Review Reports - The Effect of Clarification Protocols on the Vinification of White Wines with Papain and Bromelain Immobilized on Glutaraldehyde Activated Chitosan

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I have reviewed the manuscript titled "The impact of various immobilization protocols of proteases, on the clarification and quality of white wines". The authors explored the immobilization of Papain and Bromelain on chitosan microparticles and their application to musts and fermented wines. The manuscript is well written and organized, and it presents some interesting results. However, the study has significant gaps that limit its scientific rigor and practical applicability. Key issues include a lack of detailed comparative data, insufficient statistical analysis, and inadequate methodological clarity. Furthermore, important aspects such as enzyme purification rationale, immobilization results, and catalyst characterization are not thoroughly addressed. These issues must be resolved to strengthen the article’s contributions and make it suitable for publication. A major revision is recommended to address these concerns.

Lack of Comprehensive Comparative Data: The study compares immobilized enzymes with bentonite, but the data are limited in detail. Differences in long-term stability, flavor, and effectiveness are underexplored. Including more in-depth comparisons and statistical analysis would strengthen the findings and show if the differences are meaningful.
Limited Statistical Analysis: No statistical validation is provided to assess the differences between treatments. Statistical tests (e.g., ANOVA) should be used to demonstrate whether variations in wine characteristics are significant.
Sensory Evaluation Issues: The sensory evaluation with only 21 tasters is relatively small and lacks clear methodology (e.g., blind testing). Expanding the sample size and providing more detailed sensory testing protocols would increase the reliability of the results.
Incomplete Literature Review: The article doesn’t sufficiently engage with recent literature on wine clarification technologies. A more robust review of current research, particularly on enzyme-based alternatives, would better position this study in the broader context of winemaking advancements.
Insufficient Methodological Details: Key procedures, such as immobilization protocols and chitosan preparation, lack sufficient detail for replication. More explicit experimental descriptions, including reaction conditions, are necessary to ensure reproducibility by other researchers.
Purification of Commercial Enzymes: The paper uses commercial Papain and Bromelain but further purifies them without explaining why this step is needed. Clarifying the reason for additional purification (e.g., removal of specific impurities or performance enhancements) is essential for understanding its relevance.
Lack of Immobilization Results: There’s no data on the outcome of immobilization (e.g., SDS-PAGE results or purification efficiency). Adding these details would validate the effectiveness of the enzyme immobilization process and its impact on enzyme activity.
Characterization of Immobilized Catalysts: Almost no information is provided on immobilization efficiency (e.g., how much enzyme was immobilized per gram of support) or the impact on enzyme activity. The authors should include these metrics and assess whether the immobilization process affected enzyme stability and catalytic function.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The overall quality of the English language in the manuscript is acceptable; however, minor editing is recommended to enhance clarity and coherence. Some sentences could benefit from a smoother flow and better organization of ideas.

Author Response

We thank all Reviewers for their constructive comments and suggestions.

A general authors’ response: The title and the text have been thoroughly revised along with English language issues.

Response to Reviewer 1:

Lack of Comprehensive Comparative Data:

The study compares immobilized enzymes with bentonite, but the data are limited in detail.

(a) Differences in long-term stability, flavor, and effectiveness are underexplored.

(b) Including more in-depth comparisons and statistical analysis would strengthen the findings and show if the differences are meaningful.

Authors’ response:

(a) Differences in long-term stability, flavor, and effectiveness have been explored (within lines 126-141 of the initial version). This part of text has been accordingly edited in the revised version (subsection 2.3. Vinifications: clarification effectiveness of immobilized proteases, and bentonite of the revised version). Accordingly, the new sub-subsections 3.2.8. Sensory Evaluation, and 3.2.9. Statistical analysis, have been added in the section 3. Materials and Methods, whereas the old 3.2.9. Wine protein heat test, has been renamed to 3.2.10. Wine protein heat test.

(b) In the revised version of our manuscript comparisons of our results versus previously reported ones have been incorporated; these comparisons have been incorporated within the text (subsections and sub-subsections 2.3, 3.2.3.3, and 3.2.8).

2.3. Vinifications: clarification effectiveness of immobilized proteases, and bentonite

3.2.3.3. Total protease content and significant immobilization parameters:

3.2.8. Sensory Evaluation:

Limited Statistical Analysis: No statistical validation is provided to assess the differences between treatments. Statistical tests (e.g., ANOVA) should be used to demonstrate whether variations in wine characteristics are significant.

Authors’ response:

A second new sub-subsection (3.2.9. Statistical analysis) has been added in the section 3; one-way ANOVA tests, were applied to evaluate whether or not are significant the variations between the wines' characteristics. The results of statistical analysis are depicted in Table S4.

Sensory Evaluation Issues: The sensory evaluation with only 21 tasters is relatively small and lacks clear methodology (e.g., blind testing). Expanding the sample size and providing more detailed sensory testing protocols would increase the reliability of the results.

Authors’ response:

According to several published articles, and as far as it concerns the number of participating testers, it seems more likely that 21 evaluators are well acceptable [Chira, et al, 2011; Ligas and Kotseridis, 2024; Williamson and Francis, 2024]. All of them are habitual consumers of white wines from Debina grape variety.

Incomplete Literature Review: The article doesn’t sufficiently engage with recent literature on wine clarification technologies. A more robust review of current research, particularly on enzyme-based alternatives, would better position this study in the broader context of winemaking advancements.

Authors’ response:

The revised version of the article has been sufficiently engaged with five (5) more recent literature on wine clarification technologies and particularly on enzyme-based alternatives, the following: 23, 24, 25, 29, 34. Two more related references have been incorporated in the supplementary materials (i.e., S-References 1 and 2).

Insufficient Methodological Details: Key procedures, such as immobilization protocols and chitosan preparation, lack sufficient detail for replication. More explicit experimental descriptions, including reaction conditions, are necessary to ensure reproducibility by other researchers.

Authors’ response:

In our opinion, the protocols of both chitosan preparation and immobilization processes are analytically described (sub-subsections 3.2.4 and 3.2.5) and can be quite easily reproduced. Moreover, all reaction conditions have been also described in detail, in section “3. Materials and Methods”. Some complementary details were given in the revised version that specify the term “mild shaking” to 140 rpm.

Purification of Commercial Enzymes: The paper uses commercial Papain and Bromelain but further purifies them without explaining why this step is needed. Clarifying the reason for additional purification (e.g., removal of specific impurities or performance enhancements) is essential for understanding its relevance.

Authors’ response:

To our knowledge, all purchased enzymes, and regardless of the supplier's purity descriptions, should be purified to remove both physical impurities (according to the supplier's protocols followed) and inactive molecules whose number increases during long-term storage. The latter is mandatory for proteases, as it is well known that their self-hydrolysis occurs continuously. Furthermore, and in our opinion, the aforementioned annotation is commonly well known and it is not necessary to be incorporated in the text of our manuscript.

7. Lack of Immobilization Results: There’s no data on the outcome of immobilization (e.g., SDS-PAGE results or purification efficiency). Adding these details would validate the effectiveness of the enzyme immobilization process and its impact on enzyme activity.

