Sensory Profile, Shelf Life, and Dynamics of Bioactive Compounds during Cold Storage of 17 Edible Flowers

Round 1

Reviewer 1 Report

This paper discussed the qualitative and quantitive qualities of 17 different edible flowers.  The authors utilized a highly trained team of taste and aroma specialists that used a scale-based rating system for each flower.   The authors also isolated polyphenols from each flower and conduct 3 different antioxidant assays.  They also conducted PCA tests and found that different aromas/tastes grouped differently.  They also noted that polyphenols did not correlate well with taste and aroma except for perhaps bitterness. 

Overall, the paper is well-written and is scientifically sound.  I have a few suggestions/questions for the authors to make it more suitable for publication. 

1. Can you please include common names for each of the flowers in this study?
2. Table 7 is long and is difficult to read.  Is there a way to take the most pertinent/interesting information from it and put it in a visual/graph and then include the rest as part of the supplementary information?
3. The discussion has several grammatical errors (ex. Line 514 Corresponded to/with, line 521 "elucidate").  Please got through the discussion and correct these errors. 
4. As someone with not a lot of experience with spider graphs, is there a "perfect" or "model" flower/graph that you can show for flower taste and aroma?   Is there a way to put these on a few graphs but with different colors (or only display those that have the most "interesting" or broad flavor profiles). 
5. Ethylene is the key to post-harvest flower freshness, viability, color, etc.  The authors do not reference this at all.  Is there any evidence that ethylene production can stimulate polyphenol biosynthesis or if any of these polyphenols play a role in senescence? Can ethylene affect taste or aroma? I think this warrants at least a few sentences in the discussion. 

Author Response

REVIEWER 1

This paper discussed the qualitative and quantitive qualities of 17 different edible flowers.  The authors utilized a highly trained team of taste and aroma specialists that used a scale-based rating system for each flower.   The authors also isolated polyphenols from each flower and conduct 3 different antioxidant assays.  They also conducted PCA tests and found that different aromas/tastes grouped differently.  They also noted that polyphenols did not correlate well with taste and aroma except for perhaps bitterness. 

Overall, the paper is well-written and is scientifically sound.  I have a few suggestions/questions for the authors to make it more suitable for publication. 

AUTHORS: thank you for the positive remarks.

1. Can you please include common names for each of the flowers in this study?

AUTHORS: English common names of the studied flowers have been included in Table 1, beside the Latin name.

1. Table 7 is long and is difficult to read.  Is there a way to take the most pertinent/interesting information from it and put it in a visual/graph and then include the rest as part of the supplementary information?

AUTHORS: thank you for the suggestion. Table 7 has been split in 3 different tables (Table 7, 8, and 9) to retain all the data and easier the reading.

1. The discussion has several grammatical errors (ex. Line 514 Corresponded to/with, line 521 "elucidate").  Please got through the discussion and correct these errors. 

AUTHORS: the discussion and all the text has been revised and English edited.

1. As someone with not a lot of experience with spider graphs, is there a "perfect" or "model" flower/graph that you can show for flower taste and aroma?   Is there a way to put these on a few graphs but with different colors (or only display those that have the most "interesting" or broad flavor profiles).

AUTHORS: thank you for the suggestion. Following also the recommendation of another reviewer we decided to show each score value for every sensory descriptor and flower in Table 3. Spider graphs, that are less clear than tabular data, have been moved to the supplementary material as Figures S2, S3, and S4.

1. Ethylene is the key to post-harvest flower freshness, viability, color, etc.  The authors do not reference this at all.  Is there any evidence that ethylene production can stimulate polyphenol biosynthesis or if any of these polyphenols play a role in senescence? Can ethylene affect taste or aroma? I think this warrants at least a few sentences in the discussion. 

AUTHORS: thank you for the suggestion. The issue of ethylene has been deepened throughout the discussion (lines 646-653, 745-751) particularly in relation to visual appearance and polyphenol production. Ethylene can also affect taste and aroma in sensitive species (https://0-doi-org.brum.beds.ac.uk/10.1016/S0925-5214(98)00091-X), however it is more likely to occur during storage. In our trial, the sensory evaluation was performed at harvest thus we can hypothesize that the influence of ethylene on taste and aroma descriptors did not occur yet. Also, the role of polyphenols during senesce has been described (lines 653-658, 740-745). 

