Torulaspora delbrueckii Strain Behaviour within Different Refermentation Strategies for Sparkling Cider Production

Round 1

Reviewer 1 Report

In the paper entitled Torulaspora delbrueckii strain behavior under different refermentation strategies for sparkling cider production, the behavior of two commercial strains of T. delbrueckii in the fermentation of apple juice to produce cider and sparkling cider was studied. The influence of the interaction between strains, method and strain-method on the physico-chemical parameters of the cider was analyzed by enzymatic and chromatographic tests. Following current and subsequent research, it is proposed to use T. delbrueckii strain in cider fermentation to develop products with unique aromatic profiles.

I recommend that authors add the full name and country of origin to all materials and equipment in the Materials and Methods chapter. I also recommend introducing chromatograms for volatile organic compounds.

 

 

Author Response

Thank you the reviewer for the constructive suggestions and comments

We have addressed specific answer to each comments hoping to have improved the manuscript as requested

Q1.1 I recommend that authors add the full name and country of origin to all materials and equipment in the Materials and Methods chapter.

A1.1 Thank you for highlighting this inadvertency. We have added the full name and country of origin to all materials and equipment in the Materials and Methods chapter.

 

Q1.2 I also recommend introducing chromatograms for volatile organic compounds.

A1.2 We thank the Reviewer for the suggestion to include chromatograms for a possible comparison of the different refermentation strategies for sparkling cider production and their effect on the formation of aroma substances. However, we apply mass spectrometry detection in full scan mode, i.e. all substances that are volatile are detected, including ethanol and other substances that are present in very high concentrations. These components often overlap the substances under investigation, so that a comparison of the chromatograms is not possible. For this reason, chromatograms for similar questions are not shown in the publications.

The quantitative and qualitative determination of the investigated volatile compounds was carried out as usual via individual selected masses/ions and their typical ratio for the respective investigated substance (single-ion monitoring). A documentation and comparison in the form of special sections of the chromatograms for each of the investigated substances and a comparison between the variants would be too confusing and extensive. For this reason, it is not practicable to show specific chromatogram sections in the publication.

Reviewer 2 Report

Topic is determined and evaluated well. Introduction part is giving clear information about topic and handled issue. MM part should be improved following recommendations should be taken in consider. It is easy to feel that each part has written by different author and combined. If the manuscript will be made into a fluent whole, it is thought that it will be a good reference for future studies in this field. Tables and figures should be improved, as well. Result are evaluated well, but sample comparisons should be improved with related mechanisms and differences.

I recommend the acceptance of manuscript after minor revision.

These advises could be taken in consider for improving manuscript:

 

Line 88-89: Please insert information for BioSüdtirol. Is this, firm that apples were obtained from? So, please revise the sentence as: Topaz cultivar organic apple samples were obtained from local supplier BioSüdtirol (City name, country name).

Also, what is the equipment name for Voran? Please revise the sentence as: … were rinsed and crushed by **** (Model name, Voran, city name, country name)

Line 90: Please, insert more detail about tanks, 10 L, 25 L, ?, etc.

Line 90-91: Please revise the sentence as: 25 g/hL nutrient (Fermaid K, Lallemand, country name) was added for each tank for yeasts.

Line 92: Please indicate, how you monitored sugar consumption?

Line 120: Please revise the ‘Microbiological analysis’ sub-section.

Line 126-127: Please give ‘the pH (XS 126 digital pH MeterXS Instruments)’ with separate sentence.

Line 128: [13]

Line 138: Please add comma after ‘(HS-SPME-GC-MS)’

Line 181: 2.15 log¹⁰ cfu/mL and please check all manuscript for similar

Line 197: How you determined yeast assimilable nitrogen (YAN)? Please insert information to methods.

Please add abbreviators to methods: Pet Nat (PN), Traditional bottle fermented method (TM) and tank fermented Charmat method (CM).

Table 1: SD should be normal, statistical lettering shoud be superscript: 4.53±0.05^a instead of 4.53 ± 0.05 a

Line 346: Please correct as: Dimethyl disulphide (DMS) amount or revise the figure name as Dimethyl disulphide amount

Line 399: Please, improve the resolution of the Figure 3.

