Valorization of Wine Lees in the Production of Reduced-Lipid Nutritive Muffins

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The present manuscript prepared low lipid and high dietary fibre muffins by incorporating Wine Lees by replacing upto 50% syunflower oil. The study is well designed and has some interesting findings. The language is clear and easy to understand. My observations are as follows-

1. In title, authors may use low fat or functional muffins as the developed product is low in lipids and high in fibre in addition to other macronutrients and antioxidants due to WL incorporation.
2. Please use italics for scietific names
3. In introduction, the major focus on the WL and its nutritive quality, but why it was added in muffins was largely missing. So I would recoomend to add 1 para on muffins and highlighting the need for loweing lipid content in them and significnance of dietary fiber and macronutrients etc. It will strengthen the hypothesis and highlight the research gaps. 
4. In abstract: please indicate level of significance
5. Muffins were cooked at 180 degree, so the TVC and yeast count was due to post-processing contamination?
6. During Muffins preparation, the WL replaced upto 50% sunflower oil; please there is some preliminary studies done by the authors to check for higher levels of replacement.
7. At section 2.3; these values were also covered in 2.2.7 section;
8. L242: please indicate sample size
9. Results and discussion- well presented with suitable references

Author Response

We would like to sincerely thank the reviewer for the careful evaluation of our manuscript and for the constructive comments and valuable suggestions. We appreciate the positive assessment of the study's relevance within the framework of sustainable food production and the circular bioeconomy.

All comments have been carefully considered, and the manuscript has been revised accordingly. We believe that the revisions and additional clarifications have significantly improved the quality, clarity, and scientific value of the paper. Detailed responses to each comment are provided below.

Comments 1: In title, authors may use low fat or functional muffins as the developed product is low in lipids and high in fibre in addition to other macronutrients and antioxidants due to WL incorporation.

Response 1: We agree that the muffins present improved nutritional characteristics due to WL incorporation. However, we prefer to retain the current title, as “reduced-lipid nutritive muffins” accurately reflects the main focus of the study, namely the partial replacement of sunflower oil with wine lees. The terms “low-fat” and “functional” may imply broader claims that were not the primary objective of this work. Therefore, the title was maintained unchanged.

Comments 2: Please use italics for scientific names.

Response 2: The scientific names of microorganisms have been revised and formatted in italics throughout the manuscript, including Saccharomyces cerevisiae, S. cerevisiae, Salmonella spp., E. coli, and the scientific names mentioned in the reference list.

Comments 3: In introduction, the major focus on the WL and its nutritive quality, but why it was added in muffins was largely missing. So I would recommend to add 1 para on muffins and highlighting the need for lowering lipid content in them and significance of dietary fiber and macronutrients etc. It will strengthen the hypothesis and highlight the research gaps. 

Response 3: We agree that, although the Introduction already discusses the nutritional composition of wine lees and their potential application in different food products, the rationale for selecting muffins as the food matrix needed to be stated more clearly. Therefore, a short paragraph has been added to highlight the relevance of reducing lipid content in muffins, the importance of dietary fiber and macronutrients, and the research gap related to the use of wine lees as a partial fat replacer in muffin formulations.

Comments 4: In abstract: please indicate level of significance

 Response 4: The level of statistical significance has been indicated in the Abstract by adding “p < 0.05” to the relevant result.

Comments 5: Muffins were cooked at 180 degree, so the TVC and yeast count was due to post-processing contamination?

Response 5: We agree that, considering the baking conditions applied in this study, the detected TVC and yeast counts should not be directly attributed to microbial survival from wine lees after baking. Although survival of highly heat-resistant microbial forms cannot be completely excluded because the internal temperature of the muffins was not monitored.

 Comments 6: During Muffins preparation, the WL replaced up to 50% sunflower oil; please there is some preliminary studies done by the authors to check for higher levels of replacement.

Response 6: In the present study, WL replacement levels of 20%, 35%, and 50% were selected to represent low, intermediate, and high levels of sunflower oil substitution. A replacement level higher than 50% was not included in the experimental design because the 50% level already produced noticeable changes in muffin structure, color, texture, and sensory acceptability. This point has been clarified in the Materials and Methods section.

