Review Reports

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

This manuscript presents the first comprehensive characterization of the traditional Portuguese apple cultivar "Reineta de Fontanelas," integrating biometric, physicochemical, nutritional, bioactive, and sensory analyses across six local producers. The research topic is highly relevant to agrobiodiversity conservation and valorization of traditional crops, addressing a critical gap in scientific data for this culturally significant cultivar. The experimental design is robust, with multi-site sampling enabling the assessment of intra-cultivar variability, and the inclusion of a commercial Reineta benchmark provides valuable contextual insights. However, substantial revisions are required to address methodological ambiguities, strengthen statistical rigor, clarify comparative interpretations, and improve data presentation. The manuscript has strong merit and potential for publication after major revisions.
1.Sensory Evaluation Protocol: The sensory analysis (Section 2.1.7) uses a 5-point hedonic scale but lacks detailed attribute definitions and scoring criteria (e.g., how "firmness to the touch" or "aroma intensity" is operationalized). Provide the full sensory evaluation form as Supplementary Material, including anchor points for each attribute (e.g., 1 = "very soft" to 5 = "very firm"). Clarify whether the panel was trained specifically for apple sensory attributes and confirm inter-panelist reliability (e.g., coefficient of variation or Kappa statistics).
2.Sample Size Justification: For biometric (n=10 fruits/sample) and texture (n=3 fruits/sample) analyses, justify the sample size based on statistical power or previous literature. Explain why texture measurements used fewer replicates than other assays, and confirm if technical replicates were performed for texture analyses (e.g., 2 compressions/fruit as mentioned, but specify if this counts as technical replicates).
3.HPLC Methods Details: The phenolic compound analysis (Section 2.1.5) mentions 24 phenolic standards but does not specify which standards correspond to each compound detected (e.g., procyanidin B1 vs. other procyanidins). Provide a complete list of standards with their retention times and UV spectra in Supplementary Material. Clarify the detection limits for individual phenolic compounds, especially for minor components.
4.Post-Harvest Handling: For both local and commercial samples, detail the time between harvest/purchase and laboratory analysis (only "within 48 h" is mentioned for commercial samples). Specify storage conditions (temperature, humidity) during transport and prior to analysis, as these factors can influence physicochemical and sensory properties.
5. Intra-Cultivar Variability Analysis: The manuscript aims to assess intra-cultivar variability but lacks explicit statistical tests to quantify this. Perform a two-way ANOVA (producers as random factor, cultivar as fixed factor) to separate genetic vs. environmental contributions to variability. Alternatively, calculate coefficients of variation (CV) for key parameters across local producers and compare to CVs for the commercial sample to highlight intra-cultivar consistency.
6. Multivariate Analysis Improvements: The PCA (Section 3.9) explains only 50.2% of total variance, indicating important variability is unaccounted for. Include a scree plot to justify the number of principal components retained. In the HCA, specify the linkage method (e.g., Ward’s, complete) and distance metric (e.g., Euclidean) used. Add loading plots for PCA to clearly show which variables drive the separation between local and commercial samples.
7. Data Anomalies and Consistency: In Table 5 (Phenolic Compounds), apigenin and myricetin concentrations are unusually high (≈46 mg/100g fw) compared to typical apple values (usually <5 mg/100g fw). Verify these values with analytical standards and confirm no co-elution issues. Correct typographical errors (e.g., "Hydroxy benzoic acid" column header split, "Procya-nidin B1" typo, decimal separators as commas in some entries).
8. Correlation Analysis: The manuscript mentions Pearson correlation analysis but does not present results. Add a correlation matrix or focus on key relationships (e.g., TSS vs. sweetness score, phenolic content vs. antioxidant capacity) to strengthen links between instrumental and sensory data.
9. Commercial Sample Comparison Clarification: The manuscript repeatedly states the commercial sample is a "contextual benchmark" but often draws direct comparisons (e.g., "local apples consistently exhibited higher soluble solids"). Explicitly separate comparisons into "cultivar-related differences" and "production/system-related differences" (e.g., low-input vs. intensive cultivation, short vs. long supply chain). Discuss how factors like storage duration, harvest maturity, and orchard management could confound cultivar comparisons.
10. Bioactive Compound Significance: The discussion (Section 4) notes higher phenolic content in local samples but does not contextualize these values relative to other traditional apple cultivars. Add a comparative table with phenolic content and antioxidant capacity of similar Mediterranean heritage apples (e.g., Portuguese ‘Bravo de Esmolfe’, Italian ‘Annurca’) to highlight Reineta de Fontanelas’ uniqueness.
11. Intra-Cultivar Variability Discussion: The manuscript mentions variability among producers but does not link it to environmental or management factors (e.g., soil type, irrigation, pruning). Discuss potential drivers of variability (e.g., sandy vs. clay-rich soils mentioned in Section 2.1) and their implications for cultivar conservation (e.g., need for standardized best practices).
12. Practical Implications Specificity: The practical implications (Section 4) are overly general. Provide concrete recommendations for producers (e.g., optimal harvest maturity based on TSS/TA ratios) and policymakers (e.g., key quality parameters to include in potential GI certification).
13. Abbreviation Standardization: Define abbreviations at first mention in the abstract (e.g., TSS, TA, TPC) and ensure consistent use throughout (e.g., "fw" for fresh weight is sometimes omitted).
14. Reference Updates: The reference list includes several in-press or unpublished works (e.g., Morariu et al., 2025). Replace with published versions or remove if unavailable. Ensure consistency in citation formatting (e.g., some references lack DOI, others have inconsistent italicization of journal names).
15. Section Numbering: Correct section numbering inconsistencies (e.g., Section 3.5 is repeated for "Total Phenolic Content" and "Quantification of free sugars").

Author Response

• Comment (C): Sensory Evaluation Protocol: Provide attribute definitions, scoring criteria, sensory form, training details, and inter-panelist reliability.

 

Response (R): We thank the reviewer for this helpful and constructive comment. The sensory methodology has been revised to improve clarity and transparency. The revised manuscript now specifies that the evaluation was conducted by a trained sensory panel in accordance with ISO 8586:2012 guidelines, with all assessors having completed more than 200 h of structured training. An apple-specific calibration session was also conducted prior to evaluation to harmonise attribute interpretation across panel members. All sensory attributes assessed using the five-point hedonic scale are now accompanied by clear operational definitions and scale anchor points, which are provided in Supplementary Material S4, as recommended.

Regarding inter-panelist reliability, coefficients of variation (CV) were calculated across assessors and ranged from 8% to 14% for key attributes. CV is a widely accepted indicator of reproducibility for trained sensory panels using continuous or hedonic scales and is commonly applied in fruit quality studies. Agreement statistics such as Cohen’s kappa were not applied, as they are intended for categorical data rather than continuous intensity scores. For conciseness, the detailed calculation procedure for CV was not included, as it is primarily procedural and does not affect interpretation of the sensory results..

