Cholesterol Reduction and Vitamin B₁₂ Production Study on Enterococcus faecium and Lactobacillus pentosus Isolated from Yoghurt

Round 1

Reviewer 1 Report

the English used is not clear and understandable, must be improved. Many typos in text, must be corrected.

Line 71: "5 LAB cultures were previously selected..." BUT in results only 3 are showed. Why? 

Page2, line 85: "...due to some technicalities". Unacceptable. Which ones? Why? How?
(IMHO, technical issues are often present in many researches: if you explain well, you will make the work much more understandable and will help people to avoid or solve issues while reproducing same experiments. Quality of the research will not be affected) 

Page 6, Line 206, Table 1 - Where 7MP is gone? Why? Explain and revise.

Page 8, Line 270 - L. pentosus appears for the first time, while before it was not even mentioned. Did something get lost in the writing of the text? Explain and revise.

Other minor conceptual errors, which can be ignored after careful revision of the text.

Author Response

Reviewer 1 Comments

Comments and Suggestions for Authors

• The English used is not clear and understandable, must be improved. Many typos in text, must be corrected.

Authors’ response: The whole MSS is revised for English, grammar and typos

• Line 71: "5 LAB cultures were previously selected..." BUT in results only 3 are showed. Why? 

Authors’ response: This sentence is revised.

• Page2, line 85: "...due to some technicalities". Unacceptable. Which ones? Why? How?
  (IMHO, technical issues are often present in many researches: if you explain well, you will make the work much more understandable and will help people to avoid or solve issues while reproducing same experiments. Quality of the research will not be affected) 

Authors’ response: This phrase is revised. The entire heading 2.2.2. MALDI-TOF-MS identification is revised now

• Page 6, Line 206, Table 1 - Where 7MP is gone? Why? Explain and revise.

Authors’ response: Only two isolates EF and Chole 1 came out as potent cholesterol reducers and VIt B1 producers isolate [Line No. 215]. 7MP was not a potent isolate and hence was not subjected to identification by 16S rRNA and MALDI-TOF

• Page 8, Line 270 - L. pentosus appears for the first time, while before it was not even mentioned. Did something get lost in the writing of the text? Explain and revise.

Authors’ response: Lactobacillus pentosus is mentioned in heading, abstract, introduction, Methods, Rsults, Discussion and conclusion

• Other minor conceptual errors, which can be ignored after careful revision of the text.

Authors’ response: The entire text is revised for possible errors

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript entitled: “Cholesterol reduction and Vitamin B12 production study on Enterobacter faecium and Lactobacillus pentosus isolated from yogurt” is poorly written as a general comment, and difficult to read. The novelty and end points of the proposed work should be clearly expressed at the beginning. The experimental part should be better described avoiding abbreviations (starting from the Abstract) without defining them at first use. The lactobacilli isolated from the yogurt are novel starins? Please clarify this in the text and give details. The title mention “yogurt” and in the text it is mentioned “fermented indian food”: please be consistent and detail the source and methods of isolation. The Materials and methods section should contain more information on the bacteria isolates and culture media (see e.g. “microaerobic conditions”: please explain). Line 80 and following: please explain why one of the strains has not been analyzed and add more experimental information. Paragraph 2.4 and following: please clear the procedures and experimental details. The cholesterol removal/reduction (see Paragraph 3.2 should be clearer: the used procedure should be confirmed by a separate experiment and/or with similar literature data and by an in vitro test. The conclusion of this paragraph are not completely clear: please add some more information and justify appropriately the claim. The same consideration is valid also for the paragraph 3.3; in the Discussion is mentioned ”dahi” which appear here for the first time. A few lines above is mentioned yogurt: please rephrase and discuss in a consistent way the expected and obtained results. Lines 263-268 are not clear: please rephrase and substantiate with appropriate references. The Conclusion section should be rewritten and persective by the Authors should be added. The potential use should be better exploited and if further studies are need please outlined also the limits of the proposed work. The English language should be checked and revised carefully for better readability. Finally, please use the proper template for the Journal Sustainability since there are some inconsistencies with the template (see footnotes from page 1 and following). 