Reviewer 2 Report

The authors have completed a very thorough and detailed study of the postharvest changes in edible flowers. I appreciated the depth to which they conducted their analysis; not content to do basic phytochemical analyses (FRAP, ABTS, Folin, etc.), they took it to the next level with the trained panel for organoleptic analyses, and the correlation work between sensory traits and flower, as well as working in some of the phytochemistry. I would have enjoyed seeing an untargeted profile of the flowers, as opposed to the limited panel of 14 phytochemical compounds. I think that might have yielded some more interesting results in that arena, as there are more compounds in the flowers than just the 14 listed. However, that is a moot point, and a potential direction for future research and projects. 

The writing was clear and concise, the figures and tables also were helpful and informative in explaining the results and findings of the study. I do not have any substantive edits or changes to suggest to the manuscript at this time; I think it is an interesting and enlightening study on the potential for edible flowers to be a part of the diet, and understanding their postharvest physiological, sensory, and chemical changes are key to this. 

Author Response

REVIEWER 2

Comments and Suggestions for Authors

The authors have completed a very thorough and detailed study of the postharvest changes in edible flowers. I appreciated the depth to which they conducted their analysis; not content to do basic phytochemical analyses (FRAP, ABTS, Folin, etc.), they took it to the next level with the trained panel for organoleptic analyses, and the correlation work between sensory traits and flower, as well as working in some of the phytochemistry. I would have enjoyed seeing an untargeted profile of the flowers, as opposed to the limited panel of 14 phytochemical compounds. I think that might have yielded some more interesting results in that arena, as there are more compounds in the flowers than just the 14 listed. However, that is a moot point, and a potential direction for future research and projects. 

The writing was clear and concise, the figures and tables also were helpful and informative in explaining the results and findings of the study. I do not have any substantive edits or changes to suggest to the manuscript at this time; I think it is an interesting and enlightening study on the potential for edible flowers to be a part of the diet, and understanding their postharvest physiological, sensory, and chemical changes are key to this. 

AUTHORS: we thank for the very positive remarks. Certainly, investigating and quantifying the diverse compounds in edible flowers, such as minerals, vitamins, and other polyphenols is the further step to undertake in order to fully understand their potential and their most suitable use in the diet.

Reviewer 3 Report

The manuscript entitled „Sensory Profile, Shelf Life, and Dynamics of Bioactive Compounds During Cold Storage of 17 Edible Flowers” describes an interesting study focused on the evaluation of the sensory quality and antioxidant capacity of fresh and stored edible flowers.

Although the study is interesting, within the scope of the journal and in the interest to the readers, the manuscript itself requires substantial improvements, as listed below:

In general, the text is often wordy. It would require English editing.

What is the significance of the bioactive compounds in the edible flowers if they are consumed in very small quantities, commonly used as a decoration, not as the main component? Then the amount of the phytochemicals delivered by the flowers is relatively marginal.

In the abstract, the first sentence should describe that the study is about edible flowers.

Line 21 – what do you mean by “postharvest performance”???

Latic names should be in Italic

Line 21-23 – The strong aroma and the interest in the consumption are not necessarily associated. Please rewrite it.

Line 31 – what about the other tests? If the antioxidant capacity of flowers was changing, it should affect all the assays.

Why didn’t the authors combine the profiling of bioactive compounds (ref 13) with the antioxidant capacity? It makes more sense than combining with the sensory quality. And the presentation of phytochemicals and their activity would result in a very nice and comprehensive article.

Based on what, these 17 flowers were selected? What happened to the other nine flowers from the previous paper with phytochemicals?

Were the flowers grown by the authors or purchased? All of them bloomed at the same time? Was it repeated in two following years for each flower?

Line 178: this heading has to be changed. It does not correspond to the meaning of the section.

Section 2.4. was the phytochemicals extracted once or repeated extraction was applied?

Section 2.5.2. I do not understand this procedure. What does each of the flask mean? How was it compared to the cyanidin-3-O-glucoside? How was the calibration curve prepared? What was the range of calibration curve?

Why is FRAP assay expressed in another unit? Why not in Trolox equivalents like the other tests? It is a commonly used procedure. And important if the authors wanted to compare the changes obtained using different assays.

In the statistical section, the PCA analysis is described. Where are the results?

Line 300: do you mean “intensities”?

