Extraction Optimization and Valorization of the Cornelian Cherry Fruits Extracts: Evidence on Antioxidant Activity and Food Applications

Round 1

Reviewer 1 Report

Dear Authors, all comments and suggestions are included in the pdf file attached to this letter.

Comments for author File: Comments.pdf

Author Response

Thank you for your suggestions!

We submit the response to reviewer 1 report.

Author Response File: Author Response.docx

Reviewer 2 Report

Manuscript is interesting and has possibility ofpractical applications. Ststistical analyse gives practical results.

Author Response

Thank you for your review process. We resubmit our manuscript.

Reviewer 3 Report

Line 205 must be on the next page

Line 240 I would suggest writing the formula and deleting the picture

Lines 242 and 243 the chemical formula must have subscript

Line 265 same with line 240

Line 305 the title of the table must be on the same page as the table

Line 307 conventional what? I assume extraction

Line 346 the title of the table must be on the same page as the table

Line 393 same as 346 and 305

Line 419 same as 393, 346 and 305

I would suggest figure 3 be smaller so the title can be seen under the picture and not on a different page

The alignment of the tables and figures is too much to the left

Line 547 (Table 6) is not aligned correctly

Line 563 please move the title on a different page

 

 

Comments for author File: Comments.pdf

Author Response

Thank you for your suggestion and comments.
We resubmit our manuscript and response to the reviewer 3 report.

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear Authors,

The article has substantial flaws when describing the optimization procedure and the results. The most important remark is that there was no reason to do the optimization procedure, since further analysis, for example, vitamin C was performed not only at optimal conditions but also at other values of studied variables. Moreover, only single-response desirability function were used for the calculation of the optimal response (according to the text p. 320-331 and chapter 3.3. including data from Table 5). Authors could optimize all parameters at once, obtaining the optimal conditions that enable to maximize all responses (AA, TPC and TFC) at the same time, which would have resulted in one value of desirability function coefficient.

Another substantial remark is that all results for the optimization procedure were carried out in duplicate, nevertheless the Authors showed the SD values (of course the computer program will calculate them). As far as I know for the calculation of SD value we need at least three replicates. Authors performed the external validation of the model; however, the procedure was not described in the method section. Moreover, the validation procedure was repeated only twice (it is not enough to show the valid values of SD). Additionally, the DPPH procedure for assessing the antioxidant activity has raised my doubts, and I could not accept Authors’ explanations.

Author Response

Thank you for your suggestions!

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

In current study an attempt was made to optimize extraction from cornelian cherry fruit with high antioxidant activity. The extraction was carried out in ethanol, the authors called such method of extraction as a conventional (CE). The authors varied the concentration of ethanol (100, 80 and 60%), temperature of extraction (30, 40 and 50°C) and time of extraction (15, 30 and 45 min). The authors also conducted experiments of extraction with ultrasound (ultrasound assisted extraction, UAE) in 60% ethanol and 40°C, while varying  time of extraction (5, 12,5 and 15 min), amplitude of sonication (40, 60, 80, 100%) and sonication mode (0.5, 0.75 and 1).

For the selection of optimal parameters (for three independent variables) the authors used Box-Behnken experimental design. This allowed them to find some trends and determine the optimal parameters among those combinations that were used for the Box-Behnken experiment itself. In particular, for CE method, the best combination of parameters was: ethanol 60%, temperature 40°C and time of extraction 15 min.

The very formulation of the experiment raises questions.

1) Why was the experiment carried out only once for each combination of parameters (except for the central points)? For example, for center points U13-U15 there was no clear convergence, so it would be important to make all points in triplicate, for greater reliability. Carrying out the experiment in triplicate would also make it possible to concretize the conclusions. For example, the authors say TFC is best extracted at 30-30-60 (T-t-EtOH), sample C7. However, the value of 1.61 for TFC is only slightly higher (by 0.08) than 1.53 for TFC for sample C8 (extracted at 40-15-60 T-t-EtOH). For two samples of center point replicates C13 and C14 the dispersion of values even higher (0.1), 1.05 and 1.15, respectively.

