EGCG accounts for roughly half of the total catechins in fresh tea leaves and is prized for antioxidant, anti-inflammatory and anticancer activities. Yet it is also notoriously unstable and poorly absorbed. During fermentation it is converted into a family of derivatives, including epigallocatechin, epicatechin, catechin, theasinensins, theaflavins, fuzhuanins and teadenols, many of which show better bioavailability and distinct biological activities. The research team, led by Zexin An and Mingzhi Zhu of Hunan Agricultural University, set out to map this transformation comprehensively, combining targeted quantification with untargeted metabolomics across four experimental systems: liquid fermentation with each fungus separately, solid-state fermentation of sterile raw dark tea, an in vivo rat study, and an in vitro anaerobic culture of rat gut microbiota.

In the liquid fermentation experiments, the fungi were grown for five days before EGCG was added to a final concentration of 1 milligram per milliliter, and samples were collected over 72 hours. In a sterile medium without fungi, EGCG and gallic acid remained stable, ruling out significant auto-oxidation. Once A. cristatus was present, EGCG became undetectable within 12 hours, whereas A. niger needed a full 24 hours to finish the job. First-order kinetic modeling quantified the difference: the apparent disappearance rate constant for A. cristatus was 0.326 per hour, roughly 3.05 times that of A. niger at 0.107 per hour, with corresponding half-lives of 2.13 and 6.49 hours. Gallic acid, the primary hydrolysis product of EGCG, accumulated steadily in both systems.

Untargeted metabolomics using ultra-high-performance liquid chromatography coupled to a Q-Exactive Orbitrap mass spectrometer detected 3,402 annotated metabolic features across the fungal cultures. After filtering for features that changed more than twofold relative to controls, the team identified 235 differential metabolites in the A. cristatus system and 114 in the A. niger system. By tracking how each metabolite’s abundance rose and fell relative to EGCG and gallic acid, the researchers organized them into early-, mid- and late-stage products. Early-stage metabolites included epigallocatechin itself and complex polyflavonoids such as samarangenin A and epigallocatechin-(4β→8)-epicatechin 3,3′-digallate, showing that fungal fermentation drives not only degradation but also polymerization from the very beginning. Mid-stage products shared by both fungi included theasinensin B, oolongtheanin, theaflavin 3,3′-digallate, prodelphinidin A2 3′-gallate and isoneotheaflavin 3-O-gallate, several of which have documented antihypertensive, cholesterol-lowering, antiviral or alpha-glucosidase-inhibiting activities.

The late stages diverged sharply between the two fungi. In A. cristatus cultures, 3-hydroxyflavone emerged as a dominant late-stage EGCG-associated metabolite, accumulating at more than 100 times the level seen with A. niger, a remarkable demonstration of the species-specific conversion efficiency. A. cristatus also generated a diverse set of gallic-acid-derived products, with database-assisted annotations pointing to compounds including ellagic acid, dihydroxyphaseic acid, camonagrel and lidoflazine. A. niger, by contrast, produced fewer but distinctive derivatives, with flavonoids such as quercetin 3-(6”-caffeylgalactoside), isotheaflavin, fuzhuanins A and prodelphinidin A1 peaking at 36 hours before declining, while pelargonidin 3-sambubioside and a methylenedioxy-substituted phenylcoumarin continued rising through 72 hours.

The most surprising result came when the team moved from the simplified liquid system to real tea. Sterile raw dark tea was fermented with each fungus for 15 days, mirroring the roughly two-week “flowering” stage of Fu brick tea production. Visually, the cultures diverged dramatically: A. cristatus eventually produced the characteristic golden “golden flowers” on the leaves, while A. niger grew vigorously and turned the leaves brownish. But the kinetics of EGCG disappearance reversed. In tea fermented with A. niger, EGCG dropped to 209.5 micrograms per gram by day 5 and was nearly undetectable by day 15, whereas A. cristatus fermentation showed a slower, more gradual decline that plateaued after day 10. The authors suggest several possible explanations, including the known ability of high EGCG concentrations to inhibit A. niger by suppressing chitinase and chitin synthase expression, and the possibility that the tea matrix redirects A. cristatus toward alternative metabolic pathways. They also caution that the tea was autoclaved before inoculation, a treatment that may itself alter catechins, proteins and their interactions, and that untreated raw tea was not directly compared.

