Optimization of Ultrasonication Probe-Assisted Extraction Parameters for Bioactive Compounds from Opuntia macrorhiza Using Taguchi Design and Assessment of Antioxidant Properties

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editor,

In the paper entitled “Optimization of ultrasonication probe-assisted extraction parameters for bioactive compounds from Opuntia macrorhiza using Taguchi design and assessment of antioxidant properties” the authors analyzed the effectiveness of different variants of the ultrasound extraction method in order to determine their efficiency in extracting biologically active ingredients from one of the cactus species. Traditional extraction techniques are mainly used to extract bioactive compounds from this raw material, therefore determining the possibility of using modern, effective and ecological ultrasound technique for this purpose seems promising.

Despite the interesting research topic, before publishing this article in Applied Sciences, the authors should complete and correct it according to the following comments:

1.       The introduction to the article should provide the current state of knowledge on the use of ultrasonic techniques for the extraction of biologically active ingredients from Opuntia macrorhiza or other varieties of this raw material. It is not enough to write that mainly traditional extraction techniques based on shaking (stirring) are used for this raw material.

2.       We wstępie do pracy należy podać aktualny stan wiedzy na temat wykorzystania ekstrakcji ultradźwiękowej do wydobywania składników biologicznie aktywnych z Opuntia macrorhiza lub innych odmian kaktusów. Niewystarczające jest napisanie, że w stosunku do tego surowca wykorzystuje się głównie tradycyjne techniki ekstrakcyjne oparte na wytrząsaniu.

3.       The authors should explain why they used ethanol for ultrasonic extraction and not another organic solvent. They should also provide full conditions of classical extraction (e.g. at what time was it carried out? what solvent was used?), because without such information it is difficult to compare the efficiency of both methods, which was carried out in chapter 3.4.

4.       When planning the extraction conditions (Table 1, Table 2) - please explain in more detail what assumptions were made for the Taguchi method, as this will enable understanding of the differences between the parameters used for the individual extraction variants (e.g. why was an extraction time of 4, 12 and 20 min chosen and not, for example, 3, 6, 9 min or 5, 10, 15 min or 10, 20, 30 min?).

5.       In the methodological part of the article, I propose to present as separate points the determination of phenolic compounds and two methods for determining antioxidant activity with indication of analytical details (e.g. determination conditions, number of parallel repetitions, etc.).

6.       Looking at the methodological assumptions indicated in Table 2, I wonder whether it would not be better to design an experiment in such a way, instead of the Taguchi method, that the influence of each of the analyzed factors could be compared separately. For example, analyzing different proportions of the solvent used in relation to the same product sample and performing extraction under constant conditions (the same solvent composition, pulsation and extraction time), and then examining the effect of different solvent composition using it in the same proportion to the product sample, performing extraction at the same (selected) pulsation and the same time, etc. Although this would generate the need to perform a much larger number of analyses, I believe that it would allow for a more unambiguous determination of the effect of each factor separately. In the current version of the article, when using the Taguchi method, the methodological assumptions listed in Table 2 resemble the "random" method. I ask the authors to present their own opinion on this subject in their response to the review.

7.       In lines 238-243 the authors analyze the antioxidant activity of the extracts obtained by comparing data measured by different methods (FRAP and DPPH) for pulp and peel treated as one population. As a conclusion from such a comparison they state that pulp had higher antioxidant activity, which is absolutely not the result of the used comparison. This should be corrected.

8.       The authors performed many statistically advanced studies obtaining interesting results, but they did not discuss them sufficiently. For example, they did not indicate what caused the differences in the degree of extraction of the analyzed compounds using the ultrasonication probe-assisted extraction (UPAE)  and the stirring extraction (STE). What is the extraction mechanism of each of these techniques and how does it affect the extraction of individual compounds? Some fragments of the statistical description were prepared in a complicated way using typical statistical concepts (e.g. text from lines 405-415), probably understandable mainly to specialists in this field, and yet the statistical analysis should serve to explain the obtained results in the context of clearly relating them to the purpose of the article. Did the used statistical analysis allow for drawing unambiguous conclusions regarding the comparison of the tested extraction techniques? Is one of these techniques (UPAE and STE) better? And if so, should it be used for both pulp and peel? What are the advantages and disadvantages of each of these methods? Should the UPAE technique then be recommended for use only for ecological reasons, or is it statistically more effective in the case of Opuntia macrorhiza than stirring extraction? The answers to these questions could also be used to supplement section 4, i.e. conclusions from the conducted research.