2) If the experiment was performed only once for each point, from what data the spread of values for the TPC, TFC and TAA come from? For example, C1 sample - TPC (mg GAE/g dw) 26.68±0.52? Where did the value ±0.52  come from? Is this an instrument error?

3) For the Box-Behnken experiment, certain combinations of parameters were used, which were taken, including on the basis of literature data. As a result of AE experiment, it turned out that when ethanol was varied in the range of 100-80-60%, the extraction improves (for total of the three studied groups of extracts), the extraction also improves in the range of temperatures 50-40-30° C, and in the series in time 45-30-15 min. In this regard, it seems that the optimization of the extraction parameters has not been completed. No inflection points were found, those values of temperature, extraction time and ethanol concentration at which the extraction would deteriorate.

The work is devoted to the selection of parameters for extraction, it is not completed. It has not been shown that reducing ethanol to 55-50% will lead to a deterioration in the extraction. With the current trend in the studied material, it seems that a decrease in the concentration of ethanol, on the contrary, can lead to an increase in the yield of extracted metabolites. Likewise, it is necessary to find the extremum points for the temperature and incubation time. That is, to reduce the temperature, time, ethanol concentration and prove that this leads to a deterioration (and not an improvement) in extraction.

4) The most interesting variant of the extraction of 30-15-60 T-t-EtOH in connection with the processed results was not performed at all during the experiment. After preliminary selection of the parameters in the Box-Behnken experiment, it would be necessary to set up new experiments in order to optimize the extraction. In its current form, the work is seen not as a scientific experiment, but as an illustration of a Box-Behnken experiment. Also, not optimized from the point of view of the maximum yield of the studied components, the extracts were used to study vitamin C content.

5) The authors neither explain nor discuss the optimization results obtained. Why does lower ethanol, temperature and extraction time increase TPC, TFC and TAA? An attempt to understand what is happening would help to sightedly approach the optimization of the extraction conditions.

 6) Since the method assumes centrifugation at 9° C for 20 min, it should also be controlled whether the extraction takes place at 9°C and what contribution it brings? This is especially important for UAE, when a short extraction time of 5-15 min was used. It is possible that the extraction took place not only during 5-15 min of incubation at the given parameters, but also 20 min during the centrifugation. In this regard, it would be necessary to make an amendment.

Work requires significant improvement in its current form can not be published.

   

Other remarks:

 

Line 16

What concentration was the ethanol stock used in the work? 100% or it was 95.5% ethanol, the more commercially available? If you used 95.5% ethanol (as 100%), do you need to recalculate 80% and 60% ethanol (it turned out to be slightly lower in concentration)?

 

Line 83 “as far as the authors’ knowledge”

remove this phrase. If you write a phrase and do not refer to a literary source, it is clear that this comes from your knowledge of the problem

 

Lines 118-119: “Increasing the extraction time up to 30 min, at the temperature 30°C, allowed to obtain the highest concentration of the TFC (1.61±0.01 mg QE/g dw)”

This cannot be asserted, since sample C8 has a TFC (1.53 ± 0.02mg QE / g dw), which lies within the scatter of the method, as can be seen from the repetitions for samples of the central point С13 и С14, TFC 1.05±0.01 and 1.15±0.08, respectively.

Author Response

Please see the attachment. Thank you!

Author Response File: Author Response.pdf

Reviewer 2 Report

I have revised the manuscript art.no. HORTI32182R1

 

Title: Extraction optimization and valorization of the cornelian cherry fruits extracts: evidences on phytochemical profile and food applications

 

The authors present a research article focused on the influences of different extraction methods to retain more polyphenols after the ext.

