Increasing Amino Acids Content of White Wines with Enzymes Treatments

Round 1

Reviewer 1 Report

I am very grateful you for the invitation to review the manuscript agronomy-1741949 by Scutarașu and co-authors "Increasing Amino Acids Content Of White Wines With Enzymes Treatments". This research focuses on monitoring the effect of enzymes on amino acids concentration during the fermentation of Fetească regală and Sauvignon blanc wines. The work is interesting but needs minor adjustments to increase the quality of the material.

 

Comments:

- Page 1, line 4: Standardize the citation of authors' names.

- Abstract: insert a brief theoretical description of the problem of the study or what you want to solve.

- Abstract, Page 1, line 17: specify which are the “applied work conditions”.

- Page 1, line 22: Change the repeated keywords by different words from the title to expand the search system.

- Page 1, Line 34: Change “ranged” by “range”.

- Page 1, lines 36-37: The sentence “while the doses should be higher to low pH values” it's not clear.

- Page 1, lines 41-42; 57-58: The authors should point out that despite having nutritional components, consumption should be moderate, as it can be associated with several diseases.

- Page 2, line 88: Change the sentence “The fermentation was carried out” by “After enzymatic treatment, fermentation….”.

- Page 2: The item “2.2. Chemicals” must be presented before item “2.1. Grapes and winemaking procedures”.

- Page 3, 2.3. Amino acid identification and quantification: Item can be reduced by removing theoretical explanations.

- Page 4, lines 181-182: Authors should make it clear that wine consumption should be moderate. The presence of important nutritional components does not justify consumption in large quantities, which can cause serious diseases. This information must be clear for all amino acids.

- Page 6, line 279, 322, 350 and Other:  Check the formatting of references in sentences cited and throughout the text.

Author Response

Thanks for your time and suggestions. The manuscript was revised according to the instructions received. Thus, the citations were standardized, new information was added in the abstract, repetitive keywords were changed, all the sentences mentioned were modified according to the indications, the theoretical aspects from the ‘Amino acid identification and quantification’ section were eliminated, all the aspects related to the action amino acids on human’s health were removed and other information on the topic was introduced. The item ‘2.2. Chemicals’ is now presented before item ‘2.1. Grapes and winemaking procedures’.

I hope that the work now meets the expectations and you accept it for publication.

Reviewer 2 Report

The authors present a study describing the evolution of amino acid concentrations in two white wines in the presence of pectolytic enzymes. While the idea is generally interesting, the manuscript lacks scientific depth and a basic understanding of yeast metabolism. It should not be considered for publication in its present form.

General comments

Please refer to the original template and work with the stylistic formatting that is required for this journal. The journal logo is missing on the front page, headers and footers are gone, and figures as well as tables need to be embedded in the text.

The presentation of results is less than ideal. The authors chose to discuss each amino acid separately, which makes it incredibly hard to see the bigger picture and possible correlations between amino acids. Please consider a more holistic presentation of results.

Specific comments

Line 34  Should be lower instead of smaller

Line 35  This sentence is confusing. What doses should be higher at low pH and why?

Line 39  Avoid abbreviations like doesn't. Use does not instead.

Line 42  Wine is definitely not a nutritional drink. The concentrations of amino acids after fermentation are too low to be considered nutritional. In addition to that I would like to encourage the authors to be careful with health related claims in the context of alcoholic beverages.

Line 50  How can amino acids be used for authenticity and typicality if every yeast strain uses them differently? This is highly questionable.

Line 57  No, amino acids in wine do not have health benefits that outweigh the effect of alcohol on the human body.

Line 60  I disagree that the amino acid patterns during alcoholic fermentation are poorly studied. There are plenty of papers out there starting in the 70s and 80s. Summaries of relevant studies can be found in various text books, for example "Yeast Physiology and Biotechnology" by Graeme M. Walker.

Line 66  There is no mention in the introduction why enzymes are expected to influence the amino acid composition. Pectolytic enzymes degrade the structural components of the berry skin; why would that change the yeast usable nitrogen?

Line 67  The objective is too long and too vague. Please revise.

Line 77  It is irrelevant that the grape cultivars are highly appreciated by consumers.

Line 78 Where is that specific vineyard? GPS data would be helpful. Also, what is the "classic method"? There needs to be a lot more information here.

Line 80  This goes for all the manufacturer information throughout the manuscript. The description should specify product, manufacturer, city, country.

Line 96  This list is too long and not relevant. Instead of mentioning IDs, purities or specific properties would be helpful.