Authors’ response:

The collected pooled active fractions of both purified papain and bromelain were evaluated by SDS-PAGE. In both cases one single band was observed corresponding to either Mr = 22 kDa or Mr = 23 kDa for the papain or the bromelain, respectively, and are mentioned in the text. The procedure followed for the SDS-PAGE, as well as both images of the electrophorese (TIFF files) are depicted in the supplementary materials of the revised version (Figures S1 and S2). Moreover, a new sub-subsection (3.2.11. SDS-PAGE) has been added to the text.

Characterization of Immobilized Catalysts: Almost no information is provided on immobilization efficiency (e.g., how much enzyme was immobilized per gram of support) or the impact on enzyme activity. The authors should include these metrics and assess whether the immobilization process affected enzyme stability and catalytic function.

Authors’ response:

A purification protocol of papain and bromelain has been added in the Supplementary Materials as Table S2. Additionally, two new sub-sub-subsections were added to the text, i.e., 3.2.3.3. Total protease content and significant immobilization parameters, and 3.2.3.4. Activity measurements of proteases. Accordingly, 7.8 mg of immobilized papain and 7.3 mg of immobilized bromelain, i.e., 15.6 mg papain per one gram of support, as well as 14.6 mg bromelain per one gram of support, were calculated. These results have been incorporated within the section 2. Results and Discussion.

The overall quality of the English language in the manuscript is acceptable; however, minor editing is recommended to enhance clarity and coherence. Some sentences could benefit from a smoother flow and better organization of ideas.

Authors’ response:

The English language in revised version of the manuscript has been carefully edited according to the recommendations of the reviewer.

Finaly, we would like to thank Reviewer 1 for his/her constructive comments and suggestions.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

General comment: the research demonstrates some valuable insights into protease immobilization on chitosan particles for wine clarification, but there are several aspects of the paper that need to be revised. The title should be more specific to really demonstrate the scope of the research. There are some inconsistencies and conditions throughout the text that need to be addressed and included. The quality of the figure and scheme is low. The conclusion should include some more specific data which will improve the manuscript. My primary concern is why the authors decided to use 10 mg/mL of bromelain and 30 mg/mL of papain. Since the amount of papain used is 3 times higher, comparing the results seems improper without providing an explanation on the amounts used.

Specific comments:

Title – does the title seem a bit unspecific? “Various” implies that there is a greater number of protocols, when in reality, there are two and both of them are on chitosan particles. I would strongly recommend providing more specific title

22 – same comment with “various” as in the title

30 – sensory evaluation with 21 tasters is mentioned in the abstract but not discussed in the paper, only mentioned in line 97. Only the most important parts of the study, discussed in the main text, should be mentioned in the abstract

71 – in row 71 it is stated N175, but in row 74 it is N157, please check

88 – I think that “and/or” part should be just “and”, as reliable immobilization protocol was detected for both proteases

104 – “although” and “however” both have the same function here, only one is necessary in this sentence

108 – if crucial factors are listed (rate, shaking, temperature), I think their values should also be included here

111 – how was the stability of formed chitosan microparticles checked? It is not explained in the experimental section either

Table 1 – it is a bit unclear how the wines, presented in Table 1, were prepared. As there were two alternative procedures of preparation described (resulting in wines of A group and B group), it is unclear how were the wines presented in this table obtained. Also, the title refers to “quantitate” analysis.

126 – there are some inconsistencies in this sentence when compared to the Table 1. First, in the table measuring units are mg/L, while here in brackets mg/mL. Second, in the table value of 3.0 is listed, while in text 3.1

142 – experimental section is missing some important information. There should be a section on characterization of wines, which will specify which methods and instruments were used to characterize the wines and obtain results given in Table 1 and supplementary table

150 - Building B seems unnecessary

191 – 300 mL prepared, but only 20 mL used?

193 – I would include rpm instead of, or along with “stirred gently” in 193 and “mild shaking” in 194. The same applies for rows 205 and 211

193 – usually “t” is time and “T” is temperature. If authors decide to change, they should do it throughout the text

210 – “and/or” part here is confusing, as “and” would imply co-immobilization. I would go with only “or”, it is also consistent with “either”

Scheme 1 – it seems to be low quality, and there is no title of the scheme. I think that the scheme should be prepared again and the title should be included

240 – mL instead of ml

260 – why the authors decided to use 10 mg/mL of bromelain and 30 mg/mL of papain in both clarifying procedures? If the amount of used papain was 3 times higher, it seems a bit unfair to compare the obtained results, without any explanation or rationale behind the used amount of immobilized enzymes.

269 – the figure seems to be low quality, image in higher resolution should be included

288 – the conclusion is very broad, I would strongly recommend including some more specific numbers in the conclusion

Comments on the Quality of English Language

Language is mostly clear, minor editing is required.

Author Response

We thank all Reviewers for their constructive comments and suggestions.

A general authors’ response: The title and the text have been thoroughly revised along with English language issues.

Response to Reviewer 2:

The title should be more specific to really demonstrate the scope of the research./ Does the title seem a bit unspecific? “Various” implies that there is a greater number of protocols, when in reality, there are two and both of them are on chitosan particles. I would strongly recommend providing more specific title. / (line) 22 – same comment with “various” as in the title.

Authors’ response:

The title has been changed to the revised version. A new title was adopted i.e., “The effect of clarification protocols on the vinification of white wines with papain and bromelain immobilized on glutaraldehyde activated chitosan” which we consider as more specific, and which reflects more realistically the scope of the research. A similar correction (elimination of word “various”) was done also in the line 22 of the initial version.

The quality of the figure and scheme is low.

Authors’ response:

The quality of both Scheme 1 and Figure 1, as well as of the new Figures S1 and S2, have been improved, in the revised version.

The conclusion should include some more specific data which will improve the manuscript.

Authors’ response:

A thoroughly revised Conclusions section replaced that of the initial version, where more specific data, as well as comparison of our results to those of previously published related studies have been also included.

My primary concern is why the authors decided to use 10 mg/mL of bromelain and 30 mg/mL of papain. Since the amount of papain used is 3 times higher, comparing the results seems improper without providing an explanation on the amounts used.

Authors’ response:

We used 10 mg/mL of bromelain and 30 mg/mL, in the sub-subsection 3.2.7. Production of clarified wines, was for the following reasons, i.e., (a) it is well known that papain possesses a more active catalytic site versus that of bromelain (indicative may be the pH profiles and the estimated pK_a-values of both proteases [14-17 and references therein]), (b) this higher reactivity of papain is also reflected by an increased rate of self-hydrolysis (versus the less reactive bromelain) when the conditions favor this phenomenon, (c) in the sub-subsection 3.2.7. Production of clarified wines, it is referred that after the addition of the clarifying agents (either bentonite or immobilized proteases) the samples (fermented musts and/or ready wines) “ were incubated for 5 days at 5^oC”, (d) during the time of 5 days and at temperature of 5^oC in an aqueous medium, it is more likely that an even relatively small percentage of the immobilized proteases may be auto hydrolyzed, (e) therefor, it is reasonable to assume that this phenomenon will be more profound in the case of papain. These thoughts led us to increase the quantity of the immobilized papain, in order to be sure that we will have positive results; potentially negative results would be characterized as ambiguous.

