Antimicrobial Potential of Biosynthesized Zinc Oxide Nanoparticles Using Banana Peel and Date Seeds Extracts

Round 1

Reviewer 1 Report

 The experimental design scheme of this manuscript is not reasonable, the innovative significance is not clear. The processing of figures in the manuscript is poor, and the data analysis is shallow. The author needs to supplement the necessary characterization and experiments. The paper needs to be major revised before it can be considered for acceptance. Here are some of the reviewer's questions and suggestions about the manuscript:

1. The author should add a blank set of experiments to discuss whether ZnONPs can be formed without the addition of BP or DS.

2. What are the reasons for the difference in UV-visible absorption range between ZnONPs-BPE and ZnONPs-DSE? During the preparation of ZnONPs, were the products washed and purified? Please fill in the details of the preparation of the experiments.

3. In Fig.2, what is the reason for the negative value of the spectral signal?

4. The fitting curve of particle size distribution is inconsistent with the reality.

5.Why TEM and SEM show different morphological characteristics of materials?

6. The explanation that the difference in surface negative charges caused the size discrepancy between the two materials is far-fetched.

7. Please provide the XRD patterns of the standard cards mentioned in the manuscript.

8. Why is the molecular vibration mode of ZnO so complex in FTIR? Did the author separate and purify ZnO in the product? What is the effective bactericidal substance in the bacteriostatic performance test?

9. Please explain the antibacterial mechanism of ZnO, and add the comparison of the properties of this material with other antibacterial materials.

10. Please provide visual evidence of the bactericidal effect of two ZnONPs on different bacteria, such as comparison of colonies before and after antibacterial.

11. Biomass assisted synthesis is an interesting topic. Many researches have been done in this field. Some typical references are suggested to be cited, e.g. Journal of Bioresources and Bioproducts 2021, 6 (1), 75-81; Journal of Bioresources and Bioproducts 2021, 6 (4), 323-337.

Minor polishing and editing of English language is required.

Author Response

Dear Editor and Reviewers,

Thank you for your letter and for the reviewers’ comments concerning our manuscript. Those comments are all valuable and helpful in revising and improving our manuscript, as well as providing significant direction for our research. We carefully considered the comments and made changes that we hope will be approved. The revised sections of the document are track changed. The following are the main corrections in the manuscript and responses to the reviewers’ comments.

1# Comments and Suggestions for Authors

 The experimental design scheme of this manuscript is not reasonable, the innovative significance is not clear. The processing of figures in the manuscript is poor, and the data analysis is shallow. The author needs to supplement the necessary characterization and experiments. The paper needs to be major revised before it can be considered for acceptance. Here are some of the reviewer's questions and suggestions about the manuscript:

1. The author should add a blank set of experiments to discuss whether ZnONPs can be formed without the addition of BP or DS.

• Many thanks for your comment, but nanoparticles couldn’t be formed without any chemical or biological reducing or capping agent. We have done it according to a protocol of reference and in NPs green preparation, we didn’t use blank in our experiment.

2. What are the reasons for the difference in UV-visible absorption range between ZnONPs-BPE and ZnONPs-DSE? During the preparation of ZnONPs, were the products washed and purified? Please fill in the details of the preparation of the experiments.

• Many thanks for your comment, the green-prepared ZnONPs were not purified prior to dryness. We have prepared them as references cited in the methodology. The difference in UV between the 2 green ZnONPs was mentioned in the results and discussion section.
• There was a significant shift of spectra between ZnONPs_BPE (290nm) and ZnONPs_DSE (400nm) due to a change in their surface plasmon resonance that is affected by nanoparticle size or capping of phytochemicals present in the extract [19,23]. There is a shift of the absorption edge to a lower wavelength (UV), as the nanoparticle size decreased [23].

3. In Fig.2, what is the reason for the negative value of the spectral signal?

• It is just at the beginning of the reading of one sample, then the spectral signal becomes ok, all within the positive values.

4. The fitting curve of particle size distribution is inconsistent with the reality.

• Sorry, but I don’t understand this statment. I have measured the size of nanoparticles with ImageJ software, and it gives the average size and curve as attached in the article.

5.Why TEM and SEM show different morphological characteristics of materials?

