Antioxidant Activity of Peptide Fractions from Chickpea Globulin Obtained by Pulsed Ultrasound Pretreatment

Round 1

Reviewer 1 Report

Revisions required

Comments for author File: Comments.pdf

Author Response

Thanks to the reviewer for his great comments that helped increase the quality of the article.

1. Clearly explain the objective of the research.

 

Response 1. The objective was explained.

 

2. The research method has not been described.

 

Response 2. The research method was described.

 

3. At the beginning of writing, avoid using abbreviations.

 

Response 3. The reviewer is right. The abbreviation was described above.

 

4. What is the basis for this conclusion?

 

Response 4. The comment was made based on the results obtained that high antioxidant capacity was obtained, which could help prevent oxidation in food, and antimicrobial, with which it could preserve perishable foods.

 

5. It is necessary to explain in detail how the effect of ultrasound pulses on antioxidant activity, based on previous study.

 

Response 5. Increments in biological activity result from the cavitation phenomenon, produced by acoustic waves during the application of ultrasound, where the microbubbles generated cause conformational changes in the proteins. With hydrophobic regions exposed, efficiency of the enzymes increases, allowing the obtaining of peptides and protein hydrolysates with more favorable amino acid composition [17-19]. To improve antioxidant activity, ultrasound pretreatment has been used as part of the extraction technique, enhancing phenolic compounds extraction from chickpea seeds [23,24].

 

 

6. Summarize the research objectives so that they are easy to understand.

 

Response 6. Objectives were summarize.  

 

 

 

7. The research method is good, quite detailed, but in the abstract, the method is not explained.

 

Response 7. The methods in the abstract are only mentioned because the number of words is recorded and there is not enough space to detail it.

 

8. The results and discussion have been well written.

 

Response 8. Thanks to the reviewer.

 

9. Explain these abbreviations. Al = Albumin? Gb = Globulin ? Gt = Glutelin?

 

Response 9.  Abbreviations were described.

 

 

10. Adjust the conclusions with the research objectives.

 

Response 10. The conclusion was adjusted.

 

 

 

11. What is the basis for this conclusion?

 Response 11. The comment was made based on the results obtained that high antioxidant capacity was obtained, which could help prevent oxidation in food, and antimicrobial, with which it could preserve perishable foods.

 

 

Reviewer 2 Report

1. The subject addressed is one of interest, This document presents the antioxidant activity of chickpea protein concentrates and the improve in antioxidant activity of protein hydrolysates and peptide fractions due to ultrasound pretreatment. The electrophoretic profile, amino acid profile, and antimicrobial activity of hydrolysates were also determined.
2. The subject is original because the compounds identified in this study could be used as ingredients in the elaboration of functional foods, being necessary to evaluate their potential beneficial effect in vivo assays.
3. As a novelty for the addressed field: the application of pulsed ultrasound pretreatment to chickpea globulin allows the production of hydrolysates and peptide fractions with a high protective effect in human erythrocytes, due to the exposure of hydrophobic amino acids, abundant in chickpea globulin, improving the proton donation mechanism of hydrolysates and peptide fractions obtained from globulin.
4. References are adequate, but could be improved.
5. The conclusions are in accordance with the purpose of the work
6.  Figure 3. (a): I suggest a better resolution
7. Table 3. Amino acid profile (residues/1000 amino acid residues) of globulin hydrolysates, the statistical interpretation is not mentioned in the table, even if under the table there is the mention: Values are mean of triplicate determinations, with standard deviation less than 5%, I suggest to appear in the table.
8. In the future, I also suggest the determination of polyphenols, bearing in mind that emphasis was placed on Antioxidant activity

Author Response

Thanks to the reviewer for his great comments that helped increase the quality of the article.

1. The subject addressed is one of interest, This document presents the antioxidant activity of chickpea protein concentrates and the improve in antioxidant activity of protein hydrolysates and peptide fractions due to ultrasound pretreatment. The electrophoretic profile, amino acid profile, and antimicrobial activity of hydrolysates were also determined.

 

Response 1. Thanks to the reviewer.

2. The subject is original because the compounds identified in this study could be used as ingredients in the elaboration of functional foods, being necessary to evaluate their potential beneficial effect in vivo assays.

 

Response 2. The first stage in vitro are these results that we try to publish in this article. Subsequently, it is considered to carry out in vivo studies due to the importance of the compounds as well mentioned by the reviewer. However, only the in vitro part is considered in this article because the in vivo part is not yet started.

