Optimization of Enzymatic Assisted Extraction of Bioactive Compounds from Olea europaea Leaves
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript by Vardakas and colleagues presents some interesting ideas regarding the application of a method, Enzyme-assisted extraction, for the extraction of bioactive compounds from olive leaves. Although not innovative, this technique combines the advantages of an effective extraction of phytochemicals from olive leaves with the use of a green method.
However, there are some aspects of the manuscript that, in my opinion, need clarification.
ABSTRACT: It doesn't seem informative enough to me. For example, it does not mention the type of enzymes used during Enzyme-assisted extraction (this information could stimulate the reader to read the paper further). Furthermore, the meaning of “...were achieved after EAE (50-50%) is unclear. What do these values ​​refer to?
INTRODUCTION: This section seems too long and redundant to me. In particular, the general part on antioxidants and their properties can be summarized to make reading easier.
RESULTS AND DISCUSSION: On several occasions, the authors express the TP values ​​using mg GAE/L as the unit of measurement. Liter of what? What is the dried leaf content by volume?
On lines 211-216, the authors write: “The negative effects of the higher enzyme dose indicate that the pectinase greatly decreased total phenolics due to the loss of rutin”. I think it is better to specify that it is not the pectinase enzyme that hydrolyses rutin, but rather the probable presence of contaminating rutinase as reported by other authors.
I also have some difficulty understanding the comparison of extraction methods reported in Table 4. It is not clear to me how mg GAE/g can be compared with mg GAE/L. Not all readers have the opportunity to find the full articles reported in the References.
Finally, what is the best result for TP that can be obtained with EAE, that of 468.19 mg GAE/L reported in the Abstract or that of 605.55 mg GAE/L reported in Table 4?
Regarding Table 4, it is also difficult to understand why the result of EAE (605.55 mg GAE/L) is better than C-C co-solvents (54.33 mg GAE/g) but is worse than MAE (104.22 mg GAE/g) or UAE (80.52 mg GAE/g):
I believe that all these clarifications can direct the reader towards an easier reading of the manuscript.
Author Response
The manuscript by Vardakas and colleagues presents some interesting ideas regarding the application of a method, Enzyme-assisted extraction, for the extraction of bioactive compounds from olive leaves. Although not innovative, this technique combines the advantages of an effective extraction of phytochemicals from olive leaves with the use of a green method.
However, there are some aspects of the manuscript that, in my opinion, need clarification.
Reply: We would like firstly to thank reviewer for his/her efforts to evaluate our study. We revised the manuscript according to yours and others reviewers suggestions/comments and we believe that you would find now the revised manuscript suitable for publication in Biomass/MDPI.
Reviewer 1: ABSTRACT: It doesn't seem informative enough to me. For example, it does not mention the type of enzymes used during Enzyme-assisted extraction (this information could stimulate the reader to read the paper further). Furthermore, the meaning of “...were achieved after EAE (50-50%) is unclear. What do these values ​​refer to?
Author: Thank you for the suggestion. All your comment about the abstract were
Reviewer 1: INTRODUCTION: This section seems too long and redundant to me. In particular, the general part on antioxidants and their properties can be summarized to make reading easier.
Author: Thank you for the suggestion. All your comment about the introduction were included/added in the revised version
Reviewer 1: RESULTS AND DISCUSSION: On several occasions, the authors express the TP values ​​using mg GAE/L as the unit of measurement. Liter of what? What is the dried leaf content by volume?
Author: Thank you for the suggestion.This has been added to the revised abstract to make it sufficient for the readers.
Reviewer 1: On lines 211-216, the authors write: “The negative effects of the higher enzyme dose indicate that the pectinase greatly decreased total phenolics due to the loss of rutin”. I think it is better to specify that it is not the pectinase enzyme that hydrolyses rutin, but rather the probable presence of contaminating rutinase as reported by other authors.
Author: Thank you for the suggestion Revised.
Reviewer 1: I also have some difficulty understanding the comparison of extraction methods reported in Table 4. It is not clear to me how mg GAE/g can be compared with mg GAE/L. Not all readers have the opportunity to find the full articles reported in the References.
Author: Please see our response to your last comment, we hope that the explanation cover your question.
Reviewer 1: Finally, what is the best result for TP that can be obtained with EAE, that of 468.19 mg GAE/L reported in the Abstract or that of 605.55 mg GAE/L reported in Table 4?
Author: Thank you for the comment. The best result after final optimization is 605.55 mg GAE/L as has been added in the Abstract.