Comments 7: At section 2.3; these values were also covered in 2.2.7 section;

Response 7: The section numbering has been revised to avoid duplication. In addition, the text has been clarified to distinguish between the calculation of the nutritional value of muffin formulations and the experimental characterization of wine lees. The nutritional value of the muffins was calculated based on the composition of all ingredients, while the composition of wine lees was experimentally determined and used as input for these calculations.

Comments 8: L242: please indicate sample size

Response 8: The sample size has been indicated in the Statistical Analysis section. We clarified that analytical measurements were performed in triplicate (n = 3), while sensory evaluation was conducted using 15 trained assessors (n = 15).

 Comments 9: Results and discussion- well presented with suitable references

Response 9: Thank you for the positive evaluation of the Results and Discussion section. No changes were required.

Reviewer 2 Report

Comments and Suggestions for Authors

The study concerns the potential use of a winemaking by-product — yeast sediment remaining after wine fermentation — as a functional ingredient in muffin production. The authors evaluated sediment obtained from local Moldovan grape varieties, Viorica and Fetească Regală, as a partial substitute for sunflower oil. Overall, the topic is interesting and fits well within the concept of sustainable food production and circular bioeconomy. However, the manuscript requires further clarification and supplementation in several methodological and interpretative aspects.

Introduction

1. The research gap is not sufficiently emphasized in the Introduction. The authors should clearly indicate that limited information is available on the use of wine lees from local Moldovan grape varieties as partial fat replacers in muffins, particularly with respect to product quality, microbiological safety, and consumer or sensory acceptance.

Materials and Methods

1. Authors should provide a basic characterization of the wine lees before their incorporation into muffins.
2. The manuscript should clarify how the sensory panel was trained. Since the study is described as QDA, information on training sessions, calibration, reference samples, descriptor development, and panel performance validation should be provided.
3. The equations should be prepared using an equation editor, with careful attention to appropriate formatting, subscripts, and superscripts. In particular,
4. Equation 2 appears to be incorrectly written and should be revised.

Results and Discussion

1. The reported dry matter values should be verified, as 21–25% dry matter, corresponding to 75–79% moisture, seems unusually low for muffins.
2. Please correct the terminology in the phrase “higher yeast substitution.” The substitution concerns sunflower oil with wine lees, not yeast; therefore, “higher WL substitution” or “higher WL incorporation” would be more appropriate.
3. For the sensory evaluation results, the authors did not provide a statistical analysis of the data. This should be included to support the interpretation of differences among samples.
4. In Figure 1a, the Y-axis lacks a proper description and numerical values. This should be corrected to improve the readability and interpretation of the figure.
5. The authors should indicate statistically significant differences between means in Table 4.
6. Why the results presented in Tables 3 and 5 do not include standard deviations and statistical significance of differences between means?

Conclusions

1. The authors state too broadly that wine lees can be used as a fat replacer. It should be emphasized that wine lees may act as a partial fat replacer under the conditions tested in this study.
2. The authors should include the main limitations of the study, particularly those related to the characterization and pre-treatment of wine lees, microbiological safety at higher substitution levels, and the scope of sensory evaluation.

Author Response

We would like to sincerely thank the reviewer for the careful evaluation of our manuscript and for the constructive comments and valuable suggestions. We appreciate the positive assessment of the study's relevance within the framework of sustainable food production and the circular bioeconomy.

All comments have been carefully considered, and the manuscript has been revised accordingly. We believe that the revisions and additional clarifications have significantly improved the quality, clarity, and scientific value of the paper. Detailed responses to each comment are provided below.

Introduction:

Comments 1: The research gap is not sufficiently emphasized in the Introduction. The authors should clearly indicate that limited information is available on the use of wine lees from local Moldovan grape varieties as partial fat replacers in muffins, particularly with respect to product quality, microbiological safety, and consumer or sensory acceptance.

