Recovery of Phenolic Compounds by Deep Eutectic Solvents in Orange By-Products and Spent Coffee Grounds

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Overall, this study demonstrated the applicapability of DES on agricultural by-products upcycling. However, it needs further works to add the existing literature for significant novelty in research methodology. For this, here is my suggestion:

Abstract

-       Please change the annotation of DES1 & 2 with more scientific words. For example, it can be replaced by CM-DES or LG-DES.

-       For antioxidant unit, if it goes by several thousand or higher, please report to mol, instead of micromole.

-       Please report the comparative recovery or efficiency between treatment with DES and without DES.

-       Please update the unit of temperature to ‘ºC’ – should use right format.

Introduction

-       Line 28: does author imply the amounts based on domestic? Or worldwide? Please be specific.

-       Line 42-54: Please exemplify any commercial case for upcycling from orange or coffee. Simple comments with benefits for health care sound weak.

Material & Methods

-       Section 2.4.1: ‘Water concentration’ is unfamiliar – please be specify with more scientific information. Such as dilution effect or water fraction etc.

-       Line169: Superscript for ‘ABT⁺’

-       Line 170: Please use proper significant number for instrumental specification.

Results and discussion

-       Please update with comparative statement instead of simple reports with numbers and data.

-       Table 2: What does under-bar at letter mean?

-       Table 5: Please align the table, appropriately.

-       Please make statement for justification of the use of orange by-product and coffee-spent. Is there any correlation between those two samples? Have you found any combination effect or synergistic effect from them? Otherwise, since author didn’t include control data, I wasn’t able to find the specific reason for authors’ selection on sample. If author wanted to find the applicability of DES, the sample treatment with inert solvent, which can be considered to positive control, should be included.   

 

Author Response

Reviewer 1

Comment: Overall, this study demonstrated the applicability of DES on agricultural by-products upcycling. However, it needs further work to add the existing literature for significant novelty in research methodology. For this, here is my suggestion:

Response: We appreciate the feedback and suggestions to improve the work.

 

Abstract

Comment: Please change the annotation of DES1 & 2 with more scientific words. For example, it can be replaced by CM-DES or LG-DES.

Response: Thank you for the suggestion. We have changed this to CM-DES (citric acid and mannitol) and LG-DES (lactic acid and glucose) in the abstract and throughout the text. (Lines: 15, 16, 21, 136, 137, 155, 321, 327-330, 339, 343, 346, 374, 375, 378, 379, 384, 404, 406, 408, 409, 411, 412, 420, 423, 432, 433, 437, 445, 446, 457, 458, 460, 461, 465, 468, 475, 490, 492, 497, 517-522, 554-556, 558, 566, 569).

 

Comment: For the antioxidant unit, if it goes by several thousand or higher, please report to mol, instead of micromole.

Response: We converted the antioxidant activity from µmol to mol. (Lines: 22, 23, 25 and in Table 5).

 

Comment: Please report the comparative recovery or efficiency between treatment with DES and without DES.

Response: We report the efficiency of extraction without DES, showing works already reported in the literature. (Lines: 275-286).

 

Comment: Please update the unit of temperature to ‘ºC’ – should use right format.

Response: We made the change in the summary and throughout the text. (Lines: 17,18, 102, 117, 119, 125, 126, 138, 140, 145, 147, 161, 163, 168, 171, 175, 178, 403, 453, 456, 459, 487, 530, 535, 604).

 

Comment: Line 28: does author imply the amounts based on domestic? Or worldwide? Please be specific.

Response: This value corresponds to the generation of by-products worldwide. We have changed the sentence for better understanding. (Lines: 32, 33).

 

Comment: Line 42-54: Please exemplify any commercial case for upcycling from orange or coffee. Simple comments with benefits for health care sound weak.

Response: We have added commercial examples for upcycling from orange by-products and spent coffee grounds. (Lines: 45-51, 62-67).