Line 109 This whole paragraph is too long. There is a lot of irrelevant information listed here such as "solvent bottle" and "allows operation in optimal conditions up to 110°C". Just mention the instrument components and analytical conditions.

Line 162 There are many methods specified in the OIV collection. Please be more specific which ones you used.

Line 167 This is too short. In order to understand your sensory panel, you need to provide more information than just gender. Also, is this method published somewhere?

Line 185 Did you analyze protocatechuic acid and gallic acid? Without the data to support the claim or even literature, this is pure speculation and should be marked as such.

Line 198 I highly doubt that the yeast produced amino acids during fermentation. There are transamination reactions and leakage of stored amino acids during autolysis but those are very different things.

Line 207 Proline is not consumed under fermentation conditions. That is the main reason why we find high concentrations in wine. The whole theory of α-amino acids vs. forms that are not usable by fermenting yeast, needs to be revisited.

Line 223 This is clearly unacceptable. You cannot mention a single amino acid in the context of cancer therapy in an alcoholic beverage when ethanol is a known carcinogen. I will skip the remaining amino acids and continue in line 497 because all this discussion is not targeted towards the wine production process.

Line 497 This paragraph is not necessary in this form. Unless the chemical attributes relate to enzyme functionality or amino acid extraction, they do not add value to the manuscript.

Line 508 I am not sure that I understand the interpretation of "balanced, with good texture and acidity" from Figure 3. There is also no Figure 4 in the manuscript but it is mentioned in the text. please revise.

Line 509 This interpretation is going beyond what your data shows. You cannot say that the wines were appreciated based on quantitative sensory data. You did not perform a hedonics study. Please revise the whole paragraph.

Line 567 I do not believe that the conclusions here are supported by the data. Parts of it is pure speculation and over-interpretation.

Author Response

Thanks for your time and suggestions. The manuscript has been revised according to the instructions provided. In general, changes have been made in all sections, depending on the indications received. The document is now formatted according to the requirements of the journal. Considering the magnitude of the results and the way in which such results are presented in the specialized literature, we considered that the variant chosen for the presentation of the obtained results best correlates the explanations with the results. The theoretical aspects from the 'Amino acids identification and quantification' section were eliminated, all the aspects related to the action amino acids on human’s health were removed and other information on the topic was introduced.

I hope that the work now meets the expectations and you accept it for publication.

Reviewer 3 Report

The authors analyzed the content of amino acids in two kinds of wine fermented in a supplementary of enzymes. The results are interesting. I would like to recommend the manuscript for the publication although I have a few minor suggestions as follows:

1. The effects from the supplemented enzymes are extremely different between the two kinds of experimental wine. The authors proposed that a lot of factors such as the harvest season potentially affected the function of the enzymes. I am very curious about the possibility to figure out how these environmental conditions work. It looks too complicated to make a reliable conclusion. Therefore, I have a concern about the reproducibility.
2. Did you detect any statistic significance for the Physicochemical characteristics, since I noticed a few deviated values such as volatile acidity.
3. Did you inject the wine sample directly into the LC-MS system? Please add the information to the method section.

Author Response

Thanks for your time and suggestions. The manuscript has been modified according to the suggestions provided. There were no statistical significant differences regarding the influence of enzymes on physicochemical parameters of wines. So, at the suggestions of one the reviewers, this section was removed from the paper. New information regarding the sample preparation before the injection were added.

I hope that the work now meets the expectations and you accept it for publication.

Round 2

Reviewer 2 Report

The authors present the revised version of the manuscript that evaluates the effect of enzyme addition on the amino acid composition in fermenting white grape juice. Despite the changes that were made, I still have a hard time understanding the relevance of the research because there are still many aspects missing. Since time is limited for this review, I will just summarize my concerns without specific line numbers.

Introduction

It is still unclear why the enzyme treatment is expected to modify the amino acid composition. The introduction talks about microorganisms and enzymes being biocatalysts, which is all true, however, the connection between enzyme function and amino acid release is missing. This is important because white wines are produced without skin contact, therefore the pectic enzymes are not changing extraction. What else are they doing?

Results and Discussion

You removed the subtitles which is a great first step but the amino acids are still not discussed in a winemaking context. At the concentration range in wine, it does not matter if a single amino acid tastes sweet or not, it would be more interesting to compare their relevance for yeast metabolism. That is why I suggested a comprehensive discussion rather than one amino acid after the other.

The graphs in Figure 2 and 3 are missing error bars and significance indicators. It is unclear if those differences are actually meaningful or not. Tables 2, 3 and 4; please use the 3 letter abbreviation of the amino acids instead of numbers. It would make looking at the tables less tiring.

