Microwave-Assisted Extraction of Phenolic Compounds from Pineapple Peel Using Deep Eutectic Solvents

Round 1

Reviewer 1 Report

The presented manuscript is correctly written, but nevertheless describes the known methods. I believe that it could be accepted after major revision. The term "polyphenols" should be clarified. It is a very wide group of compounds, which include, among others: tannins, flavonides or anthocyanins. The authors write about polyphenols, but they mainly mention phenolic acids. In order to draw the correct conclusions, the chemical composition of the tested extracts in terms of compounds with a polyphenol character should be more precisely determined. Especially that both - the type of the reagent used for the extraction, and the extraction method used- may and, as reported in the literature, have an impact on the type and amount of extracted compounds. Hence, the radical scavenging activity will be different. Therefore, it is necessary to determine the qualitative and quantitative composition of the examined extarcts in the context of polyphenolic compounds.

There is no explanation as to what caused the higher extraction results for using choline chloride-glycerol than other extraction solvents. The reference to the literature results is not enough. Please refer to your tested extracts.

The first paragraph in section 3.2 is incomprehensible. What compounds were tested in these extracts? As previously indicated by the authors, the tested extracts contain polyphenols. So where does the conclusion come from: "opposite to that found for the polyphenolic content"? Besides, flavonoids are also polyphenols.

Line 186: The RSA values in this research are higher than those reported in other studies  - There is no explanation as to what this could be caused by?

 Line 191-192: I can't agree with that. Difference between EtOH 50% and MeOH 100% looks as it is lower then beetween methanol 100% and Methanol 50%. And EtOH 50% was not included in that conclusion. Why authors use notation: 100% methanol? It is pure reagent, so we do not neeed such notation. When comparing this notation to water, we should also write 100% water

 

Error bars on Fig. 4 are quite different comparing different solvents. I wonder, should it be that way?

 

In my opinion, the manuscript in its current form is too general and requires more detail and correction of the conclusions drawn.

 

Author Response

Evaluator's comments	Reply
. The authors write about polyphenols, but they mainly mention phenolic acids. In order to draw the correct conclusions, the chemical composition of the tested extracts in terms of compounds with a polyphenol character should be more precisely determined. Especially that both - the type of the reagent used for the extraction, and the extraction method used- may and, as reported in the literature, have an impact on the type and amount of extracted compounds. Hence, the radical scavenging activity will be different. Therefore, it is necessary to determine the qualitative and quantitative composition of the examined extarcts in the context of polyphenolic compounds”	The word polyphenols was changed to phenolic compounds; however, the chemical composition of the extracts was not determined.
There is no explanation as to what caused the higher extraction results for using choline chloride-glycerol than other extraction solvents. The reference to the literature results is not enough. Please refer to your tested extracts.	This is because the extraction with DESs allows the formation of hydrogen bonds with the phenolic compounds present in the pineapple, which generates a higher affinity and therefore a higher extraction rate, in addition, the microwave extraction process decreases the viscosity of the DESs facilitating their entry into the pineapple peel matrix and the interaction with the compounds to be extracted. This explanation is included in the text
The first paragraph in section 3.2 is incomprehensible. What compounds were tested in these extracts? As previously indicated by the authors, the tested extracts contain polyphenols. So where does the conclusion come from: "opposite to that found for the polyphenolic content"? Besides, flavonoids are also polyphenols.	The correction was made in the text clarifying that the compounds measured are total phenolic compounds, not flavonoids.
Line 186: The RSA values in this research are higher than those reported in other studies  - There is no explanation as to what this could be caused by?	This explanation is included in the text
Error bars on Fig. 4 are quite different comparing different solvents. I wonder, should it be that way?	Although the figure shows a difference between the error bars, the test for equality of variances was performed, which resulted in equal variances between the data groups, implying that the deviations are due to variations inherent to the extraction process that are not controllable by the researcher.
In my opinion, the manuscript in its current form is too general and requires more detail and correction of the conclusions drawn.	Further results are added to the work that allow more concrete and specific conclusions to be drawn in this regard.

Reviewer 2 Report

Dear Authors,

Below are detailed notes for the work:

1) Line 73 - change the citation (Bubalo et al., 2016) to a number,

2) Add information about the use of microwaves in crop storage, seed stimulation, etc. to the introduction. - indicate the economic aspects of the use of electromagnetic fields.

3) How was the size of a single sample determined?

4) 2.6 Statistical analysis - the description of the method shows that you used a parametric ANOVA; Was the normality of the distribution of the population of variables tested, was the homogeneity of variance in the samples tested, with what tests, what was their result? These are the prerequisites for using parametric tests - enter this information into this section.

