Properties and Characteristics of Acid-Soluble Collagen from Salmon Skin Defatted with the Aid of Ultrasonication

Round 1

Reviewer 1 Report

Overall the paper presented represents valuable information regarding properties of acid-soluble collagen from salmon skin defatted with the aid of ultrasonication. This is an interesting study and the authors have collected a valuable dataset using cutting-edge methodology. However, in my opinion, the paper has some shortcomings regarding the Introduction. In the Introduction, we can find very general information on the subject study, making it difficult to accept the work. What's the novelty of the study? Please underline this clearly in the Introduction. For that, you need a solid literature review. Among the areas that need improvement is work to provide better citations that will help place the current work in a better context of the scientific progress over the past 10+ years.
Additionally, better citations will help the authors to provide better and more accurate information. In 48 items, there are only two papers from 2021, one from 2020, and several from 2019-2018. It should be updated; otherwise, it would mean that the topic is not receiving much attention nowadays. For this reason, the authors have an incorrect number of known types of collagen (27). We now know more of them. Please also add the collagen isolation schemes in the experimental part using your methods.

Author Response

Response to reviewer 1

Comments and Suggestions for Authors

1. Overall the paper presented represents valuable information regarding properties of acid-soluble collagen from salmon skin defatted with the aid of ultrasonication. This is an interesting study and the authors have collected a valuable dataset using cutting-edge methodology. However, in my opinion, the paper has some shortcomings regarding the Introduction. In the Introduction, we can find very general information on the subject study, making it difficult to accept the work. What's the novelty of the study? Please underline this clearly in the Introduction. For that, you need a solid literature review. Among the areas that need improvement is work to provide better citations that will help place the current work in a better context of the scientific progress over the past 10+ years.

***Thank you very much for comments. Following the reviewer’s suggestion, the authors had provided the importance and novelty of the present work in “Introduction” in line 52 – 59. Authors would like clarify the novelty of our study as follows:

Point 1.

In the present study, the ultrasonication was used as the aid of defatting of salmon skin to enhance the efficiency for fat removal. Skin of salmon, which is a temperate fish, is rich in fat and the potential defatting of skin prior to collagen extraction is inevitably required. Based on the literature, the ultrasonication is mostly used for the aid of collagen extraction to increase the extraction yield from other raw materials, such as chicken cartilage, hide, fish skin, etc. 

1. Akram, A. N., & Zhang, C. (2020). Effect of ultrasonication on the yield, functional and physicochemical characteristics of collagen-II from chicken sternal cartilage. Food Chemistry, 307, 125544.
2. Wang, R., Wang, H., & Liu, P. (2020). Extraction of yak hide collagen by ultrasound-assisted lactic acid-pepsin hydrolosis method and its characterisation. Journal of the Society of Leather Technologists and Chemists, 104(3), 136-140.
3. Petcharat, T., Benjakul, S., Karnjanapratum, S., & Nalinanon, S. (2021). Ultrasound-assisted extraction of collagen from clown featherback (Chitala ornata) skin: yield and molecular characteristics. Journal of the Science of Food and Agriculture, 101(2), 648-658.
4. Schmidt, M. M., Vidal, A. R., Mello, R. O., Mazutti, M. A., Cansian, R. L., Dornelles, R. C. P., Demiate, I. M., & Kubota, E. H. (2021). Ultrasound as an alternative method to increase the extraction yield from chicken mecanically separated meatresidue collagen. Journal of Food Science and Technology, 58(7), 2487-2496.

            In summary, the novelty of this work is the use of ultrasonication for enhancement of defatting of salmon skin in order to reduce fat content before the extraction of collagen. This is stated in line 58-60. On the other hand, other works reported are mainly related with the use of ultrasonication to improve the extraction yield.

            As suggested by the reviewer, the scientific papers on fish collagen over the past 10 years have been gathered and reported in the manuscript.

Point 2

            Nowadays, acidic drinks like fruit juice fortified with native collagen are available in the market. However, no scientific report on its digestibility in gastrointestinal tract has been reported. Thus, another novelty of the present work is to investigate the hydrolysis of native collagen, especially that extracted from salmon skin defatted with the aid of ultrasonication, in simulated gastrointestinal tract. This is mentioned in line 60-62.

