Impact of Drying and Storage Conditions on the Bioactive and Nutritional Properties of Malolactic Wine Lees

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments are attached.

Comments for author File: Comments.pdf

Author Response

We thank the reviewer for the time dedicated to evaluating this work. The suggestions and observations made were of fundamental importance in improving our work. Below are our responses to the requests made.

 

1. Abstract

The abstract does not specify essential methodological parameters such as the number of replicates, assay types used for phenolic and antioxidant measurements, or storage conditions (temperature, duration, packaging). Without these, the reported values (e.g., 77.92 mg GAE/g) lack context and reproducibility. Please briefly indicate the main analytical methods and experimental setup to make the study design understandable within the abstract. A careful language edit will improve readability and professionalism.

 

Response: Thank you for the suggestion. The topic has been reviewed.

 

Samples were analyzed at 0, 45, and 90 days of storage at 25°C under light exposure conditions. Total phenolic content was determined by Folin-Ciocalteu, antioxidant activity by DPPH and FRAP assays, and phenolic groups by HPLC-DAD-FD.

 

2. Line 29-31

The claim that freeze-dried lees had higher total phenolic content while oven-dried samples showed greater antioxidant activity appears contradictory and requires clarification. The abstract should either (a) provide a brief mechanistic explanation (e.g., possible Maillard reaction products enhancing antioxidant assays) or (b) avoid drawing conflicting comparative statements that are not explained within the limited abstract space.

 

Response: The topic has been reviewed.

 

The freeze-dried lees showed a higher phenolic content (77.92 mg GAE/g), whereas the oven-dried exhibited greater antioxidant activity, likely due to the formation or release of bound phenolic compounds induced by thermal processing. Among the phenolic groups, tannins were the most favored in terms of preservation.

 

3. Line 33-35

The final statement is too broad, given that safety, sensory, and regulatory aspects were not assessed. The conclusion should be moderated to reflect the study’s actual findings, namely, the compositional and stability data, rather than extending to product application claims not directly supported by the presented results.

 

Response: The topic has been reviewed.

Storage time significantly influenced the parameters evaluated, indicating the impact of drying on stability. While freeze-drying better preserved phenolic compounds, oven drying was considered the most suitable option for large-scale application.

 

4. Introduction Line 39-55

The introduction does not clearly transition from the broad context (winemaking by-products) to the specific problem this study addresses. The text should more explicitly define the type of lees (malolactic vs. alcoholic fermentation lees) at the start and justify why malolactic lees in particular deserve attention, since this is the stated focus later in the abstract.

 

Response: We thank the reviewer for the insightful comment. This study does not aim to compare alcoholic and malolactic lees, as the differences between these fermentation processes are well established in the scientific literature. Therefore, the introduction was focused on the drying processes and on exploring malolactic lees specifically, given the limited number of studies available on this particular by-product.

 

5. Line 50-55

Condense this part to one or two lines and explicitly connect it to the study’s aim, e.g., “Valorizing WL through drying aligns with circular economy strategies by converting winery waste into functional food ingredients.”

 

Response: Thank you for the suggestion. The change has been made accordingly.

 

Despite their valuable characteristics, WL are still mainly used for low-value purposes such as animal feed and fertilizers. Valorizing WL through drying aligns with circular economy strategies by converting winery waste into functional food ingredients with nutritional and technological potential [7–9].

 

6. Line 65-76

This section does outline the key gap, limited information on WL powder stability, and clearly presents the study aim. However, one concise statement emphasizing why this is scientifically or practically important (e.g., relevance to food product development or ingredient shelf-life) would strengthen the rationale and highlight novelty.

 

Response: Thank you for the suggestion.

 

One reasons for this gap is related to the variability in the composition of WL, which depends on factors such as grape variety, winemaking process and malolactic fermen-tation conditions, making it difficult to establish standardized storage protocols [2]. Ad-ditionally, the complexity of the WL composition poses another challenge for this type of research, since the high concentration of phenolic compounds, proteins, polysaccharides and yeast cells makes it highly susceptible to oxidation reactions, enzymatic browning and microbiological changes during storage [1]. Therefore, generating reliable shelf-life data that support the development of functional ingredients and nutraceuticals derived from WL for the food and pharmaceutical industries is essential for advancing circular economy strategies in the wine sector.

 

7. Methodology

Subsection 2.3

Line 104-109

“Preparation of ethanolic extract” is a more scientifically appropriate subheading. The solvent ratio “1:5 (m/v)” and “ethanol (80%)” lack clarification of units (w/v, v/v), and “the sample was dried” does not clearly indicate whether it refers to the filtrate or residue; these should be specified to ensure methodological clarity.

 

Response: Thank you, the correction has been addressed.

 

The oven-dried and freeze-dried wine lees were extracted in a 1:5 (w/v) ratio using ethanol (80% v/v) and stirred for 2 hours at room temperature [21]. The solids were re-moved by filtering through a paper filter, and the liquid filtrate was subsequently concentrated in a rotary evaporator at 55 °C for 1 hour.

 

8. 4.2

Line 125

There is an inconsistency between the text and the citation year: AOAC (2025) is mentioned in the manuscript, whereas AOAC 2005 is listed in the references. Please verify and correct the year to ensure consistency

between in-text citation and reference list.

