Optimization of Polyphenol-Rich Extracts from Defatted Avocado Peel and Seed Residues Using Ultrasound-Assisted RSM: Antioxidant Potential and Valorization Prospects

Round 1

Reviewer 1 Report

Reviewer’s suggestions and comments on the Manuscript entitled:

Optimization of Polyphenol-Rich Extracts from Defatted Avocado Peel and Seed Residues Using Ultrasound-Assisted RSM: Antioxidant Potential and Valorization Prospects

Manuscript ID: antioxidants-3946617

From the very title, which is well thought out, it is clear that the subject of this study is the examination of the possibilities of using extracted oil from avocado peel and seed. In the Introductory section, the authors provide comprehensive information about reactive oxidative species, with a particular emphasis on their harmful effects when present in excess in the body. Following this, the authors continue to elaborate on the issue of reactive species by exploring the possibility of their elimination through the use of biological byproducts, with a focus on green technology for oil extraction from byproducts using ultrasound. At the end of the introduction, the authors explain why the waste parts of the avocado, specifically the peel and the seed, will be the subject of investigation, particularly their extracted oil, in terms of evaluating their antioxidant power and capacity. The entire Introduction is supported by relevant references.

The Materials and Methods section is described in detail, including chemicals, reagents, instruments, and software. Furthermore, a detailed description is provided of the processing of avocado peel and seed, as well as the oil extraction procedure, fatty acid analysis, and antioxidant power evaluation.

In the Results and Discussion section, an experimental design matrix is presented at the outset, along with the results of the antioxidant ability tests. The discussion of the obtained results is scientifically well-analyzed. This is followed by an analysis of the results using a second-degree polynomial model. Visualization of this model is done in the form of contour diagrams. Based on this, the authors provide an overview of the optimal extraction conditions to achieve the strongest antioxidant response. For further analysis of the results, the authors perform a Pareto plot analysis, a statistical technique that uses a Pareto chart to identify the most significant influencing factors. The potential interrelationship between the antioxidant capacities of the peel and seeds is also examined. Using Partial Least Squares (PLS) analysis, the extraction process was optimized. At the end of this section, the authors present the results of the analysis of the extracted oil in terms of fatty acid composition, oxidative stability, and pigment content.

The English language, in my opinion, is at a very high level, making the manuscript easy to read. Throughout the text, the authors refer to relevant references. This comprehensive study is well-designed, executed, and analyzed. The range of techniques used is impressive, as are the software programs employed. The reviewer did not identify any part of the text that requires correction. If published, this study has the potential for high citation rates due to its high-quality writing, highly interesting results, analyses, and discussions.

Therefore, I strongly recommend that the Editorial Office accept this manuscript in the present form.

The English language, in my opinion, is at a very high level, making the manuscript easy to read. Throughout the text, the authors refer to relevant references. This comprehensive study is well-designed, executed, and analyzed. The range of techniques used is impressive, as are the software programs employed. The reviewer did not identify any part of the text that requires correction. If published, this study has the potential for high citation rates due to its high-quality writing, highly interesting results, analyses, and discussions.

Author Response

Major comments

Reviewer’s suggestions and comments on the Manuscript entitled:

Optimization of Polyphenol-Rich Extracts from Defatted Avocado Peel and Seed Residues Using Ultrasound-Assisted RSM: Antioxidant Potential and Valorization Prospects

Manuscript ID: antioxidants-3946617

From the very title, which is well thought out, it is clear that the subject of this study is the examination of the possibilities of using extracted oil from avocado peel and seed. In the Introductory section, the authors provide comprehensive information about reactive oxidative species, with a particular emphasis on their harmful effects when present in excess in the body. Following this, the authors continue to elaborate on the issue of reactive species by exploring the possibility of their elimination through the use of biological byproducts, with a focus on green technology for oil extraction from byproducts using ultrasound. At the end of the introduction, the authors explain why the waste parts of the avocado, specifically the peel and the seed, will be the subject of investigation, particularly their extracted oil, in terms of evaluating their antioxidant power and capacity. The entire Introduction is supported by relevant references.

The Materials and Methods section is described in detail, including chemicals, reagents, instruments, and software. Furthermore, a detailed description is provided of the processing of avocado peel and seed, as well as the oil extraction procedure, fatty acid analysis, and antioxidant power evaluation.

