Novel Polyamide/Chitosan Nanofibers Containing Glucose Oxidase and Rosemary Extract: Fabrication and Antimicrobial Functionality

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article presents a study regarding the preparation of nanofibers based on nylon-Ag masterbatch, chitosan, glucose oxidase and Rosmarinus officinalis extract, their characterization from morphological, structural and antibacterial point of view and their potential applications in the food packaging area. However, it is not clear why the samples based on MBN-Ag/CS, MBN-Ag/CS/GOx, MBN-Ag/CS/RO and MBN-Ag/CS/GOx/RO having the optimum composition (according to the authors claim: 70:30 ratio, 0.05 mg GOx and 1.5 % RO) are not investigated from morphological, structural, antibacterial and enzymatic activity point of view. Moreover, it is not clear in which way the results of the “Design of Experiments” and “Identifying the impact of variables on preservation (%) values” sections are useful for the present study. Furthermore, in all figures, the number used on X-Y scales are barely visible. Consequently, the authors must provide a better explanation for their results and a better correlation between their experimental data. Also, they must increase the font size on X-Y scales. Furthermore, the English must be carefully checked in the entire manuscript. Specific comments are given bellow:

1. In the title GOx must be replaced with the glucose oxidase. Also, the chitosan must be add. The fibers investigated in the present study are based on nylon-Ag, chitosan, glucose oxidase and Rosmarinus extract. Consequently, all these components must be mentioned in the title, each of them influencing the properties of the fiber composites.

2. In the Introduction section, the authors must provide a better motivation of their study. Why they choose nylon-Ag for their experiments and not only nylon? The Ag plays a major role in the properties of the fabricated fibers?

3. The authors must provide references for their claim: “It is important to note that, in the last decade, researchers have been working on different methods to develop new packaging technologies and materials, aiming to extend the shelf-life, safety, and cost efficiency of food products.”. Excepting the electrospinning, what methods are used?

4. The authors must provide references for their claim: “However, very few researches have utilized Rosmarinus officinalis extract and glucose oxidase into electrospun mats for these applications.” Moreover, the results reported on the properties of composites based on the compounds (nylon, chitosan, glucose oxidase and Rosmarinus extract) must be mentioned in order to emphasize the novelty of the present study.

5. The authors must provide more information regarding the properties of the materials used as precursors (ex: what is the Ag amount in the commercial nylon-Ag masterbatch?; what is the molecular weight of commercial chitosan?; what is the type of the commercial glucose oxide?)

6. The authors must provide more information regarding the choose of the experimental parameters involved in the preparation of the solutions used in the elestrospinning process. Why they choose MBN-Ag (7 w%) and CS (5 w%)? Why they choose GOx (0.01, 0.03, 0.05, 0.07 mg)? Why they choose 1.5% of RO extract? These amounts were selected based on preliminary tests? The authors must clarify these aspects.

7. In the glucose oxidase activity evaluation, the authors mentioned: “For this purpose, 1 ml of glucose oxidase (1 mg/ml) and 4 ml of glucose oxidase solution (0.2 mol/l) were poured into a test tube at 37 °C for 10 min to react.” The authors mentioned only glucose oxidase. They must correct and add the amount of fiber composite that reacts with the glucose oxidase.

8. In the “Materials and Methods” section, the authors must add a paragraph regarding the food packaging experiments.

9. It is not in which way are useful the information provided by the sections “Design of Experiments” and “Identifying the impact of variables on preservation (%) values”? The “preservation (%) value” refers to the amount of each precursor from the solution intended to be electrospinning and from the fabricated fibers? It is not clear what means “preservation (%) value”? Moreover, in which experiments are used the values from Table 3 (“Optimum values of different variables”)? The authors must provide a better explanation for the importance of these sections for their study.

10. The authors claim: “In contrast to the changes by glucose oxidase (GOx), the FESEM revealed no morphological changes in MBN-Ag/CS (70:30) nanofiber composites due to the addition of RO (1.5 wt%).”. The authors must add the FESEM images in order to sustain their claim.

11. The authors must add the FESEM images of the fibers based on MBN-Ag/CS/RO and MBN-Ag/CS/GOx/RO.

12. Concerning the FESEM investigation, the values estimated “170, 185, and 197 nm” are not representative taking into account that usually electrospinning process leads to fibers with different diameters. Consequently, the authors must provide FESEM images at different magnifications (higher than those already presented in the manuscript) for all investigated samples: MBN-Ag/CS, MBN-Ag/CS/GOx, MBN-Ag/CS/RO and MBN-Ag/CS/GOx/RO. In this way, they can provide a more accurate perspective regarding the diameters of the fibers of each investigated sample and comment the influence of the addition of each compound on the variation of the dispersity of the fiber diameter (the authors must add histograms).

