Lipid Profile of Fresh and Aged Wollemia nobilis Seeds: Omega-3 Epoxylipid in Older Stored Seeds
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
The paper entitled: Lipid profile of fresh and aged Wollemia nobilis seeds: omega-3epoxylipid in older stored seeds is well done both in the Introduction section and Experimental part and for this reason I suggest to publish it in this form.
Author Response
Comment 1. The paper entitled: Lipid profile of fresh and aged Wollemia nobilis seeds: omega-3epoxylipid in older stored seeds is well done both in the Introduction section and Experimental part and for this reason I suggest to publish it in this form.
Response 1. Thank you very much for this comment.
Reviewer 2 Report
Comments and Suggestions for Authors
In the manuscript the fatty acid pattern in Wollemia nobilis seeds is determined by GC-MS. A key finding is that signals of epoxy-FA are detected by NMR.
The authors need to demonstrate more data about the presence and characterization of the epoxy-FA. Only NMR data is not sufficient.
There are several described methods for the detection of epoxy-PUFA e.g. by GC-MS as used in the present study (for example Lipids 37, 917–924).
The manuscript should be only considered for publication if this or another chromatography mass spectrometry technique has been used to support the NMR finding.
Further points
1) I am not convinced that the signal of the methyl group in the 1H NMR spectra can really be used to quantitatively determine the amount of omega-3, omega-6, omega-9 and saturated fatty acids. Why not use other signals in the spectrum that are specific for the different types of fatty acids and that are more isolated? (Or at least a combination of both signals)
2) I understand that you can prove by comparison with the NMR of the corresponding methyl ester that a 15,16-epoxy derivative is also present in WPS, but not how to rule out the absence of a 12,13-epoxy (or 9,10-epoxy) derivative. What signals would be expected for the other derivatives?
Specific points
Page 3, line 131f: Did you use “pure” CDCl3 or CDCl3 containing a specific amount of TMS?
Page 4, line 155ff: What amount of seed did you typically used for the oil extraction and isolation?
Page 5, line 203f: How did you chose on which signal the spectra are referenced?
Page 7, Figure 2: What does the peak at 3.1036 in the whole spectra mean?
Page 9, line 333: Could you show a direct comparison of the 1H NMR spectra of fresh and stored seeds and indicate the 9 “basic” signals and the 5 additional ones?
Page 9, line 339: Why did you decide to analyse your samples precisely for cis-15,16-epoxy-13-hydroxy 9Z,11E-octadecadienoic acid?
Author Response
Comments 1
In the manuscript the fatty acid pattern in Wollemia nobilis seeds is determined by GC-MS. A key finding is that signals of epoxy-FA are detected by NMR.
The authors need to demonstrate more data about the presence and characterization of the epoxy-FA. Only NMR data is not sufficient.
Response 1
Most seed oils consist of long chain fatty acid triglycerides. The purpose of the NMR data was to analyse the intact seed oil, mainly to determine if it had unusual glycerol ester content. Detection of the signals at 4.15 and 4.28 ppm and their characteristic pattern indicated that the seed oil consisted of triacyl glycerides. The 1H NMR signal at 1.06 ppm, consisting of a triplet, J = 7.5 Hz, is characteristic of an omega-epoxide [38]. The multiplet at 2.93 ppm was only detectable in seed oil samples with the signal at 1.06 ppm. Tentative identification and the omega-3 lipid as 15,16 epoxide of alpha-linolenic acid or ester was made based on the overlapping multiplets at 2.93 ppm for the H-15 and H-16 observed in the 1H NMR spectrum of the 15,16 epoxide of alpha-linolenic acid methyl ester [38]. Because of the low availability of seeds due to their high conservation status, insufficient aged seeds were available for isolation and full characterisation of the omega-3 epoxy lipid tentatively identified as the 15,16 epoxide of alpha-linolenic acid. Hydrolysis of triacyl glycerides is likely to be required to isolate the omega-3 fatty acid which may require derivatisation to achieve effective chromatographic purification.
