Indoor Space Disinfection Effect and Bioactive Components of Chamaecyparis obtusa Essential Oil

Round 1

Reviewer 1 Report

In the manuscript titled "Space disinfection effect and bioactive components of Chamaecyparis obtusa essential oil", the authors identified the bioactive compounds and analyzed the antibacterial effect of three lots of C. obtusa essential oil through a space disinfection test. The manuscript is interesting and well written. However, I have comments for the authors to address before the paper is recommended for publication.

1. Line 21: Insert the abbreviated name of the author of the species and the botanical family: Chamaecyparis obtusa (Siebold & Zucc.) Endl. (Cupressaceae).

2. Line 30: Replace keywords with others that do not appear in the title of the manuscript to increase the possibility of the article being found in databases after publication.

3. Line 40: Hydrodistillation is also an important essential oil extraction method. Please, include in the text.

4. Lines 60-61: Put the species names Citrus limon and Abies alba in italics.

5. Line 82: What was the part of the plant used and the method of extracting the essential oil from Chamaecyparis obtusa? It is important to provide these details.

6. Line 95 (2.2. Space disinfectant preparation and space disinfectant test): Apparently, the authors were not based on a methodology previously described in the literature to perform the spatial disinfection test using essential oil. Is this methodology adopted by the authors really effective? Are the authors proposing a new methodology for spatial disinfection?

7. Line 107: The number of bacterial cells was counted using which method? Please, make this clear in the text.

8. Lines 115-120: The authors reported in the text the major compounds identified in the 3 lots of the essential oil of Chamaecyparis obtusa. However, in Table 1, the authors only showed data from lot 1. I suggest including data from the GC/MS analysis of lots 2 and 3. These data can also be included as appendices.

9. Lines 124-136: The discussion of the results is very concise. I suggest that the authors seek more data in the literature on the antimicrobial effect of the major compounds identified in the essential oil of Chamaecyparis obtusa.

10. Are there studies in the literature on the investigation of the toxicity of the essential oil of Chamaecyparis obtusa? Toxicological studies are important to validate the biological safety of natural or synthetic products.

11. For future studies, I suggest that the authors identify the species of bacteria collected in environments sprayed with essential oil.

Minor editing of English language required.

Author Response

Response to Reviewer 1’s Comments

In the manuscript titled "Space disinfection effect and bioactive components of Chamaecyparis obtusa essential oil", the authors identified the bioactive compounds and analyzed the antibacterial effect of three lots of C. obtusa essential oil through a space disinfection test. The manuscript is interesting and well written. However, I have comments for the authors to address before the paper is recommended for publication.

Author response: First of all, we appreciate the reviewer's in-depth advice. The answers to the points made by the reviewer were answered one by one. Modified parts are marked in red color.

Q1. Line 21: Insert the abbreviated name of the author of the species and the botanical family: Chamaecyparis obtusa (Siebold & Zucc.) Endl. (Cupressaceae).

Author response #1: Thank you for the appreciation. The abbreviated name pointed out by the reviewer have been corrected in abstract section 

Q2. Line 30: Replace keywords with others that do not appear in the title of the manuscript to increase the possibility of the article being found in databases after publication.

Author response #2: Thank you for the appreciation. The inserted the typo ‘indoor disinfection’ in keywords 

Q3. Line 40: Hydrodistillation is also an important essential oil extraction method. Please, include in the text.

Author response #3: Thank you for the appreciation. The inserted the typo ‘Hydrodistillation’ in introduction

Q4. Lines 60-61: Put the species names Citrus limon and Abies alba in italics.

Author response #4: Thank you for the appreciation. we changed the species names in italics (line 72, 73, introduction) 

Q5. Line 82: What was the part of the plant used and the method of extracting the essential oil from Chamaecyparis obtusa? It is important to provide these details.

Author response #5: Thank you for the appreciation. We used cypress leaves, and additionally described cypress leaves in the method section 2.1(line94). 

Q6. Line 95 (2.2. Space disinfectant preparation and space disinfectant test): Apparently, the authors were not based on a methodology previously described in the literature to perform the spatial disinfection test using essential oil. Is this methodology adopted by the authors really effective? Are the authors proposing a new methodology for spatial disinfection? 

Author response #6: We appreciate the reviewer's in-depth advice. We could not obtain a lot of empirical data on disinfectant tests of essential oil. In the previous reports, antibacterial efficacy in airtight hoods or hospital rooms has been reported, so it was described in the introduction section, and we reported the results by applying essential oil components to a space that fits our reality.