Line 460: Figure 5 is very hard to read. All the words are overlapped. Please revise it.

Author Response

Thank you to the reviewer for the constructive suggestions and comments

We have addressed specific answer to each comments hoping to have inmproved the manuscript as requested

Comments and Suggestions for Authors

Topic is determined and evaluated well. Introduction part is giving clear information about topic and handled issue. MM part should be improved following recommendations should be taken in consider. It is easy to feel that each part has written by different author and combined. If the manuscript will be made into a fluent whole, it is thought that it will be a good reference for future studies in this field. Tables and figures should be improved, as well. Results are evaluated well, but sample comparisons should be improved with related mechanisms and differences.

Thank you to the Reviewer for these constructive comments, we attempted to improve the Material and Method section, fluency, tables, figures and results evaluation as in the many changes can be found in this revised version of the manuscript (in red)

 

Q2.1  Line 88-89: Please insert information for BioSüdtirol. Is this, firm that apples were obtained from? So, please revise the sentence as: Topaz cultivar organic apple samples were obtained from local supplier BioSüdtirol (City name, country name).

Also, what is the equipment name for Voran? Please revise the sentence as: … were rinsed and crushed by **** (Model name, Voran, city name, country name)

A2.1 The sentence has been revised and added the equipment and other necessary information.

 

Q2.2Line 90: Please, insert more detail about tanks, 10 L, 25 L,?, etc.  

A2.2 The tanks capacity has been added to the text.

 

Q2.3 Line 90-91: Please revise the sentence as: 25 g/hL nutrient (Fermaid K, Lallemand, country name) was added for each tank for yeasts.

A2.3 Thanks for the correction. The sentence has been modified as suggested with slight modification as in Iine 103-105.

 

Q2.4 Line 92: Please indicate, how you monitored sugar consumption?

A2.4 Thanks for the comment, we have revised a sentence describing the used analytical approach.

 

Q2.4 Line 120: Please revise the ‘Microbiological analysis’ sub-section.

A2.4 Thank you for your suggestion, the sub-section has been revised

 

Q2.5 Line 126-127: Please give ‘the pH (XS 126 digital pH MeterXS Instruments)’ with separate sentence.

A2.5 The sentence has been separated and more details give.

 

Q2.6 Line 128: [13]

Q2.6 Line has been corrected.

 

Q2.7 Line 138: Please add comma after ‘(HS-SPME-GC-MS)’

Q2.7 Comma has been added.

 

Q2.8 Line 181: 2.15 log¹⁰ cfu/mL and please check all manuscript for similar

Q2.8 Line has been corrected e all the manuscript checked.

 

Q2.9 Line 197: How you determined yeast assimilable nitrogen  (YAN)? Please insert information to methods.

Q2.9 Thanks for the observation. We have better clarified the sentence describing the method applied to measure YAN content in juice. A specification has also been added to the section “Enzymatic assays” in methods.

 

Q2.10 Please add abbreviators to methods: Pet Nat (PN), Traditional bottle fermented method (TM) and tank fermented Charmat method (CM).

A2.10 Abbreviations have been added to Methods.

 

Q2.11 Table 1: SD should be normal, statistical lettering shoud be superscript: 4.53±0.05^a instead of 4.53 ± 0.05 a 

A2.11Thanks for the observation. The table has been changed according to reviewer suggestion.

 

Q2.12 Line 346: Please correct as: Dimethyl disulphide (DMS) amount or revise the figure name as Dimethyl disulphide amount.

A2.12 It has been corrected.

 

Q2.13 Line 399: Please, improve the resolution of the Figure 3. 

A2.13 The figure resolution has been improved as suggested. 

 

Q2.14 Line 460: Figure 5 is very hard to read. All the words are overlapped. Please revise it.

A2.14The figure has been revised and divided into two to make it easier to be read.

 

Reviewer 3 Report

The present study explored the behaviour of two commercial strains of  T. delbrueckii in apple juice fermentation to produce cider and sparkling cider. At the same time, the authors chose a multifactorial approach to research, which complicates the description of the results of the experiment and the perception of the article as a whole.

Thus, there are several comments on the manuscript that I invite the authors to consider.

