Rapid Screening for Mycosporine-like Amino Acids (MAAs) of Irish Marine Cyanobacteria and Their Antioxidant Potential
Round 1
Reviewer 1 Report
The manuscript is well-written and suitable for publication in sustainability. Cyanobacteria are the most primitive and diversified organisms and are well known for producing various bioactive compounds such as MAAs. Authors have advocated 12 MAAs from different isolates, of which 4 are novel. This is a very interesting finding. I have a very short question and one suggestion which is below;
1. Fig.2 resolution is very poor. It can be transferred to a supplementary file.
2. Many MAAs have been reported from cyanobacterial species, and how do the new isolates by the author differ in production and/or quality (bioactivity)??
Author Response
Comment: The manuscript is well-written and suitable for publication in sustainability. Cyanobacteria are the most primitive and diversified organisms and are well known for producing various bioactive compounds such as MAAs. Authors have advocated 12 MAAs from different isolates, of which 4 are novel. This is a very interesting finding. I have a very short question and one suggestion which is below;
Response: Thanks for your favourable comments. Accordingly, we made necessary changes and responded to your query below.
Comment: 1. Fig.2 resolution is very poor. It can be transferred to a supplementary file.
Response: Thanks for your suggestion. Accordingly, we moved Fig 2 as supplementary figure S4.
Comment: 2. Many MAAs have been reported from cyanobacterial species, and how do the new isolates by the author differ in production and/or quality (bioactivity)??
Response: Thanks for your query. We agree with the reviewer that many cyanobacterial species were reported to produce MAAs, however there is no systematic comparison in terms of their productivity. MAAs content in these new isolates were essentially different (0.114 - 0.511 A* mg DW-1 MAAs) compared to Calothrix sp. that was reported to contain 0.320 A* mg DW-1 MAAs (Garcia-Pichel, F.; Castenholz, R.W. Occurrence of UV-absorbing, mycosporine-like compounds among cyanobacterial isolates and an estimate of their screening capacity, Appl. Environ. Microbiol. 1993, 59, 163-169). (This information was provided in sections “3.2. Determination of specific MAAs content” as well as in discussion). None of our extracts were tested for quality (bioactivity) and hence we cant compare this information relative to new isolates.
Reviewer 2 Report
The manuscript evaluated various Irish marine cyanobacteria and identified potential producers of mycosporine-like amino acids (MAAs) to meet the increasing demand for replacing chemical sunscreen with bio-sunscreen. Specific strains were found to possess desirable MAAs content as high as 0.511 A* mg DW-1. Considerable amounts of radical scavenging activity and reducing antioxidant activities were also reported for tested stains. The quality of the study could be improved in the following aspects:
Content
1. Line 97, the methanol extracts were “left at 4 degree overnight”. Any particular reasons for this step?
2. The detailed information for the mass data processing software in not included. Assuming MassHunter was used to process the “User Spectra” and “Generate Formulas from Spectrum Peaks”. As the formula generations is based on mass resolution and accuracy of the mass signal, methods and standard compounds used to calibrate the Q-TOF mass spectrometer should be included.
3. In the section of LC-MS based metabolites analysis, no literature reference where exact same experimental conditions were used nor chromatographic traces for standard compounds were included. The MS data or even MS/MS alone is not sufficient for assignment of the peaks to known compounds.
4. Consider provide geological and environmental information regarding the tested cyanobacteria isolates as in Applied and Environmental Microbiology, Jan. 1993, p. 163-16. Any correlations between the MAA yield and UV exposure of the corresponding niche would be interesting.
5. Graphic evidence for all standard curves generated in DPPH assay, FRAP assay and ORAC assay should be included to showcase the linearity and dynamic range of the assays.
6. 30 μL, 20 μL and 20 μL MAA extract samples were used in DPPH assay, FRAP assay and ORAC assay respectively. Any dosing study has been conducted to ensure they stay within the dynamic range of the assays? If so, please include the description or referred literature.
7. Figure 2, any chance to add scale bar in the microscopic photos?
8. Leptolyngbya tenuis SABC010201 was found to possess notably higher 15 MAAs content but demonstrated medium radical scavenging activity and reducing antioxidant power in DPPH assay, FRAP assay respectively. Are these bioactivities more related to any particular species of MAA? Is it possible to provide an explanation for the observation in the discussion section?
Author Response
The manuscript evaluated various Irish marine cyanobacteria and identified potential producers of mycosporine-like amino acids (MAAs) to meet the increasing demand for replacing chemical sunscreen with bio-sunscreen. Specific strains were found to possess desirable MAAs content as high as 0.511 A* mg DW-1. Considerable amounts of radical scavenging activity and reducing antioxidant activities were also reported for tested stains. The quality of the study could be improved in the following aspects:
Content
Comment: 1. Line 97, the methanol extracts were “left at 4 degree overnight”. Any particular reasons for this step?