Table 3 is not needed. It does not present anything. Better provide the table with the score values for each flower for each parameter. Moreover, the table is better than the spider graphs with this amount of flowers, especially having them of 3 pages is difficult to compare.

Line 322 – what does “well-defined profile” mean?

Was each of the flowers analysed independently by the panellists or in the groups or altogether the same day? Was comparing flowers the aim of study or just their characteristic?

Please provide the R² and Q² values of the PLS analysis.

Where is the description of Figure 6 in the manuscript?

The correlations presented in Table 5 are very weak, therefore the assumptions made in the text are incorrect. Please modify the text underlying that there are no real associations.

Table 7 – the statistic is missing for anthocyanins. If the authors consider these values as trace or below the LOQ, and that is why they didn’t calculate statistics, then please remove values and mark them as “<LOQ” or “trace”.

Line 510: This is a strange sentence. The floral and fruity aroma depends on the presence of terpenoids, esters, ketones etc., not on the lack of polyphenols.

Line 520 – if the R² value is low, then PLS should not be performed and presented.

Line approx. 539 – 551 – all these listing is not needed and does not bring any value to this study.

Line 582 – 584 – this is obvious. The flowers belonging to different generals or families will have a different profile of phytochemicals. It can be said without running any analyses.

The discussion is missing “discussion”. Comparing the results obtained to the literature is not a discussion.

The text in the discussion has to be edited. It is not clear now if the sentence is about the results obtained by the authors or comes from the references. And all is mixed together. It has to be ordered.

The discussion should contain the explanations: why these changes are obtained? Why was the content of phenolics changing during storage? What is the mechanism?

Author Response

REVIEWER 3

The manuscript entitled „Sensory Profile, Shelf Life, and Dynamics of Bioactive Compounds During Cold Storage of 17 Edible Flowers” describes an interesting study focused on the evaluation of the sensory quality and antioxidant capacity of fresh and stored edible flowers.

Although the study is interesting, within the scope of the journal and in the interest to the readers, the manuscript itself requires substantial improvements, as listed below:

In general, the text is often wordy. It would require English editing.

AUTHORS: English has been edited through the entire manuscript, and particularly, the Discussion has been reordered.

What is the significance of the bioactive compounds in the edible flowers if they are consumed in very small quantities, commonly used as a decoration, not as the main component? Then the amount of the phytochemicals delivered by the flowers is relatively marginal.

AUTHORS: the increasing research on this issue and an emergent innovative culinary approach are bringing edible flowers to be an ingredient of the recipes and not only a simple decoration. Despite available and eaten in lower quantities than fruits and vegetables, through these kinds of research they can be characterised not only for their aesthetic value, but also for their properties and thus consciously used to add benefits to the diet. A sentence on this issue has been added in the Conclusions, lines 789-793. 

In the abstract, the first sentence should describe that the study is about edible flowers.

AUTHORS: the first sentence of the abstract has been modified accordingly. “In this study, 17 edible flowers (Allium ursinum L.,……) were investigated to assess their sensory profile at harvest and their shelf life and bioactive com-pounds dynamics during cold storage.”

Line 21 – what do you mean by “postharvest performance”???

AUTHORS: the “postharvest performance” was referred to the shelf life and the trend of bioactive compounds and antioxidant activity during postharvest. The sentence has been modified to be clearer: “Generally, species had a wide range of peculiar sensory and phytochemical characteristics at harvest, as well as shelf life and bioactive compounds dynamics during postharvest”

Latic names should be in Italic

AUTHORS: thank you for the remark. The submitted .docx version had Latin names in italics. We will verify that the system maintains the italics during the submission of the revised version.

Line 21-23 – The strong aroma and the interest in the consumption are not necessarily associated. Please rewrite it.

AUTHORS: the sentence has been modified accordingly. “A strong aroma was indicated for A. ursinum, D. carthusianorum, L. angustifolia, and L. vulgare, while B. officinalis and C. officinalis had very low values for all aroma and taste descriptors, resulting in poor sensory profiles”.

Line 31 – what about the other tests? If the antioxidant capacity of flowers was changing, it should affect all the assays.

