Differences in the Proteolytic System of Lactic Acid Bacteria Affect the Release of DPP-IV Inhibitory Peptides from Whey Proteins

Round 1

Reviewer 1 Report

Please see all comments and suggestion linked in the pdf file attached.

Comments for author File: Comments.pdf

See attached pdf file.

Author Response

All suggestions and corrections provided were answered as follow:

Generat comments: First of all, this article need thorough revision for language and grammar. This is very critical before the manuscript moves forward in the review process.

The manuscript was reviewed for grammar, syntax, and general language. The most relevant changes were highlighted in blue.

Other relevant comments are provided below in the article.

Language and grammar were revised throughout all the article

This is not the correct way to start an abstract..Study with the objective of the study...

The abstract was reorganized and started with the objective. Some phrases were moved

 

What do you mean by Higher duplication rate???

Line 28. The phrase was changed to a faster duplication rate to clarify the kinetic concept.

 

were pure peptides tested in this study? I am sure not, so you cannot write this statement. Better to write low molecular weight peptide fraction....

Line 30. The word peptides was changed to peptide fractions according to the suggestion of the reviewer

 

you need to mention the outcome of some major studies done on DPP-IV inhibition using microbial fermentation..

Lines 62-68. Information from current studies related to releasing peptide fractions and peptides by fermentation of whey with lactic acid bacteria was added.

 

Has not been investigated extensively.

Line 60. The phrase was changed

 

Study.

Line 60. The word article was changed by study

 

Provide a section on "material and chemical used"

Lines 74-83. Section 2.1 was added corresponding to materials and reagents

 

What kind of whey it was, ??? Provide a separate section as 2.1 on the preparation of the whey proteins.

Adding a new section to verify the preparation of the buttermilk was unnecessary. The brand of whey powder that was used in this study was placed

 

Three systems. what does this mean?

Line 98. The phrase three different whey solutions were prepared instead of three different systems.

 

What does "kinetic parameters" mean?? Write specific information about what kind of kinetic study is this?

The section's title was completed, specifying from which experiment the kinetic parameters were analyzed, which are also specified in this section.

 

Study. Methods

Line 19. The word study was changed by methods

How was Free amino groups calculated? Provide an equation of the same.

Lines 132-135. The equation obtained from the linear regression of the glycine standard curve was placed in section 2.4.1

 

Provide details methodology for this section.

Lines 141-155. Details of the peptide separation methodology by SDS-Tris-Tricine were placed in section 2.5.2

 

Provide the protein concentration in "ug/mL"

Units were changed according to the reviewer's suggestion

 

What is this 16.5 % T gel??

When talking about polyacrylamide gel electrophoresis (SDS-PAGE), the term T is universal and corresponds to the concentration of acrylamide and bisacrylamide. However, the meaning of T according to the solution used was specified in section 2.5.2.

 

correct this?? Write a proper equation with proper explanation....

Lines 169-172. The language of the equation was corrected, and the explanation of each element in section 2.6 was expanded

 

Provide details on the statistical replicates?, How many time the experiments were replicated??

Lines 175-176. The number of replicates of the experiments was specified in section 2.7.

 

Explain how the growth rate, generation time and growth rate constant was calculated.?

The explanation of the calculations and the equations used (Eq.1, Eq. 2 and Eq. 3) are found in the section 2.4

 

What is the reason for decrease in the Free amino group in L. Rhamnosus and S. Thermophilus treated samples after 12 h and then again increase after 21 h, but in co-culture, the trend is increasing until 21 h.

Lines 268-275. The explanation is in the discussion. However, this fact has been described in a paragraph added to this section.

 

Make sure all the scientific names are italic. This is basic requirement.

It was ensured that all the names of microorganisms were italicized

 

It is important to include the front side of the Tricine page, to see how the proteins underwent hydrolysis after fermentation. Include the front side of all Tricine page.

All study information was placed as supplementary material. In it are the complete gels and the analysis by lane derived from the report produced by the Gel-Doc software.

 

This figure is not needed at all.

The figure was placed as supplementary material due to its importance in the discussion and understanding of the differences in the proteolytic system of lactic acid bacteria.

Reviewer 2 Report

 

This study investigates fermented whey and its potential to inhibit DPP-IV activity. This inhibition is attributed to release of peptides with anti-diabetic properties.

