Effects of Caffeic Acid and Chlorogenic Acid Addition on the Chemical Constituents of Lychee Wine Fermented with Saccharomyces cerevisiae DV10
Round 1
Reviewer 1 Report
the work is good like this
Author Response
Thanks for your appreciation.
Reviewer 2 Report
Positive notes and comments:
1. The topic of the manuscript is relevant, as it is related to a study of the effects of caffeic acid and chlorogenic acid addition on the chemical constituents of lychee wine fermented with Saccharomyces cerevisiae DV10. In this aspect, the article is within the scope of the scientific journal and will be of interest to its readers;
2. The working hypothesis is based on the assumption that the addition of caffeic acid will optimize the fermentation process of the wine and lead to an improvement of the technological parameters and the organoleptic qualities of the wine;
3. The previously made literature review substantiates the purpose of the study and shows the necessity of the performed comparative studies regarding the influence of chlorogenic and caffeic acids on the fermentation process caused by yeast /Saccharomyces cerevisiae DV10/;
4. The materials have been selected successfully, and the methods are modern and adequate to the study. It is clear that the authors are scientifically investigating the tradition of some communities in Asia to produce wine from the Lychee fruit (Litchi chinensis Sonn.);
5. The physico-chemical parameters of the obtained wine were studied, as well as the aromatic components produced as a result of the alcoholic fermentation;
6. The mathematical processing of the obtained data and the multifactorial analysis allow a deeper interpretation of the scientific data;
7. At the end of the scientific article, conclusions are presented as a result of the conducted studies.
Negative notes and recommendations:
1. It is not described in detail what are the technological qualities of the used S. cerevisiae DV10, which was purchased from Lallemand Inc. (Montreal, Quebec, Canada) and how they differ from other yeast strains used for alcoholic fermentation;
2. It would be useful from a technological point of view to present the effect of the addition of caffeic acid on the physicochemical indicators /especially pH/ in dynamics during the course of the three phases of alcoholic fermentation;
3. The scientific discussion of each article shows the erudition of the authors and their ability to dig deep into the scientific forest. I recommend expanding the biochemical interpretation of scientific data and explaining the influence of acid additives on the course of glycolysis and the accumulation of fermentation end products;
4. I recommend supplementing the literature reference and discussion of polyphenolic content by adding some quality articles for example: Tzanova, M., Atanassova, S., Atanasov, V., Grozeva, N.. 2020. Content of polyphenolic compounds and antioxidant potential of some Bulgarian red grape varieties and red wines, determined by HPLC, UV, and NIR spectroscopy. Agriculture (Switzerland), 2020, 10(6), pp. 1-14, 193;
5 I recommend the preparation of specific recommendations for the winemaking practice based on the results achieved in the article.
Author Response
Question 1： It is not described in detail what are the technological qualities of the used S. cerevisiae DV10, which was purchased from Lallemand Inc. (Montreal, Quebec, Canada) and how they differ from other yeast strains used for alcoholic fermentation;
Answer: Thanks for your advices. By referring to the study of Zhong et al.(2005) we found that S. cerevisiae DV10 had a better acid-reducing effect and improved the quality of lychee wine compared with other yeast strains. In addition, the reduction of volatile acid content was taken as the index in the experimental design of single factor and response surface, so we selected S. cerevisiae DV10.
Question 2：It would be useful from a technological point of view to present the effect of the addition of caffeic acid on the physicochemical indicators /especially pH/ in dynamics during the course of the three phases of alcoholic fermentation;
Answer: Thanks for your advices. Unfortunately, we did not take into account the effect of adding caffeic acid and chlorogenic acid on pH value during the experiments.
Question 3：The scientific discussion of each article shows the erudition of the authors and their ability to dig deep into the scientific forest. I recommend expanding the biochemical interpretation of scientific data and explaining the influence of acid additives on the course of glycolysis and the accumulation of fermentation end products;
Answer: Thanks for your advices. The mechanism of influence of acid additives on the course of glycolysis and the accumulation of fermentation end products should be further studied.
