Ultrasound-Assisted Extraction of Flavonoids from Kiwi Peel: Process Optimization and Bioactivity Assessment
, Maria Inês Dias 1, Ricardo C. Calhelha 1, Dejan Stojković 3, Marina Soković 3, Débora Tavares 4, Analía Laura Cánepa 2, Isabel C. F. R. Ferreira 1, Cristina Caleja 1,* and Lillian Barros 1,*Round 1
Reviewer 1 Report
The manuscript describes the development of efficient methodology for the extraction of bioactives from kiwi-peel. The ultrasound-assisted extraction protocol under various condition has been examined based on the response surface methodology. The experiments were designed and carried out appropriately, and the conclusions have been drawn based on solid experimental evidences and unambiguous interpretation of those. The depiction of the results (Tables and Figures) is very neat and understandable, and descriptions are presented for good readability. Overall this manuscript is of high quality and has significance in view of process development. Some minor concerns are as below.
- On page 3, the authors name the variables as Y1, Y2, and etc., however, they use variables such as Yyield, YBtEC, and etc. on pages 6 and 7. The way variables are named should be unified.
- It would be helpful for readers to provide HPLC chromatograms of the extract obtained under the optimized condition as a Figure in the manuscript.
- The description of antioxidant activity of the extract can be misleading. It seems that trolox shows higher IC50 values (lower activity) compared to the extract (line 411-413). Then the expression ‘lower values’ or the last sentence (line 413-415) does not match. The sentences and values should be revised carefully.
Author Response
: The manuscript describes the development of efficient methodology for the extraction of bioactives from kiwi-peel. The ultrasound-assisted extraction protocol under various condition has been examined based on the response surface methodology. The experiments were designed and carried out appropriately, and the conclusions have been drawn based on solid experimental evidences and unambiguous interpretation of those. The depiction of the results (Tables and Figures) is very neat and understandable, and descriptions are presented for good readability. Overall this manuscript is of high quality and has significance in view of process development. Some minor concerns are as below.
Answer: The authors thank the Reviewer for the careful reading and analysis of the manuscript and for the constructive observations and suggestions, which were considered during the preparation of the revised version of the manuscript.
On page 3, the authors name the variables as Y1, Y2, and etc., however, they use variables such as Yyield, YBtEC, and etc. on pages 6 and 7. The way variables are named should be unified.
Answer: Thank you for the constructive observation. The name of the independent variables was properly unified throughout the manuscript.
It would be helpful for readers to provide HPLC chromatograms of the extract obtained under the optimized condition as a Figure in the manuscript.
Answer: As suggested, a representative HPLC-chromatographic profile of phenolic compounds in the kiwi peel extract was provided in Figure S1.
The description of antioxidant activity of the extract can be misleading. It seems that trolox shows higher IC50 values (lower activity) compared to the extract (line 411-413). Then the expression ‘lower values’ or the last sentence (line 413-415) does not match. The sentences and values should be revised carefully.
Answer: Thank you for the constructive observation. The sentence was revised accordingly.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript presents the optimization by central composite rotatable design coupled with Response Surface Methodology of the ultrasound-assisted extraction (UAE) of four flavonoids from kiwi peel with etanol:water to study the bioactivity of the extracts with potential use as a food preservative ingredient. Authors consider 3 variables (time, ethanol concentration and ultrasonic power) in a 5-level experimental design, though they do not consider other solvents or extraction temperature.
Authors identify four flavonoids in kiwi peel extracts by chromatographic analysis, but they do not show the unidentified peaks, and they seem to forget the 99% components of the extracts. It is contradictory to maximize extraction yield, up to 59% w/w, and focus only in 4 components that represent 0.15% of the final extract. It would have been more logical to include other responses instead of total extraction yield. Since the amount of the 4 flavonoids is only 1.51 mg of flavonoids per g of extract, it is clear that hundreds of components of the extract could be responsible of the observed bio-activities. This fact should be considered and the manuscript revised accordingly.
Authors should add chromatograms of the extracts, including non identified peaks, and consider that other major components may be responsable of the bioactivity of the extracts, not just the 4 mentioned compounds. Moreover, HPLC-DAD-ESI/MSn method should be included and adequately described.
The English language should be improved throughout the manuscript. For instance, in Abstract, line 29: Authors write: “…were the experimental responses used in the optimization.”
They probably mean: “…where the experimental responses used in the optimization.”
There are many references included that are of difficult access, not general, and many in Portuguese. Most of them could be changed by others. For instance, Reference 8, on the potencial of fruit by-producs as sources of bioactive molecules, it is not specially relevant and it should be changed by other of the dozens previous articles on the topic.
“The study of the potentiality of fruits by-products as sources of bioactive molecules still has been little explored, thus becoming a niche of opportunities for obtaining high-added value molecules [4,8].“
Furthermore, the reference list is too long, with several articles not directly related with the topic and many self-citations that could be removed.
