PONE-D-22-07202Development and validation of reliable astaxanthin quantification from natural sourcesPLOS ONE

Dear Dr. Antje Labes,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jun 16 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Vandana Vinayak, PhD

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following in the Competing Interests section: 

"IK has worked for Sea and Sun Technology, a provider of H. pluvialis biomass for the study. All authors have declared that no other competing interests exist."

Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests).  If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. 

Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

Additional Editor Comments:

Dear Dr. Antje Labes,

I can now inform you that the reviewers and editor have evaluated the manuscript PONE-D-22-07202

Development and validation of reliable astaxanthin quantification from natural sources.

Please consider the reviews to see if revision would be feasible. Should you wish to resubmit you should explain how and where each point of the reviewers' comments has been incorporated. For this, use submission item "Revision Notes" when uploading your revision. Also, indicate the changes in marked changes version of the revised manuscript (submission item "Revision, changes marked").

Regards

Dr. Vandana Vinayak

Academic Editor

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments:

Sentence 41-43: “calibration……5%” seems incomplete and complicated statement.

Sentence 44: Rewrite it, as it seems the two di-Z forms are 9Z and 13Z mentioned in the sentence and if so why to mention the “two di-Z forms” in the same sentence again?

Sentence 47-49: only the dried biomass was processed for Astaxanthin and not the liquid culture or the fresh biomass?

Sentence 49-50: Cholesterol esterase (1.0 to 2.0 U) is per how many mg or mL of dried biomass or culture respectively?

Sentence 50-52: This line is not necessary here; it should be discussed in introduction section.

Sentence 53: “The reliability of photometric Astaxanthin estimation was assessed”, how? Name the method or explain in one sentence.

It’s not clear in the abstract if the authors are comparing two methods of extraction or estimation or both, please clarify.

Sentence 61-63: needs reference.

Sentence 68-69: what about the allergenicity of Astaxanthin if provided as food supplement.

Sentence 84-89: it gives explanation of why this technique is needed. The limitations due to which related techniques like this have not been used should also be stated.

Sentence 100: which wavelength?

Sentence 119-128: it gives the glimpse of steps involved, but in the introduction it was claimed that it is a shorter and faster method. However, Astaxanthin extraction, enzymolysis, liquid-liquid extraction and then analysis with UHPLC,UV/VIS spectrophotometry does not seem to be a short method.

Sentence 156: what was the make of beat miller, diameter of zirconium beads, time of beat milling, and the cooling time in the process?

Sentence 167: is the fresh biomass dewatered?

Sentence 172: Mention 3.3U/mL as cholesterol esterase concentration in abstract used for de-esterification.

Sentence 211: how the proportion of 69.4% was assumed?

Sentence 215: replace solved to dissolve.

Sentence 223: is 45 min. (0.75 h) is the minimum time used, why authors did not try the lower incubation period or higher incubation period than to 1.5 h.

Sentence 260-265: previously, it was mentioned that additional incubation time of 1.5 h was also used so; for detection limit and linearity, why only one incubation time is used?

Sentence 267-271: there is lot of confusion between the samples (fresh or dry), if fresh then how it was weighed and what is the incubation period in no. of days decided on what basis, which two samples were chosen and why?

I cannot follow the series of experiments performed and what samples were used what was the incubation period, was there any stress? I suggest authors to prepare an experimental design section with text and detail figure to explain precisely how the experiments are performed with each step and from which step sample were taken for which analysis and why?

Sentence 287: On what basis it was considered 4.5 % w/w of the H. pluvialis is Astaxanthin?

Sentence 300: In sentence 270-271, four days are mentioned in sentence 300 five days are mentioned, why? It’s confusing to follow.

In figure 5: It seems like there is no effect of increasing the biomass (mg) but, how it is possible if considered 4.5 % w/w of the H. pluvialis is Astaxanthin?

In figure 7: give legends with in the graph for samples like M1, M2 ETC.

In figure 9: which stress is imposed and why? Also indicate level of significance in each figure.

Conclusion is too long; make it precise and conclusive in 10-12 sentences. For instance sentence 986 seems like repetition of first line of conclusion. Comparison should be a part of discussion. Similarly sentence 990-991 should be discussed in methodology section.

Reviewer #2: Comments:

1. In line no 44-45 sentence “In extracts from H. pluvialis, the observed measurement range was extended to 30 µg/mL”. What was the amount of H. pluvialis biomass taken? It should be mentioned in the abstract also.

2. Line no 47, 48, 48 ‘The precision of all-E-astaxanthin quantification in dried H. pluvialis biomass was calculated with a coefficient of variation of maximal 1.1%, whereas it was below 10% regarding the diastereomers” What was the amount of H. pluvialis biomass?

