Laminaria hyperborea as a Source of Valuable Glyceroglycolipids—A Characterization of Galactosyldiacilglycerols in Stipe and Blade by HPLC-MS/MS
Round 1
Reviewer 1 Report
In this article paper “Laminaria hyperborea as a source of valuable glyceroglycolipids - a characterization of MGDG and DGDG in stipe and blade by HPLC-MS/MS”, mono- and digalactosyldiacylglycerols (MGDG and DGDG) have been identified and the different species distribution in stipe and blade have been discovered by using total ion chromatograms (TIC). The author did a through study into the very detailed distribution of numbers of carbons and the numbers of double bonds equivalents in the acyl chains. In the study, they proved there are clear difference in the glyceroglycolipid composition of blade and stipe. This report is of great interest and support the great potential for further valorization of biomass from L. hyperborean. The further research into the bioactivity of the different molecular species of MGDG and DGDG form the author is very promising.
Overall, the author solved the scientific question of “characterization of MGDG and DGDG in stipe and blade”. It is a well-organized, well-written research article.
However, there are some points should be considered.
Page 2, line 54: “digalatosyldiacylglycerols” should be “digalactosyldiacylglycerols”.
Page 3, line 97 and line 98: “once…, once…” should be “one…, one…”.
Page 3, line 08: “The polar phases were extracted…”, I suppose it should be “The aqueous phases were extracted…”.
Page 3, line 38: “sodium methoxide solution” should be “sodium methoxide methanol solution”.
Page 4, line 54: “… was held 3 for …” should be “… was held for 3”.
Page 7, Figure 1: On the scheme and legend “-R1COOH” and “-R2COOH”, I suppose they should be “-R1COO-” and “-R2COO-”. Please double check.
Page 7, line 83: The tittle “L. hyperborea” would be better as full name “Laminaria hyperborea”.
Please provide a more specified tittle. Same as “3.3.1”.
Page 7, line 85: “The NL and FFA fractions were not found to contain any of the predicted MGDG or DGDG.” If the author could add any comment or explanation, that would be better.
Page 7, line 94: “A higher number…” there should be a reference. Or it would be better if the author could provide quantified data like page 8 line 93.
Page 10, line 38: “The most abundant acyl pairs in these peaks correspond to MGDG(18:4/18:4), MGDG(20:5/18:4), DGDG(16:0/18:1) and DGDG(20:5/18:4) in blade only, and MGDG(14:0/18:2), MGDG(16:0/18:1), and MGDG and DGDG(14:0/18:1)”.
The author has labeled all these species in figure 2. Please add the label in the context.
Page 10, figure 2: Please keep the Y axis consistent and add the retention time of first graph if possible.
Page 11, figure 3: The legend “Figure 3:…” should keep the same as figure 1 and 2 that is dot instead of colon.
Page 12, figure 4: The picture there is VERY blur. The words there is not readable. C16 in the legend is stripped. However, there is no differences with C20 in the picture.
In the legend, “C20 (che- quered)” should be “C20 (chequered)”. Same for line 87.
Page 12, chapter 3.4: This part has been discussed well in the introduction (page 2, line 57). It is not necessary to describe again.
Comments for author File: 
Comments.pdf
Author Response
We thank the reviewer for excellent suggestions and comments on the paper. We have done our best to address your concerns. Our responses are listed below. The newly added or revised portions of text are marked in a separate file named “….compared”.
Page 2, line 54: “digalatosyldiacylglycerols” should be “digalactosyldiacylglycerols”.
Author comments: We have corrected this
Page 3, line 97 and line 98: “once…, once…” should be “one…, one…”.
Author comments: We have corrected this
Page 3, line 08: “The polar phases were extracted…”, I suppose it should be “The aqueous
phases were extracted…”.
Author comments: We have corrected this
Page 3, line 38: “sodium methoxide solution” should be “sodium methoxide methanol solution”.
Author comments: We have corrected this
Page 4, line 54: “…was held 3 for …” should be “…was held for 3”.
Author comments: We have corrected this
Page 7, Figure 1: On the scheme and legend “-R1COOH” and “-R2COOH”, I suppose they should be “-R1COO-” and “-R2COO-”. Please double check.
Author comments: We have double checked, and the original version is correct, this is also described in the figure 1 legend: …” combined neutral loss of a hexose residue and each of the acyl chains as neutral carboxylic acids”.
Page 7, line 83: The tittle “L. hyperborea” would be better as full name “Laminaria hyperborea”.
Author comments: We agree and have corrected this
Please provide a more specified tittle. Same as “3.3.1”.
Author comments: We have changed the title.