13. In the inhibitory effect of the silver on glucose oxidase mechanism, the silver is missing. In which way silver interact with the glucose oxidase? Taking into account that the silver plays a major role in the antibacterial properties, the authors must provide a better explanation for the mechanism. Moreover, in the figures representing the GOx activity at different compositions of MBN-Ag/CS and various amount of GOx, the Y scale must be plotted up to 100% in order to evaluate accurate the values mentioned by the authors. Moreover, it is not clear why “for further studies, MBN-Ag/CS nanofiber composite with a ratio of 70:30 was selected as an optimum sample.”, this sample having “the lowest GOx activity” while “the MBN-Ag/CS/GOx nanofiber composite with 0.05 GOx content was selected as an optimum sample due to the highest enzymatic activity”. The authors must clarify this important aspect.

14. If the authors claim that MBN-Ag/CS nanofiber composite (70:30) exhibited the most uniform morphology and the composite with 0.05 GOx amount was selected as an optimum sample due to the highest enzymatic activity, why the authors carried the antibacterial tests on MBN-Ag/CS/RO (60/40/3%), MBN-Ag/CS (60/40), MBN-Ag/CS/GOx (60/40/0.05), and MBN-Ag/CS/GOx/RO (60/40/0.05/3%) samples? The authors must clarify this important aspect. Moreover, they must provide de values of the inhibition zone size of each investigated samples and provide the photographs for each sample in the case of S. aureus and E. coli media. Furthermore, a better explanation of the antibacterial results including a schematic representation of a possible mechanism must be given.

15. The authors must clearly specify what is the composition of the MBN-Ag/CS, MBN-Ag/CS/GOx, MBN-Ag/CS/RO and MBN-Ag/CS/GOx/RO samples investigated by FTIR. In addition, in order to emphasize better the presence of each component in the fiber composites, the authors must add the FTIR spectra of each precursor: MBN-Ag, CS, GOx and RO extract. Moreover, in the FTIR figure on the X scale can be noted a particular aspect: the increment is 400 cm-1 in the 2000-3600 cm-1 domain, 200 cm-1 in the 600-2000 cm-1 range and 150 cm-1 between 450 and 600 cm-1. Why? The author must explain or correct this aspect. Furthermore, the FTIR spectra must be replotted for a better visibility of the number associated to the vibration modes (the font size must be increased).

16. The authors must clearly specify what is the composition of the MBN-Ag/CS, MBN-Ag/CS/GOx, MBN-Ag/CS/RO and MBN-Ag/CS/GOx/RO samples used for obtained packages. In addition what material is used as “the “regular package”? Moreover, in the figure representing the GOx activity of different packages, the Y scale must be plotted up to 100% in order to sustain the authors claim: “The results showed that MBN-Ag/CS/GOx/RO nanofiber composite had the highest activity (90.1%) with excellent preservation properties.” Furthermore, the authors must provide details regarding the preparation and properties of these “packages”. For examples the fiber density is the same in all investigated samples? This parameter can be correlated to the transmittance of the fiber mats. Furthermore, the wetting properties of these fiber mats must be evaluated taking into account the major role played by the hydrophilicity / hydrophobicity  property in the food packages applications. Consequently, the authors must add UV-VIS transmittance and contact angle measurements for MBN-Ag/CS, MBN-Ag/CS/GOx, MBN-Ag/CS/RO and MBN-Ag/CS/GOx/RO samples and the photographs of these samples as fiber mats used in the preparation of the “packages”.

17. The authors must provide a better correlation between the morphological, enzymatic activity, antibacterial and storing fruit results.

18. The Conclusions section must be rewritten in order to summarize better the results of the present study.

19. The authors must cite papers published in the 2023 in order to emphasize the novelty of the present study in relation to the state-of-the-art in the field of their work.

20. The English must be carefully checked in the entire manuscript. Examples: “fabrcation” ; “shwed” ;  “This oxidoreductase enzyme generally generated by Aspergillus niger and Penicillium spp cultures.” ; “This can another reason for the growth of microorganisms in active food packagings.” ; “Recent investigatiens” ; “wave number” 

The authors must provide a point-to-point response.

Comments on the Quality of English Language

The English must be carefully checked in the entire manuscript. 

Author Response

Reviewer 1:

The article presents a study regarding the preparation of nanofibers based on nylon-Ag masterbatch, chitosan, glucose oxidase and Rosmarinus officinalis extract, their characterization from morphological, structural and antibacterial point of view and their potential applications in the food packaging area. However, it is not clear why the samples based on MBN-Ag/CS, MBN-Ag/CS/GOx, MBN-Ag/CS/RO and MBN-Ag/CS/GOx/RO having the optimum composition (according to the authors claim: 70:30 ratio, 0.05 mg GOx and 1.5 % RO) are not investigated from morphological, structural, antibacterial and enzymatic activity point of view. Moreover, it is not clear in which way the results of the “Design of Experiments” and “Identifying the impact of variables on preservation (%) values” sections are useful for the present study. Furthermore, in all figures, the number used on X-Y scales are barely visible. Consequently, the authors must provide a better explanation for their results and a better correlation between their experimental data. Also, they must increase the font size on X-Y scales. Furthermore, the English must be carefully checked in the entire manuscript. Specific comments are given bellow:

1. In the title GOx must be replaced with the glucose oxidase. Also, the chitosan must be add. The fibers investigated in the present study are based on nylon-Ag, chitosan, glucose oxidase and Rosmarinus extract. Consequently, all these components must be mentioned in the title, each of them influencing the properties of the fiber composites.