 

Response: The revision has been applied as requested.

 

9. 4.3

Line 127-169

The temperature of hydrolysis (110°C) is fine, but the duration (22 h) should ideally be mentioned as 24 h to match the standard AOAC 994.12 method unless otherwise optimized. Furthermore, the use of “FAO/WHO (2013)” should cite the full report properly in the references (e.g., FAO/WHO/UNU Expert Consultation on Protein and Amino Acid Requirements in Human Nutrition).

 

Response: Revised as suggested.

 

10. 4.6

Line 188-196

Units and basis for DPPH (EC₅₀) and FRAP assays are unclear. EC₅₀ should be expressed as concentration (e.g., μg/mL), not “μg per g

 

Response: We appreciate your comment and have made the correction.

 

11. Line 198-220

Correct typo ‘Phenomenex’’, state exact mobile phase composition (v/v and pH), add re-equilibration step/time, explicitly state use of authentic standards for ID and the calibration/quantification procedure (standards, ranges, LOD/LOQ), and report final units (mg compound/g sample or mg/g extract).

 

Response: The correction has been duly applied.

 

The compounds were identified by comparison of retention times and UV-Vis spectra with authentic standards and confirmed by co-injection (spiking) with the corresponding standards. Quantification was performed using external calibration curves prepared with authentic standards at concentration ranges of 0.5–100 μg/mL (r² > 0.995 for all compounds). Limits of detection (LOD) and quantification (LOQ) ranged from 0.01–0.50 μg/mL and 0.05–1.50 μg/mL, respectively. The DAD detector was used to monitor: at 280 nm, gallic acid, epicatechin (-)-gallate, epigallocatechin (-)-gallate, and cis-resveratrol; at 320 nm, trans-resveratrol, caffeic acid, syringic acid, ferulic acid, chlorogenic acid, caftaric acid, piceatannol and viniferine; at 360 nm, kaempferol-3-O-glucoside, myricetin, rutin, isoquercetin and isorhamnetin-3-O-glucoside; at 520 nm, all anthocyanins (cya-nidin-3-glucoside, cyanidin-3,5-diglucoside, malvidin-3,5-di-O-glucoside, mal-vidin-3-glucoside, delphinidin-3-glucoside, peonidin-3-O-glucoside). FLD was used with excitation at 280 nm and emission at 320 nm for (+)-catechin, procyanidin B1, procyanidin B2, procyanidin A2 and (-)-epicatechin. The results were expressed as mg of compound per g of dried extract (mg/g) [31].

 

12. Line 243-244

“Lower moisture content is responsible for concentrating more nutrients.” Replace “responsible for” with “likely contributed to higher dry-basis nutrient concentrations.”

 

Response: It has been addressed.

 

The lower moisture content of the freeze-dried sample likely contributed to higher dry-basis nutrient concentrations compared to oven drying [32].

 

13. Line 258

The reported significance level “p > 0.5” appears to be a typographical error and should be corrected to “p > 0.05” to reflect the standard threshold for statistical non-significance.

 

Response: Thank you for pointing that out. The correction has been made.

 

14. Line 270-274

Attributing lipid differences solely to microbial transfer is speculative; add “possibly due to both grape skin contribution and microbial lipids.”

 

Response: The correction has been made.

 

The lipid content present in the WL was statistically different between the oven-dried and freeze-dried samples (1.20 ± 0.07 and 2.02 ± 0.08 g/100 g, p < 0.00, respectively). This difference can be attributed to the distinct mechanisms of each drying process. During oven drying at 40 °C, prolonged exposure to elevated temperatures may promote lipid oxidation and the volatilization of certain lipid fractions, resulting in lower lipid retention. In contrast, freeze-drying operates under vacuum and low temperatures, which mini-mizes lipid degradation [19]. Additionally, this difference is possibly due to both grape skin contribution and microbial lipids, as lipids are essential components of microbial cell membranes [39].

 

15. 1 and 3.2

Mean ± SD values and corresponding p-values should be reported within the text, not solely in Table 1 and Table 2, to facilitate interpretation of key statistical differences.

 

Response: The correction has been made.

 

16. Line 298

Authors are advised to include complete reference details for the cited comparison “values (72.6 and 68.7 mg GAE g⁻¹) for Pinot Noir and Riesling” to ensure proper source verification and traceability.

 

Response: The manuscript has been updated accordingly.

 

The importance of other factors for the results of this parameter, such as the stage of collection of the lees studied, is reinforced in the literature by other authors who evaluated the total phenolic content in Pinot Noir and Risieling wine lees using different wine-making techniques [42].

 

17. Line 286-309

The authors are advised to refine the language to maintain a clear distinction between data reporting and interpretation. Quantitative results (mean ± SD, p-values) should be stated more precisely, followed by a concise interpretation. This will enhance the section's scientific tone and analytical clarity.

 

Response: The correction has been made.

 

18. Table 2

“Total phenolic content (mg GAE/g) of wine lees powder stored under different conditions for 90 days” will be a better title. The title (caption) of each table should be placed above the table, following standard formatting conventions for scientific manuscripts. The unit “mg GAE/g” should be clearly indicated in the column header, not only in the footnote. The table note states “Asterisks (*) indicate statistical differences (p≤0.5)”. This appears to be a typographical error, it should be p ≤ 0.05.

