Ultrafiltration of Saithe (Pollachius virens) Protein Hydrolysates and Its Effect on Antioxidative Activity

Round 1

Reviewer 1 Report

Coments for the manuscript catalysts-1338432

The article rules out saithe rest raw material ultrafiltration as there is no improvement in bioactive properties and protein yield is low, but only two enzymes have been tested. On the other hand, the oil contained in the fish rest raw material is not removed, which surely influences the performance of the enzymes and the formation of emulsions with the proteins, reducing the final performance of the product sought, which is the hydrolysate. So, I think it should go deeper into these two aspects mentioned, try other enzymes and study the possibility of removing the oil before hydrolysis, as is done in the case of oilseed hydrolysates, like soya, chia, hemp, etc.

On the other hand, with respect to references, journal name is not in abbreviated form.

Further coments:

1.- Aminoacid composition:

In line 28-29 say that fish is a well-balanced aminoacids composition and in line 42-43 said that fish rest raw material is an excellent source of amino acids for human metabolism, but:

• An amino acid analysis method is used that does not detect all the essential amino acids (see line 188), there are methods that, combining them, can detect all amino acids, see for example:

https://doi.org/10.1016/0021-9673(92)80236-N

https://doi.org/10.1016/j.foodchem.2003.07.026

• In line 188 say Phe could not be detected but it appears in all the samples analized
• On the other hand, I miss a comparison of the amino acid composition with some standard such FAO, it is essential.

FAO/WHO/UNU. Scoring pattern mg/g protein requeriment in adults;

 FAO and FINUT, 2017. Dietary protein quality evaluation in human nutrition. FAO Food and Nutrition Paper NO. 92.

2.- Enzymatic hydrolysis.

2.1.- In line 381 say that the experimental procedure was conducted as described by Kjellnes, Rustad and Falch and this procedure is in an manuscript submitted for publication and, although I do not doubt that the process is perfectly detailed in that manuscript, as is done in another manuscript published by Falch (see doi:10.1016/j.procbio.2009.02.010), several aspects of the process are not clear, such as:

• How many grams of fish rest raw material per liter is used.
• How long the hydrolysate is kept at pH 7 before adding the enzymes, the time necessary for a first protein dissolution.
• Because the enzymes papain and bromelain have been chosen.
• How the enzyme is inactivated after the reaction time.
• How the separation of the sludge is carried out.

2.2.- In line 391 say the resulting SPH were freeze dried, is it strictly necessary to do so?, keep in mind that in an industrial scale, drying is an expensive process and if after it, is necessary to redissolve to filter it, should you try to carry out filtration after hydrolysis?.

2.3.- In line 270 say low hydrolysis degree, when has the degree of hydrolysis been measured?, ¿where are those results?

3.- Ultrafiltration

In line 405 say, SPH were resuspended in distilled water to a concentration of 1%, is that the final concentration of saithe protein hydrolysate before drying? Why is that concentration chosen?

4.- Molecular weight distribution

In line 443-444 say samples (SPH and P4) were dissolved in distilled water and filtered through a low protein binding microfilter to remove large peptides and proteins.

I consider that the samples resulting from the filtration can no longer be called SPH and P4 since, part of them has been removed. If they are filtered to be able to use Superdex column, I think it is more convenient to use another type of column with a greater range of molecular weights, such as Superose 12 10/300 GL column

5.- Antioxidant activity.

I think it is essential to compare the antioxidant activity with the starting protein

5.- Other considerations:

- In line 142 say, it is not common practice to report protein yield of ultrafiltration processing.

I think that in a manuscript in which a process is intended to be carried out on an industrial scale, a mass balance, a protein balance, as well as a study of process times is essential.

Author Response

Dear reviewer 1,

Please see the attachment

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript is written very well and clearly. The research carried out is very extensive and the results obtained are very interesting. The literature cited includes works published in recent years. I highly recommend manuscript for publication, after improving the quality of Figures 3a and 3b. 

Author Response

Dear reviewer 2, 

Please see the attachment

Author Response File: Author Response.docx

Reviewer 3 Report

In this manuscript, Hjellnes et al. investigated the feasibility of extracting and concentrating peptides from saithe rest raw material based on enzymatic hydrolysis and membrane ultrafiltration. Papain and bromelain were used to perform digestion followed by ultrafiltration on a pilot-scale system using a ceramic membrane with a cutoff of 150 kDa; and a PESH membranes with a MWCO of 4 kDa. Finally, dry matter, antioxidants, amino acid composition, protein efficiency ratio value and molecular weight distribution were determined. The subject is interesting given the scope. However, if the manuscript is generally well written, we note here and there many imprecisions. In the following, I will list just a few of the points that I think are important to correct/clarify.

First of all, the authors in the manuscript only had to test the antioxidative effects of the peptides obtained by enzymatic digestion. I would therefore suggest that the title be modified and that the terms “effect on bioactivity” be replaced by “antioxidant effects”, which here is more in line with the content of the manuscript.