Comments 2
There are several described methods for the detection of epoxy-PUFA e.g. by GC-MS as us ed in the present study (for example Lipids 37, 917–924).
Response 2
GC-MS epoxy-PUFA generally requires transesterification of triacyl glycerides. The conditions required to form the methyl esters may lead to opening of the epoxide ring and/or other degradation. The GC-MS analysis methods used could not detect epoxy lipids or polar metabolites such as hydroxy or hydroperoxyl lipid derivatives.
Comments 3
The manuscript should be only considered for publication if this or another chromatography mass spectrometry technique has been used to support the NMR finding.
Response 3
The authors believe that the NMR spectral data clearly show evidence for an omega-3 epoxy fatty acid indicated to be the 15,16 epoxide of alpha-linolenic acid by a corelating 1H NMR signals. Moreover, alpha linolenic acid was the only omega-3 fatty acid identified by GC-MS analysis of methyl esters and is likely to be the source of the omega-3 fatty acid. The results are tentative, full characterisation was not feasible due to limited amounts of seeds and resources.
Comments 4
Further points
1) I am not convinced that the signal of the methyl group in the 1H NMR spectra can really be used to quantitatively determine the amount of omega-3, omega-6, omega-9 and saturated fatty acids. Why not use other signals in the spectrum that are specific for the different types of fatty acids and that are more isolated? (Or at least a combination of both signals)
Response 4
The 1H NMR signal for the terminal methyl at 0.98 ppm clearly identifies fatty acids as omega-3, the methyl at 0.89 ppm identifies fatty acids as omega-6, omega-9 and saturated fatty acids both have their methyl at 0.88 ppm [38]. 1H NMR quantitation is reasonably accurate to determine the amount of omega-3 fatty acids. Less accurate for omega-6 fatty acids due to partial overlap with omega-9 and saturated fatty acids. For quantitation by this method omega-9 and saturated fatty acid are indistinguishable. They are quantitated as total omega-9 and saturated a fatty acid.
Comments 5
2) I understand that you can prove by comparison with the NMR of the corresponding methyl ester that a 15,16-epoxy derivative is also present in WPS, but not how to rule out the absence of a 12,13-epoxy (or 9,10-epoxy) derivative. What signals would be expected for the other derivatives?
Response 5
For the alpha-linolenic acid methyl esters the 1H NMR chemical shifts for the terminal methyl group are 1.057 for the 15,16-epoxide, 0.987 for the 12,13-epoxide and 0.975 ppm for the 9,10-epoxide [38]. Only the 15,16 epoxide can be clearly identified in a WPS oil mixture.
Comments 6
Specific points
Page 3, line 131f: Did you use “pure” CDCl3 or CDCl3 containing a specific amount of TMS?
Response 6
CDCl3 containing 0.03% TMS was used. This has been corrected in the “2.2 Reagents and Materials’ section.
Comments 7
Page 4, line 155ff: What amount of seed did you typically used for the oil extraction and isolation?
Response 7
The average seed weight was 23.8 mg, kernel weight 18.9 mg and oil weight 7.5 mg
Comments 8
Page 5, line 203f: How did you chose on which signal the spectra are referenced?
Response 8
The centre of each peak or multiplet was referenced relative to TMS.
Comments 9
Page 7, Figure 2: What does the peak at 3.1036 in the whole spectra mean?
Response 9
“The H-15 signal at 3.1 ppm observed for cis-15,16-epoxy-13-hydroxy-9Z,11E-octadecadienoic acid [39] was absent from the all samples of WPS oil indicating that this omega-3 fatty acid was not present in significant quantity in any samples. [line 339]” The mark at 3.1036 is to indicate the position of the H-15 signal at 3.1 ppm that would be observed for cis-15,16-epoxy-13-hydroxy-9Z,11E-octadecadienoic acid if it was present in observable quantity.
Comments 10
Page 9, line 333: Could you show a direct comparison of the 1H NMR spectra of fresh and stored seeds and indicate the 9 “basic” signals and the 5 additional ones?