Reviewer 1: Regarding Table 4, it is also difficult to understand why the result of EAE (605.55 mg GAE/L) is better than C-C co-solvents (54.33 mg GAE/g) but is worse than MAE (104.22 mg GAE/g) or UAE (80.52 mg GAE/g)
Author: Converting the mg GAE/L to mg GAE/g which means (60.55 mg GAE/g) the results with EAE are higher or lower than the methods as expressed. We do not mention as better or worse but as higher and lower polyphenolic obtained content comparison with the other extraction methods.
I believe that all these clarifications can direct the reader towards an easier reading of the manuscript.
Author: Thank you for all the suggestions/comments.
Reviewer 2 Report
Comments and Suggestions for AuthorsThis study is focused on the optimization of an enzymatic extraction process for obtaining extracts with high content of phenolic compounds exhibiting antioxidant activity. Although the experimental design is well-designed, the presentation of the results, as well as their evaluation and discussion are not well conducted. Conclusions are not supported by the results showed in the tables up to a point in which it does not seem to make sense to continue the investigation. Hence, I do not recommend this article for publication. Some of the biggest issues analysed in the present article include:
- Line 34: Detail some examples for these specific chemical compounds.
- Line 69-71: Rephrase this sentence to clarify.
- Figure 1: Give a more detailed caption, for example, under what conditions?
- Line 80: Specify if this statement is referred to oleuropein or what compound. Give some data about compounds’ concentration in olive leaves reported in literature.
- The introduction section should include a brief review of the current literature on the enzymatic extraction of polyphenols from olive leaves.
- Line 108: What is the residual humidity?
- Line 125: It is said that the sample in water was rehydrated and, later, placed in a 50ºC water bath for 20 minutes before adding the enzymes. However, afterwards it is said how the enzyme inactivation was carried out without explaining the enzymatic process, which is the key process of the study. Specify temperature, time, pH or buffer employed, enzyme:sample solution ratio, reconstitution of enzyme if needed…..
- Line 128: How was exactly determined the extraction yield if not lyophilized?
- Line 139: In the Folin method for TPP determination, was used a blank?
- Line 153: What was the temperature?
- Table 2: Check: state the superscript legend (a) in the correct place. Explain in the methods section in which consist the control (no enzyme) sample. How was it obtained?
- Line 186-187: Stablish the specific code for the binary enzyme combination specified in table 2 to better understanding. If this mixture is mix 3, it does not present higher yield than control.
- Lines 186-189: How is possible that control (without enzyme) exhibits a higher yield in comparison with the rest of the treatments with enzyme? If that is true, what is the sense to continue with the experiments if, just with incubation and not using enzymes, the control exhibits higher yields? Control also contains greater TPP content than some samples, and values are not significant different to those with higher content. The same for antioxidant activity (DPPH).
- Line 202: What enzyme combination was selected as the most effective combination?
- Table 3: What is the meaning of the coded values (+/-/0)?
- Line 2012: Describe in which cases the TPP decreased by increasing enzyme rate. Discussion of the possible reasons for this non-enzyme rate dependence is given as the loss of rutin, as reported in literature, however, in this study phenolics identification has not been conducted so it cannot be confirmed to be the cause for the decrease in TPP and antioxidant activity preservation.
- Lines 217-219: When using the same enzyme dose, for example 0.02, increasing time extraction did not promote a decrease in TPP, yield or antioxidant activity, on the contrary, increasing time lead to increasing values.
Author Response
This study is focused on the optimization of an enzymatic extraction process for obtaining extracts with high content of phenolic compounds exhibiting antioxidant activity. Although the experimental design is well-designed, the presentation of the results, as well as their evaluation and discussion are not well conducted. Conclusions are not supported by the results showed in the tables up to a point in which it does not seem to make sense to continue the investigation. Hence, I do not recommend this article for publication. Some of the biggest issues analysed in the present article include:
Authors: We would like firstly to thank reviewer for his/her efforts to evaluate our study. We revised the manuscript according to yours and others reviewers suggestions/comments and we believe that you would find now the revised manuscript suitable for publication in Biomass/MDPI.
- Reviewer 2: Line 34: Detail some examples for these specific chemical compounds.
Authors: Thank you for the comment/suggestion. Revised
- Reviewer 2: Line 69-71: Rephrase this sentence to clarify.
Authors: Thank you for the comment/suggestion. Revised
- Reviewer 2: Figure 1: Give a more detailed caption, for example, under what conditions?
Authors: Thank you for the comment/suggestion. In the revised version this caption has been deleted
- Reviewer 2: Line 80: Specify if this statement is referred to oleuropein or what compound. Give some data about compounds’ concentration in olive leaves reported in literature.
Authors: Thank you for the comment/suggestion. Revised
- Reviewer 2: The introduction section should include a brief review of the current literature on the enzymatic extraction of polyphenols from olive leaves.