3. In the beginning sentence of introduction “Astaxanthin (3,3´-dihydroxy-β,β´-carotene-4,4´-dione) is a secondary ketocarotenoid. It has a hydrocarbon backbone that comprises a central, delocalized π-electron system. β-ionone rings terminate the hydrocarbon chain at both ends. The presence of one hydroxy- and one oxo-group at each of these terminal rings further classify it as xanthophyll.” Add reference and for more information prefer the suggested article “Light modulates transcriptomic dynamics upregulating astaxanthin accumulation in Haematococcus: A review” https://doi.org/10.1016/j.biortech.2021.125707

4. Add new references from line no 84-89.

5. Line no 157, what was the percentage of acetone used for astaxanthin extraction? Likewise line no 174 mention the percentage of petroleum ether?

6. Line no 161 please check whether it is 10.000 x g or 10,000 x g. See at other places also.

7. Mention makes and model of the instruments/ equipments used during the experiment. For instance, vortex and ultrasonicate.

8. Line no 190 and 191 ‘Optical spectra were measured in a range of 200 to 800 nm, and astaxanthin data were analyzed and quantified at 474 nm. Lutein was quantified at 448 nm.’ Add the references for astaxanthin and lutein as well, to increase the novelty of the article.

9. Line no 215 please check whether “solved” is the right word to use here. Check in the entire manuscript.

10. Check for typographical errors in the entire manuscript.

11. Measuring units should be written clearly for instance see line no 221-224.

12. Text in the conclusion should be reduced to 8-12 lines.

Reviewer #3: In the manuscript “Development and validation of reliable astaxanthin quantification from natural sources” Authors developed enzymolysis-based astaxanthin quantification method to hydrolyse astaxanthin esters and determine free astaxanthin in all its diastereomeric forms. The investigated results could be useful for understanding the scientific knowledge. Therefore, I recommend this study for the publication in PLOS ONE after answering the following queries.

• Author said that, mostly sophisticated techniques as liquid chromatography and spectrophotometric/mass spectrometry are used for identification and quantification of astaxanthin. However author themselves used ultra-high performance liquid chromatography (UHPLC) and UV/VIS spectrometry techniques in the present study; justify

• In the calibration curves of astaxanthin standards in table 1; why different number of measurements was chosen?

• For detection limits of astaxanthin determination why selected specific biomass quantity of H. pluvialis (from 0.04 to 3.98 mg), saponification time (0.75 h) and enzyme concentration (2.0 U).

• Why astaxanthin concentration deceased in the sample exposed to ambient air (3.46±0.04% w/w) as compared to vacuumed samples (3.98±0.04% w/w) after prolong storage?

• There is poor English language at many places in the manuscript, so author needs to check thoroughly and improve.

Reviewer #4: Astaxanthin is a biomolecule with a very high added value and with promising/already established applications, including in biomedicine. For at least economic and correct dosage, an accurate method for astaxanthin quantification is required. The manuscript by Koopmann and collaborators enters in this frame and therefore appears as timely and witha high potential interest. The quantification of astaxanthin as other carotenoids is difficult because astaxanthin is actually constituted by several diastereoisomers, the relative abundance of which depending on many factors, including extraction and storing conditions. In addition, depending on which phylum the source belongs, different enantiomers are found. The manuscript is well written even if I found it difficult to read in some places. Concerning language issues, I have proposed some modifications that the authors should evaluate before an eventual validation because I am not a native English writter. A detailled llist of comments is displayed below. Altogether, I found the manuscript interesting but difficult to use. For increasing its usefulness, I suggest to the authors to add (1) an additional figure displaying a logic scheme to allow the readers to choose what to do regarding quantification objectives and (2) the corresponding finalized protocols. Both could be added as supplemental data

l88: add a citation

l100: 'suggesting using another wavelength' -> 'suggesting the use of another wavelength'

l104: already indicated above

l105: 'a similar approach' but related to what?

l110 should continue l109

l213: what is the source of lutein?

l240 and throughout the manuscript: check the use of '.' in figures. Here 3.000 = 3 x g ... Centrifuge aceleration are usually written under this format XXX x g (with g in italic to avoid confusion with the mass unit).

l253 and throughout the manuscript: I suggest to use (X) rather than x) and to avoid '.' before. Actually, the (x) indicates a sucession of items.

l268: Are the two samples arising from the same batch?

l302: which sample?

l313 and throughout the manuscript: italize 'a' of chlorophyll a'

l327: give a range for the vacuum or at least how it was performed

l362: why the linear regression does not cross the (0,0) coordinate?

l390-394: indicate the meaning of each symbol both in the graph and in the legend

l396: by 11%?

l402-405: at least a citation about this is required. Pictures comparing the phases would also help

l405-407: Is there any reason for this different behaviour?

l412-418: it would help the reader to reproduce the separation if the text would be accompanied by a table providing (1) log k', (2) the wavelength maximum/maxima in the eluting solvent and (3) in a solvent of reference

l421: 'dissolved' instead of 'solved'?

l426: would be better to indicate it as relative amount

l431: could the author observe the cis-peak?

l442: I do not understand 'these significant differences'

Fig3

- why are m/z and UV/vis bandwidths different?

