Genetic Differentiation in Anthocyanin Content among Berry Fruits

Round 1

Reviewer 1 Report

The manuscript titled ‘Genetic differentiation in anthocyanin content among berry fruits’ by Hallmann and coworkers have shown that some of the selected species and cultivars of berry fruits differ significantly in their anthocyanin contents. Among all species tested, blackberry and blackcurrant were characterized significantly by the highest content of sum of anthocyanins, whereas the redcurrant fruits had lowest content.

This manuscript will definitely convince the readers the importance of this research and would definitely help in selection and validation of most productive species.

This manuscript should be accepted with a minor revision. Authors should add figure of diphenylpropane skeleton (C6C3C6).

Author Response

Reviewer no 1.

Thank you very much for the review and for your suggestions and opinions about my manuscript in the “Current Issues in Molecular Biology” journal. All pointed comments were put into the text. All comments reply are presented below.

Comment 1: “INTRODUCTION: Authors should add figure of diphenylpropane skeleton (C6C3C6).”

Authors’ response: According to Reviewer request, appropriate Figure of diphenylpropane skeleton (C6C3C6) was add to manuscript text.

Reviewer 2 Report

The manuscript entitled “Genetic differentiation in anthocyanin content among berry fruits”, authored by Alicja Ponder, Ewelina Hallmann, Martyna Kwolek, Dominika Średnicka-Tober, and Renata Kazimierczak, deals with the identification and quantification of anthocyanins in extracts from berry fruits widely consumed in Europe. The manuscript does not contain a large amount of experimental data, however the authors have been able to comment and compare them to data previously reported in the literature. All this is very appreciable. I have some small doubts about the manuscript:

KEYWORDS: keywords should be words not contained in the title of the manuscript, and limitedly present in the abstract. I strongly suggest authors to introduce more keywords (maximum 10) and replace those that are repeated in the title. The usefulness of keywords is to make the article both more and more easily searchable visible after its publication through commonly used search engines.

REFERENCES: The bibliographic references used by the authors are appropriate, but unfortunately dated. In particular, the authors reported 53 bibliographic notes, and only 7 in a range between 2017 and 2021. Recent published manuscripts, whether review (10.3390/agriculture11030212) or original articles (doi.org/10.3390/plants10030472; 10.3389/fchem.2018.00052), treated the topic of anthocyanins, investigating the biosynthetic pathways, both from a chemical and molecular point of view, their distribution and their role in plants and human nutrition. Authors should mention these recent works in their manuscript and compare their data to that reported in these recent manuscript.

INTRODUCTION:

• Line 45 – 54 need an appropriate reference related to ecological role of anthocyanin.
• The authors correctly report the different functions of anthocyanins, also from a nutraceutical point of view. However, apart from the aforementioned actions, anthocyanins have an important function for the quality control of fruits and foods that contain claim to contain a specific anthocyanin-enriched fruits. In fact, as the authors have appreciate from their experiments, a characteristic anthocyanin pattern is specific for each fruit. Based on this "fingerprinting" it is possible to recognize the adulteration and/or sophistication of particular products that claim to contain a certain fruit (oi.org/10.3390/nu12040992; doi.org/10.1007/s11694-020-00443-y; doi.org/10.1016/j.chroma.2018.01.032). Authors should briefly treat also this aspect.

RESULTS AND DISCUSSION:

• The wording "N/A" reported in table 1 is conceptually wrong. In order to state this, the authors would have to calculate LOD and LOQ values. Furthermore, they should replace N.D. with <LOD. Please, see my comment related to Materials and Methods for other information.
• Due to the few experimental data present in the article, I might suggest to the authors to include an additional statistical analysis. In particular, they could calculate the total content of anthocyanins for each fruit (summing the punctual content of each anthocyanin) and perform a cluster analysis (or simply a histogram by ordering the bars in descending order for their own content). Then the authors can compare this result with those obtained from meta-analysis in which the anthocyanin content it has been analysed in several plant sources (10.3390/agriculture11030212).