In the Results and Discussion section, an experimental design matrix is presented at the outset, along with the results of the antioxidant ability tests. The discussion of the obtained results is scientifically well-analyzed. This is followed by an analysis of the results using a second-degree polynomial model. Visualization of this model is done in the form of contour diagrams. Based on this, the authors provide an overview of the optimal extraction conditions to achieve the strongest antioxidant response. For further analysis of the results, the authors perform a Pareto plot analysis, a statistical technique that uses a Pareto chart to identify the most significant influencing factors. The potential interrelationship between the antioxidant capacities of the peel and seeds is also examined. Using Partial Least Squares (PLS) analysis, the extraction process was optimized. At the end of this section, the authors present the results of the analysis of the extracted oil in terms of fatty acid composition, oxidative stability, and pigment content.

The English language, in my opinion, is at a very high level, making the manuscript easy to read. Throughout the text, the authors refer to relevant references. This comprehensive study is well-designed, executed, and analyzed. The range of techniques used is impressive, as are the software programs employed. The reviewer did not identify any part of the text that requires correction. If published, this study has the potential for high citation rates due to its high-quality writing, highly interesting results, analyses, and discussions.

Therefore, I strongly recommend that the Editorial Office accept this manuscript in the present form.

Detailed comments

The English language, in my opinion, is at a very high level, making the manuscript easy to read. Throughout the text, the authors refer to relevant references. This comprehensive study is well-designed, executed, and analyzed. The range of techniques used is impressive, as are the software programs employed. The reviewer did not identify any part of the text that requires correction. If published, this study has the potential for high citation rates due to its high-quality writing, highly interesting results, analyses, and discussions.

We sincerely thank the reviewer for the very positive and encouraging evaluation of our manuscript. We greatly appreciate the recognition of the study’s design, methodology, and analysis, as well as the clarity of the writing. The reviewer’s supportive comments further motivate us to continue our research in this field. We are grateful for the recommendation to accept the manuscript in its present form.

Reviewer 2 Report

The article is interesting and valuable in addressing waste. Recommendations were made to improve the reliability of the research results in 2. Materials and Methods, especially in 2.3. Avocado Fruit Handling.

It is very important, when conducting research of this kind, related to plant raw materials, to properly form a research sample that corresponds to the overall quality of avocados.

Therefore, it is especially recommended to provide a detailed description of how the avocado sample was formed:

1. At what time of day were the avocados collected (morning, noon, evening?), because further research results largely depend on this?

2. How Unripe avocado fruits were collected, i.e. the size of the research field, how many repetitions, how many fruits in each repetition?

3. It is stated that "Both peel and seed fractions were cut into small pieces". Please detail more specifically how many repetitions were selected for quantitative fractions? What is the size of each sample?

4. In section 3.2. Analysis section for better visualization (line 324 and down) it is recommended to present in a separate table The fitted models for TPC, FRAP, DPPH radical scavenging activity, and AAC;

1. Chapter 2.3. Avocado Fruit Handling : Line 138 and below 

How the avocado sample was formed? The questions were presented for authors and Editors (for familiarization)

2. In section 3.2. Analysis section for better visualization (line 324 and down) it is recommended to present in a separate table The fitted models for TPC, FRAP, DPPH radical scavenging activity, and AAC. Recommendations were presented for authors and Editors (for familiarization)

Author Response

Major comments

The article is interesting and valuable in addressing waste. Recommendations were made to improve the reliability of the research results in 2. Materials and Methods, especially in 2.3. Avocado Fruit Handling.

We would like to thank the reviewer for their valuable recommendations.

It is very important, when conducting research of this kind, related to plant raw materials, to properly form a research sample that corresponds to the overall quality of avocados.

Therefore, it is especially recommended to provide a detailed description of how the avocado sample was formed:

1. At what time of day were the avocados collected (morning, noon, evening?), because further research results largely depend on this?

Additional information about avocado collection was added, as requested.

2. How Unripe avocado fruits were collected, i.e. the size of the research field, how many repetitions, how many fruits in each repetition?

Information about the field, the repetitions and fruits collected in each repetition were added into the manuscript, as requested.

