Proposed Methods for Testing and Comparing the Emulsifying Properties of Proteins from Animal, Plant, and Alternative Sources

Round 1

Reviewer 1 Report

This paper provides a review of which properties of plant based proteins that influences their function as emulsifiers in foods, and provides a detailed suggestion of standardized protocols to test and compare such proteins. In the view of the large industrial interest of plant based formulation, and academic research in the field, such a standardized protocol is much in need. The review is comprehensive both in terms of theoretical background, characterization methods and coverage of relevant literature. Both figures and tables are clear and informative. Especially the artwork illustrations are very helpful (e.g., Fig. 13, 14 and 16). Presentation of specific empirical data to illustrate the effects and methodologies is also helpful. Some specific question and comments below:

1. As I understand the organization, Section 2 gives background and Section 3 discuss protocols. However, in some cases in Section 2 (for example p. 3, lines 131 and onwards), there seem to be protocol suggestions. I think the idea of clearly separating the two is helpful for the reader.
2. The protocol section (3) is very extensive in describing a large number of methods for characterizing each property (for example Kjeldahl, Dumas and spectroscopy for proximate composition, p. 14; many methods for protein content on p. 16 and emulsification capacity yon pp. 19). From one perspective, I like the extensive list and overview. However, if the aim is to suggest a standard protocol, it would be interesting with some recommendations about which of the methods are most appropriate (surely there must be differences in validity and reliability?). Is there a risk that different studies using different methods for (for example) emulsion stability characterization would be difficult to compare due to this?
3. Although I understand the idea, it is technically wrong to equate a force with a pressure (p. 4, lines 169-170). Could this be reformulated slightly?
4. For some figures I was wondering if it is based on empirical data or if it is a schematic illustration (mainly Fig. 6). So in general, when based on experimental data in previous study, a short reference in the figure legend (like done in Fig. 5) would help be helpful for me as a reader (both in identifying this as coming from empiric data) and for easily identifying the source to finding out more about it.

Author Response

This paper provides a review of which properties of plant based proteins that influences their function as emulsifiers in foods, and provides a detailed suggestion of standardized protocols to test and compare such proteins. In the view of the large industrial interest of plant based formulation, and academic research in the field, such a standardized protocol is much in need. The review is comprehensive both in terms of theoretical background, characterization methods and coverage of relevant literature. Both figures and tables are clear and informative. Especially the artwork illustrations are very helpful (e.g., Fig. 13, 14 and 16).

• We thank the reviewer for their constructive comments and suggestions on our manuscript. We have revised it accordingly and included a detailed list of responses below.

Presentation of specific empirical data to illustrate the effects and methodologies is also helpful. Some specific question and comments below:

1. As I understand the organization, Section 2 gives background and Section 3 discuss protocols. However, in some cases in Section 2 (for example p. 3, lines 131 and onwards), there seem to be protocol suggestions. I think the idea of clearly separating the two is helpful for the reader.

• We thank the reviewer for pointing this out. In response, we have included a discussion of the importance of solubilizing the proteins properly and then measuring the fraction of soluble and insoluble proteins present in the later section on the impact of emulsifiers on emulsion formation (Section 3.2)

 

2. The protocol section (3) is very extensive in describing a large number of methods for characterizing each property (for example Kjeldahl, Dumas and spectroscopy for proximate composition, p. 14; many methods for protein content on p. 16 and emulsification capacity yon pp. 19). From one perspective, I like the extensive list and overview. However, if the aim is to suggest a standard protocol, it would be interesting with some recommendations about which of the methods are most appropriate (surely there must be differences in validity and reliability?). Is there a risk that different studies using different methods for (for example) emulsion stability characterization would be difficult to compare due to this?

• We thank the reviewer for this comment. As mentioned, there are many different methods available that differ in their advantages and disadvantages (including costs, ease of use, availability, accuracy, and sensitivity).  In response, we have revised the manuscript to include some recommendations about the most appropriate methods for determining proximate analysis and emulsion performance. 