MATERIALS AND METHODS:

• The section related to HPLC analysis is very well described. However, this subsection lack in the evaluation of some important parameters, essential for this typology of studies. In particular, the authors should provide information regarding LOD (limit of detection) and LOQ (limit of quantification) values. These are important parameters for the validation of any LC methodology, especially if the authors claim that a specific molecule is not present. Authors can calculate LOD and LOQ using the standard deviation of the response (Sy) and the slope (m) of the calibration curves [LOD = 3.3(Sy/m); LOQ = 10(Sy/m)]. Author can easily calculate m and Sy using the MS Excel function “=SLOPE” and “=STEYX”, targeting as y-values the response (area peak) and as x-values the concentrations.

Author Response

Reviewer no. 2

Thank you very much for the review and for your suggestions and opinions about my manuscript in the “Current Issues in Molecular Biology” journal. All pointed comments were put into the text. All comments reply are presented below.

Comment 1: “INTRODUCTION: Line 45 – 54 need an appropriate reference related to ecological role of anthocyanin.”

Authors’ response: Thank you very much for the positive evaluation of our paper and valuable advice. All tips have been implemented. They definitely improved this study and made it more valuable. In fact, the proposed an appropriate reference related to ecological role of anthocyanin is definitely useful in our study. Of course, additional current reference from 2021 related to ecological role of anthocyanin has been included in the Introduction and they definitely broaden this description and thus the study has become more valuable.

Mannino, G., Gentile, C., Ertani, A., Serio, G., & Bertea, C. M. Anthocyanins: Biosynthesis, Distribution, Ecological Role, and Use of Biostimulants to Increase Their Content in Plant Foods—A Review. Agriculture, 2021, 11, 212. Doi: 10.3390/agriculture11030212

Comment 2: “INTRODUCTION: The authors correctly report the different functions of anthocyanins, also from a nutraceutical point of view. However, apart from the aforementioned actions, anthocyanins have an important function for the quality control of fruits and foods that contain claim to contain a specific anthocyanin-enriched fruits. In fact, as the authors have appreciate from their experiments, a characteristic anthocyanin pattern is specific for each fruit. Based on this "fingerprinting" it is possible to recognize the adulteration and/or sophistication of particular products that claim to contain a certain fruit (oi.org/10.3390/nu12040992; doi.org/10.1007/s11694-020-00443-y; doi.org/10.1016/j.chroma.2018.01.032). Authors should briefly treat also this aspect. “

Authors’ response: As suggested by the reviewer, in the Introduction we briefly discuss the aspect related to "fingerprints", which makes it possible to recognize adulteration and / or sophistication of particular products that claim to contain a certain fruit.

Mannino, G., Di Stefano, V., Lauria, A., Pitonzo, R., & Gentile, C. Vaccinium macrocarpon (Cranberry)-based dietary supplements: Variation in mass uniformity, proanthocyanidin dosage and anthocyanin profile demonstrates quality control standard needed. Nutrients, 2020, 12(4), 992. doi.org/10.3390/nu12040992

Krishnan, V., Rani, R., Pushkar, S., Lal, S. K., Srivastava, S., Kumari, S., ... & Sachdev, A. Anthocyanin fingerprinting and dynamics in differentially pigmented exotic soybean genotypes using modified HPLC–DAD method. Journal of Food Measurement and Characterization, 2020, 1-10. doi.org/10.1007/s11694-020-00443-y

Krüger S., Morlock G.E. Fingerprinting and characterization of anthocyanins in 94 colored wheat varieties and blue aleurone and purple pericarp wheat crosses, J Chromatogr A, 2018, 1538, 75-85. doi.org/10.1016/j.chroma.2018.01.032

Comment 3: “RESULTS AND DISCUSSION: The wording "N/A" reported in table 1 is conceptually wrong. In order to state this, the authors would have to calculate LOD and LOQ values. Furthermore, they should replace N.D. with <LOD. Please, see my comment related to Materials and Methods for other information.”