 Answer: We thank our esteemed reviewer for the comment. Changes have been made to the text.

2. In the Introduction section, the authors must provide a better motivation of their study. Why they choose nylon-Ag for their experiments and not only nylon? The Ag plays a major role in the properties of the fabricated fibers?

 Answer: We thank our esteemed reviewer for the comment. In the optimization of nanofibers, the most important part is related to the density of nylon, and compared to nylon, silver has shown little effect in optimizing the structure of nanofibers. On the other hand, we used nylon-Ag for providing an antibacterial ability to nanofibers.

3. The authors must provide references for their claim: “It is important to note that, in the last decade, researchers have been working on different methods to develop new packaging technologies and materials, aiming to extend the shelf-life, safety, and cost efficiency of food products.”. Excepting the electrospinning, what methods are used?

 Answer: We thank our esteemed reviewer for the comment. As recommended, we cited the statement.

4. The authors must provide references for their claim: “However, very few research have utilized Rosmarinus officinalis extract and glucose oxidase into electrospun mats for these applications.” Moreover, the results reported on the properties of composites based on the compounds (nylon, chitosan, glucose oxidase and Rosmarinus extract) must be mentioned in order to emphasize the novelty of the present study.

 Answer: We thank our esteemed reviewer for the comment. The following sources show the antibacterial ability of extracts and enzymes. We added them to the reference list.

Aseer Manilal, et al. Antibacterial Activity of Rosmarinus officinalis against Multidrug-Resistant Clinical Isolates and Meat-Borne Pathogens. Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2021, Article ID 6677420, 10 pages https://doi.org/10.1155/2021/6677420

279. Dobbenie, M. Uyttendaele, J. Debevere. Antibacterial Activity of the Glucose Oxidase/Glucose System in Liquid Whole Egg. Journal of Food Protection Volume 58, Issue 3, 1 March 1995, Pages 273-279.
280. The authors must provide more information regarding the properties of the materials used as precursors (ex: what is the Ag amount in the commercial nylon-Ag masterbatch?; what is the molecular weight of commercial chitosan?; what is the type of the commercial glucose oxide?)

 Answer: We thank our esteemed reviewer for the comment.  Change have been made to the text.

6. The authors must provide more information regarding the choose of the experimental parameters involved in the preparation of the solutions used in the elestrospinning process. Why they choose MBN-Ag (7 w%) and CS (5 w%)? Why they choose GOx (0.01, 0.03, 0.05, 0.07 mg)? Why they choose 1.5% of RO extract? These amounts were selected based on preliminary tests? The authors must clarify these aspects.

 Answer: We thank our esteemed reviewer for the comment.  The percentage and ratio of polymers were selected based on the initial density of polymers for electrospinning. And the percentage range of enzyme and extract was also selected based on the initial tests and also the available resources to create antibacterial capability.

7. In the glucose oxidase activity evaluation, the authors mentioned: “For this purpose, 1 ml of glucose oxidase (1 mg/ml) and 4 ml of glucose oxidase solution (0.2 mol/l) were poured into a test tube at 37 °C for 10 min to react.” The authors mentioned only glucose oxidase. They must correct and add the amount of fiber composite that reacts with the glucose oxidase.

Answer: We thank our esteemed reviewer for the comment. The percentage of enzyme in the samples was determined based on the polymer volume and was used.

8. In the “Materials and Methods” section, the authors must add a paragraph regarding the food packaging experiments.

 Answer: We thank our esteemed reviewer for the comment. Changes have been made to the text.

9. It is not in which way are useful the information provided by the sections “Design of Experiments” and “Identifying the impact of variables on preservation (%) values”? The “preservation (%) value” refers to the amount of each precursor from the solution intended to be electrospinning and from the fabricated fibers? It is not clear what means “preservation (%) value”? Moreover, in which experiments are used the values from Table 3 (“Optimum values of different variables”)? The authors must provide a better explanation for the importance of these sections for their study.

 Answer: We thank our esteemed reviewer for the comment.  Changes were added and Information provided by the “Design of Experiments” was used for experimental study of synthesis of nanofiber.

10. The authors claim: “In contrast to the changes by glucose oxidase (GOx), the FESEM revealed no morphological changes in MBN-Ag/CS (70:30) nanofiber composites due to the addition of RO (1.5 wt%).”. The authors must add the FESEM images in order to sustain their claim.

