A Study on the Stability and Carbohydrate Metabolic Traits of Starter Cultures in Response to Continuous Subculturing

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The author purport to study the Effects of Contonuous subculting on the physiological and genetic stability of starter cultures but spend most of the time describing carbon metabolism in general although milk contains mainly lactose, on the genome structure of bacterial strains that have been already sequenced and do not emphasize the genetic differences that could be relevent to their study. This should be retitled at least or rewriten to conform to the title at best.

Furthermore, the authors study main different strains as shown in the figures but focus solely on 2 strains in the text without giving any reason.

Growth curves do not show any experimental points which might suggest that they are generated. In that regard nowhere is the generatino time of the bacteria mentionned to give a sense as to how long the experiment went on.

Figure 1, the pictures are to small to see any of the details mentionned.

Table 1 is very difficult to read since columns and lines refer to different statistical significance, no clear message comes out.

Author Response

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Reviewer 2 Report

Comments and Suggestions for Authors

This paper describes difference between strains at 0 and 2000 cell divisions, describing how the strains are generally stable, which is very important for consistent production.  Comparisons between 0 and 2000 generations were done.

I don't understand the inclusion of the carbohydrate metabolic processing.  Why was this included?  What passage of strains was this on?  Was there a comparison between 0 and 2000 done?

The genomes need to be deposited into an appropriate database and referenced in the manuscript.

 

Author Response

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Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The have reframed the title of their paper to better reflect its content. However, the paper is blotted with information, although interesting, not pertinent to the discussion. Indeed, the aim of the paper is to determine how the morphology, physiology and the genome change after 2000 generations of continuous cultivation.

For the morphology, they have assessed growth on plates and observed cells with light and electron microscopy. However in the figure, which is duplicated, they correctly label each panel but fail to do it in the text where it is confusing. lines 89-119. They separate time 0 from time 2000 generations but cite all panels in the former but none in the latter.

For the metabolism, they chose to assess growth on many different carbon sources but I was expecting that they would do a comparative analysis of the time 0 and time 2000 generations here also to show if given the growth in the presence of only 1 carbon source, lactose, cells would loose certain 'useless' pathways. They also assessed growth of representative strains and showed that after continuous culture strains acidified more the medium, had a shorter lag phase, their stationary OD was higher and had higher fermentative capacity. Although they have simplified the statistical analysis, I'm still confused about the difference between a and b indices. Does 1 mean that the difference is more significant than the other? I think that the whole profiling section is too long. The information for the purpose of this paper are these strains are able to metabolize many different carbon sources and there are variations between  strains. This enables them to justify their choices for the next section.

For the genome section, they' re too many details. The authors have sequenced the starter strains and the final strains. They should summarize their findings. Are there differences between their starter strains and published strains and are there differences with the final strains in gene content. I'm not sure that after 2000 generations the GC content would change much. Unless there was doubt about the strains used, the clustering is not very useful since this is not a genomics paper.

The section about the genomic analysis does not show any difference with what was said in the intro about the 2 strains. Are there mutations affecting the carbon metabolism and if so which gene is impacted. This is also why in the metabolism section it would have been nice to have the carbon utilization after 2000 generations.

By shortening the paper, the authors would bring forward the conclusion that genomes are robust enough to insure the continuous quality of the product despite the industrial practices.

Author Response

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Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for your revisions.

Author Response

Thank you very much for your time involved in reviewing the manuscript. We also greatly appreciate your constructive comments. To facilitate this discussion, we first retype your comments in italic font and then present our responses to the comments. As requested, we have submitted a revised manuscript with all modifications highlighted in red for ease of review.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have adressed my main concerns in their latest revision. I only have some minor remarks about the manuscript.

line 22 the authors talk about acidification kinetics but if I understood correctly in the main text they refer to fermentation activity. If it is the same thing, the authors should adopt a uniform naming.

lines 52-54. I find the sentence not very clearly formulated. The authors state that environmental stresses can trigger genomic variations. In the context, I would think that environmental stresses select genomic variations which can alter... Or environmental stresses can trigger genomic variations in the sense that pathways normally active become inactive or vice versa in which case the genomic variations are regulatory variations. Could the authors clarify this point?

line 99, they describe L. bulgaricus as round however on the micrographs they are rod-shapes and not round.

Line 112, typo resulted

line 116 it's either after the 2000th generation or after 2000 generations.

lines 119-123 the sentences are not clear since it seems that there are no differences between the 2 time points yet the authors seem to want to see differences.

section 2.2, the authors should be more precise it seems they're discussing the internal pH of the cells whereas I think they mean the pH of the culture medium. Furthermore, they insist on the size of the difference, which is very small yet significant to me. A variation of .4 in pH represents a variation in H+ concentration of 2.10⁶ molar.

In table 1, I still don't see the difference in significance between superscript a and b.

line 194, remind the reader what PM (phenotypical microarray) stands for.

In section 2.3, I would reroot the tree using the node that connects the lactose group to the rest. Indeed, this group contains the most commonly used carbon sources. This will I think show that there are more clusters that are worth mentioning and that better characterize each bacterium.

line 223 there is I think a which missing.

line 229 I think we should read trehalose and not rehalose.

 

Author Response

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