Page 7, line 85: “The NLand FFA fractions were not found to contain any of the predicted MGDGorDGDG.” If the author could add any comment or explanation, that would be better.
Author comments: We have added an explanation in the manuscript.
Page 7, line 94: “Ahigher number…” there should be a reference. Or it would be better if the authorcould provide quantified data like page 8 line 93.
Author comments: We have updated and incorporated data as suggested
Page 10, line 38: “The most abundant acyl pairs in these peaks correspond to
MGDG(18:4/ 18:4), MGDG(20:5/ 18:4), DGDG(16:0/ 18:1) and DGDG(20:5/ 18:4) in blade only,
and MGDG(14:0/ 18:2), MGDG(16:0/ 18:1), and MGDGand DGDG(14:0/ 18:1)”.
The author has labeled all these species in figure 2. Please add the label in the context.
Author comments: We don’t understand what you want us to change by this comment.
Page 10, figure 2: Please keep the Yaxis consistent and add the retention time of first graph if possible
Author comments: We have corrected this
Page 11, figure 3: The legend “Figure 3:…” should keep the same as figure 1 and 2 that is dot insteadof colon.
Author comments: We have corrected this
Page 12, figure 4: The picture there is VERYblur. The words there is not readable. C16 in the legendis stripped. However, there is no differences with C20 in the picture.
In the legend, “C20 (che- quered)” should be “C20 (chequered)”. Same for line 87.
Author comments: We have double checked and exchanged all figures with error corrections
Page 12, chapter 3.4: This part has been discussed well in the introduction (page 2, line 57). It is notnecessary to describe again.
Author comments: We have deleted this chapter
Reviewer 2 Report
The manuscript is of interest to the community and presents interesting results to be reported, increasing knowledge about Laminaria hyperborea. I consider it approved after due corrections.
Suggestion:
- Change the title of paper to "Laminaria hyperborea as a source of valuable glyceroglycolipids - a characterization of galactosyldiacylglycerols in stipe and blade by HPLC-MS/MS", bacause it is necessary to avoid abbreviation in the title. I believe that MGDG and DGDG are abbreviations of terms more specific and less known than HPLC-MS/MS. Furthermore, monogalactosyldiacylglycerol and digalactosyldiacylglycerol are keywords.
Questions:
- How were all lipids identified? Was it done through putative notation and comparison with databases? If yes, which databases? Or was the identification done only through fragmentation proposals? Please, if possible, add a discussion of this in the manuscript.
Figures:
- Put "a" and "b" in the Figure 3 indicating which graph is referred to Figures 3a and 3b, respectively.
- It's necessary increase the size of Figure 3 and font size. Perhaps, put the "graphic b" (Figure 3b) below "graphic a" (Figure 3a)
- Improve the resolution of figure 4 and increase the font size in the legend and graph axes.
Minor corrections:
1-) Format "L. hyperborea" in italic style (line 83 - title of section 3.3);
2-) Correct "L. hyper- borea" to "Laminaria hyperborea" (line 337, in conclusions);
3-) Write "sp." from "Gracilaria sp." without the italic style (line 388, in the reference 9);
4-) Correct "cli- matic zones" to "climatic zones" (line 431, in the reference 25);
5-) Correct "inter- acting effects" to "interacting effects" (line 445, in the reference 31);
6-) Correct "extrac- tion method" to "extraction method" (line 452, in the reference 34);
7-) Correct "Bioprospect- ing of marine" to "Bioprospecting of marine" (line 462, in the reference 38);
8-) Correct "eukary- otic pathways" to "eukaryotic pathways" (line 505, in the reference 53).
Author Response
We thank the reviewer for excellent suggestions and comments on the paper. We have done our best to address your concerns. Our responses to address specific concerns are below. The newly added or revised portions of text are marked in a separate file named “….compared”.
Suggestion:
- Change the title of paper to "Laminaria hyperborea as a source of valuable glyceroglycolipids - a characterization of galactosyldiacylglycerols in stipe and blade by HPLC-MS/MS", bacause it isnecessary to avoid abbreviation in the title. I believe that MGDG and DGDG are abbreviations ofterms more specific and less known than HPLC-MS/MS. Furthermore,monogalactosyldiacylglycerol and digalactosyldiacylglycerol are keywords
Author comments: We agree and have corrected the title according to the reviewer´s suggestion
Questions:
How were all lipids identified? Was it done through putative notation and comparison with databases? If yes, whichdatabases? Or was the identification done only through fragmentation proposals? Please, if possible, add a discussionof this in the manuscript.