Answer: We thank our esteemed reviewer for the comment.  Changes have been made to the text.

11. The authors must add the FESEM images of the fibers based on MBN-Ag/CS/RO and MBN-Ag/CS/GOx/RO.

 Answer: We thank our esteemed reviewer for the comment.  Changes have been made to the text.

12. Concerning the FESEM investigation, the values estimated “170, 185, and 197 nm” are not representative taking into account that usually electrospinning process leads to fibers with different diameters. Consequently, the authors must provide FESEM images at different magnifications (higher than those already presented in the manuscript) for all investigated samples: MBN-Ag/CS, MBN-Ag/CS/GOx, MBN-Ag/CS/RO and MBN-Ag/CS/GOx/RO. In this way, they can provide a more accurate perspective regarding the diameters of the fibers of each investigated sample and comment the influence of the addition of each compound on the variation of the dispersity of the fiber diameter (the authors must add histograms).

 Answer: We thank our esteemed reviewer for the comment.  Changes have been made to the text.

13. In the inhibitory effect of the silver on glucose oxidase mechanism, the silver is missing. In which way silver interact with the glucose oxidase? Taking into account that the silver plays a major role in the antibacterial properties, the authors must provide a better explanation for the mechanism. Moreover, in the figures representing the GOx activity at different compositions of MBN-Ag/CS and various amount of GOx, the Y scale must be plotted up to 100% in order to evaluate accurate the values mentioned by the authors. Moreover, it is not clear why “for further studies, MBN-Ag/CS nanofiber composite with a ratio of 70:30 was selected as an optimum sample.”, this sample having “the lowest GOx activity” while “the MBN-Ag/CS/GOx nanofiber composite with 0.05 GOx content was selected as an optimum sample due to the highest enzymatic activity”. The authors must clarify this important aspect.

 Answer: We thank our esteemed reviewer for the comment. Due to the importance of uniform structure and nanofiber size, 30/70 ratio was chosen as the optimal ratio. On the other hand, both silver and enzyme have antibacterial ability, and this has caused a synergy effect and improved antibacterial ability of nanofiber. Also, chitosan/ enzyme sample did not have suitable antibacterial effect compare to the final composite sample.

14. If the authors claim that MBN-Ag/CS nanofiber composite (70:30) exhibited the most uniform morphology and the composite with 0.05 GOx amount was selected as an optimum sample due to the highest enzymatic activity, why the authors carried the antibacterial tests on MBN-Ag/CS/RO (60/40/3%), MBN-Ag/CS (60/40), MBN-Ag/CS/GOx (60/40/0.05), and MBN-Ag/CS/GOx/RO (60/40/0.05/3%) samples? The authors must clarify this important aspect. Moreover, they must provide de values of the inhibition zone size of each investigated samples and provide the photographs for each sample in the case of S. aureus and E. coli media. Furthermore, a better explanation of the antibacterial results including a schematic representation of a possible mechanism must be given.

 Answer: We thank our esteemed reviewer for the comment. Usually, the antibacterial test is performed on the optimal sample and the control samples, and for this reason, in this study, the antibacterial test was performed on the optimized sample of each step and control.

15. The authors must clearly specify what is the composition of the MBN-Ag/CS, MBN-Ag/CS/GOx, MBN-Ag/CS/RO and MBN-Ag/CS/GOx/RO samples investigated by FTIR. In addition, in order to emphasize better the presence of each component in the fiber composites, the authors must add the FTIR spectra of each precursor: MBN-Ag, CS, GOx and RO extract. Moreover, in the FTIR figure on the X scale can be noted a particular aspect: the increment is 400 cm-1 in the 2000-3600 cm-1 domain, 200 cm-1 in the 600-2000 cm-1 range and 150 cm-1 between 450 and 600 cm-1. Why? The author must explain or correct this aspect. Furthermore, the FTIR spectra must be replotted for a better visibility of the number associated to the vibration modes (the font size must be increased).

 Answer: We thank our esteemed reviewer for the comment.  In the FTIR image, different groups were identified and corrections were made in the images.

16. The authors must clearly specify what is the composition of the MBN-Ag/CS, MBN-Ag/CS/GOx, MBN-Ag/CS/RO and MBN-Ag/CS/GOx/RO samples used for obtained packages. In addition what material is used as “the “regular package”? Moreover, in the figure representing the GOx activity of different packages, the Y scale must be plotted up to 100% in order to sustain the authors claim: “The results showed that MBN-Ag/CS/GOx/RO nanofiber composite had the highest activity (90.1%) with excellent preservation properties.” Furthermore, the authors must provide details regarding the preparation and properties of these “packages”. For examples the fiber density is the same in all investigated samples? This parameter can be correlated to the transmittance of the fiber mats. Furthermore, the wetting properties of these fiber mats must be evaluated taking into account the major role played by the hydrophilicity / hydrophobicity property in the food packages applications. Consequently, the authors must add UV-VIS transmittance and contact angle measurements for MBN-Ag/CS, MBN-Ag/CS/GOx, MBN-Ag/CS/RO and MBN-Ag/CS/GOx/RO samples and the photographs of these samples as fiber mats used in the preparation of the “packages”.

