Review Reports - The Response of a Leaky Gut Cell Culture Model (Caco-2/THP-1 Co-Culture) to Administration of Alternative Protein Sources

Round 1

Reviewer 1 Report

The MS should make a story with few plate cell culture photography and few gene expression bp and their temperature.

Author Response

The authors would like to thank the reviewer for their comment. We hope this response sufficiently addresses reviewer concerns. If not, we look towards additional guidance.

The MS should make a story with few plate cell culture photography and few gene expression bp and their temperature. Unfortunately, we did not capture images of the cell culture nor did we assess gene expression. As we cannot add these on to the existing data set, we will certainly look to both for subsequent experiments.

Reviewer 2 Report

Line 9: The abstract should have a sentence for introduction and them the authors express the aim of study.

Line 39: ''extracts from soy'' can be ''soy extracts''

Line 46: the reference of the sentence is missed.

Lines 50-54: The sentences should have a reference(s).

Lines 55-59: The sentences should have a reference(s).

Lines 59-65: The sentences should have related references.

Line 68: ''porcine intestinal cell lines (IPEC)'' should be ''intestinal porcine enterocyte cell line (IPEC)''

Line 69: "Various techniques" should be "different techniques"

Lines 68, 70, : The sentences should have related references.

Line 72: full words of "IBD" should be written.

Line 101: "(Madison, Wisconsin)(Table 1)" should be (Madison, Wisconsin, Country, Table 1).

Lines 171, 185, 187-189, 191-205: The significant and insignificant differences should be indicated (p value or any defined levels)

Line 207-286: The discussion is well written but there are some sentences that should have references. Please do not declare a finding without mentioning the related references

Line 303-308: It looks that the sentences are copied.

Author Response

The authors would like to thank the reviewer for their comment. We trust that we have interpreted the guidance correctly and addressed deficiencies sufficiently. Below are specific remarks to reviewer feedback. As always, if we have not correctly interpreted the feedback, we are more than happy to revise.

Line 9: The abstract should have a sentence for introduction and them the authors express the aim of study. We have added a brief introduction to the abstract (Ln 9-11)

Line 39: ''extracts from soy'' can be ''soy extracts'' We have changed as per reviewer suggestion.

Line 46: the reference of the sentence is missed. Reference(s) have been added

Lines 50-54: The sentences should have a reference(s). We added examples to Ln 50 rather than a specific reference since this is an observation by the authors. We then added reference(s) for the next two sentences.

Lines 55-59: The sentences should have a reference(s). Reference(s) have been added.

Lines 59-65: The sentences should have related references. Reference(s) been added.

Line 68: ''porcine intestinal cell lines (IPEC)'' should be ''intestinal porcine enterocyte cell line (IPEC)'' This has been changed as per the reviewer suggestion.

Line 69: "Various techniques" should be "different techniques" This has been changed as per the reviewer suggestion.

Lines 68, 70, : The sentences should have related references. Reference has been added

Line 72: full words of "IBD" should be written. This has been changed as per reviewer suggestion.

Lines 86-90 and other lines of methods and materials: How did you find or purchase the materials? how much? You should indicate the name of companies and countries that you bought the materials. The timing of purchasing, the condition of storage should be indicated too. We have added clarifying information regarding the amount of sample and timing of the receipt of the products and their storage. The specific company information can be found in Table 1.

Line 101: "(Madison, Wisconsin)(Table 1)" should be (Madison, Wisconsin, Country, Table 1). This has been changed per reviewer suggestion.

Lines 171, 185, 187-189, 191-205: The significant and insignificant differences should be indicated (p value or any defined levels). We have added a statement of significance in Ln 153-154 and also indicated in Ln 170 (now Ln172) that there were significant differences across treatments for all cytokines (one-way ANOVA). The discussion that follows is in regards to the means separation and P values have been added.

Line 207-286: The discussion is well written but there are some sentences that should have references. Please do not declare a finding without mentioning the related references. Thank you for this feedback. This section has been broadly re-organized and revised. We trust that we have addressed the general recommendation of the reviewer.

Line 303-308: It looks that the sentences are copied. It is unclear to the authors what is meant by this feedback regarding the reference section.

Reviewer 3 Report

Although the subject of the study is relevant and interesting, the manuscript has some shortcomings and issues should be addressed.

Main comments/questions:

- Abstract, line 14: To say "NF-kB concentration" is not correct.

- Introduction, line 49: What does "Bacterial proteins may be cultivated on methane" mean?

- M&M: the preparation of aqueous extracts from the different sources has not been rigorously described. Please, provide detailed information on the preparation of powders from different sources.

- Given the procedure used to obtain the aqueous extracts (dissolution in water and centrifugation), what type of proteins will predominate in these samples?

- Table 1: please, provide full name for "DM". Units of quantification of crude protein, crude fat and carbohydrates are missing.

- Cell co/culture, lines 133-134: "Sodium butyrate was used as positive control". Positive control of which treatment? In which compartment was it added? Please explain.

- Results, item 3.1: PC analysis and K-means clustering should be moved to the end of this section (after TEER and cytokine analysis) as the results described in these items are used.

- Figure 1: the caption describes the results instead of relevant aspects of methods. Please correct accordingly.