- the resolution and the contrast of the magnified parts is very weak. Please increase

- could other keto-carotenoid be detected?

l560: 'small': really, Is not reaching up to 10%? In addition, small is a very relative term. Better to give a range in percent of the volume. No emulsion at the interface of the two phases?

l639-640: I do not understand clearly if it is the same sample or different samples.

l640: '3 and 4 different days' of what?

l659: I do not understand towhat correspond the 'differentlly cultivated'

Fig6 and the next figures: figure out the 100%, for instance by a dashed line. How the 100% was determined?

l680: 'different conditions': specify them

l684: indicate if the biomass is in DW or FW

l720: remove 'also'

l722-728: could this part summarized by writting that the optimal concentration ranges between 10 et 18%?

l725: 'other enzymes': could the authors be more preciseN

l729: '... without ethanol': this study? If not, add a citation

Fig8

- I could not find the call to this figure

- what is the meaning of (-) in the title of the X axis?

l753! 'maximum': how was it measured?

l754: ... mg oleoresin': DW or FW?

l761: replace 'exceeding' by 'an excess'?

l762: about carotenoids! astaxanthin belongs to carotenoid?

l763: why competition? Could the authors elaborate a bity on this?

l764: indicate the dilution factor

l774-775: I do not understand this sentence

l783: 'alga' instead of 'algae'

l783-784! I would replace 'and' by 'or'

l791! 'higher proportion' in O3 but not in O1 and O2. Are the fdifference significant?

l797-799: indicate the number of repetition

l808: I do not understand

l811-812: and what?

l828-830: this is strange, is not it? Please elaborate on this.

l843: 'general equation': which one?

l845-851: no statistics?

l853-856: write 'Batch A' and 'Batch B' on figure 9

l863: 616 and 662 nm are not characteristic of chloorphyll a and chlorophyll b, respectively

l870-878: rather obvious. Could this part reduced?

l916: give the solvent in which E-all-astaxanthin has been dissolved

l929: italize 'H.' in 'H. pluvialis'

l936

- of course, it cannot be the same biomass? Was it an aliquot?

- I would replace 'it' by 'the'

l953: e.g.

l965: under which type of atmosphere?

l968-973: rather complicated section

l969-970! 12% and 22% regarding what?

Missing citations

Kopecky, J., et al. (2000). "Microalgae as a source for secondary carotenoid production: a screening study." Algological Studies 98: 153-168

Schoefs, B., et al. (2001). "Astaxanthin accumulation in Haematococcus requires a cytochrome P450 hydroxylase and an active synthesis of fatty acids." FEBS LETTERS 500(3): 125-128.

Lemoine, Y. and B. Schoefs (2010). "Secondary ketocarotenoid astaxanthin biosynthesis in algae: a multifunctional response to stress." Photosynthesis Research 106: 155-177

Gateau, H., et al. (2017). "Carotenoids of microalgae used in food industry and medicine." Mini-Review in Medicinal Chemistry 17: 1140-1172

Solymosi, K., et al. (2015). Food colour additives of natural origin. Colour Additives for Foods and Beverages: Development, Safety and Applications. M. Scotter, Woodhead Publishing: 1-34.

Scarsini, M., et al. (2020). Carotenoid overproduction in microalgae: Biochemical and genetic engineering. Pigments from Microalgae Handbook. E. Jacob-Lopes, M. I. Queiroz and L. Q. Zepka. Cham, Springer International Publishing: 81-126

Schoefs, B. (2003). "Chlorophyll and carotenoid analysis in food products. A practical case-by-case view." Trends in Analytical Chemistry 22(6): 335-339.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: Comments.docx

Attachment

Submitted filename: Reviewer comments PONE-D-22-07202.docx

Development and validation of reliable astaxanthin quantification from natural sources

PONE-D-22-07202R1

Dear Dr. Labes,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Vandana Vinayak, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

authors have revised the manuscript and may be published as per Journals policy

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript addresses the problems of astaxanthin quantification, and it has significant scientific data to provide answers to the problems in quantification

Reviewer #2: Authors have answered all the comments correctly. This article is recommended for the possible publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Dr. Mrinal Kashyap

Reviewer #2: No

**********