Effects of Pulsed Electric Field Processing and Sous Vide Cooking on Muscle Structure and In Vitro Protein Digestibility of Beef Brisket

Round 1

Reviewer 1 Report

(1) The last sentence of conclusion mentions muscle protein structure. However, no experimental data are involved in the effects of PEF treatment on the protein structure.

(2) It is interesting that the microscopic ultrastructures appear unchanged after PEF treatment compared to the control (Figure 4), while PEF treatment did improve the digestibility of the muscle (Figure 3 &5). Thus it would be better for the authors, in the conclusion session, to provide some comments on the mechanism of the effects of PEF treatment on the muscle tissue.

Author Response

I would like to thank the reviewer for providing constructive comments and suggestions, which have greatly improved this manuscript. Our responses to the reviewer’s comments are given below:

(1) The last sentence of the conclusion mentions muscle protein structure. However, no experimental data are involved in the effects of PEF treatment on the protein structure.

- The last sentence of the conclusion has been removed (also as requested by reviewer 3).

(2) It is interesting that the microscopic ultrastructures appear unchanged after PEF treatment compared to the control (Figure 4), while PEF treatment did improve the digestibility of the muscle (Figure 3 & 5). Thus it would be better for the authors, in the conclusion session, to provide some comments on the mechanism of the effects of PEF treatment on the muscle tissue.

-As PEF has been reported to lead to the formation of pores in cell membranes due to the electroporation effect, the formation of pores might enhance the diffusion of digestive juices into the meat matrix, promoting the digestive proteolysis in the PEF-treated SV-cooked meat. However, in this experiment, we did not look into the structure of cell membranes, which could be done in future studies to confirm this speculation. This information has been added in results and discussion (L359-365) and conclusion sections, with appropriate citation.

Reviewer 2 Report

Dear Authors,

you have prepared a great paper. There are some aspects of it:

This paper deals with the in vitro protein digestibility of pulsed electric fields-treated of sous vide cooked beef brisket by using biochemical and microscopy approaches. Although previous papers established some aspects of pulsed electric fields-treated raw meat and sous vide cooked meat separately, this paper combine it. Authors used state-of-the-art methods, they described them thoroughly and performed the research with much attention. The presentation of the results is highly understandable, tables and figures are clear and well explained. The discussion is easy to follow, supported by relevant and adequate research but also opens areas for discussion among researchers about the utilization of the measurement of the degree of hydrolysis with the trichloroacetic acid soluble peptide concentrations for expression of protein digestibility. Results are used for simple conclusions although complex research was presented, and this all is rounded by clear language writing. This paper could be only better if the “true” control of raw beef brisket meat were evaluated also. Considering the presentation and discussion, and the paper length this is not mandatory, but it would broaden the general knowledge about the changes under sous vide and pulsed electric fields treatment.

There is only one suggestion related to the title regarding the name and the abbreviations of “pulsed electric fields” and “sous vide”: it is suggested not to use the abbreviations in the title.

Author Response

I would like to thank the reviewer for providing constructive comments and suggestions, which have greatly improved this manuscript. Our responses to the reviewer’s comments are given below:

(1) There is only one suggestion related to the title regarding the name and the abbreviations of “pulsed electric fields” and “sous vide”: it is suggested not to use the abbreviations in the title.

- The abbreviations have been removed from the title.

Reviewer 3 Report

General comments:

The aim of the study is very interesting and the paper is prepared with a care by the Authors.

I appreciate application of several complementary research methods to explain the problem of muscle proteolysis under planned conditions.

However there are some small points in the text that need improvement to make the study of better quality.

Specific comments:

• Line 49- consumers usually seeking tasty food. I do not agree that only now.
• Lines 350- 352 - the study regarding swelling of peanuts is not connected with beef. Please remove this example as well as the citation. You have enough discussion without this case.
• The section 3.2 should be divided in 3 subsection (microstructure, ultrastructure, transmission analysis). It will be more transparent.
• First sentence of conclusion is absolutely unnecessary. The same remark applies to the last one (beginning from; “overall……”)
• Part of the bibliography [15; 16; 18, 21; 31; 34] is downright historical. If they are key sources, then they should be left out but please consider this remark. I cannot indicate the specific lines because they are missing here.

