What is hidden in Pu-erh tea? From the Microbial Ecosystem to the threat of Mycotoxins
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We analyze the bacterial diversity of this most unique tea and the real danger to our health.
Pu-erh is a tea produced in Yunnan, China, by the microbial fermentation of fresh Camellia sinensis leaves using two processes: traditional raw fermentation and faster, ripened fermentation. As a product of microbial fermentation, the safety of Pu-erh tea is a subject of constant concern. Toxic microbial metabolites have been investigated from Pu-erh tea samples or fungal isolates recovered from Pu-erh, but inconsistent results have been found in the literature. Some studies have not detected mycotoxins, but other studies have detected mycotoxins such as aflatoxin B1, deoxynivalenol, and ochratoxin. Previous studies, however, have not correlated the composition of the microbial community with the production of potentially toxic microbial metabolites.
Using next-generation sequencing of DNA isolated from Pu-erh, we found many more OTUs (operational taxonomic units) at the species level, 390 fungi and 600 bacteria, than had been identified in previous studies. For example, Tian et al. used both culture-dependent gel electrophoresis and denaturing gradient gel electrophoresis methods to find ca. 20 fungal and 30 bacterial OTUs from 19 Pu-erh samples. Zhao et al. used the dilution method to find 41 fungal species from 60 Pu-erh samples. Several fungi have been identified in Pu-erh tea by culture. Aspergillus niger, an important industrial fungus, has been identified as the primary fermentation mold in Pu-erh production. We found this fungus in all raw and ripened Pu-erh samples, and its relative abundance was higher in ripened Pu-erh (second most abundant OTU, 16.1% of sequences) than in raw (15th most abundant OTU, 1.4% of sequences). In raw Pu-erh, the most dominant fungal taxonomy was an unidentified Aspergillus sp. (24.6%). In ripened Pu-erh, the most dominant fungal species was Blastobotrys adeninivorans (46.5%), which, in raw Pu-erh, was the third most abundant fungus. B. adeninivorans, another fungus of biotechnological interest, has often been isolated from Pu-erh by other researchers. Some fungal species frequently reported in the past, such as Penicillium chrysogenum and Yarrowia spp., were not found in our study. We cannot say whether the differences between our next-generation approach and these previous studies are due to differences in fungal viability, growth rate, DNA availability, or amplification efficiency, but all four aspects are likely involved. The incomplete reference database used for taxonomic classification is another significant factor because 38% (148/390) of the fungal OTUs could not be assigned to species.
One of our most significant findings is that fungal α-diversity is greater in fresh tea leaves than in Pu-erh and that bacterial α-diversity shows the opposite trend, i.e., lower in fresh leaves than in Pu-erh. With bacteria, the difference in α-diversity between leaves and tea appeared with ripened, but not raw tea, indicating that solid-state fermentation was responsible for the increase.
A second important finding is that the composition of both the fungal and bacterial community changes significantly due to Pu-erh tea fermentation, as evidenced by significant β-diversity differences for both fungal and bacterial communities in pairwise comparisons between fresh leaves, raw, and ripened Pu-erh. The reasons for these differences must be due to the microbes present on the leaves compared to those acquired during processing and fermentation. Fresh leaves contain sequences representing approximately 54% of the fungal OTUs and 22% of the bacterial OTUs found in Pu-erh. These OTUs were generally present in low abundance in fresh leaves and enriched in Pu-erh. The remaining fungal and bacterial OTUs found in Pu-erh must have their origins in production processes that allow the introduction of environmental microorganisms (e.g., pile covers, fermentation room, and workers' hands).
An interesting finding is that the most sought-after tea, aged raw Pu-erh, has a fungal community more like ripened than young raw Pu-erh, and a similar trend was observed for the bacterial community. This result indicates that the accelerated microbial fermentation of ripened Pu-erh, encouraged by the addition of water and the heat produced by microbial fermentation, results in a microbial community composition similar to that found in much older, raw Pu-erh. It also provides an ecological explanation for the rapid acceptance and widespread use of the ripened Pu-erh process.
We looked for correlations between four variables in tea production and microbial community composition: tea age, tea producer, whether the tea was pure or contaminated with other plants, and whether the tea was left loose or compressed into cakes. Tea age showed a significant correlation with fungal and bacterial community composition only for raw Pu-erh. From this result, one can conclude that raw Pu-erh is a robust but time-consuming method of product preparation and that the preparation of ripened Pu-erh is a more demanding process but does not benefit from aging. This result also seems to support the conjecture that the long transport of raw Pu-erh from Yunnan to Tibet and other remote destinations in ancient times contributed to its maturation. Aging does not significantly affect the communities of ripened tea, suggesting that ripened tea does not need to be aged. The other three variables did not have a significant effect on the microbial communities.
Regarding our discovery in ripened but not raw Pu-erh of rbcL sequences from Musa, Pinus and Brassica, we speculate that they arose from plants or plant products used by producers to cover tea during pile fermentation to delay water loss and retain heat. Naturally, contamination would also be possible from fermentation room floors, tools, or packaging materials, as well as contamination during harvesting. We were not able to determine the amount of these contaminant plants in the ripened Pu-erh samples, but the contamination was not enough to cause a significant difference in the composition of the microbial community or in the taste of the brewed tea.
