Investigating Grape Seed Extract as a Natural Antibacterial Agent for Water Disinfection in Saudi Arabia: A Pilot Chemical, Phytochemical, Heavy-Metal, Mineral, and CB-Dock Study Employing Water and Urine Samples

Response to Reviewer 1 Comments

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Point-by-point response to Comments and Suggestions for Authors

Comments 1: The abstract is too long; the first three lines are unnecessary.

Response 1: Done inside the manuscript.

Comments 2: Correct the scientific names throughout the manuscript, ensuring proper orthographic conventions, e.g., Pseudomonas aeruginosa, Staphylococcus epidermidis. The binomial nomenclature should be followed with the genus name capitalized and the species name in lowercase, both italicized. Abbreviations like P. aeruginosa can be used. Also, correct the supplementary material files and "nonpublish."

Response 2: Done inside the manuscript.

Comments 3: In the introduction, replace "micrograms" with "microorganisms."

Response 3: Done inside the manuscript.

Comments 4: Although Figure 1 is a general diagram, it should be placed in the materials section.

Response 4: With all due respect, after your permission, we propose that the content presented in Figure 1, which introduces our hypothesis, should be in the introduction section. By citing it early in the introduction, we can succinctly convey the study’s hypothesis and methods.

Comments 5: In the Methods section, detail the origin of the potable and desalinated water samples, providing more information to allow readers to associate the results with the characterization.

Response 5: In the Methods section, we refrain from providing detailed information about the origin of the potable and desalinated water samples. However, we offer sufficient data to allow readers to associate the results with the characterization. This approach helps avoid conflicts of interest and maintains confidentiality regarding specific sample details or brands.

Comments 6: Throughout the article, change "ml" to "mL."

Response 6: Done inside the manuscript.

Comments 7: A detailed description of the API is unnecessary since it is a standard technique.

Response 7: With all due respect, while I understand the theoretical perspective, I firmly believe that including mechanism of action of the API is essential. The data serves as crucial material for presenting mechanism of action, biochemistry data, color reaction, results, etc. Moreover, it aids both undergraduate and postgraduate readers who may use the paper for in-depth understanding. Reducing the level of detail would diminish the value and benefits for diverse audiences with varying scientific backgrounds.

Comments 8: Clearly state that the authors published a previous work related to this research. In subsequent citations, refer to it by the corresponding number, avoiding terms like “our previous publication” or “our previous reports” to reduce distraction for the reader. Additionally, in line 172, the word "researchers" is unnecessary.

Response 8: dear reviewer we correct accordingly, after your permission may I say few words

In the context of subsequent citations, we refer to the authors’ previous work related to this research using the corresponding reference number. The approach terms like “our previous publication” or “our previous reports referring to our previous publication in our manuscript can enhance the completeness of our work. It provides context and allows readers to connect related research.

Comments 9: Clarify the use of nutrient agar and agarose for well formation, as a modified disk diffusion technique is referred to.

Response 9: Done inside the manuscript.

Comments 10: In line 177, it is mentioned that some Petri dishes were incubated at 28 °C for other microorganisms, but only bacteria were isolated in this study. What other microorganisms are being referred to?

Response 10: Done inside the manuscript.

(Next, they applied 100 microliters of the bacterial culture (102 cells/mL of bacteria) onto nutrient agar plates to assess susceptibility using the well diffusion method. Nutrient agar supports bacterial growth, while agarose provides the structure for well diffusion assays. Circular holes with a diameter of six millimeters were created in the agarose gel, and crude grape seed extract oil was added to the wells at doses of 10, 20, 30, 50, and 100 mg/mL. The Petri dishes were then incubated for 24-48 hours at 37°C for bacterial strains and 48 hours at 28°C for other microbes such as fungi.

No other organism or fungi are detected.[32]

Comments 11: Table 2, the values are not below the detection limit?

Response 11: Corrected inside the manuscript.

Comments 12: In figure 2B, remove "Chart title" from the graph.

Response 12: Done inside the manuscript.

Comments 13: In figure 3, avoid duplicating "%" on the Y-axis.

Response 13: Done inside the manuscript.

Comments 14: In figure 6, correct the inversion of axis titles.

Response 14: After your permission, Figure 6 aids both undergraduate and postgraduate readers who may use the paper for easy in-depth understanding in this way, the inversion will not give easy understanding.

Comments 15: Discuss the characteristics of grape seed extract in comparison to other reports on metal content, phytochemistry, and nutrients.

Response 15: Done inside the manuscript.

Comments 16: Tables 1 and 5 consider presenting in the supplementary material instead of the main body of the article.

Response 16: after your permission, tables 1 and 5 are essential results for the study, I can’t consider them as supplementary. With all due respect, while I understand the theoretical perspective, I firmly believe that including the table is essential. The table serves as crucial material for presenting the main data. Moreover, it aids both undergraduate and postgraduate readers who may use the paper for in-depth understanding. Reducing the level of detail would diminish the value and benefits for diverse audiences with varying scientific backgrounds.

Response to Comments on the Quality of English Language

Point 1: It is advised that the manuscript undergo some editing adjustments to improve its readability.

Response 1:    Done by the native according to MDPI style.

Response to Reviewer 2 Comments

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Point-by-point response to Comments and Suggestions for Authors

Comments 1: According to the reviewer, 53 pages is too much for a research article. Moreover, most of it is taken up by tables 1 and 5.

Response 1: after your permission, tables 1 and 5 are essential results for the study, I can’t consider them as supplementary. With all due respect, while I understand the theoretical perspective, I firmly believe that including the table is essential. The table serves as crucial material for presenting the main data. Moreover, it aids both undergraduate and postgraduate readers who may use the paper for in-depth understanding. Reducing the level of detail would diminish the value and benefits for diverse audiences with varying scientific backgrounds.