 

Material & Methods

Comment: Section 2.4.1: ‘Water concentration’ is unfamiliar – please he specifies with more scientific information. Such as dilution effect or water fraction etc.

Response: The term water concentration was replaced by water content. This term is widely used in extraction studies with deep eutectic solvents, which can be observed in the studies described below. (Lines: 16, 17, 101, 145, 153, 156, 158, 166, 176, 291, 320, 325, 341, 344, 345, 360, 366, 370, 372, 373, 385, 391-393, 403, 491).

Pontes et al., (2021) - Choline chloride-based deep eutectic solvents as potential solvent for extraction of phenolic compounds from olive leaves: Extraction optimization and solvent characterization.

Alasalvar and Yildirim (2021) - Ultrasound-assisted extraction of antioxidant phenolic compounds from Lavandula angustifolia flowers using natural deep eutectic solvents: An experimental design approach.

 

Comment: Line169: Superscript for ‘ABT+’.

Response: Thanks for the note. It has been changed to (ABTS⁺). (Line: 194).

 

Comment: Line 170: Please use proper significant number for instrumental specification.

Response: We use the appropriate significant number for instrumental specification as ABTS⁺. (Lines: 195).

 

Results and discussion

Comment: Please update with a comparative statement instead of simple reports with numbers and data.

Response: A comparative statement of the results was made. There are other studies in the literature that report the physical-chemical composition of orange by-products and SCG, however, there is a great variability in the results. This variability is attributed to the different species of orange and coffee, growing conditions, soil types, climatic conditions, processing (type of fermentation and degree of coffee roasting), types of extraction methods applied and techniques for preparing the coffee beverage. In addition, it is worth noting that there may be a difference in the proportionality of the material, between peel, leaf, bagasse and seeds, that is, of all the fragmented material, due to the process used for extraction, which is not always reported in the studies described in the literature. (Lines: 237-245).

 

Comment: Table 2: What does under-bar at letter mean?

Response: Thanks for the observation. We have removed the bar from the Table and modified the explanation about the means test. (Table 2).

 

Comment:  Table 5: Please align the table, appropriately.

Response: Table 5 has been aligned.

 

Comment: Please make statement for justification of the use of orange by-product and coffee spent. Is there any correlation between those two samples? Have you found any combination effect or synergistic effect from them? Otherwise, since author didn’t include control data, I couldn’t find the specific reason for authors’ selection on sample. If author wanted to find the applicability of DES, the sample treatment with inert solvent, which can be considered to positive control, should be included.  

Response: The orange by-product and coffee grounds were chosen because they are by-products produced in large quantities in Brazil, which is the largest producer of oranges and coffee. In addition, they have a high content of phenolic compounds, thus allowing their valorization to recover these bioactive compounds with high added value. This justification was mentioned in the introduction. (Lines: 34-44, 51-58). There is no data on the effect of combining these by-products, and this work is a pioneer in the use of eutectic solvents to extract phenolic compounds from orange by-products and SCG. However, this effect will be considered for future work; however, now, the present study aims to perform an isolated characterization of each by-product to understand these by-products better. A controlled study of the samples (orange by-products and spent coffee grounds) was carried out using the conventional extraction method from 80% methanol described in Table 1 for comparison purposes and evaluation of the effectiveness of eutectic solvents in extracting phenolic compounds. (Lines: 145-147, 275-278).

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript entitled “Recovery of phenolic compounds by deep eutectic solvents in orange by-products and spent coffee grounds” focuses on the extraction of phenolic compounds from wastes by two kinds of DESs. It could be considered for publication after the following points are fully addressed.

1. Some abbreviations need to be explained, for example, “TPC”, “FRAP”, “DPPH” and “ABTS” in the abstract.

2. The results in Table 2 show that with the change of water content from 10% to 50%, the extraction performance of DESs for TPC is significantly reduced. The authors should study the extraction performance when the water content is 0% and 5% for comparison.