3. It is stated that "Both peel and seed fractions were cut into small pieces". Please detail more specifically how many repetitions were selected for quantitative fractions? What is the size of each sample?

Additional information was added into the manuscript, as suggested.

4. In section 3.2. Analysis section for better visualization (line 324 and down) it is recommended to present in a separate table The fitted models for TPC, FRAP, DPPH radical scavenging activity, and AAC;

We respectfully maintain the separation of the fitted models into two tables, as each one contains distinct content. This separation avoids overloading the reader with excessive information in a single table and follows common practice in RSM studies.

 

Detailed comments

1. Chapter 2.3. Avocado Fruit Handling : Line 138 and below 

How the avocado sample was formed? The questions were presented for authors and Editors (for familiarization)

Additional information was added, as suggested.

2. In section 3.2. Analysis section for better visualization (line 324 and down) it is recommended to present in a separate table The fitted models for TPC, FRAP, DPPH radical scavenging activity, and AAC. Recommendations were presented for authors and Editors (for familiarization)

This comment is identical to point 4 in Major comment section, and so, it is addressed in that section.

Reviewer 3 Report

The article presents a study on the extraction of polyphenols from the skin and seeds of defatted avocado using ultrasounds. The study is topical and makes important contributions to the processes of waste recovery from the food industry with high applied potential. Investigation and characterization techniques are modern and up-to-date. To increase the quality of the article, several improvements can be made.

Recommendations:

1. Materials and methods

– it would be useful to present the equations for the calibration curves for DPPH, TPC, FRAP, AAC.

-chapters 2.6 and 2.7 should be presented before chapter 2.5 for a better understanding.

-the specific absorption coefficient for the assigned carotenoid (2500 for carotenoids in diethyl ether) –(p. 5 row 193) requires a bibliographic reference.

-p. 5 - row 196-197 – what is FD?

2. Results and discussions

-chapter 3.6 should be presented before chapter 3.1 for a chronological order of the investigations and a better understanding.

-more details are needed regarding the structure of the identified catechin derivatives (catechin-derivatives 1-5, Table 7).

Author Response

Major comments

The article presents a study on the extraction of polyphenols from the skin and seeds of defatted avocado using ultrasounds. The study is topical and makes important contributions to the processes of waste recovery from the food industry with high applied potential. Investigation and characterization techniques are modern and up-to-date. To increase the quality of the article, several improvements can be made.

We would like to thank the reviewer for their insightful comments.

 

Detailed comments

Recommendations:

1. Materials and methods

– it would be useful to present the equations for the calibration curves for DPPH, TPC, FRAP, AAC.

Equations of calibration curves of each assay were provided, as suggested.

-chapters 2.6 and 2.7 should be presented before chapter 2.5 for a better understanding.

We kept the order of sections 2.5–2.7 to reflect the actual workflow of the study: first the experimental design was established (2.5), then the extractions were performed (2.6), and finally the analyses were conducted (2.7). This order mirrors the chronological execution of the experiments.

-the specific absorption coefficient for the assigned carotenoid (2500 for carotenoids in diethyl ether) –(p. 5 row 193) requires a bibliographic reference.

A bibliographic reference has now been added to support the specific absorption coefficient used for carotenoids in diethyl ether.

-p. 5 - row 196-197 – what is FD?

FD refers to the dilution factor. Explanation was added into the manuscript.

1. Results and discussions

-chapter 3.6 should be presented before chapter 3.1 for a chronological order of the investigations and a better understanding.

The order of the paragraphs was changed, as recommended.

-more details are needed regarding the structure of the identified catechin derivatives (catechin-derivatives 1-5, Table 7).

We thank the reviewer for highlighting the need for further clarification regarding the structure of the identified catechin derivatives (Table 7). As LC-MS or NMR instrumentation was not available to us, structural elucidation beyond UV-Vis comparison was not feasible. The designation as catechin "derivatives" was based on the similarity of their UV-Vis spectra to that of the catechin standard. To support this identification, we have included Figure A1 in the Appendix, which presents an overlay of the UV-Vis spectra (200–400 nm) for catechin and the five identified derivatives. This figure illustrates the spectral alignment and peak positions that guided our classification.

