Extraction of Total Anthocyanins from Sicana odorifera Black Peel Fruits Growing in Paraguay for Food Applications

Round 1

Reviewer 1 Report

This work describes the hydroalcoholic extraction of total anthocyanins from the peel of S. odorifera, optimised by experimental design. These extracts may have various practical applications which add will value to what would otherwise be a wasted resource. The analysis of the extracts has been well handled and described well, and in general the manuscript is detailed and well written. However the sequence of experimental design optimisation using two software packages is difficult to understand.
There are two separate experimental designs, one developed and analysed using Minitab software (design matrix in Table 4), while the other uses DesignExpert (design matrix in Table 5). These appear to cover different design spaces, for example solvent concentration (20% to 70% in Table 4, and 60% to 100% in Table 5) as well as a different range for S-L ratio. It is unclear how these two experimental designs are related to each other.
Why weren't these experimental runs handled in one software package throughout, in one integrated design? The relationship between these designs needs to be clearly established (perhaps in an expanded Section 1.7).

 

There are some minor detail and formatting concerns.

L226: give an estimate of the peel thickness. 

At L158 '1.5.2. Development of the response surface' - should be in the results section, not materials and methods.
L372-383 should go in the Conclusion

Many results, especially for GAE, are given with unjustified numbers of decimal places in tables and e.g. in Line 204 (10132.07±336.32 mg GAEs/100 g DW). Expressing this result as 10.13±0.33 g demonstrates that quoting mg amounts with 2 or even (in Tables 4 and 5) 3 places of decimals implies a level of analytical precision that is totally unrealistic.
The Liquid-Solid value is not really a ratio (a ratio is unitless e.g. 80:1); at the first use of this parameter in L27 the value is given in mg/L which is incorrect (should be mL/g).
It would be acceptable to write the abbreviation S. odorifera every time after the first use.
Also be consistent when writing numbers (L321-322: contains both '14' and 'twelve') and temperatures (space before degree sign or not).

Author Response

Comments and Suggestions for Authors

This work describes the hydroalcoholic extraction of total anthocyanins from the peel of S. odorifera, optimised by experimental design.  These extracts may have various practical applications which add will value to what would otherwise be a wasted resource. The analysis of the extracts has been well handled and described well, and in general the manuscript is detailed and well written.

The authors are thankful for your comments on the improvement of the manuscript. It has been added a line of relevance and the possible practical applications, as it was suggested
(See Line 255-265), and the entire manuscript has been reviewed and edited to improve its presentation.

However the sequence of experimental design optimisation using two software packages is difficult to understand. There are two separate experimental designs, one developed and analysed using Minitab software (design matrix in Table 4), while the other uses DesignExpert (design matrix in Table 5). These appear to cover different design spaces, for example solvent concentration (20% to 70% in Table 4, and 60% to 100% in Table 5) as well as a different range for S-L ratio. It is unclear how these two experimental designs are related to each other. Why weren't these experimental runs handled in one software package throughout, in one integrated design? The relationship between these designs needs to be clearly established (perhaps in an expanded Section 1.7).

We appreciate your comments and consider that they are relevant to improve this document. Regarding the design, the first is a screening design that allows identifying the most important variables of the process. In this case, initially, 5 variables were considered, three of which were significant.

With the intention of improving the extraction conditions, the second design of the experiments was carried out considering only the 3 most significant variables for the process (solvent concentration, solid/liquid ratio, and pH of the dissolution). The ranges studied in the second design (respond to Figure 2b where the trend of the different variables is shown). We chose the Design expert software due to the quality of the obtained graphics. Nonetheless, following this recommendation, the document was modified and the two designs are currently evaluated in Minitab.

There are some minor detail and formatting concerns.

L226: give an estimate of the peel thickness.

An estimate of the peel thickness (1-2 mm) has been incorporated in the results section following your suggestion. In addition, an explanation of the parts of the analyzed fruit has also been added and a description of the separation of the peels, previous to obtain the extract in the materials and methods section.

At L158 '1.5.2. Development of the response surface' - should be in the results section, not materials and methods.

The authors welcome your comment. The mentioned section was modified according to the suggestion.

L372-383 should go in the Conclusion

This comment has been considered by adding these results in the conclusion, thank you very much.

Many results, especially for GAE, are given with unjustified numbers of decimal places in tables and e.g. in Line 204 Line 295 (10132.07±336.32 mg GAEs/100 g DW). Expressing this result as 10.13±0.33 g demonstrates that quoting mg amounts with 2 or even (in Tables 4 and 5) 3 places of decimals implies a level of analytical precision that is totally unrealistic.

The authors are especially grateful for this observation. Certainly these “unreal” decimals have been dragged from the averages recorded in Excel spreadsheets and they have been corrected, as seen in tables 4 and 5.

The Liquid-Solid value is not really a ratio (a ratio is unitless e.g. 80:1); at the first use of this parameter in L27 the value is given in mg/L which is incorrect (should be mL/g).

Thank you very much, this inadvertent bug has been fixed.

It would be acceptable to write the abbreviation S. odorifera every time after the first use.
Also be consistent when writing numbers (L321-322: contains both '14' and 'twelve') and temperatures (space before degree sign or not).

Based on these suggestions, the entire manuscript was modified, thank you very much. Comments and suggestions are appreciated.

