Round 1

Reviewer 1 Report

I have no comments and accepts it as it stands

Author Response

Thank you for your evaluation and for taking the time to review the manuscript.

Reviewer 2 Report

In my opinion reviewed manuscript “Pilot-Scale Protein Recovery from Cold-Pressed Rapeseed Press Cake: Influence of Solids Recirculation” present decent level of the expertise in the rapeseed protein extraction technology.

I suggest accepting the manuscript after minor revision.

Below you can find detailed description.

 

1. I have found the Introduction very informative and creating well background of the study. However, I would expect the clearer explanation of the goal of the study showing its scientific value and accentuating the novelty that can be drawn from the preceding part of the Introduction.

Author Response

Comment #1: I have found the Introduction very informative and creating well background of the study. However, I would expect the clearer explanation of the goal of the study showing its scientific value and accentuating the novelty that can be drawn from the preceding part of the Introduction.

Answer: Thank you for pointing this out. The aim has been rephrased to reflect the novelty and purpose of the study (p3: lines 121-128). There are only a few studies on the pilot plant scale and to our knowledge no studies investigating the effect of recirculation on pilot scale extraction of rapeseed protein.

Reviewer 3 Report

Manuscript ID: processes-1621141

Title: Pilot-Scale Protein Recovery from Cold-Pressed Rapeseed Press 2 Cake: Influence of Solids Recirculation

Authors: Cecilia Ahlström, Johan Thuvander, Marilyn Rayner, Inger-Cecilia Mayer Labba, Ann-Sofie Sandberg, Karolina Östbring

 

Overview and general recommendation:

The aim of the manuscript was to investigate the pilot scale recover protein from cold-pressed rapeseed press cake, and to investigate the effect of recirculation of the SSF on the protein yield, protein content glucosinolate and phytate in the various process streams.

The subject of the extraction of protein form the rapeseed press cake is not a new subject, the “golden ages” of the investigation were in the 80-90s of the last century, there are even some industrial products available on the market. But still rapeseed proteins are not as popular as soy proteins that is why improvement in the quantity and quality of obtained rapeseed protein products needs to be investigated. However, in my opinion, authors of the manuscript did not stress the novelty of the experimental method that was described in the manuscript.

Below I give also some concerns that require review:

Comments:

1. In the introduction part there are some inaccuracies that need to be cleared up. In the text there is information: “The processes most commonly used for rapeseed oil production are hot-pressing and cold-pressing.” which is not true for industry methods. Still for the production of refined rapeseed oil (which is a main product) the most commonly used is extraction.
2. In the Introduction (lines from 83) part there are also described methods that were used in other experiments, were those methods also applied on the press cake, with 20% of oil residue? Or defatted one (lines 101, 105)
3. Material – there is a lack of the characteristic of the raw material – so the press cake, there is no information on the amount of residual (after pressing) oil content or protein level? It is also not clear whether the cake was delivered once from a single production or if the experiment was somehow repeated on another production batch.
4. Methodology – “2.6.5 Water Holding Capacity” – line 207 – please explain the validity of presenting the results of this method, in fact it is the only (out of many) parameters that define the technological properties and are probably not fully in line with the aim of the manuscript.
5. Results and discussion part – this part of the manuscript is also divided into chapters - such as “3.1. Extraction” – in some of them there are only presented results, without a summary to suggest what this shows or whether it is better or worse.
6. Title of the chapter 3.1 “Extraction” please specify if it is oil or protein extraction, as it is not clear.
7. Line 399 – “The fat content increased along the recovery process” - can the authors explain why the fat was not removed and whether this had a negative effect on the protein extraction process.
8. Some parts of the text are in green font.

Author Response

Comment #1: In the introduction part there are some inaccuracies that need to be cleared up. In the text there is information: “The processes most commonly used for rapeseed oil production are hot-pressing and cold-pressing.” which is not true for industry methods. Still for the production of refined rapeseed oil (which is a main product) the most commonly used is extraction.

Answer: Thank you for pointing this out. We have now changed the nomenclature to expeller pressing followed by extraction by solvents instead of hot-pressing (p1: lines 42-44 and p2: lines 45-52).

Comment #2: In the Introduction (lines from 83) part there are also described methods that were used in other experiments, were those methods also applied on the press cake, with 20% of oil residue? Or defatted one (lines 101, 105)

Answer: Thank you for your valuable comment. This is now clarified in lines 92-93 on page 2 and 99-100 on page 3.

 

Comment #3: Material – there is a lack of the characteristic of the raw material – so the press cake, there is no information on the amount of residual (after pressing) oil content or protein level? It is also not clear whether the cake was delivered once from a single production or if the experiment was somehow repeated on another production batch.

