Metabolic Activity Profiling of High-Temperature Daqu Microbiota Using Single-Cell Raman Spectroscopy and Deuterium Isotope Probing

The microbial metabolic activity of Daqu, a key factor in baijiu production, varies due to environmental and raw material diversity. Quality differences exist among Daqu batches, even within the same fermentation room, and continue to change during storage. An accurate assessment of Daqu's microbial activity is crucial for enhancing baijiu quality and production consistency, yet an in situ, nondestructive detection method remains elusive. Here, we employed Raman microspectroscopy and deuterium isotope probing (Raman-DIP) to measure the metabolic activity of high-temperature Daqu microbiota at the single-cell level. Based on the median C/D ratio (the ratio of C/D to the sum of C-D and C-H), 18 high-temperature Daqu samples from different fermentation batches were classified into three microbial activity patterns A-C (with thresholds of >70%, 45-70%, and <45%), representing high, medium, and low microbial metabolic activities. Partial least-squares analysis of the Raman characteristic spectra showed that PC-Daqu exhibited lower band intensities than did PA-Daqu and PB-Daqu. Furthermore, exploration of the correlation between Raman spectral phenotypes and physicochemical phenotypes revealed that the C/D ratio and Raman characteristic peak intensities were significantly correlated with total acidity, liquefaction enzyme activity, and saccharification enzyme activity. Finally, we developed a 2D-CNN model for discriminating high-temperature Daqu metabolic activity patterns based on the Raman characteristic fingerprint region of microbial communities at the single-cell level, achieving an accuracy of 99%. This study provides a Raman-DIP-based method to assess the in situ metabolic activity of high-temperature Daqu microbiota, facilitating the classification of high-temperature Daqu with distinct functionalities for baijiu brewing.