Response 1: We agree that the research gap should be more clearly emphasized in the Introduction. Therefore, the Introduction has been revised to indicate that limited information is available on the use of wine lees from local Moldovan grape varieties as partial fat replacers in muffin formulations, particularly with respect to product quality, microbiological safety, and sensory acceptance.

 Materials and Methods:

Comments 1: Authors should provide a basic characterization of the wine lees before their incorporation into muffins.

Response 1: The Materials and Methods section has been revised to clarify that the wine lees were characterized before their incorporation into muffin formulations. The basic physicochemical and nutritional characterization included pH, dry matter, ash, carbohydrates, lipids, proteins, β-glucans, and fractional lipid composition, as presented in Table 2.

Comments 2: The manuscript should clarify how the sensory panel was trained. Since the study is described as QDA, information on training sessions, calibration, reference samples, descriptor development, and panel performance validation should be provided.

 Response 2: The Sensory Analysis section has been revised to provide additional information on panel training and calibration. We clarified that, before the formal QDA evaluation, assessors participated in a training and calibration session, during which the sensory scale, descriptors, and reference muffin samples were discussed and harmonized. A statement on panel consistency during descriptor use was also added.

Comments 3: The equations should be prepared using an equation editor, with careful attention to appropriate formatting, subscripts, and superscripts. In particular.

Response 3: All equations have been revised using an equation editor, with attention to appropriate formatting, subscripts, and superscripts. 

Comments 4: Equation 2 appears to be incorrectly written and should be revised.

Response 4: In particular, Equation 2 was corrected and reformatted according to the standard CIELAB total color difference equation.

Results and Discussion:

Comments 1: The reported dry matter values should be verified, as 21–25% dry matter, corresponding to 75–79% moisture, seems unusually low for muffins.

 Response 1: The dry matter and moisture values were rechecked, and an error in the description of these parameters was identified. The values were incorrectly presented as dry matter values. The manuscript has been corrected to indicate that the moisture content of the muffins ranged from 21% to 25%, corresponding to dry matter values of 75% to 79%. The related text, figure legend, and discussion have been revised accordingly.

Comments 2: Please correct the terminology in the phrase “higher yeast substitution.” The substitution concerns sunflower oil with wine lees, not yeast; therefore, “higher WL substitution” or “higher WL incorporation” would be more appropriate.

Response 2: We agree that the phrase “higher yeast substitution” was inaccurate, as sunflower oil was replaced with wine lees rather than yeast. The terminology has been corrected to “higher WL incorporation” throughout the manuscript.

Comments 3: For the sensory evaluation results, the authors did not provide a statistical analysis of the data. This should be included to support the interpretation of differences among samples.

Response 3: The sensory data were statistically analyzed to support the interpretation of differences among samples. The Statistical Analysis section has been revised to clarify that sensory scores were analyzed by one-way ANOVA followed by Tukey’s HSD test at p < 0.05, using the individual assessors’ scores as replicates (n = 15). The Sensory Analysis results were also revised accordingly.

 Comments 4: In Figure 1a, the Y-axis lacks a proper description and numerical values. This should be corrected to improve the readability and interpretation of the figure.

Response 4: Figure 1a has been revised to include a clear Y-axis title and numerical values.

 Comments 5: The authors should indicate statistically significant differences between means in Table 4.

 Response 5: The color parameters in Table 4 are presented as descriptive results, expressed as mean ± standard deviation (SD), because a separate inferential statistical analysis was not performed for these measurements. To avoid overinterpretation, the discussion of color results has been revised and statements implying statistically significant differences were removed or reformulated.

 Comments 6: Why the results presented in Tables 3 and 5 do not include standard deviations and statistical significance of differences between means?

Response 6: The values presented in Table 3 were not experimentally measured in independent replicates, but were calculated based on the formulation and the nutritional composition of each ingredient. Therefore, standard deviations and statistical significance between means are not applicable for Table 3. In contrast, the quantitative microbiological results in Table 5 were obtained from triplicate analyses and have been revised to include mean ± standard deviation (SD).

Conclusions:

Comments 1:The authors state too broadly that wine lees can be used as a fat replacer. It should be emphasized that wine lees may act as a partial fat replacer under the conditions tested in this study.