Question 4：I recommend supplementing the literature reference and discussion of polyphenolic content by adding some quality articles for example: Tzanova, M., Atanassova, S., Atanasov, V., Grozeva, N.. 2020. Content of polyphenolic compounds and antioxidant potential of some Bulgarian red grape varieties and red wines, determined by HPLC, UV, and NIR spectroscopy. Agriculture (Switzerland), 2020, 10(6), pp. 1-14, 193;
Answer: Thanks a lot for your valuable suggestion. And it has been revised as your suggestion.
Question 5： I recommend the preparation of specific recommendations for the winemaking practice based on the results achieved in the article.
Answer: Thanks a lot for your valuable suggestion. And it has been revised as your suggestion.
Reviewer 3 Report
The present work had as its objective to evaluate the effect of caffeic acid and chlorogenic acid addition on the volatile acid, primary aroma, and amino acid in lychee wine.
For the background that supports the research delivered in the introduction, the justification for the addition of phenolic acids during the fermentation process should be deepened and better explained. For example point out the cause (if any) that as noted "the type and content of phenolic acids are affected by Saccharomyces cerevisiae and acetic acid bacteria" (line 44), in order to understand if they affect the metabolism of microorganisms and therefore the composition of the wine or if they affect their populations or another effect that is not explained.
Although well-described analytical techniques are addressed, there are important methodological aspects that must be explained in greater detail, since they concern the design of the experiment that are fundamental for the presentation of the results, discussions and conclusions of the work.
- In section 2.2.1, the volume of juice (must) obtained from pressing the fruits is not indicated (line 66). Neither the commercial brand nor the origin of the pectinase is provided in order to know if its activity improves at 45°C for 3 hours (which, on the other hand, affects the primary aromatic compounds of the future wine). This information must be indicated.
- In line 72, change "the control group had no phenolic acid [9]" to "phenolic acids were not added to the control treatment".
- In line 73, it is indicated that the alcoholic fermentation began at 20 ° C, but it does not indicate what was the evolution of the temperature during the 15 days of fermentation and it is not indicated if the density was monitored to know if the fermentation evolved normally. It would be advisable if this information could be provided. The fermentation temperature is directly related to the metabolism of yeasts and their ability to produce fermentative (secondary) aromas.
- Line 74 states that "clear wine samples were obtained by filtering with bentonite for later use". Bentonite is not a filtering agent but is a finning agent. The dose of bentonite used should be indicated since the concentration of aromatic compounds and some phenolic compounds may decrease due to the action of bentonite.
- In line 76 change "total acid" to "total acidity", as in the tables and text.
- Line 74 must indicate the conditions in which the samples were stored until chemical and sensory analysis.
- In item 2.2.1. it does not indicate the type of deposit where the samples were fermented, their volume, if at least the treatments were done in duplicate or triplicate. It is essential that the design of the experiment is clearly explained, since in the text it is stated on several occasions that "3 samples were analyzed" (lines 150; 297), giving the impression that there were no repetitions (only one sample per treatment).
- In line 174 it is stated that "this phenomenon might be due to the inhibition of acid-producing bacteria by phenolic acids." Considering that before the fermentation process 100 mg / L of sodium sulfite (line 67) was applied, that is, 50mg / L of total SO2 and that then the juice was heated for 3 hours at 45 ° C and also that in a medium rich in CO2 such as the fermenting must where acetic bacteria are not possible to proliferate, it will not be a better explanation that S. cerevicea may have diminished the production of acetic acid (production that is well described for this yeast). I would recommend reviewing this discussion.
- Review line 186 and the text "After chlorogenic acid fermentation".
- Review the text that starts online 202 "Ferulic acid indicated "the highest polyphenol content in the control group, while gallic acid was used in the caffeic and chlorogenic acid treatment groups". Perhaps he wanted to point out that "among the phenolic compounds identified, ferulic acid presented the highest concentration in the control treatment, while in the treatments with the addition of phenolic acids it was gallic acid that presented the highest concentration".