Sensory evaluation with 21 tasters is mentioned in the abstract but not discussed in the paper, only mentioned in line 97. Only the most important parts of the study, discussed in the main text, should be mentioned in the abstract.

Authors’ response:

A new sub-subsection (3.2.8. Sensory Evaluation) has been added in the section 3. Materials and Methods, as well as a statistical analysis is presented in the revised version; these results are depicted in Table S3. Therefore, we conformed to the Reviewer’s’ comments and suggestions.

(line) 71 – in row 71 it is stated N175, but in row 74 it is N157, please check.

Authors’ response:

This error has been accordingly corrected to N¹⁵⁷

(line) 88 – I think that “and/or” part should be just “and”, as reliable immobilization protocol was detected for both proteases.

Authors’ response:

It has been corrected to “..papain and bromelain ..”

(line) 104 – “although” and “however” both have the same function here, only one is necessary in this sentence.

Authors’ response:

The last sentence of the subsection 2.2. Purification and Immobilization of papain and bromelain, has been revised according to the to the Reviewer’s’ comments and suggestions.

(line) 108 – if crucial factors are listed (rate, shaking, temperature), I think their values should also be included here,

Authors’ response:

The rate of dropwise addition of the chitosan dispersion to the beaker containing TPP-sodium solution is 20 mL/h, and the temperature 25^oC; both of these factors are referred in the sub-subsection 3.2.4. Preparation of chitosan micro particles (within the section Materials and Methods). The mild shaking of beaker was at 140 rpm, and it has been added in the revised version, in the same sub-subsection 3.2.4. Preparation of chitosan micro particles.

(line) 111 – how was the stability of formed chitosan microparticles checked? It is not explained in the experimental section either.

Authors’ response:

The stability of formed plain chitosan microparticles (without glutaraldehyde spacer and immobilized proteases), as well as the formed chitosan microparticles carrying the glutaraldehyde spacer and the immobilized proteases, were checked after six and twelve months. The plain chitosan microparticles remained unchanged after twelve months. In the case of chitosan microparticles carrying the glutaraldehyde spacer and the immobilized proteases a decay of their proteolytic activity of about 15%, after six months, and about 40% after twelve months was observed. However, these latter changes are not macroscopically visible. We incorporated this important information within the text, as last sentence of the sub-subsection 3.2.5. Immobilization processes of papain and bromelain.

Table 1 – it is a bit unclear how the wines, presented in Table 1, were prepared. As there were two alternative procedures of preparation described (resulting in wines of A group and B group), it is unclear how were the wines presented in this table obtained. Also, the title refers to “quantitate” analysis.

Authors’ response:

A revised title has been provided for Table 1, i.e. "Table 1. Results of the quantitative analysis of white wines of A-groups produced with the indigenous yeast strain Saccharomyces cerevisiae Z622, using different clarification treatments". This title is consistent with the Reviewer's comment. All the referred measurements in Table 1 were quantitative. The followed methodology applied for the quantitative analysis has been incorporated into the Supplementary Materials, for the sake of space, as the main subject of this article is far from the development of analytical methods.

(line) 126 – there are some inconsistencies in this sentence when compared to the Table 1. First, in the table measuring units are mg/L, while here in brackets mg/mL. Second, in the table value of 3.0 is listed, while in text 3.1.

Authors’ response:

The units are mg/L, and were corrected within the text, in the revised version. The value in the last line / right column of Table 1 is 3.1, and it has been corrected.

(line) 142 – experimental section is missing some important information. There should be a section on characterization of wines, which will specify which methods and instruments were used to characterize the wines and obtain results given in Table 1 and supplementary table

Authors’ response:

This suggestion of the Reviewer has been accomplished according to the following:

(a) Addition of related detailed information in the subsection 2.2., (b) Changes in the title of Table 1. (c) addition of related details in the subsection 2.3., on the (i) turbidity measurements, (ii) quantitative analysis of volatile compounds (in the supplementary materials), and (iii) the sensory evaluation (also in the new 3.2.1. sub-subsection), (d) the addition of Tables S2, S3 and S4, and the results from the one-way of ANOVA tests, (e) addition of sub-subsection 3.2.3.3. (Total protease content and determination of significant immobilization parameters) and the related results in 2.2., as well as addition of sub-subsection 3.2.3.4. (Activity measurements of proteases), and (f) addition of sub-subsection 3.2.9, (Statistical analysis).

(line) 150 - Building B seems unnecessary // It is in line 152, and

Authors’ response:

The “Building B” has been erased in the revised version.

(line) 191 – 300 mL prepared, but only 20 mL used?

Authors’ response:

Many experiments were necessary to be carried out. Therefore, for the sake of uniformity of raw material and reliability of comparisons we had to rely on a single initial preparation of Chitosan micro particles. Besides, the prepared Chitosan micro particles remained unchanged after twelve months (please, see the Authors’ response of the comment 10).

(line) 193 – I would include rpm instead of, or along with “stirred gently” in 193 and “mild shaking” in 194. The same applies for rows 205 and 211.

Authors’ response:

All the commented fields were specified by referring the corresponding rpm values. The text has been accordingly corrected (please, see the Authors’ response of the comment 9).

(line) 193 – usually “t” is time and “T” is temperature. If authors decide to change, they should do it throughout the text.

Authors’ response:

The temperature symbol has been removed.

(line) 210 – “and/or” part here is confusing, as “and” would imply co-immobilization. I would go with only “or”, it is also consistent with “either”

Authors’ response:

It has been corrected to “or”.

Scheme 1 – it seems to be low quality, and there is no title of the scheme. I think that the scheme should be prepared again and the title should be included

Authors’ response:

Scheme 1 has been reconstructed thoroughly with higher quality, and incorporated in the text of the revised version. A title “Schematic representation of the immobilization process of papain and bromelain in Chitosan micro particles through a glutaraldehyde spacer.” has been added.

(line) 240 – mL instead of ml.

Authors’ response:

It has been corrected to “mL”.

(line) 260 – why the authors decided to use 10 mg/mL of bromelain and 30 mg/mL of papain in both clarifying procedures? If the amount of used papain was 3 times higher, it seems a bit unfair to compare the obtained results, without any explanation or rationale behind the used amount of immobilized enzymes.

Authors’ response:

It has been already given the necessary explanations in the Authors’ response of the comment 4.

(line) 269 – the figure seems to be low quality, image in higher resolution should be included

Authors’ response:

Figure 1 has been reconstructed thoroughly with higher quality and incorporated in the text of the revised version.

(line) 288 – the conclusion is very broad, I would strongly recommend including some more specific numbers in the conclusion.

Authors’ response:

A thoroughly revised Conclusions section replaced that of the initial version, where more specific data, as well as comparison based on specific numbers are included in the revised version. In addition, our results are compared to those of previous published relative works.

The text has been thoroughly revised by editing existing English language issues.

Finaly, we would like to thank the Reviewer 2 for his/her constructive comments and suggestions

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

General comments

The Abstract, Motivation and Conclusions must be consistent.

