Analyzed the data: YZ IS.
Pu-erh is a tea produced in Yunnan, China by microbial fermentation of fresh Camellia sinensis leaves by two processes, the traditional raw fermentation and the faster, ripened fermentation. We characterized fungal and bacterial communities in leaves and both Pu-erhs by high-throughput, rDNA-amplicon sequencing and we characterized the profile of bioactive extrolite mycotoxins in Pu-erh teas by quantitative liquid chromatography-tandem mass spectrometry.
We identified fungal and bacterial OTUs from leaves and both Pu-erhs. Major findings are: 1 fungal diversity drops and bacterial diversity rises due to raw or ripened fermentation, 2 fungal and bacterial community composition changes significantly between fresh leaves and both raw and ripened Pu-erh, 3 aging causes significant changes in the microbial community of raw, but not ripened, Pu-erh, and, 4 ripened and well-aged raw Pu-erh have similar microbial communities that are distinct from those of young, raw Ph-erh tea.
Twenty-five toxic metabolites, mainly of fungal origin, were detected, with patulin and asperglaucide dominating and at levels supporting the Chinese custom of discarding the first preparation of Pu-erh and using the wet tea to then brew a pot for consumption.
Tea is one of the most popular and widely consumed beverages in the world. It is normally produced from the leaves of two varieties of the tea plant, Camellia sinensis var. Tea has important physiological effects on consumers due to the presence of compounds such as polyphenols, amino acids, vitamins, carbohydrates, caffeine, and purine alkaloids, all of which can have health benefits [ 2 — 4 ]. Among the claimed effects of consuming tea are blood lipid and weight reduction, antimicrobial, antioxidant, and anticancerogenic activities, and enhanced digestion [ 4 , 5 ].
Based on processing procedures, tea can be divided into at least six different types: green, yellow, white, oolong, black called red tea in China , and post-fermented tea called dark tea in China [ 6 ]. Of them, post-fermented tea is unique due to the microbial fermentation process, which may last from several months to many years. Post-fermented Chinese teas include Fu-zhuan in Hunan, Qing-zhuan in Hubei, Liu-bao in Guangxi, and Pu-erh in Yunnan, the latter being best known, both for its taste and its political economics [ 7 ].
Pu-erh tea has been made from C.
There are two types: naturally fermented raw and purposely fermented ripened Figure A in S1 File. For raw Pu-erh, the initial processing starts with natural withering of fresh tea leaves to initiate their drying, roasting of leaves to continue drying and denature plant enzymes, rolling the leaves to remove additional moisture, and, finally, complete the drying process through direct exposure to the sun [ 8 ].
The dry, raw Pu-erh is then aged for varying periods to promote natural, solid substrate fermentation. To some extent, the quality and flavor of raw Pu-erh tea improves with age [ 9 ], and consequently aged raw Pu-erh is more valuable. Finished Pu-erh tea, both raw and ripened, can be left as loose leaves or compressed into cakes or bricks to facilitate transport and storage.
The production and quality of Pu-erh is closely related to microbial activity [ 12 ], making it important to understand the Pu-erh microbiome. Aspergillus niger and Blastobotrys adeninivorans were frequently documented as dominant lineages in Pu-erh from both culture-dependent and culture-independent studies. Some other studies have investigated the microbial diversity in Pu-erh teas of different ages [ 21 , 22 ], but none have characterized the microbial communities of fresh leaves, raw Pu-erh, and ripened Pu-erh, nor attempted to correlate bacterial and fungal community composition with environmental factors e.
As a product of microbial fermentation, the safety of Pu-erh tea is a topic of continued concern.
Toxic microbial metabolites were investigated from Pu-erh tea samples or fungal isolates recovered from Pu-erh, but inconsistent results were found in literatures. Some studies detected no mycotoxins [ 15 , 23 , 24 ], but other studies detected mycotoxins such as aflatoxin B1, deoxynivalenol, and ochratoxin A [ 25 — 28 ]. Previous studies, however, have not related microbial community composition to the production of potentially toxic microbial metabolites.
Recent advances in massively parallel, short-amplicon, sequencing technologies have launched a breakthrough in microbial ecology studies of the fermentation of wine, milk, and other foods [ 29 — 37 ]. We employed high-throughput amplicon sequencing to investigate the microbiome in fresh tea leaves, raw and ripened Pu-erh, and then performed multiplex analysis of metabolites in the tea samples.
