An Integrated Metagenomics/Metaproteomics Investigation of the Microbial Communities and Enzymes in Solid-state Fermentation of Pu-erh tea

Abstract

Microbial enzymes during solid-state fermentation (SSF), which play important roles in the food, chemical, pharmaceutical and environmental fields, remain relatively unknown. In this work, the microbial communities and enzymes in SSF of Pu-erh tea, a well-known traditional Chinese tea, were investigated by integrated metagenomics/metaproteomics approach. The dominant bacteria and fungi were identified as Proteobacteria (48.42%) and Aspergillus (94.98%), through pyrosequencing-based analyses of the bacterial 16S and fungal 18S rRNA genes, respectively. In total, 335 proteins with at least two unique peptides were identified and classified into 28 Biological Processes and 35 Molecular Function categories using a metaproteomics analysis. The integration of metagenomics and metaproteomics data demonstrated that Aspergillus was dominant fungus and major host of identified proteins (50.45%). Enzymes involved in the degradation of the plant cell wall were identified and associated with the soft-rotting of tea leaves. Peroxiredoxins, catalase and peroxidases were associated with the oxidation of catechins. In conclusion, this work greatly advances our understanding of the SSF of Pu-erh tea and provides a powerful tool for studying SSF mechanisms, especially in relation to the microbial communities present.

Figure 1

Sensory evaluation of tea leaves collected on day 0 (a) and 21 (b) during the SSF of pu-erh tea. The sensory evaluation was developed according the Chinese standard (GB/T 23776-2009) as follow: 3 g of dried tea leaves were infused in 150 ml boiled water for 5 min; the tea leaves, the tea liquor and leaves after infused were showed, respectively.

Figure 2

Proteins separated by SDS-PAGE after extraction from fermented tea leaves using various methods (a) Proteins separated by 2-DE after extraction from fermented tea leaves using the Tris-HCl/phenol and methanol precipitation (TPMP) method (b) TAP method indicates proteins extracted using TCA/acetone precipitation; TTAP method indicates proteins extracted using Tris-HCl and TCA/acetone precipitation; and UTAP method indicated protein extracted using urea/thiourea and TCA/acetone precipitation.

Figure 3

Biological process (a) and Molecular function (b) categories of identified proteins based on a gene ontology analysis. The categories including more than 1% of the total identified proteins are presented.

Figure 4

Summary of protein extractions from fermented tea leaves by trichloroacetic acid (TCA)/acetone precipitation (a), Tris-HCl/phenol and methanol precipitation (b), Tris-HCl and TCA/acetone precipitation (c) and urea/thiourea and TCA/acetone precipitation (d). ^aCentrifugation (4,000 × g at 4 °C for 5 min); ^bcentrifugation (12000 × g at 4 °C for 15 min); ^cpellets were suspended in cold acetone and kept at −20 °C for 1 h followed with centrifugation at 12,000 × g at 4 °C for 20 min.