Microbial Dynamics in a Musalais Wine Fermentation: A Metagenomic Study

Abstract

This study provides a comprehensive analysis of the microbial dynamics involved in the fermentation process of traditional Musalais wine, an intangible cultural heritage of Xinjiang. Utilizing metagenomic sequencing, we identified 2894 microbial species, of which 494 persisted throughout the fermentation process. Saccharomyces cerevisiae was the dominant species, with its prevalence increasing from 97.35% in the early phase to 99.38% in the mid phase, before slightly decreasing to 98.79% in the late phase. Additionally, 24 non-Saccharomyces yeast species, including Hanseniaspora uvarum, Lachancea thermotolerans, and Torulaspora delbrueckii, were detected. Common species associated with other fermented foods, including Wickerhamomyces anomalus, Kluyveromyces marxianus, Saccharomyces eubayanus, and Zygosaccharomyces parabailii, were also identified. Notably, species not previously used in food fermentation, such as Saccharomyces jurei, Sodiomyces alkalinus, Vanrija pseudolonga, and Moesziomyces antarcticus, were also identified in this study. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KO) and Gene Ontology (GO) revealed notable variations in metabolic pathways and enriched functional genes. In addition, a total of 82 volatile compounds were detected in the final product, with higher alcohols (60.12%), esters (37.80%), and organic acids (1.80%) being the most prevalent. These results offer important insights into microbial interactions and their influence on Musalais wine quality, laying the groundwork for optimizing the fermentation process.

Keywords: Musalais wine; metagenomics; microbial diversity; spontaneous/wild fermentation; volatile compounds.

Figure 1

Investigation of physicochemical parameters of Musalais wine across various fermentation stages. (A) Measurement of Brix value; (B) assessment of residual sugar. Each sample has three parallel treatments.

Figure 2

Alpha diversity indices of Musalais wine samples at different fermentation stages. (A) Chao1 index; (B) Shannon index. Note: F: early-fermentation period, M: middle fermentation period, E: late-fermentation period.

Figure 3

Linear discriminant analysis effect size (LEfSe) analysis delineating phase-specific microbial biomarkers in Musalais wine fermentation. (A) LEfSe histogram. (B) LEfSe analysis of the phylogenetics. Note: F: early-fermentation period, M: middle fermentation period, E: late-fermentation period.

Figure 4

Taxonomic profiling of microbial communities at the genus level and temporal variations in relative abundance across Musalais wine fermentation stages. Note: F: early-fermentation period, M: middle fermentation period, E: late-fermentation period.

Figure 5

Comparative analysis of microbial community dynamics during Musalais wine fermentation. (A) Venn diagram illustrating phase-specific species distribution. Different colors represent different groups. The number in the overlapping area indicates the number of species shared by multiple groups, and the number in the non-overlapping area indicates the number of species unique to the corresponding group. (B) Heatmap profiling for relative abundance of predominant taxa. The x-axis represents species names, and the y-axis represents groups. The color gradient of the color blocks indicates the variation in the abundance of different species in the samples. On the right side of the figure, the color gradient representing values has been shown, and the color scale above indicates whether the species belongs to bacteria or fungi. Note: F: early-fermentation period, M: middle fermentation period, E: late-fermentation period.

Figure 6

Redundancy analysis of the correlation between physicochemical parameters and core microbiota in the Musalas fermentation system. (A) Dynamic association of microbial community structure with environmental factors at different fermentation stages. (B) Species-level response of dominant genera to key physicochemical factors. Note: F: early-fermentation period, M: middle fermentation period, E: late-fermentation period.

Figure 7

Musalais wine microbial symbiosis network diagram. Ellipses, triangles, and squares represent the phyla Pseudomonadota, Ascomycota, and Firmicutes, respectively. Yellow and green connecting lines denote negative correlations (competitive interactions) and positive correlations (mutualistic symbiosis), respectively, between microbial taxa. The area of each geometric shape corresponds to the relative abundance of the respective phylum within the microbial community.

Figure 8

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. (A) Bubble chart illustrating the 20 most enriched pathways during the mid-fermentation phase relative to the early phase. (B) Bubble chart depicting the 20 most enriched pathways in the late-fermentation phase compared to the mid-fermentation phase. (C) Bubble chart representing the 20 most enriched pathways across all three fermentation phases. Statistical significance was determined at a threshold of p < 0.05.

Figure 9

The pie chart of composition ratios of volatile components in Musalais wine per the gas chromatography–mass spectrometry analysis.