5) In my opinion, the share of microwaves in the experiment should be more exposed, give the radiation frequency (presumably 2.45 GHz), justify why the power was 60%, why the exposure time was 60s?

4a) see: DOI 10.15199 / 48.2018.12.58, EFFECTS OF MICROWAVE RADIATION ON THE GERMINATION OF SOLANUM TUBEROSUM L. TUBERS, The influence of selected physical methods on the content of starch and simple sugars in stored potato tubers.

6) Figure 1.… .. The results are the average of three replicates. Error bars indicate standard deviation - you have already provided this information in the methodology (it is unnecessary here)

7) Fig. 3-4 - as in point 6 above

8) Chapter 5 - this is a summary, conclusions should be constructive, emphasize the applicability of the results obtained

Author Response

Evaluator's comments	Reply
Line 73 - change the citation (Bubalo et al., 2016) to a number,	Correction is made
Add information about the use of microwaves in crop storage, seed stimulation, etc. to the introduction. - indicate the economic aspects of the use of electromagnetic fields.	Correction is made
How was the size of a single sample determined?	Please clarify the comment you are referring to.
In my opinion, the share of microwaves in the experiment should be more exposed, give the radiation frequency (presumably 2.45 GHz), justify why the power was 60%, why the exposure time was 60s?	The conditions of the extraction process were determined by preliminary tests. The optimization process was added to the article where the microwave exposure time was evaluated.
Figure 1.… .. The results are the average of three replicates. Error bars indicate standard deviation - you have already provided this information in the methodology (it is unnecessary here) 7) Fig. 3-4 - as in point 6 above	Correction is made
Chapter 5 - this is a summary, conclusions should be constructive, emphasize the applicability of the results obtained	The conclusions are modified according to the additional aggregate results.

Reviewer 3 Report

Although the subject of the work, evaluation of deep eutectic solvents for microwave-assisted extraction of bioactive compounds from pineapple peels and their antioxidant capacity, is interesting and of potential scientific interest, the level of research and the results presented are not sufficient for publication. The use of environmentally friendly solvents with low toxicity and high capacity for the extraction of bioactive compounds deserves our attention, and therefore I think that the authors should make additional efforts to complete the results by additional studies and present them according to the suggestions of the journal Horticulturae. In addition, the paper is technically poorly prepared, which makes it appear "unfinished," e.g., the literature citations are inconsistent and do not follow the journal's instructions. The authors conclude that DESs are a viable green alternative for the extraction of phenolic compounds from pineapple peels, which is supported by the high antioxidant capacity of the extracts obtained. The conclusion is very brief, which is a consequence of the insufficient results that need to be completed both in terms of content, discussion and presentation. The authors should also complete the introduction and discussion with the results of similar studies and interpret their own results and their significance in this context. Nevertheless, I believe that the authors can complete the results and restructure the paper and submit a new version of the paper that is consistent with the general statements of the journal.

Author Response

Taking into account the comments of the evaluator, the optimization section was added where the effect of drying temperature, extraction time and solvent-solid ratio on the total content of phenolic compounds and the antioxidant capacity of the extract were evaluated, this allowed to expand the discussion of the results and to give more specific conclusions to the work. 

Reviewer 4 Report

Please, read the file.

Results can be presented better. Discussion can be improved.