Answer: Thanks for the comment. We have included the proximate analysis of the rapeseed press cake in the material section 2.1 (p3: lines 133-135). We also clarified that we used material from two different industrial batches which were very similar in proximate analysis. We have been using rapeseed press cake from the same producer for 6 years in the lab and the proximate analysis are very stable over the years.

Comment #4: Methodology – “2.6.5 Water Holding Capacity” – line 207 – please explain the validity of presenting the results of this method, in fact it is the only (out of many) parameters that define the technological properties and are probably not fully in line with the aim of the manuscript.

Answer: It was requested by another reviewer to give an indication of the state of the protein after repeated pH shifts.

Comment #5: Results and discussion part – this part of the manuscript is also divided into chapters - such as “3.1. Extraction” – in some of them there are only presented results, without a summary to suggest what this shows or whether it is better or worse.

Answer: Thank you for your comment. We have rewritten the aim (p3: lines 121-128) and the first line in the result section to emphasize the two parts of the study (p7: lines 266-269). We have merged sections 3.1-3.3 to one and added comparisons with other studies where it was lacking (p7: lines 281-283 and p8: lines 284-289). 

Comment #6: Title of the chapter 3.1 “Extraction” please specify if it is oil or protein extraction, as it is not clear.

Answer: Thank you for the valuable comment, we have now changed the title (see comment above).

Comment #7: Line 399 – “The fat content increased along the recovery process” - can the authors explain why the fat was not removed and whether this had a negative effect on the protein extraction process.

Answer: Thank you for your comment. We have clarified this in line 423 on page 12. The recovery process is water-based and therefore water-soluble substances will be enriched in the aqueous phase. In Table 2 it can be seen that the concentration of carbohydrates is decreasing with the number of cycles. Protein and fat on the other hand are enriched in the remaining precipitate. However, the total mass of the precipitate is decreasing and can be seen in Table 1. This means that both fat, protein, and carbohydrates are lost in the supernatant fraction after precipitation but to different degrees.

Comment #8: Some parts of the text are in green font.

Answer: Thank you for pointing this out. All text that was marked green in the manuscript was revised work since the last reviewer round. We have now changed all text to black font.

Reviewer 4 Report

Interesting study about protein recovery from cold-pressed rapeseed press cake on pilot scale. The revised article can be publish in Processes in its actual form.

Author Response

Thank you for your evaluation and for taking the time to review the manuscript.

Round 2

Reviewer 3 Report

Manuscript ID: processes-1621141

Title: Pilot-Scale Protein Recovery from Cold-Pressed Rapeseed Press 2 Cake: Influence of Solids Recirculation

Authors: Cecilia Ahlström, Johan Thuvander, Marilyn Rayner, Inger-Cecilia Mayer Labba, Ann-Sofie Sandberg, Karolina Östbring

 

Overview and general recommendation:

The manuscript has been improved, the authors have modified the purpose and made the changes to the text indicated in the comments. Comments that needed explanation were properly explained by the authors. In my opinion manuscript now conforms to Processes standards.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

General comments

The current manuscript by Ahlstrom and co-workers, entitled, “Pilot-Scale Protein Recovery from Cold-Pressed Rapeseed Press 2 Cake: Influence of Solids Recirculation” have used pH precipitation as a tool to recover protein that can be precipitated at acid or alkaline pH on pilot scale. The pH precipitation has been utilized as a step to get the enriched fractions of the protein of interest in the scientific literature. Hence, it is not new or novel concept, except that the authors have used pilot scale experiments in their investigation.

However, It is not clear from the MS that what is the use of this pilot scale fraction or What was the main goal of getting all these fractions? No experiments have been performed or conducted to investigate that what is present in the collected fraction (i.e., characterization). In my view, the manuscript lacks critical experiments to demonstrate that what is recovered on the pilot scale has any value to industrial application. The current MS lacks experiments on the functional aspects of protein characterization that is very important to get the complete picture. Hence, the MS in its present form is not suitable for the next step in the process of publication.

Major points

• No efforts have been devoted to study the presence of (i) antinutritional compounds, (ii) colours, (iii) amino acid composition of the protein.
• Have authors thought of studying the parameters like solubility, emulsification capacity and emulsification stability of their collected samples (proteins).
• What about testing the bioactive properties of the proteins?
• In simple terms what is the value proposition of this work. Although authors have touched on this (other’s investigators finding/work) in the introduction of the MS line 50-63).
• Material and Methods: I also have some concerns about the equation 1 and 2, what’s the difference between “mass of protein in rapessed press case” versus “mass of protein in the rape seed press cake or spent solids”.
• Material and Methods: Statistical analysis, why only duplicate samples why not triplicate, authors have a lot of sample amounts (i.e., quantity)? Please explain.
• Material and Methods: what the authors meant by samples were placed in freeze dryer for four days. After freezer drying why the samples were dried at 103 C for one day. Why not desiccator was used after freeze drying?
• Results, Table 1: Please double check the superscript symbols, some needs to be changed.
• Results line 223, after “isoelectric precipitation, please rephrase to just pH precipitation. This is a mixture of protein at the selected pH.
• Conclusion line 343 to 346: Is this a part of conclusion?