Author Response File: Author Response.docx

Reviewer 2 Report

Paper title:

Extraction of total anthocyanins from Sicana odorifera black 2 peel fruits growing in Paraguay for food applications 

 

SUMMARY

The manuscript is well written in an engaging and lively style, the subject is important and RSM is currently something of a “hot topic”, although the response analyzed should be carefully selected. The integration of high technologies such as UPLC-ESI-QqQ-MS works in both positive and negative aspects to authors, this characterization is relevant and the results will be very handy for the science community, although, the same identification could be used for quantification and optimization of the extractions tested. It does not make a lot of sense to use powerful technology such as UPLC and ultrasound while the response used could lead to many errors due to interference.

This manuscript almost ticks all the boxes we have in mind for a fingerprinting of bioactive molecules and subsequent optimization, but the response analyzed should be determined in a more reliable way.

 

There are also some editorial mistakes that have to be double-checked.

 

 

Essential issues

The robustness of the data presented in the optimization response as well as the author's choice for the optimal point should be clarified.

In order to proceed with the publication of this manuscript, authors should consider the inclusion of optimization of results from the UPLC quantification or consider another type of journal for this publication as it is.

 

Suggestions

In line 129 of section 1.3 add “followed by centrifugation and filtration” after “ultrasonic bath (15 min)”, otherwise, ultrasonic bath and centrifugation might be confusing for readers.

 

In line 130, the second extraction was made following the parameters of the first one just changing the solvent? Or was stirred and kept for 30 min at room temperature in the dark? The authors should clarify better the methodology.

 

In line 132, the authors used the word plotted for the spectrophotometric reading, and although a calibration curve is performed, there is not a real need for plotting the result, consider using words such as “read” or “registered”.

 

In line 145 consider replacing “medium” for “dissolution”

 

In line 154, the text has a reference error, please correct it.

 

In table 1, is the solid to liquid ratio ok? Authors 

 

Considering authors used an Ultrasound bath and longer times, authors should provide how they were able to control the temperature of the extraction? In my own experience, I have worked with both an ultrasound bath and ultrasound prove and external equipment is needed to overcome the heating generated by the own ultrasound. Please, provide the information.

 

In line 163, the text has a reference error, please correct it.

 

In section 1.5.2, why authors choose the box-Behnken design instead of a CCD which could provide a little more information?

 

In line 219, the text has a reference error, please correct it.

 

In line 224, the text has a reference error, please correct it.

 

In table 3, what is the difference between peel and pulp vs extract? Pulps and peels are not extracted? Provide relevant info at the bottom of the table.

 

References still displaying an error, please revise the whole document

 

In equation 2, the L-S ratio2 is not significant, remove also that term from the equation.

 

In figures 4, figure b) and c) are repetitions, please remove or add others that provide more information.

 

In lines, 296-298 authors recommend 15 minutes as optimal point, but in the screening analysis, factor time was not significant, also, if authors decided to ignore the statistics and based their criteria on a little higher amount as shown in figure 2 a), are those 3 mg important compared with the time and energy consumed?

 

It seems that line 302 information is missing.

 

In line 308 authors again refer to peel vs extract, please clarify this, for the determinations authors do not extract compounds from peel?? What is the difference between peel and extract? Please provide this information.

 

It seems that at line 319 information is missing.

 

Figure 5 is repeated in the text, please correct. Also, the colored text has to be black.

 

In lines 417-423, optimal conditions should be revised, my concern is that the authors seem not considered energy and time consuming for this optimization, but the main drawback might be the way in which the response was measured since the authors have characterized chromatographically the molecules, optimization should rely on chromatographic quantification since spectrophotometric assays are only good screening methods, but are not recommended for qualifications or optimizations, since plenty of interferences happens on this broad measurements.

 

In lines, 424-429 format is needed

 

Reference 29 is incomplete, please provide the whole information

Comments for author File: Comments.pdf

Author Response

REVIEWER 2

 

Paper title:

 

Extraction of total anthocyanins from Sicana odorifera black peel fruits growing in Paraguay for food applications

 

 SUMMARY

 

The manuscript is well written in an engaging and lively style, the subject is important and RSM is currently something of a “hot topic”, although the response analyzed should be carefully selected. The integration of high technologies such as UPLC-ESI-QqQ-MS works in both positive and negative aspects to authors, this characterization is relevant and the results will be very handy for the science community, although, the same identification could be used for quantification and optimization of the extractions tested. It does not make a lot of sense to use powerful technology such as UPLC and ultrasound while the response used could lead to many errors due to interference.

This manuscript almost ticks all the boxes we have in mind for a fingerprinting of bioactive molecules and subsequent optimization, but the response analyzed should be determined in a more reliable way.

There are also some editorial mistakes that have to be double-checked.

 

Essential issues

 

The robustness of the data presented in the optimization response as well as the author's choice for the optimal point should be clarified. In order to proceed with the publication of this manuscript, authors should consider the inclusion of optimization of results from the UPLC quantification or consider another type of journal for this publication as it is.