- Lines 204-207: It is essential to understand that the phenolic compounds present in lychee juice before fermentation come from the fruit. In this case, phenolic compounds caused by yeast metabolism such as tryptopol (from the amino acid tryptophan) or tyrosol (from the amino acid tyrosine) were not found. Considering the above, there is no phenolic compound identified in Table 2 (benzoic acid such as gallic acid; flavan-3-ols such as catechins and epicatechina or cinnamic acids such as ferulic or chlorogenic acid) that has been synthesized by Saccharomyces cereviceae. Any modification in the concentration of the compounds can be explained by their possible loss due to oxidative processes as there are no compounds with antioxidant action added to the environment such as caffeic and chlorogenic acids. For this reason it is not possible to point out for example that gallic acid increases. Nor that epicatechin is generated. There is a clear antioxidant protective effect of caffeic acid that decreases significantly both in the control treatment and in the treatment with caffeic acid, but in the latter it allows to preserve gallic acid and chlorogenic acid. It is possible that the oxidation of caffeic acid contributes to the oxidation of catechins and epicatechin (see Cheynier and Moutounet, 1992; Fernández-Zurbano et al., 1995, Singleton et al., 1985; Rodríguez-Bencomo, Andújar-Ortiz, Sánchez-Patán, Moreno-Arribas, & Pozo-Bayón, 2016). Please review what is stated in the text.
- In Table 1 how it is possible that if 100 mg / L of sodium sulfite was added to the juice (line 67), that is, 50mg / L of total SO2, values of 166.83 mg / L, 155.73mg/L and 147.2mg/L total SO2 are observed in the tratments after fermentation. This is impossible, since although S. cereviceae can synthesize SO2 during fermentation, the concentrations are very small. There is an error in the results delivered in this Table.
- In Table 2 please explain what you mean by "catechins" ((+)- catechin and what else)
- Line 317. It is very curious that it is pointed out that the amino acid proline increases after the addition of phenolic acids, since proline comes from the juice and is an amino acid that cannot be metabolized by S. cereviceae, so it should remain constant in all three treatments. The only reason for its increase could be that by the action of the medium, the yeast synthesizes it in a higher concentration releasing it to the wine product of autolysis. Review this discussion.
- Line 342. Please review the discussion which states that "the strong astringency after caffeic acid treatment"was related to the high content of total phenol". If the high phenol content refers to Table 2 (which should be corrected by changing Total to Total mono-phenols), they are clearly compounds that are bitter, but not necessarily astringent.
- In the conclusions, line 358, it is stated that "both the addition of caffeic acid and chlorogenic acid increased the total phenol with tent, decreased the production of catechins". If the catechins come from the fruit and the 3 treatments were started with the same concentration of catechins, what would be the origin of "the production of catechins". Please review the concepts on type and origin of phenolic compounds in fermented beverages.
- Line 362 indicates that wine produced with the addition of caffeic acid was the "most popular" in sensory analysis. The methodology does not indicate that "popularity" has been measured, nor has acceptability or global preference been measured. Please review this conclusion.
Author Response
Question 1：For the background that supports the research delivered in the introduction, the justification for the addition of phenolic acids during the fermentation process should be deepened and better explained. For example point out the cause (if any) that as noted "the type and content of phenolic acids are affected by Saccharomyces cerevisiae and acetic acid bacteria" (line 44), in order to understand if they affect the metabolism of microorganisms and therefore the composition of the wine or if they affect their populations or another effect that is not explained.
Answer: Thanks for your advices, and it has been revised as your suggestion. In subsequent work, we have determined the effect of adding caffeic acid and chlorogenic acid to the fermentation process on microorganisms by high-throughput sequencing.
Question 2：Although well-described analytical techniques are addressed, there are important methodological aspects that must be explained in greater detail, since they concern the design of the experiment that are fundamental for the presentation of the results, discussions and conclusions of the work.
Answer:Thanks for your advices. and it has been revised as your suggestion.
Question 3：- In section 2.2.1, the volume of juice (must) obtained from pressing the fruits is not indicated (line 66). Neither the commercial brand nor the origin of the pectinase is provided in order to know if its activity improves at 45°C for 3 hours (which, on the other hand, affects the primary aromatic compounds of the future wine). This information must be indicated.
Answer:Thanks for pointing out our mistakes. And it has been revised as your suggestion.
Question 4：- In line 72, change "the control group had no phenolic acid [9]" to "phenolic acids were not added to the control treatment".
Answer: Thanks for pointing out our mistakes. And it has been revised as your suggestion.
