Functional Properties of Egg White Protein and Whey Protein in the Presence of Bioactive Chicken Trachea Hydrolysate and Sodium Chloride

Round 1

Reviewer 1 Report

Dear authors,

I have attached my review report. Unfortunately, the current version of your manuscript is not publishable because it has many flaws. 

Comments for author File: Comments.pdf

Author Response

Response to reviewer

Reviewer #1

Thank you very much for spending your valuable time reviewing our manuscript. Your suggestions and comments help improve the quality of our manuscript. Thank you very much again.

1. Dear Authors, The manuscript: “The Effect of Egg White Protein, Whey Protein and Sodium Chloride on Functional and Bioactive Properties of Chicken Trachea Hydrolysate” contains interesting research results which, apart from scientific knowledge, also have great application value. However, the manuscript is not sound since the title is not in line with the objectives of the study. For example, the title looks on the effect of egg white protein, whey protein, and sodium chloride on functional and bioactive properties of chicken trachea hydrolase.

Response Thank you very much for your valuable suggestions. We’ve already changed the title of the manuscript to a better reflect of the result of our study (Line 1-4). Thank you very much again.

 

2. My concern here is that chicken trachea hydrolysate (CTH) at 0, 0.25 and 0.25% w/w was used, therefore, are authors saying CTH will have an effect on functional and bioactive properties of CTH?. Authors are talking about the system in line 13, under abstract. Can authors explain what is this system they are talking about?

Response Thank you very much for your kind comments. The system that referred to is the protein mixture system. However, to a better understanding and clarifying the sentence, the wording “in the system” was deleted. Thank you very much again.

 

3. Under 2.2, materials and methods, authors talked about protein mixture preparation. Can authors explain if they are checking the effect of egg white protein, whey protein, CTH and sodium chloride on functional and bioactive properties of protein mixture or CTH? This is very confusing.

Response Thank you very much for your valuable comments. We’ve already rewritten the Materials and Methods part and clarified the confusing points as shown on Page 3, Line 106-290. Moreover, the introductory section has been largely re-written to improve English and to clarify the main interest in the effect that CTH had on the functional properties of the egg white and whey protein. We studied the bioactive properties of CTH in combination with the egg and whey proteins (and salt) because if we are to use CTH as bioactive ingredient in food formulations, it is important to know if any interactions with proteins are likely to reduce this effect.  The changes of Introduction were made on Page 1, Line 31-105. Thank you very much again.

 

4. From 3.2, under materials and methods to 3.5, under results and discussion is all about protein mixture in the system with or without sodium chloride. This shows that the study is concentrating on the protein mixture rather than CTH.

Response Thank you very much for your kind comments. Yes, our study were also emphasized on the effect of protein mixture with CTH and with and without sodium chloride. To a better understanding of our manuscript, all details have been revised and rewritten as shown in Introduction, Materials and Methods and Results parts. Please kindly check all changes via track change in the file. Thank you very much again.

 

5. Under introduction, lines 70 – 79, there are contradicting statements by the authors, moreover, line 79, under introduction, “These properties directly affected the quality………is this result from this study or other studies?

Response Thank you very much for your kind suggestions and please apologize us for making you a big confuse. We’ve tried our best in revising the Introduction part and rewritten all of those sentences (Page 1, Line 31-105). Please kindly check all changes via track change in the file. We’re really appreciated your comments. It helps improving our manuscript quality. Thank you very much again.

 

6. Again, under results, Figure 1 is Foaming capacity (A) and foaming stability (B) of egg white (E) and whey protein (W) mixed with hydrolysate (H) from chicken trachea (0, 0.25, and 0.5%) and sodium chloride (S; 0 and 1.5%). Here now authors determined the foaming capacity and foam stability of egge white and whey protein, not the other way round where we need to see the effect of these egg white and whey protein on the protein mixture or CTH……..this does not relate to the topic. These make it difficult to follow the main objectives of this study.

Response Thank you very much for your valuable suggestions. We’ve already rewritten the Results and Discussion part to solve this problem (Page 7, Line 291) and already changed the title (Page 1, Line 1) to reveal our main objectives. If there is any incorrect points still shown in our manuscript, please do not hesitate to let us know. Thank you very much again.