2. Additionally, better citations will help the authors to provide better and more accurate information. In 48 items, there are only two papers from 2021, one from 2020, and several from 2019-2018. It should be updated; otherwise, it would mean that the topic is not receiving much attention nowadays. For this reason, the authors have an incorrect number of known types of collagen (27). We now know more of them. Please also add the collagen isolation schemes in the experimental part using your methods.

***Thank you for insightful suggestions. Collagen is still one of natural products, which has gained interest for research and applications. The number of papers on fish collagen have been published during year 2017-2022. Therefore, additional relevant papers published from year 2017 to 2022 have been included. Please see in text as highlighted in yellow at line 33, 138, 160, 197 – 199, 215 – 217, 272 – 277, 296, 338, 341, 350, 354, 369, 389 and 393. Those papers are included in the list of references. Please see line 485 – 487, 510 – 512, 516 – 517, 530 – 537, 546 – 548, 557 – 559, 569 – 571 and 573 – 575.

            Authors have updated the number of known types of collagens from 27 to 29. The reference has been provided. Please see line 483 – 484.

            Additionally, the scheme of collagen extraction from salmon skin defatted with two different processes has been provided in the methodology as per the reviewer’s suggestion. Please see line 103 –117. Thank you very much for the valuable suggestion.

Reviewer 2 Report

1- Acid soluble collagen and ultrasonic defatting is not a novel work, previously many researchers reported on different products.

2- There are some typographical errors need to be fixed.

3- Discussion for "Fat content, Hydroxyproline content and Color" is not adequate, need more through discussion for these.

The more elaborated comments on the review are mentioned below.
Line 231-232 : the claim is not supported with the comparison of published work.
Line 233-235: Need refences for this statement.
Line 243-244: Need supportive discussion with reference.
Line 246-248: Comparison for these interpretation with published work.
Line 250-261: Discussion is lac of any published work companions.

Author Response

Response to reviewer 2

Comments and Suggestions for Authors

1. Acid soluble collagen and ultrasonic defatting is not a novel work, previously many researchers reported on different products.

*** The use of ultrasonication has been implemented widely to increase the extraction yield of several products. Thus, it is still the potential technique to be applied, including fat extraction or fat removal from raw materials. So far, no information concerning the use of ultrasonication for fat removal from fish skin, especially high fat skin such as salmon skin, has been documented. This is the new information for effective extraction of collagen from fish skin containing high fat.  Authors found that fat removal process with the aid of ultrasonication affected the resulting collagen in different fashions. For instance, ultrasonication could enhance the fat removal, but it decreased the extraction yield of resulting collagen. Nevertheless, fat removal with the aid of ultrasonication had no effect on thermal properties, protein pattern, FTIR spectra and triple helix structure of resulting collagen. The aforementioned scientific information can provide the up-to-date knowledge to the readers, in which the process could be applied properly.

2. There are some typographical errors need to be fixed.

***The typographical errors have been checked and corrected throughout the text using ‘Grammarly’ software.

3. Discussion for "Fat content, Hydroxyproline content and Color" is not adequate, need more through discussion for these. The more elaborated comments on the review are mentioned below.

3.1 Line 231-232: The claim is not supported with the comparison of published work.

*** The statement focusing on the role of solvent in membrane lipid abundant in fish skin or tissue membrane is: ‘Solvent having slight polarity was suitable for removal of lipids in skin containing membrane lipids such as phospholipids, etc.’

The above statement was based on our previous research on fat removal of seabass skin and also from fish muscle, in which membrane lipids are present. Those defatted samples were further processed into different products.

Sae-leaw, T.; Benjakul, S.; O'Brien, N.M. Effect of pretreatments and defatting of seabass skins on properties and fishy odor of gelatin. J. Food Biochem. 2016, 40, 741-753, doi:10.1111/jfbc.12267

Thiansilakul, Y.; Benjakul, S.; Shahidi, F. Antioxidative activity of protein hydrolysate from round scad muscle using alcalase and flavourzyme. J. Food Biochem. 2007, 31, 266-287.