Authors: Thank you for the comment/suggestion. This comment helped as to revised the introduction with current literature on the enzymatic extraction of polyphenols from olive leaves. As we express in the text there is a gap on the enzymatic assisted extraction of olive leaves and this is one of the reasons that we made this optimization study. We have added as well as a paragraph explain the advantages of this method.
- Reviewer 2: Line 108: What is the residual humidity?
Author: Thank you for the comment/suggestion. The fresidual humidity determined and added in the revised text.
- Reviewer 2: Line 125: It is said that the sample in water was rehydrated and, later, placed in a 50ºC water bath for 20 minutes before adding the enzymes. However, afterwards it is said how the enzyme inactivation was carried out without explaining the enzymatic process, which is the key process of the study. Specify temperature, time, pH or buffer employed, enzyme:sample solution ratio, reconstitution of enzyme if needed…..
Authors: Thank you for the comment/suggestion. All these parameters like temperature, time and enzyme doses are part of the main experimental part and analyzed in the “2.6 experimental design” part.
- Reviewer 2: Line 128: How was exactly determined the extraction yield if not lyophilized?
Authors: Thank you for the comment/suggestion. The yield was measured after the extraction process weighting the final supernatant after paper filtration under vacuum as expressed on lines 138-139.
- Reviewer 2: Line 139: In the Folin method for TPP determination, was used a blank?
Authors: Thank you for the comment/suggestion. Revised (see line 150)
- Reviewer 2: Line 153: What was the temperature?
Authors: Thank you for the comment/suggestion. Revised (see line 155)
- Reviewer 2: Table 2: Check: state the superscript legend (a) in the correct place. Explain in the methods section in which consist the control (no enzyme) sample. How was it obtained?
Authors: Thank you for the comment/suggestion. Revised
- Reviewer 2: Line 186-187: Stablish the specific code for the binary enzyme combination specified in table 2 to better understanding. If this mixture is mix 3, it does not present higher yield than control.
Authors: Thank you for the comment/suggestion. According to your suggestionTable 2 has been revised. The mix 3 was the most efficient, indeed statistically there were no differences at this step but it gave the maximum polyphenolic content in comparison with all the other results and after the final optimization, the differences were more significant. The mix 3 in comparison with the control sample has 37% higher polyphenolic content and 8% higher antioxidants.
- Reviewer 2: Lines 186-189: How is possible that control (without enzyme) exhibits a higher yield in comparison with the rest of the treatments with enzyme? If that is true, what is the sense to continue with the experiments if, just with incubation and not using enzymes, the control exhibits higher yields? Control also contains greater TPP content than some samples, and values are not significant different to those with higher content. The same for antioxidant activity (DPPH).
Authors: Thank you for the comment/suggestion. Please check our previous comment about the differences between the control sample and the final polyphenolic and antioxidant content after the final optimization process.
- Reviewer 2: Line 202: What enzyme combination was selected as the most effective combination?
Authors: Thank you for the comment/suggestion. Revised and mentioned on line 245
- Reviewer 2: Table 3: What is the meaning of the coded values (+/-/0)?
Authors: Thank you for the comment/suggestion. Revised
- Reviewer 2: Line 2012: Describe in which cases the TPP decreased by increasing enzyme rate. Discussion of the possible reasons for this non-enzyme rate dependence is given as the loss of rutin, as reported in literature, however, in this study phenolics identification has not been conducted so it cannot be confirmed to be the cause for the decrease in TPP and antioxidant activity preservation.
Author: Thank you for the comment/suggestion. The aim of this study as explained in the Introduction is to investigate the optimum extraction conditions of EAE and this is the reason that we did not include the polyphenolic profile of the olive leaves. The main point has been achieved, to find the optimum enzyme combinations and the incubation time to obtain as much as possible of total polyphenols content. The polyphenolic profile of the extract is for future study, and it is in our plans (see also revised conclusion section).
- Reviewer 2: Lines 217-219: When using the same enzyme dose, for example 0.02, increasing time extraction did not promote a decrease in TPP, yield or antioxidant activity, on the contrary, increasing time lead to increasing values.
Author: Thank you for the comment/suggestion. The reason probably, is the very low amount of the enzymes 0.01 mL of Viscozyme plus 0.01 mL of Pectinex comparison with the optimum concentration which is 0.5 mL of each enzyme (50-50 v/v).
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript "Optimization of Enzymatic Assisted Extraction of Bioactive Compounds from Olea Europaea Leaves" presents a study on the utilization of enzyme-assisted extraction (EAE) to extract polyphenols from olive leaves. The research is well-organized, and it offers insights into the optimization of EAE for industrial applications. Nevertheless, additional clarification and enhancements are necessary before the manuscript can be considered for publication.
Abstract
- Olea europaea, should be written in Italic
- “TP optimal extraction conditions (468.19 mg GAE/L)”, the term GAE should be defined when it is first mentioned.