3. As the viscosity of DESs is always mentioned in this work, the physical property of DESs, such as density and viscosity before and after the addition of water, should be measured and listed.

4. What does the superscript of the data mean in Tables 2 to 5?

5. How to regenerate the DESs after the extraction? Can these DESs be reused?

 

Comments on the Quality of English Language

The authors should carefully check the full manuscript to avoid writing and grammar errors.

Author Response

Reviewer 2

 This manuscript titled “Recovery of phenolic compounds by deep eutectic solvents in orange by-products and spent coffee grounds” focuses on the extraction of phenolic compounds from waste by two types of DESs. It could be considered for publication after the following points are fully addressed.

Response: We appreciate the feedback and suggestions to improve the work.

 

Comment: Some abbreviations need to be explained, for example, “TPC”, “FRAP”, “DPPH” and “ABTS” in the abstract.

Response: The abbreviations have been explained in the text, such as Total Phenolic Compounds (TPC), Ferric Reducing Antioxidant Power (FRAP), DPPH radical scavenging activity (2,2-diphenyl-1-picrylhydrazyl - DPPH) and ABTS ((2,2-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid - ABTS). (Lines: 14, 15, 21-25).

 

Comment: The results in Table 2 show that by changing the water content from 10% to 50%, the extraction performance of DESs for TPC is significantly reduced. For comparison, authors should study the extraction performance when the water content is 0% and 5%.

Response: The water content of 10% was the minimum range that allowed the solubilization of the solid material in the solvent and a viscosity sufficient to permit the extraction of phenolic compounds, in addition to facilitating operational issues. Due to the high viscosity of these solvents, adding a water content below 10% makes the operation of the solvent and the extraction process difficult, making using these solvents unfeasible for future applications. (Lines: 157-159).

 

Comment: As the viscosity of DESs is always mentioned in this work, the physical properties of DESs such as density and viscosity before and after adding water should be measured and listed.

Response: Our research group has characterized the deep eutectic solvents produced, especially those solvents that have good properties in the extraction and recovery of bioactive compounds. Therefore, the characterization of these solvents will be carried out in the future.

 

Comment: What does the superscript of the data in Tables 2 to 5 mean?

Response: ^a–f Means within the same column with lowercase letters are significantly different (Tukey test, p≤0.05) between different water contents for the same DES and by-product. ^A–B Means with uppercase letters are significantly different (T-test, p≤0.05) between solvents (CM-DES and LG-DES) for the same by-product. (Table 2, Table 3, Table 4, and Table 5).

Comment: How to regenerate DESs after extraction? Can these DESs be reused?

Response: We have added the methods used for DES regeneration to the text. In addition, we report that regenerated DESs can be reused in extraction processes. (Lines: 308-316).

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Here are a few concise comments for authors’ abstract and introduction:

Abstract:

• Consider emphasizing the practical implications of using DES for phenolic compound extraction in the food industry.
• Ensure keywords are highly relevant and specific to the study's focus.

Introduction:

• Streamline background information to focus more directly on the rationale for using DES.
• Clarify the specific objectives related to evaluating DES for phenolic compound extraction.

These adjustments will help streamline and focus authors’ abstract and introduction while maintaining clarity and relevance to your study on DES extraction methods.

Your summary on the extraction of phenolic compounds using deep eutectic solvents (DES) is clear and informative. Here are a few questions that might help in further understanding and discussing the topic:

1. Water Content in DES:
• What specific deep eutectic solvents (DES) were used in the study?
• Why does a 10% water content in DES optimize the extraction of total phenolic compounds (TPC)?
2. Solid-Liquid Ratio:
• How does the solid-liquid ratio affect the efficiency of TPC extraction?
• What are the practical implications of the different optimal solid-liquid ratios for orange by-products and spent coffee grounds (SCG)?
3. Temperature:
• How does temperature influence the solubility and stability of phenolic compounds during extraction?
• Are there any drawbacks or limitations to increasing the extraction temperature up to 80ºC?
4. Substrate Characteristics:
• What specific phenolic compounds were identified in the orange by-products and SCG?
• How do the compositions of phenolic compounds differ between orange by-products and SCG?
5. Sustainability and Applications:
• How does the use of DES compare to traditional solvents in terms of environmental impact and cost?
• What potential applications do these bioactive phenolic compounds have in the food and pharmaceutical industries?

