Recovery and Characterization of Calcium-Rich Mineral Powders Obtained from Fish and Shrimp Waste: A Smart Valorization of Waste to Treasure

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1- line: 54 ( mati et al. 2016) Please replace by number

2- line 128: please add the amount of samples that were added to HCL (w:v)

3-line 141: How to measure the total protein

4- why add different working solutions in the determination of ABTS

4- the protein, ash, fat, and moisture, what about carbohydrates?

5- We suggest adding a table containing the amount of proximate in the present study and previous study, also the methods and solvents of extractions

6- Water soluble index (WSI) , how calculated please add to material and methods

7- In EDX analysis, please add a table containing the weight % of metals contents of all samples

8-line 466 peak intensities or weight % please clear

8- what is the difference between EDX and ICp-OES, and why were heavy metals detected by ICP-OES, and not detected by EDX

9-the amount of heavy metals differs according to the location of fish, so please add the traditional zone, where the fishing occurred

 

 

 

 

 

Author Response

Response to the comments of the Reviewer 1

 We greatly appreciate to the Reviewer for evaluating our manuscript. On the following text, we respond to the Reviewer’s comments point by point. The changes in the revised manuscript have been marked in red color. All line numbers mentioned in the responses to comments refer to the numbers that appear on the right margin of the text of the revised manuscript.

 

Comment 1: Line: 54 (mati et al. 2016) Please replace by number.

Response: We apologize for the mistake. We have corrected in the revised manuscript (Page: 2; Line: 54).

 

Comment 2: Line 128: please add the amount of samples that were added to HCL (w:v)

Response: We added 1 g sample and stated in the revised manuscript (Page: 3, line: 137).

 

Comment 3: Line 141: How to measure the total protein?

Response: The total protein content was measured according to lowery method as we mentioned in our manuscript (Page: 4; Line: 147-148).

 

Comment 4: Why add different working solutions in the determination of ABTS?

Response: We appreciate the query of the reviewer. Different concentrations of ABTS solution were added to evaluate the ABTS radical scavenging activity of the samples in a dose dependent manner. It helps in determining the antioxidant capacity of a sample by evaluating how the antioxidant activity changes with varying concentrations of the antioxidant. It is crucial for accurately determining, comparing, and understanding the antioxidant capacities at different concentrations. It provides a detailed profile of how antioxidants perform across a range of concentrations, which is valuable for both scientific research and practical applications.

 

Comment 5: The protein, ash, fat, and moisture, what about carbohydrates?

Response: The remaining amount, after deducting the protein, ash, fat, and moisture, is considered as carbohydrate. The carbohydrate content in fish bone is generally considered not important bioactive and is often ignored by many researchers. For reference, please see the articles:

https://doi.org/10.1016/j.apt.2020.04.015

https://doi.org/10.1080/10498850.2020.1775742

https://doi.org/10.1080/10498850.2018.1432733

DOI: 10.7763/IJBBB.2013.V3.219

 

Comment 6: We suggest adding a table containing the amount of proximate in the present study and previous study, also the methods and solvents of extractions.

Response: Thank you very much for the suggestion. Actually, our research team is preparing a review manuscript focusing on the proximate composition, including nutritional values, biofunctional properties, and applications of fish bone powders. Therefore, we are unable to provide such a table in the manuscript.

 

Comment 7: Water soluble index (WSI), how calculated please add to material and methods.

Response: Thank you for the suggestion. We have added the statements and Eq. for calculating WSI (page: 4, line: 1162-164).

 

Comment 8: In EDX analysis, please add a table containing the weight % of metals contents of all samples

Response: We appreciate the reviewer for the suggestion. We provided the values of minerals in the text, and we are concerned that adding a table containing these values would duplicate the results (Page: 12-13; Lines: 456-458, 468-469, 478-481).

 

Comment 9: Line 466 peak intensities or weight % please clear.

Response: We enlarged the figure and increased the resolution for improved visibility.

 

Comment 10: What is the difference between EDX and ICP-OES, and why were heavy metals detected by ICP-OES, and not detected by EDX?