Reviewer 4 Report

The work “Optimization of Polyphenol-Rich Extracts from Defatted Avocado Peel and Seed Residues Using Ultrasound-Assisted RSM: Antioxidant Potential and Valorization Prospects” is focused on optimizing the extraction of polyphenols from avocado by-products once peels and seeds were defatted, as their lipid fractions were also valorized. The work is generally well-described and oriented, with high scientific significance and interest for readers.

I recommend this article to be published in this journal after these following minor revision are addressed:

INTRODUCTION:

• Lines 59-65: Please, give a more detailed description of ultrasounds-assisted extraction technology, including main extraction parameters to be considered.
• Line 68-70: Please, provide information on avocado worldwide production and the volume of residues generated from it annually.
• Lines 73-76: What about AP composition? Include some specific compounds (also for AS) reported in the literature and an approximate composition (%).
• Lines 78-80: Although here is mentioned that “Until now, great importance has been given to the seed, and as a result the peel and all the beneficial substances it contains have not been studied thoroughly.”, a brief review on the background of the existing literature regarding the valorization of avocado by-products (seeds and peel) is missing.
• Line 84: “Appropriate solvent” is said to be analyzed but later, ethanol is said to be used in different proportions. Please, clarify in the objectives.
• Line 84-85: The last objective “In addition, the oil present in AS and AP will also be studied.” Is not very clear. Integrate within the others, and detail how it will be conducted.

 

MATERIALS AND METHODS:

• Section 2.2.: It results weird to talk about the equipment and software used for each analysis when specific analysis methods have not been yet described. I suggest incorporating information on each equipment/software in the corresponding analysis section to make sense with the whole text. For instance, the freeze-dryer model employed for drying avocado by-products may fix better within line 145, milling apparatus in line 147, rotary evaporator in line 156, and so on.
• Figure 1: Under my opinion, this figure is not necessary, as it does not provide any significant information (maybe in supplementary).
• Line 154-155: Specify temperature for defatting, and ratio hexane-raw material.
• Section 2.4.3.: What compound was used as standard/positive control?
• Equation 4: Specify variables meaning. If FD is for dilution factor, please homogenize; above dilution factor is named as “V”.
• Table 1: What is the meaning of the assigned letters given to the variables: E, R, C, t? (if they have assigned a corresponding code.
• Line 227, 232, 237, and 244: Describe briefly the methods.

 

RESULTS AND DISCUSSION

• Line 291-292: “TPC and FRAP were consistently higher in peel extracts, with ethanol concentration emerging as the most influential factor”. Most influential in the quadratic term but not in the single (table 3).
• Equation 7: The interaction X2X4 appears as not significant in table 3. Same in equation 8 for the quadratic term of X3. Please, check.
• Figures 2-5: Specify in the figures caption the fixed central levels for the corresponding variables. Increase the size for P/S in the graphs to properly identify peel/seed graphs. These figures are not mentioned in the discussion part in the text. Try to include them to justify the discussion on lines 290-312.
• Lines 356-361: Please check this part. Data on optimal parameters seem not to agree with those reported in table 4. For example: FRAP opt. is said to be reached at low ratio when, in fact, same ratio (70 mL/g) is the optimal for the four responses, and ethanol is said to be intermediate (20%) when is lower in comparison with TPC, in which is said to be low ethanol (39%). Same for DPPH: harsher conditions? Looking at table 4, same ratio is used, same power than for TPC, and maximum ethanol (100%) is used, but lower time (5 min).
• Figure 7: Check color for distinguishing between seed/peel.
• Figure 8: Include variable’ names with the codes in the X axe.
• Table 7: Chlorogenic acid calibration curve equation does not appear in Table A1, check. How were the catechin derivatives identified? Compound 12 (luteolin-7-glucoside) seems to appear later in the chromatogram (fig 9) compared to its retention time (fig A1).
• Lines 477-481; Cite the references in which the bioactivity of these compounds are stated.
• Line 502: Oleic acid is said to be significantly higher in seed oil, but no statistical significance is shown in table 8, check.
• Line 505: SFA in see oil are mainly constituted by palmitic, and later, by stearic. Lignoceric is present in lower than 1%, check.
• Line 509: Cite the reference for the bioactivity (anti-inflammatory potential) associated with this ratio.
• Section 6: As the first step in the article is to defat the samples to later extract polyphenols from defatted samples, this section would fit best before the UA extraction (as did in the abstract).
• Lines 547-551: Cite references.