 

 

• C: Sample Size Justification: For biometric (n = 10 fruits/sample) and texture (n = 3 fruits/sample) analyses, justify the sample size based on statistical power or previous literature. Explain why texture measurements used fewer replicates than other assays, and confirm if technical replicates were performed for texture analyses (e.g., 2 compressions/fruit as mentioned, but specify if this counts as technical replicates).

 

R: We thank the reviewer for this valuable comment. The Methods section has been expanded to clarify the rationale underlying the sample sizes used for biometric and texture analyses. For biometric measurements, a subsample of 10 fruits per producer was analysed. This sample size is consistent with commonly adopted protocols in pomological studies, where 8–12 fruits are generally considered sufficient to capture intra-orchard variability and have been widely applied in previous apple quality assessments. This approach allows representative characterisation of fruit morphology without requiring excessively large sample sets.

Regarding texture analysis, the revised manuscript now clarifies that three fruits per sample were analysed, with each fruit subjected to two independent compressions. These repeated measurements were treated as technical replicates, resulting in a total of six determinations per sample. This sampling strategy follows established postharvest practice, as instrumental firmness typically shows relatively low within-sample variability when fruits are harvested at comparable maturity. Consequently, the use of fewer biological replicates is considered adequate for reliable texture estimation, provided that technical replication is applied. These clarifications have been incorporated into the revised manuscript (Lines 156-165 and 200-208), thereby providing clearer justification for the sampling strategy and explicitly defining the use of technical replicates in texture evaluation.

 

• C: The phenolic compound analysis mentions 24 phenolic standards but does not specify which standards correspond to each compound detected. Provide a complete list of standards with retention times and UV spectra in Supplementary Material. Clarify detection limits for individual phenolic compounds, especially minor components.

 

R: We thank the reviewer for this valuable comment. In response, we have revised the description of the HPLC–PDA phenolic analysis to improve clarity and transparency (Lines 228–251). The revised text now provides a clearer overview of the chromatographic conditions, compound identification strategy, and quantification approach used in the study. All detailed information requested by the reviewer has been added to Supplementary Material S5, including the complete list of the 24 phenolic standards used for identification and quantification, together with their retention times, PDA detection wavelengths, UV–Vis absorption maxima, and the corresponding limits of detection (LOD) and quantification (LOQ) for each compound. The equations used to calculate LOD and LOQ (LOD = 3.3 Sy/S; LOQ = 10 Sy/S) are also explicitly reported. These additions ensure full methodological transparency and reproducibility, particularly for minor phenolic constituents, while keeping the main manuscript concise and focused.

 

• C: For both local and commercial samples, detail the time between harvest/purchase and laboratory analysis. Specify storage conditions (temperature, humidity) during transport and prior to analysis, as these factors can influence physicochemical and sensory properties.

 

R: We thank the reviewer for this important comment. The manuscript has been revised to clarify postharvest handling conditions for both local and commercial samples. The revised text now explicitly reports the time elapsed between harvest or purchase and laboratory analysis, as well as the temperature and relative humidity conditions during transport and short-term storage prior to analysis (Lines 129-136).

 

• C: The manuscript aims to assess intra-cultivar variability but lacks explicit statistical tests to quantify this. Perform a two-way ANOVA (producers as random factor, cultivar as fixed factor) to separate genetic vs. environmental contributions to variability. Alternatively, calculate coefficients of variation (CV) for key parameters across local producers and compare to CVs for the commercial sample to highlight intra-cultivar consistency.

 

R: We thank the reviewer for this thoughtful comment and fully agree that quantifying variability is an important aspect when studying traditional cultivars. We carefully considered the suggested statistical approaches; however, after evaluating the structure and limitations of our dataset, we concluded that neither a two-way ANOVA nor producer-level CV comparisons would yield statistically meaningful or interpretable results in this case.

The Reineta de Fontanelas samples originate from independent orchards characterised by different microclimates, soil types, and management practices. As such, these samples do not represent biological replicates of a controlled factor but rather distinct expressions of the same cultivar under heterogeneous, real-world production conditions. Consequently, variation among producers cannot be formally partitioned into genetic and environmental components using a two-way ANOVA framework. Similarly, calculating CVs across producers would not reflect true intra-cultivar variability in a statistical sense, but rather the combined effects of environmental and management heterogeneity.

In addition, the commercial Reineta apples represent a single retail lot without internal biological replication and therefore cannot be treated as a factor level suitable for variance partitioning or CV-based comparison. Given these constraints, we opted to strengthen the descriptive and integrative interpretation of variability. The Results and Discussion sections were revised to more explicitly describe patterns of dispersion among producers and to identify parameters showing greater or lesser variability (Lines 703-710 and 796-805). This approach is consistent with the study’s primary objective: to characterise the Reineta de Fontanelas cultivar as it is produced under authentic local conditions, rather than to partition variance components within a controlled experimental design.

 

• C: The PCA explains only 50.2% of total variance. Include a scree plot to justify the number of principal components retained. In the HCA, specify the linkage method and distance metric. Add loading plots for PCA to clearly show which variables drive the separation between local and commercial samples.

 

R: We thank the reviewer for these valuable and constructive suggestions. To address these points while maintaining a clear and concise presentation, we have expanded and clarified the description and justification of the multivariate analyses in the revised manuscript. Although the first two principal components explained 50.2% of the total variance, this level of explained variance is typical for multivariate analyses of biological datasets encompassing multiple correlated quality traits. The revised text now explicitly notes that inspection of the eigenvalue distribution revealed a clear inflection after the second principal component, indicating that subsequent components account primarily for minor and unstructured variation. This supports the retention of PC1 and PC2 for interpretation of the main compositional patterns among samples.

In response to the request for loading plots, we have strengthened the textual description of the PCA by explicitly summarising the main variable loadings associated with PC1 and PC2. This approach conveys the key drivers of sample separation while avoiding redundancy with the biplot already presented. Given the descriptive aim of the multivariate analysis, we considered this textual summary sufficient to support interpretation. Finally, the hierarchical cluster analysis methodology has been clarified by specifying the use of Ward’s minimum variance linkage method and Euclidean distance as the similarity metric. These details are now clearly reported in the revised manuscript (Lines 686-699).

 

• C: In Table 5, apigenin and myricetin concentrations are unusually high (≈46 mg/100 g fw) compared to typical apple values. Verify these values with analytical standards and confirm no co-elution issues. Correct typographical errors (e.g., column header splits, ‘Procya-nidin B1’ typo, decimal separators).

 

R: We thank the reviewer for this observation. The values for apigenin and myricetin were rechecked against the analytical standards used for calibration, and their quantification was confirmed using retention time and UV–Vis spectral matching. Inspection of the chromatograms showed no evidence of co-elution with neighbouring peaks, and peak purity was consistent with the corresponding standards. Although these concentrations are higher than those typically reported for commercial apple cultivars, they fall within the range described for traditional or phenolic-rich landraces, particularly when peel is included in the extraction, as was the case in this study. We have tried to correct all typographical issues identified in Table 5 (e.g., column header formatting, split words such as ‘procyanidin B1,’ and decimal separators). Because the table is extensive and contains numerous columns, some formatting artefacts were caused by automatic adjustments in the word-processing software, making perfect alignment challenging. Nevertheless, we have carefully revised and standardised the layout to the fullest extent possible, and we are confident that these artefacts will not appear in the final typeset version.