Clause 2.2 requires further clarification regarding the procedure for preparing the mash and possibly correcting its initial characteristics, which is usually used in the production of cider. Or explanations why acidity regulators, enzyme preparations, etc. were not used.

lines 215-217 the text needs to be corrected. Figure 1 does not reflect the kinetics of the fermentation process

lines 262-266 need to expand the analysis. What gives an increase in the consumption of ammonium nitrogen during the first fermentation ...

clause 3.8. the results of organoleptic analysis are presented very poorly

The conclusions section contains general phrases and does not show the solution of the tasks set in the study.

Author Response

Thank you to the reviewer for the constructive suggestions and comments

We have addressed specific answer to each comments hoping to have improved the manuscript as requested

Q3.1 Clause 2.2 requires further clarification regarding the procedure for preparing the mash and possibly correcting its initial characteristics, which is usually used in the production of cider. Or explanations why acidity regulators, enzyme preparations, etc. were not used.

A3.1 Thank you for your valuable comment, we attempted to better clarify the procedure by adding more details and therefore explain the cider production performed following the more common procedure in the Southern Tyrolean par of Italy.

Q3.2 lines 215-217 the text needs to be corrected. Figure 1 does not reflect the kinetics of the fermentation process. We refer to the fermentation kinetic in figure S1

A3.2 We attempted to better explain the intent of represent this data (Line 235-248) also changing the figure now renamed S2

Q3.3 lines 262-266 need to expand the analysis. What gives an increase in the consumption of ammonium nitrogen during the first fermentation ...

A3.3 Line 329-331 The reason for the higher consumption of ammonium nitrogen were detailed.

Q3.4 clause 3.8. the results of organoleptic analysis are presented very poorly

A3.4 We attempted to better present clause 3.8, and related methodology.

Q3.5 The conclusions section contains general phrases and does not show the solution of the tasks set in the study.

A3.5 Than you for this constructive comment, Conclusion section had be partially re-written and the focus on the task solution has been shown.

Reviewer 4 Report

The manuscript entitled “Torulaspora delbrueckii strain behaviour within different refermentation strategies for sparkling cider production” presented the results of fermentation trials using two commercial strains of this important non-conventional yeast species to produce cider. Different approaches were also tested for the second fermentation aimed at producing a sparkling beverage. Chemical and sensory analyses were then carried out to compare the products obtained with the different strategies of fermentation. Overall, the text is well-written and clearly structured, but a fine English review, preferentially by a native speaker, could be used especially in the Results and Discussion section. Nevertheless, some further points should be addressed to improve the manuscript:

1.     Lines 34-40: As you used in your trial apples sourced from the Südtirol region, it could be interesting to include in this paragraph some information about cider production in Italy

2.     Lines 47-71: There are plenty of recent articles discussing the application of non-Saccharomyces in cider production, you might check some of them to improve the introduction, more centered around cider than wine applications. Here are a few examples: DOI: 10.1016/j.ijfoodmicro.2019.108471; DOI: 10.1016/j.foodchem.2019.125623; DOI: 10.1016/j.fochms.2022.100095; DOI: 10.1016/j.foodchem.2020.128833; DOI: 10.1002/jib.512; DOI: 10.1093/femsle/fnaa093; DOI: 10.1016/j.lwt.2022.113538; DOI: 10.1016/j.lwt.2022.113233; DOI: 10.3389/fmicb.2022.1042613; DOI: 10.1016/j.fm.2020.103446

3.     Especially these ones concerning T. delbrueckii should be considered: DOI: 10.1016/j.lwt.2018.09.075; DOI: 10.1016/j.foodchem.2017.04.067; DOI: 10.1007/s00217-021-03910-y

4.     Line 59: Saccharomyces cerevisiae in italics

5.     Lines 90, 98, 102, 105, 108, 112: Please add the precise volumes used on each stage of fermentation trials. I think that a figure showing the complete experimental design could be of great value to improve the understanding about the different methods, volumes, and stages of fermentation

6.     Lines 92-93: Please describe here how sugar consumption was monitored

7.     Lines 96-97, 216: Why is there this difference in sugar between tanks A and B? Are these values an average of the triplicate?

