In Silico and In Vitro Assessment of Portuguese Oyster (Crassostrea angulata) Proteins as Precursor of Bioactive Peptides

Round 1

Reviewer 1 Report

Although the findings concerning oyster hydolysates contain inhibitory peptides for ACE and DPP IV are very interesting, a number of points need clarifying and certain statements require further justification. These are given below.

<Points>

1.        The results seem to be premature. Authors should identify at least a peptide that inhibits ACE and/or DPP IV activity. It strengthens the authors’ idea and increases the significance of the research.

2.        The enzyme assay using angiotensin I as a substrate for ACE assay and incretin as a substrate for DPP IV assay will clearly demonstrate the authors’ idea and increases the significance of the research.

3.        A part of Figures 1, Figure 2, and Table 1 is invisible. They should be placed appropriately.

4.        Concerning page 9, lines 4-14, the authors should describe according to the instruction of ILMS.

5.        In Figure 5 and 6, the authors should clearly describe what “A”, “B”, “C”, “a”, and “b” mean. In addition, “A” and “B” should be changed to another symbols because there are another “A” and “B” (it means Figure 5/6A and 6A/B).

6.        Reference list should be re-write according to IJMS style.

Author Response

Comments and Suggestions for Authors

Although the findings concerning oyster hydolysates contain inhibitory peptides for ACE and DPP IV are very interesting, a number of points need clarifying and certain statements require further justification. These are given below.

<Points>

The results seem to be premature. Authors should identify at least a peptide that inhibits ACE and/or DPP IV activity. It strengthens the authors’ idea and increases the significance of the research.

The authors greatly appreciate the suggestion and deems it necessary to increase the significance of the study, however, for this study the identification of certain bioactive peptides is not the main objective of the study. We believe this could be done later in a separate study that would focus more on the specific bioactive peptides from the raw material.

 

The enzyme assay using angiotensin I as a substrate for ACE assay and incretin as a substrate for DPP IV assay will clearly demonstrate the authors’ idea and increases the significance of the research.

The authors acknowledge that the use of natural substrates will clearly show the activities of the peptides in the raw material. However, various references in literature have preferred the use of synthetic substrates like FAPGG and Gly-pro-p-nitroanilide for screening purposes due to their convenience and cost-efficiency. Moreover, our study have focused mainly on confirmation of in silico bioactivities of oyster proteins through in vitro analyses. We believe this could be considered later in a separate study.

 

A part of Figures 1, Figure 2, and Table 1 is invisible. They should be placed appropriately.

Necessary corrections were made. Please refer to the corrected version of the paper. 

Concerning page 9, lines 4-14, the authors should describe according to the instruction of ILMS.

Necessary corrections was made. Please refer to the corrected version of the paper (line 267 to 275).

 

In Figure 5 and 6, the authors should clearly describe what “A”, “B”, “C”, “a”, and “b” mean. In addition, “A” and “B” should be changed to another symbols because there are another “A” and “B” (it means Figure 5/6A and 6A/B).

Capital letters (A and B) representing significantly different means were already replaced with capital letters (DEF) to avoid confusion. Pls. refer to figures 5 and 6.

Reference list should be re-write according to IJMS style.

Necessary corrections was made. Please refer to the corrected version of the paper

 

Reviewer 2 Report

Overall an interesting study but there are a few concerns:

Statistics: more details needed, such as the particular software used, the 'n' for each experiment and please use an acceptable post-hoc test following ANOVA (for multiple comparisons, a Tukey's post-hoc test is appropriate). Ethics: since live animals (oysters) were used in the study, it is surprising that the authors did not seek an ethics approval from their respective academic institutions. Besides, all such studies should also conform to acceptable international standards for live animal research. It is strongly recommended to use whole oyster protein hydrolysates (using the different enzymes used for individual proteins) to demonstrate the ACE inhibitor and DPPIV inhibitor activities. Since it is economically not feasible to isolate particular proteins and prepare hydrolysates for use, it is more likely that the whole oyster preparations would undergo hydrolysis to generate useful products. As such, it is critical to demonstrate similar results using whole protein hydrolysates to complement their current results. The introduction needs to provide detailed background including the significance of examining ACE- and DPPIV- inhibition from protein hydrolysates. An extensive literature is available on the role of food-derived bioactive peptides as useful enzyme inhibitors and this needs to be briefly summarized. While ACE blockers are well known as antihypertensives, less is known about the need for DPPIV inhibitors so further discussion is warranted.