Authors’ response: Following the Reviewer’s suggestion, the wording N/A has now been replaced by <LOD in the tables 1 and 2.

Comment 4: “REFERENCES: The bibliographic references used by the authors are appropriate, but unfortunately dated. In particular, the authors reported 53 bibliographic notes, and only 7 in a range between 2017 and 2021. Recent published manuscripts, whether review (10.3390/agriculture11030212) or original articles (doi.org/10.3390/plants10030472; 10.3389/fchem.2018.00052), treated the topic of anthocyanins, investigating the biosynthetic pathways, both from a chemical and molecular point of view, their distribution and their role in plants and human nutrition. Authors should mention these recent works in their manuscript and compare their data to that reported in these recent manuscript.”

Authors’ response: Of course, some additional current articles from last 10 years and additional information have been included in the Introduction and they definitely broaden this description and thus the study has become more valuable.

Mannino, G., Gentile, C., Ertani, A., Serio, G., & Bertea, C. M. Anthocyanins: Biosynthesis, Distribution, Ecological Role, and Use of Biostimulants to Increase Their Content in Plant Foods—A Review. Agriculture, 2021, 11, 212. Doi: 10.3390/agriculture11030212

Bæksted Holme, I., Dionisio, G., & Brinch-Pedersen, H. A roadmap to modulated anthocyanin compositions in carrots. Plants, 2021, 10, 472. doi.org/10.3390/plants10030472

Liu Y, Tikunov Y, Schouten RE, Marcelis LFM, Visser RGF, Bovy A. Anthocyanin Biosynthesis and Degradation Mechanisms in Solanaceous Vegetables: A Review. Front Chem. 2018, 9, 6:52. doi: 10.3389/fchem.2018.00052.

Comment 5: “MATERIAL AND METHODS: Due to the few experimental data present in the article, I might suggest to the authors to include an additional statistical analysis. In particular, they could calculate the total content of anthocyanins for each fruit (summing the punctual content of each anthocyanin) and perform a cluster analysis (or simply a histogram by ordering the bars in descending order for their own content). Then the authors can compare this result with those obtained from meta-analysis in which the anthocyanin content it has been analysed in several plant sources (10.3390/agriculture11030212).”

Authors’ response: As suggested by the reviewer, we included an additional statistical analysis and performed a cluster analysis (Figure 2.).

Comment 5: “MATERIAL AND METHODS: The section related to HPLC analysis is very well described. However, this subsection lack in the evaluation of some important parameters, essential for this typology of studies. In particular, the authors should provide information regarding LOD (limit of detection) and LOQ (limit of quantification) values. These are important parameters for the validation of any LC methodology, especially if the authors claim that a specific molecule is not present. Authors can calculate LOD and LOQ using the standard deviation of the response (Sy) and the slope (m) of the calibration curves [LOD = 3.3(Sy/m); LOQ = 10(Sy/m)]. Author can easily calculate m and Sy using the MS Excel function “=SLOPE” and “=STEYX”, targeting as y-values the response (area peak) and as x-values the concentrations.”

Authors’ response: According to Reviewer remark, all LOD and LOQ values were properly calculated and paste in table in Material and methods section

Reviewer 3 Report

I carefully reviewed the manuscript (cimb-1183720) dealing with genetic differentiation in anthocyanin content among berry fruits. The study only highlighted the anthocyanin content of berries, which is the limitation of the study and no purification technique was used.

What is the novelty of study other than anthocyanins? There are many studies exclusively focused on anthocyanins of berries.

Abstract

1. Objective of the study should be highlighted before the methodology part of the abstract.
2. Among all species tested, blackberry and blackcurrant were characterized significantly by the highest content of sum of anthocyanins. The lowest content was found in redcurrant fruits…. How much? Value should be mentioned

Introduction

L95-97: cultivar and species names should be added for clear understanding.

Why authors are selected only those cultivars?

There are many studies available in the literature dealing with anthocyanins of berries. Are these undervalued or underutilized berries in Poland?Selection basis should be highlighted

Materials and methods

L332: obtained from where? Supplied company name?