Response: Thank you for the query. EDX detects characteristic X-rays emitted from a sample and analyzes the surface layer of the sample. EDX has relatively lower sensitivity for detecting trace elements whereas ICP-OES involves ionizing the sample with an inductively coupled plasma and then detecting the emitted light from the ions. The wavelengths of this emitted light are characteristic of specific elements. ICP-OES analyzes the entire sample volume, providing a comprehensive overview of the elemental composition and has much higher sensitivity for trace elements or heavy metals. If the heavy metals are not concentrated on the surface or are present in deeper layers or in very low concentrations, they might not be detected by EDX. That’s why heavy metals were detected by ICP-OES not by EDX.

 

Comment 10: The amount of heavy metals differs according to the location of fish, so please add the traditional zone, where the fishing occurred.

Response: We absolutely agree with the reviewer’s comment. The sources of fish and shrimp are provided in the revised manuscript (Page: 3; Lines: 105-107).

 

Reviewer 2 Report

Comments and Suggestions for Authors

today aquaculture pushes research to make progress in raising awareness about the environment and the quality of fish. all the procedures performed to obtain an optimal result are clearly highlighted in the manuscript.

the tests carried out are in line with global standard procedures and highlight the perfectly adequate regulatory framework for a qualified product.

From what has been described, the chemical-physical and microbiological tests were carried out meticulously and safeguarding the characteristics of the compounds used, with particular attention also to the protein and amino acid characteristics.

in my subjective opinion, before transferring the research to the human context, in the future, I recommend proceeding with research on the animal model, in order to carefully and in detail examine the effect of this waste reduced to powder, in a living organism. this helps highlight the benefit obtained from these products on an animal's physiological performance.

Author Response

Response to the comments of the Reviewer 2

 We greatly appreciate to the Reviewer for evaluating our manuscript. On the following text, we respond to the Reviewer’s comments point by point. The changes in the revised manuscript have been marked in red color. All line numbers mentioned in the responses to comments refer to the numbers that appear on the right margin of the text of the revised manuscript.

 

Comments: Today aquaculture pushes research to make progress in raising awareness about the environment and the quality of fish. All the procedures performed to obtain an optimal result are clearly highlighted in the manuscript. The tests carried out are in line with global standard procedures and highlight the perfectly adequate regulatory framework for a qualified product.

From what has been described, the chemical-physical and microbiological tests were carried out meticulously and safeguarding the characteristics of the compounds used, with particular attention also to the protein and amino acid characteristics.

In my subjective opinion, before transferring the research to the human context, in the future, I recommend proceeding with research on the animal model, in order to carefully and in detail examine the effect of this waste reduced to powder, in a living organism. This helps highlight the benefit obtained from these products on an animal's physiological performance.

 

Response to the comments: Thank you very much for taking the time and effort to evaluate our manuscript. We also agree that the products should be tested in animal model studies before being applied to human food. We have recommended for the animal model study in the conclusion section (Page: 17; Line: 591-592).

Reviewer 3 Report

Comments and Suggestions for Authors

The study addresses the critical issue of managing aquaculture waste by developing a sustainable valorization approach for calcium-rich powders from mackerel tuna and pangas bones and shrimp shells. Various physicochemical and nutritional parameters were characterized, including proximate composition, amino acids, protein solubility, WHC, OHC, heavy metal contents, and structural examinations using FE-SEM, EDX, and FT-IR. Significant findings include high protein content in shrimp shell powder (37.78%) and notable ABTS free radical scavenging activity, suggesting potential health benefits. Overall, this study presents valuable insights into the sustainable use of aquatic biomass waste with practical applications in food and pharmaceuticals. The manuscript is suitable for the journal, but some revisions are needed. See my other comments below.

The manuscript could benefit from mentioning any study limitations and suggesting future research directions (not just in the Conclusions).

The mentioned physicochemical characteristics are not parallel, with some being parameters and others being equipment.

The authors present the Results and Discussion from nine aspects, but there is a lack of integration between results from different aspects.

In subsection 3.1, the proximate composition presented in Figure 1 is over-discussed. This subsection should be revised to be more concise, especially considering the insignificant differences in the proximate composition between treatments.