 

Thank you very much for your comments. The differential pH method was chosen to monitor anthocyanin extraction over the UHPLC method due to its availability, simplicity, and economy. Although our institution has UHPLC, the equipment is not always available and LC-MS solvents are very expensive. Further, the pH differential method is currently employed to monitor anthocyanin extraction conditions for optimization purposes (Jiang et al., 2017; Yin et al., 2017). It has been shown that although the values obtained by this method may differ from those of HPLC due to the individual components of each sample, the trend is the same. In other words, the values obtained by both methods show a high correlation among each other, therefore the samples with a high value by HPLC will show a high value per pH differential (Lee, Rennaker and Wrolstad, 2008; Lao and Giusti, 2016). Taking into account the mentioned above, the results reflect a trend that would be independent of the mentioned quantification methods.  Editorial mistakes has been edited, thank you for yours comments, they are usefull to improvement the manuscript.

 

 

Suggestions

 

In line 129 of section 1.3 add “followed by centrifugation and filtration” after “ultrasonic bath (15 min)”, otherwise, ultrasonic bath and centrifugation might be confusing for readers.

In line 130, the second extraction was made following the parameters of the first one just changing the solvent? Or was stirred and kept for 30 min at room temperature in the dark? The authors should clarify better the methodology.

In line 132, the authors used the word plotted for the spectrophotometric reading, and although a calibration curve is performed, there is not a real need for plotting the result, consider using words such as “read” or “registered”.

In line 145 consider replacing “medium” for “dissolution”

In line 154, the text has a reference error, please correct it.

In line 163, the text has a reference error, please correct it.

In line 219, the text has a reference error, please correct it.

In line 224, the text has a reference error, please correct it.

References still displaying an error, please revise the whole document

It seems that line 302 information is missing.

 

All these edit details have been corrected. Was considered and the terms was changed. Thankyou.

 

In table 1, is the solid to liquid ratio ok? Authors

 

It is correct. It was considered liquid-solid ratio using mL per g.

 

Considering authors used an Ultrasound bath and longer times, authors should provide how they were able to control the temperature of the extraction? In my own experience, I have worked with both an ultrasound bath and ultrasound prove and external equipment is needed to overcome the heating generated by the own ultrasound. Please, provide the information.

Ice cubes were added to the ultrasound bath in order to maintain the temperature at the value established in Table 1 along each experiment. In this way the bath measures the temperature in real time.

 

In section 1.5.2, why authors choose the box-Behnken design instead of a CCD which could provide a little more information?

The explanation was added, please see line L285-295.

In table 3, what is the difference between peel and pulp vs extract? Pulps and peels are not extracted? Provide relevant info at the bottom of the table.

 

The kurugua fruit has three well differentiated parts, the pulp or endocarp where the seeds are contained, it is the interior of the fruit, then the mesocarp is an intermediate layer, inedible. The outermost part; the exocarp, black, where the highest concentration of anthocyanins is.

The term "extract" in this manuscript, refers to the optimized extract, after the application of the response surface method, since in all cases a previous extraction is carried out for the anthocyanin analysis, in base to your suggestions the term extract has been edited by " optimized peel extract "to refer to the extract finally obtained under the selected conditions and which has been characterized.

To follow a logical sequence of the work performed, the skin data in Table 3 were eliminated and the corresponding results are better described in Table 6.

 

 In equation 2, the L-S ratio2 is not significant, remove also that term from the equation.

 

This has been included, since despite not being significant, the inclusion of this term in the model improves the fit of the data obtained.

 

In figures 4, figure b) and c) are repetitions, please remove or add others that provide more information.

Thankyou for your observation, it was edit.

In lines, 296-298 authors recommend 15 minutes as optimal point, but in the screening analysis, factor time was not significant, also, if authors decided to ignore the statistics and based their criteria on a little higher amount as shown in figure 2 a), are those 3 mg important compared with the time and energy consumed?

Despite not being significant, there is a trend that shows that an increase in time is useful to increase the extraction of AMT. Longer times were not used in this work due to the limitations of the equipment and the energy consumption that the application of this technology entails.

 

In line 308 authors again refer to peel vs extract, please clarify this, for the determinations authors do not extract compounds from peel?? What is the difference between peel and extract? Please provide this information.

Thank you very much for your suggestion to improve the manuscript. Certainly these terms could be confusing, which is why it has been modified in the manuscript.

The term peel refers to the exocarp of the fruit, a thin layer of 1-2 mm of black color, the results referred to peel are from the initial extraction for the characterization of the peel under the described conditions (before the optimization of the model) .

 

It seems that at line 319 information is missing.

Figure 5 is repeated in the text, please correct. Also, the colored text has to be black.

These sections has been edited, according to his suggestions.

 

In lines 417-423, optimal conditions should be revised, my concern is that the authors seem not considered energy and time consuming for this optimization, but the main drawback might be the way in which the response was measured since the authors have characterized chromatographically the molecules, optimization should rely on chromatographic quantification since spectrophotometric assays are only good screening methods, but are not recommended for qualifications or optimizations, since plenty of interferences happens on this broad measurements.

 

Once again, we greatly appreciate this comment to improve the manuscript, and taking into account the aforementioned, the results reflect a trend that would be independent of the quantification methods mentioned. To consider your suggestion, the following will be included in the discussion section:

In this work, the optimal conditions for anthocyanin extraction were achieved at t = 15 min, T = 20°C, pH = 6, S= 60% ethanol, v/v and L-S of 80 mg/L, obtaining a concentration of 61.908 mg C3G/100 g DW. “It is important to mention that although the final anthocyanin concentration might be higher due to the inherent limitations of the quantification method, the mentioned extraction conditions would remain the same regardless of the chosen quantification method (Lee, Rennaker & Wrolstad, 2008; Lao & Giusti, 2016).”