here are a few focused questions based on your summary:

1. Effect of Temperature:
• How does increasing the temperature to 80 ºC enhance the extraction efficiency of total phenolic compounds (TPC) from both orange by-products and spent coffee grounds (SCG)?
2. Antioxidant Activity and Stability:
• How do deep eutectic solvents (DES) increase electron transfer and neutralize oxidative reactions during the extraction process?
• Can you elaborate on the types of intermolecular interactions, such as hydrogen bonds, that contribute to the stability of phenolic compounds in DES?
3. Measurement of Antioxidant Activity:
• What are the main differences between the ABTS, DPPH, and FRAP methods in measuring antioxidant activity, and why do they yield different values for the same extracts?

Author Response

Reviewer 3

Comment: Here are a few concise comments for authors’ abstract and introduction.

Response: Thanks for the suggestions.

 

Abstract

Comment: Consider emphasizing the practical implications of using DES for phenolic compound extraction in the food industry.

Response: We emphasize the practical implications of using DES to remove phenolic compounds in the food industry as follows: Orange by-products and coffee grounds, rich in phenolic bioactive compounds, can be used in the food industry as antioxidants, colorants, flavorings, and additives, mainly because they are solvents that are easy to prepare, have a lower cost, are thermally stable, biodegradable, renewable, and are considered GRAS (Generally Recognized as Safe). (Lines:10-12).

 

Comment: Ensure keywords are highly relevant and specific to the study's focus.

Response: The keywords have been changed as suggested to orange by-product, spent coffee grounds, phenolic compounds, deep eutectic solvent, and bioactivity. (Lines: 28-29).

 

Introduction:

Comment: Streamline background information to focus more directly on the rationale for using DES.

Response: Relevant information justifying the use of DES in the extraction of phenolic compounds has been added in the introduction. (Lines: 90-94). However, we cannot simplify the basic information to meet the suggestions proposed by the other reviewers.

 

Comment: Clarify the specific objectives related to evaluating DES for phenolic compound extraction.

Response: The specific objectives related to the evaluation of DES for the extraction of phenolic compounds were clarified as follows: These conditions were evaluated to increase intermolecular interactions and hydrogen bond formation, responsible for raising the dissolution and extraction of phenolic compounds in DES and, consequently, improving the recovery and bioactivity of these bioactive compounds. (Lines: 102-105).      

 

Comment: These adjustments will help streamline and focus the authors’ abstract and introduction while maintaining clarity and relevance to your study on DES extraction methods.

Response: Thank you very much for your suggestions. The changes have been incorporated into the text to improve clarity and relevance to our study.

 

Comment: Your summary on the extraction of phenolic compounds using deep eutectic solvents (DES) is clear and informative. Here are a few questions that might help in further understanding and discussing the topic.

Response: Thanks for the suggestions.

 

1. Water Content in DES:

Comment: What specific deep eutectic solvents (DES) were used in the study?

Response: Two specific eutectic solvents were used in the present study: citric acid and mannitol (CM-DES) and lactic acid and glucose (LG-DES). (Lines: 135-136).

 

Comment: Why does a 10% water content in DES optimize the extraction of total phenolic compounds (TPC)?

