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Microbial diversity and component variation in Xiaguan Tuo Tea during pile fermentation.

ABSTRACT: Xiaguan Tuo Tea is largely consumed by the Chinese, but there is little research into the microbial diversity and component changes during the fermentation of this tea. In this study, we first used fluorescence in situ hybridization (FISH), next-generation sequencing (NGS) and chemical analysis methods to determine the microbial abundance and diversity and the chemical composition during fermentation. The FISH results showed that the total number of microorganisms ranges from 2.3×102 to 4.0×108 cells per gram of sample during fermentation and is mainly dominated by fungi. In the early fermentation stages, molds are dominant (0.6×102~2.8×106 cells/g, 0~35 d). However, in the late stages of fermentation, yeasts are dominant (3.6×104~9.6×106 cells/g, 35~56 d). The bacteria have little effect during the fermentation of tea (102~103 cells/g, <1% of fungus values). Of these fungi, A. niger (Aspergillus niger) and B. adeninivorans (Blastobotrys adeninivorans) are identified as the two most common strains, based on Next-generation Sequencing (NGS) analysis. Peak diversity in tea was observed at day 35 of fermentation (Shannon-Weaver index: 1.195857), and lower diversity was observed on days 6 and 56 of fermentation (Shannon-Weaver index 0.860589 and 1.119106, respectively). During the microbial fermentation, compared to the unfermented tea, the tea polyphenol content decreased by 54%, and the caffeine content increased by 59%. Theanine and free amino acid contents were reduced during fermentation by 81.1 and 92.85%, respectively.

SUBMITTER: Li H

PROVIDER: S-EPMC5819769 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Primary dark tea is used as raw material for compressed dark tea, such as Fu brick tea, Hei brick tea, Hua brick tea, and Qianliang tea. Pile-fermentation is the key process for the formation of the characteristic properties of primary dark tea, during which the microorganism plays an important role. In this study, the changes of major chemical compounds, enzyme activities, microbial diversity, and their correlations were explored during the pile-fermentation process. Our chemical and enzymatic analysis showed that the contents of the major compounds were decreased, while the activities of polyphenol oxidase, cellulase, and pectinase were increased during this process, except peroxidase activity that could not be generated from microbial communities in primary dark tea. The genera Cyberlindnera, Aspergillus, Uwebraunia, and Unclassified Pleosporales of fungus and Klebsiella, Lactobacillus of bacteria were predominant in the early stage of the process, but only Cyberlindnera and Klebsiella were still dominated in the late stage and maintained a relatively constant until the end of the process. The amino acid was identified as the important abiotic factor in shaping the microbial community structure of primary dark tea ecosystem. Network analysis revealed that the microbial taxa were grouped into five modules and seven keystone taxa were identified. Most of the dominant genera were mainly distributed into module III, which indicated that this module was important for the pile-fermentation process of primary dark tea. In addition, bidirectional orthogonal partial least squares (O2PLS) analysis revealed that the fungi made more contributions to the formation of the characteristic properties of primary dark tea than bacteria during the pile-fermentation process. Furthermore, 10 microbial genera including Cyberlindnera, Aspergillus, Eurotium, Uwebraunia, Debaryomyces, Lophiostoma, Peltaster, Klebsiella, Aurantimonas, and Methylobacterium were identified as core functional genera for the pile-fermentation of primary dark tea. This study provides useful information for improving our understanding on the formation mechanism of the characteristic properties of primary dark tea during the pile-fermentation process.

Project description:BACKGROUND:Sugarcane bagasse is a major source of lignocellulosic biomass, yet its economic potential is not fully realised. To add value to bagasse, processing is needed to gain access to the embodied recalcitrant biomaterials. When bagasse is stored in piles in the open for long periods it is colonised by microbes originating from the sugarcane, the soil nearby or spores in the environment. For these microorganisms to proliferate they must digest the bagasse to access carbon for growth. The microbial community in bagasse piles is thus a potential resource for the discovery of useful and novel microbes and industrial enzymes. We used culturing and metabarcoding to understand the diversity of microorganisms found in a uniquely undisturbed bagasse storage pile and screened the cultured organisms for fibre-degrading enzymes. RESULTS:Samples collected from 60 to 80 cm deep in the bagasse pile showed hemicellulose and partial lignin degradation. One hundred and four microbes were cultured from different layers and included a high proportion of oleaginous yeast and biomass-degrading fungi. Overall, 70, 67, 70 and 57% of the microbes showed carboxy-methyl cellulase, xylanase, laccase and peroxidase activity, respectively. These percentages were higher in microbes selectively cultured from deep layers, with all four activities found for 44% of these organisms. Culturing and amplicon sequencing showed that there was less diversity and therefore more selection in the deeper layers, which were dominated by thermophiles and acid tolerant organisms, compared with the top of pile. Amplicon sequencing indicated that novel fungi were present in the pile. CONCLUSIONS:A combination of culture-dependent and independent methods was successful in exploring the diversity in the bagasse pile. The variety of species that was found and that are known for biomass degradation shows that the bagasse pile was a valuable selective environment for the identification of new microbes and enzymes with biotechnological potential. In particular, lignin-modifying activities have not been reported previously for many of the species that were identified, suggesting future studies are warranted.

Project description:Contamination by ochratoxigenic fungi and its prevention during the pile-fermentation of post-fermented tea have always been a concern. The present study aimed to elucidate the anti-fungal effect and mechanism of polypeptides produced by B. brevis DTM05 (isolated from post-fermented tea) on ochratoxigenic fungi, and to to evaluate their use in the pile-fermentation process of post-fermented tea. The results showed that polypeptides (produced by B. brevis DTM05) with a strong antifungal effect against A. carbonarius H9 mainly had a molecular weight between 3 and 5 kDa. The Fourier-transform infrared spectra of this polypeptide extract showed that it was a mixture consisting mainly of polypeptides and small amounts of lipids and other carbohydrates. The polypeptide extracts significantly inhibited the growth of A. carbonarius H9, and its minimum inhibitory concentration (MIC) was 1.6 mg/L, which significantly reduced the survival rate of spores. The polypeptides also effectively controlled the occurrence and ochratoxin A (OTA) production of A. carbonarius H9 on the tea matrix. The lowest concentration of polypeptides that significantly inhibited the growth of A. carbonarius H9 on the tea matrix was 3.2 mg/L. The enhancement of the fluorescence staining signal in the mycelium and conidiospore showed that the polypeptides with a concentration of more than 1.6 mg/L increased the permeability of the mycelium membrane and conidial membrane of A. carbonarius H9. The significant increase in the extracellular conductivity of mycelia suggested the outward leakage of intracellular active substances, and also further indicated an increase in cell membrane permeability. Polypeptides with a concentration of 6.4 mg/L significantly down-regulated the expression level of the polyketide synthase gene related to OTA production (acpks) in A. carbonarius H9, which may be the fundamental reason why polypeptides affect OTA production. In conclusion, reasonable use of the polypeptides produced by B. brevis can destroy the structural integrity of the cell membrane, make the intracellular active substances leak outward, accelerate the death of fungal cells and down-regulate the expression level of the polyketide synthase gene in A. carbonarius; thus, they can effectively control the contamination of ochratoxigenic fungi and OTA production during the pile-fermentation of the post-fermented tea.

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Microbial diversity and component variation in Xiaguan Tuo Tea during pile fermentation.

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