- “EAE (50–50%)”, is it w/w, v/v, w/v,..etc.
Introduction
- Figure 1 is not mentioned in the text. Please refer to this figure before its location in the manuscript.
- The introduction provides a solid background on the significance of olive leaves as a source of bioactive compounds, it would be also helpful if authors have included a paragraph about the advantages of using enzymatic process.
- Kindly add recent references to highlight the latest advancements in this field.
Materials and methods
- It would be better to visualize your methods by adding a flow chart describes your methods steps.
Results and discussion
- The results are presented in a straightforward manner, utilizing the appropriate number of tables and figures. It is noteworthy to employ a simplex-centroid design and an optimal central composite design (OCCD). Nevertheless, the results section would be enhanced by a more comprehensive examination of the findings' importance. For example, the analysis would be enhanced by providing a more detailed explanation of the reasons why specific enzyme combinations were more effective and by addressing any unexpected results.
- With reference to discussions, I stress about that authors should compare their results with other similar studies and highlighting the practical implications of the optimized EAE conditions, and suggesting potential areas for future research.
-
References
- The reference list contains one reference 2019, one 2020, one, 2021, and no references for years 2022, 2023, and 2024. Kindly update your reference list.
Comments on the Quality of English LanguageThe English of this manuscript needs revision.
Author Response
The manuscript "Optimization of Enzymatic Assisted Extraction of Bioactive Compounds from Olea Europaea Leaves" presents a study on the utilization of enzyme-assisted extraction (EAE) to extract polyphenols from olive leaves. The research is well-organized, and it offers insights into the optimization of EAE for industrial applications. Nevertheless, additional clarification and enhancements are necessary before the manuscript can be considered for publication.
Authors Reply: We would like firstly to thank reviewer for his/her efforts to evaluate our study. We revised the manuscript according to yours and others reviewers suggestions/comments and we believe that you would find now the revised manuscript suitable for publication in Biomass/MDPI.
Abstract
- Reviewer 3: Olea europaea, should be written in Italic
Author : Thank you for the suggestion. Revised
- Reviewer 3: “TP optimal extraction conditions (468.19 mg GAE/L)”, the term GAE should be defined when it is first mentioned.
Author: : Thank you for the suggestion. Revised
- Reviewer 3: “EAE (50–50%)”, is it w/w, v/v, w/v,..etc.
Author: : Thank you for the suggestion. Revised
Introduction
- Reviewer 3: Figure 1 is not mentioned in the text. Please refer to this figure before its location in the manuscript.
Author: : Thank you for the suggestion. Figure 1 has been deleted
- Reviewer 3: The introduction provides a solid background on the significance of olive leaves as a source of bioactive compounds, it would be also helpful if authors have included a paragraph about the advantages of using enzymatic process.
Authors: : Thank you for the suggestion. Added see the revised introduction section.
- Reviewer 3: Kindly add recent references to highlight the latest advancements in this field.
Authors: Thank you for the suggestion. Please find our response at your last comment.
Materials and methods
- Reviewer 3: It would be better to visualize your methods by adding a flow chart describes your methods steps.
Authors: Thank you for the suggestion. In the revised version a flow chart was added.
Results and discussion
- Reviewer 3: The results are presented in a straightforward manner, utilizing the appropriate number of tables and figures. It is noteworthy to employ a simplex-centroid design and an optimal central composite design (OCCD). Nevertheless, the results section would be enhanced by a more comprehensive examination of the findings' importance. For example, the analysis would be enhanced by providing a more detailed explanation of the reasons why specific enzyme combinations were more effective and by addressing any unexpected results.
Author: Thank you for the suggestion. Some more details have been added in the conclusions
- Reviewer 3: With reference to discussions, I stress about that authors should compare their results with other similar studies and highlighting the practical implications of the optimized EAE conditions, and suggesting potential areas for future research.
Authors: Thank you for the suggestion. Up to our knowledge this is the first optimization of enzyme-assisted extraction on olive leaves field. Nevertheless, there are not even other trials with enzymes except some combinations with enzymes like enzymatic and microwave-assisted extraction which mentioned in this study and compared the results. About the future research please check the added part in conclusions.
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References
- Reviewer 3: The reference list contains one reference 2019, one 2020, one, 2021, and no references for years 2022, 2023, and 2024. Kindly update your reference list.
Authors: Thank you for the suggestion. Unfortunately, there are not references on this field the last 3-4 years. All the extraction trials on olive leaves bioactive compounds are referred in this paper up to our research.
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have improved the revised manuscript according to the reviewer's comments so the manuscript is now acceptable for publication.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors have considered the reviewer's comments. I suggest publication.