 

3. Although I understand the idea, it is technically wrong to equate a force with a pressure (p. 4, lines 169-170). Could this be reformulated slightly?

• We thank the reviewer for highlighting this. As mentioned, forces and pressures should not be equated, since pressure is force per unit area.  We have revised the manuscript to correct this (changing “force” to “stress” where appropriate).

 

 

4. For some figures I was wondering if it is based on empirical data or if it is a schematic illustration (mainly Fig. 6). So in general, when based on experimental data in previous study, a short reference in the figure legend (like done in Fig. 5) would help be helpful for me as a reader (both in identifying this as coming from empiric data) and for easily identifying the source to finding out more about it.

• We thank the reviewer for pointing this out. In response, we have revised the figure captions to clarify the origin of the data (schematic, predictions, or experimental data)

Reviewer 2 Report

Dear authors,

Thank you for writing the review. I am sure that the field will find it a useful paper that will guide them toward 'easier' design of food emulsion products.

Although I feel that your work is rather complete, I still would like to point out that in terms of dynamic effects taking place your review is lacking. An emulsion droplet size is a resultant of droplet formation and coalescence, and now you are taking this 'as one' overall effect. I feel that the work done related to elucidating these effects individually would also deserve mentioning in the review.

I am not a fan of recommending my own work when reviewing work of others, but it does happen to be work that is done within my group, so in this case it is inevitable that I do refer to that. Here are two suggestions, but please don't feel that you need to use these two.

Droplet microfluidics for food and nutrition applications (2021) K Schroen, C Berton-Carabin, D Renard, M Marquis, A Boire, ... Micromachines 12 (8), 863

The importance of interfacial tension in emulsification: Connecting scaling relations used in large scale preparation with microfluidic measurement methods (2020) K Schroen, J de Ruiter, C Berton-Carabin ChemEngineering 4 (4), 63

Kind regards,

Karin

Author Response

Thank you for writing the review. I am sure that the field will find it a useful paper that will guide them toward 'easier' design of food emulsion products.

• We thank Karin for her positive comments on our manuscript – much appreciated.

 

Although I feel that your work is rather complete, I still would like to point out that in terms of dynamic effects taking place your review is lacking. An emulsion droplet size is a resultant of droplet formation and coalescence, and now you are taking this 'as one' overall effect. I feel that the work done related to elucidating these effects individually would also deserve mentioning in the review.

I am not a fan of recommending my own work when reviewing work of others, but it does happen to be work that is done within my group, so in this case it is inevitable that I do refer to that. Here are two suggestions, but please don't feel that you need to use these two.

Droplet microfluidics for food and nutrition applications (2021) K Schroen, C Berton-Carabin, D Renard, M Marquis, A Boire, ... Micromachines 12 (8), 863

The importance of interfacial tension in emulsification: Connecting scaling relations used in large scale preparation with microfluidic measurement methods (2020) K Schroen, J de Ruiter, C Berton-Carabin ChemEngineering 4 (4), 63

• As highlighted, both oil droplet disruption and coalescence during homogenization are important physicochemical phenomena influencing the droplet size produced. In response, we have included a statement about these processes and included the above references.  Karin’s laboratory is carrying out important research in this area and we apologize for omitting this important work from the original article.

Reviewer 3 Report

This manuscript shows the fundamental theory and the methods for emulsification. Emulsification is quite an important strategy for soft material engineering. However, the reviewer cannot recommend this manuscript for publication in Colloids and Interfaces. The reviewer considered that this manuscript should be submitted to a more specialized journal, such as the Food Engineering field. Some reasons are shown below.