 

• C: The manuscript mentions Pearson correlation analysis but does not present results. Add a correlation matrix or focus on key relationships (e.g., TSS vs. sweetness score, phenolic content vs. antioxidant capacity) to strengthen links between instrumental and sensory data.

 

R: We thank the reviewer for this helpful suggestion. Rather than adding a full correlation matrix—which would be extensive and of limited relevance to the main objectives of the study—we revised the manuscript to explicitly present and discuss the most biologically meaningful Pearson correlations directly in the Results section. The revised text now highlights key relationships, including the positive association between total soluble solids and perceived sweetness, the relationship between titratable acidity and perceived acidity, and the strong correlations between total phenolic content and antioxidant capacity as measured by DPPH and ABTS assays. In addition, the correspondence between instrumental firmness and sensory texture firmness is described. Focusing on these targeted correlations strengthens the link between instrumental measurements and sensory perception while maintaining clarity and conciseness. These additions are reported in Lines 653–663 of the revised manuscript.

 

• C: The manuscript repeatedly states the commercial sample is a 'contextual benchmark' but often draws direct comparisons. Explicitly separate comparisons into cultivar-related differences and production/system-related differences (e.g., low-input vs. intensive cultivation, short vs. long supply chain). Discuss how factors like storage duration, harvest maturity, and orchard management could confound cultivar comparisons.

 

R: We thank the reviewer for this important and insightful observation. In response, the manuscript has been revised to more clearly distinguish between cultivar-related characteristics and differences arising from production and postharvest systems. The commercial Reineta apples are now consistently described and interpreted solely as a contextual retail benchmark rather than as a direct cultivar-level comparator. The revised text explicitly states that observed differences between the commercial apples and the locally produced Reineta de Fontanelas samples may primarily reflect production-system factors, including orchard management intensity, harvest maturity, storage duration, and supply-chain length, rather than intrinsic genetic differences. We further discuss how extended cold-chain storage, earlier harvest timing, and standardised commercial handling practices can influence key quality attributes such as acidity, aroma, phenolic composition, and texture, thereby potentially confounding direct cultivar comparisons. These clarifications were incorporated in both the Materials and Methods and Discussion sections (Lines 137–153 and 778-784), ensuring consistent interpretation of the commercial sample throughout the manuscript and reinforcing the descriptive, non-comparative objectives of the study.

 

• C: The discussion notes higher phenolic content in local samples but does not contextualize these values relative to other traditional apple cultivars. Add a comparative table with phenolic content and antioxidant capacity of similar Mediterranean heritage apples (e.g., Portuguese ‘Bravo de Esmolfe’, Italian ‘Annurca’) to highlight Reineta de Fontanelas’ uniqueness.

 

1. R. We appreciate the reviewer’s suggestion to further contextualize the phenolic and antioxidant values observed for Reineta de Fontanelas After careful consideration, we decided not to include an additional comparative table, as reported phenolic content and antioxidant capacity values for heritage apple cultivars vary widely across the literature depending on factors such as extraction methodology, analysed tissue (peel, pulp, or whole fruit), analytical detection conditions, harvest maturity, and storage history. As a result, direct tabular comparisons across studies using different experimental approaches could lead to misleading or non-equivalent interpretations. Instead, we strengthened the Discussion by incorporating a detailed textual comparison with representative Mediterranean heritage cultivars, including the Portuguese cultivar ‘Bravo de Esmolfe’, the Italian cultivar ‘Annurca’, and other regional landraces. These studies report phenolic and antioxidant ranges that overlap closely with the values observed in Reineta de Fontanelas apples, thereby providing appropriate context while avoiding methodological bias. This expanded discussion supports the interpretation that traditional Mediterranean cultivars frequently exhibit enriched phenolic profiles relative to commercial apples and situates Reineta de Fontanelas within this broader phenolic-rich germplasm group (Lines 816-827).

 

• C: The manuscript mentions variability among producers but does not link it to environmental or management factors (e.g., soil type, irrigation, pruning). Discuss potential drivers of variability (e.g., sandy vs. clay-rich soils mentioned in Section 2.1) and their implications for cultivar conservation (e.g., need for standardized best practices).

 

R: We thank the reviewer for this comment. This aspect has already been addressed in the revised manuscript and was further reinforced in the final part of the Discussion. The text now explicitly links the variability observed among producers to documented environmental and orchard-management factors in the Sintra region, including differences in soil type (sandy versus clay-rich soils), microclimatic conditions, and heterogeneous low-input practices such as irrigation, pruning, and fertilisation. In addition, the Discussion now includes a concise paragraph outlining the implications of this variability for cultivar conservation, emphasising that moderate heterogeneity is characteristic of traditional landraces and can coexist with the identification of stable core quality traits that may inform voluntary best-practice guidelines. These additions are presented in the revised Discussion section (Lines 778-784 and 839-846).

 

• C: The practical implications (Section 4) are overly general. Provide concrete recommendations for producers (e.g., optimal harvest maturity based on TSS/TA ratios) and policymakers (e.g., key quality parameters to include in potential GI certification).

 

R: We thank the reviewer for this helpful suggestion. In response, we have substantially revised the Practical Implications section to provide concrete, evidence-based recommendations for producers, local associations, and policymakers. The revised text now includes specific guidance on harvest maturity indicators (e.g., TSS/TA balance, firmness thresholds), handling practices that preserve phenolic quality, and parameters that could inform the development of geographical indication (GI) specifications. These additions make the section more actionable and better aligned with the applied relevance of the study (Lines 895-915).

 

• C: Abbreviation standardization: Define abbreviations at first mention in the abstract (e.g., TSS, TA, TPC) and ensure consistent use throughout (e.g., ‘fw’ for fresh weight is sometimes omitted.

 

R: We thank the reviewer for this useful comment. We have carefully reviewed the manuscript and standardised all abbreviations to ensure clarity and consistency. Abbreviations such as TSS (total soluble solids), TA (titratable acidity), TPC (total phenolic content), and fw (fresh weight) are now defined at their first occurrence in the abstract and again at first use in the main text, following journal guidelines. We also verified that all abbreviations are used consistently throughout the manuscript, and any instances where terms such as “fresh weight” or analytical units were previously written in full have now been harmonised. These revisions improve the readability and uniformity of the manuscript.

 

• C: Reference updates: The reference list includes several in-press or unpublished works (e.g., Morariu et al., 2025). Replace with published versions or remove if unavailable. Ensure consistency in citation formatting (e.g., some references lack DOI, others have inconsistent italicisation of journal names.

 

R: We thank the reviewer for this helpful observation. The reference list has been thoroughly reviewed and updated. All citations previously listed as “in press” or containing incomplete bibliographic information were verified and updated with final publication details where available, or removed when a published version could not be confirmed. In addition, the formatting of all references was standardised to ensure consistency with the journal’s guidelines, including journal name italicisation and inclusion of DOIs when available.