Author Response

Reviewer number 1

Thank you for the valuable comments. We have done our best to make the suggested changes and hope you find them satisfactory in the revised manuscript. Please find our responses to the comments below. In the revised manuscript new and significantly modified sections are indicated with track changes.

Q1: No efforts have been devoted to study the presence of (i) antinutritional compounds, (ii) colours, (iii) amino acid composition of the protein.

Response: The scope of the present study was to optimize the protein extraction and recovery yield by a recirculation approach on the pilot scale. We agree that analysis of the mentioned compounds is important and interesting and are addressing these matters in our coming studies. 

Q2: Have authors thought of studying the parameters like solubility, emulsification capacity and emulsification stability of their collected samples (proteins).

Response: Yes, functional properties such as solubility and emulsifying properties as well as long-term emulsion stability are studied in a manuscript that will be submitted very soon. The focus of the present study was the scale-up of the process.  

Q3:What about testing the bioactive properties of the proteins?

Response: This is an interesting suggestion, however is beyond the scope of the experimental framework in the present study. 

Q4: In simple terms what is the value proposition of this work. Although authors have touched on this (other’s investigators finding/work) in the introduction of the MS line 50-63).

Response: Thank you for pointing this out. The scientific value of this work is to increase the knowledge on the extraction of rapeseed protein on the pilot scale. Many studies have been performed on the laboratory scale but in order to up-scale extraction to industrial scale, a lot of challenges need to be addressed. Here pilot scale studies give valuable data on the road of process up-scaling but data in the literature on this subject is scarce. Furthermore, protein yield is always a challenge that has to be solved in order for an industrial application to be viable. One of the main findings in this work is how recirculation of the spend solids greatly impacts the protein yield which enables better utilization of the raw material.

Q5: Material and Methods: I also have some concerns about the equation 1 and 2, what’s the difference between “mass of protein in rapeseed press cake” versus “mass of protein in the rape seed press cake or spent solids”.

Response: Thank you for indicating this uncertainty. The accumulated protein yield is a sum of the extracted proteins divided by the protein in the rapeseed press cake. By using this equation, we get a measurement of the efficiency of the total process including the recirculation steps. Relative protein yield is a measurement of the efficiency in each of the recirculation steps. Here the mass of the extracted protein is divided by the starting material of the specific recirculation cycle. For the first run, the starting material is rapeseed press cake but for the following recirculation steps, the starting material is the spent solids fraction from the decanter.  We changed “mass of protein in rapeseed press cake or spent solids” to “mass of protein in starting material” and hope that this improves the clarity (line 154 and 160).

Q6: Material and Methods: Statistical analysis, why only duplicate samples why not triplicate, authors have a lot of sample amounts (i.e., quantity)? Please explain.

Response: The extraction and recirculation trials were performed in triplicates and each analysis was conducted in duplicate which gives six data points per sample and parameter. This has been clarified in line 180.

Q7: Material and Methods: what the authors meant by samples were placed in a freeze dryer for four days. After freezer drying why the samples were dried at 103 C for one day. Why not desiccator was used after freeze drying?

Response: Thank you for pointing this out. Our desiccators were unfortunately not large enough so we had to use the oven instead to ensure that the powder was not hydrated by the ambient air before the analysis.

Q8: Results, Table 1: Please double check the superscript symbols, some needs to be changed.

Response: As suggested by the reviewer, we have double-checked the superscript symbols in table 1 and corrected the letters.

Q9: Results line 223, after “isoelectric precipitation, please rephrase to just pH precipitation. This is a mixture of protein at the selected pH.

Response: Thank you for the valuable comment, we corrected it to pH precipitation (line 227).

Q10: Conclusion line 343 to 346: Is this a part of conclusion?

Response: We agree that the text line at 343-346 in the conclusion has more the character of suggestions of future work and have deleted the paragraph. 

 

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript entitled “Pilot-Scale Protein Recovery from Cold-Pressed Rapeseed Press Cake: Influence of Solids Recirculation” is dedicated to protein isolation from rapeseed press cake. The method was based on the precipitation of protein in solid and liquid fraction. There are several papers on this topic in literature, but the present work adds value by operating at a pilot scale level for the extraction of protein, by using more recirculation steps in order to increase the protein content.  

I recommend minor revision with some improvements.