Line 243: Correct: FLVAV: total content of flavonoids.
Line 386: Correct: “were fund to”
Author Response
The manuscript presents the optimization by central composite rotatable design coupled with Response Surface Methodology of the ultrasound-assisted extraction (UAE) of four flavonoids from kiwi peel with etanol:water to study the bioactivity of the extracts with potential use as a food preservative ingredient. Authors consider 3 variables (time, ethanol concentration and ultrasonic power) in a 5-level experimental design, though they do not consider other solvents or extraction temperature.
Answer: The authors thank the Reviewer for the careful reading and analysis of the manuscript and for the constructive observations and suggestions, which were considered during the preparation of the revised version of the manuscript. In this study, ethanol-water mixtures were selected as extraction solvent for being non-toxic food-grade solvents authorized in the food industry. In addition, the ultrasonic power was selected as extraction intensification factor due to the advantages over temperature that have been reported in the literature. This information was added to the introduction of the revised manuscript.
Authors identify four flavonoids in kiwi peel extracts by chromatographic analysis, but they do not show the unidentified peaks, and they seem to forget the 99% components of the extracts. It is contradictory to maximize extraction yield, up to 59% w/w, and focus only in 4 components that represent 0.15% of the final extract. It would have been more logical to include other responses instead of total extraction yield. Since the amount of the 4 flavonoids is only 1.51 mg of flavonoids per g of extract, it is clear that hundreds of components of the extract could be responsible of the observed bio-activities. This fact should be considered and the manuscript revised accordingly.
Answer: Thank you for the comment. In fact, the detected flavonoids represented a low percentage of the obtained extract. However, the author believe that the remaining fraction of the kiwi peel extract is composed mainly by fibers and other carbohydrates, given the intrinsic nature of this by-product. Our study was focused on phenolic compounds, since these secondary metabolites are commonly linked to antioxidant and other bioactive effects, which are of interest when developing natural bioactive ingredients. Therefore, given the impossibility of including further experimental responses, the following sentence was added to the revised manuscript: “compounds other than flavonoids were being extracted (possibly fibers and other carbohydrates, given the intrinsic nature of the kiwi peel) and represented the major fraction of the obtained extract”.
Authors should add chromatograms of the extracts, including non identified peaks, and consider that other major components may be responsable of the bioactivity of the extracts, not just the 4 mentioned compounds. Moreover, HPLC-DAD-ESI/MSn method should be included and adequately described.
Answer: A representative HPLC-chromatographic profile of phenolic compounds in kiwi peel extract was provided in Supplementary Material Figure S1. As can be observed in the HPLC-chromatographic profile, there are no other unidentified compounds that have quantitative significance to be considered as response variables in this optimization study. The description of the chromatographic method was improved as suggested.
The English language should be improved throughout the manuscript. For instance, in Abstract, line 29: Authors write: “…were the experimental responses used in the optimization.” They probably mean: “…where the experimental responses used in the optimization.”
Answer: The English language was carefully improved. In the abstract, we actually mean: “were the experimental responses used in the optimization”.
There are many references included that are of difficult access, not general, and many in Portuguese. Most of them could be changed by others. For instance, Reference 8, on the potencial of fruit by-producs as sources of bioactive molecules, it is not specially relevant and it should be changed by other of the dozens previous articles on the topic.
“The study of the potentiality of fruits by-products as sources of bioactive molecules still has been little explored, thus becoming a niche of opportunities for obtaining high-added value molecules [4,8].“
Furthermore, the reference list is too long, with several articles not directly related with the topic and many self-citations that could be removed.
Answer: Thank you for the observation. The reference list was double-checked and updated as suggested, as well as the citations throughout the manuscript.
Line 243: Correct: FLVAV: total content of flavonoids.
Answer: Thank you for the observation. The name of the independent variables was revised accordingly throughout the manuscript.
Line 386: Correct: “were fund to”
Answer: Thank you for the observation. The correction was made accordingly.
Author Response File: Author Response.docx
Reviewer 3 Report
Article with a lot of theoretical and experimental work involved. The results show that the methodology used is interesting to optimize the extraction of substances with potential applications. In addition, tests are carried out to demonstrate that the extract obtained has properties of interest.
Check Y3 on lines 119 and 121
Author Response
Article with a lot of theoretical and experimental work involved. The results show that the methodology used is interesting to optimize the extraction of substances with potential applications. In addition, tests are carried out to demonstrate that the extract obtained has properties of interest.
Answer: The authors thank the Reviewer for the careful reading and analysis of the manuscript and for the constructive observations and suggestions, which were considered during the preparation of the revised version of the manuscript.
Check Y3 on lines 119 and 121
Answer: Thank you for the observation. The correction was made accordingly.
Author Response File: Author Response.docx