AUTHORS: the information on the other antioxidant activity tests (DPPH and FRAP) has been added to the sentence. “During cold storage, the content of bioactive compounds differed, as total phenolics followed a different trend according to the species and anthocyanins remained almost unaltered for 14 days. Considering antioxidant activity, ABTS values were the least variable, varying in only 4 species (A. ursinum, D. carthusianorum, L. angustifolia, and P. officinalis), while both DPPH and FRAP values varied in 8 species”.

Why didn’t the authors combine the profiling of bioactive compounds (ref 13) with the antioxidant capacity? It makes more sense than combining with the sensory quality. And the presentation of phytochemicals and their activity would result in a very nice and comprehensive article.

AUTHORS: profiling the amount of each bioactive compounds in these flowers was not the purpose of this study, as they were investigated in a previous in trial (ref 13). There, the compounds were already linked to the antioxidant activity. To provide new information, we investigated the relation with the sensory properties, which is a challenging issue in the sensory science.    

Based on what, these 17 flowers were selected? What happened to the other nine flowers from the previous paper with phytochemicals?

AUTHORS: for this study we selected a group of species that was representative of the variability of colours, aromas and fragrances of flowers, with widely recognized food or medicinal use (thus excluding Dianthus pavonius, Erythonium dens-canis, Geranium sylvaticum), and excluding also those particularly niche (e.g. Mentha aquatica, Trifolium alpinum) or those already extensively studied (e.g. Bellis perennis, Primula vulgaris, Tagetes patula, Viola odorata).

Were the flowers grown by the authors or purchased? All of them bloomed at the same time? Was it repeated in two following years for each flower?

AUTHORS: for the species already available on the market (e.g. B. officinalis, C. officinalis, L. angustifolia, and T. majus), the flowers were collected in the nursery, while for the others it was necessary to collect them from wild plants. All the flowers were collected during their full bloom (March through September according to the species) and activities were performed once for each species. All the details are present in the ref 13, thus in the section 2.1 we added more information on the plant material and sampling referring to ref 13.

Line 178: this heading has to be changed. It does not correspond to the meaning of the section.

AUTHORS: the heading has been changed in “Shelf life”.

Section 2.4. was the phytochemicals extracted once or repeated extraction was applied?

AUTHORS: the phytochemicals were extracted once, and this is now specified at line 210.

Section 2.5.2. I do not understand this procedure. What does each of the flask mean? How was it compared to the cyanidin-3-O-glucoside? How was the calibration curve prepared? What was the range of calibration curve?

AUTHORS: the procedure for the evaluation of total anthocyanin content has been better describe at lines 226-249 as follows: “The total anthocyanins were estimated by pH differential method using two buffer systems: hydrochloric acid/potassium chloride buffer at pH 1.0 (25 mM) and sodium acetate buffer pH 4.5 (0.4 M), as described in the literature [45,79,80]. This method is based on the structural transformation of anthocyanins due to a change in pH (colored at pH 1.0 and colorless at pH 4.5). Briefly, 0.2 mL of each extract was diluted in a 5-mL volumetric flask with the corresponding buffers and the solution was read, after 15 min, against Milli-Q water as a blank at 510 and 700 nm. by means of a spectrophotometer (Cary 60 UV-Vis, Agilent Technologies, Santa Clara, CA, USA). Absorbance (A) was calculated as follows: A = (A510nm−A700nm)pH1.0 − (A510nm−A700nm)pH4.5. Then, the total anthocyanins (TA) of each extract were calculated by the following equation: TA=[A×MW×DF×1000]×1/ε×1,where A is the absorbance; MW is the molecular weight of cyanidin-3-O-glucoside (449.2 D); DF is the dilution factor (25); ε is the molar extinction coefficient of cyanidin-3-glucoside (26.900) and results were expressed on a fresh weight basis in milligrams of cyanidin-3-O-glucoside per 100 grams (mg C3G/100 g).The evaluation of total anthocyanins was performed in triplicate on extracts of days 0, 3, 7, 10, and 14.”

Why is FRAP assay expressed in another unit? Why not in Trolox equivalents like the other tests? It is a commonly used procedure. And important if the authors wanted to compare the changes obtained using different assays.

AUTHORS: the unit depends on which standard was used for the calibration curve. We have used FeSO4 · 7H2O as a consolidated method and therefore we evaluate the mmol of Fe2+ that are obtained. There are several papers in literature confirming that the FRAP assay measuring the ferric-to-ferrous iron reduction in the presence of antioxidants is very effective and convenient in terms of its operation (Cao & Prior, 1998; Caser et al; 2019; Demasi et al; 2021). Besides, our aim was not to compare different methods but to provide a more comprehensive view of antioxidant activity.