• The SEM photos have been updated.

6. The explanation that the difference in surface negative charges caused the size discrepancy between the two materials is far-fetched.

• Many thanks for your comment but I wrote this explanation according to the below references. If there is any other explanation, I will be grateful if you mention its reference to me and I will update it.
• Mukherjee, D. Chowdhury, R. Kotcherlakota et al., “Potential theranostics application of bio-synthesized silver nanoparticles (4-in-1 system),” Theranostics, vol. 4, no. 3, pp. 316–335, 2014. “Previously, it was reported that a high negative potential value increases nanoparticles’ repulsion force that in turn helps in their dispersal, stability, and good colloidal nature [39].”
• Punamjyoti Das, Malay K. Das, in Nanocosmeceuticals, 2022. Production and physicochemical characterization of nanocosmeceuticals. “When the particles in the suspension have a high negative or positive zeta potential, then they display an inclination to repel in order that no flocculation takes place and shows resistance to aggregation.”
• Shabnam Samimi, ... Farid Dorkoosh, in Characterization and Biology of Nanomaterials for Drug Delivery, 2019 “Lipid-Based Nanoparticles for Drug Delivery Systems” (High ZP implies highly charged particles, which prevents aggregation of the particles due to electric repulsion. If the ZP is low, attraction overcomes repulsion, and it is likely that the mixture forms coagulates [45].)

7. Please provide the XRD patterns of the standard cards mentioned in the manuscript.

• Many thanks for your suggestion, Done, and Figure 5 was updated.
• Figure 5. XRD patterns of green synthesized ZnONPs by using BP and DS extracts in comparison to JCPDS, card number (36-1451). Both patterns show sharp peaks at the same 2 Theta.

8. Why is the molecular vibration mode of ZnO so complex in FTIR? Did the author separate and purify ZnO in the product? What is the effective bactericidal substance in the bacteriostatic performance test?

• ZnONPs due to their green synthesis have biochemical groups that are found on their surfaces as capping and stabilization agents that are present in BPE and DSE extracts used in NPs synthesis.
• Bioactive molecules are mentioned in detail in the article.
• Moreover, the bactericidal potential of green synthesized ZnONPs may be due to bioactive molecules, from BP and DS extracts, adsorbed onto the NP surface. BP has high levels of pectin and phytochemicals (0.90-3.0 g/100 g dry weight) such as ascorbic acid, β-carotene, tocopherol, and gallocatechin [34,35].
• It was reported that DS contains polyphenols, flavonoids, alkaloids, tannins, and ster-oids that significantly retard microbial growth, proliferation, and infection [40]. Their antibacterial efficiency differs according to the type of date, organic solvent, target or-ganism, and plant parts used [41].

9. Please explain the antibacterial mechanism of ZnO, and add the comparison of the properties of this material with other antibacterial materials.

• Many thanks for your suggestion, Done. The mechanism was mentioned in detail within the text and Figure 7 was added as a schematic illustration of the suggested mechanism (suggested by reviewer 2).
• Figure 7. Schematic illustration of the suggested mechanism of the bactericidal potential of green synthesized ZnONPs. Created in BioRender.com.

10. Please provide visual evidence of the bactericidal effect of two ZnONPs on different bacteria, such as comparison of colonies before and after antibacterial.

• Many thanks for your suggestion.
• A supplementary file was attached showing photos of plates for different bacterial strains treated with ZnONPs (using BP and DS extracts) and the output table values of MIC. This file was cited within the text.

11. Biomass assisted synthesis is an interesting topic. Many researches have been done in this field. Some typical references are suggested to be cited, e.g. Journal of Bioresources and Bioproducts 2021, 6 (1), 75-81; Journal of Bioresources and Bioproducts 2021, 6 (4), 323-337.

