Enhancing Flavor Complexity in Craft Beer: Sequential Inoculation with Indigenous Non-Saccharomyces and Commercial Saccharomyces Yeasts

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

This study explores the potential of non-Saccharomyces (NS) yeasts in craft beer production. A total of fifty NS yeast strains were isolated and assessed for their fermentation efficiency and ethanol tolerance. Torulaspora delbrueckii BTd3 was selected for further testing. The study also included an evaluation of aroma profiles and sensory characteristics.

 

The following suggestions are offered for revision.

1. Abstract: There is no data present in the abstract.

 

2. In table 2 and 3, the numbers should be written with decimal points, not commas.

 

3. Wickerhamomyces or Wickeramomyces? Please check the correctness.

 

 

Line 91

The full name and composition of YNB should be presented.

 

Line 94

It should be 10⁶.

 

Line 103

The full name of IBU should be presented.

 

Line 115

The full name and composition of DBDM culture medium should be presented.

 

Line 151

It should be 1.4 L.

 

Line 179 (section 3.1)

The result of veil formation is not described in section 3.1.

 

Line 197

1090 OG should be 1090 Di.

 

Line 211

9 consecutive days

 

Line 225

Volatile products of the fermented beer were quantified by SPE-GC-MS. In reference [23] however, the authors used GC-MS. Please check the correctness.

 

Line 236 (Table 3)

(1) Several spelling errors in table 3. Please check the correctness.

(2) In line 228, a total of 20 volatile compounds were identified. However, only 19 were listed in Table 3.

 

Line 242

Anise should be corrected to anisole.

 

Line 252

The fermentations were carried out for 7 days.

However, according Line 154~156. “The non-Saccharomyces yeast was inoculated on day 0 at a rate of 1 x 10⁷ cells/mL and after 48 hours, 1 x 10⁷ cells/mL of commercial yeast US05 was inoculated (T1). The fermentations were conducted at 18°C for 7 days.

It took 2+7 days. Please check the correctness.

 

Line 261~264

(1) Pure BTd3 should be T2 not (T1). Line 261

(2) Mix treatment should be T1 not (T2). Line 262 and 264

 

Line 265~267

It should be Table 4, not Table 3.

 

Line 271 (Table 4)

The data of “%AA” is not consistent with Line 265. Please check the correctness.

 

Line 295 (Figure 4)

The figure legend is not clear. The three groups should be presented is different symbols or color.

 

Line 321

Reference [3] do not study the function of PAD1 and FDC1.

Please check the correctness.

 

Line 331

Reference [29] do not study the transporters. Please check the correctness.

 

Line 333

What is the reference to be cited? Please check the correctness.

Author Response

This document addresses the comments and suggestions made by the reviewers of our manuscript. We have carefully considered each point and provided responses outlining the changes made to the manuscript.

 Reviewer 1: 

1. Abstract: There is no data present in the abstract.

Relevant information has been added to the abstract. 

1. In table 2 and 3, the numbers should be written with decimal points, not commas.

 

Tables have been corrected, replacing commas with decimal points.

1. Wickerhamomyces or Wickeramomyces? Please check the correctness.

The scientific name has been reviewed and corrected to Wickerhamomyces throughout the document. 

1. Line 91: The full name and composition of YNB should be presented.

The full name of YNB (Yeast Nitrogen Base) was incorporated.

1. Line 94: It should be 106.

The number in line 94 (now line 96) has been corrected to 106.

1. Line 103: The full name of IBU should be presented. 

The full name of IBU (International Bitterness Unit) has been incorporated (line 110)

1. Line 115: The full name and composition of DBDM culture medium should be presented.

The full name of DBDM (Dekkera/Brettanomyces Differential Medium) has been included in line 123.

1. Line 151: It should be 1.4 L.

The volume has been corrected to 1.4 L. (line 162) 

1. Line 179 (section 3.1): The result of veil formation is not described in section 3.1.

The following sentence has been added to section 3.1 (line 206) to describe the result of veil formation: "Of the 50 isolates tested, only three Hanseniaspora strains displayed veil production."

1. Line 197: 1090 OG should be 1090 Di. 

We have used 'Original gravity' and 'Final gravity' throughout the text, so we request that the density be described in this way.

1. Line 211: 9 consecutive days

The number of fermentation days was corrected (line 167)

1. Line 225: Volatile products of the fermented beer were quantified by SPE-GC-MS. In reference [23] however, the authors used GC-MS. Please check the correctness.

We employed SPE for the extraction of volatile compounds, and GC-MS for the identification and quantification of these compounds.. 