 

REFERENCES

Lao, F. & Giusti, M. M. (2016). Quantification of Purple Corn (Zea mays L.) Anthocyanins Using Spectrophotometric and HPLC Approaches: Method Comparison and Correlation. Food Analytical Methods, 9, 1367-1380.

Lee, J., Rennaker, C., & Wrolstad, R. (2008). Correlation of two anthocyanin quantification methods: HPLC and spectrophotometric methods. Food Chemistry, 782-786.

Jiang, H-L., Yang, J.-L., & Shi, Y.-P. (2017). Optimization of ultrasonic cell grinder extraction of anthocyanins from blueberry using response surface methodology. Ultrasonics Sonochemistry, 34, 325–331.

Yin, Y., Jia, J., Wang, T., & Wang, C. (2017). Optimization of natural anthocyanin efficient extracting from purple sweet potato for silk fabric dyeing. Journal of Cleaner Production, 149, 673e679.

 

Author Response File: Author Response.docx

Reviewer 3 Report

I read an interesting and comprehensive research work entitled ‘Extraction of total anthocyanins from Sicana odorifera black peel fruits growing in Paraguay for food applications’. The concept of the article is interesting and suitable to publish in Applied Sciences. This manuscript is generally well written and clearly presented however still needs to address some comments, and thus require moderate revision.

1)         In the abstract authors should mention the importance of research work briefly. Give results values. Add research representing research work don’t use any abbreviation here

2)         A well addressed graphical scheme of study design should be inserted.   

3)      In the introduction section, write the novelty of the work and the problem statement clearly. Add more details about the ultrasound. It is not only for the extraction of phytochemicals but it is also found useful for the pretreatment of biomass refer and cite Industrial Crops and Products 150, 112425, 2020. Surprisingly there is no discussion about the importance of modeling.

4)      Add quantitative data of Sicana odorifera black peel fruits and their applications somewhere..

5)      The detailed discussion about the novelty, significance of your research work and research gap relative to the literature is essential.

6)      Instead of mentioning antioxidant studies, discuss their results effectively.

• This manuscript lacked substantial discussion of results with the literature authors should concentrate on this during revision.

7)      Section 1.7 changed as a statistic. Line no 302 incomplete sentence.

8)       For figure and table captions give all details which are quite expected. Don’t use any abbreviation.

9)       Techno Economic challenges of the developed system need to be addressed. What are the limitations and future research directions that need to be described by adding a new section before the conclusions section?

10)    The conclusion of the study needs to be added with the specific output obtained from the study, it could be modified with precise outcomes with a take home message. 

11)    Some English and grammar mistakes are present that need to be correct to improve the quality of the manuscript.

Author Response

Comments and Suggestions for Authors

I read an interesting and comprehensive research work entitled ‘Extraction of total anthocyanins from Sicana odorifera black peel fruits growing in Paraguay for food applications’. The concept of the article is interesting and suitable to publish in Applied Sciences. This manuscript is generally well written and clearly presented however still needs to address some comments, and thus require moderate revision.

The authors are thankful for your comments on the improvement of the manuscript. Comments and suggestions are appreciated.

1)         In the abstract authors should mention the importance of research work briefly. Give results values. Add research representing research work don’t use any abbreviation here:

The authors appreciate these observations; the abstract has been edited according to the suggestions.

2)         A well addressed graphical scheme of study design should be inserted.   

A graphical scheme of the study design has been inserted immediately after the Abstract.

3)      In the introduction section, write the novelty of the work and the problem statement clearly. Add more details about the ultrasound. It is not only for the extraction of phytochemicals but it is also found useful for the pretreatment of biomass refer and cite Industrial Crops and Products 150, 112425, 2020. Surprisingly there is no discussion about the importance of modeling.

The introduction was modified as it was pointed out. Thank you very much for the sent reference. A discussion on other applications of the ultrasound has been included, as well as the importance of the response surface modeling.

4)      Add quantitative data of Sicana odorifera black peel fruits and their applications somewhere.

Thanks for the comment to improve the manuscript. Data on fruit production and cultivation have been added in the introduction section.

5)      The detailed discussion about the novelty, significance of your research work and research gap relative to the literature is essential.

Thanks for the comment to improve the manuscript.

6)      Instead of mentioning antioxidant studies, discuss their results effectively.

• This manuscript lacked substantial discussion of results with the literature authors should concentrate on this during revision.

Discussion points on this comment have been added to the manuscript.

7)      Section 1.7 changed as a statistic. Line no 302 incomplete sentence. 8)       For figure and table captions give all details which are quite expected. Don’t use any abbreviation.

It has been corrected based on your suggestions.

 

9)       Techno Economic challenges of the developed system need to be addressed. What are the limitations and future research directions that need to be described by adding a new section before the conclusions section?

The techno-economic challenges are mainly the development of machinery to remove the peel of such a large fruit, optimize the industrial process for obtaining colorants through friendly techniques (drying in a Spray Tower, etc.), following the trend of obtaining a natural product with coloring properties for food applications, currently in high demand. Thanks for the comment to improve the manuscript.