 

• C: Section numbering: Correct section numbering inconsistencies (e.g., Section 3.5 is repeated for 'Total Phenolic Content' and 'Quantification of free sugars’).

 

R: We thank the reviewer for noting this issue. All section numbering inconsistencies have been corrected, and the Results section now follows a clear and sequential numbering scheme. All internal cross-references were updated accordingly.

 

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

This work characterized the biometric, physicochemical, nutritional, bioactive, and sensory properties of Reineta de Fontanelas apples collected from six local producers, and highlighted the potential of this traditional cultivar as a high-quality fruit for consumers. The results may be helpful for the exploration of this traditional apple cultivar. There are several issues that need to be addressed.

1. line 134: 'addition to the local samples' should be 'In addition to the local samples'?
2. line 151: '2.1' should be '2.2', and the following '2.1.1', '2.1.2', '2.1.3', ... should be revised accordingly.
3. How many apples were collected for each sample? Does the note 'n=3' below Table 1 mean only 3 apples were determined?
4. It is not necessary to show the energy values in both kJ and kcal. Please delete one of them.
5. line 440: 'in vitro' should be in italics.
6. As indicated in Table 4 and Figure 4, JC seems to be rather different from others. Please provide some discussion regarding this difference.
7. Does the 'ascobic acid' in Table 5 mean 'ascorbic acid'? Generally, ascorbic acid does not belong to phenolic compounds. Additionally, some values of total PC in Table 5 seem to be not correctly calculated. Please delete the data of ascorbic acid from phenolics and check the calculation of total PC.
8. Generally, little lactic acid and acetic acid can be detected in fresh apples. Please check the method for identification and quantification of these two acids.
9. The 'hardness' in Figure 7 should be changed to 'firmness' to be in accordance with previous presentation.
10. In the discussion, more comparison can be included with other commercial or local apples reported in previous literatures.

Author Response

We thank Reviewer 3 for the positive assessment of our work and for the constructive suggestions. We address each point below.

 

1. Comment (C): line 134: 'addition to the local samples' should be 'In addition to the local samples'?

 

Response (R): We thank the reviewer for noticing this. The sentence has been corrected to: “In addition to the local samples…” (line 137).

 

2. C: line 151: '2.1' should be '2.2', and the following '2.1.1', '2.1.2', '2.1.3', ... should be revised accordingly.

 

R: We appreciate the observation. The section numbering in the Materials and Methods was reviewed and fully updated to ensure sequential consistency throughout the manuscript.

 

3. C: How many apples were collected for each sample? Does the note 'n=3' below Table 1 mean only 3 apples were determined?

R: We thank the reviewer for this comment. Each producer provided a 3 kg lot of apples, from which 10 representative fruits were selected for biometric measurements. This information is now clearly stated in the Methods section and reflected in the revised table notes. The notation n = 3 used in other tables refers exclusively to three analytical replicates performed on homogenised pooled tissue and does not correspond to the number of individual apples analysed. All relevant descriptions and table footnotes have been revised throughout the manuscript to ensure full clarity.

 

4. C: It is not necessary to show the energy values in both kJ and kcal. Please delete one of them.

 

R: We thank the reviewer for this suggestion. To avoid redundancy, the nutritional table now reports energy only in kJ, following standard SI usage, and the kcal column has been removed.

 

5. C: Line 440: “in vitro” should be italicised

 

R: Thank you — this has been corrected.

 

6. C: As indicated in Table 4 and Figure 4, JC seems to be rather different from others. Please provide some discussion regarding this difference.

 

R: We appreciate the reviewer’s observation. The manuscript has been carefully revised to address this point, and the Discussion now includes an explicit explanation of the distinct behaviour observed among local producers, interpreted in the context of orchard-specific and microclimatic variability typical of traditional low-input production Systems (Lines 383-389 and 786-805).

 

7. C: Does the 'ascobic acid' in Table 5 mean 'ascorbic acid'? Generally, ascorbic acid does not belong to phenolic compounds. Additionally, some values of total PC in Table 5 seem to be not correctly calculated. Please delete the data of ascorbic acid from phenolics and check the calculation of total PC.

 

R: We thank the reviewer for this accurate and helpful comment. The typographical error “ascobic acid” has been corrected to ascorbic acid, and we fully agree that ascorbic acid is not a phenolic compound. Accordingly, the total phenolic content was recalculated with ascorbic acid excluded from the phenolic sum. Ascorbic acid was retained in the table because it was quantified in the same HPLC–PDA analysis and is a relevant antioxidant constituent. To avoid any ambiguity, the table title and an explicit explanatory note were added, clarifying that ascorbic acid is reported separately for completeness of the chromatographic profile and is not included in the calculation of total phenolic content. All related text, tables, and calculations were carefully revised to ensure consistency throughout the manuscript. (Lines 252-256, 524-528, 547-557 and 786-763)

 

8. C: Generally, little lactic acid and acetic acid can be detected in fresh apples. Please check the method for identification and quantification of these two acids.

 

R: We thank the reviewer for this helpful comment. We carefully rechecked the identification and quantification of lactic and acetic acids and confirmed that the detected peaks were correctly assigned using authenticated standards; therefore, no modification of the reported data was required. To clarify their presence, a brief explanatory comment was added to the Discussion, noting that lactic and acetic acids are typically present only at trace levels in fresh apples and that their detection in this study likely reflects early postharvest metabolic activity, minor contributions from natural epiphytic microflora, and the high sensitivity of the HPLC–PDA method at 210 nm. All relevant text was revised accordingly (Lines 773-777)

 

9. C: The 'hardness' in Figure 7 should be changed to 'firmness' to be in accordance with previous presentation.

 

R: Corrected. Thank you for noting the inconsistency.

 

10. C: In the discussion, more comparison can be included with other commercial or local apples reported in previous literatures.

 

R: We thank the reviewer for this suggestion. The Discussion has been revised to include additional comparisons with commercial and local apple cultivars reported in previous studies. In particular, we expanded the comparison of phenolic content, antioxidant capacity, and vitamin C levels of Reineta de Fontanelas with those described for Mediterranean heritage cultivars (e.g., Bravo de Esmolfe and Annurca) as well as values typically reported for commercial apples. Relevant published references were added to strengthen this contextualisation and provide a more robust comparative framework (Lines 816-838).

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

This manuscript has been revised and improved according to the Reviewer's suggestion, therefore, is ready for acceptance.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Unfortunatelly i do not agree with the comments. The study itself is not done properly, thus the results are missleading. They have no proper control as apples were purchased from a local supermarket, so you do not know when and how were they harvested. Furthermore peaking was used for identification which missidentifies the compounds in 90% of the cases, this is not appropriate, LC-MS should be used to identify the compounds. I do not agree with the acceptance of this article i am sorry. Please make a proper research with control and use LC-MS for identification, quantification can be on HPLC offcourse. Then i would agree you can publish your article, but at the moment no.