 

7. There are too many flaws that need to be addressed before the manuscript can be considered for publication. The current version is not publishable.

Response Thank you very much again for your kind revision. We’ve tried our best on revising this manuscript and appreciate any suggestions that may arise. We will be more than happy to correct it.

Reviewer 2 Report

Summary:

Pramualkijja et al. present experimental data on the impact of Chicken Trachea Hydrolysate (CTH) and Sodium chloride (NaCl) on the bioactive and functional properties of protein solutions containing chicken egg white and whey proteins. This included assessments of physical properties (e.g. foaming and rheological properties) and biochemical activity (antioxidant potential and ACE inhibition). Using this, the authors proposed a formulation for future study.

Broad Comments:

• The title implies that the primary focus of the article will be on CTH and how it is modified by egg white protein, whey protein, and NaCl. As it is currently written, however, the manuscript is structured around how the egg white and whey proteins are modified by CTH and NaCl individually or in combination. The title should be modified to more accurately reflect the focus of the study.
• The data and analyses are generally straightforward. However, some of the interpretations and proposed explanations are questionable and should be re-visited (this includes some points in which different sentences in the manuscript appear to contradict each other).

Specific Comments:

• Line 133: Please explain where the constant value (2.303) is derived from in addition to the variables.
• Lines 199, 211, and 212: These sentences state that “HEPES-HCl” was used. However, HEPES is acidic and is usually pH adjusted using NaOH. Did the authors intend “HEPES-NaOH”? Please verify the accuracy of this part of the Methods.
• Lines 217-219, 402-405: These paragraphs refer to experiments not performed in this study. While I appreciate the emphasis that the insights from this paper are being implemented practically, the current wording makes it seem that other experiments were performed for this study but were left out of the manuscript. It may be better to rephrase in a way that refers to more general applications and does not imply specific experiments.
• Lines 293-296: I’m not sure that this statement is fully supported by the reference, as Ref 33 evaluates both pH and salt concentration. This study does not appear to take pH into account when making the inference articulated in this sentence.
• Lines 297-300: This sentence seems to be contradicted by Lines 397-399. In general, it is expected that salt improves the folding and stability of proteins. Is there sufficient evidence that this is not the case for whey protein? The reference cited focused on Whey Protein Concentrate. Would the same mechanisms be at work in the protein solutions used in this study?
• Figure 3: Typo—“strian” should be “strain”
• Lines 355-357: This sentence states that the addition of NaCl increased the antioxidant activity of both protein solutions. In Figure 5, however, I see no convincing evidence that NaCl increases the antioxidant activity of the egg white solution. For the whey protein it appears there may be a marginal difference, but the results are mixed. Lines 416-418 also seems to contradict Lines 355-357.
• Lines 369-372 and Figure 6: How were the protein concentrations quantified? Do the numbers reflect total protein (e.g. Whey + CTH) or just and individual component?
• Lines 390-394: Glu and Asp are both acidic amino acids with charged side chains at physiological pH. Under these conditions, they do not constitute “hydrophobic” amino acids.

Author Response

Response to reviewer

Reviewer #2

Pramualkijja et al. present experimental data on the impact of Chicken Trachea Hydrolysate (CTH) and Sodium chloride (NaCl) on the bioactive and functional properties of protein solutions containing chicken egg white and whey proteins. This included assessments of physical properties (e.g. foaming and rheological properties) and biochemical activity (antioxidant potential and ACE inhibition). Using this, the authors proposed a formulation for future study.

Response: Thank you very much for spending your valuable time reviewing our manuscript. Your suggestions and comments help improve the quality of our manuscript. Thank you very much again.

 

Broad Comments:

• The title implies that the primary focus of the article will be on CTH and how it is modified by egg white protein, whey protein, and NaCl. As it is currently written, however, the manuscript is structured around how the egg white and whey proteins are modified by CTH and NaCl individually or in combination. The title should be modified to more accurately reflect the focus of the study.

Response Thank you very much for your valuable suggestion. We’ve already changed the title as shown in Page 1, Line 1. Thank you very much again.