 

Response: We appreciate. The table has been corrected.

 

19. Line 317-324

Unnecessary spacing should be removed to ensure consistency and proper formatting throughout the manuscript.

 

Response: The manuscript has been updated accordingly.

 

20. Line 332-418

The Results and Discussion section is overly extended and should be refined for conciseness and clarity. While combining these sections is acceptable, the current version contains repeated explanations and overlapping interpretations, particularly regarding the relationship between phenolic content and antioxidant activity. Descriptions of the DPPH and FRAP assays are also unnecessarily detailed and partially reiterate information already presented in the Materials and Methods. Furthermore, some interpretations merely restate data trends visible in Tables 3 and 4, contributing to redundancy. The section would benefit from more focused discussion immediately following each table, avoiding repetition and maintaining a clear, analytical tone throughout. Overall, the authors are advised to revise this part for brevity, coherence, and stronger alignment with a scientific reporting style.

 

Response: We appreciate the reviewers’ comments and have revised the manuscript accordingly.

 

Thermal drying methods can release bound phenolic compounds from the cell wall matrix, increasing antioxidant capacity [10,52]. Previous studies reported increased an-tioxidant activity despite reduced phenolic content, attributed to enhanced activity of partially oxidized phenolic compounds [53–55].

Although thermal drying methods are generally considered to be unfavorable for maintaining phenolic compounds, they can release bound phenolic compounds from the cell wall matrix, increasing antioxidant capacity [10,52]. Previous studies reported in-creased antioxidant activity despite reduced phenolic content, attributed to enhanced activity of partially oxidized phenolic compounds [53–55].

When evaluating the results related to storage time, a behavior similar to that previously described for the samples was observed for the DPPH method: despite re-duction in phenolic content, antioxidant activity increased. This phenomenon aligns with mechanisms including formation and release of antioxidant compounds during thermal processing, structural modifications in phenolic compounds, greater availability for free radical reactions, and compound complexation processes [56–58]. FRAP results showed contrasting reaction: freeze-dried samples exhibited progressive antioxidant decline, while oven-dried samples showed initial decrease followed by recovery, consistent with previous findings [21]. Methodological differences explain these divergences: DPPH measures radical scavenging capacity while FRAP assesses reducing potential via Fe³⁺ to Fe²⁺ conversion [59]. FRAP demonstrates superior reproducibility, and DPPH's purple coloration may cause spectral interference in pigmented matrices like WL [60,61].

WL applications in foods such as hamburgers and ice cream demonstrate enhanced antioxidant capacity, protecting against lipid and protein oxidation while replacing ad-ditives like sodium ascorbate [20,62].

 

21. Line 420-523

The “Identification of phenolic compounds” section is excessively long and should be substantially condensed for clarity and scientific rigor. The current version includes repetitive descriptions, detailed listings of compounds already presented in tables, and repeated explanations of freeze-drying and phenolic stability mechanisms. The narrative often restates data trends rather than interpreting their scientific relevance. The authors are advised to streamline this section by focusing on key comparative outcomes, removing redundant or descriptive content, and presenting supplementary details (e.g., compound lists and extended mechanistic explanations) in supporting materials. A more concise, analytical discussion will strengthen readability and align the section with standard scientific reporting practices.

 

Response: All suggestions from the reviewers have been carefully considered and incorporated.

 

22. Line 503-510

“Light had no statistically significant effect.” Add caution: specify storage intensity (806 lumens) and explain if 90 days may be insufficient for photodegradation.

 

Response: Thank you for the suggestion. The change has been made accordingly.

 

Light exposure (white LED lamp, 9 W, 806 lumens) showed no significant effect on the phenolic groups analyzed, suggesting good stability of these compounds under the tested conditions. However, the 90-day storage period may not capture long-term photodegradation. Practically, while drying strongly influences compound preservation, specific light protection may not be critical for maintaining phenolic integrity within the evaluated timeframe.

 

23. Line 544-545

The authors state that the result “contradicts previous authors” but provide no justification for this discrepancy. The discussion should be expanded to explain possible reasons, such as differences in pigment concentration, sample opacity, or other matrix effects influencing the assay outcome.

 

Response: We sincerely appreciate the reviewers’ insightful feedback.

 

However, the b* result for the sample that underwent the freeze-drying technique in this study (2.37) was statistically the same as that obtained by the oven-dried WL (2.12), which appears to contradict previous findings. This discrepancy may be attributed to the specific chromatic composition of malolactic wine lees, which are characterized by a high concentration of red-purple anthocyanins and tannins that dominate the color profile in both the L* and a* dimensions, thereby masking potential differences in the b* parameter [81]. Additionally, the relatively low absolute b* values observed for both samples (< 2.5) suggest that yellow pigmentation is minimal in WL, unlike in matrices such as carote-noid-rich fruits and vegetables where heat-induced isomerization and degradation sig-nificantly impact b* values [49].

 

24. Line 548-549

Authors are advised to include an interpretive statement such as “A ΔE > 3 indicates a color difference perceptible to the human eye” to clarify the practical significance of the reported ΔE value (6.08).

 

Response: Thank you for the observation. The requested information is already included in the text, where a ΔE greater than 1 is described as indicating a color difference perceptible to the human eye.