Response: Thanks for your query. This step was added to ensure improved extractability due to uniform and longer contact of cyanobacterial cells with aqueous methanol before final extraction at 45 °C. This information was added in the revised manuscript.
Comment: 2. The detailed information for the mass data processing software in not included. Assuming MassHunter was used to process the “User Spectra” and “Generate Formulas from Spectrum Peaks”. As the formula generations is based on mass resolution and accuracy of the mass signal, methods and standard compounds used to calibrate the Q-TOF mass spectrometer should be included.
Response: Thanks for the above comments. The mass data processing was carried out using Agilent Mass Hunter Workstation Qualitative Analysis B.05.00 software, which is included in the manuscript. Detail LC-MS identification method was provided in section “2.6. LC-MS identification of specific MAAs” where generation of Formulas for possible new compounds were based on the features of the software. For standard MAAs, we have used our accurate mass database created during this study based on published literature.
Comment: 3. In the section of LC-MS based metabolites analysis, no literature reference where exact same experimental conditions were used nor chromatographic traces for standard compounds were included. The MS data or even MS/MS alone is not sufficient for assignment of the peaks to known compounds.
Response: Thanks for your expertise and finding out this obvious mistake. We have now added appropriate reference that was adopted for LC-MS analysis. We agree with the reviewer that MS data or even MS/MS alone is not sufficient for assignment of the peaks to known compounds. Our analysis comprised several steps starting from spectrophotometric absorbance peak for possible UV-absorbing compounds followed by DAD profile of each extracts at 300, 310, 320, 330, 340, 350, and 360 nm to get an idea about the MAAs elution pattern. Subsequently, extracted ion chromatogram (EIC) of each known MAAs was obtained from the total ion chromatogram (TIC) by entering the accurate [M-H]- values. If any EIC peaks were detected, they were then compared with the best DAD profile to match the retention time (RT) from the literature (compiled a database). Then, the MS spectra profile of interested EIC peaks was obtained to confirm the presence of specific MAAs with accurate mass values.
Comment: 4. Consider provide geological and environmental information regarding the tested cyanobacteria isolates as in Applied and Environmental Microbiology, Jan. 1993, p. 163-16. Any correlations between the MAA yield and UV exposure of the corresponding niche would be interesting.
Response: Thanks for your valuable comment. We have previously published the geographical and environmental information and accordingly we changed the revised manuscript adding “(details of geographical location and sampling sites were published earlier [31])”. We reserved the UV-exposure data for another publication in relation to the response of MAAs content to UV lights exposure, but in general each isolate tested showed an induced response to UV exposure as that of the findings in Applied and Environmental Microbiology, Jan. 1993, p. 163-16.
Comment: 5. Graphic evidence for all standard curves generated in DPPH assay, FRAP assay and ORAC assay should be included to showcase the linearity and dynamic range of the assays.
Response: Thanks for your expert comment. Accordingly, we have now added all standard curves as supplementary figures S1, S2, and S3 which we used for the quantification using appropriately diluted samples to obtain the values within the range.
Comment: 6. 30 μL, 20 μL and 20 μL MAA extract samples were used in DPPH assay, FRAP assay and ORAC assay respectively. Any dosing study has been conducted to ensure they stay within the dynamic range of the assays? If so, please include the description or referred literature.
Response: Thanks for your expert comment. In general, we initially tested each extracts as neat and 20X diluted to find if the values obtained were within the range of the standard curve. Then, each sample was diluted to 20X or 50X as required, and used only one concentration in triplicate for each assay. The average of this triplicate value was presented. We have not carried out any concentration dependent assays. 30 μL, 20 μL and 20 μL samples were used to make up the final assay volume as well as to match the assays for generating the standard curves.
Comment: 7. Figure 2, any chance to add scale bar in the microscopic photos?
Response: Thanks for your comment. We have now moved Figure2 as supplementary figure S4 with scale bars added for each microscopic photos.
Comment: 8. Leptolyngbya tenuis SABC010201 was found to possess notably higher 15 MAAs content but demonstrated medium radical scavenging activity and reducing antioxidant power in DPPH assay, FRAP assay respectively. Are these bioactivities more related to any particular species of MAA? Is it possible to provide an explanation for the observation in the discussion section?