The results appear very interesting and could have some application in the food industry, but the analyses conducted appear too superficial to describe the changes in the phytochemical content. Indeed, only the total phenol content, total flavonoid content, ascorbic acid content, and DPPH was done. I advise further analyze the phytochemical profile changes with an HPLC or through other instruments. Moreover, DPPH analysis alone should be strengthened by other antioxidant activity analyses such as FRAP and ORAC.

Although the author reports interesting aspects (to improve further), many things need to be re-analyzed. For these reasons, I think that the ms should not be considered for publication in Molecules.

Author Response

Please see the attachment. Thank you!

Author Response File: Author Response.pdf

Reviewer 3 Report

Line 55, remove extra space between “of” and “several,” it appears that there is extra space.

Line 92, change to “is to establish”

Line 96-99, reword sentence structure (i.e., The second part of the study, was to evaluate the vitamin C content….)

Line 99-100, reword sentence (i.e., The cornelian cherry extract with the highest antioxidant activity was then selected for…)

Section 2 Results, write out the acronyms for TPC, TFC, and TAA as well as the units. I see that it is written out in the methods section, but one would have to search the entire document to understand the context.

Figure 2, the y-axis should be clearly explained in the figure caption.

In Section 3.4.6, please add a short sentence on the importance of doing a textural analysis of jelly candies.

In Section 3.3, please add a short sentence explaining why one would need to demonstrate adding cornelian cherry extracts in jelly candy formulation. Is there a limit or a criterion for jelly candy formulation that needs to be met? Would add extracts change this criterion?

Author Response

Please see the attachment. Thank you!

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors took into account the comments made. And wherever possible they made the necessary corrections.

 

1) Unfortunately, one of the main questions about the experiment, the authors were unable to resolve (“Why  was  the  experiment  carried  out  only  once  for  each  combination  of  parameters  (except  for  the central points)? For example, for center points U13-U15 there was no clear convergence, so it would be important to make all points in triplicate, for greater reliability. Carrying out the experiment in triplicate would also make it possible to concretize the conclusions” ©). The authors note that «All 15 runs were performed ones due to the lower amount of the raw material». However, the raw material itself (ripe cornelian cherry fruits) doesn't look exotic or hard to get. Just to conduct an experiment, you need to prepare it in the required amount so that each point can be represented in three repetitions. In order to demonstrate that the selected parameters are optimal for extraction from ripe cornelian cherry fruits from studied region, it would be worth repeating the experiment three times from the very beginning.

2) Major (not minor) revision of the article, which was set in the review, suggested a significant revision of the experiment, carrying it out in triplicate. Also, the determination of the extremum points in the selected parameters, that is, those values at which the extraction begins to deteriorate. However, the authors did not set up additional experiments, they just found an error in the previously published data and corrected it.

3) The authors only answer in the review, operating with literary data (“Based on previous works related to the extraction of bioactive compounds, concentrations of ethanol higher than 50% increased the extractability of these compounds. That is the reason why the experimental ranges were selected” ©). However, there is no evidence-based experimental base in the experiment itself on a specific object from a specific area. On it, the optimization of the expression is not completed (since neither the deterioration nor the improvement of extraction has been proven with a decrease in the concentration of ethanol, a decrease in the temperature and extraction time).

4) The question was: “The authors neither explain nor discuss the optimization results obtained. Why does lower ethanol, temperature and extraction time increase TPC, TFC and TAA? An attempt to understand what is happening would help to sightedly approach the optimization of the extraction conditions” ©.

The authors did not add this discussion to the text of the article, but only briefly (without references to literary sources) replied to the reviewer. If the corresponding experiments with decreasing the values of the selected parameters were not carried out, at least they should have been discussed in the article itself.

The above remarks show that the authors made not a major but a minor revision of the article. In this regard, further work is required, namely the setting of additional experiments.

Reviewer 2 Report

The authors did not resolve any issues raised in the previous comments. For these reason, once again I think that the ms should not be considered for publication in Molecules.