Although Pu-erh tea has been considered a safe beverage for hundreds of years, without reports of poisoning, the quality and safety of any product that has undergone microbial fermentation are topics of ongoing interest and concern. Among the compounds detected from Pu-erh in this study, the most frequently encountered was asperglaucide, which was detected in all samples and in high amounts in raw (6596 μg/kg) and ripe (6799 μg/kg) tea. This metabolite is reported to be produced by Aspergillus spp, including A. penicillioides, which was detected in all samples. It is also reported by some plants, e.g., Walsura yunnanensis, but in none of the plants detected in this study. Asperglaucide is reported to have anti-inflammatory activity and the ability to inhibit cysteine peptidases, which may be beneficial in protecting against cartilage degeneration. Neoechinulin A, which has anti-inflammatory activity and can be produced by certain Eurotium species, was also detected in all samples. Fumigaclavine A, an antibacterial alkaloid produced by Aspergillus spp. was detected only in ripe tea, while lotaustralin, a precursor of hydrocyanic acid, was detected in all raw tea samples, but not in ripe tea samples. The fungicide, produced by Lysobacter capsici and Alternaria alternata, was detected in 60% and 100% of the raw and ripe tea samples, respectively, but in significantly higher amounts in ripe tea. This distribution also occurred for rugulusovin, which is produced by Penicillium spp. and has been shown to have cytotoxic activity against human and mouse cancer cells. It was identified in half of the raw and all of the ripe samples, a distribution that can be explained not only by the differences in the microbiome, but also by the differences in growth conditions between the two tea categories.
Patulin was detected in 60% of the raw samples with a mean concentration of 1169 μg/kg and in only 12.5% of the ripe samples at a mean concentration of 915 μg/kg. Patulin is of concern because it is produced by a large number of fungi and is suspected to be clastogenic, mutagenic, teratogenic, genotoxic, and cytotoxic. The US FDA has set a maximum limit of 50 μg/kg for patulin in apple juice and apple juice concentrates. Although the concentration of patulin is expected to be lower in a cup of properly brewed tea than the approximately 1000 μg/kg found in dry tea leaves, patulin concentrations in brewed tea are expected to exceed the limit set by the FDA. The discrepancy between our findings of a high concentration of patulin and the healthy reputation enjoyed by Pu-erh may be explained by the regulation of patulin toxicity through the action of green tea polyphenols.
Although we detected patulin in Pu-erh, we do not know its source. Known producers of patulin are Penicillium spp. (P. expansum, P. griseofulvum, P. carneum, P. glandicola, P. coprobium, P. vulpinum, P. clavigenum and P. concentricum), Aspergillus spp. (A. clavatus, A. giganteus and A. terreus), Paecilomyces variotii and Byssochlamys nivea. However, none of these species were identified in our tea samples. Therefore, it is possible that there are species in the tea that have not yet been reported to produce patulin. Conversely, we found fungal species reported to produce ochratoxin A (Aspergillus niger in all samples and A. ochraceus in six of the ripe samples), but no ochratoxin A was detected. This result is consistent with Mogensen et al., who did not find ochratoxin A content in five Pu-erh teas investigated, but different from Haas et al., who detected ochratoxin A in four of the 36 Pu-erh samples. In the present study, a small amount of zearalenone was detected in only one sample, while no aflatoxin, fumonisins, or trichothecenes were detected. Haas et al. did not find aflatoxins or fumonisins in the 36 Pu-erh samples tested. Wu et al. investigated 70 Pu-erh samples and found that all tea samples were safe regarding fumonisin B1 and T-2 toxin; however, 8 samples showed higher aflatoxin B1 concentrations than the safety limit and 63 samples exceeded the safety limit for deoxynivalenol. An explanation for finding fungi capable of producing mycotoxins, but not detecting the toxins themselves, can be found in a recent report that tea extracts inhibited aflatoxin production by Aspergillus flavus while not inhibiting the growth of the fungus' mycelia. Inhibition of mycotoxin production without inhibition of fungal growth was also reported for plants other than tea. This situation may also apply to ochratoxin. As noted above, to drink Pu-erh safely, most Pu-erh tea producers or distributors recommend discarding the first brew, a practice that may be recommended for the removal of water-soluble or suspended contaminants.
Conclusions
Next-generation sequencing revealed high fungal and bacterial diversity in Pu-erh tea. Fungal diversity decreases and bacterial diversity increases as a result of raw or ripe fermentation. The composition of microbial communities changes significantly between fresh leaves, raw and ripe Pu-erh, with aged raw tea having a similar community to ripe tea. The age of the tea is identified as a significant variable affecting the microbial community of raw tea, but not of ripe tea. Multiple mycotoxins were detected in one or both categories of Pu-erh, but all except patulin and asperglaucide were below the safety threshold. For safe consumption, we recommend discarding the first brew.
Health Tea Path observation :
Often, it is recommended to discard the second (short) brew as well, especially with ripe Pu-erh.
Buying teas from large and well-known factories is considered safer, as they have controlled hygiene conditions.
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