Review Reports - Carotenoid-Producing Yeasts: Selection of the Best-Performing Strain and the Total Carotenoid Extraction Procedure

Round 1

Reviewer 1 Report

The submitted paper is well written and provides a very throughly analysis of carotenoid production by yeasts and the best conditions for microbial growth and compounds extraction. In methods section, the following questions arose from the reading and, if possible, should be clarified:

1) What are all the sources of the samples utilized to isolate the strains (even those where no yeasts were retrieved)? Why were they selected?

2) Media composition highly influences the production of metabolites by microbes. How cultivation medium was selected for biomass production?

In line 111: what is the composition of YPD medium? Is it different from YDP?

In lines 370-371: the term "the sum of utilization scores" appears three times inside the brackets. Please verify.

Since Results and Discussion sections were combined, I suggest to change the heading "Results" to "Results and discussion".

Apart from the minor questioning presented, the paper is a valuable contribution to the field and therefore I recommend its publication.

Author Response

Reviewer 1

The submitted paper is well written and provides a very throughly analysis of carotenoid production by yeasts and the best conditions for microbial growth and compounds extraction.

ANSWER: The Authors would like to thank the Reviewer for a quick and professional review as well as the opportunity to make essential and crucial changes in our work. All the Reviewer' remarks are accepted and the paper is changed according to their comments. The Authors believe that the changed paper would satisfy the Reviewer' criteria and that it is going to be interesting enough for publishing in the Processes.

We decided to revise the manuscript according to the Reviewer' remarks, highlighting the changes directly in the revised manuscript.

In methods section, the following questions arose from the reading and, if possible, should be clarified:

1) What are all the sources of the samples utilized to isolate the strains (even those where no yeasts were retrieved)? Why were they selected?

ANSWER: Given that the authors of the paper are also members of the accredited Laboratory for Testing Food Products, we encounter a large number of food and food-related samples every day. Through many years of analysis, we noticed that red yeasts appear sporadically as contamination in certain groups of products: fresh and dried fruits and vegetables, dairy products, juices, food samples rich in sugars, etc. When the idea of the possibility of using red yeasts in scientific frameworks was born, a selection of samples was made from which red-pigmentated yeasts were previously isolated. Therefore, we added additional detail about isolation steps in the text.

2) Media composition highly influences the production of metabolites by microbes. How cultivation medium was selected for biomass production?

ANSWER: Thank you for your observation. After reviewing the literature and trying different combinations of nutrient medium content, we decided to use the YP nutrient medium, which often is specifically used for carotenoid production from Rhodotorula strains. Therefore, we added a few references of scientific-relevant work where the Authors used this nutrient medium.

In line 111: what is the composition of YPD medium? Is it different from YDP?

ANSWER: Thank you for this observation, it is the same medium, we permuted letters.

In lines 370-371: the term "the sum of utilization scores" appears three times inside the brackets. Please verify.

ANSWER: Thank you, it is corrected, all mentioned parameters are “the sum” but different quality parameters: the sum of utilization scores, the sum of production scores, and the total sum of scores.

Since Results and Discussion sections were combined, I suggest to change the heading "Results" to "Results and discussion".

ANSWER: Thank you for this suggestion, we changed it.

Apart from the minor questioning presented, the paper is a valuable contribution to the field and therefore I recommend its publication.

ANSWER: The Authors would like to thank the Reviewer for a professional review, we hope that this revised version will be published in the Processes.

Author Response File: Author Response.docx

Reviewer 2 Report

Dear authors,

The manuscript entitled "Carotenoid-producing yeasts: Selection of the best performing strain and the total carotenoid extraction procedure” discusses the isolation of yeasts from natural resources and their selection of the most potent bio-agent for carotenoid production and the upgraded carotenoid extraction protocol, which implies testing four different methods for cell lysis. It presents scientific relevance for the area of Biology, Chemistry and Natural Products area. After consulting www.sciencedirect.com; https://pubmed.ncbi.nlm.nih.gov/ and others databases, some authors have publications related to subjects related to the theme of the manuscript. The language (English) are satisfactory (I suggest the final revision)! However, you need to change some details/information in the abstract, Introduction, Methods, results, discussion and conclusions. I request information on the procedures and interpretation of the results obtained.