 Answer: We thank our esteemed reviewer for the comment.  The composition of all samples is specified in the text of the article. Also, the preparation steps of nanofibers are completely mentioned in the text of the article.

17. The authors must provide a better correlation between the morphological, enzymatic activity, antibacterial and storing fruit results.

Answer: We thank our esteemed reviewer for the comment. The optimal sample have shown a uniform structure, and this uniformity indicates the uniform distribution of the enzyme in the nanofibers. On the other hand, with the presence of the enzyme, the antibacterial effect has increased, and this indicates a significant correlation between the uniform structure and the effect of enzyme presence in nanofibers.

18. The Conclusions section must be rewritten in order to summarize better the results of the present study.

 Answer: We thank our esteemed reviewer for the comment. Changes have been made to the text.

19. The authors must cite papers published in the 2023 in order to emphasize the novelty of the present study in relation to the state-of-the-art in the field of their work.

 Answer: We thank our esteemed reviewer for the comment. Changes have been made to the text.

20. The English must be carefully checked in the entire manuscript. Examples: “fabrcation” ; “shwed” ;  “This oxidoreductase enzyme generally generated by Aspergillus niger and Penicillium spp cultures.” ; “This can another reason for the growth of microorganisms in active food packagings.” ; “Recent investigatiens” ; “wave number” 

Answer: We thank our esteemed reviewer for the comment. The text was corrected.

Reviewer 2 Report

Comments and Suggestions for Authors

1) More information about polymers is required. What is the molar weight of Nylon and Chitosan? What is acetylation degree of chitosan?

Chitosan is water-soluble. What about the stability of the nanofibers in wet conditions? The stability of the nanofibers should be demonstrated (e.g SEM images after soaking). The efforts in characterization of Ag should be undertaken. What is it, nanoparticles or ions? (e.g. TEM of diluted Nylon-Ag if particles or SEM-EDX if ions)

2)According to Table 1. №27, 28, it looks like an increase in Rosmarinus concentration in the nanofibers, led to a decrease in preservation. It should be explained.

table 1 MBN-Ag, MBN-Ag/CS are also in %?

3) on page 7 (2.7 Antibacterial test) the colony counting method is mentioned, why not the results were included into the manuscript? why antibacterial tests were used instead of antifungial?

description of the antibacterial test is not complete. (size of the sample for disc diffusion, were the same weight used)

figure 11 it is unclear what is shown on top, please correct the capture, and number the images,

Why were the samples removed from the agar surface?

4) should be corrected:

page 2 "developed with

biopolymers. Besides the fact that they have shown a strong barrier for foods preservation 64

against humidity and oxidation,".

The biopolymers are mostly wettable. In addition, nanofibers are porous, so, the oxygen (or any gas) penetration through the nanofiber mesh is enhanced, compared to films e.g.

"Providing a high mass-transfer rate from an electrospun substrate to the active sites 

of an enzyme; " 

"Representing a highly durable performance for multiple usages; " in all cases? all fibers?

5) some papers about antibacterial nanofibers were suggested to be cited in the manuscript

https://www.sciencedirect.com/science/article/abs/pii/S0963996919308130

https://www.sciencedirect.com/science/article/abs/pii/S1381514819304833?via%3Dihub

minor comments:

Staphylococcus aureus should be italic Staphylococcus aureus

there is no point in making 3d diagrams if you don't have a third axis fi9a, fig 10

Comments on the Quality of English Language

e.g the first sentence "bioactive materals" should be materials

and many others, please check English in whole manuscript

Author Response

Reviewer 2:

1) More information about polymers is required. What is the molar weight of Nylon and Chitosan? What is acetylation degree of chitosan?

Answer: We thank our esteemed reviewer for the comment. Polyamide - Nylon 6 (PA 6), powder, max. particle size 50micron, weight 500 g, min. particle size 5micron. Chitosan (CS) medium weight (Sigma, Deacetyliertes Chitin, Poly(D-glucosamin, CAS-Nummer: 9012-76-4),

Chitosan is water-soluble. What about the stability of the nanofibers in wet conditions? The stability of the nanofibers should be demonstrated (e.g SEM images after soaking). The efforts in characterization of Ag should be undertaken. What is it, nanoparticles or ions? (e.g. TEM of diluted Nylon-Ag if particles or SEM-EDX if ions)

Answer: We thank our esteemed reviewer for the comment. Unfortunately, our department is not able to run SEM and TEM of samples at this time. The timeline for acquiring images will be two months. We will consider these tests for the next part of the paper. As reported, we have already included SEM micro results in our article.