Our goals were 1 to identify microbial diversity and composition in Pu-erh; 2 to compare microbial community structure among fresh tea leaves, and raw and ripened Pu-erh tea; 3 to identify potential factors affecting microbial communities in Pu-erh tea, and 4 to identify microbial metabolites in Pu-erh tea. Seven samples of fresh leaves of Camellia sinensis var. Fresh leaves were collected in three different tea gardens located in Pu-erh City of southern Yunnan Province, southwest China.
The 31 tea samples, 15 raw and 16 ripened, were collected from five different companies in Pu-erh City and had been stored for 0—28 years raw and 0—13 years ripened Table A in S1 File. They were either loose or compressed as cakes or bricks. All samples were subject for high throughput sequencing.
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The 31 tea samples were also analyzed for fungal and bacterial bioactive extrolites. Chromatogram files were viewed using FinchTV 1. Sequences were aligned using Muscle 3.
A nt barcode unique for each sample was included in reverse primers. PCR was carried out in the same reactions as plant fragment amplification but without the Q-Solution.
Concentrations of the pooled amplicons and length distribution were measured in an Agilent Bioanalyzer at the Functional Genomics Laboratory of UC Berkeley.
Fungal amplicons and bacterial amplicons were pooled at a ratio and sent to the Stanford University Functional Genomics Facility for bp paired-end sequencing on an Illumina Miseq platform. Fungal and bacterial sequencing primers were also pooled for each read before submission to the sequencing facility. Forward and reverse raw reads from the sequencing facility were first trimmed with CutAdapt1. For each dataset, identical sequences were de-replicated, and singleton sequences were discarded.
To discard non-target sequences, unassigned fungal sequences were further evaluated by ITSx 1. Any OTUs representing less than 0.
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To compare samples on an equal basis, all samples were rarefied to even sampling depths prior to statistical analysis. Rarefaction depths were set to maximize the number of samples included while still maintaining a reasonable number of sequences.
Specifically, when comparing among fresh leaf, raw and ripened Pu-erh samples, we rarefied the fungal dataset to 39 sequences per sample by keeping all samples; we rarefied the bacterial dataset to 1 sequences per sample by discarding three fresh leaf samples LN2, LS2, and LS4 and three raw Pu-erh samples A3, A14, and A When focusing just on raw Pu-erh samples, we rarefied the fungal dataset to 60 sequences per sample and the bacterial dataset to 1 sequences per sample, after removing from the dataset three raw Pu-erh samples A3, A14, and A When focusing just on ripened Pu-erh samples, we rarefied the fungal dataset to 94 sequences per sample and the bacterial dataset to 16 sequences per sample.
We also tested for potential correlation between fungal and bacterial community composition using a Mantel test based on Binary-Jaccard or Bray-Curtis distance matrices.
Samples were extracted for 90 min on a GFL rotary shaker GFL, Burgwedel, Germany , diluted with the same volume of extraction solvent, and the diluted extracts injected [ 57 ].
Centrifugation was not necessary due to sufficient sedimentation by gravity. Apparent recoveries of the analytes were determined by spiking five different samples with a multi-analyte standard on one concentration level. The spiked samples were stored overnight at ambient temperature to allow evaporation of the solvent and to establish equilibrium between the analytes and the sample.
The extraction, dilution and analysis were as described previously [ 57 ]. The chromatographic method and the chromatographic and mass spectrometric parameters are as described by Malacova et al. Confirmation of positive analyte identification was obtained by the acquisition of two MRMs per analyte with the exception of moniliformin which exhibited only one fragment ion.
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This approach yielded 4. In addition, the LC retention time and the intensity ratio of the two MRM transitions agreed with the related values of an authentic standard within 0. Direct sequencing of rbcL amplicons was used to test for C. For these samples, we visually inspected the heterozygous chromatogram peaks and found nucleotides characteristic of C. Microscopic examination and tasting of tea made from these three samples did not find obvious differences between them and other ripened Pu-erh samples.