Response: The water content significantly affects DES's viscosity, modifying its physical and chemical characteristics. By adding 10% water, the viscosity of these solvents can be reduced by breaking the hydrogen bonds formed between the constituents of the eutectic mixture, favoring better extraction efficiency by maximizing the extraction of phenolic compounds. Furthermore, adding water can change the solvent's polarity, increasing the DES's polarity and, consequently, the TPC extraction rate. The increase in TPC was observed with the addition of up to 10% of water, but at concentrations of 20% to 50%, there was a lower efficiency in TPC recovery. Excess water, that is, the addition of water content above 10%, can weaken the formation of hydrogen bonds between DES and TPC, resulting in lower solubility and, consequently, lower mass transfer rate and efficiency in extraction. Therefore, water content is a factor that must be considered to achieve better recovery in the extraction of TPC. (Lines: 357-368).

 

2. Solid-Liquid Ratio:

Comment: How does the solid-liquid ratio affect the efficiency of TPC extraction?

Response: The solid-liquid ratio is a crucial parameter in the extraction of phenolic compounds, as it directly influences the efficiency of the process. This increase in the extraction rate can be attributed to the concentration gradient between the solvent and the solute, resulting in a higher transfer rate of TPC from the interior of the matrix to the solvent and, consequently, a greater extraction yield. Increasing the solvent concentration favors interactions between the solute and the solvent, increasing the extraction rate until solvent saturation. Therefore, an adequate solid-liquid ratio facilitates the diffusion of phenolic compounds from the solute to the solvent. Furthermore, with a greater amount of solvent, the solubilization of the compounds is more efficient, since the concentration gradient is greater, promoting mass transfer. (Lines: 409-414, 422-427).

 

Comment: What are the practical implications of the different optimal solid-liquid ratios for orange by-products and spent coffee grounds (SCG)?

Response: Checking the best solid-liquid ratio for orange by-products and SCG, provides better solubilization of the solutes in the solvent and consequently, a greater extraction yield of phenolic compounds. The solid-liquid ratio is a crucial parameter in the extraction of phenolic compounds, as it directly influences the efficiency of the process. This increase in the extraction rate can be attributed to the concentration gradient between the solvent and the solute, resulting in a higher transfer rate of TPC from the interior of the matrix to the solvent and, consequently, a greater extraction yield. Increasing the solvent concentration favors interactions between the solute and the solvent, increasing the extraction rate until solvent saturation. Therefore, an adequate solid-liquid ratio facilitates the diffusion of phenolic compounds from the solute to the solvent. Furthermore, with a greater amount of solvent, the solubilization of the compounds is more efficient, since the concentration gradient is greater, promoting mass transfer. (Lines: 409-414, 422-427). Furthermore, it demonstrates that obtaining a high yield in TPC extraction without adding large volumes of solvents becomes advantageous by facilitating the purification of the extract and reducing the extraction cost. (Lines: 437-439).

 

3. Temperature:

Comment: How does temperature influence the solubility and stability of phenolic compounds during extraction?

Response: The higher yields obtained at higher temperatures (80 °C) may be associated with the fact that the temperature reduces the viscosity due to the weakening of the hydrogen bond networks formed between the components, reducing the surface tension and increasing the solubility of TPC to DES, the effective diffusivity and extraction efficiency. (Lines: 472-476). Furthermore, increasing temperature tends to reduce the polarity of DES, strongly influencing extraction performance. This occurs because the temperature can modulate the polarity of DES, aligning the eutectic mixture's polarity with the solute's polarity, thus favoring an increase in solubility and extraction yield. This process can improve extraction time and facilitate the transfer of TPCs to solvents (DES). (Lines: 479-484). Furthermore, studies suggest that DES can increase the stability of phenolic compounds due to intermolecular interactions, especially the formation of hydrogen bonds between DES and target compounds. The strong hydrogen bond formed between extracts and DES promotes better thermal stability when compared to conventional solvents. (Lines: 533-537).

 

Comment: Are there any drawbacks or limitations to increasing the extraction temperature up to 80 ºC?