In the actual field, the space is used after a few minutes to several hours after treating the disinfectant agent. Therefore, as described in the method section, after treatment with PO, falling bacteria not removed by PO series were measured for 8 hours to get results under conditions similar to reality. we presented experimental conditions suitable for our reality to evaluate space disinfection. It is very simple, but the antimicrobial effect of natural essential oils can be easily reproduced in the field, and it is described so that other researchers can comfortably handle candidates in the laboratory.

Q7. Line 107: The number of bacterial cells was counted using which method? Please, make this clear in the text.

Author response #7: Thank you for the appreciation. We have described the contents of line 107 in detail(line122, section2.2).

Q8. Lines 115-120: The authors reported in the text the major compounds identified in the 3 lots of the essential oil of Chamaecyparis obtusa. However, in Table 1, the authors only showed data from lot 1. I suggest including data from the GC/MS analysis of lots 2 and 3. These data can also be included as appendices.

Author response #8: Thank you for the appreciation. As the reviewer 1 pointed, we showed lot 1 data as representative data for GCMS analysis. Lot 2 and 3 also showed similar results. Since the manufacturing date of each sources is different, there are slight differences in some components, however the main components are similar. Therefore, in the results and discussion section, common ingredients are presented as analytic markers for quality control.

Q9. Lines 124-136: The discussion of the results is very concise. I suggest that the authors seek more data in the literature on the antimicrobial effect of the major compounds identified in the essential oil of Chamaecyparis obtusa.

Author response #9: Thanks for the reviewer's comments. We choose some substances from GCMS data of cypress oil as biomarker, and single substance with known antimicrobial efficacy have already been described in section 3.1(line139-150). There were no data describing specific antimicrobial efficacy evaluations for other active substances. Perhaps because individual components of cypress are difficult to obtain commercially, it seems that the antimicrobial susceptibility of all candidate components has not been studied.

Q10. Are there studies in the literature on the investigation of the toxicity of the essential oil of Chamaecyparis obtusa? Toxicological studies are important to validate the biological safety of natural or synthetic products.

Author response #10: Thanks for the reviewer's comments. one study have been reported on the effect of cypress essential oil on the human body. It has been reported that inhaling cypress essential oil for 5 minutes in 16 healthy adults showed a positive effect on the control of sympathetic nervous system dysfunction.

Considering the results of our results, 0.25L of PO100 used in the space control experiment was sprayed in a space of 60 to 90 m² for 15 minutes, and the actual amount of essential oil sprayed was about 2.5mL. Therefore, it was thought that there would be no harmful effect on the human body.

Q11. For future studies, I suggest that the authors identify the species of bacteria collected in environments sprayed with essential oil.

Author response #11: Thanks for the reviewer's comments. It was thought that a study on the analysis of controlled microorganisms would be necessary in the future, so it was described as a future study in section 3.2. (line188)

Author Response File: Author Response.pdf

Reviewer 2 Report

Please provide one pragraph that explain the following statement "The disinfectants should not only be effective in reducing the microbess present in the air but should also not be toxic for humans, allowing their continuous application" Please search statistical relevant literature that explain the consequence use of toxic disinfectant results in mortality rate or fatality rate or other.

Please explain more in one paragraph about "The antimicrobial effects of Essential oils are explained by their composition and cytotoxic effects, which cause cell membrane damage"

Please provide more reference and citation on the following statement ". The potential use of Melaleuca alternifolia (Tea tree) oil as a disinfectant has been shown in previous report for control bacteria due to terpinen- 46 4-ol (35–45%) and 1,8-cineole (1–6%). What about its role as insecticide? Is there any relationship between disinfectant and insecticide?

Please provide empirical model used in current experiment. Illustrate your experiment.  "Therefore, empirical experiments on the space control effect of various essential oil components are needed, and the need to  present the basis for the development of various natural disinfectants based on empirical data is increasing"

Please include heading of instrumentation and provide the list with source.

Please include list of chemicals and provide the list with source.

Please provide evidence of purchase of test material supplied from Earthmate Jungamjin co (Jangheung, Korea). Provide reference number.  Provide the purity percentage from source.

It is mandatory to give rational and logical on your following statement "Test samples for Space disinfectant test (HINOPHY PO) were supplied by Earthmate  Jungamjin co.kr (Jangheung, Korea) by diluting LOT3 100 times, 500 times, and 1000 times 97 (PO100, PO500, and PO1000). " 

Provide visual illustration on your experiment "visualization of falling bacterial strain" Please illustrate your specification and provide source of your experimentation methodology. If it is your own new method then explain the protocol.