10)    The conclusion of the study needs to be added with the specific output obtained from the study, it could be modified with precise outcomes with a take home message. 

Thanks, the conclusion has been edited as follows:

The peel physicochemical characteristics and composition of ripe fruits of S. odorifera black accession grown in Paraguay was rich in polyphenols compounds and anthocyanins. After the statistical validation of the theoretical model, it was possible to predict the best extraction conditions for total anthocyanins. The optimal conditions for anthocyanin extraction from the peel by the response surface method, were ultrasound-assisted extraction by 15 min at 20°C pH = 6 with 60% ethanol and L-S of 80 mg/L, where a higher concentration of total anthocyanins was obtained, up to 60 mg C3G/100 g DW.

 In the extract, fourteen main compounds were detected; twelve were tentatively identified, including five anthocyanin derivatives, five flavonols derivatives, two flavonol aglycones. We observed malvidin rhamnose shikimate and another cyanidin rutinoside isomer, this malvidin derivative is reported for the first time in S. odorifera samples. The chromatographic profile of the peel extracts from the Paraguayan S. odorifera showed intense signals of flavonols, including quercetin and kaempferol derivatives, the major was quercetin rutinoside. Other flavonols informed in this work, such as quercetin hexoside succinate and kaempferol aglycon, to the best of our knowledge, are reported for the first time in this species. This study provides important information for obtaining an anthocyanin and flavonoids based extract in the optimized conditions by ultrasound-assisted extraction from black kurugua peel, an important by-product of this autochthon fruit. This extract may be of interest for future applications as a natural ingredient in high added value foods.

11)    Some English and grammar mistakes are present that need to be correct to improve the quality of the manuscript.

Grammar and English verified with an English speaker. Comments and suggestions are appreciated.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Paper title

Extraction of total anthocyanins from Sicana odorifera black peel fruits growing in Paraguay for food applications

 SUMMARY

The manuscript is well written in an engaging and lively style, the subject is important and RSM is currently something of a “hot topic”, although the response analyzed should be carefully selected. The integration of high technologies such as UPLC-ESI-QqQ-MS works in both positive and negative aspects to authors, this characterization is relevant and the results will be very handy for the science community, although, the same identification could be used for quantification and optimization of the extractions tested. It does not make a lot of sense to use powerful technology such as UPLC and ultrasound while the response used could lead to many errors due to interference.

This manuscript almost ticks all the boxes we have in mind for a fingerprinting of bioactive molecules and subsequent optimization, but the response analyzed should be determined in a more reliable way.

There are also some editorial mistakes that have to be double-checked.

Essential issues

The robustness of the data presented in the optimization response as well as the author's choice for the optimal point should be clarified. In order to proceed with the publication of this manuscript, authors should consider the inclusion of optimization of results from the UPLC quantification or consider another type of journal for this publication as it is.

Thank you very much for your comments. The differential pH method was chosen to monitor anthocyanin extraction over the UHPLC method due to its availability, simplicity, and economy. Although our institution has UHPLC, the equipment is not always available and LC-MS solvents are very expensive. Further, the pH differential method is currently employed to monitor anthocyanin extraction conditions for optimization purposes (Jiang et al., 2017; Yin et al., 2017). It has been shown that although the values obtained by this method may differ from those of HPLC due to the individual components of each sample, the trend is the same. In other words, the values obtained by both methods show a high correlation among each other, therefore the samples with a high value by HPLC will show a high value per pH differential (Lee, Rennaker and Wrolstad, 2008; Lao and Giusti, 2016). Taking into account the mentioned above, the results reflect a trend that would be independent of the mentioned quantification methods.  Editorial mistakes has been edited, thank you for yours comments, they are usefull to improvement the manuscript.

Please provide the correlation mentioned

Suggestions

In line 129 of section 1.3 add “followed by centrifugation and filtration” after “ultrasonic bath (15 min)”, otherwise, ultrasonic bath and centrifugation might be confusing for readers.

In line 130, the second extraction was made following the parameters of the first one just changing the solvent? Or was stirred and kept for 30 min at room temperature in the dark? The authors should clarify better the methodology.

In line 132, the authors used the word plotted for the spectrophotometric reading, and although a calibration curve is performed, there is not a real need for plotting the result, consider using words such as “read” or “registered”.

In line 145 consider replacing “medium” for “dissolution”

In line 154, the text has a reference error, please correct it.

In line 163, the text has a reference error, please correct it.

In line 219, the text has a reference error, please correct it.

In line 224, the text has a reference error, please correct it.

References still displaying an error, please revise the whole document

It seems that line 302 information is missing.

All these edit details have been corrected. Was considered and the terms was changed. Thankyou.

Thank you

In table 1, is the solid to liquid ratio ok? Authors

It is correct. It was considered liquid-solid ratio using mL per g.

Thank you

Considering authors used an Ultrasound bath and longer times, authors should provide how they were able to control the temperature of the extraction? In my own experience, I have worked with both an ultrasound bath and ultrasound prove and external equipment is needed to overcome the heating generated by the own ultrasound. Please, provide the information.

Ice cubes were added to the ultrasound bath in order to maintain the temperature at the value established in Table 1 along each experiment. In this way the bath measures the temperature in real time.