• Many thanks for your suggestion.
• Therefore, the use of renewable biomass as the raw material for NPs synthesis has been considered a low-cost and sustainable option that could help in solving local and global pollution [43]. For example, rice husks (or rice hulls) contain high amounts of cellulose, hemicellulose, and lignin used to synthesize SiO₂ [44]_, nanoporous MnO₂ [45], and Mg₂SiO₄ [46] nanomaterials that have various applications such as biomedical fields [43], used as electrode material for high-performance supercapacitors, and as a green additive for sustainable concrete production [47]. In addition, sugar cane bagasse, bamboo leaf, eggshell, walnut shell, wheat straw, coconut shell, banana peel, tea waste, and other miscellaneous agricultural wastes are abundantly available that could be used in various NPs syntheses [43]. Finally, biomass-derived nano synthesis opened a novel window to innovate new routes in clean, eco-friendly, and large-scale synthesis of thoroughly purified, morphologically well-defined nanoparticles [43].
• [43] Zamani, A.; Marjani, A.; Mousavi, Agricultural waste biomass-assisted nanostructures: Synthesis and application. Green Processing and Synthesis. 2019, 8(1), 421-429. https://doi.org/10.1515/gps-2019-0010
• [44] Ghorbani, M.; Biparva, P.; Hosseinzadeh, S. Effect of colloidal silica nanoparticles extracted from agricultural waste on physical, mechanical and antifungal properties of wood polymer composite. J. Wood Prod., 2018, 76, 749-757.
• [45] Yuan, C.; Lin, H.; Lu, H.; Xing, E.; Zhang, Y.; Xie, B. Synthesis of hierarchically porous MnO₂/rice husks derived carbon composite as high-performance electrode material for supercapacitors. Energy, 2016, 178, 260-268.
• [46] Mathur, L.; Hossain, S.K.S.; Majhi, M.R.; Roy, P.K. Synthesis of nano-crystalline forsterite (Mg₂SiO₄) powder from biomass rice husk silica by solid-state route. Soc. Esp. Ceram. V., 2018, 57, 112-118.
• [47] Lim, J.L.G.; Raman, S.N.; Lai, F.C.; Zain, M.F.M.; Hamid, R., Synthesis of nano cementitious additives from agricultural wastes for the production of sustainable concrete. Clean Prod., 2018, 171, 1150-1160.

Reviewer 2 Report

 In this study, zinc oxide nanoparticles (were synthesized using extract in eco-friendly procedure. The results were characterized by related analysis of SEM, TEM, Zeta potential analysis, XRD, and FTIR spectroscopy. The antimicrobial potency of compounds was evaluated using broth microdilution assay against pathogenic strains to determine the minimum inhibitory concentration and minimum bactericidal concentration. In my opinion, authors suggest facile synthesis route for preparation of material with high performance, and can be published after focus on novelty aspects of the study.

1-The distinction of this study compared to previous studies should be highlighted in the abstract.

2-In introduction section, some of sentence are very conventional. Please check and rewrite this sentences. For example:

“Nanoparticles (NPs) are very tiny particles that their size ranges from 1 to 100 nm. NPs have large surface area in comparsion to their volume that gives them a unique phys-ical, chemical, and biological properties”

“Zinc oxide nanoparticles (ZnONPs) induce excess reactive oxygen species (ROS) within the cell such as superoxide anion (O2•–), hydrogen peroxide (H2O2), and hydroxyl radicals (•OH)”

And……………………….

3- In experimental section, the purity and Mw of reagents were missed.

4-Quality of Figures 3 and 4 is very low.

5-The characterization XRD patterns should be done by X-pert high score soft ware

6-Schematic results of antibacterial test should be added in manuscript.

7-What are the originality and the important scientific progress in this manuscript? And some related literatures were suggested to be cited for broader reader, such as:

A) doi: 10.1021/acs.jmedchem.2c01674, B) doi: 10.1128/spectrum.02311-22, C) doi: 10.1111/1462-2920.15940, D) doi: 10.2147/IJN.S397298 and E) doi: 10.1039/d2gc02467e

8-English writing need to improve by native person

English writting need to polish by native person.

Author Response

Dear Editor and Reviewers,

Thank you for your letter and for the reviewers’ comments concerning our manuscript. Those comments are all valuable and helpful in revising and improving our manuscript, as well as providing significant direction for our research. We carefully considered the comments and made changes that we hope will be approved. The revised sections of the document are track changed. The following are the main corrections in the manuscript and responses to the reviewers’ comments.