Therefore, authors would like to keep the citations in the present form.

3.2 Line 233-235: Need references for this statement.

***The reference has been provided. Please see line 237 as highlighted in green.

3.3 Line 243-244: Need supportive discussion with reference.

*** Based on the result, hydroxyproline content was slightly increased when the fat was more removed, especially with the aid of ultrasonication. To our knowledge, the effect of defatting process with the aid of ultrasonication on hydroxyproline content of resulting collagen has not been reported. Thus, no information is available for comparison with the present work.

In the present study, the ultrasonication was implemented during defatting process. The fat was removed effectively during defatting with the aid of ultrasonication. As a result, the remaining components in defatted skin particularly collagen became concentrated, as indicated by slight increase in hydroxyproline content. Hydroxyproline is unique amino acid in collagen, which can be used to indicate the presence of collagen.

For better understanding, more discussion with reference has been added. Please see line 246 – 251 as highlighted in green.

3.4 Line 246-248: Comparison for these interpretation with published work.

***Due to the lack of information about the hydroxyproline content in the collagen extracted from defatted skin with the aid of ultrasonication, this statement was made based on the obtained results. Sorry for this lack in the previously published information.

3.5 Line 250-261: Discussion is lack of any published work companions.

***Some references have been provided. Please see line 260 – 261. Thank you very much for suggestion.

Reviewer 3 Report

The aim of the study described in this article is to evaluate the effect of ultrasonication on the defatting process in the extraction of collagen from salmon skin. For this purpose, the molecular properties of acid soluble collagen (ASC) isolated from skin salmon that was previously defatted with 30% isopropanol alone or with the aid of ultrasonication are evaluated with various methodological approaches aimed at establishing the extraction yield, the percentage of fats, the amino acid composition, the molecular integrity (electrophoretic profile), the thermal stability (DSC), the molecular structure (CD Spectra, FTIR spectra) and the degree of hydrolysis in gastrointestinal  digestion model system and the antioxidant properties of derived peptides.

In my opinion, the article is methodologically complete and accurate. However, I found the result concerning the extraction yield unclear. In the materials and method section the authors wrote that it is expressed as mg Hyp content in collagen/ mg Hyp content in raw material but in results in Table 1 the yield was expressed as %, dry weight basis. The yield data shold be  typically expressed as dry weight of the final extract/ dry weight of the starting material. I strongly suggest that the authors express the yield data in this way, even because the hydroxyproline assay method can give false positives and is not very suitable on raw materials. However, if the authors want to be more accurate, they can calculate the collagen content present in their final extract using the hydroxyproline content considering that it is 5.6% in collagen (as shown in Table 2). In this way the yield data in the two samples can be expressed as mg of collagen on dry weight of raw material.

Finally, in the sentence in line 34 “Native collagen is insoluble and appropriate pretreatment is required before extraction [5].” it should specify that native collagen is organized in fibers and therefore insoluble.

Author Response

Response to reviewer 3

Comments and Suggestions for Authors

The aim of the study described in this article is to evaluate the effect of ultrasonication on the defatting process in the extraction of collagen from salmon skin. For this purpose, the molecular properties of acid soluble collagen (ASC) isolated from skin salmon that was previously defatted with 30% isopropanol alone or with the aid of ultrasonication are evaluated with various methodological approaches aimed at establishing the extraction yield, the percentage of fats, the amino acid composition, the molecular integrity (electrophoretic profile), the thermal stability (DSC), the molecular structure (CD Spectra, FTIR spectra) and the degree of hydrolysis in gastrointestinal  digestion model system and the antioxidant properties of derived peptides.

***Thank you for your kind understanding. All corrections have been provided as per the reviewer’s suggestions and comments as highlighted in blue color.