Thank you

In section 1.5.2, why authors choose the box-Behnken design instead of a CCD which could provide a little more information?

The explanation was added, please see line L285-295.

I was not able to find the answer on the lines mention, please provide an answer

In table 3, what is the difference between peel and pulp vs extract? Pulps and peels are not extracted? Provide relevant info at the bottom of the table.

The kurugua fruit has three well differentiated parts, the pulp or endocarp where the seeds are contained, it is the interior of the fruit, then the mesocarp is an intermediate layer, inedible. The outermost part; the exocarp, black, where the highest concentration of anthocyanins is.

The term "extract" in this manuscript, refers to the optimized extract, after the application of the response surface method, since in all cases a previous extraction is carried out for the anthocyanin analysis, in base to your suggestions the term extract has been edited by " optimized peel extract "to refer to the extract finally obtained under the selected conditions and which has been characterized.

To follow a logical sequence of the work performed, the skin data in Table 3 were eliminated and the corresponding results are better described in Table 6.

Thank you

 

 In equation 2, the L-S ratio2 is not significant, remove also that term from the equation.

This has been included, since despite not being significant, the inclusion of this term in the model improves the fit of the data obtained.

In figures 4, figure b) and c) are repetitions, please remove or add others that provide more information.

Thankyou for your observation, it was edit.

Thank you

In lines, 296-298 authors recommend 15 minutes as optimal point, but in the screening analysis, factor time was not significant, also, if authors decided to ignore the statistics and based their criteria on a little higher amount as shown in figure 2 a), are those 3 mg important compared with the time and energy consumed?

Despite not being significant, there is a trend that shows that an increase in time is useful to increase the extraction of AMT. Longer times were not used in this work due to the limitations of the equipment and the energy consumption that the application of this technology entails.

Actually, in figure 2 it is observed a decrease followed by a small increase compared with the low level. 5 minutes is just slightly lower than 15 min, and from table 3, it is clear that liquid to solid ratio is the most influential factor. So once more, why if you have an insignificant factor as time, authors choose the most expensive condition? I believe that optimization is not only finding target values but understanding the whole results in order to maximize results with the minimum spending.

In line 308 authors again refer to peel vs extract, please clarify this, for the determinations authors do not extract compounds from peel?? What is the difference between peel and extract? Please provide this information.

Thank you very much for your suggestion to improve the manuscript. Certainly these terms could be confusing, which is why it has been modified in the manuscript.

The term peel refers to the exocarp of the fruit, a thin layer of 1-2 mm of black color, the results referred to peel are from the initial extraction for the characterization of the peel under the described conditions (before the optimization of the model) .

 Thank you

It seems that at line 319 information is missing.

Figure 5 is repeated in the text, please correct. Also, the colored text has to be black.

These sections has been edited, according to his suggestions.

 Thank you

In lines 417-423, optimal conditions should be revised, my concern is that the authors seem not considered energy and time consuming for this optimization, but the main drawback might be the way in which the response was measured since the authors have characterized chromatographically the molecules, optimization should rely on chromatographic quantification since spectrophotometric assays are only good screening methods, but are not recommended for qualifications or optimizations, since plenty of interferences happens on this broad measurements.

 

Once again, we greatly appreciate this comment to improve the manuscript, and taking into account the aforementioned, the results reflect a trend that would be independent of the quantification methods mentioned. To consider your suggestion, the following will be included in the discussion section:

In this work, the optimal conditions for anthocyanin extraction were achieved at t = 15 min, T = 20°C, pH = 6, S= 60% ethanol, v/v and L-S of 80 mg/L, obtaining a concentration of 61.908 mg C3G/100 g DW. “It is important to mention that although the final anthocyanin concentration might be higher due to the inherent limitations of the quantification method, the mentioned extraction conditions would remain the same regardless of the chosen quantification method (Lee, Rennaker & Wrolstad, 2008; Lao & Giusti, 2016).”

 Thank you. Also, in table 5, run 3 has the highest yielding with 56 mg C3G/100 g DW with conditions of pH 4.75, why do authors choose pH 6?

REFERENCES

Lao, F. & Giusti, M. M. (2016). Quantification of Purple Corn (Zea mays L.) Anthocyanins Using Spectrophotometric and HPLC Approaches: Method Comparison and Correlation. Food Analytical Methods, 9, 1367-1380.

Lee, J., Rennaker, C., & Wrolstad, R. (2008). Correlation of two anthocyanin quantification methods: HPLC and spectrophotometric methods. Food Chemistry, 782-786.

Jiang, H-L., Yang, J.-L., & Shi, Y.-P. (2017). Optimization of ultrasonic cell grinder extraction of anthocyanins from blueberry using response surface methodology. Ultrasonics Sonochemistry, 34, 325–331.

Yin, Y., Jia, J., Wang, T., & Wang, C. (2017). Optimization of natural anthocyanin efficient extracting from purple sweet potato for silk fabric dyeing. Journal of Cleaner Production, 149, 673e679.

 

 

Author Response

Dear Reviewer 2

We appreciate your comments and suggestions. 