Illustrate the chemicals list identified using GCMS and draw the structure using chemdraw. 

Please include three heading such as study limitation, generalization of findings, future directions.

How did you validate the findings? Provide experimentation or statistical test. 

According to author "There have been no studies on the control of disinfectants containing essential oils in real spaces" Please revisit the statement and check available literature and compare findings with your results. 

Is there other study that uses Space disinfectant test? If this is the first experiment and your team develop the test then please explain and illustrate more the protocol. This is a good test. I appreciate it.

The manuscript is good and after correction can published. I recommend the manuscript. It is a quality paper.

Author Response

Response to Reviewer 2’s Comments

First of all, we appreciate the reviewer's in-depth advice.

The answers to the points made by the reviewer were answered one by one.

Modified parts are marked in red color.

Q1. Please provide one paragraph that explain the following statement "The disinfectants should not only be effective in reducing the microbes present in the air but should also not be toxic for humans, allowing their continuous application" Please search statistical relevant literature that explain the consequence use of toxic disinfectant results in mortality rate or fatality rate or other.

Please explain more in one paragraph about "The antimicrobial effects of Essential oils are explained by their composition and cytotoxic effects, which cause cell membrane damage" 

Author response #1: We appreciate the reviewer's in-depth advice. The toxicity-related review literature pointed out is attached, and the antibacterial mechanism and use of low-toxic natural materials are also added in introduction section.

Q2. Please provide more reference and citation on the following statement ". The potential use of Melaleuca alternifolia (Tea tree) oil as a disinfectant has been shown in previous report for control bacteria due to terpinen- 46 4-ol (35–45%) and 1,8-cineole (1–6%). What about its role as insecticide? Is there any relationship between disinfectant and insecticide?

Author response #2: Thanks for the reviewer's comments. Terpinene and cineol, components of Melaleuca alternifolia essential oil, are antibacterial-related components, and reference has been added. We did not handle the relationship with insecticides.

Q3. Please provide empirical model used in current experiment. Illustrate your experiment.  "Therefore, empirical experiments on the space control effect of various essential oil components are needed, and the need to present the basis for the development of various natural disinfectants based on empirical data is increasing"

Author response #3: We appreciate the reviewer's in-depth advice. We could not obtain a lot of empirical data on disinfectant tests of essential oil. In the previous reports, antibacterial efficacy in airtight hoods or hospital rooms has been reported, so it was described in the introduction section, and we reported the results by applying essential oil components to a space that fits our reality.

Q4. Please include heading of instrumentation and provide the list with source. Please include list of chemicals and provide the list with source. Please provide evidence of purchase of test material supplied from Earthmate Jungamjin co (Jangheung, Korea). Provide reference number.  Provide the purity percentage from source. It is mandatory to give rational and logical on your following statement "Test samples for Space disinfectant test (HINOPHY PO) were supplied by Earthmate  Jungamjin co.kr (Jangheung, Korea) by diluting LOT3 100 times, 500 times, and 1000 times 97 (PO100, PO500, and PO1000). " 

Author response #4: Thanks for the reviewer's comments. As pointed out by the reviewer, information about devices, column, medium, and consumables are inserted in sections 2.1 and 2.2. Also, the description of PO100, 500 and 1000 has been rewritten. PO100, 500, and 1000 were provided free of charge, and there is no official documentation. We apologize for the lack of official documentation.

Q5. Provide visual illustration on your experiment "visualization of falling bacterial strain" Please illustrate your specification and provide source of your experimentation methodology. If it is your own new method then explain the protocol. Illustrate the chemicals list identified using GCMS and draw the structure using chemdraw. 

Author response #5: Thanks for the reviewer's comments. There was no specific methodology for fallout. We assumed that the airborne bacteria were free-falling and collected the falling bacteria by placing a solid medium in the test space as described in M&M. The analysis results for GCMS are shown in Table 1. Since it is difficult to show all the structures of all identified substances, the structures of three main markers have been added to Fig. 3.

Q6. Please include three heading such as study limitation, generalization of findings, future directions.

How did you validate the findings? Provide experimentation or statistical test. 

Author response #6: Thank you for the reviewer's thoughtful comments. We deeply sympathize with the reviewer's point.

We thought that there would be a lot of variation in the types and numbers of microbes in the spaces that people actually use. In the case of PO100, statistical significance was shown in all three types of space. For PO500, R2 showed real statistical significance.

