Anthocyanins in the Berries of 123 Interspecific Black Currant Crosses: A Multi-Analytical Approach

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents a valuable and well-executed study, successfully establishing and validating a rapid, high-resolution RP-HPLC-DAD method for the comprehensive characterization of the anthocyanin profile across a large panel of 123 black currant genotypes. However, the manuscript requires attention to several points to enhance its clarity, rigor, and conciseness.

1. The Introduction section is too long and requires streamlining. I recommend condensing the general background material on health benefits and market size of black currant. Detailed production statistics are less critical for establishing the research gap and should be significantly shortened or removed, and the discussion on interspecific hybridization should also be condensed.

2. The use of 0.36 N HCl in the mobile phase is a major concern. This strong acid at a relatively high concentration is uncommon and may cause corrosion damage to standard stainless steel HPLC components (tubing, injector, frits). The authors need to justify why this acid was chosen over more common and less corrosive alternatives like low-concentration trifluoroacetic acid (TFA) or formic acid.

3. In Section 3.3.2, lines 401 & 402, the units for the reported total anthocyanin content values should be indicated.

4. In Section 3.4.1, line 457, the statement that concentration variation ranges from "two to three orders of magnitude" (100 to 1,000 fold) is inconsistent with the detailed fold-differences reported later in the paragraph. Based on these specific results, the statement should be corrected to "one to two orders of magnitude."

5. The conclusion is too long. It contains excessive detail, repeating specific results or discussion. Please significantly reduce the length to a brief summary of the main achievements and the novel utility of the developed method.

Author Response

We sincerely thank you for all the comments, remarks, and suggestions that have contributed to enhancing the manuscript and its scientific quality. The graphical abstract, manuscript, and supplementary materials have been improved accordingly. Provided changes are marked in red font.

Reviewer 1

Comments and Suggestions for Authors

Comment 1: This manuscript presents a valuable and well-executed study, successfully establishing and validating a rapid, high-resolution RP-HPLC-DAD method for the comprehensive characterization of the anthocyanin profile across a large panel of 123 black currant genotypes. However, the manuscript requires attention to several points to enhance its clarity, rigor, and conciseness.

Response 1: Thank you for the positive overview. The manuscript was improved according to the recommendations.

Comment 2: The Introduction section is too long and requires streamlining. I recommend condensing the general background material on health benefits and market size of black currant. Detailed production statistics are less critical for establishing the research gap and should be significantly shortened or removed, and the discussion on interspecific hybridization should also be condensed.

Response 2: Thank you for the comment. The Introduction section was shortened according to the recommendations. However, shortening discussion on interspecific hybridization was difficult, because it is one paragraph. We tried.

Comment 3: The use of 0.36 N HCl in the mobile phase is a major concern. This strong acid at a relatively high concentration is uncommon and may cause corrosion damage to standard stainless steel HPLC components (tubing, injector, frits). The authors need to justify why this acid was chosen over more common and less corrosive alternatives like low-concentration trifluoroacetic acid (TFA) or formic acid.

Response 3: Thank you for the comment. As a mobile phase, it was used “4% formic acid in water (A)”, not “0.36 N HCl in the mobile phase”. The 0.36 N HCl in 50% ethanol (v/v) was used for the extraction of anthocyanins. It is one of the most popular acids used for extracting anthocyanins from black currants. We added an additional paragraph about the impact of the type of acid used and the temperature of extraction on anthocyanin extractability. During the 2-year study, over 4000 injections of extracts containing 0.36 N HCl in 50% ethanol (v/v), we did not observe any symptoms of HPLC parts damage.

Comment 4: In Section 3.3.2, lines 401 & 402, the units for the reported total anthocyanin content values should be indicated.

Response 4: Thank you for noticing. Missing units were added.

Comment 5: In Section 3.4.1, line 457, the statement that concentration variation ranges from "two to three orders of magnitude" (100 to 1,000 fold) is inconsistent with the detailed fold-differences reported later in the paragraph. Based on these specific results, the statement should be corrected to "one to two orders of magnitude."

Response 5: Thank you for noticing. The correction has been done.

Comment 6: The conclusion is too long. It contains excessive detail, repeating specific results or discussion. Please significantly reduce the length to a brief summary of the main achievements and the novel utility of the developed method.

Response 6: Thank you for the comment. The Conclusion section was shortened according to the recommendations.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents a comprehensive, well-structured study combining HPLC-DAD method development, extraction optimization, and large-scale screening of 123 interspecific black currant genotypes. The authors propose a rapid, eco-friendly, and analytically robust protocol for anthocyanin profiling. The dataset is extensive, the experiments are logically designed, and the results are presented with adequate statistical analysis.

