Synergistic Effect Enhances Aromatic Profile in Beer Brewing Through Mixed-Culture Fermentation of Pichia kluyveri and Saccharomyces cerevisiae var. diastaticus
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsManuscript: Synergistic effect enhances aromatic profile in beer brewing through mixed-culture fermentation of Pichia kluyveri and Saccharomyces cerevisiae var. diastaticus
A brief summary:
The idea of this research is interesting and innovative, providing insight into the impact of these two types of yeast on the aromatic profile (enhanced aroma and taste) in beer production. This combination of yeasts and their synergistic effect sounds promising for the beer industry.
General and specific comments:
The Introduction is well written, it opens the topic to the reader and explains the research. The authors are kindly requested to clearly state the research objective (aim) in one to two sentences at the end of the Introduction and to unify the font throughout the entire paper.
It is not mandatory, but the authors may consider to cite the following two papers if they believe they have missed them (I did not see them in the reference list). But it is not obligatory.
- Schober, D.; Wacker, M.; Schmarr, H.-G.; Fischer, U. Understanding the Contribution of Co-Fermenting Non-Saccharomyces and Saccharomyces Yeasts to Aroma Precursor Degradation and Formation of Sensory Profiles in Wine Using a Model System. Fermentation 2023, 9, 931. https://doi.org/10.3390/fermentation9110931
- Hong, K., Li, C., Ai, J., Han, X., Han, B., Qin, Q., Deng, H., Wu, T., Zhao, X., Huang, W., Zhan, J., & You, Y. (2025). Biogenic amines degradation ability of Saccharomyces cerevisiae I45 and Pichia sp. NW5 & LB60 and their application in beer fermentation. Food research international (Ottawa, Ont.), 202, 115726. https://doi.org/10.1016/j.foodres.2025.115726
In the section under Materials and Methods (lines 79-81), these two sentences are not necessary. They can be moved to the introduction or discussion. This section should only include the materials used and the conditions of the method, i.e., the practical part.
In the section 2.3. Determination of sugar residue content, please specify in more details did you use a calibration curve for this determination, which concentration ranges were applied, whether internal standards were used or not, and how much time elapsed between sampling and analysis. Was the chromatographic method validated with an estimated measurement uncertainty? Additionally, please specify the duration of the HPLC-RI analysis.
In section 2.4 under Volatile Compounds, please specify the duration of the analysis (Runtime).
Line 160 – if this link is a reference please specify it like the other references.
if it is possible to enlarge or separate the words on diagram in Figure 3, due to clarity, it would be advisable to do so.
Please revise the references according to the Instructions for authors.
Author Response
|
||||||||||||||||||||||||||||||
Author Response File: Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
This study investigated the fermentation process of beer with Saccharomyces cerevisiae and Pichia kluyveri. Parameters such as pH, sugar content (including glucose, fructose, maltose, sucrose and maltotriose), and aroma compounds were analyzed. The synergistic effects were also discussed.
The following suggestions are offered for revision.
- Although 2-phenethyl acetate is equivalent to 2-phenylethyl acetate, the terminology should remain consistent. (Line 136, 340, 345, 346, Table 1, 2)
Line 19
(1) The full name of SY should be presented.
(2) The phenylethyl acetate, isoamyl acetate, and linalool increased 12.03, 12.37, and 1.17 folds than the SY monoculture, respectively.
What is the sample compared with SY group?
Phenylethyl acetate ( 0.17 vs 2.08 : 12.23 fold)
Isoamyl acetate ( 1.04 vs 13.03 : 12.53 fold)
Please check the correctness.
Line 38
The full name of P. kluyveri should be presented.
Line 74
5.8 S Internal Transcribed Spacer (ITS)
Line 75
RFLP analysis??
It should be DNA sequence analysis.
Line 78
5 × 109
Line 101
measuring CO2 evaporation using a precision balance
The machine should be described.
Line 102
“were sampled every 24 h”
It is not consistent with the data of figure 1 and 2.
