulus L. Oxidative Medicine and Cellular Longevity, 2018, 5919237, 14 pages, 2018. doi: 10.1155/2018/5919237.

[3] Stompor-Gorący M, Włoch A, Sengupta P, Bajek-Bil A, Nasulewicz-Goldeman A, Domańska M, Wietrzyk J, Machaczka M. Interactions of hop xanthohumols with model membranes and their toxicity to cancer and erythrocytes cells. Chem Biol Interact. 2026 Jul 25;435:112119. doi: 10.1016/j.cbi.2026.112119.

Comment 14: There are a few typos, hyphens, punctuation errors, and wrong spellings that should be thoroughly checked.

Response 14:

We thank the Reviewer for pointing this out. We have carefully reviewed the entire manuscript and meticulously corrected the typos, hyphenation, punctuation errors, and misspellings. Additionally, to ensure the highest linguistic quality and smooth academic flow, the final version of the text has been thoroughly proofread and corrected by a native English speaker.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The article describes five extraction methods for four different varieties of hops, with the aim of identifying the most effective method for producing a product with a high α-acid, β-acid, and iso-α-acid content and notable antibacterial activity against reference and multidrug-resistant uropathogenic bacteria. The manuscript is marginally acceptable for publication, but some corrections are required.

Abstract

The abbreviation UPEC is not defined.

Materials and methods

In the sentence «Clinical strains of uropathogenic E. coli and MRSA were obtained from the collection of the Department of Biology and Medical Parasitology, Wroclaw Medical University, Poland.»

can the name of the S. aureus culture be omitted before the word MRSA? (lines 465-467)

Were any control experiments carried out (with water and ethanol as solvents) during the study of minimum inhibitory concentrations of laboratory hop extracts? What was the procedure for preparing the solutions for the extracts in antimicrobial susceptibility testing?

Results

Table 1: the abbreviation “nd” should be explained either in the table title or immediately after the table.

Figure 2, A–F: it is necessary to increase the font size of the labels on both axes, the font size of the text in the legend, and the font size of the peak labels on the chromatogram, as the current text is unreadable.

The authors state that the Lyubelski hop variety has a low declared α-acid content (2.7%) and a high declared β-acid content (4.0%). The origin of this statement is unclear, as no source is cited. (Lines 223-225)

Table 2: there might be a typo in the column headings — one column is marked as “KI” instead of “RI”.

In the text MIC values of the ethanol extracts against S. aureus ATCC 29211 and Clinical S. aureus are mixed up and contradict the values given in Table 1. (Lines 244-246)

Author Response

Reviewer comments

Comment 1: Abstract

The abbreviation UPEC is not defined.

Response 1:

Thank you for this suggestion. The abbreviation UPEC has now been defined at its first occurrence in the manuscript.

Comment 2: Materials and methods

In the sentence «Clinical strains of uropathogenic E. coli and MRSA were obtained from the collection of the Department of Biology and Medical Parasitology, Wroclaw Medical University, Poland.» can the name of the S. aureus culture be omitted before the word MRSA? (lines 465-467)

Response 2:

Thank you for bringing this oversight to our attention. We have removed the name S. aureus.

Comment 3: Materials and methods

Response 3:

The aqueous and ethanolic hop extracts were supplied as dry preparations; therefore, there was no need to include water and ethanol controls. For the MIC assay, dry aqueous extracts were dissolved in phosphate-buffered saline (PBS), whereas dry ethanolic extracts were dissolved in 30% dimethyl sulfoxide (DMSO). Therefore, controls for DMSO were performed for all tested bacterial strains. Stock solutions of both types of extracts were prepared at a concentration of 30 mg/mL. The stock solutions were subsequently subjected to two-fold serial dilutions in MHB to obtain the required concentrations for the antimicrobial susceptibility assay. In the final test samples, the highest concentration of DMSO was 15%. Therefore, a solvent control containing 15% DMSO, without hop extract, was included to assess the potential effect of DMSO on bacterial viability. Bacterial viability in the 15% DMSO control did not differ from that observed in the control samples containing MHB alone, indicating that DMSO at the maximum concentration used in the assay did not affect bacterial growth under the experimental conditions.

Subsection 4.7. Antimicrobial Susceptibility Testing has been corrected in the manuscript. (Lines 628-637)

Comment 4: Results

Table 1: the abbreviation “nd” should be explained either in the table title or immediately after the table.

Response 4:

Thank the Reviewer for this comment. The abbreviation “nd” has been explained.

Comment 5: Results

Response 5:

Figure 2 has been corrected

Comment 6: Results

The authors state that the Lubelski hop variety has a low declared α-acid content (2.7%) and a high declared β-acid content (4.0%). The origin of this statement is unclear, as no source is cited. (Lines 223-225)

Response 6:

We thank the Reviewer for bringing this to our attention. Specifically, we received an official certificate confirming the authenticity and the specific chemical parameters of the Lubelski hop pellets used in our experiments. This certificate (No. 1PL/23/10) was officially issued by the Provincial Inspectorate of Agricultural and Food Quality (Wojewódzki Inspektorat Jakości Handlowej Artykułów Rolno-Spożywczych) in Poland.

Because this specific data sheet and the certificate are proprietary and not publicly available online, we could not include a standard reference link or citation in the bibliography. However, for your reference, we have attached the original certificate and the accompanying data sheet to this response. Please see the attachment.

To ensure clarity for readers, we have revised the manuscript to state that the reported values were declared by the supplier of the raw material. We have also revised the description of our experimental results to make them clearer. (Lines 146-152)

Comment 7: Results

Table 2: there might be a typo in the column headings — one column is marked as “KI” instead of “RI”.

Response 7:

Thank you for this detailed comment. We changed the explanation under Table 2 to KI: Kovàts retention index.

Comment 8: Results

In the text MIC values of the ethanol extracts against S. aureus ATCC 29211 and Clinical S. aureus are mixed up and contradict the values given in Table 1. (Lines 244-246)

Response 8:

Thank you for this valuable comment. We have corrected this mistake and revised the description of this subsection in accordance with the suggestions provided by another Reviewer. (Lines 231-247)

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The resubmitted manuscript has addressed most of my previous concerns, and the revisions have substantially improved the clarity and overall quality of the manuscript. I have only one remaining minor point. Replace the current Figure 2 with a representative chromatogram obtained from a mixed standard solution containing all standards.

Author Response

We sincerely thank the Reviewer for accepting our previous corrections and for the valuable suggestions and constructive comments provided during the review process. We greatly appreciate the time and effort devoted to improving our manuscript.

Figure 2 has been modified. It now shows a representative chromatogram obtained from a mixed standard solution containing all standards.

Reviewer 2 Report

Comments and Suggestions for Authors

line 557. sentence is duplicated
check refrence 8,14,24.
check author initial in author contribution
authors have completed all the given suggestions completly.

Author Response

Line 557 – the duplicated sentence has been removed

References 8, 14, and 24 have been checked and corrected

Author initials in author contributions have been corrected

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for considering all the comments on the content of your manuscript and making the necessary edits. There are no further remarks.

Best regards,

Reviewer

Author Response

We sincerely thank the Reviewer for accepting our revisions and for the valuable suggestions and constructive comments provided during the review process. We greatly appreciate the time and effort devoted to improving our manuscript.