Abstract: Adequate, but I suggest rewrite and add information:

- The abstract is well written. However, I suggest inserting details of the most efficient method and results obtained, more relevant. There are no quantitative data on the results!

- None of the keywords are mentioned in the title or abstract! I suggest review!

* Introduction section: It is well written, but I suggest:

- As the manuscript aims to discuss and compare extractive methodologies, I suggest inserting 1 or 2 paragraphs on this topic. There is no more detailed information about this. Idem for ultrasound!

- I suggest highlighting the "innovative" proposal of the study, as well as the advantages / disadvantages, at the end of the introduction.

* Material and Methods section: The methodological proposal is appropriate to the manuscript, but I suggest:

- Page 2, lines 89-90, in “2.1. Yeast isolates and their characteristics” section: I suggest indicating more information about the origins of red yeast. I believe that such information is relevant to strengthen the idea proposed in the study. Please indicate place of acquisition, period, storage, pre-treatment, etc. , as well as storage until the moment of the analyzes.

- Page 3, line 126, in “2.1. Yeast isolates and their characteristics” section: To replace “g/L” by “g L^-1". To review throughout the manuscript, if necessary.

- “2.1. Yeast isolates and their characteristics” section, in general: I suggest indicating the references used for the procedures adopted.

- Page 3, in “2.2. Biomass production” section: I suggest indicating the references used for the procedures adopted.

- Pages 3-4, in “2.3. Experimental design for extraction of total carotenoids from yeast cells” section:

I suggest writing (before this item) a section on the extraction procedures used in more detail.

- Three different qualitative variables were studied: method for cell lysis (X1), carotenoids extraction method (X2), and solvent extraction (X3). And the quantitative variables?

- Page-4, lines 152-172, in “2.3. Experimental design for extraction of total carotenoids from yeast cells” section: How were these conditions defined? Did you use any references? If yes, I suggest indicating!

- Page 4, in “2.4. Determination of carotenoid yield” section Did you use any references? If yes, I suggest indicating!

- Page 5, in “2.5.2. Cluster analysis of temperature and pH growth profiles of yeast isolates” section: I suggest expanding this subsection. Wouldn't it be Table 1, in the order of presentation in the manuscript?

* Results section

There is no "Discussion" section! Wouldn't it be more interesting to combine the "results” with the "discussion" to better describe the findings and compare them with other works published in the literature? I suggest expanding the discussions!

- Page 6: I think table 1 is unnecessary; information can be throughout the text.

- Page 6: When indicating Figure 1, highlight which image it is - A, B, C, D, (E or F ???).

- Page 7, in figure 2: Has a more detailed study been done between 0 and 100 ^oC? In Page 8, line 311, the authors wrote; “Temperature and pH are the most important environmental factors influencing the growth of microorganisms”. So, wouldn't it be interesting to study this range up to 50 ^oC? I suggest expanding the discussions!

- Page 9, line 319: How was the pH range defined? Did you follow any ref? I suggest expanding the discussions!

- Page 11: I suggest expanding the discussions of the data presented in Table 4.

- Also, expand on the analytical validation of the spectrophotometric method.

- At the end of the “results and discussion” section, I suggest writing 1 or 2 paragraphs ending the discussions about the ideas and results.

* Conclusion section

Long paragraph! I suggest splitting! I suggest rewriting, improving the conclusions based on some comments. I suggest highlighting the advantages of the method and the study!

* Tables and Figures: Adequate!

* References: Please, check if the references are in accordance with the journal's rules.

Author Response

Reviewer 2

Dear authors,

We decided to revise the manuscript according to the Reviewer' remarks, highlighting the changes directly in the revised manuscript.