Line 110
The column size should be presented.
Line 120
(the final concentration was 2.08 μL/L)
It is not a unit of concentration. Please check the correctness.
Line 179
A~B shall be labeled in the figure 1.
Line 189
Fig. 2 A, B
Line 221 (Figure 2)
(1)The method for the determination of total fermentable sugars shall be presented.
(2) A~F shall be labeled in the figure.
Line 240~241
3-methyl-1-butanol in SY pure fermentation at the end of fermentation were 24.3% and 91.9% higher than those of the mixed fermentation (SE-3-PKL/SY and SI-PKL/SY), respectively.
The numbers of 24.3% and 91.9% are not correct.
Please check the correctness.
Line 242~243
the content of 2-phenylethanol in SY co-fermentation was elevated by 23.5% and 20.0% (Table S1).
(1)The numbers of 23.5% and 20.0% are not correct.
(2) It shall be Table 1.
Please check the correctness.
Line 257
it increased by 13.39% over SI-PKL/SY
The numbers of 13.39% is not correct.
Please check the correctness.
Line 261 (Table 1)
The changes of ethyl laurate during different fermentation shall be discussed in the text.
Line 265~270
“Octanoic acid, 9-decenoic acid, decanoic acid, and dodecanoic acids were produced more significantly by both coculture groups than SY monoculture (p<0.05).” Further analysis of the differences between the two cocultures revealed that levels of octanoic acid and dodecanoic acid in SE-3-PKL/SY coculture were 1.25 and 1.82 times higher than those in the SI-PKL/SY coculture, respectively. Conversely, the content of 9-decenoic acid in the SI-PKL/SY coculture was 1.83 times higher than that in the SE-3-PKL/SY coculture (Table S1).
The data shall be presented in Table 1.
Line 275~278
the yield of isoamyl acetate fermented with the SE-3-PKL/SY and SI-PKL/SY was approximately 12.03 and 7.02 folds higher than the SY monoculture. For the content of 2-phenylethyl acetate, the increasing folds were 12.37 and 7.88.
The numbers of “12.03, 7.02 and 12.37, 7.88” are not correct.
Please check the correctness.
Line 283~286
the SE-3-PKL/SY produced the highest concentration of ethyl octanoate, ethyl nonanoate, ethyl trans-4-decenoate, ethyl decanoate, ethyl dodecanoate, ethyl tetradecanoate, and ethyl hexadecanoate, followed by SI-PKL/SY
The data shall be presented in Table 1.
Line 291~292
the lowest amount of 3-methyl-1-butanol and 2-phenylethanol.
The concentration of 2-phenylethanol in SE-PKL/SY group is not lowest.
Please check the correctness.
Line 300~301
In addition, the content of geranyl acetate fermented with SE-3-PKL/SY was 4.71 and 3.99 times higher than SY and SI-PKL/SY, respectively (Table S1).
The data should be presented in Table 1.
Line 318 (Figure 3)
The resolution of Figure 3 is not enough. The authors should provide higher resolution figure.
Line 338
The full name of VIP should be presented.
Line 344~346
The result represented the sensory score for ‘intensity’ was significantly relevant to the content of isoamyl acetate, 2-phenethyl acetate, ethyl laurate and linalool. The ‘fruit’ score was significantly relevant to the content of isoamyl acetate, linalool and 2-phenethyl acetate.
It is not consistent with the data of table 2. Please check the correctness.
Line 358 (Table 2)
“2-Phenethyl acetate” should be corrected to 2-phenylethyl acetate.
Line 367~368
a 12.03-fold increase in phenylethyl acetate, a 12.37-fold increase in isoamyl acetate.
Please check the correctness.