The sensory results are still unclear. I doubt that the sensory properties are related to an enzyme treatment. I rather believe that the enzyme treatment altered the amino acid profile (for reasons that still need to be explained) and that led to a modified yeast metabolism, which then led to a different aroma composition. It would be too simple to assume that the enzymes had a direct effect on aroma. You mention studies that found positive and negative descriptors depending on the nutrient status. This is exactly the type of correlation you need to show.

Author Response

Thanks for the feedback. The manuscript has now and in the previous revision been completed with information to satisfy the requirements of the reviewers, to be in accordance with the topic of the journal and the specialized literature.

Additional explanations have been added in the introduction section.
We believe that they are sufficient, given that this research, among others, aims to support winemakers with information on how enzymes overall influence the quality of final products. It is known that enzymes, mostly with a protein structure, can be a source of nutrients for yeast and can influence their activity. Yeast can also release certain enzymes under certain technological conditions. Enzymes are quite controversial products but they are widely used in the food industry.

The present research is part of a broader research. Some results showing the influence of enzymatic products on the physico-chemical characteristics of wine quality have been published and cited in this manuscript. We wanted to complete this information.

The tables have been updated using the abbreviations for the amino acids names.

The amino acid profile does not have a direct and singular influence on the aroma profile of the analyzed samples. It wouldn't even work, considering that there are over 1000 chemical compounds in the wine. Enzymes are involved, in addition to other mechanisms, in the activity of yeasts, which have a major influence on organoleptic characteristics, which was mentioned in the manuscript. These results supplement what we have previously published on this topic.

We will try in further tests to bring even more clarification about the correlation of amino acids and the organoleptic characteristics of wines. The paper was made in accordance with the requirements of the journal and respecting the topics.

Round 3

Reviewer 2 Report

Thank you addressing some of the concerns I raised. However, I still do not agree with some of the statements.

Line 33  Should be proteins or enzymes instead of amino acids as a source of amino acids. However, this point is really controversial. Enzymes and proteins by themselves are too big to be taken up by the yeast as a nutrient source. Peptides are feasible but functional technical enzymes are not. So unless you are suggesting that the enzymes are digested into peptides and amino acids (which would make them non-functional in your scenario), they are not providing nutritional support directly. The statement on page 2 lines 9 and 10 needs to be revised or supported by a reference.

Figure 2 and 3  As I suggested earlier, it would be great to have the significance indicators in the graphs in order to see if the differences are actually significant. I realize that you have the information in tables but given the number of graphs and attributes, I think combining the information is necessary for understanding it.

Page 11, line 21  It is not relevant that leucine contributes to insulin formation. This is not related to wine production or yeast metabolism.

Page 12, line 26  Is there an explanation why proline is decreasing if it is not consumed by the yeast?

Page 13, line 22  Glycine might have a sweet taste as a pure substance but certainly not in the concentrations found in wine. Sugars, ethanol, and glycerol are much more potent. 

Page 14, line 5  Melanin is produced by lactic acid bacteria?

Page 15, line 41  I am wondering if the "considerable formation of amino acids by yeast, under the action of endogenous enzymes" is really a direct correlation. What are the enzymes doing to encourage transamination reactions for example?

Page 16, line 24  Why would there be a correlation between incomplete fermentation and higher concentrations of glutamate?

Page 16, line 47  β-glycosides can produce effective results in Sauvignon blanc wines? The exact same wording is repeated on page 18, line 15.

Author Response

The manuscript was revised according to the suggested instructions, as follows:

Line 33 – ‘Amino acids’ was replaced with ‘enzymes’. The statement on page 2 lines 9 and 10 was revised and reference was added.

Figures 2 and 3 – The statistical test were applied for the final samples (resulting wines). The significance value and homogenous groups were added to the graphs.

Page 11, line 21 – The phrase was removed from the paper.

Page 12, line 26 – An explanation was included in the text.

Page 13, line 22 – The text was revised. Even if sugars and ethanol are much potent in defining the sweet taste, the physicochemical results highlighted that samples with high residual sugar values did not influence the sensory perception of tasters. Moreover, the alcoholic strength value was similar for all variants of each variety.

Page 14, line 5 – The phrase was revised.

Page 15, line 41 – The phrase was revised.

Page 16, line 24 – The mentioned idea was not found in the paper.

Page 16, line 47 – The phrase was revised.

All changes are marked with yellow in the text. We hope that the manuscript now meets the expectations and will be accepted for publication.