In the main text, when introducing the results, showing just the means is sufficient. To make the reading smoother, it’s unnecessary to show the standard deviation.

Line 14, “A single With” needs revision.

The full name of ABTS should be given.

Lines 62-64 need references to support the statement.

Line 77, “by-products provides” needs revision.

Lines 78-79, “studies”, but only one reference is cited.

Lines 163-173, the exact heavy metals that were measured should be listed.

Line 232, “Bubel et al.” should be deleted.

In Figure 2, the numerical values on the vertical and horizontal coordinates should be integers.

Comments on the Quality of English Language

The language is acceptable.

Author Response

Response to the comments of the Reviewer 3

 

We greatly appreciate to the Reviewer for evaluating our manuscript. On the following text, we respond to the Reviewer’s comments point by point. The changes in the revised manuscript have been marked in red color. All line numbers mentioned in the responses to comments refer to the numbers that appear on the right margin of the text of the revised manuscript.

 

Comment 1: The study addresses the critical issue of managing aquaculture waste by developing a sustainable valorization approach for calcium-rich powders from mackerel tuna and pangas bones and shrimp shells. Various physicochemical and nutritional parameters were characterized, including proximate composition, amino acids, protein solubility, WHC, OHC, heavy metal contents, and structural examinations using FE-SEM, EDX, and FT-IR. Significant findings include high protein content in shrimp shell powder (37.78%) and notable ABTS free radical scavenging activity, suggesting potential health benefits. Overall, this study presents valuable insights into the sustainable use of aquatic biomass waste with practical applications in food and pharmaceuticals. The manuscript is suitable for the journal, but some revisions are needed. See my other comments below.

Response: We cordially thank the reviewers for their valuable efforts and comments to improve the manuscript.

 

Comment 2: The manuscript could benefit from mentioning any study limitations and suggesting future research directions.

Response: The study limitations and future research suggestions are provided in the conclusion sections according to the comments of the reviewer (Page: ; Line: )..

 

Comment 3: The mentioned physicochemical characteristics are not parallel, with some being parameters and others being equipment.

Response: We appreciate the reviewer for the comment. The physicochemical characteristics are written homogenously in the revised manuscript (Page: 17; Line: 589-593).

 

Comment 4: The authors present the Results and Discussion from nine aspects, but there is a lack of integration between results from different aspects.

Response: Thank you very much for the query. Actually, there are some deviations in the materials and methods, as well as in the results and discussions, to facilitate easier and more convenient understanding for the authors.

 

Comment 5: In subsection 3.1, the proximate composition presented in Figure 1 is over-discussed. This subsection should be revised to be more concise, especially considering the insignificant differences in the proximate composition between treatments.

Response: Thank you very much for the opinion. We have deleted the statements for insignificant differences in the proximate composition.

 

Comment 6: In the main text, when introducing the results, showing just the means is sufficient. To make the reading smoother, it’s unnecessary to show the standard deviation.

Response: We have corrected throughout the manuscript.

 

Comment 7: Line 14, “A single With” needs revision.

Response: Thank you for your comment. We have revised the sentence and made the correction (Page: 1; Line: 14).

“The increasing in global aquaculture production has made management of waste from aquatic biomass a significant concern.”

 

Comment 8: The full name of ABTS should be given.

Response: The full name of ABTS is provided in the revised manuscript (Page: ; Line: ).

 

Comment 9: Lines 62-64 need references to support the statement.

Response: Many thanks for the comment. We have provided reference in the revised manuscript (Page: 5; Line: 216).

 

Comment 10: Line 77, “by-products provides” needs revision.

Response: Correction has been done in the revised manuscript (Page: 2; Line: 79).

 

Comment 11: Lines 78-79, “studies”, but only one reference is cited.

Response: Corrected in the revised manuscript (Page: 2; Line: 80).

 

Comment 12: Lines 163-173, the exact heavy metals that were measured should be listed.

Response: The list of the heavy metals analyzed is provided in the revised manuscript (Page: 4; Lines: 185-186).

 

Comment 13: Line 232, “Bubel et al.” should be deleted.