 

25. Line 562

Caption “Source: Author (2025)” should be removed. Journal captions shouldn’t reference authorship.

 

Response: Updated accordingly.

 

26. Line 582

Authors are advised to place the figure title above the figure, before its description or legend, in accordance with standard formatting conventions.

 

Response: Updated accordingly.

 

27. Line 583

Authors are advised to introduce a distinct subheading (e.g., “Principal Component Analysis”) for clarity. This will improve logical flow and section organization.

 

Response: The manuscript has been updated accordingly.

 

28. Table 7 and table 8

Authors are advised to replace commas with decimal points in numerical values (e.g., 188,83 to 188.83) to maintain consistency with standard

scientific notation.

 

Response: The manuscript has been updated accordingly.

 

29. Line 662 vs 668

AAS description repeated twice (Lines 662–669 and 668–671). Remove duplication.

 

Response: The change has been made in the abstract. Thank you.

 

30. Line 609-663

The section inconsistently states that only the freeze-dried sample was analyzed, yet data for both drying methods appear, which must be clarified. The text is overly descriptive and repetitive, particularly regarding amino acid functions and FAO/WHO criteria. Revise for conciseness and focus on the interpretation of findings.

 

Response: The section has been revised.

 

The amino acid profile was determined only for the freeze-dried wine lees powder, as both drying methods showed no significant difference in protein content (p > 0.05). Therefore, this analysis was performed to establish the reference nutritional composition of the powder at time zero, before any storage-related transformations occurred.

 

31. Line 655

The reported EAAI value (1.55) appears inconsistent with typical scales. Please confirm the calculation method and units: EAAI is normally computed as the geometric mean of (aa_test / aa_reference) across essential amino acids, yielding a ratio (commonly presented between 0 and 1) or expressed as a percentage (×100). If you intend a ratio, re-evaluate the calculation; if you intend a percentage, report it clearly (e.g., EAAI = 155%). Also confirm that both test and reference amino acid values were normalized per g of protein and that the correct reference pattern was used.

 

Response: We thank the reviewer for this observation. The EAAI was calculated as the geometric mean of the ratios between the essential amino acid content in the sample and the FAO/WHO (2013) reference pattern, with all values normalized per gram of protein. The reported value (1.55) corresponds to 155% of the reference. This result indicates that the wine lees protein has a higher relative proportion of essential amino acids than the reference, confirming its high nutritional quality. It is worth noting that similar results have been reported in the literature, where EAAI values above 1 were also observed, for example, in Foods 2023, 12, 24090, https://doi.org/10.3390/foods12224090, further supporting the plausibility of the result obtained in this study.

 

32. Line 685-687

Sentence repeats content from the Abstract almost verbatim; authors are advised to condense or rephrase to avoid redundancy.

 

Response: The change has been made in the abstract. Thank you.

 

33. Line 694

Delete “section is not mandatory…” this is a leftover from the journal template.

 

Response: Thank you for the warning.

 

34. Conclusion

Authors are advised to refine it by focusing on the main outcomes and their broader implications, avoiding repetition of detailed findings. The comparative statement on drying methods should be expressed more objectively, and future perspectives should be summarized to enhance clarity and scientific conciseness.

 

Response: We thank the reviewer for the helpful suggestion. The conclusion has been refined for clarity and conciseness.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The topic “Impact of Drying and Storage Conditions on the Bioactive and Nutritional Properties of Malolactic Wine Lees” is interesting. The authors conclude that freeze drying method preserves phenolic compounds while oven drying method enhances the stability of antioxidant activity, making oven drying at 40°C is suitable for large scale production in terms of cost efficiency. These findings are essential for future applications of Malolactic Wine Lees  in food and pharma industries . However, I want to suggest the following  areas to be improved.

Abstract

1. Lines 22-23, please revise the abstract introduction to mention the concentration of phenolic compounds and antioxidant activity (high or low?). (eg. revise … high phenolic and ….)
2. Please conclude the abstract by indicating which method is more suitable for future scalable applications in the food and pharmaceutical.

Introduction

3. Please clarify why oven drying and freeze-drying methods were selected among different drying methods. Please briefly describe the underlying mechanisms of these selected processes.
4. Lines 68-71, the authors combine two ideas (i) there are studies on the utilization of wine lees powder in food products, (ii) studies focusing on its shelf life are rare. I recommend revising this sentence for clarity and including examples of specific products in which the powder has been utilized. Also, regarding the phrase “few records” in Line 70, please discuss the main challenges associated with this type of study and identify the existing gaps. Additionally, what is the significance of your current research, and what are the expected novel findings?

Materials and Methods

5. There are several minor errors related to spelling (eg. Line 86, Folin-Ciocalteu), and to superscript and subscript formatting (see Line 83, 20°C, Line 98, 40°C, Line 108, 55°C, Line 149, Na₂HPO₄, etc…….). Please recheck the entire manuscript.
6. Please mention the model number, manufacturer, and country of origin for every equipment used in this experiment. Also, note that this information should appear after the equipment name, not after temperature or operating conditions. (eg. HPLC (xx, xx, xx) and muffle furnace (xx, xx, xx).
7. For the preparation of wine lees powder, how did the authors determine the operating parameters? (eg. Based on preliminary testing or reference from other sources?). Please provide additional details. Did the authors analyze and compare particle size after drying? Additionally, what was the yield percentage of particles after drying and grinding for each method?
8. For stability testing, why did the authors choose the time frame of 90 days? Please provide supporting references or additional explanation.
9. Line 188-196, The description of the antioxidant activity method is not sufficiently detailed. I suggest adding more information, including relevant equations.
10. What extraction solvent was used for Soxhlet extraction? I suggest adding the specific solvent in the methods.