- Table 1: please define N/A. The results presented in the table are somewhat confusing. Which is the difference between CM and LPS+ conditions?

- Results, lines 169-173: "All products increased the TEER compared to the CM control and were able to maintain the TEER at its initial levels, although soybean meal and E. coli were not significantly different. (Table 3). Moreover, torula 171 yeast, dried yeast, Chlorella, fish meal, black soldier fly, Methylobacterium, and Bio-Mos 172 increased the TEER, compared to the initial value". This section is confusing. Are the TEER values statistically different or not after 24h incubation with samples?

- Results, item 3.3: this section is a repetitive and somewhat tedious description of the results presented in Table 2. Please rewrite the section in a more conceptual and reader-friendly way.

- The discussion section could be improved. As suggestion, discussion about soybean meals should be mentioned first as it is a well known source of alternative protein.

- Discussion, lines 228-229: What does "negative components" mean?

Minor points:

- M&M, line 85: What does "Ingredients" mean?

- M&M, line 87: please define CP.

- M&M, line 142: the concentration of HC assessed in missing.

Author Response

- Abstract, line 14: To say "NF-kB concentration" is not correct. We have corrected this state NF-kB activity.

- Introduction, line 49: What does "Bacterial proteins may be cultivated on methane" mean? We have revised this to state propagation rather than cultivation and clarified that this describes growth.

- Introduction, lines 66 to 79: in this paragraph some models used to study intestinal permeability have been mentioned. However, in vitro and in vivo assays should be described separately to avoid confusion. Thank you for this feedback. The intention of this paragraph is to only include in vitro techniques, including using DSS. It is unclear to the authors how to remedy this as it appears that we do not highlight in vivo techniques here. However, we have applied this feedback broadly, particularly in the discussion section where both in vitro and in vivo references are discussed.

- M&M: the preparation of aqueous extracts from the different sources has not been rigorously described. Please, provide detailed information on the preparation of powders from different sources. It is unclear to the authors what specific information is missing on the preparation of the powders. Does the question relate to preparation in the laboratory or preparation by the manufacturer? We have, however, added some language to indicate the the protein powders were tested as received except for black soldier fly. We have also added some additional information requested including date of receipt and storage conditions.

- Given the procedure used to obtain the aqueous extracts (dissolution in water and centrifugation), what type of proteins will predominate in these samples? Great question and it will likely vary extensively from product to product. We could hypothesize that for plant proteins, it is likely storage proteins, whereas for animal proteins, it is likely structural proteins. For single cell proteins, certainly there will be cytosolic proteins that would be released but cell wall proteins may also be released. We would point out that proteins only made up to ~15% of total solids in the extracts. So, soluble carbohydrates, minerals, and other components may be the driving force behind the observations, including well-described bioactives. We have added some minor language in the discussion section regarding the soluble protein contribution relative to total solids. However, we don't have particular insight relating to the types of protein.

- Table 1: please, provide full name for "DM". Units of quantification of crude protein, crude fat and carbohydrates are missing. This has been revised as per reviewer recommendation.

- Cell co/culture, lines 133-134: "Sodium butyrate was used as positive control". Positive control of which treatment? In which compartment was it added? Please explain. The role of sodium butyrate has been clarified and a reference added. We have also added the concentration of sodium butyrate and into which compartment it was added.

- Results, item 3.1: PC analysis and K-means clustering should be moved to the end of this section (after TEER and cytokine analysis) as the results described in these items are used. This has been revised as per reviewer suggestion.

- Figure 1: the caption describes the results instead of relevant aspects of methods. Please correct accordingly. It appears unorthodox to convey methodological information in the figure caption (based on a perusal of similar figures within mdpi and other journals). Nonetheless, we have added some additional minor methodological information regarding the PCA and clustering methodology. If there is specific methodological information the reviewer would like to see, the authors are happy to include such information.

- Table 1: please define N/A. The results presented in the table are somewhat confusing. Which is the difference between CM and LPS+ conditions? We take it that this feedback is in reference to Table 2. We have added a definition to N/A. We have combined the CM and LPS+ lines, and reframed as LPS+ only; the same was done with Table 3. For this model, all treatments are stimulated by LPS. What is considered the LPS positive control is a treatment in which no protein is added. For TEER measurement, we considered it confusing to say LPS+ since they are all LPS stimulated. Instead, CM is used to describe a treatment with no protein. For cytokines reporting, this was less problematic. Nonetheless, we can see the confusion

- Results, item 3.3: this section is a repetitive and somewhat tedious description of the results presented in Table 2. Please rewrite the section in a more conceptual and reader-friendly way. This has been revised to, hopefully, be more clear and concise. In particular, we eliminated mentioned of cytokines that were not significantly different.

- The discussion section could be improved. As suggestion, discussion about soybean meals should be mentioned first as it is a well known source of alternative protein. We have revised this section, including the rearrangement suggested by the reviewer, .

- Discussion, lines 228-229: What does "negative components" mean? Revised to read as "detrimental components"

Minor points:

- M&M, line 85: What does "Ingredients" mean? Revised to products, unless speaking about ingredients within a diet.

- M&M, line 87: please define CP. Corrected as suggested by reviewer

- M&M, line 142: the concentration of HC assessed in missing. Concentration has been added (as has the concentration of sodium butyrate).