• The data and analyses are generally straightforward. However, some of the interpretations and proposed explanations are questionable and should be re-visited (this includes some points in which different sentences in the manuscript appear to contradict each other).

Response Thank you very much for your valuable suggestion. We’ve already revised and rewritten through out our manuscript for a better understanding of objectives and findings (Page 7, Line 291). Please kindly check all changes via track change in the file. Thank you very much again.

Specific Comments:

• Line 133: Please explain where the constant value (2.303) is derived from in addition to the variables.

Response Thank you very much for kind comments. The explanation is shown below:

Line 133 – the constant 2.303 is introduced to convert the absorbance(A) to a turbidity (T), using the relationship

T=(2.303×A×dilution)/(light path length)

This has been added to the manuscript text.

• Lines 199, 211, and 212: These sentences state that “HEPES-HCl” was used. However, HEPES is acidic and is usually pH adjusted using NaOH. Did the authors intend “HEPES-NaOH”? Please verify the accuracy of this part of the Methods.

Response Lines 199, 211, and 212: Hepes-HCl has been changed to HEPES-NaOH. Thank you very much for your kind suggestions.

• Lines 217-219, 402-405: These paragraphs refer to experiments not performed in this study. While I appreciate the emphasis that the insights from this paper are being implemented practically, the current wording makes it seem that other experiments were performed for this study but were left out of the manuscript. It may be better to rephrase in a way that refers to more general applications and does not imply specific experiments.

Response  Lines 217-219, 402-405: These have been deleted and we’ve already rephased the sentence to a better understanding. Thank you very much for your suggestions.

• Lines 293-296: I’m not sure that this statement is fully supported by the reference, as Ref 33 evaluates both pH and salt concentration. This study does not appear to take pH into account when making the inference articulated in this sentence.

Response Thank you very much for your valuable comments the sentence was deleted and rewritten as shown in Page 7, Lines 293-296. Thank you very much again.

• Lines 297-300: This sentence seems to be contradicted by Lines 397-399. In general, it is expected that salt improves the folding and stability of proteins. Is there sufficient evidence that this is not the case for whey protein? The reference cited focused on Whey Protein Concentrate. Would the same mechanisms be at work in the protein solutions used in this study?

Response Thank you very much for your valuable comments. The contradiction of these two paragraphs was resolved as follows:

Lines 297-300: This section has been modified (and reference [34] deleted) to emphasize that the differences between protein+CTH emulsions are likely due to differences in the way in which CTH and egg white or whey protein interact with each other.

Lines 397-399: This section has been clarified to explain better how increasing solubility of the hydrolysate might improve bioactive properties. Please kindly check all changes via track change in the file. Thank you very much again.

• Figure 3: Typo—“strian” should be “strain”

Response Thank you very much for your kind comment. We’ve already corrected the typo in Figure 3. The work “strain” was changed to “strain”. Thank you very much again.

• Lines 355-357: This sentence states that the addition of NaCl increased the antioxidant activity of both protein solutions. In Figure 5, however, I see no convincing evidence that NaCl increases the antioxidant activity of the egg white solution. For the whey protein it appears there may be a marginal difference, but the results are mixed. Lines 416-418 also seems to contradict Lines 355-357.

Response Thank you very much for your valuable comments. We’ve clarified the effect of NaCl on antioxidant activity as shown on Page 13, Line 432. We’ve also rewritten these paragraphs to minimize the contradiction as shown on Page 13, line 453. Please kindly check all changes via track change in the file. Thank you very much again.

• Lines 369-372 and Figure 6: How were the protein concentrations quantified? Do the numbers reflect total protein (e.g. Whey + CTH) or just and individual component?

Response: Thank you very much for your kind comments. The IC50 is calculated relative to the total protein content (WPC or EW + CTH). The protein concentration was fixed during the preparation of the mixture and the sentences were modified as shown in the manuscript on Page 13, Line 444. Please kindly check all changes via track change in the file. Thank you very much again.

• Lines 390-394: Glu and Asp are both acidic amino acids with charged side chains at physiological pH. Under these conditions, they do not constitute “hydrophobic” amino acids.