Comments for author File: Comments.pdf

Author Response

Evaluator's comments	Reply
Authors wanted to show that so called green solvents like DES are more convenient to extract polyphenols from pineapple peel waste. They demonstrated a higher yield of polyphenols after extraction with choline chloride –glycerol but not with choline-chloride malic acid. What was reason for the difference?	The DESS used in the present work have as a common factor the use of choline chloride as hydrogen acceptor and vary in the hydrogen donor, this leads to the behavior of the DESs not being the same. It has been demonstrated in other studies that the nature of the compound to be extracted influences the capacity of the DES to facilitate the extraction process. The results presented in this work demonstrate that the DESs with choline-glycerol chloride have greater facility for the formation of hydrogen bonds with the antioxidant compounds present in pineapple.
Preparation of DES is energy, time, money consuming. What happens with the waste? Of course, it is not a problem in laboratory, but in industrial application?	The DES used in this work are considered in the group of natural deep eutectic solvents (NADESs), this means that the compounds used are common in the cosmetic, pharmaceutical and food industries, which allows these extracts to be used directly in a product without the need for purification processes, which implies less cost, production time and waste generation.
Figure 2 does not show anything new, the yield is proportional to polyphenolics content.	Correction is not taken into account as Yield is an important factor for the selection of a solvent in an extraction process.
Figure 3 RSA % should by recalculated by dry weight or by phenolic content similarly to	As mentioned by the evaluator, this result can be presented in units of mg/GDw, however both have different meanings since the % inhibition indicates the capacity of the extract to inhibit a concentration of the DPPH radical, and is a function of the bioactivity of the extract, while the antioxidant capacity measured as trolox equivalents is a measure of the amount of antioxidant compounds as a function of the dry matter of the pineapple peel. As authors, we consider it important to show the inhibition capacity of the extracts, however, more results were added to the paper where this response variable was analyzed from the perspective of the amount of phenolic compounds.
Figure 4 for ORAC. In Figure 4 missing results for CC-malic acid.	No results were obtained for this extract because it interfered with the measurement technique.
Conclusions are set up only for yield of phenolic compounds with CC-glycerol, but their antioxidant activity recalculated by dry matter showing that better solvent is a mixture water-ethanol. So, what is the aim, to get more various polyphenols or to have higher biological activity? Firstly, polyphenols due to their chemical variability react with radical by different mechanisms. Secondly, the other compounds (not phenols) can contribute also to biological effects. Thirdly, polyphenolics are not only antioxidants but also pro-oxidants.	The conclusions were modified according to the new results analyzed.
Some mistakes are found in citing references – line 30, ref. 1, line 73, 176, 177,181…It should be unified. Fig. 1 and line 146 using DW for dry weight, but in Fig. 4 using dm for dry matter or mass ..it should be unified. Line 102 50% water is unknown at least for some of us.	Correction was made

Round 2

Reviewer 2 Report

Additional explanations for authors:

Thank you for taking into account some of the comments.

1) size of a single sample

Minimum sample size - how much data are needed for the maximum estimation error (confidence level 1 - \ alpha) to be "d" or less. The more observations, the more accurate the estimate is (thus the confidence interval will be narrower).

2)      Anderson-Darling test and Bonferroni test; no values obtained in the result part

Author Response

Dear reviewer, thank you very much for your comments.
Regarding comment 1. It is still not clear for us that correction, for the experimental design 3 replicates were used for each treatment as usual in this type of work. 
2. The results of the normality test and equal variances were added to the text.

 

Reviewer 3 Report

Dear Authors,

Below are some of the errors observed, which mainly belong to the technical errors, but also affect the overall impression of the work. Please correct the above as well as similar unspecified irregularities...:
Line 32: in several places (e.g., in line 32) the space between the last word in the sentence and the reference is missing
Line 35, 239: the dot should be at the end of the sentence (after the reference number)
Line 36, 74, 91, 111-112, 233: spaces between paragraphs of the same chapter are unnecessary
Line 73: dot is missing
Line 105: reference 22 is quoted twice in the same sentence
Line 100 - 110: In my opinion, the English should be improved in several places, e.g., The general principle of DES is its ability to donate and absorb hydrogen. This ability allows the formation of hydrogen bonds with the components to be extracted and facilitates the extraction process [22]. DESs are a mixture of a quaternary ammonium salt, such as choline chloride, and a compound that can act as a hydrogen donor, such as sugars, vitamins, amines, alcohols, and carboxylic acids. The mixture of these compounds results in a solution with optimal properties for the extraction of natural products such as polyphenols [22]. DESs based on the use of choline chloride, known as CH -DESs, are characterized by significant retention of bioactive target compounds, better biodegradability compared to other DESs, low cost, low toxicity, and environmental compatibility. These properties make them a promising alternative for food, pharmaceutical and cosmetic applications [23].
Line 156: References describing the technique should be cited at the end of the sentence. Deviations from the technique should only be stated if they exist.
Line 220: A space is missing after 3.1.
Line 266, 344: dot is missing
Line 307: reference number 30 is cited twice. As I can see, reference 29 is missing from the main text
Line 309: "In Figure 5, the behavior of total phenolic compound content from pineapple peels is observed" - this should be reworded, the authors should convey more clearly and precisely to the reader the meaning of the new chapter and the results presented
Lines 321-322: The text under Figure 5 needs to briefly explain the meaning of Figures a, b, and c.
Lines 377-380: The authors should conclude the last chapter with their conclusions from the presented experiments and end the discussion with the general meaning of the presented results. In the way presented, I have the impression that the discussion is unfinished.

Author Response

Dear reviewer, thank you very much for your comments.
Regarding the comments related to the form of the document, they were taken into account and corrected in the text. 
Regarding the comment on line 309, an explanation of the optimization chapter was added, and the conclusion of the optimization chapter was expanded as suggested. 

Reviewer 4 Report

Better than before.

Author Response

Thank you very much for your comments.