Transcriptional Analysis of Mixed-Culture Fermentation of Lachancea thermotolerans and Saccharomyces cerevisiae for Natural Fruity Sour Beer

Round 1

Reviewer 1 Report

The paper explores the intriguing realm of yeast-yeast interactions in mixed-culture fermentation, catering to the growing interest in this area driven by both market demands and the pursuit of biotechnological solutions for enhanced sensory characteristics in beer. The study employs a combination of Lachancea thermotolerans and Saccharomyces cerevisiae, with a specified cell population ratio, in the fermentation process for sour beer. Notably, the study adheres to traditional brewing industry conditions, providing a practical application of the proposed method. One commendable aspect of the paper is its focus on natural processes, with L. thermotolerans contributing lactic acid production without the addition of foreign flavors. The resulting sour beer exhibits a distinctive fruity profile and a gentle sourness, offering a novel sensory experience. The exploration of double-yeast mixed-culture fermentation proves advantageous, as it generates a richer array of flavor substances compared to single-culture processes. The presence of isobutyl acetate and isoamyl acetate contributes to an augmented fruit aroma, effectively balancing the sour notes and imparting a refreshing taste to the beer. The study not only enhances beer quality but also presents an alternative mass production solution. The controlled processes and reduced fermentation time achieved through the developed double-yeast mixed-culture fermentation offer practical advantages for large-scale production. Furthermore, the incorporation of RNA sequencing (RNA-Seq) and metabolite data provides insights into the stress responses of L. thermotolerans during fermentation. This integration enables a comprehensive understanding of the metabolic flux distribution from S. cerevisiae and non-Saccharomyces yeasts, paving the way for potential strategies to modulate transcriptional responses associated with aroma compounds. In conclusion, the paper significantly contributes to the field by presenting a viable and innovative approach to sour beer fermentation, backed by empirical evidence and insights into the molecular aspects of yeast interactions. Given its practical applications, scientific rigor, and potential impact on the brewing industry, I recommend accepting this paper for publication. A valuable suggestion would be to improve the introduction, making the paper's novelty clearer and including a good literature review.

Author Response

The Introduction and Conclusions sections were revised to enhance clarity and accessibility for the target audience. This involved restructuring the content to emphasize key points and ensuring a more logical flow of information.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Author:

Lachancea thermotolerans has been deeply studied in wine but no so well studied in beer and no specifically in sour beer. I do not know any previous study that use RNA sequencing to determine transcriptome changes of L. thermotolerans in sour beer. So, I think that the manuscript posses some novelty. I think that Figures 5 to 8 gives good information.

I introduce several points to improve the manuscript from its original form:

Introduction

It could be indicated that L. thermotolerans is one of the few non-Saccharomyces with enough fermentative power to properly ferment beer up to ethanol levels up to close to 100 %. Only three non-Saccharomyces (T. delbrueckii, S. pombe and L. thermotolerans) possess that ability.

Line 38 => write Sacharomyces in itallics like in line 35.

Line 45 => write Sacharomyces in itallics like in line 35.

Note: look in all the text for the word “Saccharomyces” using software Word or similar seeker tool and change it to italics.

Material and Methods

2.4 Analytical Determinations

Indicate the models of the equipment in a similar way as it is done in section 2.5 for the Agilent Bioanalyzer

Line 121 => Although it is indicated a reference it could be included the model of HPLC employed.

Line 125 => Refer the used ion Chromatographer model (Company, country)

Authors can also include pH determination in this point

Results and Analysis

Line 178 => Saccharomyces must be written in italics.

Table 1 => introduce statistical differences. Most article indicate them with letters. For example it is clear that L. thermotolerans produce statistically significant differences in lactic acid when compared to K. marxianus that is the lowest produce in lactic acid. It can be performed with an easy ANOVA test.

Lines 190 to 192 => Authors refer to alcohols (2,1 % vol and 6,75 % vol), it is not clear if they refer to ethanol, those values could be also been included in a graph or table as I do not find them out of the text. Same for lines 260 to 263.

References

Latin names of microorganisms should be written in italics in all cited references.

Best Regards.

Author Response

1. Analytical Determinations (Section 2.4):The models of the HPLC, ion chromatograph, and pH meter used in the study were included.

2. Data Analysis (Section 3.1 and 3.6):

• Table 1 in Section 3.1 wasfollowed by a sentence to explain the statistical difference.
• A dedicated section titled "3.6 Statistical Analysis" was incorporated to further describe the analysis method.

3. Concentration Units (Lines 189-190):The originally reported concentrations of 2.1% vol and 6.75% vol were revised to show their corresponding values in g/L (1.66 g/L and 5.33 g/L, respectively). 

4. The Latin names of Saccharomyces, L. thermotolerans, and other microorganisms have been consistently italicized, both in the paper and all cited references.

Author Response File: Author Response.pdf