Response: In the present study, the best results were obtained at the maximum temperature studied (80 °C), with an increase in TPC as the extraction temperature increased, which may be related to the fact that higher temperatures favor a more significant reduction in the viscosity of the DES studied, allowing better mass transfer and better extraction efficiency. However, temperatures above 80 °C can lead to phenolic compounds' degradation due to the sensitivity of these bioactive compounds to high temperatures. In addition, the higher the temperature, the greater the energy expenditure and the probability of degradation of other bioactive compounds present in the by-products. Therefore, the use of 80 °C was the ideal temperature to obtain the maximum yield of phenolic compounds in the orange by-products and SCG. Furthermore, studies suggest that DES can increase the stability of phenolic compounds due to intermolecular interactions, especially the formation of hydrogen bonds between DES and target compounds. The strong hydrogen bond formed between extracts and DES promotes better thermal stability when compared to conventional solvents. However, further studies are needed to investigate the effect of DES on the stability of bioactive compounds in plant matrices. (Lines: 529-541).

 

4. Substrate Characteristics:

Comment: What specific phenolic compounds were identified in the orange by-products and SCG?

Response: Although the analysis for the identification and quantification of phenolic compounds in the studied by-products was not carried out, it is expected that the orange by-products will mainly contain hesperidin, naringin, naringenin, catechin and phenolic acids such as ferulic, coumaric and caffeic acid, while for SCG, chlorogenic acid, caffeic acid, ferulic acid, gallic acid and coumaric acid. (Lines: 507-513).

 

Comment: How do the compositions of phenolic compounds differ between orange by-products and SCG?

Response: The chemical composition of orange by-products and SCG about the content of phenolic compounds, proteins, lipids, and other macronutrients and micronutrients may differ from each other due to the following factors such as different species of orange and coffee, growing conditions, soil types, climatic conditions, processing (kind of fermentation and degree of coffee roasting), types of extraction methods applied, and coffee beverage preparation techniques. (Lines: 237-242).

 

5. Sustainability and Applications:

Comment: How does the use of DES compare to traditional solvents in terms of environmental impact and cost?

Response: DES has advantages such as biodegradability, biocompatibility, recyclability, easy preparation, lower cost, low volatility, and lower toxicity than organic solvents. (Line 85-87).

 

Comment: What potential applications do these bioactive phenolic compounds have in the food and pharmaceutical industries?

Response: Therefore, extracts rich in phenolic compounds can be applied in the formulation of food products as ingredients or food additives and in the production of bioactive packaging, aiming to obtain products rich in natural antioxidants, safe and with a longer shelf life. In addition, their biological effects such as neuroprotective, cardioprotective, anti-aging, anticancer, and anti-inflammatory, allow their application in the production of medicines and products in the cosmetic area. (Lines: 281-286).

 

Comment: Here are a few focused questions based on your summary:

Response: Thanks for the suggestions

 

1. Effect of Temperature:

Comment: How does increasing the temperature to 80 ºC enhance the extraction efficiency of total phenolic compounds (TPC) from both orange by-products and spent coffee grounds (SCG)?

Response: In the present study, the highest results were obtained at the maximum temperature studied (80 °C), with an increase in TPC as the extraction temperature increased, which may be related to higher temperatures favoring a more significant reduction in the viscosity of the DES was studied, allowing more excellent mass transfer and better extraction efficiency. (Line 526-533).

 

2. Antioxidant Activity and Stability:

Comment: How do deep eutectic solvents (DES) increase electron transfer and neutralize oxidative reactions during the extraction process?

Response: Eutectic solvents extract solvents for phenolic compounds in the plant matrix. The extracted phenolic compounds have high antioxidant activity and can neutralize free radicals. Free radicals are highly reactive compounds that trigger a series of oxidative reactions in proteins, lipids, and nucleic acids (RNA and DNA), resulting in the oxidation of food products. At the same time, in human cells, several cancerous, cardiovascular, and aging diseases occur. On the other hand, antioxidants are compounds that, even in small quantities, can inhibit/neutralize free radicals. Antioxidants act by transferring a hydrogen atom or electron to the radical with an unpaired electron atom in the last electron layer. These unpaired electrons are highly unstable, and therefore, the donation of an electron by antioxidants makes it more stable. Thus, extracts rich in antioxidant activity allow the delay of lipid peroxidation, the leading cause of food deterioration, and protect human cells, slowing the progression of many chronic diseases.

