Pontastacus leptodactylus (Eschscholtz, 1823) and Faxonius limosus (Rafinesque, 1817) as New, Alternative Sources of Chitin and Chitosan

Round 1

Reviewer 1 Report

The submitted manuscript is perceived as a proper report of competently conducted research. However, two key factors are not included in the report. One is the gross composition of the isolated chitins. In particular, the protein and inorganic matter content of the chitin samples obtained would provide relevant elements to discuss most of the reported observations. Another is the determination of the degree of deacetylation (DD) of the chitins and chitosans produced. This parameter is recognized as fundamental to explain the functional properties of these polysaccharides. The authors acknowledge this in the Introduction and Discussion sections. It could be estimated from the FTIR measurements and considered in the discussion of the observed results.

Please also take in account the following suggestions:

Lines 90-92. Some questions about the described procedure. What is the water to crayfish mass ratio used to boil them? Why are boiled several hours? What is the reason of changing the boiling water five times?

Section 2.2.3. Please specify what you use to heat the reaction vessel. Also, verify that 100 mL of water where used to neutralize 20 mL of NaOH 60%

Section 2.3. Has any kind of software correction been applied to the FTIR spectra?

Section 2.7.2. The description of the experimental conditions for the determination of MIC and MBC is in need of some clarification. First, please specify if a particular reference is used as guide for this procedure. The choice of solvent seems odd, DMSO is not a good solvent for chitosan. This could affect the dispersion of the chitosan in the culture medium; also, since DMSO is an aprotic solvent, it cannot generate the polycationic charges in the chitosan that are closely related to its antimicrobial effect. Another observation is the use of “extract(s)” regarding the chitosan along this section

Section 2.9. Please list the packages and their versions used for R.

Section 3.1. Consider the use of ‘yield’ or ‘recovery’ instead of ‘content’ in this section

Table 1. The same applies as in the previous point. That is, the chitin content in one species of crayfish does not vary, what changes is the amount of chitin isolated by the different processes. Also, a respectful suggestion is to include the treatments as a column in the table.

Lines 258-259. The presence of the band centered at 1414 cm^-1 is associated to proteins that remains in the chitin sample isolated from F. limosus. There is another band, centered at 874 cm^-1, not present in the chitin spectra that is not consistent with the presence of protein in the sample.

Line 444. Please provide the reference used or the criteria for the proposed molecular mass classification.

Section 5. The first conclusion that is stated is not in line with the experimental approach and the results that are reported. There are no elements to evaluate economic viability. .

Improve all the Figures. E.g. better resolution in graphs, bigger axis numbers fonts, translate Italian legends, etc.

 

Careful proofreading of the manuscript is advisable to improve redaction and correct some mistyping (e.g. lines 121, 126, 266, 338 and others)

Author Response

Dear Reviewer,

thank You for Your questions, suggestions, and comments. They were really helpful and contributed to the manuscript's improvement. 

Please see the response in the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Remarks:

Line 113: Are the authors sure that the demineralization was carried out before deproteination step?

Line 125: Extraction is the extraction of a substance from a solution or dry mixture using a solvent (extractant) that dissolves the substance. Here, the term "separation" should be used;

Line 184-185: Chitosan is insoluble in both DMSO and water. Please, clarify the method of chitosan dissolution;

Line 236: "...did not result in the complete chitin purification (isolation)";

Line 242: Please, clarify that are the b-NADES, c-NADES?

Lines 351-352: number (M_n) and weight (M_w) average molecular weight. D_m (molecular dispersity) should be used instead of PDI following IUPAC;

Fig.7: Please, explain the bimodal molecular weight distribution of chitosan, molecular weight calculation  and a high molecular dispersity of chitosan samples;

Line 409: from about to 26%;

Discussion: Please, use MW (molecular weight) instead of M_w (weight average molecular weight).

Conclusion: manuscript requires a minor revision.

Minor editing of English language required

Author Response

Dear Reviewer,

thank You for Your questions, suggestions, and comments. They were really helpful and contributed to the manuscript's improvement. 

Please see the response in the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

The only thing I disagree with is using indigenous species for such experiments. Although I understand that you aimed to compare species as the potential sources of chitin and chitosan, you performed that very well. Nevertheless. There are many more invasive species with even potentially higher biomass, such as signal crayfish (Poland), red-swamp crayfish (Italy, Spain), marbled crayfish.