The title of the paper The impact of various immobilization protocols of proteases, on the clarification and quality of white wines does not reflect the essence of the study and the content of the text. The proposed title such as Immobilization of papain and bromelain on activated chitosan and testing prepared biocatalysts in vinification of white vines seems more appropriate for the following reasons: 1) only one type of biocatalysts prepared using activated by glutaric aldehyde chitosan and purified plant protease was studied in more details; 2) information about other immobilization protocols, as well as testing prepared biocatalysts are presented very sparingly (lines 90–91, lines 103-105 Attempts aimed at armless immobilizations of both the aforementioned proteases, although successful, however, their activity decreased in a relatively short period of time; lines 221-228) 3) there is no information about the biocatalytic properties of “armless immobilized” proteases, except “their activity decreased in a relatively short period of time”

Therefore, keywords also do not reflect the essence of the study. The proposed keywords can be arranged in the following order: Immobilization of Papain; Immobilization of Bromelain; Activated Chitosan; Clarification of Wines; Protein Haze Removal

*Clarification of wines and protein haze were analyzed using photometer and nephelometer respectively.

Since the experimental results are not convincing and do not support the conclusions, it is necessary to obtain and present additional data.

It should also be noted that there is a complete lack of information about “the organoleptic characteristics of ready wines were compared with traditionally produced white wine controls treated with bentonite, through sensory evaluation (twenty-one tasters)” and improvement of “sensory and stability characteristics” (lines 26-31)

Comments and questions

Line 27. “per clarifying agent…. and per treatment, on” …Sentence needs editing for clarification

Line 30. What is sensory evaluation (twenty-one tasters)? What parameters are evaluated?

Section 3.2.1. Buffers As a rule, buffers are called like this: phosphate buffer, acetate buffer, tartrate buffer (and not buffer phosphate, buffer acetate, etc.)

Line 102 and so on in the text. The phrase “chitosan particles equipped with glutaraldehyde arms is not entirely successful. Better to use “chitosan particles activated (or modified, or functionalized) by glutaraldehyde spacers”

Line 142. Section 3. Materials and Methods It is recommended to collect the description of all devices and equipment in a separate section.

Line 159 and 182. рН value of Tris-HCl buffer is different, pH 5.00 and 8.00 respectively. Check please.

Line 177 and 187. Substrates for proteases (Suc-Phe-Leu-pNA and Cbz-Phe-Arg-pNA for papain and bromelain respectively) were described in the same ref. [14]. But the title is “Insight into the Catalysis of Hydrolysis of Four Newly Synthesized Substrates by Papain: A Proton Inventory Study” and the information may only concern papain. It is recommended to decipher the abbreviations Suc, Cbz, pNA and briefly describe the procedure for measuring activity

Line 179. Сheck the information from the ref. [25] and in the text for consistency with each other

Lines 206-207. Really, stable Schiff bases were formed between the free primary amino groups of chitosan and/or amino acid residues in protein and one of the aldehyde groups of glutaraldehyde (glutaric dialdehyde, GA). How was it proven that GA did not form cross-links within the chitosan polymer between the two neighboring amino groups?

Line 244. What is sensor control? What parameters are controlled?

Table 1. This table is recommended to be supplemented with all the results from Table S1 (columns for W_S^Bentonite, W_s^Bromelain , W_s^Papainor explain why this cannot be done. It is recommended to add data on changes in turbidity to the table.

Line 133. It should be explained what effect on the varietal character of white wines was observed.

Fig.1. It is recommended to make this figure clearer and more distinct. Use in same manner, for example, FERMENTATION (instead Fermentation or Fermented blank).

It should be indicated which parameter formed the Conclusion that immobilized Papain is the best. From all data presented in tables and described in text it is clear that there is no particular advantages for application of immobilized proteases. Preparing biocatalysts requires reagents, time and labor, including obtaining chitosan as a support for immobilization. Bentonite can certainly be such a support. The main advantage of immobilization, namely, the reuse of biocatalysts, is not used or discussed in any way in this paper. Are immobilized proteases consumables and not reused??

The typos are corrections are highlighted in color pdf-file attached.

Comments for author File: Comments.pdf

Author Response

We thank all Reviewers for their constructive comments and suggestions.

A general authors’ response: The title and the text have been thoroughly revised along with English language issues.

Response to Reviewer 3:

The Abstract, Motivation and Conclusions must be consistent.

Authors’ response:

Both the Abstract and Conclusions sections have been thoroughly and carefully revised so that the aim of this article, its design, applied methods, results and conclusions are consistent and understandable as a whole.

The title of the paper The impact of various immobilization protocols of proteases, on the clarification and quality of white wines does not reflect the essence of the study and the content of the text. The proposed title such as Immobilization of papain and bromelain on activated chitosan and testing prepared biocatalysts in vinification of white wines seems more appropriate for the following reasons: 1) only one type of biocatalysts prepared using activated by glutaric aldehyde chitosan and purified plant protease was studied in more details; 2) information about other immobilization protocols, as well as testing prepared biocatalysts are presented very sparingly (lines 90–91, lines 103-105 Attempts aimed at armless immobilizations of both the aforementioned proteases, although successful, however, their activity decreased in a relatively short period of time; lines 221-228) 3) there is no information about the biocatalytic properties of “armless immobilized” proteases, except “their activity decreased in a relatively short period of time”

Authors’ response:

According to the Reviewer's comments and suggestions (1 and 2 in this page), the title of the article was changed to "The effect of clarification protocols on the vinification of white wines with papain and bromelain immobilized on glutaraldehyde activated chitosan", as only one of the three prepared biocatalysts were used to clarify white wines.

Two more biocatalysts were prepared with papain and bromelain immobilized on activated chitosan micro particles without a spacer (described in sub-subsection 3.2.5. Papain and bromelain immobilization procedures). Although in both these cases papain and bromelain were successfully immobilized, however, their activity declined relatively fast over time (described in subsection 2.2. Purification and Immobilization of papain and bromelain). Therefore, the immobilized protocols of these two cases of immobilization without use of a spacer, are described in details within the text. Nevertheless, unfortunately, cannot be further tested and/or used in wine clarification processes.

Therefore, keywords also do not reflect the essence of the study. The proposed keywords can be arranged in the following order: Immobilization of Papain; Immobilization of Bromelain; Activated Chitosan; Clarification of Wines; Protein Haze Removal.

Authors’ response:

Keywords have been changed to “Activated Chitosan; Immobilization of papain; Immobilization of bromelain; Clarification of wines; Protein Haze Removal”, according to the suggestion of Reviewer.

*Clarification of wines and protein haze were analyzed using photometer and nephelometer respectively.

Authors’ response:

In this study the Double beam spectrophotometer Perkin-Elmer Lambda 25 UV-Vis, equipped by digital driving and recording system (hardware and software), has been used, as well as another photometric device i.e., a specific wine analyzer (CDR WineLab® Junior Analyzer), and a turbidimeter (Eutech™ TN-100, portable digital turbidimeter). All of these devices are referred in the subsection 1.2. Specific apparatus, in the revised version of the manuscript.

