Rapid characterization of hypanthium and seed in wild and cultivated rosehip: application of Raman microscopy combined with multivariate analysis

Rosehip (pseudo-fruit) of dog rose (Rosa canina L.) is highly valued, and owing to nutritional and sensory properties it has a significant place in the food industry. This work represents an innovative report focusing on the evaluation of the phytochemical composition of rosehips (hypanthium and seed) grown in different locations in Serbia, using Raman microspectroscopy combined with multivariate data analysis. Some significant differences arose between the analysed rosehip samples with regard to the chemical profile of both hypanthium parenchyma cells and seed, although no evident discrimination was recorded between the samples of wild and cultivated rosehip. The differences between the hypanthium samples compared were mainly determined by the content of carotenoids, phenolic compounds and polysaccharides, whereas phenolics, polysaccharides (pectin, cellulose and hemicellulose) and lipids (to a lower extent) contributed to the seed sample discrimination. The differences observed between the rosehip samples may be attributed to abiotic factors (growing, ripening and storage conditions), which had a significant impact on the carotenoid and polyphenols biosynthesis.


Recommendation?
Major revision is needed (please make suggestions in comments)

Comments to the Author(s)
In this article, authors reported that multivariate analysis of the fingerprint region of Raman spectra of the fresh ripe rosehip pulp can tell if the roship is a cultivar or a wild type. However, the similar analysis on the seeds could not distinguish a cultiver from the wild type. How the sample was prepared lacks clarity. Choice of different lasers to record the spectra for seed and pulp is not also clear. Many conclusions have been drawn that are not substantiated by the results. Specific comments are given below: 1) The title says "characterization of storage reserves", however, no temporal data is given. It is not even clear from the methods, how long the rosehips were stored and in which condition before the spectra were measured. It is important as the samples were collected from different geographical locations.
2) Regarding the preparation of the seeds two statements were made: a) "for Raman spectroscopy analysis, seeds were separated from hypanthium for each sample, and grinded to fine powder in a mill." b) "Raman microspectroscopy of rose hip fruit was focusing on direct measurement of rosehips hypanthium parenchyma cells and its seeds. The seeds were longitudinally cut at room temperature…" Please clarify how the seeds were prepared for Raman measurement.
3) Wouldn't it be better to record spectra from homogenized pulp? 4) Why 532 nm laser was used for the fleshy part and 785 nm for seeds? The fleshy part contains more light active molecules and fluorophores. Any fluorescence would suppress weak signals in Raman spectra. Wouldn't it be better to use 785 nm laser for the fleshy parts as well.
5) The authors claimed the method to be a "novel screening tool" developed by the authors, however, PCA coupled with Raman spectroscopy is already a widely used method.
Many conclusions have been drawn which are not substantiated by the results and purely hypothetical: 6) The authors conclude that the method could be used "...fast selection of rosehips...without fruit destruction." It would be relevant for sorting individual fruits even from the same harvest. However, the authors undertook no such study to show that the method can actually distinguish different quality fruits from the same harvest. Instead, they averaged over many samples from the same harvest.
7) The authors conclude that the method could "optimize the production of high-quality fruit." This study could only distinguish wild and cultivated varieties. From this study it cannot be concluded if the low and high quality fruit could be distinguished by this method. The seeds contained widely different levels of phytochemicals as evident from the Raman spectra of the seeds. However, PCA could not distinguish the seeds. It can be argued that the method shall fail to distinguish high and low quality fruits. 8) On "fruit development and maturation," as well, no study was undertaken. All the results are on mature fruits.
Additional experiments maybe performed: 9) FT-IR is complementary to Raman spectroscopy. It would be interesting to see if PCA of FT-IR spectra can lead to the similar results.
Minor comments: 10) Figure 3 and 5 may be moved to supporting information. 11) Authors may tabulate the peak positions of figure 3 to highlight the peak shift. 12) "The ancient Romans believed that...bite of rabid dogs." Should accompany a statement that no scientific association has been found. 13) "...used in folk medicine in wound-healing and skin disease treatments [5]." Should accompany a statement if there is any scientific proof and cite the primary sources.
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Dear Professor Popović-Djordjević: Title: Rapid characterisation of storage reserves in wild and cultivated rosehip: application of Raman microscopy combined with multivariate analysis Manuscript ID: RSOS-202064 Thank you for your submission to Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