In all other respects, the idea of this study is very interesting and well-realized, so I gladly recommend this manuscript for publication after some minor revisions.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

thank You for Your questions, suggestions, and comments. They were really helpful and contributed to the manuscript's improvement. 

Please see the response in the attachment

Author Response File: Author Response.pdf

Reviewer 4 Report

I have read the manuscript. I have a few questions and suggestions.
1. In the "Introduction" section, it is necessary to consider methods for obtaining chitin and, accordingly, chitosan, from the shells of hydrobionts.
2. In section 2.2.4, please describe the "standard" method for obtaining chitin.
3. Please explain why heading 2.2.4 is called "Preliminary NADES chitin extraction"?
4. In Table 1, include the chitosan content from chitin and shells for A. leptodactylus and F. limosus. Give a statistical scatter of data or it was a single experiment? Compare the received data. Discuss the relevance of the chosen method.
5. In Figures 1 and 2, show the FTIR for NADES.
6. In Figure 7, please show the spectrum of the reference sample.
7. What was the ash content of the samples? Provide data.
8. In section 2.2.1, please indicate the conditions for demineralization.
How much ash was before and after demineralization?
9. What is the purity of the obtained samples of chitin and chitosan?
10. In what areas do the authors plan to apply their products? It must be indicated in the conclusion.

Author Response

Dear Reviewer,

thank You for Your questions, suggestions, and comments. They were really helpful and contributed to the manuscript's improvement. 

Please see the response in the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

It is possible to estimate the DA with the FTIR spectra already included in the manuscript. I believe that there is no need for additional analysis to obtain a suitable calculation. This parameter will provide a necessary dimension for a deeper discussion of the observed results. However, it is up to you.

Author Response

Dear Reviewer,
thank You for your time spent on our work, and all valuable comments and questions in each round. Please see our response uploaded in the attachment

Author Response File: Author Response.pdf

Reviewer 4 Report

I have read the revision manuscript. The authors partially answered my questions. Some questions require additional work and answers.
1: The authors in their manuscript propose a new source of chitin. Comparison with a reference sample is necessary to confirm that new sources of chitin provide a quality product. Please provide figure 7 of the spectra of the reference sample.
2: Ash is a key indicator of the purity of the resulting product. Without knowledge of this indicator, it is impossible to judge the purity and characteristics of chitins obtained by the authors of the article. What was the ash content of the samples? How much ash was before and after demineralization?
Authors should complete the experiment and provide updated data. Authors should compare the data with the literature.
3: Where do the authors plan to use their products? This should be stated in the Conclusion. Otherwise, what was the purpose of the work and why was all this done?

Author Response

Dear Reviewer,

thank You for your time spent on our work, and all valuable comments and questions in each round. Please see our answers below:

Point 1: In Figure 7, please show the spectrum of the reference sample.

Response 1: The Authors would like to thank the Reviewer for the suggeston. Figure 7 shows the size exclusion chromatograms of both chitosan samples. Unlike adsorption chromatographies, like GC and HPLC, or even TLC, in size exclusion chromatography (SEC) molecules are eluted by volume. Therefore, the standards one uses in this technique are polymers of known molecular weight, not necessarily with the same structure of the analyzed samples. In order to get the closest possible relation between molecular weight and molecular volume, the structure of the standards should be as close as possible to the one of the sample. But, normally, a polymer of the exact same structure cannot be used, because it is extremely difficult to get low molecular dispersity (Dm) polymers that can be used as standards in SEC. This is patent in the elution profiles of the PSS standards used, some of which present several peaks, and the nominal MW (supplied by the manufacturer) used for the calculations is that of the major component.
Therefore, for example, for neutral polysaccharides, normally pullulan standards are used for calibration. But for charged polysaccharides, a standard also charged is better. That is why we used polystyrene sulfonate for the calibration curve. This curve is a representation of LOG Mw versus elution volume for a number of standard samples (please see the attached PDF file). In this case, standards between 208 Da and 33500 Da (peak mass) were used. From this representation, the fit equation y = 0.03172 x^2 - 1.66403 x + 24.137, with x = elution volume, was derived. These fitting equations are seldom linear, being normally second (as in this case) or third grade.
For sample analysis, the software divides the peak in slices and calculates the MWs for each one from the equation, and then the MWs (Mw, Mn, Mz, Mv) for the whole sample.