Author Response

Major comments

The work “Optimization of Polyphenol-Rich Extracts from Defatted Avocado Peel and Seed Residues Using Ultrasound-Assisted RSM: Antioxidant Potential and Valorization Prospects” is focused on optimizing the extraction of polyphenols from avocado by-products once peels and seeds were defatted, as their lipid fractions were also valorized. The work is generally well-described and oriented, with high scientific significance and interest for readers.

We would like to thank the reviewer for their insightful comments.

 

Detailed comments

I recommend this article to be published in this journal after these following minor revision are addressed:

INTRODUCTION:

• Lines 59-65: Please, give a more detailed description of ultrasounds-assisted extraction technology, including main extraction parameters to be considered.

More details about ultrasonication were added into the manuscript, as requested.

• Line 68-70: Please, provide information on avocado worldwide production and the volume of residues generated from it annually.

Information about globally avocado production and waste emerging by it was added into the manuscript, along with their corresponding references.

• Lines 73-76: What about AP composition? Include some specific compounds (also for AS) reported in the literature and an approximate composition (%).

Information about AP composition was added, as suggested.

• Lines 78-80: Although here is mentioned that “Until now, great importance has been given to the seed, and as a result the peel and all the beneficial substances it contains have not been studied thoroughly.”, a brief review on the background of the existing literature regarding the valorization of avocado by-products (seeds and peel) is missing.

Information was added, as asked.

• Line 84: “Appropriate solvent” is said to be analyzed but later, ethanol is said to be used in different proportions. Please, clarify in the objectives.

Clarification was made, as requested.

• Line 84-85: The last objective “In addition, the oil present in AS and AP will also be studied.” Is not very clear. Integrate within the others, and detail how it will be conducted.

Clarification was made, as asked.

 

MATERIALS AND METHODS:

• Section 2.2.: It results weird to talk about the equipment and software used for each analysis when specific analysis methods have not been yet described. I suggest incorporating information on each equipment/software in the corresponding analysis section to make sense with the whole text. For instance, the freeze-dryer model employed for drying avocado by-products may fix better within line 145, milling apparatus in line 147, rotary evaporator in line 156, and so on.

Modifications were made, as suggested.

• Figure 1: Under my opinion, this figure is not necessary, as it does not provide any significant information (maybe in supplementary).

The authors would like to retain the image in the main text, as it provides information on the anatomy of the fruit and gives an idea of how the separation of the individual components analyzed was carried out.

• Line 154-155: Specify temperature for defatting, and ratio hexane-raw material.

Specifications were added, as requested.

• Section 2.4.3.: What compound was used as standard/positive control?

Trolox was used as a positive control. Specification was added into the manuscript.

• Equation 4: Specify variables meaning. If FD is for dilution factor, please homogenize; above dilution factor is named as “V”.

FD actually is the dilution factor; an explanation was added in the manuscript. Variables were not changes, as they derive from well-established equations and laws.

• Table 1: What is the meaning of the assigned letters given to the variables: E, R, C, t? (if they have assigned a corresponding code.

These letters refer to the independent variables; explanation was added into the manuscript.

• Line 227, 232, 237, and 244: Describe briefly the methods.

The description of these methods in the main text was avoided as it elevates the similarity of this study with already published articles. However, a description of them was added in the manuscript, as asked.

 

RESULTS AND DISCUSSION

• Line 291-292: “TPC and FRAP were consistently higher in peel extracts, with ethanol concentration emerging as the most influential factor”. Most influential in the quadratic term but not in the single (table 3).

Quadratic terms are generally more influential than the single ones, that’s why this conclusion was conducted.

• Equation 7: The interaction X2X4 appears as not significant in table 3. Same in equation 8 for the quadratic term of X3. Please, check.

We thank the reviewer for this valuable observation. We carefully re‑checked the regression models against the ANOVA tables. In the revised version, non‑significant terms (p > 0.05) were removed through stepwise regression, resulting in reduced quadratic equations. To preserve model hierarchy, linear terms were retained when their corresponding quadratic or interaction terms were significant. The final equations (11–14) now include only statistically significant predictors, and are fully consistent with the ANOVA results. This adjustment improves clarity and avoids confusion regarding the role of non‑significant factors.