The editor assigned to your manuscript has now received comments from reviewers. We would like you to revise your paper in accordance with the referee and Subject Editor suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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(ii) The information on the value of Rosehip in the Republic of Serbia is more suited to the introduction than the summary. Is it valued in the rest of Europe? The world? (iii) The acronym PCA should be explained in the Summary, and all acronyms should be spelled out when first used in the text. (iv) "The absence of interference with water molecules...". Water does have a Raman scattering signal. It is, however relatively weak, although it is not absent! (v) "rosehips hypanthium parenchyma cells" are mentioned for the first time in Section 3.2. They should be mentioned earlier.
(vi) "a frequency-doubled Nd/YAG laser.." normally has a wavelength of 532 nm (although in an Xplora system, a semiconductor diode laser of the same wavelength is usually employed). A 785 nm laser is also usually a semiconductor diode laser. (vii) How often was the instrument calibrated? Was it an "autocalibration" (I think Labspec 6) or a manual calibration (I think Labspec 5)? (viii) With reference to (vii), the shifts in spectra in Figure 2 and Figure 3 may be an indication that the calibration was different for different measurements. This is also supported by the loading of PC2 in Figure 6, which looks like a first derivative of PC1. It should be confirmed that the calibration for all measurements was the same. (ix) the loading of PV1 of Figure 6 is mainly dominated by a baseline drift below ~950cm-1. apart from this baseline drift, all other identifiable spectral features are positive, and therefore do not identify an difference between samples. (x) the Intensity axis of Figure 5 should show numbers... where is the zero? This may who the origin of the baseline drift. (xi) All spectra, rather than just the means, should be input into the PCA plot, to show the intraand inter sample variability.
Reviewer: 2 Comments to the Author(s) In this article, authors reported that multivariate analysis of the fingerprint region of Raman spectra of the fresh ripe rosehip pulp can tell if the roship is a cultivar or a wild type. However, the similar analysis on the seeds could not distinguish a cultiver from the wild type. How the sample was prepared lacks clarity. Choice of different lasers to record the spectra for seed and pulp is not also clear. Many conclusions have been drawn that are not substantiated by the results. Specific comments are given below: 1) The title says "characterization of storage reserves", however, no temporal data is given. It is not even clear from the methods, how long the rosehips were stored and in which condition before the spectra were measured. It is important as the samples were collected from different geographical locations.
2) Regarding the preparation of the seeds two statements were made: a) "for Raman spectroscopy analysis, seeds were separated from hypanthium for each sample, and grinded to fine powder in a mill." b) "Raman microspectroscopy of rose hip fruit was focusing on direct measurement of rosehips hypanthium parenchyma cells and its seeds. The seeds were longitudinally cut at room temperature…" Please clarify how the seeds were prepared for Raman measurement.
3) Wouldn't it be better to record spectra from homogenized pulp? 4) Why 532 nm laser was used for the fleshy part and 785 nm for seeds? The fleshy part contains more light active molecules and fluorophores. Any fluorescence would suppress weak signals in Raman spectra. Wouldn't it be better to use 785 nm laser for the fleshy parts as well.
5) The authors claimed the method to be a "novel screening tool" developed by the authors, however, PCA coupled with Raman spectroscopy is already a widely used method.
Many conclusions have been drawn which are not substantiated by the results and purely hypothetical: 6) The authors conclude that the method could be used "...fast selection of rosehips...without fruit destruction." It would be relevant for sorting individual fruits even from the same harvest. However, the authors undertook no such study to show that the method can actually distinguish different quality fruits from the same harvest. Instead, they averaged over many samples from the same harvest.
7) The authors conclude that the method could "optimize the production of high-quality fruit." This study could only distinguish wild and cultivated varieties. From this study it cannot be concluded if the low and high quality fruit could be distinguished by this method. The seeds contained widely different levels of phytochemicals as evident from the Raman spectra of the seeds. However, PCA could not distinguish the seeds. It can be argued that the method shall fail to distinguish high and low quality fruits. 8) On "fruit development and maturation," as well, no study was undertaken. All the results are on mature fruits.
Additional experiments maybe performed: 9) FT-IR is complementary to Raman spectroscopy. It would be interesting to see if PCA of FT-IR spectra can lead to the similar results.
Minor comments: 10) Figure 3 and 5 may be moved to supporting information. 11) Authors may tabulate the peak positions of figure 3 to highlight the peak shift. 12) "The ancient Romans believed that...bite of rabid dogs." Should accompany a statement that no scientific association has been found. 13) "...used in folk medicine in wound-healing and skin disease treatments [5]." Should accompany a statement if there is any scientific proof and cite the primary sources.
Author's Response to Decision Letter for (RSOS-202064.R0) See Appendix A.