There are many pieces of information missing from the introduction. For example, on the lines 34-35, it is written: "Fish RRM has a high protein content". Do the authors have any idea of this protein content? If so, please add the value and give the reference. Same on the line 36, it would be nice to give the readers the composition of fish RRM. Also, in line 72, the authors state that "The Norwegian whitefish industry generates huge amounts of RRM each year" without giving any idea of the quantities.

Lines 99-101: "5 grams of SPH was dissolved in 500 mL of distilled water and filtrated through a ceramic membrane with 150 kDa MWCO in order to remove high molecular weight peptides". The question is whether at 150 kDa it would still be peptides or proteins? So, after application of the 4 kDa cut-off PESH membrane, the retentate obtained R4 contains not only peptides, but most certainly many proteins with MW up to 150 kDa, since no information was provided on whether the enzymatic digestion of proteins was complete in just 60 minutes. All this must be clearly clarified by the authors.

Figure 1 does not contain the error bars. How many times were the experiments repeated (n=?). The same goes for Figures 4a and 4b.

Please check the layout and the presentation of Table 1. Also, the titles of the Tables are usually given above the tables. This does not seem to be the case in the manuscript (Table 2 for example), please check that.

Figures 3a and 3b do not contain the legend and it is not clear what each of the colors refers to.

The permeate fluxes during the ultrafiltration processes were not given. It would be nice to add this data to show the efficiency of the ultrafiltration operation.

Line 388: please add the specific activity of the enzymes used (papain and bromelain). How was the pH neutralized?

Line 445: please add the information about the Superdex column as well as the separation conditions: solvents used, separation mode (isocratic/gradient), elution program, flow rate, separation time.

In the method part, it is not explained how the different peptide fractions (> 20; 20-15; 15-10; 10-5; 5-2; 2-1; 1-0.5; 0.5-0.2; <0.2 kDa) were obtained.

Author Response

Dear reviewer 3, 

Please see the attachment. 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Thank you for responding to my comments, all points discussed have been perfectly clarified.

Best luck for future research.

Author Response

Dear Reviewer 1, 

Thank you for your time and effort. Your comments were highly appreciated and was very helpful to improve our manuscript. 

Best of luck for future research for you as well. 

Reviewer 3 Report

Dear authors, thank you for trying to answer the points raised. As mentioned in my previous report, I had underlined only some of the points (among many shortcomings) that seemed to me important to correct/clarify to bring the manuscript into an acceptable form for publication. Unfortunately, the answers given so far do not seem to be satisfactory and it appears that there is a lack of scientific rigor in what is done.

All the changes made are not clearly identified in the manuscript and in the cover letter, also the lines where the changes were made are not given, which makes it difficult to follow the changes made.  An example is "300,000 tones" which has been added but is not marked in the text, and so on.

In point 3, peptides are short chains of amino acids while proteins are large macromolecules that comprise one or more long chains of amino acid residues. This is a simple definition to differentiate the two. Therefore, large fragments up to 150 kDa (fractions R4, R150 and P150) cannot be considered as peptides. Furthermore, if proteins are not water soluble as the authors would suggest, then why did they dissolve the SPH in distilled water rather than in a buffer solution? Also, there are several ways to determine the protein content of a solution. I remain convinced that fractions R4, R150 and P150 would be more a protein fraction than a peptide fraction, especially as it would be difficult under the conditions presented in this manuscript to perform the complete digestion of proteins. This remains a major issue that needs to be properly clarified both in the method part as well as in the discussion of the different results.

Point 4, if the experiments have not been duplicated/triplicated, how can the reproducibility of the experiments be confirmed? This alone could be a reason to reject the manuscript. Furthermore, if the SPH filtration is a time-consuming process as the authors point out in their answer, this raises the question of the efficiency of their process, especially for an industrial scale application

Point 7, the authors give an average of the flux, this probably means that they had to measure the flux at different times during the filtration to obtain the average, is this correct? If so, why not produce the data in a way that shows the drop in permeate flux? Rather than giving an average of the flux, it might be more appropriate to estimate the steady state permeate flux.  Also, a schematic diagram of experimental systems would have allowed the reader to better understand the implementation of these processes.

Points 9 and 10, for a research work, as explained in the instructions for authors, the methods and protocols should be described in detail with sufficient information to allow others to replicate the experiments performed. Not having any information about the characteristics of the column used, the types of solvents or even the mode of elution (isocratic/gradient), for example, how to understand the results presented?  This question also remains open.

Good luck.

Author Response

Dear Reviewer 3, 

Please see the attachment.

Author Response File: Author Response.docx

Round 3

Reviewer 3 Report

Dear authors, thank you for the answers provided. Congratulations for the work and good luck in the continuation of your project.