In addition to the remarks presented above, I would also like to ask the authors to read the comments that I included in the manuscript attached to the review.

 

 

Comments for author File: Comments.pdf

Author Response

Dear Editor,

In the paper entitled “Optimization of ultrasonication probe-assisted extraction parameters for bioactive compounds from Opuntia macrorhiza using Taguchi design and assessment of antioxidant properties” the authors analyzed the effectiveness of different variants of the ultrasound extraction method in order to determine their efficiency in extracting biologically active ingredients from one of the cactus species. Traditional extraction techniques are mainly used to extract bioactive compounds from this raw material, therefore determining the possibility of using modern, effective and ecological ultrasound technique for this purpose seems promising.

We would like to acknowledge the reviewer for his/her positive feedback on our manuscript.

Despite the interesting research topic, before publishing this article in Applied Sciences, the authors should complete and correct it according to the following comments:

1. The introduction to the article should provide the current state of knowledge on the use of ultrasonic techniques for the extraction of biologically active ingredients from Opuntia macrorhiza or other varieties of this raw material. It is not enough to write that mainly traditional extraction techniques based on shaking (stirring) are used for this raw material.

The recovery of Opuntia spp. was the subject of numerous investigations that employed ultrasonication baths. The authors believe that further research was required to improve the extraction process of Opuntia macrorhiza, given its nutritional significance, prospective benefits, and significant applications in the food and pharmaceutical industries. Given the scarcity of studies that utilize ultrasonication probe-assisted extraction, the primary goal of this study was to examine the specific extraction technique in OM fruit peels and pulp. The Introduction section has been revised accordingly.

2. We wstępie do pracy należy podać aktualny stan wiedzy na temat wykorzystania ekstrakcji ultradźwiękowej do wydobywania składników biologicznie aktywnych z Opuntia macrorhiza lub innych odmian kaktusów. Niewystarczające jest napisanie, że w stosunku do tego surowca wykorzystuje się głównie tradycyjne techniki ekstrakcyjne oparte na wytrząsaniu.

Google Translate: 2. The introduction to the work should include the current state of knowledge on the use of ultrasonic extraction to extract biologically active ingredients from Opuntia macrorhiza or other varieties of cacti. It is insufficient to write that traditional extraction techniques based on shaking are mainly used for this raw material.

The specific comment has been previously responded to.

3. The authors should explain why they used ethanol for ultrasonic extraction and not another organic solvent. They should also provide full conditions of classical extraction (e.g. at what time was it carried out? what solvent was used?), because without such information it is difficult to compare the efficiency of both methods, which was carried out in chapter 3.4.

Water and ethanol are recognized as food-grade solvents, which are suitable for use in the food industry as they are safe for human consumption. The closing sentence of Section 3 of the manuscript elucidates the rationale for selecting these solvents for extraction. Regarding conventional extraction, it is stated in section 2.4. that the extraction was carried out based on an optimized method using water as a solvent, 20 mL/g liquid-to-solid ratio through stirring hotplate at 500 rpm, at 40 °C for 2 h.

4. When planning the extraction conditions (Table 1, Table 2) - please explain in more detail what assumptions were made for the Taguchi method, as this will enable understanding of the differences between the parameters used for the individual extraction variants (e.g. why was an extraction time of 4, 12 and 20 min chosen and not, for example, 3, 6, 9 min or 5, 10, 15 min or 10, 20, 30 min?).

It is crucial to address this information in the text, and we are grateful to the reviewer for the chance to elaborate on it. It has been reported that the UPAE belongs to the green extraction techniques and that it is substantially faster than conventional extraction (STE). The rationale for employing the UPAE time interval (4-20 min) is that the maximal extraction time is considerably shorter than the typical limit for conventional extractions (1 or 2 h). Furthermore, a four-minute interval between extraction design points was deemed sufficient in comparison to a three-minute interval, which was considered short, and a ten-minute interval, which was considered longer. However, a five-minute interval could have also been applied. To that end, the manuscript has been revised to incorporate the reviewer's suggestions.

5. In the methodological part of the article, I propose to present as separate points the determination of phenolic compounds and two methods for determining antioxidant activity with indication of analytical details (e.g. determination conditions, number of parallel repetitions, etc.).