Since the experimental results are not convincing and do not support the conclusions, it is necessary to obtain and present additional data.

Authors’ response:

Additional data are presented in the revised version that support the conclusions. More specifically, all the necessary documentation for the organoleptic characteristics of ready white wines clarified with immobilized proteases has been incorporated in the text. These white wines were compared through sensory evaluation to both a commercial sample of Debina white wine and the white wines produced in this study and clarified with bentonite. Subsequently, a new subsection (3.2.8. Sensory Evaluation) was added to section 3. Materials and Methods.

Line 27. “per clarifying agent…. and per treatment, on” …Sentence needs editing for clarification.

Authors’ response:

Two consecutive sentences are replaced in the text (Abstract), as follows: “The results showed the effect of immobilized proteases and bentonite, in the removal of haze from the ready white wines produced in this study. Moreover, the organoleptic characteristics of the ready wines clarified with immobilized proteases were compared with a commercial sample of Debina white wine, but also with the bentonite-clarified white wines prepared in this work, through sensory evaluation.”

Line 30. What is sensory evaluation (twenty-one tasters)? What parameters are evaluated?

Authors’ response:

A new subsection (3.2.8. Sensory evaluation) has been added to the text, where the composition of the panel of sensory evaluators (testers) is referred in details, as well as the detailed process of the sensory evaluation is also referred. In this work the used grape musts were also subjected to a specific sensory evaluation. In the case of sensory evaluation of white wines, evaluators were asked to compare specific characteristics of white wines, namely the flavors (a) fruity or (b) woody or (c) acidic, odors (a) unpleasant or (b) aromatic , as well as the clarity of the wines. References to relevant published articles have also been given in subsection 3.2.8. Sensory Evaluation.

Section 3.2.1. Buffers As a rule, buffers are called like this: phosphate buffer, acetate buffer, tartrate buffer (and not buffer phosphate, buffer acetate, etc.).

Authors’ response:

We agree with the Reviewer. The sub-subsection 3.2.1. Buffers, has been corrected.

Line 102 and so on in the text. The phrase “chitosan particles equipped with glutaraldehyde arms is not entirely successful. Better to use “chitosan particles activated (or modified, or functionalized) by glutaraldehyde spacers”.

Authors’ response:

We agree with the Reviewer. The text has been thoroughly corrected.

Line 142. Section 3. Materials and Methods It is recommended to collect the description of all devices and equipment in a separate section.

Authors’ response:

We agree with the Reviewer. A new sub-subsection 3.1.2. Specific apparatus has been added.

Line 159 and 182. pH value of Tris-HCl buffer is different, pH 5.00 and 8.00 respectively. Check please.

Authors’ response:

The pH-value of the referred Tris-HCl buffer is 8.00. The text has been corrected. Besides, the buffering capacity of Tris-HCl buffers is between the values pH 7.0 and 9.2.

Line 177 and 187. Substrates for proteases (Suc-Phe-Leu-pNA and Cbz-Phe-Arg-pNA for papain and bromelain respectively) were described in the same ref. [14]. But the title is “Insight into the Catalysis of Hydrolysis of Four Newly Synthesized Substrates by Papain: A Proton Inventory Study” and the information may only concern papain. It is recommended to decipher the abbreviations Suc Cbz, pNA and briefly describe the procedure for measuring activity.

Authors’ response:

The substrates for papain and bromelain, N-Succinyl-Phe-Leu-p-nitroanilide, and N-Carbobenzoxy-Phe-Arg-p-nitroanilide, respectively, have been used in this work; the text has been accordingly corrected. The use of these substrates is reported within references [14] and [16]. Unintentionally, only reference [14] was cited in the original version, and it has been corrected in the revised version.

Line 179. Сheck the information from the ref. [25] and in the text for consistency with each other

Authors’ response:

In reference [24] are described procedures for purification of proteins by both Mono-S cation-exchange chromatography and Mono-Q anion-exchange chromatography. In reference [25] are given details about significant precautions that could improve the purification of bromelain, as well as of other cysteine proteases. Therefore, both references [24] and [25] should be referred in the text; the text has been corrected.

Lines 206-207. Really, stable Schiff bases were formed between the free primary amino groups of chitosan and/or amino acid residues in protein and one of the aldehyde groups of glutaraldehyde (glutaric dialdehyde, GA). How was it proven that GA did not form cross-links within the chitosan polymer between the two neighboring amino groups?

Authors’ response:

The formation of stable Schiff bases was successful, as well as the immobilization of both proteases on chitosan particles activated by glutaraldehyde spacers, for three reasons:

(a) We used a dilute solution of glutaraldehyde, 3% (^v/_v) in phosphate buffer pH 7.00 (in the sub-subsection 3.2.2, a correction was occurred: the pH-value 7.00 of the used phosphate buffer was added in the revised version).

(b) Schiff bases can be formed [1] and stabilized at pH values ≥ 7.00 [2], where (in our case) only a part of the free animo groups of chitosan, whose pKa-values vary from 6.20 to 7.00, are deprotonated and function as nucleophiles, i.e., those exposed to the solvent H₂O but not all. Therefore, the reaction of Schiff base formation in neutral solutions does not favor a cross-linking.

(c) In addition to the dilute glutaraldehyde solution, we used a relatively large amount of chitosan micro particles (500 mg).

These three limiting conditions contributed to avoid a cross-linking of glutaraldehyde within the chitosan polymer; it could have occurred only to at small extent. The resulting biocatalysts demonstrate that cross-linking was largely avoided and successful immobilizations were achieved.

[1] Phadnaik, G. A Short Review: Methodologies for the Synthesis of Schiff’s Bases, Ind. J. Adv. Chem. Sci. 8, 2020, 163-176

[2] Santiago de Alvarenga, Ε. Characterization and Properties of Chitosan, in Biotechnology of Biopolymers, Ed. by Elnashar, M., 2011, InTech, Rijeka, Croatia, Ch.5, pp. 91-108.

Line 244. What is sensor control? What parameters are controlled?

Authors’ response:

Please consider the same response, as in the above 7 (your comment “Line 30. What is sensory evaluation (twenty-one tasters)? What parameters are evaluated?”.

Table 1. This table is recommended to be supplemented with all the results from Table S1 (columns for WSBentonite, WsBromelain , WsPapain or explain why this cannot be done. It is recommended to add data on changes in turbidity to the table.

Authors’ response:

Detailed data on the changes in turbidity have been presented within the text (subsection 2.3. Vinifications: clarification effectiveness of immobilized proteases, and bentonite). These data were enriched in the revised version (within subsection 2.3.); two new related references have been also added [Di Bella, et all; Vázquez-Pateiro, I et al.]. After a serious consideration of the Reviewer’s suggestion to supplement Table 1 with all results from Table S1, we thought that Table 1 will be a little bit boring and may be confusing to the reader. The basic reason is that the estimated differences between the columns, by one-way ANOVA test, were found not significant; thus, they could not add value to the main objective of this work.

Additionally, a revised title has been provided for Table 1, i.e. "Table 1. Results of the quantitative analysis of white wines of A-groups produced with the indigenous yeast strain Saccharomyces cerevisiae Z622, using different clarification treatments".