Are the interpretations and conclusions justified by the results? Yes
Is the language acceptable? Yes Do you have any ethical concerns with this paper? No

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
Although the authors have addressed many of the issues raised during the initial review, there remain some concerns, particularly in relation to the PCA. Figure 4 indicates that, those samples which score positively according to PC1 (a) have strong carotenoid features (b), and that those that score negatively have a strong background tail between (in (b) this is "Variable 0-250"). There are no clearly identifiable chemical features which are associated with the negatively scoring samples in Fig 4(a). This suggests an issue of baselining the spectra.

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation?
Accept as is

Comments to the Author(s)
The authors adequately addressed all the issues in the revised manuscript. It is suitable for publication in its current form.

Decision letter (RSOS-202064.R1)
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Dear Professor Popović-Djordjević: Title: Rapid characterisation of hypanthium and seed in wild and cultivated rosehip: application of Raman microscopy combined with multivariate analysis Manuscript ID: RSOS-202064.R1 Thank you for submitting the above manuscript to Royal Society Open Science. On behalf of the Editors and the Royal Society of Chemistry, I am pleased to inform you that your manuscript will be accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the reviewers' comments at the end of this email.
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RSC Subject Editor:
Comments to the Author: (There are no comments.) ************************************** Reviewer comments to Author: Reviewer: 1 Comments to the Author(s) Although the authors have addressed many of the issues raised during the initial review, there remain some concerns, particularly in relation to the PCA. Figure 4 indicates that, those samples which score positively according to PC1 (a) have strong carotenoid features (b), and that those that score negatively have a strong background tail between (in (b) this is "Variable 0-250"). There are no clearly identifiable chemical features which are associated with the negatively scoring samples in Fig 4(a). This suggests an issue of baselining the spectra.

Reviewer: 2
Comments to the Author(s) The authors adequately addressed all the issues in the revised manuscript. It is suitable for publication in its current form.

See Appendix B.
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Dear Professor Popović-Djordjević: Title: Rapid characterisation of hypanthium and seed in wild and cultivated rosehip: application of Raman microscopy combined with multivariate analysis Manuscript ID: RSOS-202064.R2 It is a pleasure to accept your manuscript in its current form for publication in Royal Society Open Science. The chemistry content of Royal Society Open Science is published in collaboration with the Royal Society of Chemistry.
The comments of the reviewer(s) who reviewed your manuscript are included at the end of this email.
Please see the Royal Society Publishing guidance on how you may share your accepted author manuscript at https://royalsociety.org/journals/ethics-policies/media-embargo/. After publication, some additional ways to effectively promote your article can also be found here https://royalsociety.org/blog/2020/07/promoting-your-latest-paper-and-tracking-yourresults/.  Figure 6, which looks like a first derivative of PC1. It should be confirmed that the calibration for all measurements was the same.

Authors:
In figures 2 and 3 the small shifts are a consequence of heterogeneous plant material and there are regularly present interactions of carotenoids with other cell constituents (Schulz et al., 2006;Baranska et al., 2006). The shifts are not-connected with the already done calibration. All Raman shifts bellow 3cm-1 (i.e. bellow used resolution) cannot be considered for interpretation since they are below resolution. Autocalibration (i.e. procedure include check all lasers and gratings using a silicone as reference) was done every time before starting the process of spectral recording for all laser and all available gratings in combination with objectives.
As we pointed out above, calibration procedure was performed frequently and in accordance with manufacturer recommendation. We used basic spectra for PCA analysis (without transformation into derivative form). Sharp peaks in loadings plots are results of relatively high resolution used in our measurements as well as the good initial spectra.

Authors:
We have presented raw spectra of all hypantium and seed individual samples in the revised supporting material document. Intensity numbers are included in those spectra.
(xi) All spectra, rather than just the means, should be input into the PCA plot, to show the intra-and inter sample variability.
Authors: Raman spectra of analyzed samples were recorded as repeated measurements, but we used mean values as representative of each sample, in order to examine the similarities and differences between the samples. The main goal of this paper was the characterization of hypanthium and seed samples. While, intra-sample variability requires a different approach to data processing, and this was not the subject of our research.