Recommendations:

1. The Abstract adequately describes the work and emphasizes the most important results.
2. The Introduction section is well written and clear. It would be useful to specify the scientific name of rapeseed, when first mentioned in the text. At line 80 please add the references after “Bérot et al.” not at the end of the sentence. The same at line 85-86 “Chabanon et al.”.
3. Please specify the concentration of citric acid, line 139.
4. In equations 1, 2 and 3 please add x 100 at the end of each equation.
5. Please add the reference number after “Yoshie-Stark et al.”, line 248.
6. Line 258, add reference number “Bérot et al.”, also “Fetzer et al.”, “Östbring et al.”. Line 278 “Pedroche et al.”.
7. Please add pages at reference 2, line 360.

The paper is well-organized and the methodology is well-explained. I recommend publishing the manuscript in the Processes journal, with minor revision, as it makes a valuable contribution to literature in this research domain.

Comments for author File: Comments.pdf

Author Response

Reviewer number 2

 

Thank you for the valuable comments. We have done our best to make the suggested changes and hope you find them satisfactory in the revised manuscript. Please find our responses to the comments below. In the revised manuscript new and significantly modified sections are indicated with track changes.

 

Q1: The Abstract adequately describes the work and emphasizes the most important results.

Response: We thank the reviewer for the comment!

Q2: The Introduction section is well written and clear. It would be useful to specify the scientific name of rapeseed, when first mentioned in the text. At line 80 please add the references after “Bérot et al.” not at the end of the sentence. The same at line 85-86 “Chabanon et al.”.

Response: Thank you for these suggestions. We have added the scientific name of rapeseed and its closest relatives which together constitute the second-largest cultivated oilseed crop (line 35). We also changed the reference number directly after “Bérot et al.” instead of at the end of the sentence (line 81). The same thing was changed for “Chabanon et al” on line 86.

Q3: Please specify the concentration of citric acid, line 139.

Response: We thank the reviewer for pointing this out. Citric acid was added in powder form (>95% purity) and the exact amount was unfortunately not measured. The powder was added under stirring until pH 3.5 was reached. The purity of the citric acid has been added to line 113.

Q4: In equations 1, 2 and 3 please add x 100 at the end of each equation.

Response: We thank the reviewer for pointing this out and have added x 100 at the end of equations 1, 2 and 3 (lines 159, 160 and 161).

Q5: Please add the reference number after “Yoshie-Stark et al.”, line 248.

Response: We agree and have updated the reference number after “Yoshie-Stark et al.” (line 252).

Q6: Line 258, add reference number “Bérot et al.”, also “Fetzer et al.”, “Östbring et al.”. Line 278 “Pedroche et al.”.

Response: Thank you for pointing this out. We have changed the reference number to directly after the author name on lines 263, 265, 268, 270 and 283.

Q7: Please add pages at reference 2, line 360.

Response: We have fixed the error and added pages to reference number 2 (line 367).

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors claim that the their work is complete, as presented and is valuable. However (after review of revised version), in my view critical data about the functional properties of the proteins in question are missing. The functional data information is not only important and interesting; it is critical to present along with this data. For any purification or enrichment strategy, the authors have to provide the specific activity of the protein of interest. This could be increased enzyme activity (e.g., acidic, basic etc.), removal of antinutritional of compounds or enrichment of certain amino acids (any protein). The functional data would tell the purity achieved in terms of fold purity or enrichment at each step of the process. I am still of the opinion that authors have to show functional data in this MS to make a complete story (i.e., their process has a value for end users). After careful reading, I can’t support his manuscript for publication..

Author Response

Reviewer number 1

We have discussed your suggestions for improvements of the manuscript and agree that data of characterization is important. We have two suggestions:

#1 We have some unpublished data from a parallel experiment were rapeseed protein have been isolated by the same conditions (leaching at pH 10.5, precipitation at pH 3.5) conducted on laboratory scale. For this material we have:

- Content of glucosinolate in rapeseed press cake and protein precipitate

- Content of phytic acid in rapeseed press cake and protein precipitate

- Emulsifying properties of rapeseed protein in the precipitate

However, the existing data is for single extractions and no data are currently available for the re-circulation. (we had plenty of material from the experiment series but since the material have been in the oven it is hard to analyse functional properties of the precipitate)

#2 We can re-run the re-circulation experiment on the pilot scale and analyse the following parameters for “precipitate” and “light phase containing unprecipitated protein” from the various recirculation runs

- Color

- Water holding capacity

- Emulsifying properties and emulsion stability (1 week stability in 4°C)

We are currently prohibited to visit the lab and the pilot plant because of corona restrictions, but Lund University has decided to withdrawn the restrictions for staff from Aug 16th.

So – in your opinion, what is the best choice for the manuscript?

Author Response File: Author Response.pdf