The research team, led by Xuechun Wang and Jian-Lin Wu, took a traditional Liu-Pao tea product as its starting material and fermented it further with one of three single strains: the golden flower fungus Eurotium cristatum (strain 8730), and two strains of the probiotic bacterium Bacillus coagulans, designated No. 10059 and JZ-1. Fermentation ran for seven days at 50 degrees Celsius, with leaf piles 80 to 100 centimeters high and moisture contents between roughly 10 and 25 percent. The choice of organisms was deliberate. E. cristatum is famed for the golden flower fungus aroma of brick teas, producing volatile compounds such as methyl salicylate and linalool oxide, while B. coagulans secretes proteases and lipases that can break down proteins and fats into flavor-active fragments. What remained unclear before this study was precisely how each strain drives the formation and transformation of the metabolites behind taste.

The first line of evidence came from human panels. Six trained tasters, working blind with randomly coded samples across three independent sessions, scored the teas on sweetness, umami, bitterness, sourness, astringency, and aged flavor using a ten-point scale under a protocol approved by an ethics committee. The unfermented control scored 5.50 for sweetness and 4.25 for bitterness. After fermentation with B. coagulans No. 10059, bitterness and astringency fell to 3.00 and 3.75 respectively, while the JZ-1 strain produced a more mellow profile with a noticeable cheese-like note. But the standout was E. cristatum: sweetness climbed to 7.50, umami jumped from 1.50 to 3.75, and bitterness dropped to 2.75, yielding the most pronounced mellow-sweet character of all the samples, accompanied by the fungus’s signature fungal aroma.

Human palates can be subjective, so the team turned to an SA-402B electronic tongue equipped with six lipid-membrane sensors that quantify sourness, bitterness, astringency, umami, saltiness, and sweetness, along with aftertaste attributes. The instrument confirmed the panel’s verdicts with striking precision. Sweetness values rose from 3.654 in the traditional tea to 4.102 and 4.302 in the two B. coagulans samples, and reached 5.989 in the E. cristatum tea, the highest of all. Bitterness moved in the opposite direction, falling from 3.484 to 2.500 in the fungal sample. Umami intensity increased across all fermented teas, from 2.776 in the control to 4.687 in the E. cristatum product. Sourness, saltiness, and astringency aftertaste registered below their tasteless thresholds and were excluded, leaving sweetness as the single most discriminative taste parameter between strains.

To trace these sensory shifts back to chemistry, the researchers quantified the tea’s major constituents. Total protein dropped significantly in the B. coagulans teas, most notably with JZ-1, consistent with the bacterium’s proteolytic enzymes, but rose significantly in the E. cristatum sample, likely reflecting fungal protein secretion and the conversion of insoluble proteins into soluble forms and peptides. Total polyphenols, the most abundant chemical class in the tea, fell significantly in the bacterial fermentations, from about 362 milligrams per gram to roughly 297, while remaining unchanged in the fungal sample. Flavonoids, a polyphenol subclass, trended upward with E. cristatum, and soluble sugar showed no significant change in any group. These bulk measurements hinted that each microbe was running a fundamentally different biochemical program on the same tea base.

The centerpiece of the study was a feature-based molecular networking approach, or FBMN, run on the GNPS platform and coupled to ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. After preprocessing the raw data with MS-DIAL, the team generated a molecular network of 18,342 mass spectral nodes, each representing a detected chemical feature, clustered by spectral similarity and visualized in Cytoscape with pie charts showing each compound’s abundance across samples. The strategy allowed the researchers to see at a glance which metabolites were associated with which fermentation strain, dramatically accelerating the discovery of microbial-derived compounds that conventional targeted analysis would have missed.

In total, 358 compounds were tentatively identified, including 249 flavonoids, 21 peptides, 6 amino acids, 32 lipids, and 50 alkaloids or nucleosides, with 10 compounds flagged as potentially novel. Among the flavonoids were catechins, flavonol glycosides, biflavonoids, and a distinctive group called flavan-3-ol B-ring fission analogs, or FBRFAs, including teadenol A, planchol A, and fuzhuanin A. The network also revealed a cluster of ethyl-pyrrolidinone-substituted flavonoid alkaloids, the puerins, and led to the characterization of a potential new compound, eps-teadenol, whose fragmentation pattern matched that of known pyrrolidinone-substituted flavan-3-ols. From this cluster, ten previously unreported eps-FBRFAs were tentatively annotated. On the peptide side, the network yielded 16 linear peptides, including the pentapeptide Gly-Pro-Phe-Pro-Leu/Ile, and five indole-containing cyclic dipeptides such as neoechinulin A and echinulin, three of which had never been reported in Liu-Pao tea.