Response Thank you very much for your valuable comments. It helps improve the weak point in our manuscript. As you mentioned, we’ve already checked the results and rewritten this paragraph as shown in Page 14, Line 472.  Please kindly check all changes via track change in the file. Thank you very much again.

 

Reviewer 3 Report

Although this is an interesting study with appropriate experimental design, the experimental results and their importance are not adequately conveyed to the readers. For example, the CHT clearly demonstrates more significant antihypertensive activity as compared to other samples, adding whey protein or egg protein will lead to reduced activity. If this is the case, what is the point of preparing CHT/other protein mixtures? What are the trade-offs when preparing protein mixtures? Also, on the antihypertensive activity, adding salt clearly leads to reduced activity, yet the authors stated that NaCl showed no effect on antihypertensive activity, this conclusion needs to be revisited. 

In addition to the above mentioned point, grammatical errors are populated throughout the manuscript. The authors are highly recommended to make corrections accordingly so that the manuscript can be easily understood by the readers.

Author Response

Response to reviewer

Reviewer #3

Thank you very much for spending your valuable time reviewing our manuscript. Your suggestions and comments help improve the quality of our manuscript. Thank you very much again.

1. Although this is an interesting study with appropriate experimental design, the experimental results and their importance are not adequately conveyed to the readers. For example, the CHT clearly demonstrates more significant antihypertensive activity as compared to other samples, adding whey protein or egg protein will lead to reduced activity. If this is the case, what is the point of preparing CHT/other protein mixtures? What are the trade-offs when preparing protein mixtures?

Response: Thank you very much for your kind comments. We’ve already rewritten and added more clarification of this point as shown in Page, line 367-373.

It was clearly shown that CTH had higher antihypertensive activity among those three proteins showing the ability of collagen peptides as a good angiotensin I inhibitor. However, we studied the mixture of these protein since we want to understand the interaction between these proteins and receive the information for further application in the high protein meat product. Egg white and whey protein are normally used for elevating protein in food, the understanding of properties of the protein mixtures will help us selecting the suitable ingredient. From our study, the mixture composed of 10%w/w whey protein, 0.5%w/w CTH and 1.5%w/w NaCl showed the best activity when considering various properties and was selected for further study in real meat system.

2. On the antihypertensive activity, adding salt clearly leads to reduced activity, yet the authors stated that NaCl showed no effect on antihypertensive activity, this conclusion needs to be revisited.

Response: Thank you very much for your kind comments. The effect of salt was studied because we want to use these protein mixture in high protein meat product which salt is one of the important ingredient. Moreover, we’ve already rewritten the conclusion to explain more on this point as shown in the conclusion part (page 15, line 490-515). Thank you very much for your suggestions.

 

3. In addition to the above mentioned point, grammatical errors are populated throughout the manuscript. The authors are highly recommended to make corrections accordingly so that the manuscript can be easily understood by the readers.

Response: Thank you very much for spending your valuable time reviewing our manuscript. Your suggestions are useful for us. We’ve already corrected the grammatical errors through out the manuscript by the native speaker. If there’s still incorrect points, please do not hesitate to let us know. Thank you very much again.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear authors,

Thank you for submitting the revised manuscript and it has been improved. Attached are just few editorial comments that need to be addressed.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer

Reviewer#1

Comments and Suggestions for Authors

Dear Authors,

The revised manuscript: “Functional Properties of Egg White Protein and Whey Protein in the presence of Bioactive Chicken Trachea Hydrolysate and Sodium Chloride ” is sound. However, the following few editorial issues should be done on the manuscript:

Response Thank you very much for kindly reviewing our manuscript. The merit and correctness of our manuscript are much improved. Thank you very much again.

(1). In the abstract, line 17, should be “CTH and NaCl promoted the gelling properties of those….”

Response Thank you very much for your kind suggestions. We’ve already corrected the sentence as shown in the abstract, line 17. Thank you very much again.

 

(2). Under abstract, line 20, should be “The addition of 1.5%w/w NaCl improved antioxidant…….

Response Thank you very much for your kind suggestions. We’ve already corrected the sentence as shown in the abstract, line 20. Thank you very much again.