Response: Thanks for your valuable comment. We believe it is due to specific type of MAAs and other compounds present in the tested extracts. We have added an explanation in the discussion as suggested as “Surprisingly, although Leptolyngbya tenuis SABC010201 possessed notably higher MAAs content demonstrated medium radical scavenging activity and reducing anti-oxidant power in DPPH and FRAP assays. This possibly indicates the types of specific MAAs and other unknown compounds present in the tested extracts.”
Reviewer 3 Report
The manuscript reported the extraction of MAAs from 53 Irish marine cyanobacteria. The authors found that the Leptolyngbya tenuis SABC010201 is the highest producer of MAAs content. Besides, 8 types of MAAs were founded using LC-MS analysis ((myco- sporine-glutamicol, mycosporine-glutaminol-glucoside, mycosporine-serinol, mycosporine-tau- rine, palythine, palythine-threonine-sulphate, porphyra-334 and usujirene) in eight cyanobacteria.). The manuscript shows new data but needs a revision before publication.
Please see the following comments:
1- The claim part at the end of the introduction is too short and didn’t show your work and expected data. It is better to redesign this part by adding more information.
2- The Figure's quality is too low. It is highly recommended to redesign all figures with high resolution and specific programs such as Origin. Besides, the X and Y labels are low.
3- It is better to put the chemical structure of MAAs.
4- FT-IR, and HNMR are useful techniques for better understanding the functionality of MAAs as antioxidant materials.
5- The conclusion part is too short. It Is highly recommended to redesign this section by focusing on the experimental analysis, results, and future prospects.
Author Response
The manuscript reported the extraction of MAAs from 53 Irish marine cyanobacteria. The authors found that the Leptolyngbya tenuis SABC010201 is the highest producer of MAAs content. Besides, 8 types of MAAs were founded using LC-MS analysis ((myco- sporine-glutamicol, mycosporine-glutaminol-glucoside, mycosporine-serinol, mycosporine-tau- rine, palythine, palythine-threonine-sulphate, porphyra-334 and usujirene) in eight cyanobacteria.). The manuscript shows new data but needs a revision before publication.
Please see the following comments:
Comment: 1- The claim part at the end of the introduction is too short and didn’t show your work and expected data. It is better to redesign this part by adding more information.
Response: Thanks for your valuable comment. We have revised the end of introduction part as suggested as “In the present investigation, we, therefore, evaluated Irish marine cyanobacteria as potential producers of MAAs and tested their antioxidant activities (DPPH, FRAP, and ORAC) from the point of view of future commercial applications. We have also characterised the extracts for the identification of known MAAs and potentially new UV-absorbing compounds.”
Comment: 2- The Figure's quality is too low. It is highly recommended to redesign all figures with high resolution and specific programs such as Origin. Besides, the X and Y labels are low.
Response: Thanks for your comment. We have moved this figure as supplementary figure S4 as per other reviewers comment. We have used Inkscape for organising these images and added scale bars in the supplementary figure. We have published high resolution figures for these isolates (Biology 2019, 8, 59; doi:10.3390/biology8030059).
Comment: 3- It is better to put the chemical structure of MAAs.
Response: Thanks for your suggestion. Accordingly, we have added chemical structures of identified MAAs as Figure 2 of the revised manuscript.
Comment: 4- FT-IR, and HNMR are useful techniques for better understanding the functionality of MAAs as antioxidant materials.
Response: Thanks for your comment. We are now privileged with FTIR and NMR facilities, and hopefully in our future studies we will use those precise methods.
Comment: 5- The conclusion part is too short. It Is highly recommended to redesign this section by focusing on the experimental analysis, results, and future prospects.
Response: Thanks for your expert comment. We have now improved our conclusion part considering your suggestions. The revised conclusion include the following.
“This is the first comprehensive evaluation of UV-screening compounds in Irish marine cyanobacteria, which identified eight cyanobacteria as potential MAAs producers with a specific content of 0.114-0.511 A* mg DW-1. However, Leptolyngbya africana SABC021601 can be considered as the best both in terms of specific content and antioxidant activities. To the best of our knowledge, this study also reports for the first time known MAAs, such as mycosporine-glutamicol, mycosporine-serinol, and palythine-threonine-sulphate from marine cyanobacteria. This study identified a total of eight different types of known MAAs (mycosporine-glutamicol, mycosporine-glutaminol-glucoside, mycosporine-serinol, mycosporine-taurine, palythine, palythine-threonine-sulphate, porphyra-334 and usujirene) and four unknown UV-absorbing compounds named as M-314, M-326, M-330, and M-346. These unknown MAAs are novel findings and need future studies related to their structural and bio-functional properties.”
Round 2
Reviewer 3 Report
The authors have raised most of the comments. So, I recommend accepting the manuscript in its current form.