• Figures 2-5: Specify in the figures caption the fixed central levels for the corresponding variables. Increase the size for P/S in the graphs to properly identify peel/seed graphs. These figures are not mentioned in the discussion part in the text. Try to include them to justify the discussion on lines 290-312.

We thank the reviewer for these constructive comments. In the revised version, the captions of Figures 3–6 have been updated to specify the fixed central levels of the non‑plotted variables. The sample type (X₁: peel or seed) is now clearly indicated in the figure panels, ensuring that the distinction between peel and seed extracts is unambiguous. The labels have been adjusted for clarity and readability. In addition, Figures 3–6 are now explicitly referenced in the Discussion section to support the interpretation of the interaction effects and to justify the trends described in the text. These changes improve both the clarity and the consistency of the presentation.

• Lines 356-361: Please check this part. Data on optimal parameters seem not to agree with those reported in table 4. For example: FRAP opt. is said to be reached at low ratio when, in fact, same ratio (70 mL/g) is the optimal for the four responses, and ethanol is said to be intermediate (20%) when is lower in comparison with TPC, in which is said to be low ethanol (39%). Same for DPPH: harsher conditions? Looking at table 4, same ratio is used, same power than for TPC, and maximum ethanol (100%) is used, but lower time (5 min).

This table shows the optimal values for each assay separately. Given that these materials contain a multitude of bioactive compounds with different chemical structures and, consequently, properties, it is perfectly normal for the assays to have different optimal conditions. A key objective of the study was to use experiments and statistical tools to find the optimal conditions that would yield the highest possible performance in each assay simultaneously. Therefore, later, when the optimal conditions are determined after extensive statistical analysis, they are generally referred to in all assays and in some cases, they differ from those in Table 4.

• Figure 7: Check color for distinguishing between seed/peel.

We thank the reviewer for pointing this out. The colors in Figure 7 have been revised to clearly distinguish between peel and seed extracts. The caption has also been corrected accordingly, and no reference to hierarchical clustering is now included.

• Figure 8: Include variable’ names with the codes in the X axe.

We thank the reviewer for this helpful suggestion. In the revised Figure 8, the variable names have been added alongside their codes on the X-axis (e.g., X1 – Waste, X2 – Power, X3 – Ratio, X4 – Ethanol, X5 – Time) to improve clarity.

• Table 7: Chlorogenic acid calibration curve equation does not appear in Table A1, check. How were the catechin derivatives identified? Compound 12 (luteolin-7-glucoside) seems to appear later in the chromatogram (fig 9) compared to its retention time (fig A1).

Calibration curve of chlorogenic acid was added in Table A1. Retention time of compound 12 was corrected and the authors would like to apologize as it was a typographical error. The designation as a catechin "derivative" was assigned after comparing their ultraviolet spectra with a catechin standard. The UV spectra of the standard and the compounds under study are provided in the Appendix.

• Lines 477-481; Cite the references in which the bioactivity of these compounds are stated.

References were added, as requested.

• Line 502: Oleic acid is said to be significantly higher in seed oil, but no statistical significance is shown in table 8, check.

We thank the reviewer for this observation. We have corrected the text to clarify that although seed oil contained a higher proportion of oleic acid (48.09 ± 2.31%) compared to peel oil (42.05 ± 3.11%), this difference was not statistically significant, in agreement with Table 2.

• Line 505: SFA in see oil are mainly constituted by palmitic, and later, by stearic. Lignoceric is present in lower than 1%, check.

We appreciate the reviewer’s careful reading. The text has been revised to state that the higher SFA content in seed oil is mainly due to stearic acid (4.70%) and, to a lesser extent, lignoceric acid (0.94%), while palmitic acid (~20%) was present at comparable levels in both oils. This correction ensures consistency with the data in Table 2.

• Line 509: Cite the reference for the bioactivity (anti-inflammatory potential) associated with this ratio.

Citations were added, as requested.

• Section 6: As the first step in the article is to defat the samples to later extract polyphenols from defatted samples, this section would fit best before the UA extraction (as did in the abstract).

We would like to thank the reviewer for this insightful remark. Section 3.6 has been moved to the beginning of section 3, as requested.

• Lines 547-551: Cite references.

References were cited, as asked.