Review Reports

In Figures 1 and 2, have exhibited structural damage of the Gram-positive bacterium Streptococcus mutans and Gram-negative bacterium Klebsiella oxytocaupon. However, the images are not clear enough. No specific characteristics of the injury were provided (such as "cell membrane rupture", "cytoplasm leakage", "cell morphology shrinkage"). Therefore, appropriate annotations and guidance can be provided on the images to help readers understand.

Signs are added to Figures 1 and 2 to differentiate between a) cell membrane, b) cytoplasm, and c) reduction in size.

The signs are explained in the text at the bottom of the figures.

The choice of microorganisms was primarily based on the availability of bacteria and yeasts in the laboratory, verification of adequate growth of the microorganisms, and having a homogeneous representation of Gram-positive bacteria, Gram-negative bacteria, and yeasts.

At the end of section 4.2, a paragraph has been added that explains the choice of microorganisms in general terms.

Two paragraphs are added to comply with the reviewer's request, which are:

Although the results are not as effective as those of the control antibiotics, they are still produced at low concentrations, suggesting that they could be incorporated into systemic formulations such as capsules or syrups and, above all, into topical antimicrobial and antifungal formulations. Another interesting application could be to prevent the growth of bacteria and yeasts in processed foods and cosmetic products as preservatives to extend the shelf life of products without causing harm to human health, replacing synthetic antimicrobials such as parabens.

Ensure consistent use of terms throughout the text. For example, "E-2-docenal" and "E-2-dodecenal" should represent the same substance in different forms, and the terms must be standardized.

In line 63 of the text, it is mentioned that "The antibacterial research on E-2-dodecenal is scarce. This study is the first to assess its antibacterial activity", but the breakthroughs in the research on E-2-dodecenal (such as the first determination of its MIC values for various strains, and the first observation of its mechanism of damage to cell structure) were not emphasized in this article. Moreover, due to the lack of data, the antibacterial advantages of this compound could not be demonstrated, and the innovation points were obscured.

Considering the observations also made by reviewer 3, we were able to locate two studies on E-2-dodecenal conducted by Kubo et al. in 2003 and 2004, which have been analyzed in the introduction, thus considering two previous investigations of activity in two strains of bacteria and yeasts.

Lack of microbial culture conditions: In Section 4.2 of the article, the culture conditions for the strains were not specified (such as the type of culture medium, whether the bacteria were cultured in LB medium or other media; the culture temperature, 37℃ or other temperatures, etc.). The unclear culture conditions made it impossible to repeat the experiments and the content lacked operability.

Depending on the observations, the text of section 4.2 was added and modified. In the end, the section was as follows.

4.2 Antimicrobial activity by microdilution plate assay

Antimicrobial activity was performed following the protocol described by Noriega et al. 2023 for essential oils [78], with modifications in the concentrations of secondary metabo-lites, since this study does not work with mixtures of molecules as in an essential oil.

The process began with the preparation of the microorganism inoculum at concentrations of 10⁸ CFU/mL for bacteria (7.5 x 10⁷ CFU/mL in the wells after deposition) and 10⁶ CFU/mL for yeasts (5.0 5 x 10⁷ CFU/mL in the wells after deposition), The medium used for activation was Mueller-Hinton agar, the temperatures for activating bacteria were 37°C and for yeast 25°C, without modification of pH.

The inoculum was placed in the microplate wells in a volume of 150 µL, along with 20 µL of a metabolite solution in descending concentrations and 30 µL of 1% w/w 2,3,5-Triphenyl-tetrazolium chloride reagent (TTC). Secondary metabolites and positive control were dissolved in DMSO at the following concentrations: 10%, 5%, 2.50%, 1.25%, 0.63%, 0.31%, 0.16%, 0.08, and 0.04%. The incubation time was 24 hours, after which the absorbances were read in an Epoch Biotek microplate reader at a wavelength of 405 nm. Chloramphenicol was used as a positive control for bacterial activity and clotrimazole for yeast activity. The negative control was executed bacteria and TTC dye without the secondary metabolite.

The choice of microorganisms was primarily based on the availability of bacteria and yeasts in the laboratory, verification of adequate growth of the microorganisms, and having a homogeneous representation of Gram-positive bacteria, Gram-negative bacteria, and yeasts.

In the steps of Section 4.4, methyl blue was added as the staining reagent to each sample, but the specific staining time was not specified.

The staining time information, which was 5 minutes, is included.

Clarify inoculum units and final concentration. The authors state “10^8 CFU (bacteria), 150 μL per well, total 200 μL” but do not specify CFU/mL vs CFU per well.

If 10^8 CFU/mL in 150 μL: final = 7.5 x 10^7 CFU/mL

If 10^8 CFU/well: final = 5 x 10^8 CFU/mL

The broth microdilution reference method specifies a final inoculum of 5 x 10^5 CFU/mL. Please explanin the rationale and validity for the bacterial inoculum, or realign with the reference method.

All concentrations used are added by increasing the following paragraph.

Please add medium (name, pH), temprature, incubation time, and pre-culture conditions used for each organisms.

Depending on the observations, the text of section 4.2 was added and modified. In the end, the section was as follows.