8.     Line 120: Please be more specific about how many samples and sampling times

9.     Line 145: Model wine solutions? Or cider?

10.  Line 171: It is not clear the meaning of the level “no yeast”

11.  Line 181: YPD

12.  Lines 183-187: This paragraph is not clear regarding which data correspond to which sampling day and which strain, please rewrite. Or you might prepare a table or chart

13.  Lines 190-191: Why did you calculate the average of three different media? Did you find different numbers and colony morphologies on each medium?

14.  Lines 198-200: This comparison is not relevant, and the same unit of measure must be used

15.  Lines 202: Did you measure the CO₂ production during fermentation of base cider? The results of Figure S1 show only ºBrix for the base cider and CO₂ for the second fermentation

16.  Lines 208-209: Table 1 actually shows significant differences for the sugar consumption among the trials with Strain B

17.  Lines 217-223: How come did you observe a difference of about 200 kPa between Strain A and Strain B in both Pet Nat and Champenoise? Where are the results for Charmat?

18.  Lines 236-242 and 271-281: The discussion of total acidity and the organic acids should be put together. Do you have information regarding the consumption or production of malic acid related to T. delbrueckii? Is the metabolism of malic and lactic acid only associated with the lactic acid bacteria already present in the apple juice? Or to which other organic acids are you referring to when discussing the synthesis or consumption by Strains A and B?

19.  Lines 245-250: It is not clear what is the relation between the ethanol content and the pH. To compare the ethanol production with the literature, it is more important to consider the initial sugar content and the amount consumed, and not only the residual content

20.  Lines 260-261: There is a strong variation on glycerol levels between strains and production methods, and you did not perform a control fermentation with S. cerevisiae, hence it is difficult to draw conclusions. Please use articles that talk specifically about T. delbrueckii, even better if concerning cider fermentation

21.  Lines 262-267: Did you add nitrogen nutrients to the bottles when aliquoting the fermenting ciders to produce the Pet Nat?

22.  Lines 268: I think it is important to comment on the production of acetic acid, as Strain B produced significantly more than Strain A and this parameter can have a relevant effect on the cider quality

23.  Lines 283-284 and 292-293: Which was the level of catechins in the apple juice?

24.  Lines 365-367: Values of PC1 and PC2 indicated in the text are different than those in the graph

25.  Figures 2 and 3: Why were there 6 replicates of each fermentation?

26.  Lines 410 and 437: Is these 66% and 33% of the quantified compounds referring to the number of compounds or their relative concentration? What about the fatty acids?

27.  Figure 4: Please check the letters indicating the differences, especially for volatile acids and esters they do not seem correct

28.  Lines 498-506: Please check again the references and rewrite the paragraph, does a higher pressure increase or decrease ester biosynthesis?

 

Author Response

Thank you to the Reviewer for the constructive suggestions and comments

We have addressed specific answer to each comments hoping to have improved the manuscript as requested

Comments and Suggestions for Authors

The manuscript entitled “Torulaspora delbrueckii strain behaviour within different refermentation strategies for sparkling cider production” presented the results of fermentation trials using two commercial strains of this important non-conventional yeast species to produce cider. Different approaches were also tested for the second fermentation aimed at producing a sparkling beverage. Chemical and sensory analyses were then carried out to compare the products obtained with the different strategies of fermentation. Overall, the text is well-written and clearly structured, but a fine English review, preferentially by a native speaker, could be used especially in the Results and Discussion section. Nevertheless, some further points should be addressed to improve the manuscript:

 

Thank you for the comment, English was reviewed by a native speaker

Q4.1.     Lines 34-40: As you used in your trial apples sourced from the Südtirol region, it could be interesting to include in this paragraph some information about cider production in Italy

A4.1 More details have been added in order to describe the more common procedure used in the Südtirol region. No specific rules are followed in cider production in Italy, since there is not such diffused tradition or specific cider apple variety as in other European country, such in France or Spain

Q4.2.     Lines 47-71: There are plenty of recent articles discussing the application of non-Saccharomyces in cider production, you might check some of them to improve the introduction, more centered around cider than wine applications. Here are a few examples: DOI: 10.1016/j.ijfoodmicro.2019.108471; DOI: 10.1016/j.foodchem.2019.125623; DOI: 10.1016/j.fochms.2022.100095; DOI: 10.1016/j.foodchem.2020.128833; DOI: 10.1002/jib.512; DOI: 10.1093/femsle/fnaa093; DOI: 10.1016/j.lwt.2022.113538; DOI: 10.1016/j.lwt.2022.113233; DOI: 10.3389/fmicb.2022.1042613; DOI: 10.1016/j.fm.2020.103446

A4.2 Thanks for the valuable comment. The introduction has been improved according to the reviewer’s suggestions and the valuable literature considered.