The determination of phenolic compounds and two methods for antioxidant activity assessment are now separated with further details provided, as recommended by the reviewer. Regarding the number of experiments repetitions, it is stated that all experiments had been conducted in triplicate in section 2.9.

6. Looking at the methodological assumptions indicated in Table 2, I wonder whether it would not be better to design an experiment in such a way, instead of the Taguchi method, that the influence of each of the analyzed factors could be compared separately. For example, analyzing different proportions of the solvent used in relation to the same product sample and performing extraction under constant conditions (the same solvent composition, pulsation and extraction time), and then examining the effect of different solvent composition using it in the same proportion to the product sample, performing extraction at the same (selected) pulsation and the same time, etc. Although this would generate the need to perform a much larger number of analyses, I believe that it would allow for a more unambiguous determination of the effect of each factor separately. In the current version of the article, when using the Taguchi method, the methodological assumptions listed in Table 2 resemble the "random" method. I ask the authors to present their own opinion on this subject in their response to the review.

The Taguchi method has become a widely used technique in fields such as technology and engineering because of its capacity to improve processing quality, reduce the number of experiments required, mitigate processing duration, and promote quality and stability. This approach has become a commonly used approach for evaluating the impact of interactions among a variety of controllable parameters during the ranking and screening process. In addition, the rationale for selecting the most impactful extraction conditions was to investigate their extreme values within a reasonable range through a limited number of experiments. Section 2.5. has been revised accordingly.

7. In lines 238-243 the authors analyze the antioxidant activity of the extracts obtained by comparing data measured by different methods (FRAP and DPPH) for pulp and peel treated as one population. As a conclusion from such a comparison they state that pulp had higher antioxidant activity, which is absolutely not the result of the used comparison. This should be corrected.

We acknowledge that we compared results from two different parts of the fruit in order to examine the overall antioxidant capacity of the fruit, which is not necessarily correct. In order to fully clarify this comparison to the general public, the manuscript has been revised accordingly.

8. The authors performed many statistically advanced studies obtaining interesting results, but they did not discuss them sufficiently. For example, they did not indicate what caused the differences in the degree of extraction of the analyzed compounds using the ultrasonication probe-assisted extraction (UPAE) and the stirring extraction (STE). What is the extraction mechanism of each of these techniques and how does it affect the extraction of individual compounds? Some fragments of the statistical description were prepared in a complicated way using typical statistical concepts (e.g. text from lines 405-415), probably understandable mainly to specialists in this field, and yet the statistical analysis should serve to explain the obtained results in the context of clearly relating them to the purpose of the article. Did the used statistical analysis allow for drawing unambiguous conclusions regarding the comparison of the tested extraction techniques? Is one of these techniques (UPAE and STE) better? And if so, should it be used for both pulp and peel? What are the advantages and disadvantages of each of these methods? Should the UPAE technique then be recommended for use only for ecological reasons, or is it statistically more effective in the case of Opuntia macrorhizathan stirring extraction? The answers to these questions could also be used to supplement section 4, i.e. conclusions from the conducted research.

It is mentioned in section 3 that the rationale for the comparison between UPAE and STE was that the target molecules can be effectively extracted using cavitation. Figure 5 illustrates the findings related to the bioactive components and antioxidant capability of OM pulp and peels. Specifically, it is feasible to analyze which approach is more effective for each section of the fruit. For the recovery of total polyphenols from OM, UPAE is more efficient than STE, and peels are superior to pulp as input material. Conversely, optimal ferric-reducing capacity necessitates STE in OM pulp. The application of STE in OM peel is more effective for the extraction of total betalains. The efficiency of each technique is a matter of extraction duration, “green” nature, and overall cost. To that end, the discussion of 3.4. section and the Conclusions have been revised correspondingly.

In addition to the remarks presented above, I would also like to ask the authors to read the comments that I included in the manuscript attached to the review.

Please read below the responses to your comments.

 

From PDF file:

L125: Why ethanol?

The benefits of using ethanol and water were mentioned earlier and are further highlighted in the manuscript.

L131: What were these assumptions based on for conventional extraction? Was it a single extraction? Over what time period was it conducted? What solvent was used?

Regarding conventional extraction (STE), it is stated in section 2.4. that the extraction was carried out using a 20 mL/g solid-to-liquid ratio through stirring hotplate at 500 rpm, at 40 °C for 2 h. Water was used as a solvent, which is now indicated in the manuscript.