 

(3). Under abstract, line 23, authors should check because they are saying the lowest IC50 value of…….was resulted as the highest inhibitory activity? Not clear

Response Thank you very much for your kind suggestions. We’ve already corrected and rewritten the sentences as shown in the abstract, line 22-24. Thank you very much again.

 

(4). Under abstract, line 24, should be “The mixture is suited……..

Response Thank you very much for your kind suggestions. We’ve already corrected and rewritten the sentences as shown in the abstract, line 22-24. Thank you very much again.

 

(5). Under materials and methods (M&M, lines 68-69, authors should make correction on Sarbon et al. (2018).

Response Thank you very much for your kind suggestions. We’ve already corrected this point as shown in the materials and methods, line 68. The author’s name was substituted by “Another study also reported …”. Thank you very much again.

 

(6). Under materials and methods (M&M), line 95, remove (2021) after Pramualkijja et al.

Response Thank you very much for your kind suggestions. We’ve already corrected this point as shown in the materials and methods, line 95. The word “2021”) was removed. Thank you very much again.

 

(7). Under M&M, line 113, should be Nomana et al. [22]

Response Thank you very much for your kind suggestions. We’ve already corrected this point as shown in the materials and methods, line 113. We’ve already changed to Nomana et al. [22]. Thank you very much again.

 

(8). Under M&M, line 117, should be min instead of minutes for consistency.

Response Thank you very much for your kind suggestions. We’ve already corrected this point as shown in the materials and methods, line 117. The “minutes” was changed to “min”. Thank you very much again.

 

(9) Under M&M, line 209, should be one instead of on, also remove to before ab- in line 195.

Response Thank you very much for your kind suggestions. We’ve already corrected this point as shown in the materials and methods, line 195 and 210. Thank you very much again.

 

            Thank you very much again for your kindness and your valuable suggestions.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors have sufficiently addressed my primary concerns. Regarding the constant 2.303 that is used in the equations, my hope was that this might be defined in a similar manner as the variables that were designated (e.g. A, dil, theta, etc.). On the protein quantification, the clarification is good, but it would be helpful if the original means of measuring the protein concentrations were included in the Methods. Is it based on the protein content of 80% as determined by Kjeldahl nitrogen analysis?

Author Response

Response to Reviewer

Reviewer#2

Comments and Suggestions for Authors

The authors have sufficiently addressed my primary concerns. Regarding the constant 2.303 that is used in the equations, my hope was that this might be defined in a similar manner as the variables that were designated (e.g. A, dil, theta, etc.). On the protein quantification, the clarification is good, but it would be helpful if the original means of measuring the protein concentrations were included in the Methods. Is it based on the protein content of 80% as determined by Kjeldahl nitrogen analysis?

Response Thank you very much for kindly reviewing our manuscript. The merit and correctness of our manuscript are much improved. Thank you very much again.

            According to your comments, the constant of 2.303 was designated in the equation as shown in Materials and Methods, the line 134. For the protein quantification, it is based on the Kjeldahl nitrogen analysis as described in Materials and Methods, the line 89. This information was provided as the specification of the egg white and whey protein that we’ve received from the company.

 

Thank you very much again for your kindness and your valuable comments.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Previous concerns from the reviewer have been properly addressed. One minor suggestion for the author: the resolution for all figures can be improved for better visualization. It should also be stated in the figure captions that how the error bars are calculated (especially for all bar graphs).

Author Response

Response to Reviewer

Reviewer#3

Comments and Suggestions for Authors

Previous concerns from the reviewer have been properly addressed. One minor suggestion for the author: the resolution for all figures can be improved for better visualization. It should also be stated in the figure captions that how the error bars are calculated (especially for all bar graphs).

 

Response Thank you very much for kindly reviewing our manuscript. The merit and correctness of our manuscript are much improved. Thank you very much again.

            According to your suggestions, the figures were corrected to a higher resolution and the error bars for all bar graphs was clarified as shown in the figure captions of Figure 1, 2 and 5.

Thank you very much for your kindness and your valuable suggestions.

Author Response File: Author Response.pdf