2)According to Table 1. №27, 28, it looks like an increase in Rosmarinus concentration in the nanofibers, led to a decrease in preservation. It should be explained.

Answer: We thank our esteemed reviewer for the comment. In the design expert software, we used a 95% statistical confidence limit at the software, that’s why we have the same recipe for each variable with different conditions with others, this process can help the software to control the 5% missing. This is the style of the software and itself standard. So, the software can analyze the results and give us the optimum. That's why we have 3 optimum recipes in Table 3. So, if the variables increase or decrease, the results will be changed, like numbers 27 and 28 the difference is just MBN-Ag, if others are the same, and we should change the Rosmarinus and MBN-Ag, this is the result, But the software will be analyzing how we can reach the optimum if we change all the parameters. This is the advantage of the software.

table 1 MBN-Ag, MBN-Ag/CS are also in %?

Answer: We thank our esteemed reviewer for the comment. Changes have been made to the text.

3) on page 7 (2.7 Antibacterial test) the colony counting method is mentioned, why not the results were included into the manuscript? why antibacterial tests were used instead of antifungial?

Answer: We thank our esteemed reviewer for the comment. Changes have been made to the text.

description of the antibacterial test is not complete. (size of the sample for disc diffusion, were the same weight used)

Answer: We thank our esteemed reviewer for the comment. Changes have been made to the text.

figure 11 it is unclear what is shown on top, please correct the capture, and number the images,

Why were the samples removed from the agar surface?

Answer: We thank our esteemed reviewer for the comment. Changes have been made to the text.

4) should be corrected:

page 2 "developed with biopolymers. Besides the fact that they have shown a strong barrier for foods preservation 64 against humidity and oxidation,".

The biopolymers are mostly wettable. In addition, nanofibers are porous, so, the oxygen (or any gas) penetration through the nanofiber mesh is enhanced, compared to films e.g. "Providing a high mass-transfer rate from an electrospun substrate to the active sites of an enzyme; " "Representing a highly durable performance for multiple usages; " in all cases? all fibers?

Answer: We thank our esteemed reviewer for the comment. As recommended, we corrected to: “the majority of electrospun nanofibers”.

5) some papers about antibacterial nanofibers were suggested to be cited in the manuscript

https://www.sciencedirect.com/science/article/abs/pii/S0963996919308130

https://www.sciencedirect.com/science/article/abs/pii/S1381514819304833?via%3Dihub

Answer: We thank our esteemed reviewer for the comment. Changes have been made to the text.

minor comments:

Staphylococcus aureus should be italic Staphylococcus aureus

Answer: We thank our esteemed reviewer for the comment. Changes have been made to the text.

there is no point in making 3d diagrams if you don't have a third axis fi9a, fig 10

Answer: We thank our esteemed reviewer for the comment. Changes have been made to the text.

Reviewer 3 Report

Comments and Suggestions for Authors

1. Avoid abbreviations in the title. Write the full name of GOx.

2. Line 36 and 37 and other place, please write the bacteria name in italics. Also check other places in the whole manuscript.

3. Lines 60 and 61 have gaps, please double-check.

4. Line 93-98 avoid points make it simple text.

5. Line 129, it should be wt%?

6. Fig. 1 is not visible improve the resolution. Texts are stretchable.

7. Write the equations in equation format.

8. Fig. 3 is not visible.

9. Present the inhibition zone of the diameter of each sample for the antibacterial test.

10. How authors calculated the antibacterial activity. Present the formula.

11. MIC and IC 50 need to be present for bacterial growth.

12. Fig. 12 is not acceptable for scientific publications. It should be drawn by scientific software and presented in a clean way.

13. The vibration band in FTIR should be marked in the figures. The X-axis should be wavenumbers (cm-1) and transmittance (a.u.).

14. FTIR or FT-IR, choose one and make it consistent in the whole manuscript.

15. Fig. 13 is also of poor quality and not visible.

16. In conclusion not significant findings were highlighted need to rewrite.

17. Update the recent references especially 2022, 2023 and 2024. Indicate the following recent references in the text Progress in Organic Coatings 183 (2023) 107749,  https://doi.org/10.1016/j.apsusc.2023.159215 and J. Mater. Chem. A, 2023, 11, 26230-26241.

18. Authors need to provide histogram curve for average diameter.

Comments on the Quality of English Language

 Extensive editing of English language required

Author Response

Reviewer 3:

1. Avoid abbreviations in the title. Write the full name of GOx.

 Answer: We thank our esteemed reviewer for the comment. Changes have been made to the text.

2. Line 36 and 37 and other place, please write the bacteria name in italics. Also check other places in the whole manuscript.

 Answer: We thank our esteemed reviewer for the comment. Changes were made in the text.

3. Lines 60 and 61 have gaps, please double-check.

 Answer: We thank our esteemed reviewer for the comment. The distance (gap) has been corrected.