L133: Maybe it is worth to put this sentence immediately after the sentence from lines 131-132?

The specific sentence has been rearranged, as recommended by the reviewer.

L138: Please explain in more detail what assumptions were made in the Taguchi method, as this will allow for understanding the chosen parameters, especially extraction times. Why are the differences between the individual variants the way they are and not others (e.g. 10, 20 and 30 min? Or 3, 6 and 9 min)?

The specific comment has been previously responded.

L148: The title of this table is stylistically incorrect.

The title of Table 1 has been changed following the reviewer’s suggestion.

L149: The sound of this title is unusual, I suggest making a stylistic change.

The title of Table 2 has been changed correspondingly.

L154: Why not the same reagent which was used for extraction?

The most used solvent in the specific Folin-Ciocalteu protocol demands methanol probably due to the greater solubility of gallic acid which is now mentioned in the manuscript.

L156: I think it would be better if the determination of antioxidant activity was discussed as two separate methodological points (each method should be discussed separately). Analytical details should also be indicated, e.g. measurement conditions, number of parallel repetitions, etc.

The specific comment has been previously responded.

L207: I wonder if this entire fragment is necessary and whether it would not be worth starting the discussion of the results from point 3.1?

This text explains the rationale behind why the optimization of OM peel and pulp extraction was necessary through Taguchi design, by using green and food-grade solvents. In addition, the use of these solvents causes the extraction of certain pigments, as the magenta-colored betacyanins are water-soluble, while the greenish-colored betaxanthins are more than an alcoholic solvent. However, this text has been moved appropriately so that section 3 of the manuscript starts with subsection 3.1, as suggested by the reviewer.

L212: This was given for the purpose of the work, is there a need to repeat it at the beginning of the discussion of results? Maybe it is worth referring to these assumptions only after characterizing your own results?

The specific text has been revised appropriately in order to avoid repetition.

L238: This fragment should start with a new paragraph, because it is not known whether the authors are writing about the results of Aruwa et al. [28] or their own results.

To provide a clearer picture of our results, the specific fragment now starts in a new paragraph, as per the reviewer’s suggestion.

L254: And what about Opuntia macrorhiza?

The manuscript has been revised appropriately referring to the same trend that applies to O. macrorhiza, which has higher betalain content (BC) than O. ficus-indica.

L257: If all betalains are soluble in water, why do ethanol extracts contain betaxanthins belonging to betalains?

Betacyanins, a chemical subclass of betalains, are water-soluble and have a magenta color, as stated several times throughout the manuscript. The authors meant “betacyanins” instead of “betalains” and have now corrected the typographical error.

L269: This fragment should also start from the new paragraph

The specific fragment now starts in a new paragraph, as suggested by the reviewer.

L294: but factor x4 was more important in peels

It is now highlighted that the X4 parameter (i.e., extraction duration) was found to have a significant impact on OM peels rather than pulps, as revealed by Partial Least Squares analysis.

L296: Similar to pulp? Or to X2 factor?

It is now highlighted in the manuscript that the X2 factor had a negative effect on both peels and pulp.

L354: It is worth stating these conditions again, or referring to table 5

The analyzed data from Partial Least Squares analysis gave the presented optimum conditions for each part of OM fruit, in which it is highlighted that are different regarding the UPAE duration.

L355: Under what conditions was this extraction performed (solvent composition, time, amplitude)?

The optimized method study by Prakash Maran and Manikandan was previously cited in section 2.4.; however, the conditions of the extraction are now mentioned again to provide a clearer picture to the readers.

L360: diverse?

The term “diverge” has been removed. The authors believe that the term "deviate" is more appropriate to express that experimental and predicted values were close.

L361: This sentence is not clear. What is being compared to what?

The sentence has been rewritten to highlight that the predicted TPC values from Partial Least Squares analysis were slightly higher by 6 and 10% in pulp and peels when compared to the experimental values, respectively.

L373: It is worth deciding whether differences between these values were found or not? Because if they were not found statistically, it means that these values are statistically the same and there is no need to write that one value is higher or lower.

The authors refer to this finding to emphasize that no significant difference in the extraction of total betalains was observed. However, the specific sentence has been corrected.

L389: This is strange, because usually the content of phenolic compounds in individual anatomical parts of plant materials is different. What could be the reason for this? What do other authors write about it?

It is now clarified that OM peel had higher polyphenolic content than pulp, regardless of the extraction technique.