Therefore, what we want to emphasize through Fig2 is that companies should produce PO100 in its most desirable product form. Based on statistical confidence, actual products were produced with PO100.

During preparation of our manuscript, we debated a lot about whether to show the data for PO1000 to readers, however in the case of PO1000, we attached the data to get the reliability of product distributors.

In addition, the expression airbone microorganism simply means something floating in the air. It is thought that the volatile and non-volatile components of PO kill airborne microorganisms, but which fraction actually reduces airborne microorganisms requires future research, so it is described in section 3.2.

Q7. According to author "There have been no studies on the control of disinfectants containing essential oils in real spaces" Please revisit the statement and check available literature and compare findings with your results. 

Is there other study that uses Space disinfectant test? If this is the first experiment and your team develop the test then please explain and illustrate more the protocol. This is a good test. I appreciate it

Author response #7: Thank you for the reviewer's thoughtful comments. In the sentence above, C. obtusa was missing. Therefore, the following changes were made.

 (~ disinfectants containing C. obtusa essential oils ~ )

In our study, we presented experimental conditions suitable for our reality to evaluate space disinfection. It is very simple, but the antimicrobial effect of natural essential oils can be easily reproduced in the field, and it is described so that other researchers can comfortably handle candidates in the laboratory.

Author Response File: Author Response.pdf

Reviewer 3 Report

Suggestion: table 1 is too big, it should present only the compounds of interest.

The authors should write a short description of the kinds of bacteria in indoor environments.

 The sedimentation method does not permit exact quantitative determination. To demonstrate the disinfection efficiency of this oil extract, the number of experiments should increase in order to have a good statistical significance.    A good idea is to use air samplers.

Minor spelling mistakes like: voletile . What is the meaning of “the space control effect” and IW leaf”?’  “Space should be changed in ‘’indoor space’’ for more clarity. The ref 1 style should be the same as other references. Reference 12 is the same whit 14.

Suggestion: table 1 is too big, it should present only the compounds of interest.

The authors should write a short description of the kinds of bacteria in indoor environments.

 The sedimentation method does not permit exact quantitative determination. To demonstrate the disinfection efficiency of this oil extract, the number of experiments should increase in order to have a good statistical significance.    A good idea is to use air samplers.

Author Response

Response to Reviewer 3’s Comments

First of all, we appreciate the reviewer's in-depth advice.

The answers to the points made by the reviewer were answered one by one.

Modified parts are marked in red color.

Q1 Suggestion: table 1 is too big, it should present only the compounds of interest.

Author response #1: Thank you for the reviewer's thoughtful comments.  We also strongly agree with the length of Table 2 pointed out by the reviewer. As pointed out by the reviewer, quality, mol weight, and Cas number were deleted for visual convenience.

Q2. The authors should write a short description of the kinds of bacteria in indoor environments.

Author response #2: Thank you for the reviewer's thoughtful comments. We described the information of airborne microorganisms in introduction section with red color

Q3. The sedimentation method does not permit exact quantitative determination. To demonstrate the disinfection efficiency of this oil extract, the number of experiments should increase in order to have a good statistical significance. A good idea is to use air samplers.

Author response #3: Thank you for the reviewer's thoughtful comments. We fully agree with the reviewer's proposed use of an air sampler.

If an air sampler is used, it is thought that there will be an advantage in obtaining additional fine dust and total carbon in the air.

However, we did not use an air sampler in this experiment. In the actual field, the space is used after a few minutes to several hours after treating the disinfectant agent. Therefore, as described in the method section, after treatment with PO, falling bacteria not removed by PO series were measured for 8 hours to get results under conditions similar to reality. we presented experimental conditions suitable for our reality to evaluate space disinfection. It is very simple, but the antimicrobial effect of natural essential oils can be easily reproduced in the field, and it is described so that other researchers can comfortably handle candidates in the laboratory.

Q4. Minor spelling mistakes like: voletile . What is the meaning of “the space control effect” and IW leaf”?’  

“Space should be changed in ‘’indoor space’’ for more clarity. The ref 1 style should be the same as other references. Reference 12 is the same whit 14.

Author response #4: Thank you for the appreciation. The typos pointed out by the reviewer have been corrected as follows.

Voletile -> volatile (Introduction section)

IW leaf -> Lot 1 ~ 3 (GCMS method section)

Space -> indoor (Tiltle, Introduction, title of 3.2 section, conclusion)

References have also been rearranged.