Author comments: Identification was performed on the bases of MS/MS fragmentation pattern as described in Materials and Methods, section 2.6 and in Results and discussion, section 3.2.
Figures:
- Put "a" and "b" in the Figure 3 indicating which graph is referred to Figures 3a and 3b, respectively.
Author comments: We have corrected this
- It's necessary increase the size of Figure 3 and font size. Perhaps, put the "graphic b" (Figure 3b)below "graphic a" (Figure 3a)
Author comments: This has been corrected
- Improve the resolution of figure 4 and increase the font size in the legend and graph axes.
Author comments: All figures have been updated.
Minor corrections:
1-) Format "L. hyperborea" in italic style (line 83 - title of section 3.3);
Author comments: This has been corrected
2-) Correct "L. hyper- borea" to "Laminaria hyperborea" (line 337, in conclusions);
Author comments: This has been corrected
3-) Write "sp." from "Gracilaria sp." without the italic style (line 388, in the reference 9);
Author comments: This has been corrected
4-) Correct "cli- matic zones" to "climatic zones" (line 431, in the reference 25);
Author comments: This has been corrected
5-) Correct "inter- acting effects" to "interacting effects" (line 445, in the reference 31);
Author comments: This has been corrected
6-) Correct "extrac- tion method" to "extraction method" (line 452, in the reference 34);
Author comments: This has been corrected
7-) Correct "Bioprospect- ing of marine" to "Bioprospecting
of marine" (line 462, in the reference 38);
Author comments: This has been corrected
8-) Correct "eukary- otic pathways" to "eukaryotic pathways" (line 505, in the reference 53).
Author comments: This has been corrected
Reviewer 3 Report
This is an interesting paper where the authors characterize MGDG and DGDG in the stipe and blade of Laminaria hyperborea for using it as a possible source of glyceroglyolipids. The study is well designed and executed, and I would suggest the revisions below to further improve the paper.
1) The authors are strongly advised to add a graphical abstract as it would help readers easily understand the paper, improve the readability of the paper, and increase its impact.
2) The conclusion section needs to be improved to include a detailed explanation of what the findings of the study mean, what the impacts are and what possible applications this may lead to.
3) The references section seems to be formatted incorrectly. Please double-check this to ensure that the spacing is correct and there is no extra white space in the last 4 pages.
4) For the LC part of the LCMS, what photodetector (UV, PDA, etc) was used? and what are the detector settings (Wavelength, etc)? Please include that.
5) Figure 2 needs to be corrected. Peaks IV, V, and VII in the top and bottom chromatogram do not align. Is there a shift in RT? If so, why? Please provide extracted ion chromatograms in the supplementary. Also, provide the mass spectra of the annotated peaks.
6)Sample Chamber temperature for LC?
Author Response
We thank the reviewer for excellent suggestions and comments on the paper. We have done our best to address your concerns. Our responses are listed below. The newly added or revised portions of text are marked in a separate file named “….compared”.
1) The authors are strongly advised to add a graphical abstract as it would help readers easily understand the paper,improve the readability of the paper, and increase its impact.
Author comments: We have added Graphical abstrac into the manuscript. However, this was also uploaded in the first submition and we don´t know why it could not be seen by the reviewer.
2) The conclusion section needs to be improved to include a detailed explanation of what the findings of the study mean, what the impacts are and what possible applications this may lead to.
Author comments: In our study we have performed a characterization of the glyceroglycolipids in L.hyperborea, a kelp native to the North Atlantic region. We have not managed to find other studies with the same extensive characterization of these lipids. Some MGDG and DGDG from seaweed have already shown positive bioactive properties in other studies and we believe that our study can be useful to all current and future kelp biomass users. This is already stated in the conclusion.
3) The references section seems to be formatted incorrectly. Please double-check this to ensure that the spacing iscorrect and there is no extra white space in the last 4 pages.
Author comments: This has been corrected
4) For the LC part of the LCMS, what photodetector (UV, PDA, etc) was used? and what are the detector settings (Wavelength, etc)? Please include that.
Author comments: We have not used a photodetector, only MS-detection
5) Figure 2 needs to be corrected. Peaks IV, V, and VII in the top and bottom chromatogram do not align. Is there a shift in RT? If so, why? Please provide extracted ion chromatograms in the supplementary. Also, provide the massspectra of the annotated peaks.
Author comments: The chromatograms are now aligned and corrected. There might be a small shift in these two chromatograms because we bougth a new column between these experiments. We don’t see that mass spectra improve the understanding of the study and the two other rewievers did not ask for this.
6) Sample Chamber temperature for LC?
Author comments: This has been added to the manuscript.