Response 1: We agree that the statement should not be generalized beyond the experimental conditions tested in this study. The manuscript has been revised to indicate that wine lees may act as a partial fat replacer in muffin formulations under the tested substitution levels and processing conditions, rather than as a general fat replacer.

Comments 2: The authors should include the main limitations of the study, particularly those related to the characterization and pre-treatment of wine lees, microbiological safety at higher substitution levels, and the scope of sensory evaluation.

 Response 2: A limitations paragraph has been added to the Conclusions section. The revised text now clarifies that the findings are limited to wine lees from the two local Moldovan grape varieties and to the substitution levels and processing conditions tested in this study.

Reviewer 3 Report

Comments and Suggestions for Authors

This study evaluates the potential of wine lees (WL), a residual oenological yeast sediment from two Moldovan grape varieties (Viorica and Fetească Regală), as a multifunctional ingredient and partial fat replacer in muffins. Sunflower oil was substituted with WL at levels of 20%, 35%, and 50%. The authors analyzed the physicochemical, nutritional, microbiological, colorimetric, and sensory properties of the resulting muffins. The research presented is very interesting and provides a valuable contribution to the search for alternatives for the use and valorization of WL. However, several improvements and additional justifications are necessary. Below are my comments:

 

Lines 44–46: The statement presented here would benefit from supporting data. Please consider providing specific figures on global wine production and the scale of the wine agro‑industry to substantiate this claim. Including relevant statistics (e.g., annual production volumes, number of processing facilities, or economic impact) would strengthen the contextual foundation of the study.

 

Lines 47–48: Please provide data on the production of WL at local, regional, and global levels. Including this information would help readers understand the scale of this by‑product stream and contextualize the relevance of the study.

 

Lines 62–63: Could the authors provide the average percentage of macro‑ and micronutrients present in wine lees (WL)? Including this information would help readers better understand the nutritional composition of this by‑product and assess its potential for further valorization.

 

Lines 131–134: To confirm that a representative sample was used for the analyses, it would be appropriate to indicate how many muffins were obtained per treatment. Including the number of replicates or samples per experimental condition would enhance the transparency and reproducibility of the study.

 

Section 3.1.2: The authors should provide a justification for why a fatty acid profile was not included in the study, considering that fatty acids may also contribute to the functional properties of WL. 

 

Figure 1: The resolution and overall quality of the images should be improved.

 

Section 3.2.1: Please explain the observed reduction in moisture content, considering that the composition of WL includes substances that may help retain water. Clarifying this aspect would help readers understand whether the drying process affected the water‑holding capacity of the matrix and how the presence of such compounds might influence the final moisture content.

 

Section 3.2.4: The results presented here appear to contradict those observed in Section 3.2.1. Therefore, an adequate explanation should be provided in the previous section to resolve this inconsistency.

 

Lines 407–415: Please explain the opposite relationship observed between moisture content and titratable acidity. 

 

Section 3.3: I suggest including a paragraph discussing the increase in dietary fiber content and its relevance to the functional properties of the muffins. Addressing this point would help readers understand how the incorporation of wine lees affects the nutritional profile of the final product and whether this increment contributes to potential health‑promoting attributes (e.g., improved digestive health, satiety). Furthermore, comparing the observed values with those of similar fortified bakery products would strengthen the discussion.

 

Table 4: Please edit the table for improved visualization.

 

Lines 464–465: This statement is valid only if there is evidence that WL contribute amino acids (e.g., lysine) or monosaccharides (e.g., pentoses, particularly xylose) with high reactivity during the Maillard reaction. I suggest verifying this information and providing adequate support for the discussion presented. Including appropriate citations or experimental data to confirm the presence of these reactive compounds would strengthen the validity of the claim.

 

Line 502: It is important to specify the grape variety from which the pomace was derived. This information is relevant because the color properties of grape pomace can vary significantly depending on the grape variety.

 

Lines 535–537: The authors should also consider that the oxidation of phenolic compounds can produce quinones and subsequently melanoidins, which generate dark shades in food products. Acknowledging this alternative pathway would provide a more comprehensive discussion of the potential mechanisms underlying color changes in the fortified muffins.