So far, the study is well performed, but the text is not always completely clear or easy to follow. Please make sure that you explain all abbreviations upon first use, to make the text easy to understand also for readers less experienced in the field.

Please mind phrasing: in the abstract you refer to “peptides with lower molecular weight (> 2 kDa)”, while later on (line 232) you refer to low molecular weight peptides (< 10 kDa)”. How about giving kDa-ranges?

Line 136: please translate to English

Line 160: “empty” brackets

You use the TNBS technique for the detection of free amino groups. Is there information whether the method detects single amino acids as well as peptides and small proteins (in terms of sensitivity, spatial accessibility)?

Figure 2 shows the MW distribution of the different samples in course of culture time and bacterium. However, those are already processed data and the sample lanes seem to show different gel sections, even within the same bacterial species (at least suggested by the labelling, e.g. in B). Bands in the gel pattern do not completely correspond to the curves above (bands in blue, peaks in green), but this may be specific for the software used. I would like to see the (unprocessed) SDS-PAGE patterns just after scanning, as a supplement. I assume that pattern changes of the bands upon treatment should be well visible there?

Names of the bacteria is sometimes in italics, sometimes not; please unify.

This is basic research. Can you give a suggestion about possible future implication of your results – in which way can those inhibitory peptides (after further study of the mechanism) be used – as food additives/medication (either as fermented whey or in purified form)? Or will they be modified (inactivated) on their way through the body to their target site and will need to be applied locally?

 

Author Response

Please make sure that you explain all abbreviations upon first use, to make the text easy to understand also for readers less experienced in the field.

All abbreviations were specified and reviewed throughout the text.

Please mind phrasing: in the abstract you refer to “peptides with lower molecular weight (> 2 kDa)”, while later on (line 232) you refer to low molecular weight peptides (< 10 kDa)”. How about giving kDa-ranges?

Line 279. The observation is correct, that is why the expression has been changed to Peptides between 10 kDa and 2 kDa

In the abstract the symbol ">2" has been changed to "<2" because that would be correct within the information of the results obtained

Line 136: please translate to English

Line 172. The phrase has been translated

Line 160: “empty” brackets

Line 199. The symbol that identifies the kinetic parameter of the duplication rate was placed within the brackets

You use the TNBS technique for the detection of free amino groups. Is there information whether the method detects single amino acids as well as peptides and small proteins (in terms of sensitivity, spatial accessibility)?

All spectrophotometric techniques for peptide detection have some disadvantages. In the case of TNBS it turns out to be very reactive for some aromatic amino acids, especially with tryptophan. However, the technique is useful to mark the degree of hydrolysis existing during fermentation. Even so, the analysis must be completed at least with another that can be related to either electrophoresis or HPLC. This is carried out in order to complete the proteolytic profile. This was the case in our study

Figure 2 shows the MW distribution of the different samples in course of culture time and bacterium. However, those are already processed data and the sample lanes seem to show different gel sections, even within the same bacterial species (at least suggested by the labelling, e.g. in B). Bands in the gel pattern do not completely correspond to the curves above (bands in blue, peaks in green), but this may be specific for the software used. I would like to see the (unprocessed) SDS-PAGE patterns just after scanning, as a supplement. I assume that pattern changes of the bands upon treatment should be well visible there?

All electrophoresis analysis by GEl.Doc has been uploaded as supplementary material. In it, it is possible to observe the complete gel and the analysis of volumes and molecular weights in the original file obtained from the equipment.

Names of the bacteria is sometimes in italics, sometimes not; please unify.

All the names of the microorganisms were italized

This is basic research. Can you give a suggestion about possible future implication of your results – in which way can those inhibitory peptides (after further study of the mechanism) be used – as food additives/medication (either as fermented whey or in purified form)? Or will they be modified (inactivated) on their way through the body to their target site and will need to be applied locally?

Despite the basic nature of the study, this is the only one that places the proteolytic system as the main actor in obtaining antidibaetic peptides obtained from co-culture fermentation. Additionally, lines 396 to 400 contain a type of perspective and importance of the study for future research. From 400 a 404 a new phrase was placed.

Round 2

Reviewer 1 Report

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