 

Q4.3.     Especially these ones concerning T. delbrueckii should be considered: DOI: 10.1016/j.lwt.2018.09.075; DOI: 10.1016/j.foodchem.2017.04.067; DOI: 10.1007/s00217-021-03910-y.

A4.3 We have improved the introduction by adding concepts deriving from the suggested references.

 

Q4.4.     Line 59: Saccharomyces cerevisiae in italics

A4.4 The yeast scientific name has ben properly formatted.

 

Q4.5.     Lines 90, 98, 102, 105, 108, 112: Please add the precise volumes used on each stage of fermentation trials. I think that a figure showing the complete experimental design could be of great value to improve the understanding about the different methods, volumes, and stages of fermentation

A4.5 Thank you for the suggestion we added more detail in the main text of the manuscript and also a schematic representation of the experiment design in Figure S1 of the supplementary material.

Q4.6.     Lines 92-93: Please describe here how sugar consumption was monitored

A4.6 A better description has been added

Q4.7.     Lines 96-97, 216: Why is there this difference in sugar between tanks A and B? Are these values an average of the triplicate?

A4.7 The values are an average of triplicates and different sugar concentrations may be due to different sugar consumption by different yeast strain.

Q4.8.     Line 120: Please be more specific about how many samples and sampling times

A4.8 Because in this section we describe only methodology and instrument, sampling time was described in the experiment design section Iine 106

Q4.9.     Line 145: Model wine solutions? Or cider?

A.4.9 now line 161: The calibration for the quantitative determination of each of the aroma compounds under investigation was carried out in model wine solutions at two different alcohol levels. It is important to take into account the alcohol content of the samples during quantification, as it has an influence on the gas chromatographic-mass spectrometric quantification. The calibration curves obtained were then applied to the quantitative determination of the aroma compounds under investigation in the sparkling cider samples. This is a common procedure in the application of gas chromatography for the quantification of aroma compounds that occur naturally in the sample/matrix under investigation.

Q4.10.  Line 171: It is not clear the meaning of the level “no yeast”

A4.10 “no yeast” is referred to the juice before yeast inoculation. The sentence has been modified.

Q4.11.  Line 181: YPD

A4.11 thanks for the correction. It has been modified in text.

Q4.12.  Lines 183-187: This paragraph is not clear regarding which data correspond to which sampling day and which strain, please rewrite. Or you might prepare a table or chart

Q4.13.  Lines 190-191: Why did you calculate the average of three different media? Did you find different numbers and colony morphologies on each medium?

A4.12 &13 Thank you for the valuable suggestion, the all section has been rewritten

Q4.14.  Lines 198-200: This comparison is not relevant, and the same unit of measure must be used

A4.14 Thank you for the suggestion, the comparison has been deleted.

Q4.15.  Lines 202: Did you measure the CO₂ production during fermentation of base cider? The results of Figure S1 show only ºBrix for the base cider and CO₂ for the second fermentation

A4.15 For the first fermentation CO2 was not measured and the fermentation was daily monitored recording Brix values.

Q4.16.  Lines 208-209: Table 1 actually shows significant differences for the sugar consumption among the trials with Strain B.

A4.16 Thanks for the observation. The sentence has been modified in text.

Q4.17. Lines 217-223: How come did you observe a difference of about 200 kPa between Strain A and Strain B in both Pet Nat and Champenoise? Where are the results for Charmat?