Results and Discussion

11. Lines 270-275, for the discussion of different lipid content after different drying, the discussion and interpretation seem to be mismatched. The authors should focus on explaining why the lipid content differs between the two drying methods, linking this difference to the underlying mechanisms of each drying process.
12. Lines 290-291, please extend the discussion to explain the reasons and underlying mechanisms responsible for the reduction of phenolics compounds during the oven-drying process.
13. I suggest adding a deeper mechanistic discussion to explain the higher phenolic content observed in the oven-drying method under light exposure compared with without light at 45 days. Please link this explanation directly to the results shown in Table 2.
14. Lines 541-546, please explain the possible reason why the current results contradict previous findings for b* value.

Conclusion

15. I suggest adding a comparison of cost savings, indicating how many times or what percentage the oven drying method is cheaper than freeze drying.
16. Lines 694-695, Please check and remove the last sentence of the conclusion.

Figures

17. Please check the quality of Figs 1 and 3, as the current version needs tick marks on both the X and Y axis, as well as proper axis labels.

Tables

18. Table 2 – Please include the unit of phenolic content (mg GAE/g) in the column. Also, column headings are unclear. (eg. column 1 title should be “drying methods and storage conditions” while column 2 title should be “Total phenolic content (mg GAE/g) at different storage time”. Please check and correct if necessary for Tables 3,4,5 too.
19. Tables 7 and 8, I suggest changing the commas to decimal points.

Author Response

We thank the reviewer for the time dedicated to evaluating this work. The suggestions and observations made were of fundamental importance in improving our work. Below are our responses to the requests made.

The topic “Impact of Drying and Storage Conditions on the Bioactive and Nutritional Properties of Malolactic Wine Lees” is interesting. The authors conclude that freeze drying method preserves phenolic compounds while oven drying method enhances the stability of antioxidant activity, making oven drying at 40°C is suitable for large scale production in terms of cost efficiency. These findings are essential for future applications of Malolactic Wine Lees in food and pharma industries. However, I want to suggest the following areas to be improved.

Abstract

1. Lines 22-23, please revise the abstract introduction to mention the concentration of phenolic compounds and antioxidant activity (high or low?). (eg. revise … high phenolic and ….)

Response: We appreciate the comment and rectified the section.

Wine lees, a winemaking by-product, have high potential for reuse due to their significant phenolic content and antioxidant capacity.

 

1. Please conclude the abstract by indicating which method is more suitable for future scalable applications in the food and pharmaceutical.

Response: The section has been revised.

Storage time significantly influenced the parameters evaluated, indicating the impact of drying on stability. While freeze-drying better preserved phenolic compounds, oven drying was considered the most suitable option for large-scale application.

 

Introduction

1. Please clarify why oven drying and freeze-drying methods were selected among different drying methods. Please briefly describe the underlying mechanisms of these selected processes.

Response: The request has been reviewed and completed

Among the various conventional drying methods, oven drying is the most common due to its simplicity, low cost, ease of implementation, and recommendations for large-scale production. It is a process based on convective heat transfer: heated air promotes the evaporation of moisture from the material. However, this technique often causes thermal degradation of bioactive compounds [11,12]. As an alternative, the freeze-drying, technique that operates at low temperatures and acts through the sublimation of water present in the material, has been widely studied, as it offers superior protection to thermosensitive compounds, preserving nutritional properties, despite its higher energy cost and processing time [13,14].

1. Lines 68-71, the authors combine two ideas (i) there are studies on the utilization of wine lees powder in food products, (ii) studies focusing on its shelf life are rare. I recommend revising this sentence for clarity and including examples of specific products in which the powder has been utilized. Also, regarding the phrase “few records” in Line 70, please discuss the main challenges associated with this type of study and identify the existing gaps. Additionally, what is the significance of your current research, and what are the expected novel findings?

Response: Thank you for the suggestion. We have made the necessary changes.

Although several studies indicate the benefits of applying powders from wine by-products to food ingredient in terms of preservation and extending shelf-life, few studies have addressed the stability of WL powder itself during storage [19,20]. One reasons for this gap is related to the variability in the composition of WL, which depends on factors such as grape variety, winemaking process and malolactic fermentation conditions, making it difficult to establish standardized storage protocols [2]. Additionally, the complexity of the WL composition poses another challenge for this type of research, since the high concentration of phenolic compounds, proteins, polysaccharides and yeast cells makes it highly susceptible to oxidation reactions, enzymatic browning and microbiological changes during storage [1]. Therefore, generating reliable shelf-life data that support the development of functional ingredients and nutraceuticals derived from WL for the food and pharmaceutical industries is essential for advancing circular economy strategies in the wine sector.

