Dynamic analysis of volatile metabolites and microbial community and their correlations during the fermentation process of traditional Huangjiu (Chinese rice wine) produced around Winter Solstice

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TL;DR

This study analyzed the dynamic changes in volatile aroma compounds and microbial communities during the 90-day low-temperature fermentation of traditional Huangjiu (Chinese rice wine) produced around Winter Solstice. Using GC–IMS, GC–MS, and high-throughput sequencing, the researchers identified 54 aroma compounds and found that Saccharomyces, Aspergillus, Saccharopolyspora, Staphylococcus, and Lactobacillus were the core functional microorganisms driving flavor formation. The findings provide a scientific basis for the traditional practice of winter brewing and offer theoretical guidance for flavor regulation through microbial augmentation.

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**Background:** Traditional Huangjiu (Chinese rice wine) is brewed under low ambient temperatures around Winter Solstice in a process lasting approximately 90 days. Empirical production experience suggests that wine produced near Winter Solstice has superior quality and more harmonious flavor, but scientific explanations for this phenomenon and the relationships between microbial community and flavor compounds during fermentation were lacking. This study aimed to analyze the dynamics of aroma compounds using GC–IMS and GC–MS, compare microbial community diversity via high-throughput sequencing, and reveal correlations between dominant microbial genera and key aroma compounds.

**Methods:** Samples were collected from Zhejiang Pagoda Brand Huangjiu Co., Ltd. (Shaoxing, China) at five fermentation time points: 0d (C1), 22d (C2), 45d (C3), 67d (C4), and 90d (C5) during secondary fermentation at medium–low temperatures (5.76 ± 0.68 °C to 18.06 ± 0.66 °C). Volatile compounds were analyzed by GC–IMS (Flavorspec®) and GC–MS (Agilent 7890 GC/5973C MS) using HS–SPME and SAFE extraction methods. Microbial DNA was extracted using TIANamp Stool DNA Kit; bacterial 16S rRNA V3–V4 regions and fungal ITS1 regions were amplified and sequenced on an Illumina MiSeq platform. Alpha diversity (Chao1, Shannon, Simpson, ACE) and beta diversity (PCoA based on weighted UniFrac) were analyzed. Pearson correlation coefficients between dominant genera and aroma compounds (OAV > 1) were calculated using R and visualized with Cytoscape.

**Key Results:** A total of 54 aroma compounds were detected (21 esters, 14 alcohols, 6 acids, 4 aldehydes, 3 ketones, 4 phenols). Alcohols constituted 73.01%–87.83% of total aroma content. Phenylethanol (326.21 mg/kg at C1) and 3-methylbutanol were the most abundant alcohols. Ethyl lactate increased approximately five-fold from C1 (12.52 mg/kg) to C5 (61.76 mg/kg). Twenty-one compounds had OAV > 1, including ethyl octanoate (OAV: 203–370), 4-vinyl-2-methoxyphenol (OAV: 348–642), and phenylacetaldehyde (OAV: up to 1006 at C5). For bacteria, Actinobacteriota (49.01%–68.81%) and Firmicutes (21.04%–38.22%) were dominant phyla; Saccharopolyspora (47.28%–67.71%) was the absolute dominant genus, decreasing with fermentation time. Staphylococcus reached 25.63% by fermentation end. For fungi, Ascomycota (96.60%–98.33%) dominated; Saccharomyces was the most abundant genus (97.03% at C1, 95.09% at C5). Aspergillus increased to 3.46% at day 67. LEfSe analysis identified 71 bacterial and 21 fungal clades with LDA scores ≥ 3.0. Correlation networks showed Saccharomyces positively correlated with phenylethanol, phenylacetaldehyde, 3-methylthiopropanol, and phenylethyl acetate. Saccharopolyspora positively correlated with 2-ethylhexanol, phenylacetaldehyde, 3-methylthiopropanol, phenylethanol, phenylethyl acetate, and isoamyl acetate. Staphylococcus positively correlated with ethyl butyrate, ethyl laurate, γ-nonanolactone, ethyl decanoate, and 4-vinyl-2-methoxyphenol. Five genera (Saccharomyces, Aspergillus, Saccharopolyspora, Staphylococcus, Lactobacillus) were identified as core functional microorganisms.

**Clinical Implications:** This study provides the first scientific explanation for the traditional winter brewing practice of Huangjiu, demonstrating that low-temperature fermentation promotes the growth of core microorganisms (Saccharomyces, Aspergillus, Saccharopolyspora, Staphylococcus, Lactobacillus) that drive the accumulation of characteristic aroma compounds. The findings offer theoretical guidance for flavor regulation through microbial community management and bioaugmentation with identified beneficial strains. However, the exact contributions and specific metabolic pathways of core microorganisms require further investigation.