Author Response

I would like to thank the reviewer for providing constructive comments and suggestions, which have greatly improved this manuscript. Our responses to the reviewer’s comments are given below:

(1) Line 49- consumers usually seeking tasty food. I do not agree that only now.

- The sentence has been modified to “As consumers usually seek food products that are tasty and nutritious, it is important to understand the effects of the different food processing combinations, such as the combined PEF-SV process, on the nutritional value of meat products, for example, the protein digestibility.”

(2) Lines 350- 352 - the study regarding swelling of peanuts is not connected with beef. Please remove this example as well as the citation. You have enough discussion without this case.

- The discussion and citation of swelling of peanuts have been removed from the manuscript.

(3) Section 3.2 should be divided into 3 subsections (microstructure, ultrastructure, transmission analysis). It will be more transparent.

- Section 3.2 has been subdivided into two subsections:

• 2.1 Microstructure studied using histochemical analysis
• 2.2 Ultrastructure studied using TEM analysis

(4) The first sentence of the conclusion is absolutely unnecessary. The same remark applies to the last one (beginning from; “overall……”)

-The first and last sentences of the conclusion have been removed.

(5) Part of the bibliography [15; 16; 18, 21; 31; 34] is downright historical. If they are key sources, then they should be left out but please consider this remark. I cannot indicate the specific lines because they are missing here.

-References 12, 16, and 18 were cited in the materials and methods section which would provide more information about the methods used to the readers. As for references 21, 31, and 34, they are cited in the discussion parts to support the discussion made. We think that providing these historical references in the manuscript is appropriate and would be beneficial to the readers.

Reviewer 4 Report

Q1) Freezing and thawing are much harsher process that PEF treatment.  I suppose that your sample was damaged much more during freezing and thawing process compared to that of PEF treatment, and these can mask the PEF effect. Why don’t you use freshly slaughtered meat in your experiment? Please discuss this point in revised manuscript.

Q2 Line 90-95) Check the specific energy of PEF treatment again. The specific energy of 99 kJ/kg is quite high, and it should increase the temperature of meat around 30ºC if the specific heat of meat is assumed as 3.0 kJ/kg⋅ºC. Was the temperature of meat increased such after PEF treatment?

Author Response

I would like to thank the reviewer for providing constructive comments and suggestions, which have greatly improved this manuscript. Our responses to the reviewer’s comments are given below:

(1) Freezing and thawing are much harsher processes than PEF treatment. I suppose that your sample was damaged much more during the freezing and thawing process compared to that of PEF treatment, and these can mask the PEF effect. Why don’t you use freshly slaughtered meat in your experiment? Please discuss this point in the revised manuscript.

- As this experiment is part of a much larger study, the same batch of frozen brisket was used across coordinated experiments done for this project by several different organizations, so as to have a fair comparison between them. Although freezing and thawing have been reported to lead to muscle structure disruption, Faridnia et al. (2015) reported that PEF had a significant effect on beef ultrastructure when compared to freezing. However, as the PEF treatment used in this experiment is milder (~100 kJ/kg vs 250 kJ/kg), it might not have the same observation as Faridnia et al. (2015). Thus, it would be good to use a fresh sample in the future to better determine the effect of PEF alone and the PEF-SV process on meat structure. This suggestion has been added in section 3.2.2.

(2) Line (90-95) Check the specific energy of PEF treatment again. The specific energy of 99 kJ/kg is quite high, and it should increase the temperature of meat by around 30ºC if the specific heat of meat is assumed as 3.0 kJ/kg⋅ºC. Was the temperature of meat increased as such after PEF treatment?

- In this experiment, the temperature of the meat was increased by around 14 ℃ post PEF processing. The temperature increase was lower than the theoretical value of around 30 ℃ (assuming the specific heat capacity of meat is 3.0 kJ/kg⋅ºC) which might be due to the heat loss to the surroundings. Alahakoon et al. (2018) reported a temperature increment of around 11 – 12 ℃ in PEF-treated beef brisket with a specific energy of 90 - 100 kJ/kg. Faridnia et al. (2016) also found that the temperature of beef Biceps femoris increased by around 16 ℃ post-PEF (specific energy of 185 kJ/kg) processing.