#2 Comments and Suggestions for Authors

 In this study, zinc oxide nanoparticles (were synthesized using extract in eco-friendly procedure. The results were characterized by related analysis of SEM, TEM, Zeta potential analysis, XRD, and FTIR spectroscopy. The antimicrobial potency of compounds was evaluated using broth microdilution assay against pathogenic strains to determine the minimum inhibitory concentration and minimum bactericidal concentration. In my opinion, authors suggest facile synthesis route for preparation of material with high performance, and can be published after focus on novelty aspects of the study.

1-The distinction of this study compared to previous studies should be highlighted in the abstract.

• Done “The present results are consistent with previous studies that have reported the antimicrobial potential of green ZnONPs through ROS induction that in turn damage microbial DNA.”

2-In introduction section, some of sentence are very conventional. Please check and rewrite this sentences. For example:

• “Nanoparticles (NPs) are very tiny particles that their size ranges from 1 to 100 nm. NPs have large surface area in comparsion to their volume that gives them a unique phys-ical, chemical, and biological properties”

• “Zinc oxide nanoparticles (ZnONPs) induce excess reactive oxygen species (ROS) within the cell such as superoxide anion (O2•–), hydrogen peroxide (H2O2), and hydroxyl radicals (•OH)”

• And……………………….

3- In experimental section, the purity and Mw of reagents were missed.

• (Zn(C₂H₃O₂)₂.2H₂O) (100% Grade, Mwt: 219.5 g/mol, Sigma-Aldrich, Saint-Louis, MO, USA).

4-Quality of Figures 3 and 4 is very low.

• High-resolution of Figures 3 and 4 are attached.

5-The characterization XRD patterns should be done by X-pert high score soft ware.

• Many thanks for your suggestion.
• I have used an alternative software “OriginLab” and Figure 5 was updated by adding JCPDS, card number (36-1451) as a reference for comparison.
• Figure 5. XRD patterns of green synthesized ZnONPs by using BP and DS extracts in comparison to JCPDS, card number (36-1451). Both patterns show sharp peaks at the same 2 Theta.

6-Schematic results of antibacterial test should be added in manuscript.

• Figure 7. Schematic illustration of the suggested mechanism of the bactericidal potential of green synthesized ZnONPs. Created in BioRender.com.

7-What are the originality and the important scientific progress in this manuscript? And some related literatures were suggested to be cited for broader reader, such as:

10. A) doi: 10.1021/acs.jmedchem.2c01674, B) doi: 10.1128/spectrum.02311-22, C) doi: 10.1111/1462-2920.15940, D) doi: 10.2147/IJN.S397298 and E) doi: 10.1039/d2gc02467e

• Zinc nanoparticles have strong bactericidal capabilities against both gram-positive and gram-negative bacteria [32,33]. ZnONPs release zinc ions that impair the cell membrane function by causing ion concentration differences, which block material transport. In which, zinc oxide (ZnO) that is positively charged directly binds to the negatively charged bacterial cell walls that in turn denature proteins by reacting with functional groups. In addition, ZnO induces a large number of free radicals (OH−, H₂O₂, and O²⁻) under UV or visible light. Those free radicals destroy the bacterial cell wall, enter the cell, damage DNA, and finally lead to bacterial death [30].
• [31] Devanand Venkatasubbu, G.; Ramakrishnan, V.; Kumar J. Nanocrystalline hydroxyapatite and zinc-doped hydroxyapatite as carrier material for controlled delivery of ciprofloxacin. 3 Biotech. 2011, 1(3), 173–186.
• [32] Wang, Y.W.; Cao, A.; Jiang, Y, et al.Superior antibacterial activity of zinc oxide/graphene oxide composites originating from high zinc concentration localized around bacteria. ACS Appl Mater Interfaces. 2014, 6(4), 2791–2798.
• [33] Li, Y.; Xia, X.; Hou, W.; Lv, H.; Liu, J.; Li, X. How Effective are Metal Nanotherapeutic Platforms Against Bacterial Infections? A Comprehensive Review of Literature. Int J Nanomedicine, 2023, 18, 1109–1128. https://doi.org/10.2147/IJN.S397298.

8-English writing need to improve by native person

• The whole manuscript has been checked.

