Quality Attributes of Ultra-High Temperature-Treated Model Beverages Prepared with Faba Bean Protein Concentrates

Round 1

Reviewer 1 Report

The topic of this article is interesting and indeed there is very few studies in the literature with emulsions with protein concentration above 2%. Yet, there are still a couple of clarifications that need to be done. For instance, authors should make it more clear that one of the aims was to produce a model plant protein beverage, this only becomes clear in the last section of discussion when authors talk about the volatiles. Authors should include in the methodology that this was done.

I suggest re-structuring the abstract as it is a bit confusing. For example, it only talks about the faba bean emulsion and then out of nowhere it compares to soy, then analysis of volatiles, and then to a model beverage. Therefore, I suggest making a clear statement of what were the aims of this study and what analysis were carried out before reporting the results so that readers can first understand what was done.

The introduction on the other hand is quite well structured and provides enough info on what has been done and what is going to be addressed.

By biggest concern is in the methodological section. Immediately after mixing the materials authors used vacuum filtering to remove undissolved material. If there was a large amount of undissolved material how can authors affirm that the emulsions indeed had 5,6,7 and 8% of protein concentration? Didn´t this filtering step possibly remove a good number of proteins? Authors should also make clear why there was a need to homogenize the emulsions after a previous processes that is already supposed to homogenize the solutions? Also a brief explanation of why pH 8 was chosen should be added.

Authors should also explain why the particle size distribution measurements were carried out with a different formulation than the prepared emulsion (with Deionized water and without lecithin). It is misleading that it comes under the “Physicochemical properties of the emulsion” section since it was not carried out on the previously prepared emulsions. Same goes for Flocculation index (FI) and coalescence index (CI) measurements, in which emulsions where they performed?

In section 2.3.7., it is not clear what was the purpose for mixing protein isolates, concentrates and soy, authors should make that clear.

Discussion on the creaming index and SDS-PAGE is missing, author only reported the results.

Conclusion should also be re-written. First author should talk about the study on the emulsions itself. Then talk about the model beverage and only then compare it to soy.

 

Some other minor comments:

Title: remove high from title as it is too vague, or use high concentration protein

Line 16: Lower

Line 19: Define SDS-19 PAGE

Line 36: How about coconut milk?

Line 39: Contribute to and why use but?

Section 2.2. I suggest putting figure 1 later as at this point readers do understand the details written in this Figure.

Line 118: Explain the reason why emulsions were made using 5mM phosphate buffer and not deionized or distilled water.

Line 227: punctuation

Section 2.4: PCA analysis was not mentioned in the methodology

Line 232: were not are

Line 235: remove s from Tukeys

What is this 3.1. section? Isn’t this methodology?

Figure 2: Improve figure colors are to light and the legends are blurry

Figure 3: The scale bar is not visible.

Line 315: depended.

Figure 4: Improve figure, the regression formulas are not visible, what does RFI stand for? What is the measuring unit for protein concentration?

Author Response

My responses are attached.

Author Response File: Author Response.docx

Reviewer 2 Report

I have reviewed this manuscript

Ultra-high temperature treatment on quality attributes of high  protein faba bean emulsions

Malik Adil Nawaz, Tanoj Kumar Singh, Regine Stockmann, Hema Jegasothy, Roman Buckow

In this study, the authors prepared a variety of emulsions (5-8% faba bean protein concentrate), homogenized them, and then subjected them to a system of ultra-high temperatures (UHT) and characterized them using various methods. In reviewing a manuscript, the tendency is to focus on the negatives; however, in this case, there are many positive aspects that I want to emphasize, including excellent structure and presentation of the methodology.

Major Issues

The authors leave unanswered one of the main questions. What is the purpose of conducting the research? There is a brief literature review in line 59 ff-line 85 about faba beans in various emulsion systems. The authors do not provide any information on the protein concentration of faba beans in these studies or how the emulsions were used. The authors only mention in line 86 that there has been less than 2% concentration in most of these reports. If the novelty of this work is the higher concentration of faba bean protein concentrate, then this should be clarified and elaborated. Why has it not been done in the past? Could it be related to different applications that the authors have considered (plant-based milk beverage)? If so, what applications have been considered in the reported emulsion systems?

The authors do not indicate what and how much undissolved material was trapped in the vacuum filtration step. How do they know the final concentration of faba bean protein concentrate is the same as the nominal one?

The stability of the emulsion is one important factor in emulsions. It is unclear whether these emulsions are stable. Particularly when it comes to the particle size analysis (sections 2.3.1 and 3.2.1) this is very important. The authors did not say how soon after emulsion preparation they measured the particle size.  Was this timing (preparing the emulsion to measure particle size) more or less the same for all of these emulsions?

The reviewer found some experiments such as SDS-PAGE or Surface hydrophobicity not to be justified. More details would be appreciated.

More discussion on Figure 7 would be appreciated.

The conclusion is modest for the number of experiments and methods used. Please amend.

Minor Issues

Figure 1: “multimixing” is better to be changed to “high shear mixing”

Different notations (D4,3 and D^4,3) have been used for D [4, 3] (and D [3, 2]). I would suggest using only one.

Captions of Figure 1 and Figure 7 could be improved.

 

Author Response

My responses are attached

Author Response File: Author Response.docx

Reviewer 3 Report

The paper tries to cover the use of faba bean protein for the development of O/W food grade emulsions, however, there are dozens of really nice published papers in this regard and I haven't found any new achievements in this paper. In fact, in an introductory section are cited several articles such as Felix et al. even with the same oil.

Here are some points to think about it.