L405: Please rephrase this fragment to clearly present the statistical verification of the obtained results in relation to the assumptions of the paper?

The specific comment has been previously responded to.

Reviewer 2 Report

Comments and Suggestions for Authors

The study revolves around the optimisation of extraction parameters of bioactive compounds from cactus fruit (Opuntia macrorhiza) by ultrasonic probe-assisted extraction (UPAE) technique and the evaluation of their antioxidant properties, which is innovative and of applied value, but there are some areas for improvement in the elaboration of the research motivation and some experimental designs.

 

Major concerns:

1) The authors provide a more detailed description of the cactus in the introductory section, but there is insufficient elaboration as to why Opuntia macrorhiza, a specific cactus species, was chosen for the study. It is recommended to further emphasise the uniqueness of this species compared to other cactus species in terms of bioactive compound content or potential application value to better illustrate the relevance and necessity of the study.

2) Although the authors used a Taguchi design for the optimisation of the extraction parameters, the application of this method in plant extract research has become more common in the current research context. The authors should have highlighted more the unique and innovative points of this study in terms of the extraction target (Opuntia macrorhiza), the combination of bioactive compounds of interest (polyphenols, betaines, and ascorbic acid), and the results in comparison with other extraction techniques.

3) In the ultrasound probe-assisted extraction experiments, the authors examined parameters such as liquid-solid ratio, extraction time, ethanol concentration and pulse frequency. However, the range of settings and the basis for selecting the levels of some parameters were not clearly described. For example, the ethanol concentration was only set at three levels of 0%, 50% and 100%, which may not fully reflect the effect of ethanol concentration on the extraction effect. It is suggested that the authors should refer to more related studies to optimise the parameter settings more reasonably and explain the basis of the settings in detail.

4) The authors compared UPAE with stirred extraction (STE), but set up only one set of experimental conditions for STE (40°C, 500 rpm, 2 h). Different extraction temperatures, stirring speeds and times may significantly affect the extraction results, so a single control condition may not provide a comprehensive and accurate comparison of the advantages and disadvantages of the two extraction methods. It is recommended that the authors increase the experimental conditions set for STE to more fully evaluate the advantages of UPAE over STE.

5) The authors used a variety of data analysis methods such as Taguchi design, multivariate factor analysis (MFA), multivariate correlation analysis (MCA) and partial least squares (PLS). However, there is a lack of clarity as to why these particular data analysis methods were chosen and how they complement each other to better interpret the experimental results. It is recommended that the authors provide a detailed explanation in the data analysis section of the rationale for and advantages of each method's selection and how the combined application of these methods can lead to more reliable conclusions.

6) In the discussion of results section, the authors have analysed the experimental results to some extent, but the discussion of some key results could be more in-depth. For example, when comparing the extraction effects of UPAE and STE, although it is mentioned that UPAE has advantages in the extraction of some bioactive compounds, the mechanism of this difference is not discussed deeply enough. In addition, there was a lack of in-depth discussion on the relationship between the changes in antioxidant activity of bioactive compounds and the structure and content of the compounds under different extraction conditions. It is recommended that the authors further explore the mechanisms behind the experimental results to improve the scientific validity and depth of the study.

 

Comments on the Quality of English Language

none

Author Response

The study revolves around the optimisation of extraction parameters of bioactive compounds from cactus fruit (Opuntia macrorhiza) by ultrasonic probe-assisted extraction (UPAE) technique and the evaluation of their antioxidant properties, which is innovative and of applied value, but there are some areas for improvement in the elaboration of the research motivation and some experimental designs.

We would like to thank the reviewer for his/her contribution to improving the readability of our manuscript.

Major concerns:

1) The authors provide a more detailed description of the cactus in the introductory section, but there is insufficient elaboration as to why Opuntia macrorhiza, a specific cactus species, was chosen for the study. It is recommended to further emphasise the uniqueness of this species compared to other cactus species in terms of bioactive compound content or potential application value to better illustrate the relevance and necessity of the study.

Opuntia macrorhiza is a cactus species with fruits known for its antioxidant, antifungal, and antimicrobial properties. The fruit contains bioactive compounds such as polyphenols and unique compounds that provide the reddish color of its peels and pulp, betalains. Other Opuntia species including O. ficus-indica, O. dillenii, and O. engelmanii have also been studied through ultrasonication bath techniques. The aim of the study has been adjusted highlighting the absence of research studies including valorization of O. macrorhiza fruit peels and pulp and optimization of its extraction using ultrasonication probe technique. To that end, the Introduction section has been revised, as suggested by the reviewer.

