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The research aims to enhance monitoring of yeast dynamics in Baijiu fermentation using GFP labeling technology.

January 14, 2026Fermentation0 citationsOpen Access

Single-Cell Tracking of Brewing Yeast Dynamics in Baijiu Fermentation Using GFP-Labeled Engineered Saccharomyces cerevisiae FSC01

Key Points

The research aims to enhance monitoring of yeast dynamics in Baijiu fermentation using GFP labeling technology.
Introduced GFP labeling into Saccharomyces cerevisiae to create the engineered strain FSC01.
Developed a recombinant plasmid containing the GFP and genetic resistance genes.
Used flow cytometry to verify the engineered strain's efficacy in detecting yeast numbers.
Conducted solid-state fermentation simulations to evaluate the impact of FSC01 on original yeast growth.
FSC01 showed high linear responses in both pure and complex systems with detection limits of 102 cells/mL.
Dynamic monitoring revealed a yeast population peak on day 7 at 1.2 × 107 cells/g, correlating with a 26.7% alcohol yield.
GFP labeling enabled real-time detection, surpassing limitations of traditional metagenomic methods.

Abstract

In view of the technical bottleneck of microbial dynamic monitoring during the solid-state fermentation of traditional Baijiu, this study introduced green fluorescent protein (GFP) labeling technology into the dominant Saccharomyces cerevisiae of Jiang-flavored Baijiu to construct the chromosomal integration engineering strain named FSC01. By designing an integrated recombinant plasmid containing the GFP gene and the geneticmycin resistance gene, an engineered strain that stably expresses fluorescent proteins was obtained by electroconversion. Flow cytometry verification showed that FSC01 showed excellent linear responses in the pure microbial system (R2 = 0.998) and the complex matrix of Baijiu jiupei (R2 = 0.981), with a detection limit of 102 cells/mL, and the detection cycle was shortened to 10 min. Solid-state fermentation simulation experiments show that the inoculation volume of FSC01 of 105 cells/kg can not only ensure the effective identification of fluorescence signals, but also does not significantly interfere with the growth and growth patterns of the original yeast (p > 0.05), which is highly consistent with the results of the traditional plate counting method. Dynamic monitoring shows that Saccharomyces cerevisiae during fermentation presents a typical succession pattern of “increase first and then decrease”, reaching a peak on the 7th day (1.2 × 107 cells/g), which is positively correlated with the base alcohol yield rate (26.7%). Compared with metagenomic (72 h) and PMA-qPCR (4 h) methods, this technology breaks through the limitations of specificity and timeliness of live bacteria detection, and provides a single-cell-level dynamic analysis tool for the digitization of traditional brewing processes. In the future, it will be expanded to monitor key functional microorganisms such as lactic acid bacteria through a multi-color fluorescent labeling system, and optimized pretreatment to eliminate starch granule interference, and promote the in-depth application of synthetic biology technology in the traditional fermentation industry.

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