A4.17 We attempted to better explain the intent of represent this data (Line 235-248) also changing the figure now renamed S2

 

Q4.18. Lines 236-242 and 271-281: The discussion of total acidity and the organic acids should be put together. Do you have information regarding the consumption or production of malic acid related to T. delbrueckii? Is the metabolism of malic and lactic acid only associated with the lactic acid bacteria already present in the apple juice? Or to which other organic acids are you referring to when discussing the synthesis or consumption by Strains A and B?

A4.18 Thank you for the observation, the two paragraphs have been put together and the results have been better discussed. We improved the discussion according to the reviewer’ suggestion. The sections on total acidity and organic acids have been put together and more details about malic acid metabolism in T. delbrueckii have been added. Considerations about the co-occurrence of spontaneous lactic acid bacteria and yeasts have been reported.

Q4.19. Lines 245-250: It is not clear what is the relation between the ethanol content and the pH. To compare the ethanol production with the literature, it is more important to consider the initial sugar content and the amount consumed, and not only the residual content

A4.19Thanks for the observation. It was a typo, and it has been corrected. No relation between pH and alcohol content was meant.

 

Q4.20. Lines 260-261: There is a strong variation on glycerol levels between strains and production methods, and you did not perform a control fermentation with S. cerevisiae, hence it is difficult to draw conclusions. Please use articles that talk specifically about T. delbrueckii, even better if concerning cider fermentation

A4.20 Even if very little information has been found in relation to glycerol production in cider making, we attempted a more detailed discussion focusing on the yeast specie T. delbrueckii used in this study (Line 314-321)

 

Q4.21.Lines 262-267: Did you add nitrogen nutrients to the bottles when aliquoting the fermenting ciders to produce the Pet Nat?

A4.21 No nutrients were added to produce Pet Nat ciders.

 

Q4.22.Lines 268: I think it is important to comment on the production of acetic acid, as Strain B produced significantly more than Strain A and this parameter can have a relevant effect on the cider quality.

A4.22 We thank you the reviewer for the valuable observation. A more detailed discussion was added including in the other acid metabolism.

 

QA.23.Lines 283-284 and 292-293: Which was the level of catechins in the apple juice?

A4.23 In the apple juice 12.5 mg/l catechins were measured. The value has been added to the text.

 

Q4.24.Lines 365-367: Values of PC1 and PC2 indicated in the text are different than those in the graph.

A4.24 Thank you for the observation, the correctio was added.

 

Q4.25.Figures 2 and 3: Why were there 6 replicates of each fermentation? 2 technical replicates and 3 biological replicates have been considered.

A4.25 The analysis have been redone considering only biological replicates and are now reported in the text and figures.

 

Q4.26.Lines 410 and 437: Is these 66% and 33% of the quantified compounds referring to the number of compounds or their relative concentration? What about the fatty acids?

A4.26 The data are referred to the relative concentration of each class of compounds (expressed as mean value among the samples) in relation to the total concentration of the detected VOCs. For acids, the relative concentration was of 1%.

 

Q4.27.Figure 4: Please check the letters indicating the differences, especially for volatile acids and esters they do not seem correct.

A4.27 Thank you for the observation, the figure 4 has been corrected.

 

Q4.28.Lines 498-506: Please check again the references and rewrite the paragraph, does a higher pressure increase or decrease ester biosynthesis?

A4.26 Thanks for the observation. The paragraph has been modified and a clarification of the sentence has been written.

Round 2

Reviewer 1 Report

For the article, Torulaspora delbrueckii strain behaviour within different refermentation strategies for sparkling cider production, I once again recommend the authors, the introducing chromatograms for volatile organic compounds.

 

 

Author Response

REVIEWER 1

 

Q1.1 I once again recommend the authors, the introducing chromatograms for volatile organic compounds.

 

A1.1 Following the reviewer recommendation we included two introducing chromatograms demonstrating the detection of aroma compounds formed during fermentation, such as higher alcohols, esters, fatty acids, etc. by HS-SPME-GC-MS in two sparkling cider samples, for publication as supplementary material (cited in Line 412 as Figure S3a and Figure S3b).

Reviewer 4 Report

Thanks for the corrections following the reviewers’ suggestions, the text was greatly improved. Nevertheless, there are still some important points that were not sufficiently addressed and should be reviewed to further improve the manuscript.

