Analyses of fungal community by Illumina MiSeq platforms and characterization of Eurotium species on Liupao tea, a distinctive post-fermented tea from China

Highlights

• The first study to characterise the microbial community in aged Liupao tea by Next-generation sequencing.

• Genus Eurotium were obtained from all Liupao tea aging fermentation.

• Eurotium species were monitored by the multilocus sequence typing method

• Mycobiota characterization is required to avoid the contamination from potentially toxic fungi.

Abstract

Liupao tea is a distinctive Chinese dark tea obtained by indigenous tea fermentation facilitated by the symbiotic association of bacteria and fungi. The composition of fungal community in 4 Liupao tea samples stored for several years under natural microbial fermentation was evaluated by MiSeq sequencing. Taxonomic analysis revealed 3 phyla, 6 families, 8 genera. The genera Eurotium and Aspergillus were dominant fungi in almost all the samples. A total of 85 strains found in 41 other tea samples were species of Eurotium. amstelodami, Eurotium. niveoglaucum, Eurotium. repens, Eurotium. rubrum, Eurotium. tonophilum and Eurotium. cristatum by culture-dependent method. Of these species, E. repens, E. rubrum and E. tonophilum have not been previously associated with Liupao tea. This report is the first to reveal fungal flora composition using Illumina-based sequencing and provide useful information for relevant studies on the isolation of Eurotium species in Liupao tea. The predominant molds are Eurotium species, and the comparison of fungal diversity in dark teas is worth considering. The taxonomic analysis of the microbial community would also aid the further study of functional genes and metabolic pathways of Liupao tea fermentation.

Introduction

Chinese tea is a popular beverage that has attracted consumers worldwide for centuries. Current general interest in such widely consumed beverage is growing rapidly because of scientific evidence on the health benefits and chronic disease prevention that can be attained by drinking tea (Fu et al., 2011, Lee and Foo, 2013, Wu et al., 2015, Zhang et al., 2013). Based on the various production areas, the manufacturing technologies and the degree of fermentation, Chinese dark tea is conventionally classified into Yunnan Pu'er tea, Hunan Fuzhuan brick tea, Sichuan Kangzhuan tea, Anhui Guyi dark tea, Shanxi Jingwei Fu tea, Hubei Qangzhuan tea, Xizang Ya'an Tibetan tea and Guangxi Liupao tea (Fig. 1A).

Liupao tea, similar to Pu'er tea, is a representative Chinese dark tea named after its place of origin Liupao Town in China. Liupao tea has been well-known since approximately 1500 years ago. It was initially introduced to northwestern China as trade merchandise for the tea-horse trade (Shao, Powell, & Clifford, 1995), and has also been introduced to Hong Kong, Macao, and many countries in Southeast Asia through the Maritime Silk Road (Su & Kong, 2011). Presently, Liupao tea is a national product of geographical indication (AQSIQ, 2011), and the production areas are confined to certain regions in the Wuzhou Region of Guangxi Province in 2011 (Fig. 1B). The Wuzhou Region has unique geographic conditions with varied landscapes and climates, particularly in Liupao Town, which is located in the mountains with abundant sunlight and substantial precipitation (CMA, 2016).

The Liupao tea material is produced from the large-leaf tea species, such as Camellia sinensis (L.) O. Kuntze, which differs from other dark teas' (Keller et al., 2013, Lv et al., 2013). The industrial production process of Liupao tea is illustrated in Fig. 1C. Bud leaves (Fig. 1a) with several tea stalks are properly selected and evenly inactivated by enzymes. For the natural fermentation process, raw tea is piled up to 1 m height lasts for 15 days. The temperature may increase up to 55 °C overnight and the leaf heaps are manually agitated to prevent an excessive increase in temperature and ensure homogeneity of the fermented tea. The rolled leaves are transformed into semi-finished tea (Fig. 1b). In the post-fermentation process, the pretreated tea is screened and then being placed in pile room for five weeks (Fig. 1c). After steaming (Fig. 1d), the tea is compressed into a bamboo basket for ease of transportation (Fig. 1e). The compressed tea is moved to caves or dry warehouses with 80–90% relative humidity and 23–28 °C for aging over six months (Fig. 1f). The mellow aroma and stable flavor components in dark tea are generated during the entire fermentation process, including decomposition of raw materials via fungi, enzymes, metabolism of microbes, as well as the complex interactions between all kinds of metabolites from microbes and tea materials (Chen et al., 2010, Liang et al., 2005).

As a post-fermented tea, the classical process of Liupao tea is based on natural fermentation facilitated by the symbiotic association of bacteria and fungi (Lyu et al., 2013, Tian et al., 2013, Zhang et al., 2016b, Zhang et al., 2016a). Given dark tea products with low water activity that can inhibit the reproduction of bacteria, most researchers believe that fungi are likely to be key players responsible for dark tea fermentation (Beuchat et al., 2013, Haas et al., 2013, Mo et al., 2008). The investigation of the mycoflora that account for the special characteristics of Liupao tea, which are responsible for either health benefits or the sensory attributes, will enable strategies for controlling these major microbes and be important guidance for improving the production of tea with higher quality. In addition, the presence of unknown fungi from natural fermentation is commonly assumed to pose safety risks when drinking the beverage due to the possibility of mycotoxin formation. Certain fungi discovered in dark teas are the main genera capable of producing toxic secondary metabolites. Many previous studies have reported on the isolation and identification of fungi from dark tea, whereas only few works have conducted investigations on Liupao tea. Therefore, the global characterization and a thorough mycological survey of the fungal diversity in Liupao tea is a needed. This kind of study can provide useful information for the targeted and expanded screening of valuable indigenous fungi.

A typical phenomenon occurs as a spread of yellow mycelial growth forming numerous macroscopic scattered spots (Fig. 2A) called “golden flowers”, traditionally judged as a quality standard of Liupao tea, appear on the surface of tea leaves. The unique yellow fungal growth phase is considered to be the key process point in the manufacture of dark tea, and critical for characteristic aroma, typical taste and unique flavor (Peng et al., 2014). It is known that these yellow dots are the typical ascomata of the genus Eurotium. The “golden flowers” have been isolated from Liupao tea and identified, phenotypically and genotypically, as Eurotium. niveoglaucum by our research group (Deng et al., 2014, Mao et al., 2013). Previous studies, however, have not discussed the diversity of “golden flowers”, correlating the results with the enumeration of the yellow colonies.

In the current study, four Liupao tea products obtained from the same plant were collected. From the analysis of the community compositions of the mycobiota and the comparison of fungal diversity by Illumina sequencing, the dominant fungi at the genus level responsible for Liupao tea can be acquired. Meanwhile, we also aimed to investigate the presence and levels of Eurotium species on the surfaces of the other 41 tea samples collected in three different tea gardens.