Author Response

Reviewer 2 comments

Comments and Suggestions for Authors

The manuscript entitled: “Cholesterol reduction and Vitamin B12 production study on Enterobacterfaecium and Lactobacillus pentosus isolated from yogurt” is poorly written as a general comment, and difficult to read.

• The novelty and end points of the proposed work should be clearly expressed at the beginning.

Authors’ response: The novelty of the work is expressed in the beginning in the revised abstract. [Line No. 23-30]

• The experimental part should be better described avoiding abbreviations (starting from the Abstract) without defining them at first use.

Authors’ response: The experimental part is now improved. All abbreviations (starting from the Abstract) have been defined at their first appearance.

• The lactobacilli isolated from the yogurt are novel starins? Please clarify this in the text and give details.

Authors’ response: The lactobacilli isolated from the yogurt are not novel strains and not claimed so in the work but their dual properties of vitamin production and cholesterol reduction are novel.

• The title mention “yogurt” and in the text it is mentioned “fermented Indian food”: please be consistent and detail the source and methods of isolation.

Authors’ response: Fermented Indian food has been replaced with yoghurt throughout the manuscript

• The Materials and methods section should contain more information on the bacteria isolates and culture media (see e.g. “microaerobic conditions”: please explain).

Authors’ response: The whole Materials and Methods section is revised. Micro-aerobic conditions has been described [Line No. 97-99]

• Line 80 and following: please explain why one of the strains has not been analyzed and add more experimental information.

Authors’ response: Since only two isolates exhibited potent cholesterol reduction ad Vitamin B12 production while third isolate exhibited less cholesterol reduction and poor vitamin B12 production and hence was not identified [Line No. 215-216]

• Paragraph 2.4 and following: please clear the procedures and experimental details.

Authors’ response: Paragraph 2.4 is revised

• The cholesterol removal/reduction (see Paragraph 3.2 should be clearer: the used procedure should be confirmed by a separate experiment and/or with similar literature data and by an in vitro test.

Authors’ response: This paragraph is now revised

• The conclusion of this paragraph are not completely clear: please add some more information and justify appropriately the claim.

Authors’ response: Conclusion is rewritten and additional information is provided. [Line No. 422-424]

• The same consideration is valid also for the paragraph 3.3; in the Discussion is mentioned ”dahi” which appear here for the first time. A few lines above is mentioned yogurt: please rephrase and discuss in a consistent way the expected and obtained results.

Authors’ response: Yoghurt is used throughout the MSS

• Lines 263-268 are not clear: please rephrase and substantiate with appropriate references.

Authors’ response: These lines are rephrased 409-412

• The Conclusion section should be rewritten and persective by the Authors should be added.

Authors’ response: The conclusion section is rewritten and perspective of the authors is added.

• The potential use should be better exploited and if further studies are need please outlined also the limits of the proposed work.

Authors’ response: The potential use of these isolates as probiotics with the hypocholestemia potential is mentioned in conclusion

• The English language should be checked and revised carefully for better readability.

Authors’ response: The English language and grammar has been thoroughly revised

• Finally, please use the proper template for the Journal Sustainability since there are some inconsistencies with the template (see footnotes from page 1 and following). 

Authors’ response: The MSS is now revised as per the templates of sustainability. Header and footer of all the pages are now revised

Author Response File: Author Response.pdf

Reviewer 3 Report

sustainability-1181257-peer-review-

This is an interesting, but quite simple work. In my opinion performed experiments can be organized in an descent paper, but authors will need to perform a very extensive revision of the manuscript, and pay special attention on correct description of the applied methods, informative presentation of the obtained and appropriate, deep discussion.

Some specific comments:

L21-22: The sentence is speculative. Please, rewrite it.

Ln25: Please, correct to: ... food adds value to the products.

Ln26: Please, provide identity to the EF, Chole 1 and 7MP.

Ln26 and Ln30: Please, use same way to identified Chole1 or Chole 1.

Ln52-53: Please, express yourself in better way.

Please, start paragraph on Ln54 with a better link to the previous information.

Ln74: Please, avoid repetition of "These cultures"

Lm79: I do not see need to have MALDI-TOF-MS identification for the previously identified strains by 16S rRNA. I am suggesting that this part can be taken out. However, if authors have good arguments, then they can keep the segment.