Line 133. It should be explained what effect on the varietal character of white wines was observed

Authors’ response:

We are referred, primarily, on the comparison of the results depicted in the column "Wine^{bentonite treated}”, of Table 1, versus those either of the columns "Wine^{bromelain treated}“ and “Wine^{papain treated}^”. Moreover, the results from the sensory evaluation of the ready white wines at the end of fermentation, as well as after six and after twelve months confirmed the sentence “Nevertheless, Bentonite was found to be more effective as a clarifying agent than immobilized Bromelain, although it exhibited a negative effect on the varietal character of white wines as compared to those treated with immobilized Proteases”, in lines 131-134, of the initial version.

Furthermore:

(a) Please consider our responses to the above 5 and 7.

(b) Common specific varietal characteristics of the Debina grape variety (produced in the region of Epirus – Greece) are fruity aromas reminiscing a mix of apple accompanied by a refreshing acidity.

(c) The results of the sensory evaluation by taking also into account the statistical analysis) are depicted in Tables S3 and S4.

Fig.1. It is recommended to make this figure clearer and more distinct. Use in same manner, for example, FERMENTATION (instead Fermentation or Fermented blank).

Authors’ response:

Figure 1 was processed and changed according to the comments and suggestions of the Reviewer.

It should be indicated which parameter formed the Conclusion that immobilized Papain is the best. From all data presented in tables and described in text it is clear that there is no particular advantages for application of immobilized proteases. Preparing biocatalysts requires reagents, time and labor, including obtaining chitosan as a support for immobilization. Bentonite can certainly be such a support. The main advantage of immobilization, namely, the reuse of biocatalysts, is not used or discussed in any way in this paper. Are immobilized proteases consumables and not reused??

Authors’ response:

Indicative are the results of the quantitative analysis of the volatile compounds of Table 1, according to the above 16, and more specifically of the last row of "Total Concentration of Esters with pleasant Aromas (mg/L)". Decisive were also the results depicted in Table S4 [(c) of the 16]. There are some strong and substantial arguments proving the advantages for the application of immobilized proteases. Of course, the work reported in our manuscript is a first approach to the removal of haze with immobilized proteases (papain) and has been developed on a laboratory scale. Therefore, the cost in time, money and work labor will be evaluated and resolved in future upscaling efforts. On the other hand, the activity of bentonite is that of a reservoir that absorbs water, alcohols, phenolic compounds and esters, including naturally the natural aromas of white wines [AIDIC CONFERENCE SERIES 2013, 11, 201-210, DOI: 10.313103]. Our results, both these depicted in Table 1 and that from the sensory evaluation (Table S3) confirmed this disadvantage of bentonite.

Both prepared biocatalysts in this work were not tested for a potential reuse. Our main concern focused on the clarification effect of these immobilized proteases on glutaraldehyde activated chitosan; we did not study other properties of the prepared biocatalysts. This will be in a future work, as it is mentioned on the Conclusions of the revised version.

Finaly, the preparations of both papain and bromelain have been affirmed as GRAS listed in 21 CFR 184 [Enzyme Preparations Used in Food (Partial List), Table 3. Enzyme preparations affirmed as GRAS listed in 21 CFR 184, https://www.fda.gov/food/generally-recognized-safe-gras/enzyme-preparations-used-food-partial-list]. Additionally, Chitosan has been characterized as nontoxic natural antimicrobial polymer and is approved by GRAS [Yan, D.; Li, Y.; Liu, Y.; Li, N.; Zhang, X.; Yan C. Antimicrobial Properties of Chitosan and Chitosan Derivatives in the Treatment of Enteric Infections, Molecules, 2021, 26, 7136.]

Finaly, we would like to thank Reviewer 3 for his/her constructive comments and suggestions

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript "The impact of various immobilization protocols of proteases, on the clarification and quality of white wines" by Papamichael et al. investigates immobilized papain and bromelain to clear haze in white wine and analyses quality parameters of the resulting wines. The topic is meaningful and the established immobilization protocol appears to be valuable although a rather minor advancement compared to previous studies. For example, some details of immobilization of bromelain on a chitosan support to reduce white wine protein haze have been analyzed by Benucci et al. (doi: 10.1111/ajgw.12093).

One main concern related to the manuscript under review is that there is nearly no discussion about the results in the context of the literature. There is no reference citation in the "results and discussion" section. I encourage the authors to expand the text on the applicability and advantages/disadvantages of the method based on the provided analytical data and on the variations of the protocol that were tested. The variations and the optimization process of the method are only briefly described (mainly in the method section), but it is not clear to me how well these variations actually worked. It might be valuable for other people to adjust the protocol further in the future, so some more detail in that regard would be very much appreciated. Especially because the title implies that "various immobilization protocols" were tested. I suggest to invite the authors to provide a revised version.

Further more detailed comments:

-How exactly did the authors determine the values in table 1? A clear reference to the method section or a short sentence on the methodology of the "quantitative analysis" close to table 1 might help to make the data more accessible for the reader.

-In table 1 "Total Concentration of Esters with pleasant Aromas (mg/L)" is 3.0 for bentonite, but in the text below it is 3.1 mg/L. Please verify the numbers.

-Line 71: "…C25, H159, D158, and/or N175, for Papain and stem Bromelain respectively": Is it really H159 for both proteases? Line 75 indicates that H158 is the catalytic histidine of bromelain? Further, is it N175 or N157 in bromelain?

-Line 111: "The formed chitosan micro particles were stable for about one year, when stored in double-distilled water (DDW), in Falcon tubes, at refrigerator." Was there a method to test that, was it verified by eye?

-Line 217: "Estimations of the immobilization yields, and enzymatic activities of the immobilized proteases were calculated as previously [9,26,27]": In my opinion it is not sufficiently clear to refer to three individual publications for immobilization yield and activity. Please describe the procedure in the manuscript, at least briefly.

-Conclusion: "Immobilized Papain proved superior vs immobilized Bromelain under the herein applied experimental conditions." It would be helpful to mention again which values are in favor of papain compared to bromelain.

-Which impact did the "sensory evaluation" that is mentioned in the abstract have for the evaluation and conclusion of the study?

-For the substances that are classified as GRAS by the FDA, please provide a link or database ID related to the FDA

-The authors may want to check uncommon capitalization of some words throughout the manuscript. Overall, spelling, style and grammar appear to be fine. I did not come across any major issues.

Author Response

We thank all Reviewers for their constructive comments and suggestions.

A general authors’ response: The title and the text have been thoroughly revised along with English language issues.

Response to Reviewer 4:

The manuscript "The impact of various immobilization protocols of proteases, on the clarification and quality of white wines" by Papamichael et al. investigates immobilized papain and bromelain to clear haze in white wine and analyses quality parameters of the resulting wines. The topic is meaningful and the established immobilization protocol appears to be valuable although a rather minor advancement compared to previous studies. For example, some details of immobilization of bromelain on a chitosan support to reduce white wine protein haze have been analyzed by Benucci et al. (doi: 10.1111/ajgw.12093) [Benucci et al used Chitopearl BCW-3010 !!!! to immobilize Bromelain].