Response: Bubel et al has been removed in the revised manuscript (Page: 5; Line: 244).

 

Comment 14: In Figure 2, the numerical values on the vertical and horizontal coordinates should be integers.

Response: Figure 2 is modified according to the reviewer’s comment.

 

Reviewer 4 Report

Comments and Suggestions for Authors

Reusing waste to create valuable products has recently been a significant concern of many researchers. Therefore, this study should be considered for publication in Sustainability. However, there are issues that the authors need to improve before the manuscript is considered for the next steps. Here are the points:

- The most critical issue this manuscript has not addressed is the application of fish bone meal and shrimp shell powder. Therefore, the authors are advised to include the necessary information in the Introduction section.

- The authors are asked to revise the title: "Calcium-rich" should be used in place of "Calcium rich". The phrase “A Smart Conversion of Waste to Treasure” does not clearly convey the content of the study. Therefore, the title should be replaced with a more appropriate one.

- Line 14: Is “A single” a mistake?

- The methods in section 2.2 need to be described more specifically.

- Lines 241-249: The authors discussed the impact of alkaline; how is this related to the findings in this study?

- Figures 2&3 need to be changed from line charts to column charts. Additionally, the error bars need to be added.

- Line 349-351: How are these contents linked to the results of this study?

- Line 497: "my current study" should be replaced with the appropriate phrase.

- Figure 7: FT-IR results are not really good. The authors are recommended replacing it with better results. In addition, the spectrum of TBP has peaks in the region of 2300 cm-1. The authors should also analyze it.

- The legend of Figure 8 needs additional information. For example, what is "nd"? What are the characters a, b, c? What do the error bars represent?

- The antioxidant results of fish bone and shrimp shell powders are too primary. It does not show whether the antioxidant activity of the powders is good or not and where this activity comes from.

- Lines 555-556: The authors are asked to consider the statement in this sentence. To my knowledge, fish bone powder does not contain significant amounts of chitin or chitosan.

- There are multiple outdated references in this manuscript. The use of outdated references detracts from the research's relevance. Thus, authors are kindly asked to include newer references, if possible.

 

Author Response

Response to the comments of the Reviewer 4

 

We greatly appreciate to the Reviewer for evaluating our manuscript. On the following text, we respond to the Reviewer’s comments point by point. The changes in the revised manuscript have been marked in red color. All line numbers mentioned in the responses to comments refer to the numbers that appear on the right margin of the text of the revised manuscript.

 

Comment 1: Reusing waste to create valuable products has recently been a significant concern of many researchers. Therefore, this study should be considered for publication in Sustainability. However, there are issues that the authors need to improve before the manuscript is considered for the next steps.

Response: We cordially appreciate the reviewer for the valuable time and efforts to review our manuscript.

 

Comment 2: The most critical issue this manuscript has not addressed is the application of fish bone meal and shrimp shell powder. Therefore, the authors are advised to include the necessary information in the Introduction section.

Response: We appreciate the suggestions. The applicability of fish bones and shrimp shell powders are addressed in the introduction section (Page: 2; Lines: 66-68).

 

Comment 3: The authors are asked to revise the title: "Calcium-rich" should be used in place of "Calcium rich". The phrase “A Smart Conversion of Waste to Treasure” does not clearly convey the content of the study. Therefore, the title should be replaced with a more appropriate one.

Response: Many thanks for the comment. The title is modified according to the suggestion of the reviewer.

 

Comment 4:  Line 14: Is “A single” a mistake?

Response: Yes, it was a mistake and corrected in the revised manuscript (Page: 1; Line: 14).

 

Comment 5: The methods in section 2.2 need to be described more specifically.

Response: We appreciate the comment. This section is specifically described (Page: 3; Lines: 122-127).

Comment 6:  Lines 241-249: The authors discussed the impact of alkaline; how is this related to the findings in this study?

Response: We appreciate the reviewer for the important query. The samples were prepared with 0.2% alkaline treatment in this study. Alkaline causes reduction on the protein content of fish bone /bone powder. That’s why the impact of alkaline has been discussed in this study.