Ln109: Please, correct to "Sterile"

Ln111: Centrifugation needs to be as "xg", not a "rpm"

Ln120, 121, and etc: Please, correct to "et al."

Ln122: E. coli (please add interval). Provide strain number for applied E. coli strain.

Ln128: Please, provide species names, not only strain identifications.

Ln131: Slurry is appropriate word? Maybe suspension is better? Please, check with linguist and correct if needed.

Ln128-135: Please, add symbol for the Celsius temperature (oC ). Please, check entire manuscript for similar error.

Ln136: Vitamin B12 Bioassay

Ln157: change to spp.

Ln157, 158: spp. (add ".")

Ln163: Please, correct spelling of Enterococcus facial.

Ln176: please, add italics for "in vitro".

Entire manuscript needs to be carefully check for the accordance with the Instructions for authors. There are several spelling and formatting errors.

Ln179: If you have done identification, then you supposed to call them strains, not isolates. And please, add the appropriate species names.

Ln179-180. The sentence need to be corrected. It is not clear.

All the experiments presented in the material and methods needs to be link to appropriate results and discussion sections. And vice versa, For example, effect of glycerol was on Vit B12 was not very well stated in the material and Methods sections.

Authors suggested that maybe other concentrations of glycerol may have effect on studied strains, but why this was not explored as option by authors in their research.

Table 1: Why not info about 7MP was presented. In fact, table 1 can be part of the text, there is no need for a separated table with this results. Most of the figures well can be clearly presented as part of the text.

Clear separation of the results and discussion needs to be taken in account in preparation of revised version.

Discussion is very basic and not really gives scientific contribution to the paper. Authors will need to upgrade extensively the discussion part of the paper.

Author Response

Comments and Suggestions for Authors

sustainability-1181257-peer-review-

• This is an interesting, but quite simple work. In my opinion performed experiments can be organized in an descent paper, but authors will need to perform a very extensive revision of the manuscript, and pay special attention on correct description of the applied methods, informative presentation of the obtained and appropriate, deep discussion.

Authors’ response: The whole MS is revised right from Abstract to the conclusion. Methodology Results and Discussion are now properly organized

Some specific comments

• L21-22: The sentence is speculative. Please, rewrite it.

Authors’ response: This sentence is now revised

• Ln25: Please, correct to: ... food adds value to the products.

Authors’ response: This sentence is deleted during revision of the abstract

• Ln26: Please, provide identity to the EF, Chole 1 and 7MP.

Authors’ response: Identity of these isolates Enterococcus faeciium (EF), Enterococcus faecium (Chole1), and Lactobacillus pentosus (7MP) is provided [Line 32-33]

• Ln26 and Ln30: Please, use same way to identified Chole1 or Chole 1.

Authors’ response: This sentence is deleted during revision of the abstract

• Ln52-53: Please, express yourself in better way.

Authors’ response: This sentence is deleted during revision of introduction

• Please, start paragraph on Ln54 with a better link to the previous information.

Authors’ response: This para and previous para are now revised for proper link

• Ln74: Please, avoid repetition of "These cultures"

Authors’ response: Repetition is omitted

• Lm79: I do not see need to have MALDI-TOF-MS identification for the previously identified strains by 16S rRNA. I am suggesting that this part can be taken out. However, if authors have good arguments, then they can keep the segment.

Authors’ response: The isolates were identified based on 16S rRNA sequencing and MALDI_TOF MS analysis was carried out as a confirmatory method. Sometimes identification by single method (16 S rRNA) may not distinguish between species and hence MALDI TOF can add value to the identification.

• Ln109: Please, correct to "Sterile"

Authors’ response: Corrected [Line 136]

• Ln111: Centrifugation needs to be as "xg", not a "rpm"

Authors’ response: Corrected [Line 138]

• Ln120, 121, and etc: Please, correct to "et al."

Authors’ response: Corrected throughout the MSS

• Ln122: E. coli (please add interval). Provide strain number for applied E. coli strain.

Authors’ response: Interval added and strain No provided

• Ln128: Please, provide species names, not only strain identifications.