Comments on the Quality of English Language
no comments
Author Response
Comments 1: Although 2-phenethyl acetate is equivalent to 2-phenylethyl acetate, the terminology should remain consistent. (Line 136, 340, 345, 346, Table 1, 2) |
Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript and have unified it as 2-phenethyl acetate. |
Comments 2: Line 19 (1) The full name of SY should be presented. (2) The phenylethyl acetate, isoamyl acetate, and linalool increased 12.03, 12.37, and 1.17 folds than the SY monoculture, respectively. What is the sample compared with SY group? Phenylethyl acetate ( 0.17 vs 2.08 : 12.23 fold) Isoamyl acetate ( 1.04 vs 13.03 : 12.53 fold) Please check the correctness. |
Response 2: Thank you for pointing this out. We agree with this comment. Therefore, we have recalculated these components based on the revised standard curve, and have revised the table 1. The sample was from SE-3-PKL/SY compared with SY group. Lines 19-21: “The 2-phenethyl acetate, isoamyl acetate, and linalool in SE-3-PKL/SY increased 12.03, 12.37, and 1.17 folds than the SY monoculture, respectively.” |
Comments 3: Line 38 The full name of P. kluyveri should be presented. |
Response 3: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript. Lines 38-40: “The Pichia kluyveri (P. kluyveri) has been applied to beverage fermentation under the above background while licensed with Generally Recognized As Safe (GRAS, GRN No. 938).” |
Comments 4: Line 74 5.8 S Internal Transcribed Spacer (ITS) |
Response 4: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript. Lines 74-76: “Identified by 5.8S-Internal Transcribed Spacer (ITS) and DNA sequence analysis (Fig. S1), it has been preserved in the China General Microbiological Culture Collection Centre (CGMCCNo.27659).” |
Comments 5: Line 75 RFLP analysis?? It should be DNA sequence analysis. |
Response 5: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript. Lines 74-76: “Identified by 5.8S-Internal Transcribed Spacer (ITS) and DNA sequence analysis (Fig. S1), it has been preserved in the China General Microbiological Culture Collection Centre (CGMCCNo.27659).” |
Comments 6: Line 78 5 × 109 |
Response 6: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript. Lines 78-79: “The percent solids were 93%-97%, living yeast cells ≥ 5 × 109 per gram of dry yeast.” |
Comments 7: Line 101 measuring CO2 evaporation using a precision balance The machine should be described. |
Response 7: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript. Lines 85-87: “A lab-scale fermentation was carried out in 500 mL Erlenmeyer flasks containing artisanal wort at 25°C with an “S” shape airlock which can let CO2 out.” Lines 99-102: “The weight loss of the entire device was measured by a ten-thousandth balance and recorded as the amount of CO2 released due to the function of the “S” shape airlock. The pH was measured by using a portable pH meter.” |
Comments 8: Line 102 “were sampled every 24 h” It is not consistent with the data of figure 1 and 2. |
Response 8: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript. Lines 102-104: “Five milliliters of fermenting wort were sampled at 12h, 24h, 3d, 5d, 7d, 9d, 11d and the cell-free supernatants were stored at −20°C for the analysis of physical and chemical indicators.” |
Comments 9: Line 110 The column size should be presented. |
Response 9: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript. Lines 107-112: “The residual sugar content of the samples including glucose, fructose, sucrose, maltose, and maltotriose, as well as ethanol in fermentation broth, were analyzed by high-performance liquid chromatography (Agilent 1100 HPLC, Agilent Technologies, Santa Clara, CA), using an Aminex HPX-87H ion exchange column (300×7.8mm, BioRad, Richmond, CA) coupled to a refractive-index detector and eluted with 0.5 mM H2SO4 at 0.6 mL/min and at 60°C, each sample was analyzed for 30 min [15].” |
Comments 10: Line 120 (the final concentration was 2.08 μL/L) It is not a unit of concentration. Please check the correctness. |
Response 10: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript. Lines 121-123: “Briefly, sodium chloride (1.5 g) was placed in the headspace bottle, followed by the addition of a 5 mL fermentation sample and 5 μL of 2-octanol as the internal standard (the final concentration was 1.71 mg/L).” |
Comments 11: Line 179 A~B shall be labeled in the figure 1. |
Response 11: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the figure 1. |
Comments 12: Line 189 Fig. 2 A, B |