Q5. Suggestion: table 1 is too big, it should present only the compounds of interest.

Author response #5: Thanks for the reviewer's comments. We also strongly agree with the length of Table 1 pointed out by the reviewer. As pointed out by the reviewer, quality, mol weight, and Cas number were deleted for visual convenience. Since it is difficult to show all the structures of all identified substances, the structures of three main markers have been added to Fig. 3(new).

Author Response File: Author Response.pdf

Reviewer 4 Report

Space disinfection effect and bioactive components of Chamaecyparis obtusa essential oil

1.     What is the whole word of “PO”?

2.     What are the “actual conditions”? If Petri dishes were exposed to an open-air environment, then how much are the room temperature, humidity, and other relevant environmental conditions?

3.     In the abstract, the statement “In a previous report, we reported that volatile and non-volatile substances have excellent antimicrobial activities against Gram-negative, Gram-positive, and antibiotic-resistant strains.”, does this mean this study also introduced the same type of bacteria for the test in the open air? If so then the type of bacteria must be specified in the manuscript. Otherwise, I think this study is nothing related to the previous anti-bacterial study because there is no knowledge about the kind of bacteria falling on Petri dishes.

4.     It seems that the authors only provided examples of GC-MS analysis of essential oil from C. obtusa in LOT 1. How about LOT 2 and 3?

5.     Table 1 is too lengthy to read. I strongly recommend authors to revise the format of Table 1. It may be better to present the data according to the intensity of the m/z ratio or percentages in the sample. The current bold-faced words are not a good presentation.

6.     Please specify the meaning of sample names such as CONR1, CORN2, etc in Fig. 2. Also, I notice that the standard deviations of some samples in Fig. 2 are way too large. For example, in samples R2 and CORN3 treated by PO500. Large standard deviations can cause large uncertainties and thus much less trustable hypothesis tests.

7.     Authors may need to provide their data as credential evidence to support their surveys.

8.     Please rewrite the conclusion and abstract. Conclusions must be specific. For example, “As a result of the analysis, it was found that common ingredients were identified for lots 1 ~ 3.” The common ingredients must be specified in the conclusion. Also in the abstract, “In addition, we firstly report space disinfectant effects of products containing C. obtusa essential oil (PO100, PO500, PO1000).” The words “firstly” is somehow wired and confused, And “It was confirmed that PO100 and PO500 could effectively remove airborne microorganisms in space.” Does this mean the oil has a kind of automatic mechanism to take off bacteria from space? I think this is a test about the anti-bacterial effects of germs falling on dishes, right?

9.     Authors need to seek professional editing services to improve their writing. Misspelling words and grammatical errors have to be corrected.

Please see the above comment.

I think an editing service is needed for this manuscript.

Author Response

Response to Reviewer 4’s Comments

First of all, we appreciate the reviewer's in-depth advice.

The answers to the points made by the reviewer were answered one by one.

Modified parts are marked in red color.

Q1. What is the whole word of “PO”?

Author response #1: Thank you for the appreciation. PO is not an abbreviation. Earthmate Jungamjin, the supplier of this product, has assigned a product code on its own.

Q2. What are the “actual conditions”? If Petri dishes were exposed to an open-air environment, then how much are the room temperature, humidity, and other relevant environmental conditions?

Author response #2: Thank you for the reviewer's thoughtful comments. We only considered the collection of falling bacteria when the petri dish was left in the test space after sample processing. Indoor temperature and humidity could not be recorded electronically. Therefore, the word ‘actual conditions’ has been deleted in section 2.2. We hope the reviewer's understanding for the mistake on our part.

Q3. In the abstract, the statement “In a previous report, we reported that volatile and non-volatile substances have excellent antimicrobial activities against Gram-negative, Gram-positive, and antibiotic-resistant strains.”, does this mean this study also introduced the same type of bacteria for the test in the open air? If so then the type of bacteria must be specified in the manuscript. Otherwise, I think this study is nothing related to the previous anti-bacterial study because there is no knowledge about the kind of bacteria falling on Petri dishes.

Author response #3: Thank you for the reviewer's thoughtful comments.  Since this study is an real/field study of a product made based on the original source used in previous studies, it is stated that we previously reported the in-tube - antimicrobial effect of essential oil in the abstract to help readers easy understanding.

Q4. It seems that the authors only provided examples of GC-MS analysis of essential oil from C. obtusa in LOT 1. How about LOT 2 and 3?