Authors’ response: This sentence is now revised

• Ln131: Slurry is appropriate word? Maybe suspension is better? Please, check with linguist and correct if needed.

Authors’ response: Since it is not intact cell preparation, slury would be better term to mention.

• Ln128-135: Please, add symbol for the Celsius temperature (oC ). Please, check entire manuscript for similar error.

Authors’ response: Symbol for Celsius temperature (^oC) is now added

• Ln136: Vitamin B12 Bioassay

Authors’ response: Corrected

• Ln157: change to spp.

Authors’ response: Changed [Line 306,307]

• Ln157, 158: spp. (add ".")

Authors’ response: Added [Line 306,307]

• Ln163: Please, correct spelling of Enterococcus facial.

Authors’ response: Corrected [Line 312]

• Ln176: please, add italics for "in vitro".

Authors’ response: Revised [Line 379]

• Entire manuscript needs to be carefully check for the accordance with the Instructions for authors. There are several spelling and formatting errors.

Authors’ response: Spelling and formatting errors have been rectified

• Ln179: If you have done identification, then you supposed to call them strains, not isolates. And please, add the appropriate species names.

Authors’ response: Revised as suggested [Line 279]

• Ln179-180. The sentence need to be corrected. It is not clear.

Authors’ response: Revised [Line 279-280]

• All the experiments presented in the material and methods needs to be link to appropriate results and discussion sections. And vice versa, For example, effect of glycerol was on Vit B12 was not very well stated in the material and Methods sections.

Authors’ response: All the experiments presented in the material and methods are now linked properly to appropriate results and discussion sections. Effect of glycerol on Vit B12 is presented under separate heading in Materials [Subheading 2.4.2], results [Subheading 3.4.] and Discussion [Line 404-405].

• Authors suggested that maybe other concentrations of glycerol may have effect on studied strains, but why this was not explored as option by authors in their research.

Authors’ response: This concentration was chosen based on the previous study of author [Reference No.8]

• Table 1: Why not info about 7MP was presented. In fact, table 1 can be part of the text, there is no need for a separated table with this results. Most of the figures well can be clearly presented as part of the text.

Authors’ response: Isolate 7MP was not found as potent cholesterol reducer and Vit B12 produces, so it was not subjected to identification [Line 204-205].Table 1 is now presented as text

• Clear separation of the results and discussion needs to be taken in account in preparation of revised version.

Authors’ response: Results and Discussion section is are thoroughly revised and well separated

• Discussion is very basic and not really gives scientific contribution to the paper. Authors will need to upgrade extensively the discussion part of the paper.

Authors’ response: Discussion part is revised

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

1- binomial name of bacteria must be both in italic

2-  line 280 Enterococcus faecium, not Enterococci

3- the structure of the discussion is still not very cohesive and the conclusions are still quite concise, however the work is now much easier to understand.  If possible, some minor improvements will be strongly appreciated.

4- minor spell and grammar corrections in the text

Author Response

Reviewer 1 Round 2 Report

Comments and Suggestions for Authors

• The binomial name of bacteria must be both in italic

Authors response: Agreed. Binomial names of bacteria are cross-checked and corrected accordingly.

• line 280 Enterococcus faecium, not Enterococci

Authors response: Agreed and corrected as Enterococcus faecium [Line No. 274, 277, 292]

• the structure of the discussion is still not very cohesive, and the conclusions are still quite concise. However, the work is now much easier to understand. If possible, some minor improvements will be strongly appreciated.

Authors response: The discussion part is now improved. All the aspects are now discussed in-depth and in a proper sequence. Additional discussion is mentioned at Line No. 252-260, 267-271, 279-289, 300-303, 313-315, and 326-331]

• minor spell and grammar corrections in the text

Authors response: Spell check and grammar corrections are done with a licensed copy of professional software

Reviewer 2 Report

The manuscript has been modified nonetheless it is still lacking of clarity regarding end points and findings. The discussion of the results should be improved and experimental details aimproved. Conclusions should be better assessed and justified. The novelty and limits of the study better outlined. In its present form needs improvement for possible publication on the Journal.

Author Response

Reviewer 2 Round 2 Report

• The manuscript has been modified. Nonetheless, it still lacks clarity regarding endpoints and findings.