2) Although the authors used a Taguchi design for the optimisation of the extraction parameters, the application of this method in plant extract research has become more common in the current research context. The authors should have highlighted more the unique and innovative points of this study in terms of the extraction target (Opuntia macrorhiza), the combination of bioactive compounds of interest (polyphenols, betaines, and ascorbic acid), and the results in comparison with other extraction techniques.

A key aspect of the research was the ability to target polyphenol extraction or betalain recovery using statistical tools such as Partial Least Squares analysis and Canonical Plot, which provide a graphical representation of our results. The model would then recommend the most suitable extraction technique and from which part of the fruit, as revised and further addressed in the Discussion (section 3.4).

3) In the ultrasound probe-assisted extraction experiments, the authors examined parameters such as liquid-solid ratio, extraction time, ethanol concentration and pulse frequency. However, the range of settings and the basis for selecting the levels of some parameters were not clearly described. For example, the ethanol concentration was only set at three levels of 0%, 50% and 100%, which may not fully reflect the effect of ethanol concentration on the extraction effect. It is suggested that the authors should refer to more related studies to optimise the parameter settings more reasonably and explain the basis of the settings in detail.

The rationale behind optimizing the specific extraction parameters is further discussed in the Methodology section, also highlighting the use of water and ethanol for their food-grade applicability. In addition, these two solvents could extract different types of betalains; betacyanins which have a reddish color and are water-soluble, and betaxanthins which have a greenish hue and are extracted through ethanol, as also illustrated in Figure 1. The study also included a Partial Least Squares analysis in which the optimum conditions (including ethanol concentration) were identified. The manuscript is now further enhanced with several studies using aqueous or alcoholic extracts to provide a clearer picture of the solvent selection in Opuntia species.

4) The authors compared UPAE with stirred extraction (STE), but set up only one set of experimental conditions for STE (40°C, 500 rpm, 2 h). Different extraction temperatures, stirring speeds and times may significantly affect the extraction results, so a single control condition may not provide a comprehensive and accurate comparison of the advantages and disadvantages of the two extraction methods. It is recommended that the authors increase the experimental conditions set for STE to more fully evaluate the advantages of UPAE over STE.

An optimized conventional extraction technique using a stirring hotplate and water as solvent was used to compare the obtained results from UPAE and was initially stated in the manuscript. In addition, we stated that ethanol and water were used for their food-grade applicability in the food industry. The advantages of UPAE and STE methods including the swift process and green nature are further elucidated in the manuscript. However, we also added that different extraction techniques could be used in either OM peels or pulp in order to target polyphenols or betalains extraction or generate an extract with antioxidant activity in the Discussion section through the canonical plot.

5) The authors used a variety of data analysis methods such as Taguchi design, multivariate factor analysis (MFA), multivariate correlation analysis (MCA) and partial least squares (PLS). However, there is a lack of clarity as to why these particular data analysis methods were chosen and how they complement each other to better interpret the experimental results. It is recommended that the authors provide a detailed explanation in the data analysis section of the rationale for and advantages of each method's selection and how the combined application of these methods can lead to more reliable conclusions.

Information about Taguchi's design of the experiment has been provided in detail within the Methodology and Discussion section. PLS was used to investigate the impact of each extraction parameter, with the results also being graphically illustrated in Figure 4A-D and provided a model with the optimized extraction conditions. Regarding the two correlation analyses, MFA interprets the relationship between extraction conditions and variables under investigation (i.e., TPC, FRAP, DPPH, etc.) as it discriminates them correspondingly. MCA simply compares the correlation of the measured variables between them. The canonical plot visually represents those findings and serves as a foundation for identifying various targets in a prospective extraction. For instance, targeting polyphenol recovery from OM fruit is best achieved through the use of peels with UAE, whereas for betalains recovery, employing peels with STE is more suitable. All of the above explanations are now inserted in the manuscript to shed more light on these statistical tools, as suggested by the reviewer.

6) In the discussion of results section, the authors have analysed the experimental results to some extent, but the discussion of some key results could be more in-depth. For example, when comparing the extraction effects of UPAE and STE, although it is mentioned that UPAE has advantages in the extraction of some bioactive compounds, the mechanism of this difference is not discussed deeply enough. In addition, there was a lack of in-depth discussion on the relationship between the changes in antioxidant activity of bioactive compounds and the structure and content of the compounds under different extraction conditions. It is recommended that the authors further explore the mechanisms behind the experimental results to improve the scientific validity and depth of the study.