Paper title

Extraction of total anthocyanins from Sicana odorifera black peel fruits growing in Paraguay for food applications

 SUMMARY

The manuscript is well written in an engaging and lively style, the subject is important and RSM is currently something of a “hot topic”, although the response analyzed should be carefully selected. The integration of high technologies such as UPLC-ESI-QqQ-MS works in both positive and negative aspects to authors, this characterization is relevant and the results will be very handy for the science community, although, the same identification could be used for quantification and optimization of the extractions tested. It does not make a lot of sense to use powerful technology such as UPLC and ultrasound while the response used could lead to many errors due to interference.

This manuscript almost ticks all the boxes we have in mind for a fingerprinting of bioactive molecules and subsequent optimization, but the response analyzed should be determined in a more reliable way.

There are also some editorial mistakes that have to be double-checked.

Essential issues

The robustness of the data presented in the optimization response as well as the author's choice for the optimal point should be clarified. In order to proceed with the publication of this manuscript, authors should consider the inclusion of optimization of results from the UPLC quantification or consider another type of journal for this publication as it is.

• Thank you very much for your comments. The differential pH method was chosen to monitor anthocyanin extraction over the UHPLC method due to its availability, simplicity, and economy. Although our institution has UHPLC, the equipment is not always available and LC-MS solvents are very expensive. Further, the pH differential method is currently employed to monitor anthocyanin extraction conditions for optimization purposes (Jiang et al., 2017; Yin et al., 2017). It has been shown that although the values obtained by this method may differ from those of HPLC due to the individual components of each sample, the trend is the same. In other words, the values obtained by both methods show a high correlation among each other, therefore the samples with a high value by HPLC will show a high value per pH differential (Lee, Rennaker and Wrolstad, 2008; Lao and Giusti, 2016). Taking into account the mentioned above, the results reflect a trend that would be independent of the mentioned quantification methods.  Editorial mistakes has been edited, thank you for yours comments, they are usefull to improvement the manuscript.

Please provide the correlation mentioned

• Thank you for your observation. We added the following line to the manuscript in the discussion section (Line 481-482), as you suggested.
• It is important to mention that although the final anthocyanin concentration might be higher due to the inherent limitations of the pH differential method, “correlation (R) values equal to 0.925 or higher among the results obtained by this method and HPLC measurements has been informed”. Therefore, the optimal extraction conditions would remain the same regardless of the quantification method.

Suggestions

In line 129 of section 1.3 add “followed by centrifugation and filtration” after “ultrasonic bath (15 min)”, otherwise, ultrasonic bath and centrifugation might be confusing for readers.

In line 130, the second extraction was made following the parameters of the first one just changing the solvent? Or was stirred and kept for 30 min at room temperature in the dark? The authors should clarify better the methodology.

In line 132, the authors used the word plotted for the spectrophotometric reading, and although a calibration curve is performed, there is not a real need for plotting the result, consider using words such as “read” or “registered”.

In line 145 consider replacing “medium” for “dissolution”

In line 154, the text has a reference error, please correct it.

In line 163, the text has a reference error, please correct it.

In line 219, the text has a reference error, please correct it.

In line 224, the text has a reference error, please correct it.

References still displaying an error, please revise the whole document

It seems that line 302 information is missing.

• All these edit details have been corrected. Was considered and the terms was changed. Thankyou.

Thank you

In table 1, is the solid to liquid ratio ok? Authors

• It is correct. It was considered liquid-solid ratio using mL per g.

Thank you

Considering authors used an Ultrasound bath and longer times, authors should provide how they were able to control the temperature of the extraction? In my own experience, I have worked with both an ultrasound bath and ultrasound prove and external equipment is needed to overcome the heating generated by the own ultrasound. Please, provide the information.

• Ice cubes were added to the ultrasound bath in order to maintain the temperature at the value established in Table 1 along each experiment. In this way the bath measures the temperature in real time.

Thank you

• In section 1.5.2, why authors choose the box-Behnken design instead of a CCD which could provide a little more information?

The explanation was added, please see line L285-295.

I was not able to find the answer on the lines mention, please provide an answer.

• There was a mistake referring the lines where the explanation was added, please see line L117-L129.

“The extraction process has many variables that could influence the efficiency of the process. Consequently, an appropriate design of experiments is essential to determine the best operational conditions. In this sense, some designs aim to find some significant factors from many potential ones. One of them is the screening design. Usually, after some factors are discarded, the significant ones are analyzed again either to find the best operational conditions or even the optimal ones. For this application, designs such as Box-Behnken are proposed. This allows the development of a response surface that might help to find the optimal operational condition [17]. Box-Behnken allows running fewer experiments than others such as central composite design, which results in a less expensive optimization methodology. Further, the method for response monitoring should be carefully selected to complement an adequate design and to accomplish the optimization. In this sense, spectrophotometric methods have been successfully employed for this purpose due to their speed and reliability [18–20]”

 

In table 3, what is the difference between peel and pulp vs extract? Pulps and peels are not extracted? Provide relevant info at the bottom of the table.

• The kurugua fruit has three well differentiated parts, the pulp or endocarp where the seeds are contained, it is the interior of the fruit, then the mesocarp is an intermediate layer, inedible. The outermost part; the exocarp, black, where the highest concentration of anthocyanins is.
• The term "extract" in this manuscript, refers to the optimized extract, after the application of the response surface method, since in all cases a previous extraction is carried out for the anthocyanin analysis, in base to your suggestions the term extract has been edited by " optimized peel extract "to refer to the extract finally obtained under the selected conditions and which has been characterized.
• To follow a logical sequence of the work performed, the skin data in Table 3 were eliminated and the corresponding results are better described in Table 6.