Materials and Methods

1. There are several minor errors related to spelling (eg. Line 86, Folin-Ciocalteu), and to superscript and subscript formatting (see Line 83, 20°C, Line 98, 40°C, Line 108, 55°C, Line 149, Na₂HPO₄, etc…….). Please recheck the entire manuscript.

Response: The section has been revised.

1. Please mention the model number, manufacturer, and country of origin for every equipment used in this experiment. Also, note that this information should appear after the equipment name, not after temperature or operating conditions. (eg. HPLC (xx, xx, xx) and muffle furnace (xx, xx, xx).

Response: The section has been revised.

1. For the preparation of wine lees powder, how did the authors determine the operating parameters? (eg. Based on preliminary testing or reference from other sources?). Please provide additional details. Did the authors analyze and compare particle size after drying? Additionally, what was the yield percentage of particles after drying and grinding for each method?

Response: The section has been revised.

1. For stability testing, why did the authors choose the time frame of 90 days? Please provide supporting references or additional explanation.

Response: The section has been revised.

1. Line 188-196, The description of the antioxidant activity method is not sufficiently detailed. I suggest adding more information, including relevant equations.

Response: We appreciate the reviewer’s valuable suggestion. The methodologies used to evaluate antioxidant activity in this study are well-established and widely applied in the scientific literature. Therefore, to avoid excessive detail and maintain a concise description, we followed the standard format adopted by previous publications (doi: 10.1002/fsn3.98; doi: 10.3390/molecules25163701), while providing the corresponding references that fully describe the procedures and equations used.

1. What extraction solvent was used for Soxhlet extraction? I suggest adding the specific solvent in the methods.

Response: The section has been revised.

Results and Discussion

1. Lines 270-275, for the discussion of different lipid content after different drying, the discussion and interpretation seem to be mismatched. The authors should focus on explaining why the lipid content differs between the two drying methods, linking this difference to the underlying mechanisms of each drying process.

Response: We appreciate the reviewer's contribution and have included additional information in the discussion.

The lipid content present in the WL was statistically different between the oven-dried and freeze-dried samples (1.20 ± 0.07 and 2.02 ± 0.08 g/100 g, p < 0.00, respectively). This difference can be attributed to the distinct mechanisms of each drying process. During oven drying at 40 °C, prolonged exposure to elevated temperatures may promote lipid oxidation and the volatilization of certain lipid fractions, resulting in lower lipid retention. In contrast, freeze-drying operates under vacuum and low temperatures, which minimizes lipid degradation [19]. Additionally, this difference is possibly due to both grape skin contribution and microbial lipids, as lipids are essential components of microbial cell membranes [39].

 

1. Lines 290-291, please extend the discussion to explain the reasons and underlying mechanisms responsible for the reduction of phenolics compounds during the oven-drying process.

Response: We appreciate this comment. The discussion has been revised accordingly.

In contrast, under the oven dring, the application of heat promotes thermal degradation of thermolabile phenolic compounds, particularly anthocyanins and flavonols, through deglycosylation and oxidation reactions [14,41]. Also, prolonged exposure to oxygen during convective drying facilitates non-enzymatic oxidation of phenolics, further re-ducing their extractability [43].

1. I suggest adding a deeper mechanistic discussion to explain the higher phenolic content observed in the oven-drying method under light exposure compared with without light at 45 days. Please link this explanation directly to the results shown in Table 2.

Response: The discussion has been enhanced as requested.

At 45 days, oven-dried samples exposed to light (58.20 ± 0.00 mg GAE/g, p < 0.04) showed slightly higher total phenolic content than samples without light (57.30 mg GAE/g). This can be explained by light catalyzing the release of bound phe-nolics from thermally-weakened cell walls and the formation of intermediate oxi-dation products detected by the Folin-Ciocalteu method, temporarily increasing apparent phenolic content [52,53,54]. The subsequent decline at 90 days (52.10 ± 0.01 vs 55.80 ± 0.08 mg GAE/g, p < 0.05) confirms that photodegradation ultimately predominates [43].

1. In contrast, under the oven dring, the application of heat promotes thermal degradation of thermolabile phenolic compounds, particularly anthocyanins and flavonols, through deglycosylation and oxidation reactions [14,41]. Also, prolonged exposure to oxygen during convective drying facilitates non-enzymatic oxidation of phenolics, further re-ducing their extractability [43].
2. Lines 541-546, please explain the possible reason why the current results contradict previous findings for b* value.

Response: The discussion has been enhanced as requested.

However, the b* result for the sample that underwent the freeze-drying technique in this study (2.37) was statistically the same as that obtained by the oven-dried WL (2.12), which appears to contradict previous findings. This discrepancy may be attributed to the specific chromatic composition of malolactic wine lees, which are characterized by a high concentration of red-purple anthocyanins and tannins that dominate the color profile in both the L* and a* dimensions, thereby masking potential differences in the b* parameter [81]. Additionally, the relatively low absolute b* values observed for both samples (< 2.5) suggest that yellow pigmentation is minimal in WL, unlike in matrices such as carotenoid-rich fruits and vegetables where heat-induced isomerization and degradation significantly impact b* values [49].

Conclusion

1. I suggest adding a comparison of cost savings, indicating how many times or what percentage the oven drying method is cheaper than freeze drying.

Response: We appreciate the revision. All requested modifications have been implemented.