To check for the presence of C. From these samples, which provided homozygous and heterozygous chromatograms of C. No variation in C. We successfully amplified and sequenced DNA from fungal and bacterial communities from all samples. After removing primers and low-quality ends, merging paired reads and de-multiplexing, each sample provided more than 42 fungal ITS sequences 7 in total and 29 bacterial SSU sequences 6 in total. Further processing to remove singleton sequences, chimeras, non-target sequences and low-abundance OTUs reduced the yield to between 39 and fungal sequences per sample 7 total and 66 to bacterial sequences per sample 2 total.
The low number of bacterial sequences detected in fresh leaf and some raw Pu-erh samples was due to amplification of competing chloroplast SSU DNA, which accounted for These chloroplast sequences were excluded prior to further analysis. The final number of OTUs passing abundance filtering at 0.
The most commonly observed fungal taxa belonged to Ascomycota OTUs; To evaluate the effect on microbial community similarity of the many rare OTUs found in each sample type, we analyzed OTUs shared among sample types using just the most abundant fungi and bacteria. Focusing on these most abundant microbes, the fraction of shared fungal and bacterial OTUs rose dramatically e. Venn diagrams illustrate the number of unique and shared fungal a, c and bacterial b, d OTUs.
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For bacteria, ripened Pu-erh showed significantly higher richness than either fresh leaves or raw Pu-erh with the observed species or Chao1 estimates, but not with Shannon or Simpson-e indices Figure E e-h in S1 File. Fungi were rarefied at 39 sequences to keep all samples, while bacteria at sequences to exclude three fresh leaf samples LN2, LS2, and LS4 and three raw Pu-erh samples A3, A14, and A Most interestingly, the oldest Pu-erh sample A6 tended to cluster with ripened Pu-erh samples, especially when considering the fungal community Fig 3a.
The oldest raw Pu-erh sample A6, 28 years old , indicated by an arrow in both PCoA analyses, is more similar to ripened Pu-erh than to other raw Pu-erh samples. Many indicator taxa were found for fresh leaf, raw and ripened Pu-erh samples Table 2. As expected, lineages of thermophilic or thermotolerant fungi e.
Two of the indicator taxa found for Pu-erh raw and ripened , Aspergillus niger and Blastobotrys adeninivorans , have been considered to be dominant fungal lineages in Pu-erh from both culture-dependent and culture-independent studies [ 15 , 16 ]. We investigated four variables age, producer, pure tea vs. To investigate the effect of aging, we binned our raw and ripened Pu-erh samples in either two young and old or three young, middle aged, and old age stages Table A in S1 File.
A Mantel test was used to examine the correlation between fungal and bacterial communities. We found no correlation between communites of the two types of microbes in raw Pu-erh based on either Binary-Jaccard or Bray-Curtis distance matrices Table D in S1 File.
Reasoning that common and abundant fungi are more likely to pose a possible mycotoxin problem to consumers, we further reduced the number of fungi to a set of the 15 most abundant OTUs in a sample type, i. Several of the fungi found in our Pu-erh samples are known mycotoxin producers, such as Aspergillus niger [ 59 ], Aspergillus restrictus [ 60 ], and Penicillium citrinum [ 61 ].
It is possible that some bacteria in Pu-erh produce toxins, but bacterial toxins are not addressed in this study. Fortunately, some frequently documented toxin-producing bacterial genera, such as Clostridium , Escherichia , Vibrio , and Salmonella [ 62 ], were not detected in our Pu-erh samples Table E in S1 File. They were revised according to online Blast search.
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All together 25 compounds were detected, most of them at low concentrations: alternariolmethylether, andrastin A, asperglaucide, aspterric acid, brevianamid F, chlorocitreorosein, citreorosein, cladosporin, cyclo L-Pro-L-Tyr , emodin, festuclavine, fumigaclavine A, fumigaclavine C, lotaustralin, malformin C, methylsulochrin, mycophenolic acid, neoechinulin A, patulin, physcion, quinocitrinin, rugulusovin, skyrin, usnic acid, and zearalenone Fig 4. Festuclavine, fumigaclavine A, methylsulochrin, chlorocitreorosein, and skyrin were detected in ripened samples only, while lotaustralin was found only in raw samples.
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Raw Pu-erh samples are indicated by circles, and ripened samples by squares. Mean concentrations and standard deviations of each metabolite in raw in blue and ripened in orange Pu-erh samples are marked.
Using next generation sequencing of DNA isolated from Pu-erh, we found many more species-level OTUs, fungal and bacterial, than had been identified in previous studies. For example, Tian et al.