To further elucidate the effects of ultrasonication in the solid matter which resulted in elevated TPC values from the UPAE method compared to STE, a possible mechanism has been introduced in section 3.4. However, the relationship between the chemical structure of the identified polyphenols and the antioxidant activity of the extracts cannot be determined with high precision, as quantification of most polyphenols was not feasible (only ~40% of the total polyphenols were quantified). Nonetheless, this limitation is addressed in the manuscript within the same section.

Reviewer 3 Report

Comments and Suggestions for Authors

The purpose of the article was the optimization of opuntia’s ultrasound extraction with the use of Taguchi designs. I find the publication interesting and shows a new approach to the topic of extraction.

The authors performed several tests, described them in quite detail, analyzed the obtained results, and discussed them extensively. However, the work contains shortcomings and requires some changes.

I am asking that the authors address the following comments and make appropriate changes to the text:

-          - Introduction. Add the description of Taguchi design, including the main idea/principles, and why this method was used in this study.

-           - 2.5. Experimental Design - Did the authors measure temperature changes during the process (longer use of ultrasound causes sample heating)?

-          - 2.7. Lack of validation data (standard curve, LOD, LOQ). Were content determinations performed for all compounds simultaneously?

-          - 3. Results – check rounding of numbers – the general rule is that the uncertainty should be rounded to a maximum of two significant figures. e.g. in Table 6 (BC) – 4 significant digits!

https://www.nist.gov/system/files/documents/2019/05/14/glp-9-rounding-20190506.pdf

- Table 7 – units of concentration are missing.

 - Conclusion – The data in Table 7 don’t support this statement. Results from Table 7 are similar for UAE and STE methods (actually, for STE they are even better).

Author Response

The purpose of the article was the optimization of opuntia’s ultrasound extraction with the use of Taguchi designs. I find the publication interesting and shows a new approach to the topic of extraction.

We would like to appreciate the reviewer’s recommendation for improving the clarity of our paper.

The authors performed several tests, described them in quite detail, analyzed the obtained results, and discussed them extensively. However, the work contains shortcomings and requires some changes.

I am asking that the authors address the following comments and make appropriate changes to the text:

- Introduction. Add the description of Taguchi design, including the main idea/principles, and why this method was used in this study.

The advantages offered by Taguchi design (limited number of experiments, swift process, low solvent consumption, robust results, etc.) are now mentioned in the Introduction section, as suggested by the reviewer.

- 2.5. Experimental Design - Did the authors measure temperature changes during the process (longer use of ultrasound causes sample heating)?

The authors initially stated that the temperature did not rise above 40 °C. It is now mentioned that the temperature was measured before, during, and after the procedure in section 2.4.

- 2.7. Lack of validation data (standard curve, LOD, LOQ). Were content determinations performed for all compounds simultaneously?

Detailed information about the simultaneous quantification of individual polyphenols is now incorporated in the Methodology section, as recommended by the reviewer.

- 3. Results – check rounding of numbers – the general rule is that the uncertainty should be rounded to a maximum of two significant figures. e.g. in Table 6 (BC) – 4 significant digits!

https://www.nist.gov/system/files/documents/2019/05/14/glp-9-rounding-20190506.pdf

We have rounded the numbers in the table.

- Table 7 – units of concentration are missing.

The units of measurement have now been added to Table 7 (now Table 8).

 - Conclusion – The data in Table 7 don’t support this statement. Results from Table 7 are similar for UAE and STE methods (actually, for STE they are even better).

No statistically significant differences were observed in the individual polyphenol concentration between the two methods in Table 7 (now Table 8). The STE method was used to compare the obtained results using the UPAE method. However, it is now stated in the Discussion section that in this study it was possible to evaluate which method is more effective for each part of the OM fruit. UPAE is more effective than STE for the extraction of total polyphenols from OM, with peels being a superior material compared to pulp. Conversely, optimal ferric-reducing capacity requires STE in OM pulp. The utilization of STE in OM peel is more efficacious for the extraction of complete betalains. The efficacy of each technique depends on extraction duration, environmental sustainability, and total cost. The discussion in section 3.4 is further expanded and the Conclusions have been updated accordingly.