Some clarifications are needed:

1. Ultrasonication parameters (60°C, 15 min) raise concerns regarding anthocyanin thermal degradation. The authors should cite relevant stability data or provide a justification for why degradation is unlikely under these conditions.
2. Regarding the statement that “smaller berries mostly contain higher anthocyanin concentrations,” the explanation based on surface-area-to-volume ratio is plausible but should reference studies in similar fruit systems. The authors should also emphasize that berry size is not an independent variable, as genetic background strongly covaries with berry morphology.
3. Table 1 should be extended to the paper margins so that the content fits better. Figure 12 is currently misplaced, and part of it is cut off. It should be extended to the margins because it contains a lot of information in small text.

Typographical errors that should be corrected:

• Line 13: “…for analysis anthocyanin’s in black currants.” → Change anthocyanin’s to anthocyanins.
• The manuscript uses units inconsistently: mg 100 g⁻¹ FW, mg, and mg 100 g^-1 fw.
• Line 600: “mg 100 g^-1 fw,” lowercase fw.
• Lines 83–84: “mg 100 g^-1 FW,” uppercase FW.
• Lines 343–350: The referencing style should be improved for clarity and consistency. Currently, the text reads: “For example, delphinidin derivatives were reported by [35]; peonidin 3-O-rutinoside (peo 3-O-rut) and malvidin 3-O-rutinoside by [36]; …”

This structure is difficult to follow, and relying solely on reference numbers makes the sentence ambiguous. I recommend rewriting it as “For example, delphinidin derivatives were reported by Obón et al. [35]; peonidin 3-O-rutinoside (peo 3-O-rut) and malvidin 3-O-rutinoside by Frøytlog et al. [36]; …” The authors can choose another style of commenting. This improves readability and aligns with standard scientific writing style.

Overall, the manuscript is of high scientific quality and can be accepted after the authors address the comments above. I recommend minor revision.

Author Response

We sincerely thank you for all the comments, remarks, and suggestions that have contributed to enhancing the manuscript and its scientific quality. The graphical abstract, manuscript, and supplementary materials have been improved accordingly. Provided changes are marked in red font.

Reviewer 2

Comments and Suggestions for Authors

Comment 1: The manuscript presents a comprehensive, well-structured study combining HPLC-DAD method development, extraction optimization, and large-scale screening of 123 interspecific black currant genotypes. The authors propose a rapid, eco-friendly, and analytically robust protocol for anthocyanin profiling. The dataset is extensive, the experiments are logically designed, and the results are presented with adequate statistical analysis.

Response 1: Thank you for the positive overview.

Comment 2: Some clarifications are needed: Ultrasonication parameters (60°C, 15 min) raise concerns regarding anthocyanin thermal degradation. The authors should cite relevant stability data or provide a justification for why degradation is unlikely under these conditions. Response 2: Thank you for the comment. We added an additional paragraph about the impact of the type of acid used and the temperature of extraction on anthocyanin extractability.

 Comment 3: Regarding the statement that “smaller berries mostly contain higher anthocyanin concentrations,” the explanation based on surface-area-to-volume ratio is plausible but should reference studies in similar fruit systems. The authors should also emphasize that berry size is not an independent variable, as genetic background strongly covaries with berry morphology.

Response 3: Thank you for the comment. An additional explanation was added to emphasize that berry size is not an independent variable, as genetic background strongly covaries with berry morphology. “However, it should be emphasized that berry size is not an independent variable, as genetic background strongly covaries with berry morphology.”

Comment 4: Table 1 should be extended to the paper margins so that the content fits better. Figure 12 is currently misplaced, and part of it is cut off. It should be extended to the margins because it contains a lot of information in small text.

Response 4: Thank you for noticing. The correction has been done.

 Comment 5: Typographical errors that should be corrected:

• Line 13: “…for analysis anthocyanin’s in black currants.” → Change anthocyanin’s to anthocyanins.
• The manuscript uses units inconsistently: mg 100 g⁻¹ FW, mg, and mg 100 g^-1 fw.
• Line 600: “mg 100 g^-1 fw,” lowercase fw.
• Lines 83–84: “mg 100 g^-1 FW,” uppercase FW.
• Lines 343–350: The referencing style should be improved for clarity and consistency. Currently, the text reads: “For example, delphinidin derivatives were reported by [35]; peonidin 3-O-rutinoside (peo 3-O-rut) and malvidin 3-O-rutinoside by [36]; …”

This structure is difficult to follow, and relying solely on reference numbers makes the sentence ambiguous. I recommend rewriting it as “For example, delphinidin derivatives were reported by Obón et al. [35]; peonidin 3-O-rutinoside (peo 3-O-rut) and malvidin 3-O-rutinoside by Frøytlog et al. [36]; …” The authors can choose another style of commenting. This improves readability and aligns with standard scientific writing style.

Response 5: Thank you for noticing. All highlighted issues were corrected according to the recommendations.

 Comment 6: Overall, the manuscript is of high scientific quality and can be accepted after the authors address the comments above. I recommend minor revision.

Response 6: Thank you for the positive overview. The manuscript was improved according to the recommendations.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed my comments.