Author response #4: Thank you for the appreciation. As the reviewer 1 pointed, we showed lot 1 data as representative data for GCMS analysis. Lot 2 and 3 also showed similar results. Since the manufacturing date of each sources is different, there are slight differences in some components, however the main components are similar. Therefore, in the results and discussion section, common ingredients are presented as analytic markers for quality control.

 Q5. Table 1 is too lengthy to read. I strongly recommend authors to revise the format of Table 1. It may be better to present the data according to the intensity of the m/z ratio or percentages in the sample. The current bold-faced words are not a good presentation.

Author response #5: Thank you for the reviewer's thoughtful comments.  We also strongly agree with the length of Table 1 pointed out by the reviewer. As pointed out by the reviewer, quality, mol weight, and Cas number were deleted for visual convenience.

Q6.  Please specify the meaning of sample names such as CONR1, CORN2, etc in Fig. 2. Also, I notice that the standard deviations of some samples in Fig. 2 are way too large. For example, in samples R2 and CORN3 treated by PO500. Large standard deviations can cause large uncertainties and thus much less trustable hypothesis tests.

Author response #6: Thank you for the reviewer's thoughtful comments. We deeply sympathize with the reviewer's point.

We thought that there would be a lot of variation in the types and numbers of microbes in the spaces that people actually use. In the case of PO100, statistical significance was shown in all three types of space. For PO500, R2 showed real statistical significance.

Therefore, what we want to emphasize through Fig2 is that companies should produce PO100 in its most desirable product form. Based on statistical confidence, actual products were produced with PO100.

During preparation of our manuscript, we debated a lot about whether to show the data for PO1000 to readers, however in the case of PO1000, we attached the data to get the reliability of product distributors.

Additionally, descriptions of CONR1, etc. have been added to the figure title.

Q7.  Authors may need to provide their data as credential evidence to support their surveys.

Author response #7: Thank you for your practical advice. Our study was limited to research for optimization of PO products as an actual test in a small space. Experiments for actual certification will have to be commissioned by the company to an accredited institution.

Q8.  Please rewrite the conclusion and abstract. Conclusions must be specific. For example, “As a result of the analysis, it was found that common ingredients were identified for lots 1 ~ 3.” The common ingredients must be specified in the conclusion. Also, in the abstract, “In addition, we firstly report space disinfectant effects of products containing C. obtusa essential oil (PO100, PO500, PO1000).” The words “firstly” are somehow wired and confused, and “It was confirmed that PO100 and PO500 could effectively remove airborne microorganisms in space.” Does this mean the oil has a kind of automatic mechanism to take off bacteria from space? I think this is a test about the anti-bacterial effects of germs falling on dishes, right?

 Author response #8: I agree with the reviewer's deep consideration. Names of common ingredients have been added to the conclusions section. And to reduce confusion, the word ‘first’ has been deleted. The expression airborne microorganism simply means something floating in the air. It is thought that the volatile and non-volatile components of PO kill airborne microorganisms, but which fraction actually reduces airborne microorganisms requires future research, so it is described in section 3.2.

Q9.  Authors need to seek professional editing services to improve their writing. Misspelling words and grammatical errors have to be corrected.

Author response #9: Thanks for the reviewer's comments. MDPI journals also recommend receiving editing services after acceptance for publication. If our MS is accepted after revision, we will request additional English editing services.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Thank you for the response. I read the answers and the authors responded to all the previously formulated observations. 

I agree with the publication of the article in its current form.

Author Response

Thank you for the full consideration of the reviewer

Reviewer 4 Report

Space disinfection effect and bioactive components of Chamaecyparis obtusa essential oil

1.     What is the whole word of “PO”?

Please explicitly indicate that

“PO is not an abbreviation. Earthmate Jungamjin, the supplier of this product, has assigned a product code on its own.”

2.     What are the “actual conditions”? If Petri dishes were exposed to an open-air environment, then how much are the room temperature, humidity, and other relevant environmental conditions?

Please explicitly indicate that

“We only considered the collection of falling bacteria when the petri dish was left in the test space after sample processing. Indoor temperature and humidity weren’t recorded.”

3.     In the abstract, the statement “In a previous report, we reported that volatile and non-volatile substances have excellent antimicrobial activities against Gram-negative, Gram-positive, and antibiotic-resistant strains.”, does this mean this study also introduced the same type of bacteria for the test in the open air? If so then the type of bacteria must be specified in the manuscript. Otherwise, I think this study is nothing related to the previous anti-bacterial study because there is no knowledge about the kind of bacteria falling on Petri dishes.