Thank you

• In equation 2, the L-S ratio2 is not significant, remove also that term from the equation.

This has been included, since despite not being significant, the inclusion of this term in the model improves the fit of the data obtained.

In figures 4, figure b) and c) are repetitions, please remove or add others that provide more information.

Thank you for your observation, it was edit.

Thank you

In lines, 296-298 authors recommend 15 minutes as optimal point, but in the screening analysis, factor time was not significant, also, if authors decided to ignore the statistics and based their criteria on a little higher amount as shown in figure 2 a), are those 3 mg important compared with the time and energy consumed?

Despite not being significant, there is a trend that shows that an increase in time is useful to increase the extraction of AMT. Longer times were not used in this work due to the limitations of the equipment and the energy consumption that the application of this technology entails.

Actually, in figure 2 it is observed a decrease followed by a small increase compared with the low level. 5 minutes is just slightly lower than 15 min, and from table 3, it is clear that liquid to solid ratio is the most influential factor. So once more, why if you have an insignificant factor as time, authors choose the most expensive condition? I believe that optimization is not only finding target values but understanding the whole results in order to maximize results with the minimum spending.

• We appreciate your comment. We are aware that longer times imply a higher operational cost. However, time is important to allow sufficient contact between solute and solvent to achieve extraction of the component of interest. More information about this was added in section 4. Discussion, as you suggested.
• “For the Box-Behnken design, the temperature was set at 20 °C and the time at 15 min. As can be seen in Fig. 2, lower temperatures are more efficient for anthocyanin removal. Regarding time, even though longer times imply a higher operational cost, in this work this time was chosen to allow an enough exposure time of the solute to the solvent. In the literature, extraction time of phenolic compounds from 10 to 90 min are reported (13).”

In line 308 authors again refer to peel vs extract, please clarify this, for the determinations authors do not extract compounds from peel?? What is the difference between peel and extract? Please provide this information.

Thank you very much for your suggestion to improve the manuscript. Certainly these terms could be confusing, which is why it has been modified in the manuscript.

The term peel refers to the exocarp of the fruit, a thin layer of 1-2 mm of black color, the results referred to peel are from the initial extraction for the characterization of the peel under the described conditions (before the optimization of the model).

 Thank you

It seems that at line 319 information is missing.

Figure 5 is repeated in the text, please correct. Also, the colored text has to be black.

These sections has been edited, according to his suggestions.

 Thank you

In lines 417-423, optimal conditions should be revised, my concern is that the authors seem not considered energy and time consuming for this optimization, but the main drawback might be the way in which the response was measured since the authors have characterized chromatographically the molecules, optimization should rely on chromatographic quantification since spectrophotometric assays are only good screening methods, but are not recommended for qualifications or optimizations, since plenty of interferences happens on this broad measurements.

Once again, we greatly appreciate this comment to improve the manuscript, and taking into account the aforementioned, the results reflect a trend that would be independent of the quantification methods mentioned. To consider your suggestion, the following will be included in the discussion section:

In this work, the optimal conditions for anthocyanin extraction were achieved at t = 15 min, T = 20°C, pH = 6, S= 60% ethanol, v/v and L-S of 80 mg/L, obtaining a concentration of 61.908 mg C3G/100 g DW. “It is important to mention that although the final anthocyanin concentration might be higher due to the inherent limitations of the quantification method, the mentioned extraction conditions would remain the same regardless of the chosen quantification method (Lee, Rennaker & Wrolstad, 2008; Lao & Giusti, 2016).”

Thank you. Also, in table 5, run 3 has the highest yielding with 56 mg C3G/100 g DW with conditions of pH 4.75, why do authors choose pH 6?

• We appreciate your comment and interest. This is because, by maximizing the equation that the model represents (Equation 2), the maximum concentration of TMA that can be reached within the ranges studied is given for a LS ratio of 80 mg / L at T = 20 °C, pH equal to 6 and a 60% aqueous ethanol solution.

Thankyou.

REFERENCES

Lao, F. & Giusti, M. M. (2016). Quantification of Purple Corn (Zea mays L.) Anthocyanins Using Spectrophotometric and HPLC Approaches: Method Comparison and Correlation. Food Analytical Methods, 9, 1367-1380.

Lee, J., Rennaker, C., & Wrolstad, R. (2008). Correlation of two anthocyanin quantification methods: HPLC and spectrophotometric methods. Food Chemistry, 782-786.

Jiang, H-L., Yang, J.-L., & Shi, Y.-P. (2017). Optimization of ultrasonic cell grinder extraction of anthocyanins from blueberry using response surface methodology. Ultrasonics Sonochemistry, 34, 325–331.

Yin, Y., Jia, J., Wang, T., & Wang, C. (2017). Optimization of natural anthocyanin efficient extracting from purple sweet potato for silk fabric dyeing. Journal of Cleaner Production, 149, 673e679.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors have substantially revised the manuscript according to the comments.

The present form of the manuscript can be accepted for publication.

Author Response

The authors especially appreciate your interest, and your suggestions to improve the manuscript.