 

Point 2: What was the ash content of the samples? What is the purity of the obtained samples of chitin and chitosan?

Response 2: The Authors agree with the reviewer concerning the importance of ash and especially on purity of chitin and chitosan but the aim of this preliminary investigation is to understand the suitability of a green alternative method for the extraction of chitin (and chitosan) from these two species and for this reason, only qualitative analyses have been carried out. Further studies are in progress including the quantitative analysis.

 

Point 3: In what areas do the authors plan to apply their products? It must be indicated in the conclusion.

Authors should do additional research and compare their data with the literature.

Response 3: As reported in the answer above, this is only a preliminary work thus without a specific area of application. Additional research (not object of this work) is in progress and the final aim of our studies on these species is the application on circular economy since we would recycle chitosan from waste.

Author Response File: Author Response.pdf

Round 3

Reviewer 4 Report

I have read the revision manuscript. The authors did not answer fundamental questions.
The authors were unable to prove that they obtained chitosan that can be used for cosmetics, medicine or food. Without data on ash, spectra, in comparison with the reference sample, it is impossible to draw a conclusion about applicability.
I repeat my questions:
1: In Figure 7, please show the spectrum of the reference sample.
2: What was the ash content of the samples? provide data.
How much ash was before and after demineralization?
What is the purity of the obtained samples of chitin and chitosan?
3: In what areas do the authors plan to apply their products? It must be indicated in the conclusion.
Authors should do additional research and compare their data with the literature.

Author Response

Dear Reviewer,

thank You for your time spent on our work, and all valuable comments and questions in each round. Please see our answers below:

Point 1: In Figure 7, please show the spectrum of the reference sample.

Response 1: The Authors would like to thank the Reviewer for the suggestion. Figure 7 shows the size exclusion chromatograms of both chitosan samples. Unlike adsorption chromatographies, like GC and HPLC, or even TLC, in size exclusion chromatography (SEC) molecules are eluted by volume. Therefore, the standards one uses in this technique are polymers of known molecular weight, not necessarily with the same structure of the analyzed samples. In order to get the closest possible relation between molecular weight and molecular volume, the structure of the standards should be as close as possible to the one of the sample. But, normally, a polymer of the exact same structure cannot be used, because it is extremely difficult to get low molecular dispersity (Dm) polymers that can be used as standards in SEC. This is patent in the elution profiles of the PSS standards used, some of which present several peaks, and the nominal MW (supplied by the manufacturer) used for the calculations is that of the major component.
Therefore, for example, for neutral polysaccharides, normally pullulan standards are used for calibration. But for charged polysaccharides, a standard also charged is better. That is why we used polystyrene sulfonate for the calibration curve. This curve is a representation of LOG Mw versus elution volume for a number of standard samples (please see the attached PDF file). In this case, standards between 208 Da and 33500 Da (peak mass) were used. From this representation, the fit equation y = 0.03172 x^2 - 1.66403 x + 24.137, with x = elution volume, was derived. These fitting equations are seldom linear, being normally second (as in this case) or third grade.
For sample analysis, the software divides the peak in slices and calculates the MWs for each one from the equation, and then the MWs (Mw, Mn, Mz, Mv) for the whole sample.

 

Point 2: What was the ash content of the samples? What is the purity of the obtained samples of chitin and chitosan?

Response 2: The Authors agree with the reviewer concerning the importance of ash and especially on the purity of chitin and chitosan but the aim of this preliminary investigation is to understand the suitability of a green alternative method for the extraction of chitin (and chitosan) from these two species and for this reason, only qualitative analyses have been carried out. Further studies are in progress including the quantitative analysis.

 

Point 3: In what areas do the authors plan to apply their products? It must be indicated in the conclusion.

Authors should do additional research and compare their data with the literature.

Response 3: As reported in the answer above, this is only a preliminary work thus without a specific area of application. Additional research (not the object of this work) is in progress and the final aim of our studies on these species is the application on the circular economy since we would recycle chitosan from waste.

Author Response File: Author Response.pdf