Decoding the Benzaldehyde Pharmacophore: Structural Determinants for Enhancing Antibacterial Efficacy and Food Safety

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The topic is relevant to food safety, natural preservatives, and antimicrobial resistance, and the work fits well within the scope of Foods. However, the following issues should be addressed before further considerations:

1. While benzaldehyde derivatives have been previously reported as antimicrobials, the claim that the benzaldehyde core itself represents the “minimal pharmacophore” should be better contextualized against existing literature. Please explicitly state what is mechanistically new compared to prior SAR or phenolic antimicrobial studies, particularly in the Introduction and Discussion.
2. The rationale for selecting only ten derivatives should be explained more clearly. Why were electron-donating vs electron-withdrawing groups emphasized, and why were additional substituents (e.g., di-substituted or meta-substituted derivatives) not included? A brief justification would strengthen the SAR logic.
3. MICs are relatively high (128–1024 µg/mL). Please discuss: Whether these concentrations are realistically achievable in food systems. How volatility, solubility, and sensory impact might affect practical application as preservatives. A short discussion on regulatory or formulation challenges would be valuable.
4. The manuscript sometimes states that Gram-negative bacteria are less responsive, yet time-kill results suggest rapid killing of E. coli. Please reconcile these observations more clearly and avoid apparent contradictions.
5. The Hammett analysis is interesting, but: Correlation coefficients (R² values) should be explicitly reported. Statistical significance of correlations should be stated. Clarify whether σ values derived from para-substituents are appropriate for ortho-substituted compounds like SAL and HMB.
6. The MD work is a strength, but: Justify the high ligand concentration (0.089 M) used in simulations relative to experimental conditions. Clarify whether ligand aggregation occurred. Please explicitly link free energy profiles to experimental permeability and depolarization data in the Discussion.
7. While Vero cell viability is maintained, please: Compare cytotoxicity concentrations with effective MIC/MBC values. Include a selectivity index (SI) if possible. This would strengthen the safety claim.

 

Author Response

Comment 1: While benzaldehyde derivatives have been previously reported as antimicrobials, the claim that the benzaldehyde core itself represents the “minimal pharmacophore” should be better contextualized against existing literature. Please explicitly state what is mechanistically new compared to prior SAR or phenolic antimicrobial studies, particularly in the Introduction and Discussion.

Author Response: We thank the reviewer for this important observation. We have revised both the Introduction and Discussion to clearly distinguish our work from prior phenolic antimicrobial studies. While benzaldehyde derivatives have been reported previously as antimicrobials no study has:

1. Define the minimal aldehyde pharmacophore experimentally,
2. Integrated Hammett σ correlations with biological activity,
3. Mechanistically validated substituent-dependent membrane interaction using atomistic MD simulations;

                We have now explicitly clarified the novelty in the revised manuscript. Please vide the page no. 3 and line no. 85-90; page no. 17 and line no. 623-628.

Comment 2: The rationale for selecting only ten derivatives should be explained more clearly. Why were electron-donating vs electron-withdrawing groups emphasized, and why were additional substituents (e.g., di-substituted or meta-substituted derivatives) not included? A brief justification would strengthen the SAR logic.

Author Response: The selected derivatives were intentionally limited to mono-substituted and structurally minimal analogues in order to isolate the independent electronic contribution of the single substituents without steric complexity introduced by di- or meta-substituted derivatives. Para-substituted compounds were prioritized because Hammett σ constants are most reliably defined for para positions, enabling quantitative correlation analysis. The panel therefore represents a controlled electronic gradient rather than an exhaustive chemical library. These details were now included in the revised manuscript. Please refer page no. 3 and line no. 120

Comment 3: MICs are relatively high (128–1024 µg/mL). Please discuss: Whether these concentrations are realistically achievable in food systems. How volatility, solubility, and sensory impact might affect practical application as preservatives. A short discussion on regulatory or formulation challenges would be valuable.

Author Response: Complied. As per the suggestion of the reviewer, a short discussion on regulatory or formulation changes is now included in the revised manuscript. Please refer page no. 18 and line no. 645.

 

Comment 4: The manuscript sometimes states that Gram-negative bacteria are less responsive, yet time-kill results suggest rapid killing of E. coli. Please reconcile these observations more clearly and avoid apparent contradictions.

Author Response: We clarified that the MIC plateau reflects a penetration barrier, while time-kill reflects rapid killing once the threshold concentration is reached. Please refer page no. 12 and line no. 459 in the revised manuscript.

 

Comment 5: The Hammett analysis is interesting, but: Correlation coefficients (R² values) should be explicitly reported. Statistical significance of correlations should be stated. Clarify whether σ values derived from para-substituents are appropriate for ortho-substituted compounds like SAL and HMB.

Author Response: We have now included R² values and clarified limitation of para σ extrapolation for ortho derivatives. Please refer page no. 9 and line no. 361 in the revised manuscript.

 

Comment 6: The MD work is a strength, but: Justify the high ligand concentration (0.089 M) used in simulations relative to experimental conditions. Clarify whether ligand aggregation occurred. Please explicitly link free energy profiles to experimental permeability and depolarization data in the Discussion.

Author Response: In deference to the Reviewer comment, the clarification is now added in the revised manuscript. Please refer the page no. 7 and line no. 265, and page no.17 and line no. 612.