 

Author Response

Comments and Suggestions for Authors

Overall an interesting study but there are a few concerns:

Statistics: more details needed, such as the particular software used, the 'n' for each experiment and please use an acceptable post-hoc test following ANOVA (for multiple comparisons, a Tukey's post-hoc test is appropriate).

 

Necessary corrections were made. Pls. refer to the corrected version of the paper.

 

Ethics: since live animals (oysters) were used in the study, it is surprising that the authors did not seek an ethics approval from their respective academic institutions. Besides, all such studies should also conform to acceptable international standards for live animal research.

Since the raw materials used in the study were considered as market samples, no ethics approval was required. These oysters are cultured for selling purposes (either for processing or to be eaten raw) and preserved (by icing) for days prior to shipment. Moreover, according to published guides and regulations for use of live animals in scientific research (http://www.nap.edu/catalog/12910.html), “laboratory animals” or “animals” are limited only to any vertebrate animal (e.g., traditional laboratory animals, wildlife, and aquatic species) produced for or used in research, testing, or teaching, thus, the main focus of these regulations and ethical considerations. In addition, the focus of Animal Protection Acts and guidelines for animal use enforcement in Taiwan is mainly vertebrates including agricultural animals and pets (cats, dogs, birds, etc.). 

 

It is strongly recommended to use whole oyster protein hydrolysates (using the different enzymes used for individual proteins) to demonstrate the ACE inhibitor and DPPIV inhibitor activities. Since it is economically not feasible to isolate particular proteins and prepare hydrolysates for use, it is more likely that the whole oyster preparations would undergo hydrolysis to generate useful products. As such, it is critical to demonstrate similar results using whole protein hydrolysates to complement their current results.

The researchers have been able to analyze the ACE and DPP-IV inhibitory activities of whole protein hydrolysates produced using pepsin, bromelain, and papain (pls. see figures 5/6A). Among the three products, pepsin hydrolysate exhibited highest inhibitory activity towards ACE and DPP-IV, therefore subjected to further fractionation. The ACE and DPP-IV inhibitory activities of PEH fractions are shown in figures 5/6B.   

 

The introduction needs to provide detailed background including the significance of examining ACE- and DPPIV- inhibition from protein hydrolysates. An extensive literature is available on the role of food-derived bioactive peptides as useful enzyme inhibitors and this needs to be briefly summarized. While ACE blockers are well known as antihypertensives, less is known about the need for DPPIV inhibitors so further discussion is warranted.

 Necessary corrections were made. Please refer to the corrected version of the paper (line 41 to 50).

 

 

Round 2

Reviewer 1 Report

Judged by the responses from the authors, most of the problems are resolved in the revised version. However, there remains a number of points need clarifying and certain statements require further justification. These are given below.

<Points>

1. Concerning Figures 1, Figure 2, and Table 1, necessary corrections were made. However, the caption of Table 4 and Table 4 itself in the revised version were separately placed.

2. In Figure 3, please add label of Y-axis.

3. Although the authors responded, “Necessary corrections was made.”, reference list is not JIMS style. Reference list should be re-write according to IJMS style. For example, “nature”, “Food Chem”, etc. should be changed to “Nature”, “Food Chem.”, etc.

Author Response

Judged by the responses from the authors, most of the problems are resolved in the revised version. However, there remains a number of points need clarifying and certain statements require further justification. These are given below.

<Points>

Concerning Figures 1, Figure 2, and Table 1, necessary corrections were made. However, the caption of Table 4 and Table 4 itself in the revised version were separately placed.

Necessary correction was made. Please refer to lines 279-281.

In Figure 3, please add label of Y-axis.

Necessary correction was made. Please refer to the corrected version of the paper.

Although the authors responded, “Necessary corrections was made.”, reference list is not JIMS style. Reference list should be re-write according to IJMS style. For example, “nature”, “Food Chem”, etc. should be changed to “Nature”, “Food Chem.”, etc.

Necessary correction was made. Please refer to the corrected version of the paper.

Reviewer 2 Report

The sentence in introduction suggesting that DPP-iv mediated degradation of GLP is causative for type II diabetes is wrong and misleading. Please mention something like DPP-iv could be involved in the pathogenesis of type II diabetes or similar (and provide multiple, recent, references to support it).

Other concerns have been addressed.

Author Response

The sentence in introduction suggesting that DPP-iv mediated degradation of GLP is causative for type II diabetes is wrong and misleading. Please mention something like DPP-iv could be involved in the pathogenesis of type II diabetes or similar (and provide multiple, recent, references to support it).

Other concerns have been addressed.

Necessary correction was made. Please refer to lines 41-61.