Authors response: MSS is thoroughly revised and improved for clarity

• The discussion of the results should be improved, and experimental details improved.

Authors response: The discussion part improved. In-depth discussion is mentioned, additional discussion is mentioned at Line No. 252-260, 267-271, 279-289, 300-303, 313-315, and 326-331]. The methodology part is also improved

• Conclusions should be better assessed and justified.

Authors response: The conclusion part is revised. Additional text is mentioned at Line No. 353-364.

• The novelty and limits of the study are better outlined.

Authors response: The novelty and limits of the work are mentioned in Line No. 28-29, 335-340, and 353-356.

• In its present form needs improvement for possible publication in the Journal.

Authors response: Improved as per the above suggestion to make the article understandable.

Reviewer 3 Report

sustainability-1181257-peer-review-V2

Authors have corrected some of the problematic parts, however, corrected version need additional attention. Moreover, some of the previous problematic parts have not been appropriately addressed and corrected.

Ln19: remove one of the intervals after "are"

Ln21: lactc acid bacteria - change to "Lactic acid bacteria" L needs to be capital.

Ln21-22:  Suggestion for correction: Lactic acid bacteria (LAB) are the pre dominant bacteria present in fermented food products and some of them may exert probiotic effects.

Ln22-23:  Some of the probiotic properties of LAB have been linked to the reduction of the  cholesterol contents in the host.

Ln25-27: Sentence is not clear, need to be corrected.

Several parts of the manuscript require language corrections. Maybe authors can look for help from professional linguist.

In fact abstract can clearly start from Ln29.

Ln29-30: Sentence needs to be corrected.

Ln36-37: “Significant increase in cholesterol production was obtained with supplementation of glycerol (0.5% v/v) in the growth medium.” _- Is this sentence correct???  

Ln35:  15S rRNA ? or 16S rRNA?

Authors have corrected the manuscript. However, during the correction process there are even more generated errors and misunderstanding. I am strongly recommending to the authors to read with high attention their manuscript and carefully correct the work. Several of the corrections (red text) do not make logic. Please, look for help from senior colleague and from professional linguist.

Work has potential, however, to many errors in the text (corrected version) and still problems in the language.

Please pay attention when you can use isolate and when strain. There is difference.

As I have mention in first round of reviewing, discussion is very basic and needs to be extended and present in better way.

Please, be sure that you do not repeat results from previous works. Maybe a self check of the manuscript for plagiarism will be a good idea before author’s submit the revised version of the manuscript.

Author Response

Reviewer 3 Round 2 Report

Comments and Suggestions for Authors

The authors have corrected some of the problematic parts. However, the corrected version needs additional attention. Moreover, some of the previous problematic parts have not been appropriately addressed and corrected.

• Ln19: remove one of the intervals after "are."

Authors response: This sentence is deleted as per your comment No. 7

• Ln21: lactic acid bacteria - change to "Lactic acid bacteria" L needs to be capital.

Authors response: Agreed and revised as suggested. [Ln No. 19]

• Ln21-22:  Suggestion for correction: Lactic acid bacteria (LAB) are the predominant bacteria present in fermented food products, and some of them may exert probiotic effects.

Authors response: This sentence is deleted as per your comment No. 7.

• Ln22-23: Some of the probiotic properties of LAB have been linked to reducing the cholesterol contents in the host.

Authors response: as per your comment No. 7

• Ln25-27: Sentence is not clear; need to be corrected.

Authors response: This sentence is deleted as per the suggestion of reviewer 2

• Several parts of the manuscript require language corrections. Maybe authors can look for help from a professional linguist.

Authors response: Language corrections have been made through licensed linguistic software.

• In fact, the abstract can clearly start from Ln29.

Authors response: Agreed. The abstract is started from Line 29; earlier lines have been deleted.

• Ln29-30: Sentence needs to be corrected.

Authors response: The sentence is revised.

• Ln36-37: “Significant increase in cholesterol production was obtained with supplementation of glycerol (0.5% v/v) in the growth medium.” _- Is this sentence correct???.