1. Line 236 (Table 3): Several spelling errors in table 3. Please check the correctness.

All spelling errors in Table 3 have been corrected.

1. In line 228, a total of 20 volatile compounds were identified. However, only 19 were listed in Table 3.

The number of volatile compounds has been corrected to 19.

1. Line 242: Anise should be corrected to anisole.

"Anise" has been corrected to "methoxybenzene"  , the systematic IUPAC name (as suggested from reviewer  3) in line 253. 

1. Line 252: The fermentations were carried out for 7 days. However, according Line 154~156. “The non-Saccharomyces yeast was inoculated on day 0 at a rate of 1 x 107 cells/mL and after 48 hours, 1 x 107 cells/mL of commercial yeast US05 was inoculated (T1). The fermentations were conducted at 18°C for 7 days. It took 2+7 days. Please check the correctness.

The total fermentation time was 8 days, with 2 days for non-Saccharomyces yeast and 6 days for S. cerevisiae. There was an error in the graphs, where the x-axis was shifted, but this has been corrected in both the graph and the text.

1. Line 261~264. Pure BTd3 should be T2 not (T1). Line 261 Mix treatment should be T1 not (T2). Line 262 and 264

The treatment designations have been corrected. Pure BTd3 is now labeled as T2, and the Mix treatment is labeled as T1.

 

1. Line 265~267. It should be Table 4, not Table 3.

The table reference has been corrected.

1. Line 271 (Table 4). The data of “%AA” is not consistent with Line 265. Please check the correctness.

The data in the text has been corrected to be consistent with the data in Table 4.

1. Line 295 (Figure 4). The figure legend is not clear. The three groups should be presented is different symbols or color.

Different colors have been added to the three treatments in Figure 4. We are open to adding circles to unify the parameters with the treatments if you consider it necessary.

1. Line 332. Reference [3] do not study the function of PAD1 and FDC1. Please check the correctness.

There was a typographical error, where "3" should have been "30." The reference has been corrected (now ref 29; line 338)

1. Line 331. Reference [29] do not study the transporters. Please check the correctness.

 

this was corrected

 

1. Line 333. What is the reference to be cited? Please check the correctness.

The reference was added in line 344

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The author aims to enhance the diversity of beer flavors by screening for new yeast strains and developing novel fermentation processes. The author evaluated the fermentation characteristics of 50 yeast strains and compared their metabolic capabilities to optimize strain selection. This work provides insights for the development of new beer products.

However, as the author notes, the development of new beer products can face suppression from traditional brewing practices. Therefore, I suggest that the author explain to readers why the development of new products encounters prejudice and how to overcome this bias among traditionalists.

 

Regarding other minor issues:

The content of Table 1 and Table 2 is unclear.

Line 94, “106 cells/mL”, superscript.

Line 164, capitalize the first letter.

Line 265, repeated items.“In Table 3, the chemical parameters of the beers obtained from the three 267 evaluated treatments are detailed”

Line 333, There is no content inside the parentheses. “but not maltotriose ().”

Author Response

 Reviewer 2: 

In the introduction, we address how we can mitigate bias regarding the use of non-conventional yeasts in the craft brewing industry.

 Regarding other minor issues:

1. The content of Table 1 and Table 2 is unclear. 

Table 1 has been corrected by adjusting the margins and line spacing.

Table 2 was designed to present the screening of yeast strains according to the tested parameters.

1. Line 94, “106 cells/mL”, superscript.

The number in line 94 (now line 96) has been corrected to 106.

1. Line 164, capitalize the first letter.

The first letter has been capitalized in line 175.

1. Line 265, repeated items.“In Table 3, the chemical parameters of the beers obtained from the three 267 evaluated treatments are detailed”

The repeated sentence has been removed.

1. Line 333, There is no content inside the parentheses. “but not maltotriose ().”

The parentheses have been removed from line 350.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The present research evaluated 50 NS yeast strains isolated from Argentina's Cuyo wine region. Torulaspora delbrueckii was selected for sequential fermentations due to its promising fermentative and physiological characteristics. The results demonstrated a significant increase in fruity and spicy aromas, suggesting that T. delbrueckii can contribute to a more complex flavor profile. These findings highlight the potential of NS yeasts to enhance the sensory characteristics of beers. The manuscript is interesting, but it needs revision according to the following remarks.

 The most important experimental results should be mentioned in the abstract, not only general conclusions and observations.

 At the end of introduction part there is need to state more clearly what is the novelty of preformed research. What was done for the first time? In addition, there is need to state the research hypothesis and how is it to be experimentally tested?

 Paragraph 2.3.1. How the volatiles were extracted before GC-MS analysis? There is need to describe the procedure of the volatiles extraction and introduction into the GC-MS system.