From an economic standpoint, oven drying proved to be substantially more cost-effective, with operational expenses approximately 3-5 times lower than freeze-drying, primarily due to reduced energy consumption and shorter processing requirements.

1. Lines 694-695, Please check and remove the last sentence of the conclusion.

Response: Thank you. The text has been removed as suggested.

Figures

1. Please check the quality of Figs 1 and 3, as the current version needs tick marks on both the X and Y axis, as well as proper axis labels.

Response: We sincerely thank the reviewer for this thoughtful observation. The figures were prepared in high resolution and adjusted to meet the journal’s template size requirements, which may have slightly reduced their apparent clarity in the review version. Both figures already include all necessary information in the captions and footnotes and were designed using grouped bar formats where traditional X and Y tick marks were intentionally omitted to maintain visual clarity and avoid redundancy. All details are clearly visible when zoomed in. We truly appreciate the reviewer’s attention to detail and the opportunity to clarify this point.

Tables

1. Table 2 – Please include the unit of phenolic content (mg GAE/g) in the column. Also, column headings are unclear. (eg. column 1 title should be “drying methods and storage conditions” while column 2 title should be “Total phenolic content (mg GAE/g) at different storage time”. Please check and correct if necessary for Tables 3,4,5 too.
2. Response: The section has been revised.
3. Tables 7 and 8, I suggest changing the commas to decimal points.
4. Response: The section has been revised.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

1. The abstract lacks key data, such as the content of phenolic substances, and the introduction lacks a conclusion.
2. The research background in the introduction is insufficient. It fails to mention the compositional differences between wine lees from the lactic acid fermentation stage and those from other fermentation stages, nor does it explain the specific technical problems existing in the drying and storage research of this type of lees.
3. Sections 3.3 and 3.4 in the results and discussion lack logical coherence.
4. Many studies have shown that color is related to the content of phenolic substances such as anthocyanins in grapes. However, section 3.5 states that light has no significant effect on color, while section 3.2 mentions that total phenols in the light-exposed group degrade faster, which presents a logical contradiction.
5. In the experiment, the wine lees powder was stored at 25℃. If the low-temperature storage scenarios in the actual food industry are considered, would the quality change trends of the lees powder under the two drying methods be altered?
6. The total phenol content in the 40℃ drying group is lower than that in the freeze-drying group, but the antioxidant activity is higher. Whether non-phenolic antioxidant substances are generated during the drying process needs to be discussed.

Author Response

We thank the reviewer for the time dedicated to evaluating this work. The suggestions and observations made were of fundamental importance in improving our work. Below are our responses to the requests made.

 

1. The abstract lacks key data, such as the content of phenolic substances, and the introduction lacks a conclusion.

Response: Thank you for your valuable feedback. Some additional information has been included as suggested. However, the abstract is limited to 200 words, which restricts the level of detail that can be added.

2. 
   The research background in the introduction is insufficient. It fails to mention the compositional differences between wine lees from the lactic acid fermentation stage and those from other fermentation stages, nor does it explain the specific technical problems existing in the drying and storage research of this type of lees.

Response: Thank you for your constructive comments. Revisions have been made to the introduction, as highlighted in green, to strengthen the research background and clarify the technical challenges related to the drying and storage of wine lees. However, the differences between lees from alcoholic and malolactic fermentation stages were not discussed in detail, since the winemaking process is already well established and extensively covered in the literature, particularly regarding alcoholic lees. Therefore, we chose to maintain a concise and focused approach, emphasizing the characteristics and relevance of malolactic lees.

Among the various conventional drying methods, oven drying is the most common due to its simplicity, low cost, ease of implementation, and recommendations for large-scale production. It is a process based on convective heat transfer: heated air promotes the evaporation of moisture from the material. However, this technique often causes thermal degradation of bioactive compounds [11,12]. As an alternative, the freeze-drying, technique that operates at low temperatures and acts through the sublimation of water present in the material, has been widely studied, as it offers superior protection to thermosensitive compounds, preserving nutritional properties, despite its higher energy cost and processing time [13,14].

Although several studies indicate the benefits of applying powders from wine by-products to food ingredient in terms of preservation and extending shelf-life, few studies have addressed the stability of WL powder itself during storage [19,20]. One reasons for this gap is related to the variability in the composition of WL, which depends on factors such as grape variety, winemaking process and malolactic fermentation conditions, making it difficult to establish standardized storage protocols [2]. Additionally, the complexity of the WL composition poses another challenge for this type of research, since the high concentration of phenolic compounds, proteins, polysaccharides and yeast cells makes it highly susceptible to oxidation reactions, enzymatic browning and microbiological changes during storage [1]. Therefore, generating reliable shelf-life data that support the development of functional ingredients and nutraceuticals derived from WL for the food and pharmaceutical industries is essential for advancing circular economy strategies in the wine sector.

3. 
   Sections 3.3 and 3.4 in the results and discussion lack logical coherence.

Response: Thank you for your helpful feedback. Sections 3.3 and 3.4 have been carefully revised to improve their logical coherence and ensure clearer connections between the results and their interpretation.

4. 
   Many studies have shown that color is related to the content of phenolic substances such as anthocyanins in grapes. However, section 3.5 states that light has no significant effect on color, while section 3.2 mentions that total phenols in the light-exposed group degrade faster, which presents a logical contradiction.