1. In my opinion, the article is methodologically complete and accurate. However, I found the result concerning the extraction yield unclear. In the materials and method section the authors wrote that it is expressed as mg Hyp content in collagen/ mg Hyp content in raw material but in results in Table 1 the yield was expressed as %, dry weight basis. The yield data should be typically expressed as dry weight of the final extract/ dry weight of the starting material. I strongly suggest that the authors express the yield data in this way, even because the hydroxyproline assay method can give false positives and is not very suitable on raw materials. However, if the authors want to be more accurate, they can calculate the collagen content present in their final extract using the hydroxyproline content considering that it is 5.6% in collagen (as shown in Table 2). In this way the yield data in the two samples can be expressed as mg of collagen on dry weight of raw material.

***Thank you very much for valuable comments. Actually, the wrong equation of extraction yield (1) was given. Therefore, the correction has been made. Please see line 121. The yield was calculated based on dry weight of collagen powder/dry weight of skin used for extraction. Sorry for this mistake.

2. Finally, in the sentence in line 34 “Native collagen is insoluble and appropriate pretreatment is required before extraction [5].” it should specify that native collagen is organized in fibers and therefore insoluble.

***Thank you very much for valuable suggestion. The sentence has been specified following the reviewer’s suggestion. Please see line 34 – 36 as highlighted in blue.

Reviewer 4 Report

V

1. The work is devoted to the isolation of collagen from salmon skin. The introduction notes the important role of fish collagen in the development of modern science and technology. Indeed, it is. Hundreds of publications are published on this subject every year. However, when discussing the use of collagen in the food, nutraceutical, pharmaceutical and biomedical industries, the authors have given only 2 references:

• Lafarga, T.; Hayes, M. Bioactive peptides from meat muscle and by-products: generation, functionality and application as 475 functional ingredients. Meat Science. 2014, 98, 227-239, doi: https://doi.org/10.1016/j.meatsci.2014.05.036. 476
• Kim, S.-K.; Mendis, E. Bioactive compounds from marine processing byproducts–a review. food res. Int. 2006, 39, 383-393.

The first reference is about meat collagen, the second work was done in 2006.

You should specify references for the last 5-7 years. I think that there should be much more of them. For the same reasons, references 6 and 7 in the list of references should be changed.

The molecular weight characteristics of collagen were analyzed by electrophoresis in a dodecyl sulfate polyacrylamide gel. In the text of the article, it is necessary to give the values of the molecular weights of polymers according to these data. These data are always given for comparison with already known literature data.

2. Molecular mass characteristics of collagen were analyzed by dodecyl sulfate-polyacrylamide gel electrophoresis. In the text of the article, it is necessary to give the values ​​of the molecular weights of polymers according to these data. These data are always given for comparison with already known literature data.
3. We can recommend the authors to evaluate not only the degree of collagen hydrolysis in percent, but also the molecular weight of collagen hydrolysates using electrophoresis.
4. I would like to see the authors' opinion why the amino acid composition of collagen isolated using the proposed method in the work differs from the amino acid composition of fish collagen given in other publications. Over the past 5-7 years, other authors have published several papers with data on the amino acid composition of collagen isolated from fish skin. They also differ from the data given in this paper. Perhaps there is some explanation?
5. There are comments on the design of the article:

• Abbreviations are always used in the text when a word is used repeatedly and are introduced when the word is first used at the beginning of the text. So the authors indicate acid soluble collagen (ASC), ASC from skin defatted with aid of ultrasonication (U-ASC), etc. However, the abbreviation "fourier transform infrared (FTIR)" is introduced on page 4 of the text, although it is used earlier - on the first page , Differential scanning calorimetry (DSC) – the abbreviation is introduced twice, on pages 4 and 5, and is no longer used in the text.
• In figures, there can be one “0” at the origin of coordinates, or if the axes do not originate from zero, then “0” is not indicated. It is necessary to correct the drawing.

Author Response

Response to reviewer 4

Comments and Suggestions for Authors

The work is devoted to the isolation of collagen from salmon skin. The introduction notes the important role of fish collagen in the development of modern science and technology. Indeed, it is. Hundreds of publications are published on this subject every year. However, when discussing the use of collagen in the food, nutraceutical, pharmaceutical and biomedical industries, the authors have given only 2 references:

Lafarga, T.; Hayes, M. Bioactive peptides from meat muscle and by-products: generation, functionality and application as 475 functional ingredients. Meat Science. 2014, 98, 227-239, doi: https://doi.org/10.1016/j.meatsci.2014.05.036. 476

Kim, S.-K.; Mendis, E. Bioactive compounds from marine processing byproducts–a review. food res. Int. 2006, 39, 383-393.