Author Response

Reviewer Comment: Unfortunately, I do not agree with the comments. The study itself is not done properly, thus the results are misleading. They have no proper control as apples were purchased from a local supermarket, so you do not know when and how they were harvested. Furthermore, peaking was used for identification which misidentifies the compounds in 90% of the cases, this is not appropriate, LC-MS should be used to identify the compounds. I do not agree with the acceptance of this article, I am sorry. Please make a proper research with control and use LC-MS for identification, quantification can be on HPLC of course. Then I would agree you can publish your article, but at the moment no.

 

Author Response (AR). We thank the reviewer for their critical assessment and for highlighting important methodological considerations. We acknowledge that the use of commercially sourced apples as a reference sample presents certain limitations in terms of full traceability of harvest and postharvest conditions. However, our objective was not to evaluate the effect of specific production practices, but rather to obtain a comparative compositional and qualitative overview between the locally produced Reineta de Fontanelas cultivar and a commonly available commercial Reineta variety. This approach, which has been used in several comparative fruit studies, allows the identification of distinctive quality attributes of traditional cultivars under real market conditions. The sampling procedure was described transparently in the Materials and Methods section, and all samples were handled, processed, and analyzed under identical laboratory conditions to minimize variability.

Regarding phenolic compound identification, we agree that LC–MS would provide more precise structural confirmation, especially for glycosylated flavonoids. In the present study, however, the focus was on comparative profiling rather than exhaustive structural elucidation. Compounds were identified tentatively by HPLC-PDA through retention times and UV–Vis spectra compared with authentic standards and literature data, a method widely accepted for preliminary phenolic characterization in apples and other fruits. Quantification was based on external calibration using aglycone standards, as described.

We have now made these methodological limitations explicit in the revised manuscript (Sections 4), noting that future work will employ LC–MS/MS to confirm compound identities and further strengthen the analytical robustness. We believe that, despite these constraints, the study provides meaningful and valid insights into the physicochemical, nutritional, and bioactive distinctiveness of the Reineta de Fontanelas apple cultivar, contributing valuable baseline data for future, more detailed research.

Reviewer 2 Report

Comments and Suggestions for Authors

As I wrote in the first review, the manuscript is based on very thorough analysis of very broad range of apple quality parameters. Authors did not limit their analysis with traditional apple fruit quality analysis, but presented fruit biochemical and nutritional value, along with sensorial evaluation. All the results are clearly presented and based on solid statistical evaluation.

Authors expanded Materials and Methods section, expanded and reformulated Limitations and Practical Implications sections, thus fully addressed my concerns raised in the first review.

Though the manuscript presents description of apple cultivar which has limited value and only in local regional aspect, it could serve as an excellent example how to conduct a thorough analysis of fruit quality.

Some minor remarks:

Choose either American or English spelling. Table 4. ‘Color’ in the title, and ‘colour’ in the table caption.

Correct reference list.

Reference 1 not understandable. ‘Dulloo, M.& R.E.& R.M.& D.A.& P.S.& M.N.& S.B.& G.D.& R.N.& S.J.& R.M.& R.M.& T.I.& G.Hannes. 816 Conserving Agricultural Biodiversity for Use in Sustainable Food Systems. . In; Arwen Bailey, A. del 817 C.N.C.M.M.S.C.P.G., Ed.; 201’

References 10, 47. Authors names written in capital letters.

Comments on the Quality of English Language

Choose either American or English spelling. Table 4. ‘Color’ in the title, and ‘colour’ in the table caption.

Author Response

Comments and Suggestions for Authors

• As I wrote in the first review, the manuscript is based on very thorough analysis of very broad range of apple quality parameters. Authors did not limit their analysis with traditional apple fruit quality analysis, but presented fruit biochemical and nutritional value, along with sensorial evaluation. All the results are clearly presented and based on solid statistical evaluation.
• Authors expanded Materials and Methods section, expanded and reformulated Limitations and Practical Implications sections, thus fully addressed my concerns raised in the first review.
• Though the manuscript presents description of apple cultivar which has limited value and only in local regional aspect, it could serve as an excellent example how to conduct a thorough analysis of fruit quality.

Some minor remarks:

• Choose either American or English spelling. Table 4. ‘Color’ in the title, and ‘colour’ in the table caption.
• Correct reference list.
• Reference 1 not understandable. ‘Dulloo, M.& R.E.& R.M.& D.A.& P.S.& M.N.& S.B.& G.D.& R.N.& S.J.& R.M.& R.M.& T.I.& G.Hannes. 816 Conserving Agricultural Biodiversity for Use in Sustainable Food Systems. In; Arwen Bailey, A. del 817 C.N.C.M.M.S.C.P.G., Ed.; 201’
• References 10, 47. Authors names written in capital letters.

 

Author Response (AR) to Reviewer

We sincerely thank the reviewer for their very positive and encouraging evaluation of our revised manuscript. We greatly appreciate the recognition of the thoroughness of our analytical approach and the improvements implemented after the first review. We also thank the reviewer for their careful reading and for pointing out minor editorial issues that have now been corrected. Our detailed responses are provided below.

AR 1. We thank the reviewer for noticing this inconsistency. We have carefully reviewed the entire manuscript and standardized the spelling to British English, as it is more consistent with the conventions used throughout the paper. Accordingly, “Color” has been changed to “Colour” in Table 4 and all relevant sections.

AR 2. We thank the reviewer for identifying this issue. The reference list has been carefully revised and corrected. Reference [1] has been fully rewritten to ensure clarity and accuracy. It now reads as follows:

AR 3. We appreciate the reviewer’s attention to formatting details. References [10] and [47] have been corrected to follow standard capitalisation (sentence case for authors’ names and title case for article titles). The full reference list has been rechecked to ensure uniform formatting throughout. All other references have been rechecked for format consistency and completeness according to MDPI reference style guidelines.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript presents a comprehensive characterization of the Reineta de Fontanelas apple cultivar, comparing it with a commercial Reineta sample. The study is well-structured and provides valuable data on biometric, physicochemical, nutritional, bioactive, and sensory properties. However, there are several areas where the manuscript could be improved, including methodological clarity, statistical rigor, and contextualization within the existing literature. The following points should be addressed before publication.

Comments to the Author:

Question 1：The manuscript is generally scientific, employing standardized methods for physicochemical, nutritional, and bioactive analyses. However, the lack of detailed statistical reporting (e.g., p-values in tables, ANOVA results) and insufficient description of sample preparation for HPLC analysis weaken its scientific rigor.

Question 2：The manuscript lacks a clear hypothesis or research question in the introduction, which is essential for framing the study’s scientific contribution.

Question 3：The introduction effectively highlights the importance of preserving agricultural biodiversity and introduces the Reineta de Fontanelas apple as a understudied cultivar. However, it lacks a critical review of recent studies on traditional apple cultivars, especially from Mediterranean regions.

Question 4：The introduction would benefit from a clearer statement of the study’s novelty---specifically, how it advances beyond existing characterizations of traditional apples.

Question 5：The methods are generally appropriate but require more detail in key areas, such as the HPLC conditions (e.g., gradient elution program) and sample extraction procedures for phenolic compounds.

Question 6：The description of the commercial sample (MA) is vague—details about harvest date, storage duration, and specific orchard origin are missing, which may confound comparisons.