Reviewer: 2
Comments to the Author(s) In this article, authors reported that multivariate analysis of the fingerprint region of Raman spectra of the fresh ripe rosehip pulp can tell if the roship is a cultivar or a wild type. However, the similar analysis on the seeds could not distinguish a cultiver from the wild type. How the sample was prepared lacks clarity. Choice of different lasers to record the spectra for seed and pulp is not also clear. Many conclusions have been drawn that are not substantiated by the results. Specific comments are given below:

Authors:
We appreciate your valuable insight into the manuscript.
1) The title says "characterization of storage reserves", however, no temporal data is given. It is not even clear from the methods, how long the rosehips were stored and in which condition before the spectra were measured. It is important as the samples were collected from different geographical locations.
Authors: Thank you for this observation. Accordingly, we clarified samples preparation procedure in M&M section.
2) Regarding the preparation of the seeds two statements were made: a) "for Raman spectroscopy analysis, seeds were separated from hypanthium for each sample, and grinded to fine powder in a mill." b) "Raman microspectroscopy of rose hip fruit was focusing on direct measurement of rosehips hypanthium parenchyma cells and its seeds. The seeds were longitudinally cut at room temperature…" Please clarify how the seeds were prepared for Raman measurement.
Authors: Samples preparation has been described in M&M, subsection 3.1.
3) Wouldn't it be better to record spectra from homogenized pulp?
Authors: Yes of course. We clarified it in M&M, subsection 3.1.
5) The authors claimed the method to be a "novel screening tool" developed by the authors, however, PCA coupled with Raman spectroscopy is already a widely used method.
Authors: This is a misunderstanding…We did not certainly develop the method. The application of the method for screening phytochemical composition of rosehip is a new approach. No literature data was found except the paper of Prof. de Oliveira (2008). 6) The authors conclude that the method could be used "...fast selection of rosehips...without fruit destruction." It would be relevant for sorting individual fruits even from the same harvest. However, the authors undertook no such study to show that the method can actually distinguish different quality fruits from the same harvest. Instead, they averaged over many samples from the same harvest.
7) The authors conclude that the method could "optimize the production of high-quality fruit." This study could only distinguish wild and cultivated varieties. From this study it cannot be concluded if the low and high quality fruit could be distinguished by this method. The seeds contained widely different levels of phytochemicals as evident from the Raman spectra of the seeds. However, PCA could not distinguish the seeds. It can be argued that the method shall fail to distinguish high and low quality fruits. 8) On "fruit development and maturation," as well, no study was undertaken. All the results are on mature fruits. Authors: Thank you for this valuable comment. According to comments 6, 7 and 8, we have re-written the Conclusion section. Some "claims" were more consideration of our research in the future.
Additional experiments maybe performed: 9) FT-IR is complementary to Raman spectroscopy. It would be interesting to see if PCA of FT-IR spectra can lead to the similar results.

Authors:
We absolutely agree with this observation. Unfortunately, in this situation and due to Covid-19, our Faculty is closed until 11 th of January and we are not able to perform additional analyses. But FT-IR will be considered in future research.
In addition, the FT-IR technique could be useful for seed material in a case on fatty acids rich Authors: The Raman shifts are already mentioned in the text 12) "The ancient Romans believed that...bite of rabid dogs." Should accompany a statement that no scientific association has been found.

Authors:
The sentence has been amended as suggested.
13) "...used in folk medicine in wound-healing and skin disease treatments [5]." Should accompany a statement if there is any scientific proof and cite the primary sources.
Authors: Correction has been made in the text and the relevant reference was cited.

Editor-in-Chief
Dear editor, I would to acknowledge you and the reviewers for dedicated work on our manuscript. Accordingly, we accepted suggestions given and replied to the comments.
Changes are highlighted in yellow in the manuscript file and Supplementary material.
In the following lines you may find detailed responses to the reviewers' comments.

Responses to Reviewers comments
Comments to the Author(s)

Reviewer: 1
Although the authors have addressed many of the issues raised during the initial review, there remain some concerns, particularly in relation to the PCA. Figure 4 indicates that, those samples which score positively according to PC1 (a) have strong carotenoid features (b), and that those that score negatively have a strong background tail between (in (b) this is "Variable 0-250"). There are no clearly identifiable chemical features which are associated with the negatively scoring samples in Fig 4(a). This suggests an issue of baselining the spectra.