4. Line 93-98 avoid points make it simple text.

 Answer: We thank our esteemed reviewer for the comment. Changes were made in the text.

5. Line 129, it should be wt%?

 Answer: We thank our esteemed reviewer for the comment. Changes were made in the text.

6. Fig. 1 is not visible improve the resolution. Texts are stretchable.

 Answer: We thank our esteemed reviewer for the comment. Changes were made in the text.

7. Write the equations in equation format.

 Answer: We thank our esteemed reviewer for the comment. Changes were made in the text.

8. Fig. 3 is not visible.

 Answer: We thank our esteemed reviewer for the comment. Changes were made in the text.

9. Present the inhibition zone of the diameter of each sample for the antibacterial test.

 Answer: We thank our esteemed reviewer for the comment. Changes were made in the text.

10. How authors calculated the antibacterial activity. Present the formula.

 Answer: We thank our esteemed reviewer for the comment. Changes were made in the text.

Antibacterial activity indicated using MIC and disc diffusion assay.

11. MIC and IC 50 need to be present for bacterial growth.

Answer: We thank our esteemed reviewer for the comment. Changes were made in the text and

Percentage of antibacterial activity reported.

12. Fig. 12 is not acceptable for scientific publications. It should be drawn by scientific software and presented in a clean way.

Answer: We thank our esteemed reviewer for the comment. With respect to the reviewer's opinion, the data is normal (original) and can be used for a scientific publication.

13. The vibration band in FTIR should be marked in the figures. The X-axis should be wavenumbers (cm-1) and transmittance (a.u.).

Answer: We thank our esteemed reviewer for the comment. The correction was carried out.

14. FTIR or FT-IR, choose one and make it consistent in the whole manuscript.

 Answer: We thank our esteemed reviewer for the comment. The correction was carried out.

15. Fig. 13 is also of poor quality and not visible.

 Answer: We thank our esteemed reviewer for the comment. We replaced new images.

16. In conclusion not significant findings were highlighted need to rewrite.

 Answer: We thank our esteemed reviewer for the comment. Changes have been made to the text.

17. Update the recent references especially 2022, 2023 and 2024. Indicate the following recent references in the text Progress in Organic Coatings 183 (2023) 107749, https://doi.org/10.1016/j.apsusc.2023.159215 and J. Mater. Chem. A, 2023, 11, 26230-26241.

 Answer: We thank our esteemed reviewer for the comment. Changes have been made to the text.

18. Authors need to provide histogram curve for average diameter.

 Answer: We thank our esteemed reviewer for the comment. Changes have been made to the text.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revised manuscript is only slightly modified in comparison to the initial manuscript. The authors still need to improve their work. Specific comments are given bellow:

1. As it is already mentioned, in order to emphasize clearly the novelty of their work, the authors must add a comment in the Introduction section concerning the previous studies reported in literature on the electrospinning and properties of nylon-Ag fibers and nylon-chitosan fibers.

2. As it is already mentioned, the authors must mention what is the Ag amount in the commercial nylon-Ag masterbatch?

3.  As it is already mentioned, it is not clear in which experiments are used the values from Table 3 (“Optimum values of different variables”) taking into account that other values are used in the electrospinning process?

4. Taking into account that the diameters of fibers evaluated from the FESEM images given in the manuscript seems to be higher than those specified by the authors, as it is already mentioned, the authors must provide FESEM images at different magnifications (higher than those already presented in the manuscript) for all investigated samples: MBN-Ag/CS, MBN-Ag/CS/GOx, MBN-Ag/CS/RO and MBN-Ag/CS/GOx/RO.

5. In the current form, the FTIR figure can not be suitable for publication (its resolution is lower than that presented in the initial manuscript). As it is already mentioned, in order to emphasize better the presence of each component in the fiber composites, the authors must add the FTIR spectra of each precursor: MBN-Ag, CS, GOx and RO extract. In addition, the authors must use the same increment on the X scale. Furthermore, the FTIR spectra must be replotted for a better visibility of the number associated to the vibration modes (the font size must be increased).

6. As it is already mentioned, the authors must provide UV-VIS transmittance and contact angle measurements for MBN-Ag/CS, MBN-Ag/CS/GOx, MBN-Ag/CS/RO and MBN-Ag/CS/GOx/RO samples and correlate these data with the other properties.

7. As it is already mentioned, the authors must add a paragraph providing a better correlation between the morphological, enzymatic activity, antibacterial and storing fruit results. The authors must improve the scientific explanation concerning their results.

The authors must insert their modifications made as response to the reviewer comment in both “Responses to Reviewer” document and revised manuscript (do not use only the sentence "Changes have been made to the text” in the “Responses to Reviewer”). A point-to-point response is required.

Comments on the Quality of English Language

English must be revised in the manuscript.