 

1. In the Introduction, the authors described the importance of protein for emulsification but did not describe the detailed explanation for the importance of emulsification in the field of the food industry. Some readers of Colloids and Interfaces do not understand why this review article is required.
2. Basic explanations of theories and methods shown in this manuscript are referenced from books. Ref. 18 is repeatedly referenced. Hence, this manuscript is like a textbook of colloids and interfacial science, not a review article.
3. The authors did not explain how they prepared some Figures. For example, Figure 4 may be made using Equation 2. However, a detailed explanation was not shown. Was another Figure 9 reprinted from an already published paper?
4. In section 3.1.2, the authors described the SDS-PAGE. This is also the famous method for protein separation and is not a method at the front line of science and engineering.
5. The manuscript can be summarized as the application of colloids and interfacial science to food engineering but is not a valuable review in colloids and interfacial science and/or engineering.

Author Response

This manuscript shows the fundamental theory and the methods for emulsification. Emulsification is quite an important strategy for soft material engineering. However, the reviewer cannot recommend this manuscript for publication in Colloids and Interfaces. The reviewer considered that this manuscript should be submitted to a more specialized journal, such as the Food Engineering field. Some reasons are shown below.

• We thank the reviewer for their comments. One of the major trends in the food, and other industries, is the utilization of alternative proteins as functional ingredients to replace more environmentally damaging synthetic ingredients or animal proteins.  Proteins are often used as emulsifiers in foods and other products.  However, there are currently no standardized methods to compare the functional performance of proteins from different sources under standardized conditions.  This paper aims to address this issue.  We believe that it could be a highly cited paper that would be valuable to the colloids and interfaces field, as well as the food science field.

1. In the Introduction, the authors described the importance of protein for emulsification but did not describe the detailed explanation for the importance of emulsification in the field of the food industry. Some readers of Colloids and Interfaces do not understand why this review article is required.

• We thank the reviewer for this suggestion. As requested, we have revised the introduction to highlight the importance of protein-based emulsifiers in the food industry.  This area has become increasingly important because of the recent growth of the plant-based foods sector.

 

2. Basic explanations of theories and methods shown in this manuscript are referenced from books. Ref. 18 is repeatedly referenced. Hence, this manuscript is like a textbook of colloids and interfacial science, not a review article.

• The main purpose of this article is to propose a series of standardized methods that can be used to characterize and compare the functional performance of proteins from different sources. The use of these methods would allow proteins to be compared so that the most appropriate one for a particular application could be selected.  Thus, it is mainly meant to be a methods paper, rather than a traditional review article.

 

3. The authors did not explain how they prepared some Figures. For example, Figure 4 may be made using Equation 2. However, a detailed explanation was not shown. Was another Figure 9 reprinted from an already published paper?

• This figure was drawn again for this paper – In response to the reviewers comment, we have included new data from the authors lab and stated this in the figure caption.

 

4. In section 3.1.2, the authors described the SDS-PAGE. This is also the famous method for protein separation and is not a method at the front line of science and engineering.

• We agree with the reviewer’s comment, SDS-PAGE is one of the most important analytical tools in protein chemistry for separating and identifying proteins. It was included in this article because it is also a critical tool for understanding protein composition and behavior in emulsions.  Protein-based emulsifiers contain different kinds of proteins, which influences their performance and so it is important to know which ones are present.

 

5. The manuscript can be summarized as the application of colloids and interfacial science to food engineering but is not a valuable review in colloids and interfacial science and/or engineering.

• We thank the reviewer for their comments. As mentioned earlier, a major trend in the food, and other industries, is the utilization of alternative proteins as functional ingredients to replace more environmentally damaging synthetic ingredients or animal proteins.  However, there is currently no standardized methods that can be used to compare the functional performance of proteins from different sources under standardized conditions.  This paper aims to address this issue. 

 

Round 2

Reviewer 1 Report

All my questions and comments on the original version have been addressed and answered. I have no further comments and suggest that the paper is accepted in its present form.

Reviewer 3 Report

The reviewer thinks that this revised version of the manuscript is appropriate for publication.