 

Comment 7: Figures 2&3 need to be changed from line charts to column charts. Additionally, the error bars need to be added.

Response: We appreciate the comment and have converted Figure 3 to column charts. However, we modified Figure 2 based on the comments from the other reviewers.

 

Comment 8:  Line 349-351: How are these contents linked to the results of this study?

Response: We apologize for the mistake. We have corrected in the revised manuscript (Page: 9; Line: 356).

 

Comment 9:   Line 497: "my current study" should be replaced with the appropriate phrase.

Response: Thank you for the comment. We have replaced the phrase with “The present study” (Page: 14; Line: 489).

 

Comment 10:  Figure 7: FT-IR results are not really good. The authors are recommended replacing it with better results. In addition, the spectrum of TBP has peaks in the region of 2300 cm-1. The authors should also analyze it.

Response: Thank you for the comment. We compared the obtained FTIR peaks with those in reference articles, and they show a similar pattern. The peak at around 2300 cm⁻¹ in TBP typically signifies the presence of carbon dioxide (CO₂) or other similar molecules in the sample.  

https://doi.org/10.1016/j.apt.2020.04.015

https://doi.org/ 10.15578/squalen.601

 

Comment 11: The legend of Figure 8 needs additional information. For example, what is "nd"? What are the characters a, b, c? What do the error bars represent?

Response: We appreciate the comment. “nd” represent not detected in the sample and we have added in the figure caption. The characters a, b, c and error bars indicate the significant difference (P≤ 0.05) for each group of heavy metals and it is provided in the figure caption. Error bars denote the standard deviation/ standard error of the data, providing a visual representation of the variability or uncertainty in the measurements.

 

Comment 12:  The antioxidant results of fish bone and shrimp shell powders are too primary. It does not show whether the antioxidant activity of the powders is good or not and where this activity comes from.

Response: The results show comparative ABTS free radical scavenging activity of fish bone and shrimp shell powders. The antioxidant activity was good, as the ABTS working solution (OD value: 1.1 at 734 nm) decreased in a dose dependent manner with the sample solution of the identified amount. Peptides derived from collagen and other proteins found in fish bones also demonstrate antioxidant properties by scavenging free radicals. Additionally, shrimp shell powder contains chitin and chitosan, polysaccharides with antioxidant attributes capable of scavenging free radicals (Page: 16; Lines: 564-568).

 

Comment 13:  Lines 555-556: The authors are asked to consider the statement in this sentence. To my knowledge, fish bone powder does not contain significant amounts of chitin or chitosan.

Response: We appreciate the comment. We agree with the reviewer and apologize for the unintentional mistakes. We have corrected the statement as:

“Additionally, shrimp shell powder contains chitin and chitosan, polysaccharides with antioxidant attributes capable of scavenging free radicals”

 

Comment 14:  There are multiple outdated references in this manuscript. The use of outdated references detracts from the research's relevance. Thus, authors are kindly asked to include newer references, if possible.

Response: Thank you very much for the important suggestion. We have searched carefully to include recent references and added (Page: 19; Line: 721-722).

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Accept in present form

Author Response

Reviewer 1, Comment 1# Accept in present form.

Response: The authors are grateful for your valuable time and efforts in reviewing and improving the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have considerately improved the manuscript, which is almost ready for acceptance. I encourage the author to further improve the writing. For example, in Line 79, add "a" before "previous study".

Comments on the Quality of English Language

Can be further improved.

Author Response

Reviewer 3, Comment 1: Is the content succinctly described and contextualized with respect to previous and present theoretical background and empirical research (if applicable) on the topic?

Response: We have discussed the relevant reports in the manuscript.

 

Reviewer 3, Comment 2: The authors have considerately improved the manuscript, which is almost ready for acceptance. I encourage the author to further improve the writing. For example, in Line 79, add "a" before "previous study".

Response: Thank you very much for the comment. We have thoroughly checked the manuscript and make corrections in the revised version.

 

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript has been improved.

Author Response

Reviewer 4, Comment 1# The manuscript has been improved.

Response: The authors are grateful for your valuable time and efforts in reviewing and improving the quality of the manuscript.