 

Comment 7: While Vero cell viability is maintained, please: Compare cytotoxicity concentrations with effective MIC/MBC values. Include a selectivity index (SI) if possible. This would strengthen the safety claim.

Author Response: We have complied with the reviewer’s suggestion and calculated the Selectivity Index (SI) based on the highest non-cytotoxic concentration tested in the Vero cell assay. The SI values were determined to be 1.17 for SAL and 2.34 for NIT. It should be clarified that these values were calculated using the maximum tested non-toxic working concentration (300 µg/mL), rather than a true IC₅₀ value, as no 50% reduction in cell viability was observed within the tested range. The primary objective of the cytotoxicity assessment in this study was to evaluate compound safety at antibacterial working concentrations rather than to establish an exact IC₅₀. Nevertheless, we appreciate the reviewer’s insightful comment, which has allowed us to clarify this point explicitly in the revised manuscript. Please refer page no. 15 and line no. 558.

Reviewer 2 Report

Comments and Suggestions for Authors

The article received for evaluation is titled 'Decoding the benzaldehyde...' and has as main author C. Shi. It is submitted to Foods journal, which is suitable for the journal purpose.

The Abstracts seems a little bit too long, I think it can reduced in size, there are some irrelevant information there.

Introduction is well developed, however there are only 8 references cited, and this is quite unusual, as the introduction sshould provide a glance of the recent literature data.

Materials and method section is well writen and provides all the requsted information to make the work reproducible. It contains a lot of sections describing in details the actual procedures. 

It is followed by the Results section which presents the experimental findings. The correlation between the Hammett electronic parameters and antibacterial potency is interesting and well known also. Tables 1 and 2 do not respect the journal recommandations. Figure 1 should show all the points, i.e. there are 3 red dots, 1 green and 2 blue.

The Discussion section analyse the previous results and make a correclation between the  Hammett constants and the antibacterial potency, showing a direct link between substituent electronics and antibacterial outcome. The simulations are welcome and support the general data. Conclusions summarizes the relevant findings. For the supplementary material it should be stated what kind of information is presented.

The paper end with 41 references in correct format, however the DOI number should be included and also the number of references increased.

Finnaly, I suppose that the work is sutable for publicatioon after these improvements, It is good thta there is a supplementary file.

Some other issues:

-please increase the size of all Figures in Supplementary Material

-please correct the Figure S1 to make the classical hexagons of chemical structures 

Author Response

Comment 1: The Abstracts seems a little bit too long, I think it can reduced in size, there are some irrelevant information there.

Author Response: The abstract has been reduced and redundant background removed. Please refer the abstract in the revised manuscript.

 

Comment 2: Introduction is well developed, however there are only 8 references cited, and this is quite unusual, as the introduction should provide a glance of the recent literature data.

Author Response: We have expanded the Introduction and added 8 recent references increasing literature coverage. Please refer the abstract in the revised manuscript.

 

Comment 3: Materials and method section is well written and provides all the requested information to make the work reproducible. It contains a lot of sections describing in details the actual procedures. 

Author Response: We thank the reviewer for the positive assessment.

 

Comment 4: It is followed by the Results section which presents the experimental findings. The correlation between the Hammett electronic parameters and antibacterial potency is interesting and well-known also. Tables 1 and 2 do not respect the journal recommendations. Figure 1 should show all the points, i.e. there are 3 red dots, 1 green and 2 blue.

Author Response: We respectfully disagree with the reviewer’s observation that Figure 1does not display all experimental points. All experimentally obtained MIC values used for the Hammett correlation analysis are included in the figure. Specifically, three data points are presented for S. aureus (red), two for E. coli (blue), and two for L. monocytogenes (green) corresponding exactly to the number of substituents evaluated for each organism.

                    The apparent discrepancy may rise from visual overlap between certain data points and the fitted regression lines. However, no experimental data have been omitted. Therefore, while we appreciate the reviewer’s careful examination of the figure, we confirm that all relevant data points are already included and no additional points are missing.

 

Comment 5: The Discussion section analyses the previous results and make a correlation between the Hammett constants and the antibacterial potency, showing a direct link between substituent electronics and antibacterial outcome. The simulations are welcome and support the general data. Conclusions summarizes the relevant findings. For the supplementary material it should be stated what kind of information is presented.

Author Response: Supplementary materials include structural representation (Figure S1), metabolic viability assay (Figure S30, Hydrogen bonding distribution analyses (Figure S4), Spot assay validation (Figure S5), membrane potential imaging (Figure S6), and DNA interaction assay (Figure S7). This statement is now included in the revised manuscript. Please refer the supplementary material section in the page no. 19.

 

Comment 6: The paper end with 41 references in correct format, however the DOI number should be included and also the number of references increased.

Author Response: All references updated with DOI numbers and total references are increased. Please refer the reference section in the revised manuscript.

 

Comment 7: Finally, I suppose that the work is suitable for publication after these improvements, It is good that there is a supplementary file.

Author Response: We thank the reviewer for the positive evaluation.

 

Comment 8: Some other issues:

-please increase the size of all Figures in Supplementary Material

-please correct the Figure S1 to make the classical hexagons of chemical structures 

Author Response: All Supplementary Figures resized to journal specifications.

                                Figure S1 chemical structures redrawn using ChemDraw with Classical aromatic hexagonal representation

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have carefully revised the manuscript in accordance with the reviewers’ comments, and the revised version is now suitable for acceptance for publication.