Authors’ response:

We would like to mention that in some published articles describing immobilization methods for either papain or bromelain and/or both of these proteases, important conditions seem to be overlooked [Enzyme Research, 2015, 573721, doi.org/10.1155/2015/573721 (2.3 - insufficient details – pH 7.00]. Generally, when proteases are found in contact with a liquid phase, a self-hydrolysis phenomenon occurs. In fact, although immobilization offers a kind of protection against possible protease auto-hydrolysis, the extent of auto-hydrolysis is highly dependent on the immobilization protocol, also affecting the lifetime of such a biocatalyst to remain active. This was one of two motivations for the present work to take immobilization protocols into serious account. Another reason was that some authors have proposed methods of preparing chitosan microparticles using diluted acetic acid preparations, e.g. 1% (^v/_v) to 3% (^v/_v). We used 5% (^v/_v) acetic acid solution. The reason is that the pK_a-values of the free amino groups of chitosan vary from 6.20 to 7.00. Therefore, a successful immobilization of proteases in chitosan microparticles should be based on an efficient method of their preparation. Prerequisite for this latter is to achieve the dissolution of as high a quantity of chitosan powder as possible, in acidic aqueous solutions that could not damage its structure and swelling properties, achieving at the same time a high degree of deprotonation of the free amino groups of chitosan. We achieved a higher dissolution of chitosan powder in 5% (^v/_v) acetic acid solution [Adv. Mat. Sci and Eng. 2019, 8781013, doi.org/10.1155/2019/8781013].

In the case of immobilization of bromelain on a chitosan support to reduce white wine protein haze have been analyzed by Benucci et al. (doi: 10.1111/ajgw.12093), authors do not used chitosan powder but Chitopearl BCW-3010, among other matrices; Chitopearl BCW-3010 is a quite different material whose sales had been canceled when our own investigation began.

One main concern related to the manuscript under review is that there is nearly no discussion about the results in the context of the literature. There is no reference citation in the "results and discussion" section. I encourage the authors to expand the text on the applicability and advantages/disadvantages of the method based on the provided analytical data and on the variations of the protocol that were tested.

Authors’ response:

We conformed to this comment and suggestions of the Reviewer. In the revised version, we have expanded the section “Results and Discussion” to include more details on our results, as well as to highlight the applicability and the advantages of the applied methods, citing relevant references.

The variations and the optimization process of the method are only briefly described (mainly in the method section), but it is not clear to me how well these variations actually worked. It might be valuable for other people to adjust the protocol further in the future, so some more detail in that regard would be very much appreciated. Especially because the title implies that "various immobilization protocols" were tested. I suggest to invite the authors to provide a revised version.

Authors’ response:

In the revised version all methods have been re-written, as well as new sub-subsections have been added to ensure the all methods are clear. The title of the manuscript has been also changed to “The effect of clarification protocols on the vinification of white wines with papain and bromelain immobilized on glutaraldehyde activated chitosan”.

How exactly did the authors determine the values in table 1? A clear reference to the method section or a short sentence on the methodology of the "quantitative analysis" close to table 1 might help to make the data more accessible for the reader.

Authors’ response:

In the revised version have been included the following sub-sub-subsections:

(a) 3.2.3.3. Total protease content and significant immobilization parameters:

(b) 3.2.3.4. Activity measurements of proteases:

(c) A detailed description of the “Quantitative analysis of volatile compounds” has been added in the Supplementary Materials

In table 1 "Total Concentration of Esters with pleasant Aromas (mg/L)" is 3.0 for bentonite, but in the text below it is 3.1 mg/L. Please verify the numbers.

Authors’ response:

The value in the last line / right column of Table 1 is 3.1, and it has been corrected.

-Line 71: "…C25, H159, D158, and/or N175, for Papain and stem Bromelain respectively": Is it really H159 for both proteases? Line 75 indicates that H158 is the catalytic histidine of bromelain? Further, is it N175 or N157 in bromelain?

Authors’ response:

This error has been accordingly corrected to N¹⁵⁷ .

Line 111: "The formed chitosan micro particles were stable for about one year, when stored in double-distilled water (DDW), in Falcon tubes, at refrigerator." Was there a method to test that, was it verified by eye?

Authors’ response:

We applied a simple method, i.e., (a) we performed (per four months) immobilization processes of papain according to sub-subsection 3.2.5., through the use of glutaraldehyde spacer, and (b) we assayed the activity of the immobilized papain. A decrease of less than 7% (mean value of three independent runs) of the activity of immobilized papain was observed after one year.

-Line 217: "Estimations of the immobilization yields, and enzymatic activities of the immobilized proteases were calculated as previously [9,26,27]": In my opinion it is not sufficiently clear to refer to three individual publications for immobilization yield and activity. Please describe the procedure in the manuscript, at least briefly.

Authors’ response:

As we stated in 4. (above), the following three sub-sub-subsections have been included in the revised version:

(a) 3.2.3.3. Total protease content and significant immobilization parameters:

(b) 3.2.3.4. Activity measurements of proteases:

-Conclusion: "Immobilized Papain proved superior vs immobilized Bromelain under the herein applied experimental conditions " It would be helpful to mention again which values are in favor of papain compared to bromelain.

Authors’ response:

A thoroughly revised “Conclusions” section replaced that of the initial version, where more specific data, as well as comparisons based on specific numbers are included. Additional data have been also provided. All the necessary documentation for the organoleptic characteristics of ready white wines clarified with immobilized proteases has been incorporated in the text. Therefore, the results depicted in Table 1 and that from the sensory evaluation (Table S3) confirmed a superiority of papain versus bromelain (Table S4 – results from one-way ANOVA tests). Besides, it is well known that papain possesses s a more active catalytic site versus that of bromelain (indicative may be the pH profiles and the estimated pK_a-values of both these two enzymes [14-17 and therein references])

-Which impact did the "sensory evaluation" that is mentioned in the abstract have for the evaluation and conclusion of the study?

Authors’ response:

A significant new sub-subsection (3.2.8. Sensory Evaluation) has been added in the section 3 Materials and Methods, where the detailed process of the sensory evaluation is referred. Furthermore, all the necessary documentation for the organoleptic characteristics of ready white wines clarified with immobilized proteases and bentonite has been incorporated in the text. Accordingly, the results from the sensory evaluation of the ready white wines after six months through comparisons between commercial sample of Debina white wine and the white wines produced in this work, are depicted in Table S3. Therefore, the impact of the sensory evaluation on the conclusions is decisive, along with the results that are depicted in Table 1, and Table S1, as well.

-For the substances that are classified as GRAS by the FDA, please provide a link or database ID related to the FDA.

Authors’ response:

The preparations of both papain and bromelain have been affirmed as GRAS listed in 21 CFR 184 [Enzyme Preparations Used in Food (Partial List), Table 3. Enzyme preparations affirmed as GRAS listed in 21 CFR 184, https://www.fda.gov/food/generally-recognized-safe-gras/enzyme-preparations-used-food-partial-list]. Additionally, Chitosan has been characterized as nontoxic natural antimicrobial polymer and is approved by GRAS [Yan, D.; Li, Y.; Liu, Y.; Li, N.; Zhang, X.; Yan C. Antimicrobial Properties of Chitosan and Chitosan Derivatives in the Treatment of Enteric Infections, Molecules, 2021, 26, 7136.].