Author Response

Reviewer 1:

The revised manuscript is only slightly modified in comparison to the initial manuscript. The authors still need to improve their work. Specific comments are given bellow:

1. As it is already mentioned, in order to emphasize clearly the novelty of their work, the authors must add a comment in the Introduction section concerning the previous studies reported in literature on the electrospinning and properties of nylon-Ag fibers and nylon-chitosan fibers.

Answer: We thank our esteemed reviewer for the comment. Corrections were made in the text:

Previous studies on Nylon-6/chitosan nanofibers have shown efficient antibacterial effect and sustainable drug release, and Nylon-Ag nanofibers showed higher antibacterial efficacy compared to nylon nanofibers against Gram-negative microorganisms.

Reference below were added:

Keirouz, A., Radacsi, N., Ren, Q. et al. Nylon-6/chitosan core/shell antimicrobial nanofibers for the prevention of mesh-associated surgical site infection. J Nanobiotechnol 18, 51 (2020). https://doi.org/10.1186/s12951-020-00602-9

2015. Xu, S. Mahalingam, J. L. Rohn, G. Ren, and M. Edirisinghe. Physio-chemical and antibacterial characteristics of pressure spun nylon nanofibres embedded with functional silver nanoparticles. Materials Science and Engineering C, vol. 56, pp. 195–204, 2015.
2016. As it is already mentioned, the authors must mention what is the Ag amount in the commercial nylon-Ag masterbatch?

Answer: We thank our esteemed reviewer for the comment. It was master batch had 1% Ag content. It was added to the text.

3. As it is already mentioned, it is not clear in which experiments are used the values from Table 3 (“Optimum values of different variables”) taking into account that other values are used in the electrospinning process?

Answer: We thank our esteemed reviewer for the comment. The optimum value of Table 3 was used for optimal synthesis of nanofibers for antibacterial effect evaluation and packaging studies.

4. Taking into account that the diameters of fibers evaluated from the FESEM images given in the manuscript seems to be higher than those specified by the authors, as it is already mentioned, the authors must provide FESEM images at different magnifications (higher than those already presented in the manuscript) for all investigated samples: MBN-Ag/CS, MBN-Ag/CS/GOx, MBN-Ag/CS/RO and MBN-Ag/CS/GOx/RO.

Answer: We thank our esteemed reviewer for the comment. With respect to the opinion of the respected reviewer, the scale bar is specified in all microscopic images to determine the size of nanofibers. We also thank our esteemed reviewer for asking for FESEM images. Unfortunately, we do not have access to this equipment for acquiring FESEM images of our samples currently. We will include this test in our future studies.

5. In the current form, the FTIR figure cannot be suitable for publication (its resolution is lower than that presented in the initial manuscript). As it is already mentioned, in order to emphasize better the presence of each component in the fiber composites, the authors must add the FTIR spectra of each precursor: MBN-Ag, CS, GOx and RO extract. In addition, the authors must use the same increment on the X scale. Furthermore, the FTIR spectra must be replotted for a better visibility of the number associated to the vibration modes (the font size must be increased).

Answer: We thank our esteemed reviewer for the comment. We would like to mention that our FTIR data were originally extracted from FTIR spectroscopy. Unfortunately, our FTIR spectroscopy is out of order, and we have no access to improve the quality of the results.

6. As it is already mentioned, the authors must provide UV-VIS transmittance and contact angle measurements for MBN-Ag/CS, MBN-Ag/CS/GOx, MBN-Ag/CS/RO and MBN-Ag/CS/GOx/RO samples and correlate these data with the other properties.

Answer:  We thank our esteemed reviewer for the comment. Unfortunately, our institutes do not have UV-VIS transmittance and contact angle to evaluate our samples. We will include these tests in our future studies.

7. As it is already mentioned, the authors must add a paragraph providing a better correlation between the morphological, enzymatic activity, antibacterial and storing fruit results. The authors must improve the scientific explanation concerning their results.

Answer: We thank our esteemed reviewer for the comment. According to the suggestion of another respected referee, this section was added to the conclusion.

Reviewer 2 Report

Comments and Suggestions for Authors

Staphylococcus aureus (S. aureus) page 8. line 204 should be also italic, please check 

Author Response

Reviewer 2:

Staphylococcus aureus (S. aureus) page 8. line 204 should be also italic, please check

Answer: We thank our esteemed reviewer for the comment. The text has been corrected.

Reviewer 3 Report

Comments and Suggestions for Authors

authors improved the manuscript. Still, I have more concerns about the figure resolution. At prespent figures are not the SCI paper publication standard.

Comments on the Quality of English Language

Minor checking is needed.

Author Response

Reviewer 2:

Authors improved the manuscript. Still, I have more concerns about the figure resolution. At present figures are not the SCI paper publication standard.

 Answer: We thank our esteemed reviewer for the comment. We replaced some of these figures with new figures of higher resolutions. Unfortunately, our FTIR spectroscopy is out of order, and we have no access to the raw data.