Question 5：- In line 73, it is indicated that the alcoholic fermentation began at 20 ° C, but it does not indicate what was the evolution of the temperature during the 15 days of fermentation and it is not indicated if the density was monitored to know if the fermentation evolved normally. It would be advisable if this information could be provided. The fermentation temperature is directly related to the metabolism of yeasts and their ability to produce fermentative (secondary) aromas.
Answer: Thanks for pointing out our mistakes. And it has been revised as your suggestion.
Question 6：- Line 74 states that "clear wine samples were obtained by filtering with bentonite for later use". Bentonite is not a filtering agent but is a finning agent. The dose of bentonite used should be indicated since the concentration of aromatic compounds and some phenolic compounds may decrease due to the action of bentonite.
Answer: Thanks for pointing out our mistakes. And it has been revised as your suggestion.
Question 7：- In line 76 change "total acid" to "total acidity", as in the tables and text.
Answer: Thanks for pointing out our mistakes. And it has been revised as your suggestion.
Question 8：- Line 74 must indicate the conditions in which the samples were stored until chemical and sensory analysis.
Answer: Thanks for your advices. And it has been revised as your suggestion.
Question 9：- In item 2.2.1. it does not indicate the type of deposit where the samples were fermented, their volume, if at least the treatments were done in duplicate or triplicate. It is essential that the design of the experiment is clearly explained, since in the text it is stated on several occasions that "3 samples were analyzed" (lines 150; 297), giving the impression that there were no repetitions (only one sample per treatment).
Answer: Thanks for pointing out our mistakes. And it has been revised as your suggestion.
Question 10：- In line 174 it is stated that "this phenomenon might be due to the inhibition of acid-producing bacteria by phenolic acids." Considering that before the fermentation process 100 mg / L of sodium sulfite (line 67) was applied, that is, 50mg / L of total SO2 and that then the juice was heated for 3 hours at 45 ° C and also that in a medium rich in CO2 such as the fermenting must where acetic bacteria are not possible to proliferate, it will not be a better explanation that S. cerevicea may have diminished the production of acetic acid (production that is well described for this yeast). I would recommend reviewing this discussion.
Answer: Thanks for pointing out our mistakes. And it has been revised as your suggestion.
Question 11：- Review line 186 and the text "After chlorogenic acid fermentation".
Answer: Thanks for pointing out our mistakes. And it has been revised as your suggestion.
Question 12：- Review the text that starts online 202 "Ferulic acid indicated "the highest polyphenol content in the control group, while gallic acid was used in the caffeic and chlorogenic acid treatment groups". Perhaps he wanted to point out that "among the phenolic compounds identified, ferulic acid presented the highest concentration in the control treatment, while in the treatments with the addition of phenolic acids it was gallic acid that presented the highest concentration".
Answer: Thanks for your advices. And it has been revised as your suggestion.
Question 13：- Lines 204-207: It is essential to understand that the phenolic compounds present in lychee juice before fermentation come from the fruit. In this case, phenolic compounds caused by yeast metabolism such as tryptopol (from the amino acid tryptophan) or tyrosol (from the amino acid tyrosine) were not found. Considering the above, there is no phenolic compound identified in Table 2 (benzoic acid such as gallic acid; flavan-3-ols such as catechins and epicatechina or cinnamic acids such as ferulic or chlorogenic acid) that has been synthesized by Saccharomyces cereviceae. Any modification in the concentration of the compounds can be explained by their possible loss due to oxidative processes as there are no compounds with antioxidant action added to the environment such as caffeic and chlorogenic acids. For this reason it is not possible to point out for example that gallic acid increases. Nor that epicatechin is generated. There is a clear antioxidant protective effect of caffeic acid that decreases significantly both in the control treatment and in the treatment with caffeic acid, but in the latter it allows to preserve gallic acid and chlorogenic acid. It is possible that the oxidation of caffeic acid contributes to the oxidation of catechins and epicatechin (see Cheynier and Moutounet, 1992; Fernández-Zurbano et al., 1995, Singleton et al., 1985; Rodríguez-Bencomo, Andújar-Ortiz, Sánchez-Patán, Moreno-Arribas, & Pozo-Bayón, 2016). Please review what is stated in the text.
Answer: Thanks for pointing out our mistakes. Thank you for your suggestion, which gave us a new idea to explain the change of mono-phenol content in lychee wine after the addition of phenolic acid. We are also struggling with the analysis of this part, but there are few relevant studies on this aspect. We really appreciate it.