Question 7： Figure 1 provides useful geographical context but lacks scale bars or GPS coordinates, which are important for reproducibility.

Question 8：The study adds value by providing the first comprehensive characterization of Reineta de Fontanelas, but it does not sufficiently contrast its findings with existing studies on other traditional apple cultivars (e.g., Serra et al., 2010; Morariu et al., 2025).

Question 9：The multivariate analysis (PCA, HCA) is a strength, but its interpretation is superficial. A deeper discussion of how these results differentiate Reineta de Fontanelas from other cultivars would enhance novelty.

Question 10：The discussion effectively links results to broader themes (e.g., biodiversity, sensory quality) but often repeats results without critical interpretation. For example, higher phenolic content is noted but not contextualized within health implications or cultivar-specific metabolism.

Question 11：The discussion overlooks contradictory findings---e.g., why some local samples (HV) clustered closer to MA in HCA---which should be explored as limitations or sources of variability.

Question 12：The conclusions are generally supported by the data but overstate superiority (e.g., “superior nutritional, bioactive, and sensory qualities”) without acknowledging variability among local samples or limitations in sample comparability.

Question 13：The claim that Reineta de Fontanelas has “high market potential” is not directly evidenced by the study---consumer preference is absent.

Question 14：The conclusions address the primary goal of characterizing Reineta de Fontanelas but fail to explicitly tie back to the biodiversity conservation theme raised in the introduction.

Question 15：Recommendations for future work (e.g., LC-MS analysis, controlled agronomic trials) are appropriate but should be integrated into the conclusions to highlight translational impact.

Question 16：English grammar.

Author Response

Comments and Suggestions for Authors. This manuscript presents a comprehensive characterisation of the Reineta de Fontanelas apple cultivar, comparing it with a commercial Reineta sample. The study is well-structured and provides valuable data on biometric, physicochemical, nutritional, bioactive, and sensory properties. However, there are several areas where the manuscript could be improved, including methodological clarity, statistical rigour, and contextualization within the existing literature. The following points should be addressed before publication.

 

Author Response (AR) to Reviewer #3

We sincerely thank the reviewer for the thorough and thoughtful evaluation of our manuscript. We appreciate the constructive suggestions regarding methodological detail, statistical reporting, and contextualization within the literature. In response, we have carefully revised the manuscript to address each of the reviewer’s concerns point by point, as outlined below.

 

Q 1. The manuscript is generally scientific, employing standardised methods for physicochemical, nutritional, and bioactive analyses. However, the lack of detailed statistical reporting (e.g., p-values in tables, ANOVA results) and insufficient description of sample preparation for HPLC analysis weaken its scientific rigour.

AR 1. We appreciate the reviewer’s comment regarding the statistical reporting. We respectfully note that all tables in the manuscript already include statistical analysis results, as indicated by the statement “Samples containing different letters are significantly different (p ≤ 0.05).” This approach reflects the conventional format used in Horticulturae and similar journals in the field, where one-way ANOVA is followed by Tukey’s HSD post-hoc test, and significance is represented by letter-based groupings rather than explicit p-values. To clarify this, we have revised the Statistical Analysis section to explicitly state that one-way ANOVA and Tukey’s HSD test were used for all comparisons, with significance set at p ≤ 0.05, and that differences are represented by distinct letters in the tables. We believe this standard and widely accepted format provides sufficient statistical detail to support the reported results and maintain readability. (Lines 300-312)

 

In response, the Materials and Methods section has been thoroughly revised and expanded, particularly the subsections describing sample extraction and preparation for HPLC analysis of phenolic compounds, free sugars, and organic acids. The HPLC analysis section has also been detailed further to specify extraction solvents, reagent concentrations, chromatographic conditions, and detection parameters, ensuring full methodological transparency and reproducibility. All these additions and clarifications are highlighted in red in the revised manuscript.

 

Q 2. The manuscript lacks a clear hypothesis or research question in the introduction, which is essential for framing the study’s scientific contribution.

AR 2. We thank the reviewer for this valuable observation. We agree that the Introduction would benefit from a more explicit statement of the research hypothesis guiding the study. In response, we have revised the end of the Introduction to clearly present both the research question and hypothesis as follows (lines 97-103):

“Specifically, this work addresses the research question of whether the Reineta de Fontanelas apple cultivar differs significantly from a commercial Reineta variety in its compositional and sensory attributes. The authors hypothesise that, owing to its local adaptation and traditional low-input cultivation practices, the Reineta de Fontanelas apple exhibits superior nutritional, bioactive, and sensory qualities, defining a unique compositional fingerprint that supports its scientific and economic valorization.”

 

Q 3 & 4. The introduction lacks a critical review of recent studies on traditional apple cultivars, especially from Mediterranean regions, and would benefit from a clearer statement of novelty.

AR 3 &4. We thank the reviewer for this constructive comment. We have revised the Introduction to include a more critical review of recent studies on traditional and Mediterranean apple cultivars, highlighting how our study differs from and extends previous work. Specifically, we now discuss relevant findings from Portugal, Spain, Italy, and other Mediterranean regions, emphasising the lack of integrated analyses combining physicochemical, nutritional, bioactive, and sensory attributes. Additionally, we have clarified the study’s novelty by explicitly stating that this is the first comprehensive characterisation of the Reineta de Fontanelas cultivar, integrating multivariate statistical approaches (PCA, HCA) to define its compositional fingerprint in comparison with a commercial Reineta variety. (Lines 78-90)

 

“Although several studies have evaluated the nutritional and bioactive characteristics of traditional apple cultivars, most have focused on Central or Northern European varieties, with limited attention to those from Mediterranean regions. Recent works from Italy, Spain, and Greece have reported that local apples often display higher phenolic content, stronger antioxidant activity, and more complex flavor profiles than commercial types [18]. However, comprehensive assessments that integrate physicochemical, nutritional, bioactive, and sensory dimensions remain scarce, particularly for Portuguese cultivars. In Portugal, studies have mainly addressed physicochemical traits or postharvest behaviour of traditional varieties such as ‘Bravo de Esmolfe’ and ‘Reineta de Alcobaça’ [19], but no detailed compositional and sensory characterisation has yet been published for the Reineta de Fontanelas apple. Therefore, the present work provides novel insights by offering the first integrated and multivariate characterisation of this cultivar, contributing to the scientific basis for its valorisation and conservation.”

• Cice, Danilo & Ferrara, Elvira & Magri, Anna & Adiletta, Giuseppina & Capriolo, Giuseppe & Rega, Pietro & Matteo, Marisa & Petriccione, Milena. (2023). Autochthonous Apple Cultivars from the Campania Region (Southern Italy): Bio-Agronomic and Qualitative Traits. Plants. 12. 1160. 10.3390/plants12051160.
• Teixeira JD, Soares Mateus AR, Sanchez C, Parpot P, Almeida C, Sanches Silva A. Antioxidant Capacity and Phenolics Profile of Portuguese Traditional Cultivars of Apples and Pears and Their By-Products: On the Way to Newer Applications. Foods. 2023 Apr 5;12(7):1537. doi: 10.3390/foods12071537.
• Arnold, M., Gramza-Michalowska, A. Recent Development on the Chemical Composition and Phenolic Extraction Methods of Apple (Malus domestica)—A Review. Food Bioprocess Technol17, 2519–2560 (2024). https://doi.org/10.1007/s11947-023-03208-9

 

Q 5. Methods require more detail in key areas, such as HPLC conditions and sample extraction for phenolics.