L341: how authors confirmed the species of the different cultivars? Any authentication was performed? Since this study is closely related to species and cultivars, I suggest to mention their authenticity by the curator or botanist.

L357: Anthocyanin separation and identification: Are authors performed purification?

Figure 2 is in material and methods. It should be in results  

Why authors selected uneven cultivar sample size for each species? On what basis?

Results and discussion

Figure 1 (top figure) is repeated in Figure 1 (below figure). Generally, biplot is the combination of PC scores of samples (dots…..) and loadings of variables (vectors →). I suggest to represent with biplot.

PCA discussion should be improved with correlation

Statistical analysis and comparison (column or row wise) should be mentioned in Table footnotes.

Author Response

Reviewer no. 3

Thank you very much for the review and for your suggestions and opinions about my manuscript in the “Current Issues in Molecular Biology” journal. All pointed comments were put into the text. All comments reply are presented below.

Comment 1: “INTRODUCTION: L95-97: cultivar and species names should be added for clear understanding.

Authors’ response: Thank you very much for the positive evaluation of our paper and valuable advice. All tips have been implemented. They definitely improved this study and made it more valuable. In the introduction, the names of cultivars and species were added.

Comment 2: “RESULTS AND DISCUSSION: Figure 1 (top figure) is repeated in Figure 1 (below figure). Generally, biplot is the combination of PC scores of samples (dots…..) and loadings of variables (vectors →). I suggest to represent with biplot.”

Authors’ response: We have now left as Figure 1 biplot as suggest reviewer 3.

Comment 3: “RESULTS AND DISCUSSION: PCA discussion should be improved with correlation.”

Authors’ response: PCA discussion has now been added in the manuscript text and relevant bibliography has been cited.

Comment 4: “RESULTS AND DISCUSSION: Statistical analysis and comparison (column or row wise) should be mentioned in Table footnotes.”

Authors’ response: This has now been added and explained in Table 1 and 2 footnotes.

Comment 5: “MATERIAL AND METHODS: L332: obtained from where? Supplied company name?”

Authors’ response: Missing chemical company and deliver name was add to manuscript text.

Comment 6: “MATERIAL AND METHODS: how authors confirmed the species of the different cultivars? Any authentication was performed? Since this study is closely related to species and cultivars, I suggest to mention their authenticity by the curator or botanist. “

Authors’ response: Authors want to explain about plants cultivars confirmation. Experimental farms are belong to organic farming system. According to law, the owners have qualified plant material as well certificate validated by plants material producers in their farm. Appropriate information about the plant material, qualification system has been add to the sub-section "Plant material origin".

Comment 7: “MATERIAL AND METHODS: L357: Anthocyanin separation and identification: Are authors performed purification?”

Authors’ response: The quantification and qualification of anthocyanins in presented experiment was according to pure standards obtained from chemical company. All identified and quantified compounds were in glycoside form as used standards. All used anthocyanins standards are present in sub-section "Chemicals and reagents"

Comment 8: “MATERIAL AND METHODS: Figure 2 is in material and methods. It should be in results”  

Authors’ response: According to Reviewer suggestion, Figure 2 with new number was move to Results section.

Comment 9: “MATERIAL AND METHODS: Why authors selected uneven cultivar sample size for each species? On what basis?”

Authors’ response: As Authors explain in response for comment no. 2 experimental material was produce in organic farms. It was impossible to find all cultivars in the same number for all species. Authors are grateful to Reviewer for pointing a new direction of future experiment. Appropriate cooperation have been made not with producing farms , but scientific institute of Horticulture, who have experimental orchards with qualified cultivars of berry fruits.  On the other hand used for presented experiment berry fruit cultivars are one of the most common in Poland. So the obtained results will be useful for fruits producers and consumers as well.

Round 2

Reviewer 3 Report

Authors are carefully answered and included all suggestions in revised manuscript. Now the quality of manuscript is improved. I recommend acceptance in the present form. However, in the future, authors should consider sample purification studies in order to obtain purified anthocyanins.