Question 14：- In Table 1 how it is possible that if 100 mg / L of sodium sulfite was added to the juice (line 67), that is, 50mg / L of total SO2, values of 166.83 mg / L, 155.73mg/L and 147.2mg/L total SO2 are observed in the tratments after fermentation. This is impossible, since although S. cereviceae can synthesize SO2 during fermentation, the concentrations are very small. There is an error in the results delivered in this Table.
Answer: Thanks for pointing out our mistakes. It should be that 300 mg / L of sodium hydrogen sulfite was added to the juice.
Question 15：- In Table 2 please explain what you mean by "catechins" ((+)- catechin and what else)
Answer: Thanks for pointing out our mistakes. And it has been revised as your suggestion.
Question 16：- Line 317. It is very curious that it is pointed out that the amino acid proline increases after the addition of phenolic acids, since proline comes from the juice and is an amino acid that cannot be metabolized by S. cereviceae, so it should remain constant in all three treatments. The only reason for its increase could be that by the action of the medium, the yeast synthesizes it in a higher concentration releasing it to the wine product of autolysis. Review this discussion.
Answer: Thanks for pointing out our mistakes. And it has been revised as your suggestion.
Question 17：- Line 342. Please review the discussion which states that "the strong astringency after caffeic acid treatment"was related to the high content of total phenol". If the high phenol content refers to Table 2 (which should be corrected by changing Total to Total mono-phenols), they are clearly compounds that are bitter, but not necessarily astringent.
Answer: Thanks for pointing out our mistakes. And it has been revised as your suggestion.
Question 18：- In the conclusions, line 358, it is stated that "both the addition of caffeic acid and chlorogenic acid increased the total phenol with tent, decreased the production of catechins". If the catechins come from the fruit and the 3 treatments were started with the same concentration of catechins, what would be the origin of "the production of catechins". Please review the concepts on type and origin of phenolic compounds in fermented beverages.
Answer: Thanks for pointing out our mistakes. Lei et al. (2020) found that Feizi Xiao litchi is rich in catechins, and in this experiment, the content of catechins showed a decreasing trend after the addition of caffeic acid and chlorogenic acid, but there was no relevant study, and the influencing mechanism was not clear.
Question 19：- Line 362 indicates that wine produced with the addition of caffeic acid was the "most popular" in sensory analysis. The methodology does not indicate that "popularity" has been measured, nor has acceptability or global preference been measured. Please review this conclusion.
Answer: Thanks for pointing out our mistakes. And it has been revised as your suggestion.
Reviewer 4 Report
The manuscript entitled Effects of caffeic acid and chlorogenic acid addition on the chemical constituents of lychee wine fermented with Saccharomyces cerevisiae DV10, presents information related to the effect of adding caffeic acid and chlorogenic acid to the fermentation process for the production of lychee wine. The topic is sound but the authors must revise the manuscript in order to address some issues. Below are the comments.
-Introduction. What is the rationale for using caffeic acid and chlorogenic acid? Clarify.
-Lines 41-46. Revise the coherence of the idea. How do S. cerevisiae and acetic acid bacteria affect the type and content of phenolic acids? Where?
-Line 70-72. What about the experimental designs (single factor and response surface optimization)? There are no methods and results shown in the manuscript.
-Section 2.2.2. Provide the full depiction of the methods.
-Section 3.2. How are synthesized the epicatechin and chlorogenic acid? From catechins and ferulic acid?
-Conclusion. Revise the writing. The ideas are not clear.
Author Response
Question 1：-Introduction. What is the rationale for using caffeic acid and chlorogenic acid? Clarify.
Answer: Thanks for your advices. And it has been revised as your suggestion.
Question 2：-Lines 41-46. Revise the coherence of the idea. How do S. cerevisiae and acetic acid bacteria affect the type and content of phenolic acids? Where?
Answer: Thanks for pointing out our mistakes. And it has been revised as your suggestion.
Question 3：-Line 70-72. What about the experimental designs (single factor and response surface optimization)? There are no methods and results shown in the manuscript.
Answer: Thanks for your advices.However, the experimental design and results of the single factor and response surface are being submitted to other journals. In order to prevent academic misconduct, only the results are presented here.