AR 5. Detailed chromatographic parameters (mobile-phase composition, gradient profile, injection volume, and run time) and extraction conditions (solvent ratio, temperature, and centrifugation settings) have been added to Section 2.1.5 (Extraction and Analysis of Phenolic Compounds).

 

Q 6. Description of the commercial sample (MA) is vague—details about harvest date, storage duration, and orchard origin are missing.

AR 6. We have clarified that the commercial apples were IGP-certified “Reineta de Alcobaça”, harvested and marketed during September–October 2024, under standardised cold-chain conditions. This information, together with a discussion of its implications, has been added in Materials and Methods → Plant material (lines 136–150) and reiterated in the Limitations section.

 

Q 7. Figure 1 lacks scale bars or GPS coordinates.

AR 7. We thank the reviewer for this observation. We would like to clarify that the GPS coordinates of the sampling area (38°50′50″ N, 9°26′14″ W) are already provided in the text of Section 2.1 (“Plant Material”). To further improve clarity and visual accuracy, we have now updated Figure 1 to include a scale bar.

 

Q 8 & 9. The study adds value but needs a stronger contrast with existing studies and a deeper interpretation of PCA/HCA results.

AR 8&9. We thank the reviewer for the positive feedback and constructive suggestions. In response, we have strengthened the Discussion of the PCA and HCA results (Section 3.7) by providing a deeper interpretation of the variables contributing to the observed clustering and separation between samples. The revised text now clearly identifies the physicochemical, nutritional, and bioactive parameters that most influenced the principal components and clusters, offering a more comprehensive explanation of the multivariate patterns. Furthermore, we have enhanced the contrast with existing studies by comparing our findings with previously published research on traditional Mediterranean apple cultivars, such as ‘Bravo de Esmolfe’ (Portugal) and ‘Annurca’ (Italy), which similarly reported higher phenolic content and antioxidant capacity relative to commercial varieties (Teixeira et al., 2023; Cice et al., 2023). These additions clarify the novelty and broader relevance of our results, highlighting the distinct compositional fingerprint and valorization potential of the Reineta de Fontanelas cultivar (Lines 631-639).

Q 10 & 11. Discussion repeats results without critical interpretation and overlooks contradictory findings (e.g., HV sample clustering near MA).

AR 10 &11. We thank the reviewer for this insightful comment. We agree that the Discussion section benefited from a deeper critical interpretation. Accordingly, we have revised the text to emphasise the underlying causes and implications of the observed patterns rather than merely restating the results.

In particular, we now address the partial clustering of the HV sample with the commercial Reineta (MA) observed in the HCA (Figure 7A). This overlap is discussed as possibly resulting from environmental or postharvest factors influencing certain physicochemical traits such as acidity and firmness, which are known to vary with orchard microclimate and ripening stage. The revised discussion acknowledges these nuances, providing a more balanced interpretation that integrates both expected and divergent findings (Lines 724-731).

 

Q 12 & 13. Conclusions overstate superiority and claim market potential without direct evidence.

AR 12 &13. We thank the reviewer for this valuable comment. We agree that the Conclusions section required a more balanced tone. Accordingly, we have revised it to avoid overstating the superiority or market potential of the Reineta de Fontanelas cultivar. The conclusions now focus strictly on the evidence obtained in this study—its distinctive compositional, nutritional, and antioxidant characteristics—without making claims beyond the scope of the data. Any references to market potential have been rephrased to emphasise possible applications or future perspectives rather than confirmed outcomes.

 

Q 14 & 15. Conclusions should reconnect to biodiversity conservation and integrate future research recommendations.

AR 14 &15. We thank the reviewer for this valuable suggestion. We have revised the Conclusions to strengthen the connection with biodiversity conservation and to include clearer recommendations for future research. The updated text emphasises the relevance of preserving traditional cultivars like Reineta de Fontanelas as reservoirs of genetic diversity and cultural heritage. In addition, we now propose future research directions, including genetic characterisation, consumer studies, and postharvest and environmental performance assessments, to support both conservation strategies and potential valorisation pathways.

 

Q 16. English grammar.

AR 16. The entire manuscript has undergone a comprehensive English language revision by a professional editor to ensure grammatical accuracy and stylistic consistency in British English.

Reviewer 4 Report

Comments and Suggestions for Authors

This study provides the comprehensive evaluation of the Reineta de Fontanelas apple cultivar by comparing  with a commercial Reineta sample (MA), and the results reinforce the importance of preserving traditional cultivars as reservoirs of biodiversity and as premium products with high market potential. However, major revision is recommended, questions and suggestions are as follows.

1 the design of the control experiment is reasonable. However, for the step where "peaking was used for identification" , phenolic compounds was based on comparison of retention times and UV–Vis spectra with standards. This method is not robust. Because most of the natural phenoilc components in apples, especially the flavonoids,  are glycosides. While, most of the standards used maybe aglycone, such as apigenin, quercetin.

2 how many phenoilc standards was used in your study? Please provide in the material and methods section. In addition, other standards and chemicals used also should be provided, such as organic acid and monosaccharide standards, HPLC chemicals.

3 The chromatographic profiles (Figure S1) confirmed these differences visually, in line 449. But I can not find this supplentary file.

4 the other chromatograms of phenolics and sugars are provided in supplentary files, for the Figure 5, this chromatograms of organic acids are suggested to be moved to supplentary files.

5 some mistakes should be checked, for example, line 386, TFC should be TPC. In addition, for apple phenolic determiantions, TFC are suggested be measured.

6 line 373, the subtitle should be revised as the total phenolic content and antioxidant capacity of Reineta Apples.

7 in the table 5, what is procyanidin? Procyanidin B?

8 in the table 7 ,the unit used is wrong.

9 For  the Hierarchical cluster analysis (HCA) and principal component analysis (PCA), why these factors were included? The contents of major indiviudal organic acid and phenoilc compound are suggested to be considered.

 

Author Response

Comments and Suggestions for Authors. This study provides a comprehensive evaluation of the Reineta de Fontanelas apple cultivar by comparing it with a commercial Reineta sample (MA), and the results reinforce the importance of preserving traditional cultivars as reservoirs of biodiversity and as premium products with high market potential. However, a major revision is recommended; questions and suggestions are as follows.

Author Response (AR) to Reviewer #4

We sincerely thank the reviewer for their careful and detailed evaluation of our manuscript and for the constructive recommendations to improve its methodological and analytical clarity. We have carefully revised the manuscript to address each point raised. Our responses are presented below.

 

C1. The design of the control experiment is reasonable. However, for the step where “peaking was used for identification”, phenolic compounds were identified based on comparison of retention times and UV–Vis spectra with standards. This method is not robust. Because most natural phenolic components in apples, especially the flavonoids, are glycosides, while most of the standards used may be aglycones, such as apigenin, quercetin.

