Enhancing Efficiency of Enzymatic-Assisted Extraction Method for Evaluating Bioactive Compound Analysis in Mulberry: An Optimization Approach

Round 1

Reviewer 1 Report

The topic of the manuscript (agronomy-2614229-peer-review-v1) is an interesting because mulberry is widely used in traditional medicine in Asian and daily fruit in many European countries. In the study, the authors optimized an enzyme-assisted extraction method for the efficient retrieval of bioactive compounds from mulberry. The results showed the extracted mixed compounds showcased significant antioxidant and antimicrobial properties. Meanwhile, some concepts should be carefully revised. There are many problems that have not been explained clearly. 1) As we know, anthocyanins belong to phenolic, are anthocyanins the main components of mixed extracts? 2) Anthocyanins and phenolics which play an important role in antioxidant activity and antimicrobial activity?

Author Response

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Reviewer 2 Report

I have carefully read and checked the manuscript entitled Enhancing Efficiency of Enzymatic-Assisted Extraction Method for Evaluating Bioactive Compound Analysis in Mulberry: An Optimization Approach. Although lengthy, there are some major points missing:

1. The whole study design lacks a comparison sample, extracted without the enzyme. This is a crucial point to be referred to, otherwise the whole study is incomplete.

2. The results from chromatographic analysis: It is not clear why the study has three methods for analysis, on different machines, when an UHPLC-MS was available. None of the methods was neither re-used, as stated by a reference, nor validated, as seen from the text. All of the qualitative and quantitative methods must be properly validated, when developed and newly-reported, in order to prove suitable for the purpose.

How is it possible with one reference standard for anthocyanins, to identify four glycosides, two of which, diglycosides? This identification is not at all acceptable, moreover, as seen from fig. 1, there is no separation in TOF-MS. What about the isobars of the reported compounds in this extract? Fig 2 is not a good proof, either. Peaks 2 and 4 are, with peak high, not quite distinguishable from the baseline noise. All the methods are twice as long, as the presented chromatograms. Why is this? If a separation is, as reported 9 minutes, the chromatogram illustrating the separation should at least have those identifiable peaks, with a time axis long, as the gradient elution. This remark concerns all the chromatography-related text in Material and methods and in Results. In my opinion, the analyses must be run again, with authentic reference substances for all the compounds mentioned. In addition, there are concentrations missing. The description of material and methods should be re-written in such a way, so the results could be reproduced, in order to clear the manuscript of any doubts on analysis. 

2. How the authors will explain the high percentage of ethanol used? Seventy percent is questionable in respect to enzyme stability. Were there any experiments, proving that the enzyme is not inactivated? How about the same on its thermostability? Why the results are not compared to an extract, made only with ethanol, without the enzyme? Why there were no experiments with ethanol with concentration below 70%? Anthocyanins, as flavilium salts, are quite soluble in plain water. 

3. The whole statistics is excessive, when the input reference data is missing. See my remark on the sample without the enzyme. This must be combined with statistics from validation of all of the analytical methods used.

4. The discussion has to be re-written in aspect of comparison with non-enzyme treated sample. 

5. The conclusion is to be revised with comparison to non-enzyme treated sample, also.

6. Supply chromatograms for each separation, following the methods that you describe. All the peaks, corresponding to identified compounds, should be annotated, and the chromatogram of each standard solution must be supplied for comparison for the positive identification. If the authors insist on using MS, they must supply the fragmentation spectra for each identified compound and the same for the reference substance for undoubtful identification.

7. The introduction has to be checked again for generalised statements as "valuable for the pharmaceutical industry and medicine". If something is reported as a background or justification of the study, please, be precise.

The study has its merit. I am afraid that it has to be thoroughly revised, so the lacking comparison be added, the identification to be justified, the chromatograms - full and comprehensive.

English needs minor re-check from authors. Overall quality is good.

Author Response

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Round 2

Reviewer 1 Report

The author made careful revisions.

Author Response

The authors express their gratitude to the reviewer.

Reviewer 2 Report

The manuscript was clearly improved.

However, the identification of metabolites by LC-MS is speculative in this form. There are no clear peaks to identify, the compounds are eluting in a very narrow segment (2 to 3 min), and are overlapping. My suggestion is this part to be omited from the manuscript. Reference 52 is only on flavonoids, with no particular information on anthocyanins. 

The control sample without the enzyme is still missing. Please, refer to my previous report. Without this sample, the information is partial, and the results and discussion not practically justified.

The problems with figure S3 remain - see my remark for peaks 2 and 4.

 

 

 

Author Response

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Round 3

Reviewer 2 Report

The manuscript is improved, omitting the speculative ,, identification " by LC-MS. Please, remove section 2.4. from Material and methods, dealing with the removed section.

Please, give at least a full set of PDA chromatograms, recorded at the proper wavelength, of a representative sample with both anthocyanins and phenolics labelled above peaks, and compared with the standard compounds used for quantitation, with the respective tR clearly visible.

The purpose was not to delete, but to replace with informative ones.

 

 

Author Response

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