In Table 3 there is need to explain the used abbreviations. In addition, there is need to use IUPAC nomenclature for the simple compound names in overall manuscript; 3-methylbutan-1-ol instead of 3-methyl-1-butanol, octan-2-one instead of 2-octanone and similar. In the experimental part there is need to explain how the concertation of the volatiles were determined by GC-MS.

 The discussion about the individual compounds distribution from Table 3 should be enriched and compared to other studies on this topic.

Author Response

Reviewer 3:

 

1. In Table 3 there is need to explain the used abbreviations. 

 

The explanation of the OAV (Odor Activity Value) has been added to the table footnote.

 

1. In addition, there is need to use IUPAC nomenclature for the simple compound names in overall manuscript; 3-methylbutan-1-ol instead of 3-methyl-1-butanol, octan-2-one instead of 2-octanone and similar. 

 

The IUPAC nomenclature has been corrected in Table 3 as requested by the reviewer.

 

1. The most important experimental results should be mentioned in the abstract, not only general conclusions and observations.

 

The abstract has been expanded to include more detailed results regarding the aromatic fraction obtained in the fermentations.

 

1.  At the end of the introduction part there is a need to state more clearly what is the novelty of preformed research. What was done for the first time? 

 

A paragraph has been added in line 66 to highlight the novelty of the research and how it contributes to the regional brewing industry.

 

1. In addition, there is a need to state the research hypothesis and how is it to be experimentally tested?

 

The research hypothesis and how it will be tested are described in lines 66-80.

 

 

1. Paragraph 2.3.1. How were the volatiles extracted before GC-MS analysis? There is a need to describe the procedure of the volatiles extraction and introduction into the GC-MS system.

 

The procedure for solid-phase extraction of aromatic compounds has been detailed in line 136.

 

1. In the experimental part there is need to explain how the concentration of the volatiles were determined by GC-MS.

 

The method for determining volatile concentration using GC-MS has been explained in line 142.

 

1. The discussion about the individual compounds distribution from Table 3 should be enriched and compared to other studies on this topic.

 

The discussion about the individual compound distribution from Table 3 has been expanded as suggested by the reviewer.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

In table 3 and 4, the numbers should be written with decimal points, not commas

Author Response

tables 3 and 4 were modified

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript was revised, but not all comments were answered and requested corrections made.

In Table 3 there is need to explain the used abbreviations. - The explanation of the OAV (Odor Activity Value) has been added to the table footnote. – What about other used abbreviations in the table?

At the end of the introduction part there is a need to state more clearly what is the novelty of preformed research. What was done for the first time? - A paragraph has been added in line 66 to highlight the novelty of the research and how it contributes to the regional brewing industry. Added paragraph is to general and not precise regarding the novelty of performed research? What was done for the first time in present research?

Paragraph 2.3.1. How were the volatiles extracted before GC-MS analysis? There is a need to describe the procedure of the volatiles extraction and introduction into the GC-MS system. - The procedure for solid-phase extraction of aromatic compounds has been detailed in line 136. However, there are no enough details about the absorption and elution from the cartridge as well details about the eluent concentration before GC-MS analysis.

In the experimental part there is need to explain how the concentration of the volatiles were determined by GC-MS. Line 142 is not explaining this issue.

Author Response

In Table 3 there is need to explain the used abbreviations. - The explanation of the OAV (Odor Activity Value) has been added to the table footnote. – What about other used abbreviations in the table? 
abbreviations in table 3 were explained line 288

At the end of the introduction part there is a need to state more clearly what is the novelty of preformed research. What was done for the first time? - A paragraph has been added in line 66 to highlight the novelty of the research and how it contributes to the regional brewing industry. Added paragraph is to general and not precise regarding the novelty of performed research? What was done for the first time in present research?

this issue was addressed from line 67 to 87 

Paragraph 2.3.1. How were the volatiles extracted before GC-MS analysis? There is a need to describe the procedure of the volatiles extraction and introduction into the GC-MS system. - The procedure for solid-phase extraction of aromatic compounds has been detailed in line 136. However, there are no enough details about the absorption and elution from the cartridge as well details about the eluent concentration before GC-MS analysis.

In the experimental part there is need to explain how the concentration of the volatiles were determined by GC-MS. Line 142 is not explaining this issue.

The procedure was explained in more depht in section 3.1 line 141 

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

Although the authors made improvments, they doid not answer to the important remark:

In the experimental part there is need to explain how the concentration of the volatiles were determined by GC-MS. - The method was not described in the paragraph 3.1.5. Without this explanation the results presented in the corresponding table are not clear.