3. As a novelty for the addressed field: the application of pulsed ultrasound pretreatment to chickpea globulin allows the production of hydrolysates and peptide fractions with a high protective effect in human erythrocytes, due to the exposure of hydrophobic amino acids, abundant in chickpea globulin, improving the proton donation mechanism of hydrolysates and peptide fractions obtained from globulin.

 

Response 3. Thanks to the reviewer for highlighting the innovation of the work.

 

4. References are adequate, but could be improved.

 

Response 4. Thanks for your observation, references have been improved.

5. The conclusions are in accordance with the purpose of the work

 

Response 5.  Thanks to the reviewer.

6. Figure 3. (a): I suggest a better resolution.

 

Response 6. Thanks for the observation, the resolution was improved to its best possible quality.

 

7. Table 3. Amino acid profile (residues/1000 amino acid residues) of globulin hydrolysates, the statistical interpretation is not mentioned in the table, even if under the table there is the mention: Values are mean of triplicate determinations, with standard deviation less than 5%, I suggest to appear in the table.

 

Response 7. The standard deviations and the statistic were added in Table 3.

8. In the future, I also suggest the determination of polyphenols, bearing in mind that emphasis was placed on Antioxidant activity.

 

Response 8. Thanks to the reviewer. We will consider your suggestion.

 

Reviewer 3 Report

This study investigated pulsed ultrasound to obtain chickpea peptide fractions with biological activity. It is interesting and valuable to the journal’s readers. However, the manuscript needs revisions and the followings are some comments and suggestions for authors to consider and improve the manuscript.

1.       1. Novelty of the present manuscript could be stated more clearly in the introduction section in the manuscript.

2.       The methods for some experiment could be condensed if they were comprehensive used. For instance, ABTS, FRAP.

3.       The results of ABTS inhibition should be expressed as IC50.

4.       As the compounds are mixture, which compound are main responsible for the biological activity observed? I suggest the authors identify the hydrolysates and peptide fractions by LC-MS/MS.

5.       The references are too much, please consider to remove unnecessary references.

6.       The full name of MIC (Line 236) should be provided when it first appeared.

7.       There are some grammars errors in the manuscript, please check throughout the manuscript and to ensure the grammars are correct.

Author Response

Thanks to the reviewer for his great comments that helped increase the quality of the article.

 

This study investigated pulsed ultrasound to obtain chickpea peptide fractions with biological activity. It is interesting and valuable to the journal’s readers. However, the manuscript needs revisions and the followings are some comments and suggestions for authors to consider and improve the manuscript.

1. Novelty of the present manuscript could be stated more clearly in the introduction section in the manuscript.

Response 1. Thanks for the observation, the suggested was added.

2. The methods for some experiment could be condensed if they were comprehensive used. For instance, ABTS, FRAP.

Response 2. Thanks for the observation, the suggested change was made.

3. The results of ABTS inhibition should be expressed as IC50.

Response 3. The hydrolyzates presented inhibition percentages in ABTS greater than 50%, therefore the IC50 of these samples could be calculated. It was placed in the results within this section (in the text). For the peptides, it was not possible to calculate the IC50 due to the inhibition percentages, at a concentration of 200 ug/mL it was less than 50% and that amount of sample is considered high, so it is not worth calculating an IC50 that will give a very high concentration.

4. As the compounds are mixture, which compound are main responsible for the biological activity observed? I suggest the authors identify the hydrolysates and peptide fractions by LC-MS/MS.

Response 3. Thanks for the reviewer's comment. Of course, it would be ideal to identify the amino acids directly responsible for the bioactivity, however, we were only able to obtain the amino acid profile of the samples by HPLC, which gives us an idea based on the references of what type of amino acids can confer the bioactivity, such as the hydrophobic type. This is seen in the discussions in the section on antioxidant capacity. In the future we will carry out the individual identification in detail, since it also takes a lot of work and at the moment we do not have an LC-MS/MS available.

5. The references are too much, please consider to remove unnecessary references.

Response 5. Thanks for your observation, references’ number has been reduced.

6. The full name of MIC (Line 236) should be provided when it first appeared.

Response 6. Thanks for the observation, the full name has been written.

7. There are some grammars errors in the manuscript, please check throughout the manuscript and to ensure the grammars are correct.

Response 7. English was reviewed by a language expert.

 

Round 2

Reviewer 3 Report

The authors well revised the manuscript.