 

Comment: Can you elaborate on the types of intermolecular interactions, such as hydrogen bonds, that contribute to the stability of phenolic compounds in DES?

Response: The increase in antioxidant activity may be related to the fact that DES increases electron transfer and neutralizes oxidative reactions. This is due to the ability of DES to increase the stability of phenolic compounds during the extraction process, due to the existence of intermolecular interactions, mainly the formation of hydrogen bonds between DES and the target compounds. These

intermolecular interactions can reduce oxidative degradation by reducing the movement of target compounds and the contact of oxygen on the DES surface and air, providing stability and protection to target compounds. (Lines: 569-573).

 

3. Measurement of Antioxidant Activity:

Comment: What are the main differences between the ABTS, DPPH, and FRAP methods in measuring antioxidant activity, and why do they yield different values for the same extracts?

Response: The different antioxidant activity values obtained for the three methods studied, namely ABTS, DPPH, and FRAP, can be attributed to differences in the mechanisms of action between the methods, which allow the identification of different antioxidant compounds. FRAP is a simple method that measures the ability of samples to donate electrons to reduce the ferric ion complex (Fe³⁺) into the ferrous form (Fe²⁺) with an intense blue color at acidic pH (pH 3.6). The acidic environment allows the solubility of iron ions and favors better electron transfer. The ABTS method is based on the ability of antioxidants to neutralize the ABTS radical (2,2'-azinobis (3-ethylbenzenethiazoline6-sulfonic acid) and has solubility in water and organic solvents, allowing the detection of lipophilic and hydrophilic antioxidant compounds in a wide pH range. The DPPH method consists of neutralizing the DPPH radical (1,1-diphenyl-2-picrylhydrazyl) in the last electronic layer, making the molecules stable and unavailable for the formation of chain oxidation reactions. (Lines: 581-593). Antioxidants are abundant components in plant matrices known to delay/inhibit the oxidative reactions of biomolecules caused by reactive species. No single method for determining antioxidant activity evaluates the effect of all antioxidants present in a matrix that makes up different types of bioactive compounds. Therefore, it is necessary to select more than one method to determine antioxidant activity due to the various mechanisms of action of antioxidant agents, origin of antioxidant compounds, and reactive species. (Lines: 542-548).

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Overall, the quality of manuscript has been improved (Minor revision). Here is some minor comments and suggestion as follow:

1. Table 4 - degree of Celsius needs to be in same font.

2. Please provide one scheme for the suggested mechanism for DES against orange by product and(or) Spent coffee grounds as a Figure 1.

 

 

 

Author Response

Reviewer 1

Overall, the quality of manuscript has been improved (Minor revision). Here is some minor comments and suggestion as follow:

Response: We appreciate the reviewer insights, which were pivotal in refining the quality of the final manuscript and bolstering the robustness of the research presented.

 

Comment: 1. Table 4 - degree of Celsius needs to be in same font.

Response: We appreciate the feedback and have made the necessary modifications.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I still think that the viscosity of DESs before and after adding water should be included in this work.

Author Response

Comment: I still think that the viscosity of DESs before and after adding water should be included in this work.

Response: We appreciate the observation and acknowledge the importance of this parameter, but unfortunately, we cannot provide viscosity values ​​for the DES studied now. However, our group will report viscosity values soon in another study. The study reported in the literature has shown that DES formed by the mixture of organic acids and sugars has a high viscosity. Therefore, adding water can reduce the viscosity, thus improving the extraction performance. (Lines: 358-365).

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Authors modified the manuscript according to reviewers' suggestions. So it can be accepted in its present form.

Author Response

Comment: Authors modified the manuscript according to reviewers; suggestions. So it can be accepted in its present form.

Response: Thank you very much for accepting our article.