AR 1. We thank the reviewer for this valuable technical observation. We acknowledge that the identification of phenolic compounds based solely on retention times and UV–Vis spectra, using mainly aglycone standards, has limitations, particularly given that many phenolics in apples occur as glycosides. In this study, our aim was to obtain a comparative phenolic profile between the Reineta de Fontanelas and the commercial cultivar rather than a complete structural elucidation of individual compounds.

To improve clarity, we have revised the Methods section (Section 2.1.5) to specify that phenolic compounds were identified by comparison with authentic standards and literature data (retention times and UV–Vis characteristics), and thus represent tentative.

 

C2. How many phenolic standards were used in your study? Please provide in the Materials and Methods section. In addition, other standards and chemicals used should also be provided, such as organic acid and monosaccharide standards, HPLC chemicals.

AR 2. We thank the reviewer for this valuable comment. In the revised version, we have specified in the Materials and Methods section that calibration curves were prepared for approximately 23 phenolic standards, representative of the main phenolic classes found in apples. Mixed standard solutions were analysed at detection wavelengths of 280, 325, and 340 nm, corresponding respectively to flavan-3-ols, hydroxycinnamic acids, and flavonols. The selected HPLC column (Synergi Hydro, 250 × 4.6 mm, 4 µm) provided good resolution and peak separation for all tested standards.

We have also added information on the organic acid and monosaccharide standards and other HPLC-grade chemicals used, as requested, to improve the completeness of the Materials and Methods section.

C3. The chromatographic profiles (Figure S1) confirmed these differences visually, in line 449. But I cannot find this supplementary file.

AR 3. We appreciate the reviewer pointing this out. The chromatographic profiles (Figure S1) were inadvertently omitted during the initial submission. They have now been included as part of the Supplementary Material and correctly cited in the text

C4. Other chromatograms of phenolics and sugars are provided in supplementary files. For Figure 5, the chromatogram of organic acids is suggested to be moved to the supplementary files.

AR 4. We agree with the reviewer’s suggestion. To streamline the main text and improve readability, the chromatogram of organic acids (formerly Figure 5) has been moved to the Supplementary Materials (now Figure S3). The main text now includes a simplified figure referencing these chromatograms (lines XXX–XXX).

 

C5. Some mistakes should be checked, for example, line 386, TFC should be TPC. In addition, for apple phenolic determinations, TFC are suggested to be measured.

AR 5. We thank the reviewer for detecting this error. The incorrect abbreviation (“TFC”) has been corrected to “TPC” (Total Phenolic Content).

We thank the reviewer for this helpful suggestion. We agree that measuring total flavonoid content (TFC) would provide useful complementary information to total phenolic content (TPC). Although TFC was not determined in this study, the HPLC-PDA analysis included flavonoid compounds, and future work will include TFC measurement to give a more complete picture of the phenolic composition.

 

C6. Line 373, the subtitle should be revised as the total phenolic content and antioxidant capacity of Reineta apples.

AR 6. We thank the reviewer for this suggestion. The subtitle on line 373 has been revised to “Total Phenolic Content and Antioxidant Capacity of Reineta Apples” to more accurately describe the section’s content.

 

C7. In Table 5, what is procyanidin? Procyanidin B?

AR 7. We thank the reviewer for requesting clarification. The compound listed as “procyanidin” refers to procyanidin B, which was confirmed based on retention time and UV–Vis spectra compared with the authentic standard. We have corrected the compound name in Table 5.

 

C8. In Table 7, the unit used is wrong.

AR 8. We thank the reviewer for pointing this out. The unit in Table 7 is incorrect. As stated in Section 2.1.6, organic acids were expressed as mg/100 g fw, not g/100 g fw. We have corrected Table 7 accordingly (including the header and the “Total OA” column) and harmonised the unit in the table caption and related text.

 

C9. For the Hierarchical Cluster Analysis (HCA) and Principal Component Analysis (PCA), why were these factors included? The contents of major individual organic acids and phenolic compounds are suggested to be considered.

AR 9. We thank the reviewer for this valuable and insightful suggestion regarding the inclusion of additional variables in the multivariate analyses. The variables included in the HCA and PCA were selected to represent key physicochemical, nutritional, and bioactive quality parameters that were consistently measured across all samples, ensuring data comparability and avoiding overfitting given the limited sample size. Our objective was to obtain a global overview of compositional and quality differences between the Reineta de Fontanelas and the commercial Reineta (MA) samples, rather than to model variability at the individual compound level.

To maintain statistical robustness and interpretative clarity, only the most representative aggregated parameters—total soluble solids (TSS), titratable acidity (TA), total phenolic content (TPC), antioxidant activity (AA), colour parameters, and firmness—were included. These variables capture the main physicochemical and bioactive traits that define overall fruit quality and consumer perception. Including numerous correlated variables (e.g., individual phenolic and organic acid contents) could inflate dimensionality and introduce multicollinearity, potentially obscuring major discrimination patterns.

We agree that incorporating the contents of major individual organic acids and phenolic compounds could provide additional resolution and valuable insights into the factors driving sample clustering. This will be considered in future work to refine the multivariate interpretation and strengthen the link between compositional markers and cultivar differentiation. The main discriminative trends observed—higher TSS, TPC, and antioxidant activity in Reineta de Fontanelas—were consistent with univariate comparisons of individual compounds, confirming that the selected variables adequately captured sample differentiation.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Q1: Try not to display the Table (Table 2) in pagination.

Q2:  The fonts, A or (A),  and pixel in all Figures should comply with the requirements of the journal horticulturae.

Q3: The abstract is too long and needs to be concise.

Author Response

Q1: Try not to display the Table (Table 2) in pagination.

R1:
We thank the reviewer for this observation. Table 2 has been reformatted to ensure it appears as a single, continuous table without pagination breaks, in accordance with the journal’s layout requirements. The revised version preserves clarity and readability within one page whenever possible.

Q2:  The fonts, A or (A),  and pixel in all Figures should comply with the requirements of the journal horticulturae.

R2:We appreciate the reviewer’s comment. All figures have been carefully revised to comply with Horticulturae formatting guidelines. Specifically, font type, font size (including “A” or “(A)” in panels), resolution (minimum 300 dpi), and overall figure layout have been standardized and updated. All figures now meet the journal’s graphical quality and style requirements.

Q3: The abstract is too long and needs to be concise.

R3: We thank the reviewer for this important recommendation. The abstract has been thoroughly revised and significantly condensed to improve clarity and conciseness while retaining the essential scientific content. The new abstract complies with Horticulturae word-length guidelines and provides a more focused summary of the study.

 

Reviewer 4 Report

Comments and Suggestions for Authors

The authors have addressed all my comments, and the manuscript has been improved for acceptance.

Author Response

We sincerely thank the reviewer for the positive assessment and for acknowledging the improvements made during the revision process. We appreciate the constructive feedback provided, which contributed to strengthening the quality and clarity of the manuscript.