Abstract: Adequate, but I suggest rewrite and add information:

- The abstract is well written. However, I suggest inserting details of the most efficient method and results obtained, more relevant. There are no quantitative data on the results!

ANSWER: Thank you for this suggestion, we expanded the Abstract with the obtained results adding the details about the gained maximum yield and optimized parameters.

- None of the keywords are mentioned in the title or abstract! I suggest review!

ANSWER: Thank you for this suggestion, we changed keywords.

* Introduction section: It is well written, but I suggest:

- As the manuscript aims to discuss and compare extractive methodologies, I suggest inserting 1 or 2 paragraphs on this topic. There is no more detailed information about this. Idem for ultrasound!

ANSWER: Thank you for this suggestion, we added information about extraction methods.

- I suggest highlighting the "innovative" proposal of the study, as well as the advantages / disadvantages, at the end of the introduction.

ANSWER: Thank you for this suggestion, we added one paragraph to highlight this part of our study.

* Material and Methods section: The methodological proposal is appropriate to the manuscript, but I suggest:

- Page 3, line 126, in “2.1. Yeast isolates and their characteristics” section: To replace “g/L” by “g L^-1". To review throughout the manuscript, if necessary.

ANSWER: Corrected.

- “2.1. Yeast isolates and their characteristics” section, in general: I suggest indicating the references used for the procedures adopted.

ANSWER: Given that the yeast manipulation shown in this section was performed using generally accepted microbiological techniques, the Authors have added only a few references to these paragraphs. It is not necessary to emphasize these steps because they are nevertheless essential microbiological steps in the isolation and selection of microorganisms from the natural environment.

- Page 3, in “2.2. Biomass production” section: I suggest indicating the references used for the procedures adopted.

ANSWER: Done.

- Pages 3-4, in “2.3. Experimental design for extraction of total carotenoids from yeast cells” section:

I suggest writing (before this item) a section on the extraction procedures used in more detail.

ANSWER: Thank you for this suggestion, we highlighted the mentioned part of the Materials and Methods, separating it from Section 2.3., but after this section. After the formation of section 2.4. for cell lysis methodology and 2.5. for carotenoid extraction methods, we added in brief detail about the extraction procedure (the differences and the advantage of it we added in the Introduction part).

- Three different qualitative variables were studied: method for cell lysis (X1), carotenoids extraction method (X2), and solvent extraction (X3). And the quantitative variables?

ANSWER: Thank you for this observation, the output of the experiments was carotenoid yield, which we additionally highlithed in this Subsection.

ANSWER: Added, thank you for this suggestion.

- Page 4, in “2.4. Determination of carotenoid yield” section Did you use any references? If yes, I suggest indicating!

ANSWER: Added.

ANSWER: We added detail about cluster analysis in this subsection and corrected the connection with obtained results.

* Results section

ANSWER: Thank you for this suggestion. We plan to have one subsection for Results and Discussion, but we leave only Results in the Section name. So, we corrected it and expanded the explanation of the obtained results.

- Page 6: I think table 1 is unnecessary; information can be throughout the text.

ANSWER: We deleted the Table, but added information about the primary strain name in brackets after identified names in the text to avoid misunderstanding. According to this change, all Tables get the new numeration throughout the text.

- Page 6: When indicating Figure 1, highlight which image it is - A, B, C, D, (E or F ???).

ANSWER: We corrected and added better guidelines for the mentioned Figure.

ANSWER: Figure 2 represent growth curves of yeast isolation (yeast number) in the function of incubation time at 30 °C. A temperature of 30 °C is an optimal value for biomass production as well as carotenoid production for almost all carotenoid-producing yeast. Also, the tested temperature of 44 °C in the next part of the Results and Discussion indicates that the tested isolates cannot grow at this temperature. Testing the higher value is not properly because all of them are out of the biokinetic zone for growth. Additionally, carotenoids are thermolabile compounds, and decomposition of them is inevitable at a temperature under 45 °C.