Response: Thank you for your insightful comment. The sections have been revised (highlighted in green) to clarify the relationship between phenolic degradation and color stability. The text in Section 3.2 now explains that light exposure can initially increase the apparent phenolic content due to the release of bound compounds, followed by gradual photodegradation over time. In Section 3.5, it is emphasized that the drying method exerts a stronger influence on color parameters than light exposure, and that the complex anthocyanin–tannin composition of malolactic wine lees likely masks minor chromatic variations. These adjustments resolve the apparent contradiction and improve the logical coherence between the two sections.

At 45 days, oven-dried samples exposed to light (58.20 ± 0.00 mg GAE/g, p < 0.04) showed slightly higher total phenolic content than samples without light (57.30 mg GAE/g). This can be explained by light catalyzing the release of bound phe-nolics from thermally-weakened cell walls and the formation of intermediate oxi-dation products detected by the Folin-Ciocalteu method, temporarily increasing apparent phenolic content [52,53,54]. The subsequent decline at 90 days (52.10 ± 0.01 vs 55.80 ± 0.08 mg GAE/g, p < 0.05) confirms that photodegradation ultimately predominates [43].

       Light exposure (white LED lamp, 9 W, 806 lumens) showed no significant effect on the phenolic groups analyzed, suggesting good stability of these compounds under the tested conditions. However, the 90-day storage period may not capture long-term pho-todegradation. Practically, while drying strongly influences compound preservation, specific light protection may not be critical for maintaining phenolic integrity within the evaluated timeframe.

5. 
   In the experiment, the wine lees powder was stored at 25℃. If the low-temperature storage scenarios in the actual food industry are considered, would the quality change trends of the lees powder under the two drying methods be altered?

Response: We appreciate your comment. The study was designed to simulate typical storage conditions for dry food ingredients, which are generally kept at room temperature (approximately 25 °C). It is acknowledged that lower storage temperatures could slow down oxidative and degradative reactions, potentially altering the rate of quality changes observed for both drying methods. However, evaluating refrigerated or frozen storage scenarios was beyond the scope of this work. This point has now been addressed in the text as a suggestion for future studies.

6. 
   The total phenol content in the 40℃ drying group is lower than that in the freeze-drying group, but the antioxidant activity is higher. Whether non-phenolic antioxidant substances are generated during the drying process needs to be discussed.

Response: Thank you for the review. The discussion in Section 3.3 has been revised to address this point.

This suggests that thermal processing may release bound phenolic compounds from the cell wall matrix or promote the formation of compounds with enhanced antioxidant properties [10,52]. Previous studies reported increased antioxidant activity despite reduced phenolic content, attributed to enhanced activity of partially oxidized phenolic compounds [53–55].

When evaluating the results related to storage time, a behavior similar to that previously described for the samples was observed for the DPPH method: despite re-duction in phenolic content, antioxidant activity increased. This phenomenon aligns with mechanisms including formation and release of antioxidant compounds during thermal processing, structural modifications in phenolic compounds, greater availability for free radical reactions, and compound complexation processes [56–58]. FRAP results showed contrasting reaction: freeze-dried samples exhibited progressive antioxidant decline, while oven-dried samples showed initial decrease followed by recovery, consistent with previous findings [21]. Methodological differences explain these divergences: DPPH measures radical scavenging capacity while FRAP assesses reducing potential via Fe³⁺ to Fe²⁺ conversion [59]. FRAP demonstrates superior reproducibility, and DPPH's purple coloration may cause spectral interference in pigmented matrices like WL [60,61].

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

No More Comments.

Author Response

There were no comments from reviewer 1.

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for revising the manuscript. However, I suggest that the authors improve some minor points mentioned below.

1. Please correct the figures according to the previous suggestions. ( Comment No 17 - Please check the quality of Figs 1 and 3, as the current version needs tick marks on both the X and Y axis, as well as proper axis labels).
2. The authors responded to comment No.10, but the extraction solvent still appears to be missing in the method section. Please recheck.
3. The authors have responded well to comment No.7. However, some parts regarding the yield(%) of powders after drying are still missing. If these values are available, I recommend adding them in the R&D section to compare which drying method resulted in lower material loss.
4. Line – 263. Please recheck the typing error.

Author Response

Reviewer 2

Dear reviewer,

 

Thank you for your effort in making the corrections. The suggested corrections are as follows.

 

1. Please correct the figures according to the previous suggestions. ( Comment No 17 - Please check the quality of Figs 1 and 3, as the current version needs tick marks on both the X and Y axis, as well as proper axis labels).

Response: Thank you for the suggestion, figures 1 and 3 adjusted.

1. The authors responded to comment No.10, but the extraction solvent still appears to be missing in the method section. Please recheck.

Response: Thank you for the suggestion, solvent of the method added to the text.

1. The authors have responded well to comment No.7. However, some parts regarding the yield(%) of powders after drying are still missing. If these values are available, I recommend adding them in the R&D section to compare which drying method resulted in lower material loss.

Response: Thanks for the suggestion, data added.

1. Line – 263. Please recheck the typing error.

Response: Thank you for the suggestion, correction made.

Author Response File: Author Response.pdf