The issue is this statement only appears in the abstract. It is misleading information that this study is directly related to the anti-bacterial tests for all 3 types of micro-organisms. I think authors have to revise the statement to make it relevant to the current study.

4.     It seems that the authors only provided examples of GC-MS analysis of essential oil from C. obtusa in LOT 1. How about LOT 2 and 3?

Please explicitly indicate that

“Lot 2 and 3 also showed similar results. Since the manufacturing date of each source is different, there are slight differences in some components, however, the main components are similar.”

5.     Table 1 is too lengthy to read. I strongly recommend authors to revise the format of Table 1. It may be better to present the data according to the intensity of the m/z ratio or percentages in the sample. The current bold-faced words are not a good presentation.

The revision is a good one.

6.     Please specify the meaning of sample names such as CONR1, CORN2, etc. in Fig. 2. Also, I notice that the standard deviations of some samples in Fig. 2 are way too large. For example, in samples R2 and CORN3 treated by PO500. Large standard deviations can cause large uncertainties and thus much less trustable hypothesis tests. 

Please check the caption of Fig. 2:

…CONR1 ~ 3 means the state before PO treatment in the experimental space (R1 ~ 3), p-value, #<0.05 and ##<0.02.

What is the meaning of “state”? I think it should be “untreated samples”, right? 

Please provide the raw data of Fig. 2. The data can be put in the appendix to shorten this manuscript. I don’t think there are propriety issues if the data can be presented in a figure but data can’t be revealed. Please note that large variations in data are an issue we must face with. I’m not questioning the correctness of the data. 

7.     Authors may need to provide their data as credential evidence to support their surveys.  

As explained previously. 

8.     Please rewrite the conclusion and abstract. Conclusions must be specific. For example, “As a result of the analysis, it was found that common ingredients were identified for lots 1 ~ 3.” The common ingredients must be specified in the conclusion. Also in the abstract, “In addition, we firstly report space disinfectant effects of products containing C. obtusa essential oil (PO100, PO500, PO1000).” The words “firstly” is somehow wired and confused, And “It was confirmed that PO100 and PO500 could effectively remove airborne microorganisms in space.” Does this mean the oil has a kind of automatic mechanism to take off bacteria from space? I think this is a test about the anti-bacterial effects of germs falling on dishes, right?

9.     Authors need to seek professional editing services to improve their writing. Misspelling words and grammatical errors have to be corrected. 

I’m asking for rewriting the abstract and conclusion based on scientific logic and academic rigor. The English editing can be done afterward, but some correct statements must be provided first. Please don’t cherry-pick a few words to change. Since both the abstract and conclusion are short, it is not difficult to make the necessary revisions.

Please use “anti-bacterial” instead of “anti-infective”. Anti-infection means “ agents used to prevent or treat infection”. There are no infected living organisms involved in this study.

As pointed out in the last round of review, I strongly suggest authors use professional English editing services to improve the writings. 

Author Response

Response to Reviewer 4’s secondary Comments

First of all, we appreciate the reviewer's in-depth advice.

The answers to the points made by the reviewer were answered one by one.

Modified parts are marked in blue color.

1. What is the whole word of “PO”?

Please explicitly indicate that

“PO is not an abbreviation. Earthmate Jungamjin, the supplier of this product, has assigned a product code on its own.”

Author response #1: Thank you for the appreciation. in section 2.2, we added product code PO100-001, PO500-001, PO1000-001

2. What are the “actual conditions”? If Petri dishes were exposed to an open-air environment, then how much are the room temperature, humidity, and other relevant environmental conditions?

Please explicitly indicate that

“We only considered the collection of falling bacteria when the petri dish was left in the test space after sample processing. Indoor temperature and humidity weren’t recorded.”

Author response #2: Thank you for the appreciation. in section 2.2, we added that We added the wording the reviewer specified (blue color)

3. In the abstract, the statement “In a previous report, we reported that volatile and non-volatile substances have excellent antimicrobial activities against Gram-negative, Gram-positive, and antibiotic-resistant strains.”, does this mean this study also introduced the same type of bacteria for the test in the open air? If so then the type of bacteria must be specified in the manuscript. Otherwise, I think this study is nothing related to the previous anti-bacterial study because there is no knowledge about the kind of bacteria falling on Petri dishes.