Response 12: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript. Lines 191-194: “During the fermentation process, it was discovered that the maltose and sucrose with SY monoculture were almost consumed during the first three days of fermentation, reaching a final concentration of 1.82 g/L and 0.83 g/L, respectively (Fig. 2A, B).” |
Comments 13: Line 221 (Figure 2) (1)The method for the determination of total fermentable sugars shall be presented. (2) A~F shall be labeled in the figure. |
Response 13: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript. Lines 112-115: “The total fermentable sugar content was determined as the sum of all residual fermentable sugars. The concentrations of individual fermentable sugar in all samples were quantified using external calibration curves established with standard solutions ranging from 10 to 100 ppm.” |
Comments 14: Line 240~241 3-methyl-1-butanol in SY pure fermentation at the end of fermentation were 24.3% and 91.9% higher than those of the mixed fermentation (SE-3-PKL/SY and SI-PKL/SY), respectively. The numbers of 24.3% and 91.9% are not correct. Please check the correctness. |
Response 14: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript. Lines 243-247: “Comparing the higher alcohol yields of these three mixed fermentation strategies, the concentrations of 3-methyl-1-butanol in SY pure fermentation at the end of fermentation were 24.3% and 91.9% higher than those of the mixed fermentation (SI-PKL/SY and SE-3-PKL/SY), respectively, and the content of 2-phenylethanol in SY monoculture was higher by 23.5% and 20.0%, respectively (Table 1).” |
Comments 15: Line 242~243 the content of 2-phenylethanol in SY co-fermentation was elevated by 23.5% and 20.0% (Table S1). (1)The numbers of 23.5% and 20.0% are not correct. (2) It shall be Table 1. Please check the correctness. |
Response 15: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript. Lines 243-247: “Comparing the higher alcohol yields of these three mixed fermentation strategies, the concentrations of 3-methyl-1-butanol in SY pure fermentation at the end of fermentation were 24.3% and 91.9% higher than those of the mixed fermentation (SI-PKL/SY and SE-3-PKL/SY), respectively, and the content of 2-phenylethanol in SY monoculture was higher by 23.5% and 20.0%, respectively (Table 1).” |
Comments 16: Line 257 it increased by 13.39% over SI-PKL/SY The numbers of 13.39% is not correct. Please check the correctness. |
Response 16: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the table 1. |
Comments 17: Line 261 (Table 1) The changes of ethyl laurate during different fermentation shall be discussed in the text. |
Response 17: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript. Lines 287-292: “Except for the acetyl esters, co-inoculation of PKL with SY significantly improved the production of ethyl esters, the SE-3-PKL/SY produced the highest concentration of ethyl octanoate, ethyl nonanoate, ethyl trans-4-decenoate, ethyl decanoate, ethyl laurate, ethyl tetradecanoate, and ethyl hexadecanoate, followed by SI-PKL/SY. For instance, the yield of ethyl laurate fermented with the SE-3-PKL/SY and SI-PKL/SY was approximately 29.05 and 10.58 folds higher than the SY monoculture.” |
Comments 18: Line 265~270 “Octanoic acid, 9-decenoic acid, decanoic acid, and dodecanoic acids were produced more significantly by both coculture groups than SY monoculture (p<0.05).” Further analysis of the differences between the two cocultures revealed that levels of octanoic acid and dodecanoic acid in SE-3-PKL/SY coculture were 1.25 and 1.82 times higher than those in the SI-PKL/SY coculture, respectively. Conversely, the content of 9-decenoic acid in the SI-PKL/SY coculture was 1.83 times higher than that in the SE-3-PKL/SY coculture (Table S1). The data shall be presented in Table 1. |
Response 18: Thank you for pointing this out. The content of these substances is a semi quantitative result and has not been compared with its threshold. In this study, their contribution to the aroma of the sample was not analyzed in depth,instead we focused on the compounds which OAVs were reported more than 1 as shown in table 1. Thank you very much for your valuable suggestions. We will further analyze the contribution of the increase in the content of these substances to the aroma of fermented beer in future research. |
Comments 19: Line 275~278 the yield of isoamyl acetate fermented with the SE-3-PKL/SY and SI-PKL/SY was approximately 12.03 and 7.02 folds higher than the SY monoculture. For the content of 2-phenylethyl acetate, the increasing folds were 12.37 and 7.88. The numbers of “12.03, 7.02 and 12.37, 7.88” are not correct. Please check the correctness. |