The authors may want to check uncommon capitalization of some words throughout the manuscript.

Authors’ response:

All uncommon capitalizations have been corrected throughout the manuscript.

Finaly, we would like to thank Reviewer 4 for his/her constructive comments and suggestions.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have improved the manuscript according to the comments in the first round of review. I have only minor additional comments:

324 - "though" is misspeled word

367 - track changes red markings should be removed

391 - the sentence is not finished, and are...?

Figure S1 and Figure S2: I suggest using less concentrated MW standard and more concentrated enzyme of interest when preforming SDS-PAGE. Also, the labels below the photos of the gel (sample, MW standard) need to be adjusted on both Figure S1 and S2.

Author Response

We thank all Reviewers. We prepared the second revision by based on their new constructive comments and suggestions.

Response to Reviewer 2:

324 - "though" is misspeled word.

Authors’ response:

It has been corrected accordingly.

367 - track changes red markings should be removed.

Authors’ response:

It has been corrected. Red marking haw been deleted.

391 - the sentence is not finished, and are...?

Authors’ response:

It has been corrected. The text “, and are” has been deleted.

Figure S1 and Figure S2: I suggest using less concentrated MW standard and more concentrated enzyme of interest when preforming SDS-PAGE. Also, the labels below the photos of the gel (sample, MW standard) need to be adjusted on both Figure S1 and S2.

Authors’ response:

All authors agree, and will take into serious account the comments and suggestions of Reviewer 2 in our future works. The labels below the photos of the gel (sample, MW standard) have been properly adjusted in both Figure S1 and S2, in the second revised version.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Section 3 is very large in terms of text volume. Section 2 lacks data for discussion. In particular, there is a lack of data for characterization of the prepared biocatalysts, their activity, stability, possibility of isolation and reuse.

Typos and comments are highlighted in blue in pdf-fila and appear in pop-up windows/

Comments for author File: Comments.pdf

Author Response

We thank all Reviewers. We prepared the second revision by based on their new constructive comments and suggestions.

Response to Reviewer 3:

Section 3 is very large in terms of text volume. Section 2 lacks data for discussion. In particular, there is a lack of data for characterization of the prepared biocatalysts, their activity, stability, possibility of isolation and reuse.

Authors’ response:

Section 3 was extensively reworked in the revised version (1), as we (all authors) agreed to conform with the comments and suggestions of all four Reviewers. Therefore, we added significant subsections to improve our “Methods”, as well as to include additional information requested of us, regarding key apparatus we used. In the second revised version of our manuscript under review, we added also few more details (are described in details below).

Section 2 has been improved in the second revised version, according to new additions in the section 3 (more details are described below).

This manuscript under review has been renamed in the revised version and is entitled “The effect of clarification protocols on the vinification of white wines with papain and bromelain immobilized on glutaraldehyde activated chitosan”. This title expresses the objectives of this study more clearly than that of the initial submission. Besides, the aim of the present study is the implementation of reliable, inexpensive and practical clarification methods of white wines, without affecting their traditional organoleptic characteristics, through treatment of musts and/or white wines with immobilized papain and bromelain (first sentence of the Abstract). Therefore, an exhaustive analysis of the catalytic properties of the prepared immobilized proteases is beyond the scope of the present study. Nevertheless, significant analytical tests on the preparation (yields and activities) of the immobilized proteases are described in the “Methods” section, with more details in the second revised edition, and are reported and discussed in the “Results and Discussion” section.

In the sub-subsection 3.2.6, of the second revised version, as well as in the 3.2.5 of the first revised version, is referred that: “The plain chitosan micro particles remained unchanged after twelve months. In the case of chitosan micro particles carrying the glutaraldehyde spacer and the immobilized proteases a decay of their proteolytic activity of about 15%, after six months, and about 40% after twelve months was observed.”. Therefore, there is not lack of data for characterization of the prepared immobilized proteases concerning their activity and stability. We have not investigated possibility of reuse of the immobilized proteases; besides, this study was performed at laboratory scale.

The 'Results and discussion" section contains only one table. The calculation data of the primary experimental data from the section 3.2.3.3. are not presented and are not discussed in any way.

Authors’ response:

The calculation data measured as described in subsection 3.2.4.1 of the second revised edition (3.2.3.3 in the first revised edition), were incorporated into a few lines of text in the Results and Discussion section (subsection 2.2.). Therefore, and based on our previous response (1) and the arguments mentioned therein, as well as the objectives of the present study, we considered it unnecessary to discuss these data further and to add another Table to the manuscript.

Section 3.2.3.3. Equation for calculation of percentage of the retained proteolytic activity due to the immobilization process is absent. In the "Results' section, the calculated data are not presented and discussed.

Authors’ response:

According to this comment, we added to the second revised edition the mentioned, but missing equation (3) for calculating the “Retained proteolytic activity (%).” In our response to (2) above is included a response to the second sentence of this comment, of the Reviewer 3.

Section 3.1.2 "Equipment" is usually placed in the "Methods" section (not in "Materials"). This section describes the equipment, apparatus, and conditions of analysis (possibly with references).

Authors’ response:

We conformed to the comments and suggestions of Reviewer 3. The text of the second revised version has been corrected accordingly.

Sections 3.2.1 and 3.2.2 list the solutions used in the work, but not the methods of their preparation. Therefore, it is recommended to move the text of these sections to section 3.1.1 under the title "Reagents and solutions"

Authors’ response:

We conformed to the comments and suggestions of Reviewer 3. The text of the second revised version has been corrected accordingly.

The Authors work for the same organization. This is essentially a list of email addresses

Authors’ response:

The numerical superscripts of the affiliations of the authors' names have been appropriately corrected.

We work in different Departments of the University of Ioannina. However, we have united as a group of Enzyme Biotechnology and Genetic Engineering.

Typos and comments are highlighted in blue in pdf-fila and appear in pop-up windows.

Authors’ response:

We conformed to the comments and suggestions of Reviewer 3. The text of the second revised version has been corrected accordingly. However, for the abbreviated nomenclature of bacteria we adopted the suggestions for Authors and Publishers in Chapter 4 Advisory Notes, of the International Code of Nomenclature of Bacteria: Bacteriological Code, 1990 Revision (https://www.ncbi.nlm.nih.gov/books/NBK8816/), and therefore we refer, within the text, the abbreviation of Saccharomyces cerevisiae as S. cerevisiae.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The authors did a good job in revising and optimizing the manuscript. Description of the methods and the discussion section have substantially been improved and extended in the current version. This particularly improved the clarity of the manuscript, helps to highlight the novelty and allows a broader applicability of the protocols. I consequently recommend the manuscript for publication in its current form.

Author Response

We thank all Reviewers. We prepared the second revision by based on their new constructive comments and suggestions.

Response to Reviewer 4:

Authors’ response:

Authors would like to thank very much Reviewer 4 for his/her comments and suggestions.

Author Response File: Author Response.pdf