Comparative Analysis of Fermentation Conditions on the Increase of Biomass and Morphology of Milk Kefir Grains

Round 1

Reviewer 1 Report

Dear Authors,

 This study aimed to perform growth kinetics during milk kefir fermentation to determine the best fermentation conditions, using monosaccharides, disaccharides and polysaccharides at different temperatures to determine their influence on the microbial content and morphological changes of kefir granules under environmental scanning electron microscopy (ESEM), confocal laser scanning microscopy (CLSM) and IDA.

The introduction provides a good, generalized background of the topic, that quickly gives the reader   appreciation of the scientific relevance and timeliness of the research theme. The article is very well prepared and written clearly. I think the findings of this study are sufficiently described in the context of the published literature.  The conclusions are supported by appropriate evidence, and equire only a slight amplification of the text to cover the aspects of the potential application character of the obtained results.

 I have some suggestions for Authors to improve further their study. These follow the text sequence:

• Figures: 1; 2: The figures are very rich in content but unclear to read.  It needs to be improved.
• Line 236: please correct the recording “The R2….”
• In the subsection 2.9,  please complete information about statistical program (manufacturer, town, country).
• Please try to clearly specify which statistical tests have been carried out, for which p –values (0.01 or 0.05?)? There are some disagreements between information in subsection 2.9 and line  381: “…Duncan´s multiple range test; Average ± SD; n = 3. Values with different letters in the same column showed significant difference (p < 0.01)…”
• Please add to subsection Conclusions the description of the potential application character of the obtained results.

The presented manuscript is appropriate for publication in Journal Applied Sciences, nevertheless based on the above comments, I suggest a minor revision prior to the article publication.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

I am very grateful you for the invitation to review the manuscript applsci-1610625 by Avila-Reyes and co-authors "Comparative Analysis of Fermentation Condition on Composition and Morphology of Milk Kefir Grains". This study aimed to perform growth kinetics during milk kefir fermentation to determine the best fermentation conditions, using monosaccharides, disaccharides and polysaccharides at different temperatures to determine their influence on the microbial content and morphological changes of kefir granules under environmental scanning electron microscopy (ESEM), confocal laser scanning microscopy (CLSM) and IDA. The work is interesting but needs adjustments to increase the quality of the material.

 

Comments:

– Abstract, Page 1: Authors should review the sentence, some information is missing: “The biomass of this group, after successive fermentations but the mechanism of growth and/or optimal conditions for production are not well understood”.

– Abstract, Page 1: Authors must include the initial microbial count in the fermentation.

– Abstract, Page 1, Line 27: Inform if the cell count refers to specific microorganisms or to the increase in biomass - “An increase in cell counts…”.

– Abstract, Page 1, Line 30-31: Include more specific and detailed information in the sentence “Agave fructans affected the morphology of the kefir granules, exopolysaccharide production and microbiological compositions,”.

– Abstract, Page 1, Line 27: In the sentence “An increase in cell counts and a change in the morphology of the granules” authors should clarify which changes and whether this compromises feasibility or is beneficial.

- Page 1, Line 38: Insert the verified biological activities and functional properties.

- Page 1, Line 44: Specify the characteristics conferred by kefir in relation to product flavor.

- Page 1-2, Introduction: Authors should insert information regarding the hypothesis of the alteration of the morphology of the biomass by the use of different substrates.

- Page 3, Line 100: References must be modified as recommended in the guide for authors.

- Introduction, Page 1: Include information about the difference and source of kefir, milk, or water for example.

- Page 3, Item 2.2. Fermentation Process: Specify more details of the fermentation, including flasks, agitation, reactor, among others, according to the procedure performed.

- Page 3, Line 124: References must be modified as recommended in the guide for authors.

- Page 5, Line 210: Specify evaluated pH range.

- Page 6, Figure 1: Low resolution and image size make viewing difficult.

- Page 6, Line 229-230: Review the sentence, inappropriate pH at the beginning of fermentation can disrupt the fermentation process. At the end of fermentation, it can reduce the viability of microorganisms, but it is also related to the conservation and sensorial of the product.

- Page 7, Figure 2: Low resolution and image size make viewing difficult.

- Page 7, Line 244-250: The discussion must be considered not only in terms of biomass increase but also in terms of practical application. At certain production levels, shorter lead times are expected because of operational costs and feasibility.

- Page 8, Line 273: Change “organoleptic” by “sensorial”.

- Page 9, Line 308-311: The reference, despite providing information on the enzyme-substrate relationship, refers to the growth of microorganisms in another environment. Please review the reference citation.

- Discussion section: The discussion section, especially related to image evaluation, is very well written.

- Discussion: Indicate how the morphological alteration can affect the practical application of kefir in the development of products, for example.

- The title should be revised, since the term composition refers to the verification of the chemical composition in each fermentation using different substrates.

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

In the opinion of the Reviewer, equation 1 is incorrect. If the kefir grains increase in weight over time, the result of the equation will be negative.

The Authors report that they used the analysis of variance to compare the growth of kefir biomass under different conditions. Where is this data in the manuscript?

What do the authors understand by Tukey's methodology? Are these Tukey post hoc tests? If so, in which part of the article were they used? The authors in Chapter 3.3 only use Duncan's test. Perhaps it would be reasonable to state in the methodology part that post-hoc tests were used.

It would also be possible to state that only Duncan's test was used if Tukey's tests were not used for the calculation.

Post-hoc tests would also be useful to evaluate comparisons of kefir biomass growth under different conditions. The analysis of variance itself could be biased by errors resulting from its limitations.

In the opinion of the reviewer, Figure 1 and 2 are hardly legible. Would it be possible to improve their graphic quality?

In the opinion of the Reviewer, the Authors misspell the names of microorganisms. Latin names should be written in italics. When naming the species of microorganisms, they should be written in two parts, eg Escherichia coli or Escherichia sp. In the absence of the second part of the name, we indicate that we are not writing the name of the species. In this case, it is reasonable to write e.g. bacteria of the genus Escherichia.

Author Response

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Author Response File: Author Response.docx