Response 19: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript. Lines 279-283: “In this study, acetyl esters from both coculture fermentations were detected at higher levels than the SY monoculture, the yield of isoamyl acetate fermented with the SE-3-PKL/SY and SI-PKL/SY was approximately 12.37 and 7.02 folds higher than the SY monoculture. For the content of 2-phenylethyl acetate, the increasing folds were 12.03 and 7.88.” |
Comments 20: Line 283~286 the SE-3-PKL/SY produced the highest concentration of ethyl octanoate, ethyl nonanoate, ethyl trans-4-decenoate, ethyl decanoate, ethyl dodecanoate, ethyl tetradecanoate, and ethyl hexadecanoate, followed by SI-PKL/SY The data shall be presented in Table 1. |
Response 20: Thank you for pointing this out. The content of these substances is a semi quantitative result and has not been compared with its threshold. In this study, their contribution to the aroma of the sample was not analyzed in depth,instead we focused on the compounds which OAVs were reported more than 1 as shown in table 1. Thank you very much for your valuable suggestions. We will further analyze the contribution of the increase in the content of these substances to the aroma of fermented beer in future research. |
Comments 21: Line 291~292 the lowest amount of 3-methyl-1-butanol and 2-phenylethanol. The concentration of 2-phenylethanol in SE-PKL/SY group is not lowest. Please check the correctness. |
Response 21: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript. Lines 296-298: “For instance, the fermentation broth of the SE-3-PKL/SY group had the largest quantity of isoamyl acetate and 2-phenethyl acetate but the lowest amount of 3-methyl-1-butanol.” |
Comments 22: Line 300~301 In addition, the content of geranyl acetate fermented with SE-3-PKL/SY was 4.71 and 3.99 times higher than SY and SI-PKL/SY, respectively (Table S1). The data should be presented in Table 1. |
Response 22: Thank you for pointing this out. The content of these substances is a semi quantitative result and has not been compared with its threshold. In this study, their contribution to the aroma of the sample was not analyzed in depth,instead we focused on the compounds which OAVs were reported more than 1 as shown in table 1. Thank you very much for your valuable suggestions. We will further analyze the contribution of the increase in the content of these substances to the aroma of fermented beer in future research. |
Comments 23: Line 318 (Figure 3) The resolution of Figure 3 is not enough. The authors should provide higher resolution figure. |
Response 23: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the figure 3. |
Comments 24: Line 338 The full name of VIP should be presented. |
Response 24: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript. Lines 343-348: “To further elucidate the correlation of the aroma compounds with the sensory characteristics, several aroma compounds were obtained that were responsible for this separation (Variable Importance in Projection>1) and have Odor Activity Values (OAVs) over one including 3-methyl-1-butanol, isoamyl acetate, 2-phenylethanol, ethyl octanoate, 2-phenethyl acetate, linalool, geraniol and ethyl laurate (Table 1).” |
Comments 25: Line 344~346 The result represented the sensory score for ‘intensity’ was significantly relevant to the content of isoamyl acetate, 2-phenethyl acetate, ethyl laurate and linalool. The ‘fruit’ score was significantly relevant to the content of isoamyl acetate, linalool and 2-phenethyl acetate. It is not consistent with the data of table 2. Please check the correctness. |
Response 25: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript. Lines 351-355: “The result represented the sensory score for ‘intensity’ was significantly relevant to the content of isoamyl acetate, 2-phenethyl acetate and ethyl laurate. The ‘fruity’ score was significantly relevant to the content of isoamyl acetate, ethyl octanoate, 2-phenethyl acetate, ethyl laurate, linalool and geraniol.” |
Comments 26: Line 358 (Table 2) “2-Phenethyl acetate” should be corrected to 2-phenylethyl acetate. |
Response 26: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript and have unified it as 2-phenethyl acetate. |
Comments 27: Line 367~368 a 12.03-fold increase in phenylethyl acetate, a 12.37-fold increase in isoamyl acetate. Please check the correctness. |
Response 27: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the table 1. |
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsDear authors,
the paper about beer breewing and mixed cultures use is appropriate for the journal Fermentation.