In the statistical section, the PCA analysis is described. Where are the results?

AUTHORS: the PCA results are described in lines 382-394, and shown as Figure 2.

Line 300: do you mean “intensities”?

AUTHORS: yes, the meaning is intensities. The word “scores” has been changed to “intensities”.

Table 3 is not needed. It does not present anything. Better provide the table with the score values for each flower for each parameter. Moreover, the table is better than the spider graphs with this amount of flowers, especially having them of 3 pages is difficult to compare.

AUTHORS: thank you for the suggestion. In Table 3 the score value for each sensory descriptor and each flower are now present. Spider graphs have been moved to the supplementary material as Figures S2, S3, and S4.

Line 322 – what does “well-defined profile” mean?

AUTHORS: the language of the entire manuscript has been revised, avoiding wordy sentences and maintaining simple and direct information.

Was each of the flowers analysed independently by the panellists or in the groups or altogether the same day? Was comparing flowers the aim of study or just their characteristic?

AUTHORS: According to the blooming season, there could be one or more species in a day, which were analysed independently by each panellist. The aim was to provide the sensory profiles of poorly studied species by trained panellists and lay the groundwork for future studies on food applications.

Please provide the R² and Q² values of the PLS analysis. Where is the description of Figure 6 in the manuscript?

AUTHORS: description of former Figure 6 and R² values were listed in lines 404-410, to which also the Q² values have been added. However, as suggested below, PLS analyses has been deleted and is now only mentioned in the text.

The correlations presented in Table 5 are very weak, therefore the assumptions made in the text are incorrect. Please modify the text underlying that there are no real associations.

AUTHORS: despite weak, some correlations are significant. Description of Table 5 has been modified accordingly (lines 422-425).

Table 7 – the statistic is missing for anthocyanins. If the authors consider these values as trace or below the LOQ, and that is why they didn’t calculate statistics, then please remove values and mark them as “<LOQ” or “trace”.

AUTHORS: the statistics for anthocyanins are present, however, there were no significant differences during time in almost all the species, except for Taraxacum officinale and Tropaeolum majus. This is why the letters of post-hoc test are present only besides these two species. 

Line 510: This is a strange sentence. The floral and fruity aroma depends on the presence of terpenoids, esters, ketones etc., not on the lack of polyphenols.

AUTHORS: this was an observation deriving from ref 29. The sentence has been modified to better address the issue. “The fruity and floral aromas are, for example, due to the presence of volatile compounds such as ethyl octanoate, 1-hexanol, ρ-cymene, or β-myrecene [29]”.

Line 520 – if the R² value is low, then PLS should not be performed and presented.

AUTHORS: nonetheless, low values of Q2 and R2 are results indicating that the phytochemicals we found cannot currently being related to sensory traits, worthing to be mentioned for future investigation.

Line approx. 539 – 551 – all these listing is not needed and does not bring any value to this study.

AUTHORS: thank you for the remark. These lines have been deleted from the text.

Line 582 – 584 – this is obvious. The flowers belonging to different generals or families will have a different profile of phytochemicals. It can be said without running any analyses.

AUTHORS: the sentence has been deleted from the text.

The discussion is missing “discussion”. Comparing the results obtained to the literature is not a discussion. The text in the discussion has to be edited. It is not clear now if the sentence is about the results obtained by the authors or comes from the references. And all is mixed together. It has to be ordered. The discussion should contain the explanations: why these changes are obtained? Why was the content of phenolics changing during storage? What is the mechanism?

AUTHORS: thank you for the remark. Polyphenols include several hundreds of compounds, still poorly characterized for all plant species, thus determine the key variables that are responsible for their variability is extremely difficult (https://0-doi-org.brum.beds.ac.uk/10.1093/ajcn/79.5.727). We added throughout the Discussion some possible explanations of the differences and variation observed at harvest and during storage (Lines 646-658, 701-706, 740-751), and the Discussion has been fully revised.

Round 2

Reviewer 3 Report

All the suggestions have been sufficiently addressed.

Author Response

We thank you for the constructive comments received which allowed us to improve the manuscript.