Statistical modeling then separated the strain-specific signatures. Orthogonal partial least squares discriminant analysis identified 71 differential compounds in the E. cristatum tea, 33 up-regulated and 38 down-regulated. Nine FBRFAs and three eps-FBRFAs rose significantly, and the prenylated cyclic peptides neoechinulin A, echinulin, and tardioxopiperazine A surged from trace levels to marked accumulation, a hallmark of fungal secondary metabolism. Critically, non-ester-type catechins such as epicatechin and epigallocatechin increased dramatically while their gallate-esterified counterparts, including EGCG and ECG, declined, suggesting that fungal enzymes hydrolyze esterified catechins into their non-esterified forms. In the B. coagulans teas, the story was proteolytic: 38 differential compounds emerged in the JZ-1 fermentation, 15 of them up-regulated, including four amino acids and eleven peptides, with representative peptides such as Pro-Glu-Val reaching their highest levels in the JZ-1 group.

Linking chemistry back to the palate, redundancy analysis showed that the first two axes explained over 93 percent of the variance in flavor attributes, with sweetness aligning strongly with non-esterified catechins, FBRFAs, and eps-FBRFAs, while bitterness tracked with the ester-type catechins. This fits established taste science: non-ester catechins like EGC and EC contribute to sweet aftertaste, whereas gallated catechins are among the most bitter and astringent compounds in tea. The linear peptides enriched by B. coagulans correlated positively with aged flavor, plausibly explaining the bacterial teas’ deeper aged character. Pushing the mechanistic question further, the team docked twelve FBRFAs and eps-FBRFAs into the Venus flytrap domains of the human sweet taste receptor TAS1R2/TAS1R3, the same pocket that recognizes sucralose. All twelve bound with predicted energies between minus 7.1 and minus 9.0 kilocalories per mole, and eleven contacted key residues including Tyr-103, Asp-142, and Asp-278. The authors are careful to note that docking alone does not prove receptor activation, and functional validation remains to be done.

Finally, the study proposed a biosynthetic route for the eps-FBRFAs, a class reported here for the first time in Liu-Pao tea. The pathway begins with the de-esterification of gallated catechins such as EGCG into non-ester forms, followed by cyclic peroxide formation, meta-cleavage, reduction, oxidation, and lactonization to yield FBRFAs. In parallel, theanine undergoes Strecker degradation to aldehyde intermediates that cyclize into ethyl pyrrolidone, which then conjugates with FBRFAs at the C-8 position. Supporting this route, theanine levels fell most sharply in the E. cristatum fermentation. Because eps-FBRFA enrichment appeared only in the fungal samples despite identical fermentation conditions, the strain itself appears essential to the transformation. Together, the findings give tea producers a molecular toolkit: E. cristatum for sweetness and reduced bitterness, B. coagulans for aged flavor depth, and a validated analytical pipeline for engineering the next generation of fermented teas.

Subject of Research: Microbial fermentation and flavor metabolites in Liu-Pao dark tea

Article Title: Integrated sensory evaluation and feature-based molecular networking elucidate flavor improvement and associated metabolites in dark tea fermented by Eurotium cristatum and Bacillus coagulans

Article References: Integrated sensory evaluation and feature-based molecular networking elucidate flavor improvement and associated metabolites in dark tea fermented by Eurotium cristatum and Bacillus coagulans. (n.d.). https://doi.org/10.1016/j.fochx.2026.104544

Image Credits: AI Generated

DOI: 10.1016/j.fochx.2026.104544

Keywords: dark tea, Liu-Pao tea, Eurotium cristatum, Bacillus coagulans, fermentation, metabolomics, molecular networking, flavor chemistry, catechins, peptides, sweet taste receptor, food science