Comments 2: What was the initial concentration of the bacterial cultures (in Colonies/ml)?

Response 2: Done inside the manuscript.

(Next, they applied 100 microliters of the bacterial culture (102 cells/mL of bacteria) onto nutrient agar plates to assess susceptibility using the well diffusion method. Nutrient agar supports bacterial growth, while agarose provides the structure for well diffusion assays. Circular holes with a diameter of six millimeters were created in the agarose gel, and crude grape seed extract oil was added to the wells at doses of 10, 20, 30, 50, and 100 mg/mL. The Petri dishes were then incubated for 24-48 hours at 37°C for bacterial strains and 48 hours at 28°C for other microbes such as fungi [32].

No other organism or fungi are detected.[32]

Comments 3: Gas chromatography data should be presented in the form of a figure.

Response 3: After your permission, Gas chromatography in table 1 help both undergraduate and postgraduate readers who may use the paper for easy in-depth understanding in this way, the separation in the form of a figure will not give many figures and loading results.

Comments 4: Do not duplicate the data from Table 4 in Figure 4. Choose one format for presenting the data!

Response 4: Done inside the manuscript.

Comments 5: Table 5 should be moved to the Supplementary Materials file. And the Results and Discussion sections should be combined. This will make the results clearer.

Response 5: after your permission, tables 5 is essential results for the study, I can’t consider them as supplementary. we cannot combine Results and Discussion section Keeping them separate allows for focused discussions, beside we follow the journal style

The comments provided do not reduce the level of the work but allow us to present interesting data better.

Thank you for your efforts, and appreciated your review, we did our best.

Response to Reviewer 3 Comments

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files.

Point-by-point response to Comments and Suggestions for Authors

Comments 1: Errors need to be shown in the each Figures.

Response 1: Done inside the manuscript. Thank you for very important advice we will include in our next funding project but after your permission, for current study:

In our study, we calculated errors using the standard deviation (SD) for urine biological samples (n=100) during metals analysis. These results are detailed in Table 3, which has been relocated to the supplementary material. Conversely, for non-biological samples (e.g., water), we determined parameters using a triplicate assay. The tightly clustered data points in this case allow us to focus solely on highlighting differences between samples

Comments 2: Some novel nano-antibacterial materials have also been applied, which cannot be ignored in the background introduction. It is recommended that the author add discussion to this section and cite it appropriately.

Response 2: Done inside the manuscript.

Comments 3: Table 3 and 4, The font used in the table is inconsistent

Response 3: Done inside the manuscript.

Comments 4: Section 3.7, An incorrect segmentation has occurred, the author should add the photos of bacterial culture, which is very important, because the antibacterial effect can be observed more directly

Response 4: Dear reviewer,

I appreciate your feedback. I agree that including photos of the bacterial culture is crucial. These images allow for a more direct observation of the antibacterial effect. However, due to our limited experience in this field, we unfortunately lost a significant amount of data and photos. Currently, we only have the percentage data from the lab. As this study is self-funded and serves as a pilot, we plan to include these initial results in our next funding round. Moving forward, we will conduct further investigations to enhance our understanding. As it a pilot study in purpose of analytical chemistry with proposals applied analytical process in further investigations such as

(Investigate the specific antibacterial mechanism. Is it inhibiting cell wall synthesis, disrupting protein synthesis, or affecting DNA replication, Observe the antibacterial effect over time. Collect samples at different intervals (e.g., 1 hour, 3 hours, 24 hours) and analyze bacterial growth inhibition) that for fruther fund studie.

Comments 5: L396 and L378, The reference does not conform to the specification

Response 5: -Dear Editor,

With your permission, the references have been cited in the appropriate locations, supporting several proven points. These references align with the context and demonstrate the presence of mimic molecules shared between the plant oil extract and grape seed oil. Furthermore, they substantiate the specified agreement across context, methodology, hypothesis and results.

Comments 6: The authors demonstrate the potential of grape seed extracts as antimicrobial agents; however, systematic discussion of isolated and identified phytochemical components is inadequate. It is recommended that authors clearly list the specific phytochemicals tested and their concentrations.

Response 6: Dear reviewer,

I appreciate your feedback. I agree with you. However, due to our limited experience in this field, we unfortunately lost a significant amount of data and photos. As this study is self-funded and serves as a pilot, we plan to include these initial results for our next funding round. Moving forward, we will conduct further investigations to enhance our understanding. As it a pilot study in purpose of analytical chemistry with proposals applied analytical process in further investigations such as specific phytochemicals tested and their concentrations for our next publication.

Comments 7: It is suggested that the authors add more experimental details to enhance the reliability and reproducibility of the study results. For example, the specific calculation method of binding energy between GSE and bacterial protein receptors.

Response 7: Dear reviewer,

Binding energy calculated automatically on the site that mentioned in material and methods and discussion sections.

(we used the protein–ligand docking method from the CB-Dock: a web server for cavity de-tection-guided protein–ligand blind docking. The protein–ligand method on this website (http://cao.labshare.cn/cb-dock/) is based on AutoDock Vina, accessed on 15 may 2024. The program design to apply accurate detection of blind protein cavities and sort them according to binding energy rather than docking of protein surface only. The ex-hausting trial is nine times sort accordingly to binding energy and cavity size and then docking according to AutoDock Vina that give us more chances for best binding cavity size-detection with highest binding energy show as vina score in ordering.)

Response to Comments on the Quality of English Language

Point 1: it's ok

Response 1: Thank you.