The first reference is about meat collagen, the second work was done in 2006.

You should specify references for the last 5-7 years. I think that there should be much more of them. For the same reasons, references 6 and 7 in the list of references should be changed.

***Thank you very much for insightful and valuable suggestions. All mentioned references have been removed and replaced with the updated and related references as guided by the reviewer. Please see line 34, 39, 485 – 494 as highlighted in gray color. Coincidentally, another reviewer also recommended the authors to cite the updated references, especially those recently published (year 2020-2022). Thus, more information on collagens from different fish have been included in the manuscript.

2. Molecular mass characteristics of collagen were analyzed by dodecyl sulfate-polyacrylamide gel electrophoresis. In the text of the article, it is necessary to give the values ​​of the molecular weights of polymers according to these data. These data are always given for comparison with already known literature data.

***Thank you for valuable suggestion. MW of alpha 1 chain and alpha 2 chain were calculated and reported in the text following the reviewer’s suggestion. Please see line 291 – 292 as highlighted in gray.

3. We can recommend the authors to evaluate not only the degree of collagen hydrolysis in percent, but also the molecular weight of collagen hydrolysates using electrophoresis.

***Thank you for insightful recommendation. We do understand that the molecular weight of peptides in collagen hydrolysate is an important information. Since, the degree of hydrolysis was sufficient for indicating the formation of small peptides in digested collagen produced under the simulated gastrointestinal digestion system. In addition, most of hydrolyzed proteins or peptides appear as the smear bands. Thus, it is hard to determine the MW of peptides using SDS-PAGE. We have on-going research for measurement of peptide size using gel filtration techniques. The antioxidant activities of each fraction related with its MW is also monitored.

Therefore, MW of collagen hydrolysates using electrophoresis was not determined using SDS-PAGE in the part of digestibility for this study.

4. I would like to see the authors' opinion why the amino acid composition of collagen isolated using the proposed method in the work differs from the amino acid composition of fish collagen given in other publications. Over the past 5-7 years, other authors have published several papers with data on the amino acid composition of collagen isolated from fish skin. They also differ from the data given in this paper. Perhaps there is some explanation?

***Thank you so much for the insightful comment. The biological factors such as species, habitat, feed, etc. as well as the experimental treatments/process used could affect the amino acid composition of fish collagen. More discussions have been provided in text. Please see line 394 – 395. Based on literature, the amino acid composition of collagen in the present study was not different from that reported in other fish collagen, in which glycine is the dominant amino acid (almost 1/3 of total amino acids) and it also contained imino acids (proline and hydroxyproline), which can vary with fish species.  

5. There are comments on the design of the article:

5.1 Abbreviations are always used in the text when a word is used repeatedly and are introduced when the word is first used at the beginning of the text. So the authors indicate acid soluble collagen (ASC), ASC from skin defatted with aid of ultrasonication (U-ASC), etc. However, the abbreviation "fourier transform infrared (FTIR)" is introduced on page 4 of the text, although it is used earlier - on the first page , Differential scanning calorimetry (DSC) – the abbreviation is introduced twice, on pages 4 and 5, and is no longer used in the text.

***Thank you for the useful comment to make the text consistent. The full name of FTIR has been mentioned in the abstract. Please see line 20. Authors have cross-checked for the rest, in which the abbreviation was defined for the first use.

Additionally, the abbreviation of differential scanning calorimetry has been used in the text after the first description in line 136. Please see line 269.

5.2 In figures, there can be one “0” at the origin of coordinates, or if the axes do not originate from zero, then “0” is not indicated. It is necessary to correct the drawing.

***The corrections have been done for figure 6. Thank you very much for suggestion.

Round 2

Reviewer 1 Report

This revision has significantly improved the manuscript. I would recommend the manuscript for publication.