4.2 Antimicrobial activity by microdilution plate assay

Antimicrobial activity was performed following the protocol described by Noriega et al. 2023 for essential oils [78], with modifications in the concentrations of secondary metabo-lites, since this study does not work with mixtures of molecules as in an essential oil.

The process began with the preparation of the microorganism inoculum at concentrations of 10⁸ CFU/mL for bacteria (7.5 x 10⁷ CFU/mL in the wells after deposition) and 10⁶ CFU/mL for yeasts (5.0 5 x 10⁷ CFU/mL in the wells after deposition), The medium used for activation was Mueller-Hinton agar, the temperatures for activating bacteria were 37°C and for yeast 25°C, without modification of pH.

The inoculum was placed in the microplate wells in a volume of 150 µL, along with 20 µL of a metabolite solution in descending concentrations and 30 µL of 1% w/w 2,3,5-Triphenyl-tetrazolium chloride reagent (TTC). Secondary metabolites and positive control were dissolved in DMSO at the following concentrations: 10%, 5%, 2.50%, 1.25%, 0.63%, 0.31%, 0.16%, 0.08, and 0.04%. The incubation time was 24 hours, after which the absorbances were read in an Epoch Biotek microplate reader at a wavelength of 405 nm. Chloramphenicol was used as a positive control for bacterial activity and clotrimazole for yeast activity. The negative control was executed bacteria and TTC dye without the secondary metabolite.

The choice of microorganisms was primarily based on the availability of bacteria and yeasts in the laboratory, verification of adequate growth of the microorganisms, and having a homogeneous representation of Gram-positive bacteria, Gram-negative bacteria, and yeasts.

In Tables 1–3, SD values of 0 for chloramphenicol/cloterimazole are implausible across independent experiments. Please clarify n, the original readings, and any rounding or censoring applied.

In fact, the SD data for the positive control were rounded up because they were very small. At the reviewer's request, they are included as exponentials so as not to have too many decimal places.

The authors mentioned a negative control (bacteria + TTC without compound), but no negative control values appear in Tables 1–3. For antimicrobial testing, paired negative and positive controls are required; please add the growth control, solvent control, and ideally no-cells blank (to check dye/solvent interference) to the tables.

The authors state readings at 405 nm, yet no absorbance data are presented. Please provide the 24 h raw A405 value, and state the MIC call criterion.

The microplate method involves absorbance readings at nine concentration positions ranging in our case from 10% (10,000 ug/mL) to 0.04% (40 ug/mL) for the solutions. To this must be added the three repetitions, five molecules, and the calibration curve, followed by the calculation of the MIC. It was not included in the manuscript due to the size of the information, as there are about 20 pages of data and calculations. We suggest that the reviewer prepare a specific supplementary document for each type of microorganism, three in total (Gram +, Gram-, and yeast), so that it can be read by researchers and better describe the technique.

Nomenclature and typographical issues.
-Unify the coupound name e.g. E-2-dodecenal; avoid mixed E-2-decenal/E-2-dodecenal.
-Correct ATTC to ATCC throughout.
-Italicize genus-species names e.g. Staphylococcus aureus and keep ATCC/strain codes in roman.

The antimicrobial activity of the terpenoids and their action on the cell membrane have been widely documented. Many of these literature findings were presented by the authors in the manuscript. The authors claim that this is the first study conducted on the antimicrobial activity of E-2-dodecenal (Lines 246-247). However, this is not valid as the following article reported the anti-salmonella activity of E-2-dodecenal:

• Kubo, K. Fujita, K. Nihei, A. Kubo, Anti‐Salmonellaactivity of (2E)‐alkenals, Journal of Applied Microbiology, Volume 96, Issue 4, 1 April 2004, Pages 693–699, https://doi.org/10.1111/j.1365-2672.2003.02175.x

Antifungal activity was also reported:

• Kubo I, Fujita KI, Kubo A, Nihei KI, Lunde CS. Modes of antifungal action of (2 E)-alkenals against Saccharomyces cerevisiae. Journal of agricultural and food chemistry. 2003 Jul 2;51(14):3951-7.

Moreover, following articles on the antimicrobial activity of essential oils where E-2-dodecenal is the main compound diminishes the novelty of the work:

Silalahi M. Essential oils and uses of Eryngium foetidum L. GSC Biological and Pharmaceutical Sciences. 2021;15(3):289-94.

Kumar S, Ahmad R, Saeed S, Azeem M, Mozūraitis R, Borg-Karlson AK, Zhu G. Chemical composition of fresh leaves headspace aroma and essential oils of four Coriander cultivars. Frontiers in Plant Science. 2022 Feb 17;13:820644

With regard to the molecule E-2-dodecenal, the information suggested by the reviewer has been incorporated with the two studies proposed by Kubo et al. Despite this, it can still be said that antimicrobial research with this molecule is scarce.

The antimicrobial study on Eryngium foetidum oil, conducted by Silalahi in 2021, has already been considered in the manuscript, and Kumar's 2022 study was not mentioned as it only evaluates the chemical composition and does not conduct antimicrobial studies.

An important consideration of our research group is that the E-2-dodecenal molecule is still novel enough to warrant further antibiotic research.

Authors use ‘E-2-docenal’ throughout manuscript, however compound is named as ‘E-2-dodecenal’ in Figure 4, which I believe is the correct one.