Authors response: The sentence is now revised as "A significant reduction in cholesterol production was obtained with supplementation of glycerol (0.5% v/v) in the growth medium". [Line No 26].

• Ln35:  15S rRNA ? or 16S rRNA?

Authors response: It is 16S rRNA. Corrected at Line No 27.

• The authors have corrected the manuscript. However, during the correction process, there are even more generated errors and misunderstandings. I am strongly recommending that the authors read with high attention their manuscript and carefully correct the work. Several of the corrections (red text) do not make logic. Please, look for help from a senior colleague and a professional linguist.

Authors response: Yes, a thorough reading of the manuscript is done. All the errors have been rectified. We have gone through the manuscript, and the flaws are now corrected. At many places in the manuscript, the presented work is revised/reframed/corrected/ deleted added/ to increase the clarity of the work. (reference 13 replaced by proper and more reliable one, references are cross-checked and altered references are correctly placed, the text is properly placed as per the presentation flow, etc.).

• However, work has the potential to many errors in the text (corrected version) and still problems in the language.

Authors response:  All the errors that have been corrected related to the grammar and language have been corrected with a licensed copy of language and grammar software.

• Please pay attention when you can use isolate and when strain. There is a difference.

Authors response: Agreed, these terms are now used properly Ln 205, 206.

• As I have mentioned in the first round of review, the discussion is very basic and needs to be extended and present better.

Authors response: The discussion is improved and made more clear. The discussion part is now mentioned in a proper sequence. The extended discussion part is mentioned at Line No. 252-260, 267-271, 279-289, 300-303, 313-315, and 326-331].

• Please, be sure that you do not repeat results from previous works. Maybe a self-check of the manuscript for plagiarism will be a good idea before authors submit the revised version of the manuscript.

Authors response: The authors have thoroughly checked the MSS. No previous results were mentioned.

Round 3

Reviewer 2 Report

The manuscript is still poorly written nowthitstanding the modifications done to the text. In particular the experimental part (see cholesterol reduction experiments, etc.) should be substantiated and compared with other techniques to assess the results obtained as well as the conclusions. The ability of cholesterol reduction as well as vitamin B12 production is to be checked against standard methods to assess the reported results. The reported data seem qualitative premilinary tests. The mechanism which is behind the reported data should be tentatively explained to better define the context. Moreover the Materials and Methods section should be improved and details adedd to the text. In its present form it does not seem ready for publication in the Journal.

Author Response

Reviewer 2 Round 3

Comments and Suggestions for Authors

• The manuscript is still poorly written, with no withstanding the modifications done to the text. In particular, the experimental part (see cholesterol reduction experiments, etc.) should be substantiated and compared with other techniques to assess the results obtained and the conclusions.

Authors response

Misleading sentences are removed (line 25 in the abstract), and modifications are done with the cholesterol reduction experiment (line 90) to make it more understandable.

• The ability of cholesterol reduction and vitamin B12 production is to be checked against standard methods to assess the reported results.

Authors response

The cholesterol reduction ability and vitamin B12 production was checked by standard references and methods to assess the properties of the isolates.

• The reported data seem qualitative preliminary tests.

Authors response

In the present study report, qualitative and quantitative data are discussed; for example, for cholesterol reduction, the BSH activity was done by plate method qualitative (semi-quantitative) method and enzymatic quantitative method with standard methodologies the standard references.

• The mechanism which is behind the reported data should be tentatively explained to define the context better. Moreover, the Materials and Methods section should be improved and details added to the text.

Authors response: The possible underlying mechanism of cholesterol reduction is mentioned (line 281 to 284 in discussion part). Moreover, the obtained results were also compared in the same paragraphs with the other recent study reports to support the present study. The material and method section is improved in the second revision.  In the future, prospects of the work could be continued with more modern and sophisticated methodologies.

Author Response File: Author Response.pdf

Reviewer 3 Report

In my opinion authors have improved the manuscript.

Author Response

Reviewer 3 Report Round 3

Comments and Suggestions for Authors

In my opinion, the authors have improved the manuscript.

Authors response: The authors are very much thankful to the reviewer for the critical but excellent and valuable suggestions that helped in significant improvement of the MSS

Author Response File: Author Response.pdf