Question 4：-Section 2.2.2. Provide the full depiction of the methods.
Answer: Thanks for your advices. We used this method without modifications, thus the determination method in detail can refer to GB/T 15037-2006. National Standards of the People's Republic of China-Wines (China).
Question 5：-Section 3.2. How are synthesized the epicatechin and chlorogenic acid? From catechins and ferulic acid?
Answer: Thanks for your advices. There is a clear antioxidant protective effect of caffeic acid that decreases significantly both in the control treatment and in the treatment with caffeic acid, but in the latter it allows to preserve gallic acid and chlorogenic acid. It is possible that the oxidation of caffeic acid contributes to the oxidation of catechins and epicatechin (Cheynier and Moutounet, 1992; Fernández-Zurbano et al., 1995; Rodríguez-Bencomo, Andújar-Ortiz, Sánchez-Patán, Moreno-Arribas, & Pozo-Bayón, 2016).
Refer to:[1] Cheynier, V. , & Moutounet, M. . (1992). Oxidative reactions of caffeic acid in model systems containing polyphenol oxidase. J.agric.food Chem, 40(11), 60-69.
[2] Fernandez-Zurbano, P. , Ferreira, V. , Pena, C. , Escudero, A. , Serrano, F. , & Cacho, J. . (1995). Prediction of oxidative browning in white wines as a function of their chemical composition. Journal of Agricultural & Food Chemistry, 43(11), 2813-2817.
[3] Rodriguez-Bencomo, J. J., Andujar-Ortiz, I., Sanchez-Patan, F., Moreno-Arribas, M. V. , & Pozo-Bayon, M. A. (2016). Fate of the glutathione released from inactive dry yeast preparations during the alcoholic fermentation of white musts. Australian Journal of Grape & Wine Research, 22(1), 46-51.
Question 6：-Conclusion. Revise the writing. The ideas are not clear.
Answer: Thanks for pointing out our mistakes. And it has been revised as your suggestion.
Reviewer 5 Report
The manuscript: Effects of caffeic acid and chlorogenic acid addition on the chemical constituents of lychee wine fermented with Saccharomyces cerevisiae DV10, describes an study on how some phenyl propanoid acids as caffeic or chlorogenic acid could indicate that its addition could enhance the flavor character and improve the quality of lychee wine.
The manuscript is well described and seems that could be reproduced, but in the section of the analysis of organic acids, there is a doubt related to the method used to quantify such kind of compounds. In the reference of Fu, et al 2015, it is not detailed the HPLC column used. How was decided that the column indicated in this paper is the best to get the retention and separation of the different organic acids?
Author Response
Question 1：The manuscript: Effects of caffeic acid and chlorogenic acid addition on the chemical constituents of lychee wine fermented with Saccharomyces cerevisiae DV10, describes an study on how some phenyl propanoid acids as caffeic or chlorogenic acid could indicate that its addition could enhance the flavor character and improve the quality of lychee wine.
Answer: Thanks for your appreciation.
Question 2：The manuscript is well described and seems that could be reproduced, but in the section of the analysis of organic acids, there is a doubt related to the method used to quantify such kind of compounds. In the reference of Fu, et al 2015, it is not detailed the HPLC column used. How was decided that the column indicated in this paper is the best to get the retention and separation of the different organic acids?
Answer: Thanks for your advices.Although the column used was not specified in the reference of Fu, et al 2015, the C18 column was the most commonly used column for the determination of organic acid content according to the reading of other literature. However, in the actual determination,The zorbax SB-Aq is the better to get the retention and separation of the different organic acids in lychee wine than C18 column,so we chose the zorbax SB-Aq.
Round 2
Reviewer 4 Report
The authors have addressed almost all the comments. However, there still are some issues to attend to. Below are the comments.
-The language of the manuscript must be revised. There are some ideas that are difficult to follow.
-Introduction. The authors must justify why they decided to use chlorogenic acid and caffeic acid.
-Lines 53-63. Revise the writing. The ideas are not clear and are difficult to follow.
-Line 56. What is the reaction between hydroxycinnamic acids and volatile compounds? How volatile substances are retained?
-Response to question 3. Include the proper citation for the information related to the experimental designs. Include only the optimal conditions in the manuscript.
