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Microorganisms
Volume 13
Issue 8
10.3390/microorganisms13081857
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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Article

Integrated Microbiome–Metabolome Analysis and Functional Strain Validation Reveal Key Biochemical Transformations During Pu-erh Tea Pile Fermentation

by
Mengkai Hu
1,
Huimin Zhang
1,
Leisa Han
1,
Wenfang Zhang
1,
Xinhui Xing
2,
Yi Wang
2,
Shujian Ou
3,
Yan Liu
1,
Xiangfei Li
1,* and
Zhenglian Xue
1,*
1
Engineering Laboratory for Industrial Microbiology Molecular Beeding of Anhui Province, College of Biologic & Food Engineering, Anhui Polytechnic University, 8 Middle Beijing Road, Wuhu 241000, China
2
Department of Chemical Engineering, Tsinghua University, Beijing 100084, China
3
Yunnan Shujian Tea Co., Ltd., Xishuangbanna 650501, China
*
Authors to whom correspondence should be addressed.
Microorganisms 2025, 13(8), 1857; https://doi.org/10.3390/microorganisms13081857
Submission received: 1 July 2025 / Revised: 1 August 2025 / Accepted: 4 August 2025 / Published: 8 August 2025
(This article belongs to the Special Issue Resource Utilization of Microorganisms: Fermentation and Biosynthesis)
Review Reports Versions Notes

Abstract

Fermentation plays a pivotal role in shaping the flavor and overall quality of Pu-erh tea, a microbially fermented dark tea. Here, we monitored physicochemical properties, chemical constituents, and microbial succession at 15 fermentation time points. Amplicon sequencing identified Staphylococcus, Bacillus, Kocuria, Aspergillus, Blastobotrys, Thermomyces, and Rasamsonia as dominant genera, with prokaryotic communities showing greater richness and diversity than eukaryotic ones. Beta diversity and clustering analyses revealed stable microbial structures during late fermentation stages. Non-targeted metabolomics detected 347 metabolites, including 56 significantly differential compounds enriched in caffeine metabolism and unsaturated fatty acid biosynthesis. Fermentation phases exhibited distinct metabolic patterns, with volatile aroma compounds (2-acetyl-1-pyrroline, 2,5-dimethylpyrazine) and health-beneficial fatty acids (linoleic acid, arachidonic acid) accumulating in later stages. OPLS-DA and KEGG PATHWAY analyses confirmed significant shifts in metabolite profiles relevant to flavor and biofunctionality. RDA revealed strong correlations between microbial taxa, environmental parameters, and representative metabolites. To functionally verify microbial contributions, 17 bacterial and 10 fungal strains were isolated. Six representative strains, mainly Bacillus and Aspergillus, exhibited high enzymatic activity on macromolecules, confirming their roles in polysaccharide and protein degradation. This integrative multi-omics investigation provides mechanistic insights into Pu-erh tea fermentation and offers a scientific basis for microbial community optimization in tea processing.
Keywords: Pu-erh tea; pile fermentation; microbial communities; non-targeted metabolomics analysis; macromolecule degradation Pu-erh tea; pile fermentation; microbial communities; non-targeted metabolomics analysis; macromolecule degradation

Share and Cite

MDPI and ACS Style

Hu, M.; Zhang, H.; Han, L.; Zhang, W.; Xing, X.; Wang, Y.; Ou, S.; Liu, Y.; Li, X.; Xue, Z. Integrated Microbiome–Metabolome Analysis and Functional Strain Validation Reveal Key Biochemical Transformations During Pu-erh Tea Pile Fermentation. Microorganisms 2025, 13, 1857. https://doi.org/10.3390/microorganisms13081857

AMA Style

Hu M, Zhang H, Han L, Zhang W, Xing X, Wang Y, Ou S, Liu Y, Li X, Xue Z. Integrated Microbiome–Metabolome Analysis and Functional Strain Validation Reveal Key Biochemical Transformations During Pu-erh Tea Pile Fermentation. Microorganisms. 2025; 13(8):1857. https://doi.org/10.3390/microorganisms13081857

Chicago/Turabian Style

Hu, Mengkai, Huimin Zhang, Leisa Han, Wenfang Zhang, Xinhui Xing, Yi Wang, Shujian Ou, Yan Liu, Xiangfei Li, and Zhenglian Xue. 2025. "Integrated Microbiome–Metabolome Analysis and Functional Strain Validation Reveal Key Biochemical Transformations During Pu-erh Tea Pile Fermentation" Microorganisms 13, no. 8: 1857. https://doi.org/10.3390/microorganisms13081857

APA Style

Hu, M., Zhang, H., Han, L., Zhang, W., Xing, X., Wang, Y., Ou, S., Liu, Y., Li, X., & Xue, Z. (2025). Integrated Microbiome–Metabolome Analysis and Functional Strain Validation Reveal Key Biochemical Transformations During Pu-erh Tea Pile Fermentation. Microorganisms, 13(8), 1857. https://doi.org/10.3390/microorganisms13081857

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