Reviewer 3 Report

1.     Title: The use of "green synthesized" could be replaced with "biosynthesized" or "biogenic" for clarity.

2.     Abstract: Significance and novelty of the work could be highlighted.

3.     Introduction: introduction should provide a clear reason for why the study was conducted (research gap) and why it is important

4.     Introduction: Suggestion is for the author to include more information on mechanism of antimicrobial activity of the zinc oxide nanoparticles.

5.     Result and discussion: Line 278, missing citation for statement “Previous studies have reported…”

Author Response

Dear Editor and Reviewers,

Thank you for your letter and for the reviewers’ comments concerning our manuscript. Those comments are all valuable and helpful in revising and improving our manuscript, as well as providing significant direction for our research. We carefully considered the comments and made changes that we hope will be approved. The revised sections of the document are track changed. The following are the main corrections in the manuscript and responses to the reviewers’ comments.

#3 Comments and Suggestions for Authors

1. Title: The use of "green synthesized" could be replaced with "biosynthesized" or "biogenic" for clarity.

• Antimicrobial potential of biosynthesized zinc oxide nanoparticles using banana peel and date seeds extracts

2. Abstract: Significance and novelty of the work could be highlighted.

• Consequently, the present results support the use of different biowastes in NPs synthesis, which is a simple way, sustainable, helps in waste management, and decreases environmental pollution.

3. Introduction: introduction should provide a clear reason for why the study was conducted (research gap) and why it is important.

• Done

4. Introduction: Suggestion is for the author to include more information on mechanism of antimicrobial activity of the zinc oxide nanoparticles.

• Briefly mentioned in the introduction section and more detailed referred in the results and discussion section with a schematic illustration of the suggested mechanism of the bactericidal potential of green synthesized ZnONPs was added.

5. Result and discussion: Line 278, missing citation for statement “Previous studies have reported…”

• Previous studies have reported that the bactericidal activity of ZnONPs could be due to their ability to perforate the microbial cell membrane to produce ROS that damage microbial DNA [30].

Reviewer 4 Report

Overall comment:

In the work, the author synthesized zinc oxide nanoparticles (ZnONPs) by using banana peel (BPE) and date seeds (DSE) extracts reaction with Zinc acetate, denoted as ZnONPs_BPE and ZnONPs_DSE. The ZnONPs_BPE and ZnONPs_DSE were confirmed by using an ultraviolet-visible spectrophotometer (UV-VIS), Scanning (SEM), transmission electron mi-10 croscope (TEM), Zeta potential analysis, X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy. Besides, the antimicrobial potency of the ZnONPs_BPE and ZnONPs_DSE was evaluated using broth microdilution assay against pathogenic strains to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The biosynthesized ZnONPs demonstrated a good antimicrobial potential against gram-positive (Staphylococcus aureus, and Bacillus subtilis) and gram-negative (Escherichia coli, Salmonella enteritidis) strains. Especially, ZnONPs-BPE has a higher antimicrobial effect against Salmonella enteritidis (MIC= 0.75 mg/ml, MBC= 1.5 mg/ml), while ZnONP _DSE has a higher effect against Staphylococcus aureus with (MIC= 0.75 mg/ml, MBC= 3 mg/ml).

The paper shows an interesting story with a green method for ZnO nanoparticle synthesis. The ZnO possesses great promise for its antimicrobial properties. 

Author Response

#4

The paper shows an interesting story with a green method for ZnO nanoparticle synthesis. The ZnO possesses great promise for its antimicrobial properties. 

• Many thanks for your comment and recommendation.

Reviewer 5 Report

This manuscript reported a green method for synthesizing ZnO NPs with different extracts., which was interesting. There are several points need to be addressed for improving this work.

1) It was mentioned in the introduction part that the other co-friendly extracts were already employed for harvesting ZnO NPs. What's their difference and disadvantages?

2) What's the roles of these two extracts and what's the difference? like pH? or other chemicals difference in the extract? The manuscript didn't give convinced evidence to support the synthesis mechanism and how these extracts affect the product of ZnO NPs.