Response 10
Figure S2 has been included in the revised supplementary materials to show “a direct comparison of the 1H NMR spectra of fresh and stored seeds and indicate the 9 “basic” signals and the 5 additional ones.”
Comments 11
Page 9, line 339: Why did you decide to analyse your samples precisely for cis-15,16-epoxy-13-hydroxy 9Z,11E-octadecadienoic acid?
Response 11
cis-15,16-epoxy-13-hydroxy-9Z,11E-octadecadienoic acid with a chemical shift of 1.06 ppm for the terminal methyl group was a possible omega-3 lipid in the stored Wollemi pine seed. This omega-3 lipid was shown to be not present by the absence of the H-15 signal at 3.1 ppm.
Reviewer 3 Report
Comments and Suggestions for Authors
The authors screened many acessions of Wollemia nobilis which has not been done prevoiusly. In addition, they identified epoxylipid in it which is rare information during storage. Thus, this manuscript can be published after minor corrections.
Introduction provided us with sufficient information and background as well as justificaition of this study.
Method of lipid profiling was detailed enough for reproducible study.
It is good idea to add fine tree picture and seeds for information for those who is not familiar with them.
In Table S3, there is inconsistent decimal points in valuesl; they should be corrected.
Author Response
Comments 1
The authors screened many acessions of Wollemia nobilis which has not been done prevoiusly. In addition, they identified epoxylipid in it which is rare information during storage. Thus, this manuscript can be published after minor corrections.
Introduction provided us with sufficient information and background as well as justificaition of this study.
Method of lipid profiling was detailed enough for reproducible study.
Response 1
Thank you very much for these comments.
Comments 2
It is good idea to add fine tree picture and seeds for information for those who is not familiar with them.
Response 2
Thank you for this suggestion. Pictures of trees and seeds have been included as Figure S3 and Figure S4.
Comment 3
In Table S3, there is inconsistent decimal points in values; they should be corrected.
Response 3
The inconsistency in the number of significant figures shown on Table S3 has been corrected.
Reviewer 4 Report
Comments and Suggestions for Authors
On the whole, the paper is well prepared and presents interesting results which can be considered as innovative in the presented area of science. Some small correction can be considered. Please see my suggestions:
Abstract: informative and readable
Line 29: Please remove the sentence: Supporting information
Introduction: well prepared based on up-to-date references, all important information are included
Methods: described in details, all information are provided, methodology adequate to the aim of studies
Results: Why SD is so high in the case of some of samples presented in Table 1 (i.e. >43% for 26.6 ± 11.6)?
On the wgole, Results and Discussion is well prepared. Authors presented most important results along with GC-MS and H NMR analysis. There is no significant errors. Statistical analysis is provided.
Conslusions: provided, informative based on most important results
Author Response
Comments 1
On the whole, the paper is well prepared and presents interesting results which can be considered as innovative in the presented area of science. Some small correction can be considered. Please see my suggestions:
Abstract: informative and readable
Response 1
Thank you very much for these comments and suggestions.
Comments 2
Line 29: Please remove the sentence: Supporting information
Response 2
Line 29 has been removed.
Comments 3
Introduction: well prepared based on up-to-date references, all important information are included
Methods: described in details, all information are provided, methodology adequate to the aim of studies
Results: Why SD is so high in the case of some of samples presented in Table 1 (i.e. >43% for 26.6 ± 11.6)?
Response 3
The SD is high because wild seeds were used with a wide range of seed sizes. At the time of the study seeds were in limited supply due to high demand for germination and cultivation to preserve the species.
Comments 4
On the wgole, Results and Discussion is well prepared. Authors presented most important results along with GC-MS and H NMR analysis. There is no significant errors. Statistical analysis is provided.
Conslusions: provided, informative based on most important results
Response 4
Thank you very much for these comments.
Round 2
Reviewer 2 Report
Comments and Suggestions for Authors
The authors did not improve the quality of the manuscript based our comments after the first submission.
Comments on the Quality of English Language
can be improved