• Abstract. Although I assume it is in percentage by weight, please specify the units of all percentages.
• As a suggestion for a better understanding of the results, if the letters FPC is in all the acronyms of the samples, it is useless. For example, replace hFPC5 by h-5.
• Line 247. If authors are analyzing droplets and not particles, authors should describe droplet size distributions.
• Line 250. In my opinion, the sentence "The D4,3 and D3.2 represents the proportion of large and small particles, respectively." is not correct. Each of these calculated mean diameters have their interpretation of the average size of all droplets in the emulsion, but that phrase can lead to an error in their meaning.
• Table 1. Some of the emulsions show D_3,2 higher than D_4,3, which is physically and mathematically is not possible. Therefore, in addition to reviewing these results, I suggest reviewing the full interpretation of those results.
• Line 267. If it is an emulsion and not a suspoemulsion, why are drop and particle sizes analyzed? What are the particles?
• Line 287. This phrase is incorrect. Are flocculation and coalescence very important destabilization mechanisms, but and ostwald ripening? What about the cremate?
• Figure 4. I don't understand the fit to a straight line of that experimental data.
• The calculation of the cremate index is valid although somewhat rudimentary, but the duration of the study is not indicated, nor whether it was performed at various dates or the aging time of the rationed CI.
• Although the authors of Felix et al. paper use thatmethod for coalescence and flocculation, I do not consider it a very reliable method for evaluating such destabilization mechanisms. For coalescence, it is simpler to perform measurements of droplet size distributions over time and evaluate whether it is coalescence or Ostwald ripening. For flocculation, CLSM images are sufficient.

Author Response

My responses are attached.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Considerable changes were made to the manuscript and now the main aim and the whys as of each analysis was carried out is now clear. However, one point is still very much intriguing and for that reason I still suggest major review until this point is clarified. Authors still must provide further info on the way protein solutions were prepared. It is common knowledge that the solubility of commercial pulse proteins is very low and that the protein at very low concentrations can be dispersed, but at high protein concentrations it is really hard to obtain dispersed solutions. The insoluble nature of the protein would greatly limit its use to prepare “soluble” dispersions. Therefore, authors should make clear how these high concentrated faba beans protein dispersions were produced. It was not left overnight to hydrate for example? It I still unclear to me how directly mixing the protein powders with buffer and oil and adjusting the pH made this sample soluble. Moreover, if there was no visible unhydrated particles then why did the authors filter the solution in the first place? If this were the case, author should provide results for solubility of the protein they used as the solubility of this material would be way above solubility of any other commercial pulse protein isolates/concentrates.

Other minor observation:

Abstract: There should still be made a link between the beverage and emulsion. Authors should state that the beverages were produced with these emulsions for example.

Line 211: Is 1% oil considered high lipid concentration?

Section 3.2. remains out of place

Author Response

Authors’ response to the reviewer’s comments

We thank the editor and reviewer for their careful reading of the manuscript and their constructive comments. We have taken the comments on board to improve and clarify the manuscript. Please find below a detailed point-by-point response to all comments (reviewers’ comments in black, our replies in red).

Major observation:

Authors still must provide further info on the way protein solutions were prepared. It is common knowledge that the solubility of commercial pulse proteins is very low and that the protein at very low concentrations can be dispersed, but at high protein concentrations it is really hard to obtain dispersed solutions. The insoluble nature of the protein would greatly limit its use to prepare “soluble” dispersions. Therefore, authors should make clear how these high concentrated faba beans protein dispersions were produced. It was not left overnight to hydrate for example? It I still unclear to me how directly mixing the protein powders with buffer and oil and adjusting the pH made this sample soluble.

Response: We agree with the reviewer that commercial plant-protein concentrates/isolates have lower protein solubility at higher concentrations. Therefore, we did not claim that the all added protein was solubilised in the phosphate buffer (pH 8) prior to emulsification.

The faba protein concentrate and 0.2 % (w/w) sunflower lecithin (for co-stabilisation) was mixed with phosphate buffer pH 8 at ~40°C for 10 min (information of operating temperature added in the revised version, Line 149-155) to ensure full hydration of protein. After hydration, the speed of the high shear mixer was increased to 10000 rpm and the sunflower oil (1 % w/w) was added slowly to make coarse emulsion.

For more clarification to the readers, this information is added in the revised methodology of coarse emulsion preparation and Figure 1.

Moreover, if there was no visible unhydrated particles then why did the authors filter the solution in the first place? If this were the case, author should provide results for solubility of the protein they used as the solubility of this material would be way above solubility of any other commercial pulse protein isolates/concentrates.

Response:

The reason of vacuum filtration.

After the preparation of the coarse emulsion, no unhydrated particles were visible. However, samples were vacuum filtered to ensure the solution only contains fully solubilised ingredients. Unhydrated particles can block the pressure homogeniser, which was used in the next step to the preparation.

Nitrogen analysis of filter papers.

We already reported that no visible particles were observed on the filter paper; therefore, we did not conduct nitrogen analysis of the filter papers (this statement is clearly added in revised version, Line 159). However, we acknowledge the reviewer’s point and will conduct protein analysis of filter papers in future studies. 

Other minor observation:

Abstract: There should still be made a link between the beverage and emulsion. Authors should state that the beverages were produced with these emulsions for example.

Response: The abstract was revised to make a link between emulsions and model beverages according to reviewer suggestions (Line 15-18).

Line 211: Is 1% oil considered high lipid concentration?

Response: Our statement was corrected “to minimise the artifact of other minor constituents of the model beverage” and supported with relevant citation (No. 48) from literature Line 216.    

Section 3.2. remains out of place

Response: The results of section 3.2 have been moved to the methodology section 2.3.3 (Line 170-173). 

 

Reviewer 2 Report

I do not have any further adjustments. Thank you

Author Response

Thanks again for reviewing our manuscript.

Reviewer 3 Report

Taking into account the review carried out by the authors, I consider that the manuscript is of sufficient quality to be published on Foods.