3) The UV-Vis spectra look weird as both peaks suddenly dropped at 300nm. Need to re-measure it. Otherwise, it can't conclude the UV-Vis spectrum of ZnO NPs_BPE peaks at 300nm, which could be even smaller than 300nm.

4)The author mentioned the negativity charges may lead to more aggregation of NPs and give a larger size distribution. Is there any literature to support this hypothesis? It seems that the zeta potential distribution was based on the product rather than intermediate during the synthesis. The post-aggregation after synthesis can't change their sizes at all, but the nuclei kinetics and growth speed will be.

5) how about the FTIR spectra of both extracts? It would be better for comparison if provided except the FTIR spectra of products.

Some sentences were confused. Should be revised and check their spelling.

Author Response

Dear Editor and Reviewers,

Thank you for your letter and for the reviewers’ comments concerning our manuscript. Those comments are all valuable and helpful in revising and improving our manuscript, as well as providing significant direction for our research. We carefully considered the comments and made changes that we hope will be approved. The revised sections of the document are track changed. The following are the main corrections in the manuscript and responses to the reviewers’ comments.

#5 Comments and Suggestions for Authors

This manuscript reported a green method for synthesizing ZnO NPs with different extracts., which was interesting. There are several points need to be addressed for improving this work.

1) It was mentioned in the introduction part that the other co-friendly extracts were already employed for harvesting ZnO NPs. What's their difference and disadvantages?

• The use of the most abundant biomass, banana peels (BP) and date seeds (DS), in NPs synthesis represents a low-cost, renewable, and eco-friendly route that could help in solving the pollution problem. In addition, biomass could be used in large-scale production of various NPS. This route is a good alternative to NPs synthesis instead of physicochemical methods that are more expensive, need more instruments, and can generate toxic and hazardous effects in the environment.

2) What's the roles of these two extracts and what's the difference? like pH? or other chemicals difference in the extract? The manuscript didn't give convinced evidence to support the synthesis mechanism and how these extracts affect the product of ZnO NPs.

• Done

3) The UV-Vis spectra look weird as both peaks suddenly dropped at 300nm. Need to re-measure it. Otherwise, it can't conclude the UV-Vis spectrum of ZnO NPs_BPE peaks at 300nm, which could be even smaller than 300nm.

• Many thanks for your comment. Done, and data was updated.

4)The author mentioned the negativity charges may lead to more aggregation of NPs and give a larger size distribution. Is there any literature to support this hypothesis? It seems that the zeta potential distribution was based on the product rather than intermediate during the synthesis. The post-aggregation after synthesis can't change their sizes at all, but the nuclei kinetics and growth speed will be.

• Many thanks for your comment but I wrote this explanation according to the below references. If there is any other explanation, I will be grateful if you mention its reference to me and I will update it.
• Mukherjee, D. Chowdhury, R. Kotcherlakota et al., “Potential theranostics application of bio-synthesized silver nanoparticles (4-in-1 system),” Theranostics, vol. 4, no. 3, pp. 316–335, 2014. “Previously, it was reported that a high negative potential value increases nanoparticles’ repulsion force that in turn helps in their dispersal, stability, and good colloidal nature [39].”
• Punamjyoti Das, Malay K. Das, in Nanocosmeceuticals, 2022. Production and physicochemical characterization of nanocosmeceuticals. “When the particles in the suspension have a high negative or positive zeta potential, then they display an inclination to repel in order that no flocculation takes place and shows resistance to aggregation.”
• Shabnam Samimi, ... Farid Dorkoosh, in Characterization and Biology of Nanomaterials for Drug Delivery, 2019 “Lipid-Based Nanoparticles for Drug Delivery Systems” (High ZP implies highly charged particles, which prevents aggregation of the particles due to electric repulsion. If the ZP is low, attraction overcomes repulsion, and it is likely that the mixture forms coagulates [45].)

5) how about the FTIR spectra of both extracts? It would be better for comparison if provided except the FTIR spectra of products.

• Many thanks for your suggestion, I will try to do that in our next work.

Comments on the Quality of English Language

Some sentences were confused. Should be revised and check their spelling.

• The whole manuscript has been checked.

Round 2

Reviewer 1 Report

The manuscript could be accepted now.

Reviewer 2 Report

The final version is acceptable.