The issue is this statement only appears in the abstract. It is misleading information that this study is directly related to the anti-bacterial tests for all 3 types of micro-organisms. I think authors have to revise the statement to make it relevant to the current study.

Author response #3: Thank you very much for the reviewer's advice. As a result of our careful review, it was determined that it would be desirable to delete the sentnces pointed out in the abstract. Therefore, problematic sentences in the abstract were deleted.

4. It seems that the authors only provided examples of GC-MS analysis of essential oil from C. obtusa in LOT 1. How about LOT 2 and 3?

Please explicitly indicate that

“Lot 2 and 3 also showed similar results. Since the manufacturing date of each source is different, there are slight differences in some components, however, the main components are similar.”

Author response #4: Thank you for the appreciation. in section 3.1, we added that We added the wording the reviewer specified (blue color)

5. Table 1 is too lengthy to read. I strongly recommend authors to revise the format of Table 1. It may be better to present the data according to the intensity of the m/z ratio or percentages in the sample. The current bold-faced words are not a good presentation.

The revision is a good one.

Author response #5: Thank you for the appreciation.

6. Please specify the meaning of sample names such as CONR1, CORN2, etc. in Fig. 2. Also, I notice that the standard deviations of some samples in Fig. 2 are way too large. For example, in samples R2 and CORN3 treated by PO500. Large standard deviations can cause large uncertainties and thus much less trustable hypothesis tests. 

Please check the caption of Fig. 2:

…CONR1 ~ 3 means the state before PO treatment in the experimental space (R1 ~ 3), p-value, #<0.05 and ##<0.02.

What is the meaning of “state”? I think it should be “untreated samples”, right? 

Please provide the raw data of Fig. 2. The data can be put in the appendix to shorten this manuscript. I don’t think there are propriety issues if the data can be presented in a figure but data can’t be revealed. Please note that large variations in data are an issue we must face with. I’m not questioning the correctness of the data. 

Author response #6: Thank you for the appreciation. we changed the figure title in detail

Figure 2. Inhibition of falling bacteria of PO100, PO500, PO1000 containing C. obtusa essential oil. CONR1 ~ 3 means the colonies of falling bacteria before not treated with PO series in the experimental space (R1 ~ 3), p value, #<0.05 and ##<0.02.

7. Authors may need to provide their data as credential evidence to support their surveys.  

As explained previously. 

Author response #7: Thank you for the appreciation. We attach the contract related to this study.

Under this agreement, we co-author Professor Min Suk Bae, an expert in the field of atmospheric environment research, to jointly review and conduct this study with us. please find attached file (response to reviewer 4-2nd)

8. Please rewrite the conclusion and abstract. Conclusions must be specific. For example, “As a result of the analysis, it was found that common ingredients were identified for lots 1 ~ 3.” The common ingredients must be specified in the conclusion. Also in the abstract, “In addition, we firstly report space disinfectant effects of products containing C. obtusa essential oil (PO100, PO500, PO1000).” The words “firstly” is somehow wired and confused, And “It was confirmed that PO100 and PO500 could effectively remove airborne microorganisms in space.” Does this mean the oil has a kind of automatic mechanism to take off bacteria from space? I think this is a test about the anti-bacterial effects of germs falling on dishes, right?

Author response #8: I agree with the reviewer's deep consideration. Names of common ingredients have been added to the conclusions section. And to reduce confusion, the word ‘first’ has been deleted. The expression airborne microorganism simply means something floating in the air. It is thought that the volatile and non-volatile components of PO kill airborne microorganisms, but which fraction actually reduces airborne microorganisms requires future research, so it is described in section 3.2.

Again, since there are various antibacterial substances in essential oil components, there is no exact mechanism for controlling microorganisms in space, so future research is needed.

9. Authors need to seek professional editing services to improve their writing. Misspelling words and grammatical errors have to be corrected. 

I’m asking for rewriting the abstract and conclusion based on scientific logic and academic rigor. The English editing can be done afterward, but some correct statements must be provided first. Please don’t cherry-pick a few words to change. Since both the abstract and conclusion are short, it is not difficult to make the necessary revisions. 

Please use “anti-bacterial” instead of “anti-infective”. Anti-infection means “ agents used to prevent or treat infection”. There are no infected living organisms involved in this study.

Author response #9: Thank you for your deep consideration.

MDPI journals also recommend receiving editing services after acceptance for publication. If our MS is accepted after revision, we will request additional English editing services.

We used the word anti-infective once in line 203. Therefore, this part was corrected with anti-bacterial.

Author Response File: Author Response.pdf