Instant dark teas (IDTs) were individually liquid-state fermented using the fungi Aspergillus cristatus, Aspergillus niger, and Aspergillus tubingensis. To understand how the chemical constituents of IDTs were affected by the fungi, samples were collected and measured by liquid chromatography-tandem mass-tandem mass spectrometry (LC-MS/MS). Untargeted metabolomics analysis revealed that 1,380 chemical constituents were identified in positive and negative ion modes, and 858 kinds of chemical components were differential metabolites. Through cluster analysis, IDTs were different from the blank control, and their chemical constituents mostly included carboxylic acids and their derivatives, flavonoids, organooxygen compounds, and fatty acyls. And the metabolites of IDTs fermented by A. niger and A. tubingensis had a high degree of similarity and were classified into one category, which showed that the fungus used to ferment is critical to the formation of certain qualities of IDTs. The biosynthesis of flavonoids and phenylpropanoid, which involved nine different metabolites such as p-coumarate, p-coumaroyl-CoA, caffeate, ferulate, naringenin, kaempferol, leucocyanidin, cyanidin, and (-)-epicatechin, were significant pathways influencing the quality formation of IDTs. Quantification analysis indicated that the A. tubingensis fermented-IDT had the highest content of theaflavin, theabrownin, and caffeine, while the A. cristatus fermented-IDT had the lowest content of theabrownin, and caffeine. Overall, the results provided new insights into the relationship between the quality formation of IDTs and the microorganisms used in liquid-state fermentation.

• Comprehensive analysis of RDT metabolites and microbiome under different RH. • High RH promote the accumulation of O-methylated catechin derivatives, polymerized catechins, and flavonols/flavones in RDT. • Aspergillus fungi and significant bacterial genera (Gluconobacter, Kluyvera, and Pantoea) highlights their roles in dark tea storage.

Microbes are critical in the Sichuan South-road Dark Tea (SSDT) organoleptic quality development during pile-fermentation. Piled tea center at fermenting metaphase is crucial for the conversion of its quality components. In this study, we investigated the microbial community of piled SSDT center below the stacked tea surface of 15 cm (SSDTB), 50 cm (SSDTX), and 85 cm (SSDTH) on the second turning time of pile-fermentation, respectively. Results showed that SSDTH and SSDTB had a higher similarity in the microbial community. Pantoea (36.8%), Klebsiella (67.7%), and Aspergillus (35.3%) were the most abundant in SSDTH, SSDTB, and SSDTX, respectively. We found 895 species were common among all samples, but 86, 293, and 36 species were unique to SSDTB, SSDTX, and SSDTH, respectively. Aspergillus niger showed high co-occurrence and was positively correlated with numerous microbes in SSDT samples, and Aspergillus niger M10 isolated from SSDTX was excellent at enhancing soluble sugar (SS), amino acids (AAs), theaflavin (TF), and thearubigins (TR) contents, while decreasing catechin (Cat), tea polyphenols (TPs)/AA, Caf/SS, Cat/SS, TPs/SS, and (TPs + Caf)/SS levels in AM10 post-fermentation, as compared with the control. Moreover, it also produced a noticeable difference in the CIELab parameters in dried, liquor, and infused tea colors between AM10 and control during fermentation. When it was further inoculated on differential mediums, we detected glycoside hydrolases, namely, β-glucosidase, mannosidase, pectinase, cellulase, amylase, and α-galactosidase being secreted by Aspergillus niger M10. Taken together, SSDXT presented a more unique microbial community. Aspergillus niger M10 probably improved the sweet and mellow taste, and the yellow brightness and red color of SSDT during fermentation. It also provided new insights into the microbial profile and organoleptic quality development mechanism of SSDT during pile-fermentation.

In this study, the dynamic changes of different anthocyanins in the processing of 'Ziyan' tea wine were investigated quantitatively. Results showed that six types of anthocyanins, namely petunidin, malvidin, pelargonidin, delphinidin, cyanidin and peonidin, as well as two co-pigmented substances, procyanidins and flavonoids, were detected in 'Ziyan' tea wine. As fermentation proceeded, the contents of petunidin, pelargonidin, delphinidin, cyanidin and peonidin decreased. Among them, petunidin, peonidin and pelargonidin showed a tendency of decreasing first, then increasing and finally decreasing, whereas delphinidin and cyanidin continued to decrease during fermentation. Variation trend of procyanidins and flavonoids was consistent with those of petunidin. Furthermore, metabolism of delphinidin, cyanidin and pelargonidin were main pathways responsible for the anthocyanin changes during 'Ziyan' tea wine processing. These findings suggested that the color of 'Ziyan' tea wine was achieved by the combination of various anthocyanins in different ratios and the co-pigmentation of procyanidins and flavonoids.

In recent years, there has been an increasingly heated debate on whether Chinese dark tea is contaminated with mycotoxins and whether it poses health risks to consumers. In this study, a rapid method based on high-performance liquid chromatography was used to detect ochratoxin A (OTA) in Chinese dark tea samples from different regions of China and different years. Of the 228 Chinese dark tea samples tested, 21 were detected for OTA contamination, with a concentration ranging from 2.51 ± 0.16 to 12.62 ± 0.72 μg/kg. Subsequently, a dark tea drinking risk assessment was conducted, and the hazard quotient for each group was far below the acceptable level of 1.0. Of the 12 Aspergillus spp. strains isolated, one strain of Aspergillus niger had the ability to produce OTA. We also found that tea polyphenols and epigallocatechin gallate inhibited the growth of ochratoxin-producing Aspergillus niger and the expression of non-ribosomal peptide synthetase (NRPS), a key gene for ochratoxin synthesis. Thus, OTA contamination of dark tea is at an acceptable risk level, and the inhibition of ochratoxigenic Aspergillus niger by polyphenols provides new insights into the safety of dark tea consumption.