1.     Line 59: S. cerevisiae [in italics]

2.     Lines 95, 128: Montreal

3.     Sections 2.2.1 and 2.2.2: for some reason, the only Supplementary Material available for download in the reviewer’s page was Table S3, thus unfortunately I couldn’t check the scheme with the experimental design. However, the information regarding number of replicates, bottles, and volumes is still missing for the PetNat, Charmat and Champenoise refermentations. For instance, in the Results you mention that 8 bottles were used for PetNat, but this information should be already clear in the M&M

4.     Line 143: The yeast countable population was verified in the apple juice before and after inoculation, at the bottling day for PN, at the end of base cider fermentation, and after inoculation for second fermentation in TM and CM. Plate count of serially diluted samples was performed on…

5.     Line 197: the VOCs (volatile organic compounds) profile… [please define the abbreviation where it’s first mentioned in the text]

6.     Line 236: The measurement of sugar consumption showed that both fermentations of base cider had a regular kinetic, being completed… [since you didn’t measure developed CO₂ in the 1^st fermentation]

7.     Lines 329-337: If you did not add nutrients to produce PetNat ciders, how do you explain that organic and inorganic nitrogen is significantly higher in PN than base cider and comparable to CM and TM?

8.     Lines 341-351: this paragraph should be deleted as these results were discussed previously (Lines 289-298)

9.     Figure 1 and Figure 4: It’s still not clear where the letters above the bars come from. It’s not possible to arbitrarily put different letters following the order in which the data is presented in the chart. The letters must reflect the statistical difference among the results but respecting the ranking of values

 

 

Author Response

Thank you to the reviewers for the comments. Following your suggestion we have added few changes hoping to have properly replied to the request

Q4.1.     Line 59:S. cerevisiae [in italics].

A4.1- It has been corrected.

Q4.2.     Lines 95, 128: Montreal.

A4.2 Montreal has been added.

Q4.3.     Sections 2.2.1 and 2.2.2: for some reason, the only Supplementary Material available for download in the reviewer’s page was Table S3, thus unfortunately I couldn’t check the scheme with the experimental design. However, the information regarding number of replicates, bottles, and volumes is still missing for the PetNat, Charmat and Champenoise refermentations. For instance, in the Results you mention that 8 bottles were used for PetNat, but this information should be already clear in the M&M

A4.3 Thank you to the Reviewer for noticing this problem. Two files were added in the Supplementary Material, but it seems that only one was saved. We prepared a unique file with all the material Including Figure S1 (the experiment design), and Table S3) In addition the number of bottles was added in the M&M.

Q4.4 Line 143: The yeast countable population was verified in the apple juice before and after inoculation, at the bottling day for PN, at the end of base cider fermentation, and after inoculation for second fermentation in TM and CM. Plate count of serially diluted samples was performed on…

A4.4 Thank you to the reviewer for the suggestion: the sentence has been reviewed according to reviewer’s suggestion.

Q4.5. Line 197: the VOCs (volatile organic compounds) profile… [please define the abbreviation where it’s first mentioned in the text]

A4.5 The abbreviation has been defined.

Q4.6. Line 236: The measurement of sugar consumption showed that both fermentations of base cider had a regular kinetic, being completed… [since you didn’t measure developed CO₂ in the 1^st fermentation]

A4.6The sentence has been corrected.

Q4.7.     Lines 329-337: If you did not add nutrients to produce PetNat ciders, how do you explain that organic and inorganic nitrogen is significantly higher in PN than base cider and comparable to CM and TM?

A4.7 Thank you to the reviewer suggestion: we attempted to explain our finding also linking the fact to the available literature (as in Line 307-317)

 

Q4.8. Lines 341-351: this paragraph should be deleted as these results were discussed previously (Lines 289-298)

A4.8 Thanks for highlighting this repetition. We have deleted the paragraph.

Q4.9. Figure 1 and Figure 4: It’s still not clear where the letters above the bars come from. It’s not possible to arbitrarily put different letters following the order in which the data is presented in the chart. The letters must reflect the statistical difference among the results but respecting the ranking of values.

A.4.9 Thank you for this suggestion. Two new version of both Figure 1 and Figure 4 have been added and letters have been assigned according to ranking value.