 

Author Response

We would like to sincerely thank the reviewer for the careful evaluation of our manuscript and for the constructive comments and valuable suggestions. We appreciate the positive assessment of the study's relevance within the framework of sustainable food production and the circular bioeconomy.

All comments have been carefully considered, and the manuscript has been revised accordingly. We believe that the revisions and additional clarifications have significantly improved the quality, clarity, and scientific value of the paper. Detailed responses to each comment are provided below.

 Comments 1: Lines 44–46: The statement presented here would benefit from supporting data. Please consider providing specific figures on global wine production and the scale of the wine agro‑industry to substantiate this claim. Including relevant statistics (e.g., annual production volumes, number of processing facilities, or economic impact) would strengthen the contextual foundation of the study.

Response 1: The Introduction has been revised to support the statement on the global relevance of viticulture and winemaking with specific statistical data. An OIV report was added as a reference, including data on global vineyard surface area and world wine production.

Comments 2: Lines 47–48: Please provide data on the production of WL at local, regional, and global levels. Including this information would help readers understand the scale of this by‑product stream and contextualize the relevance of the study.

 Response 2: We added recent global wine-sector statistics from the OIV to strengthen the contextual foundation of the study. However, official and reliable statistical data specifically reporting wine lees production at the national or local level were not available.

 Comments 3: Lines 62–63: Could the authors provide the average percentage of macro‑ and micronutrients present in wine lees (WL)? Including this information would help readers better understand the nutritional composition of this by‑product and assess its potential for further valorization.

 Response 3: We agree that information on the nutritional composition of wine lees is important for understanding their valorization potential. In the present study, the wine lees used for muffin formulation were experimentally characterized, and their macronutrient composition, including carbohydrates, lipids, proteins, β-glucans, and fractional lipid composition, is presented in Table 2. However, we avoided adding generalized average values from the literature because the composition of wine lees varies considerably depending on grape variety, wine type, fermentation conditions, and post-fermentation handling. In addition, the mineral and micronutrient profile was not the main focus of this study. Therefore, the manuscript emphasizes the experimentally determined composition of the specific wine lees used in this work.

Comments 4: Lines 131–134: To confirm that a representative sample was used for the analyses, it would be appropriate to indicate how many muffins were obtained per treatment. Including the number of replicates or samples per experimental condition would enhance the transparency and reproducibility of the study.

 Response 4: In this study, the experimental unit was the muffin formulation batch rather than the number of individual muffins, as the latter may vary depending on mold size and batter portioning. Each formulation was prepared in triplicate, and samples for analysis were randomly collected from the independent batches to ensure representativeness.

 Comments 5: Section 3.1.2: The authors should provide a justification for why a fatty acid profile was not included in the study, considering that fatty acids may also contribute to the functional properties of WL. 

 Response 5: We agree that the fatty acid profile may provide additional information regarding the functional properties of wine lees. In the present study, lipid characterization was limited to total lipid content and fractional lipid composition, while the detailed fatty acid profile was not included because it was beyond the main objective of this work. The study focused primarily on evaluating wine lees as a partial fat replacer in muffins in terms of technological, nutritional, microbiological, colorimetric, and sensory properties. This limitation has been acknowledged in the revised manuscript, and future studies will include fatty acid profiling to better understand the contribution of WL lipids to product functionality.

 Comments 6: Figure 1: The resolution and overall quality of the images should be improved.

 Response 6: Figure 1 has been revised to improve its resolution and overall readability. The diagrams were re-exported at higher quality and adjusted to fit the manuscript format without compromising the visibility of axes, labels, and numerical values. If required, the original high-resolution versions of the figures can be provided as separate files.

 Comments 7: Section 3.2.1: Please explain the observed reduction in moisture content, considering that the composition of WL includes substances that may help retain water. Clarifying this aspect would help readers understand whether the drying process affected the water‑holding capacity of the matrix and how the presence of such compounds might influence the final moisture content.