The paper will be of interest to readers in the beer breewing sector.
The introduction is appropriate and literature is suitable.
The materials and methods are extensively described and therefore are appropriate.
The results are widely explaining the findings and are appropriate.
in the results in the begining part the acronyms can be written in their whole names SY , SI_PKL/SY so it is easier to follow.
The figure 3 should be enlarged so it can be readable,
now the letters are too small.
Some minor technical details should be checked e.g in conclusion the term var. diastaticus is no in palatyno linotype.
kind regards, the reviewer
Author Response
Comments 1: in the results in the begining part the acronyms can be written in their whole names SY , SI_PKL/SY so it is easier to follow. |
Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have revised the manuscript. Lines 169-178: “During the initial four days of fermentation, the rate of carbon dioxide released by the monoculture of Saccharomyces cerevisiae var. diastaticus yeast (SY) surpassed that observed in co-fermentations. (Fig.1A), and pH of SY monoculture decreased significantly (Fig.1B). The mixed fermentation of simultaneous (SI-PKL/SY) coculture started immediately on the first day, but the change of CO2 evaporation and pH was at a relatively slow rate compared with SY, and the final pH was slightly lower than the SY monoculture. For the co-fermentation of sequential approach (SE-3-PKL/SY), the variation of the CO2 evaporation and pH value in the initial three days of fermentation is relatively small and started accelerating after inoculation with S. cerevisiae SY after three days.” |
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsLine 19 and Table 1
“The 2-phenethyl acetate, isoamyl acetate, and linalool in SE-3-PKL/SY increased 12.03, 12.37, and 1.17 folds than the SY monoculture”
However, in table 1:
Phenylethyl acetate ( 0.18 vs 2.16 : 12.00 fold)
Please check the correctness.
Comments on the Quality of English LanguageNo comments
Author Response
Comments 1: Line 19 and Table 1 “The 2-phenethyl acetate, isoamyl acetate, and linalool in SE-3-PKL/SY increased 12.03, 12.37, and 1.17 folds than the SY monoculture” However, in table 1: Phenylethyl acetate ( 0.18 vs 2.16 : 12.00 fold) Please check the correctness. |
Response 1: Thank you for pointing this out. This is due to the different decimal places retained in the original data. Thank you very much for your careful review. Considering the revised data in Table 1, we have calculated and changed 12.03 to 12.00. |
Lines 19-21:
“The 2-phenethyl acetate, isoamyl acetate, and linalool in SE-3-PKL/SY increased 12.00, 12.37, and 1.17 folds than the SY monoculture, respectively.”
Lines 282-283:
“For the content of 2-phenethyl acetate, the increasing folds were 12.00 and 7.88.”
Lines 374-377:
“In particular, SE-3-PKL/SY exhibited the capacity to enhance fruity aromas, notably leading to a 12.00-fold increase in 2-phenethyl acetate, a 12.37-fold increase in isoamyl acetate, and a 1.17-fold increase in linalool compared to SY single inoculation in beer brewing.”
Author Response File: Author Response.pdf