- Page 9, line 319: How was the pH range defined? Did you follow any ref? I suggest expanding the discussions!

ANSWER: Given that yeasts can grow at different pH values in the environment, but also have a wide biokinetic zone, the authors decided to monitor the range of pH values between 3 and 9. We added the reference where a similar study was conducted.

- Page 11: I suggest expanding the discussions of the data presented in Table 4.

ANSWER: Thank you for this observation, some connected changes were made according to this suggestion. Figure 4 was rearranged to the Reviewer’s comment, and the text was changed to this suggestion. As mentioned in the manuscript, the ranking procedure was based on discriminating criteria described in Figure 4, while the results are presented in Table 3. According to the ranking procedure, yeast isolate R. mucilaginosa top 30 achieved the best performances, showing the highest number of positive reactions (13) and the number of negative reactions (9). The positive reactions for yeast isolate R. mucilaginosa top 30 were observed for D-glucose, glycerol, L-arabinose, D-xylose, xylitol, D-galactose, D-maltose, D-sucrose, D-melesitosis, D-raffinose assimilation as well as protease, lipase and cellulase production. Similar results were obtained for isolate R. glutinis 4_34 (which showed 11 positive and 11 negative reactions) and isolate R. mucilaginosa FK3 (which showed 12 positive and 10 negative reactions).

- Also, expand on the analytical validation of the spectrophotometric method.

ANSWER: Thank you for this suggestion, we added an additionally explanation of this step.

- At the end of the “results and discussion” section, I suggest writing 1 or 2 paragraphs ending the discussions about the ideas and results.

ANSWER: Thank you for this suggestion, we added paragraphs as the final summary of the obtained results and conclusion section was changed to connect with this part.

* Conclusion section

Long paragraph! I suggest splitting! I suggest rewriting, improving the conclusions based on some comments. I suggest highlighting the advantages of the method and the study!

ANSWER: Thank you for this comment, we separate the main highlights and added new insight into methodology advantages.

* Tables and Figures: Adequate!

ANSWER: Thank you so much!

* References: Please, check if the references are in accordance with the journal's rules.

ANSWER: Thank you for this suggestion, we did double-checking of references and the list.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The authors have satisfactorily answered my questions.

I suggest only 2 structural modifications so that the manuscript is able to be accepted for publication:

1 - The keywords (Rhodotorula glutinis; extraction optimization; red-pigmented yeasts; yeast biotechnology; and, total carotenoid) are not included in the title or abstract. I suggest that you use words contained in the abstract or title, as they attract the attention of readers. For example, using the genus Rhodotorula (instead of the species); carotenoid extraction; artificial neural network, etc.

2 - In figure 1 (Page 8), is the correct letter: e) or f)? See lines 339 and 342; and, the image itself. Please correct the figure and text!

Once again, I congratulate you for the manuscript, which is relevant to the strengthening of science in several areas of knowledge.

Regards,

Author Response

Reviewer 2

Dear authors,

The authors have satisfactorily answered my questions.

ANSWER: The Authors would like to thank the Reviewer for a quick and professional review as well as the opportunity to make essential and crucial changes in our work.

We decided to revise the manuscript according to the Reviewer' remarks, highlighting the changes directly in the revised manuscript.

I suggest only 2 structural modifications so that the manuscript is able to be accepted for publication:

ANSWER: Thank you for this suggestion, we changed keywords according to this suggestion.

2 - In figure 1 (Page 8), is the correct letter: e) or f)? See lines 339 and 342; and, the image itself. Please correct the figure and text!

ANSWER: Thank you for this observation! We made a mistake, so we changed f) to e).

Once again, I congratulate you for the manuscript, which is relevant to the strengthening of science in several areas of knowledge.

ANSWER: Thank you for all your help to improvement the paper’ quality and presentation.

Author Response File: Author Response.docx