 Response 7:After rechecking the data, we identified an error in the presentation of moisture and dry matter values. The values of 21–25% correspond to moisture content, not dry matter, while dry matter values correspond to 75–79%. Section 3.2.1 and Figure 1a have therefore been revised, and the discussion has been corrected to clarify the effect of WL incorporation on the water-related properties of the muffin matrix.

Comments 8: Section 3.2.4: The results presented here appear to contradict those observed in Section 3.2.1. Therefore, an adequate explanation should be provided in the previous section to resolve this inconsistency.

Response 8: We agree that the previous version created an apparent inconsistency between Sections 3.2.1 and 3.2.4. After rechecking the data, we identified an error in the presentation of moisture and dry matter values in Section 3.2.1. This section has been revised accordingly, and the discussion has been corrected to align with the water absorption capacity results presented in Section 3.2.4.

 Comments 9: Lines 407–415: Please explain the opposite relationship observed between moisture content and titratable acidity. 

 Response 9: After rechecking the data, we identified that the apparent opposite relationship between moisture content and titratable acidity was partly related to the previously incorrect presentation of moisture and dry matter values.

Comments 10: Section 3.3: I suggest including a paragraph discussing the increase in dietary fiber content and its relevance to the functional properties of the muffins. Addressing this point would help readers understand how the incorporation of wine lees affects the nutritional profile of the final product and whether this increment contributes to potential health‑promoting attributes (e.g., improved digestive health, satiety). Furthermore, comparing the observed values with those of similar fortified bakery products would strengthen the discussion.

 Response 10: We agree that the increase in dietary fiber content is relevant for the nutritional profile of the muffins and may contribute to their potential functional value. However, an extended discussion comparing the fiber content with other fortified bakery products and addressing specific health-promoting effects, such as satiety or digestive health, would broaden the scope and length of the manuscript. Since these physiological effects were not directly evaluated in the present study, we avoided making unsupported health-related claims. This aspect will be considered in future studies focused specifically on the functional and health-related properties of WL-enriched bakery products.

Comments 11: Table 4: Please edit the table for improved visualization.

Response 11: Since the table contains several color parameters measured for both wine lees and muffin samples, the complete dataset was retained to avoid loss of relevant information. However, the formatting, alignment, column headings, and table note were improved to make the data easier to read and interpret.

Comments 12: Lines 464–465: This statement is valid only if there is evidence that WL contribute amino acids (e.g., lysine) or monosaccharides (e.g., pentoses, particularly xylose) with high reactivity during the Maillard reaction. I suggest verifying this information and providing adequate support for the discussion presented. Including appropriate citations or experimental data to confirm the presence of these reactive compounds would strengthen the validity of the claim.

 Response 12: We agree that the original statement regarding the contribution of WL to the Maillard reaction required more specific evidence on reactive amino acids or sugars. Since the detailed profile of free amino acids and reactive monosaccharides was not determined in the present study, the statement has been revised to avoid overinterpretation. The discussion now refers more generally to possible thermal browning reactions and pigments derived from WL. The identification of specific Maillard-reactive compounds, such as lysine or pentoses, will be included in future studies to better clarify their contribution to color development in WL-enriched muffins.

Comments 13: Line 502: It is important to specify the grape variety from which the pomace was derived. This information is relevant because the color properties of grape pomace can vary significantly depending on the grape variety.

 Response 13: The wine lees used in the present study were already specified in the Materials and Methods section as originating from the local Moldovan grape varieties Viorica and Fetească Regală.

Comments 14: Lines 535–537: The authors should also consider that the oxidation of phenolic compounds can produce quinones and subsequently melanoidins, which generate dark shades in food products. Acknowledging this alternative pathway would provide a more comprehensive discussion of the potential mechanisms underlying color changes in the fortified muffins.

 Response 14: We agree that oxidation of phenolic compounds may represent an additional pathway contributing to color changes in WL-enriched muffins. Since color measurements were performed shortly after baking, prolonged storage-related oxidation was not considered. However, thermal processing during baking may promote non-enzymatic oxidation of phenolic compounds and the formation of darker pigments.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors corrected the manuscript accordingly.