To untangle the chemistry of the tea matrix, the researchers applied weighted correlation network analysis to 5,144 detected compounds. In the A. cristatus fermentations, the data sorted into 15 color-coded modules, with the Meblue module of 651 metabolites showing a highly significant positive correlation with EGCG content. Within it, 585 core metabolites passed stringent thresholds, including 13 flavans such as catechin, epigallocatechin, taxifolin, neotheaflavin and methylated epigallocatechin gallates. A network built around EGCG linked it to 244 highly associated metabolites. Molecular networking on the GNPS platform then clustered structurally similar compounds sharing a common fragment ion at m/z 170.02, revealing a putative EGCG dimer at m/z 915.161, a potential epigallocatechin dimer at m/z 611.14, and other catechin relatives whose mass shifts hint at hydroxymethylation and related modifications. A. niger fermentations yielded their own module structure and unique core metabolites, including 4-gallocatechol, which accumulated significantly by day 15 and may represent a core late-stage product.

The study then followed EGCG into the body. Rats received a single oral dose of 500 milligrams per kilogram of body weight, and fecal samples were collected over 24 hours. EGCG first appeared in feces at 6 hours post-gavage, peaked at 440.27 micrograms per gram at 10 hours, and had fallen to just 6.14 micrograms per gram by 24 hours, pinpointing a 6-to-12-hour window as the key intestinal transit phase. Metabolomics of 106 available fecal samples yielded 14,931 annotated features, from which 683 EGCG-associated candidates were selected by chemical-class filtering and 59 core metabolites were prioritized using statistical models. Among 13 core flavans, the team found not only monomeric catechins and their derivatives but also a high-molecular-mass EGCG-associated product and putative theaflavin-related compounds, suggesting that oxidative transformations, potentially involving both non-enzymatic reactions and gut microbial interactions, occur within the intestine. A dimethylated catechin derivative also appeared, a modification that may alter polarity, stability and metabolic kinetics.

Perhaps the most instructive contrast was between the living gut and the petri dish. When rat gut microbiota were cultured anaerobically with EGCG at three concentrations, the bacteria reduced EGCG levels over time but unexpectedly failed to degrade it completely, even after 72 hours. Only nine flavans were detected among 5,942 annotated features, mostly monomeric catechins such as epigallocatechin, catechin and epicatechin, along with sulfated derivatives including epicatechin 4′-sulfate and 7′-sulfate. Seven of these nine metabolites also appeared in the fungal fermentations, and epicatechin 4′-sulfate showed similar concentration trends in vivo and in vitro. The far richer metabolic profile seen in living rats therefore cannot be explained by fecal microbiota alone; host enzymatic reactions, intestinal absorption, dynamic pH, bile, oxygen gradients and the mucus-associated microenvironment all likely contribute. 16S rDNA sequencing of the cultures showed Ligilactobacillus, Lactobacillus, Streptococcus and Escherichia-Shigella as dominant genera, with exploratory correlations between microbial abundance and EGCG levels that did not remain statistically significant after correction for multiple testing.

The authors are candid about the limits of the work: without isotope-labelled EGCG, the proposed pathways remain putative, since endogenous fungal metabolites, native tea constituents and host-derived compounds may change in parallel with EGCG. Future studies using carbon-13-labelled EGCG with high-resolution tandem mass spectrometry will be needed to confirm precursor-product relationships. Even so, the study delivers a clear message with practical implications. EGCG transformation is not a single uniform degradation pathway but a strain- and substrate-dependent process, meaning that fungal strain selection and fermentation conditions could be deliberately tuned to control how much EGCG is depleted and which catechin derivatives accumulate in dark tea. Given that many of the identified derivatives carry documented health-promoting activities, that tuning could shape not just the flavor of fermented tea but its functional value, while deepening our understanding of what happens to tea polyphenols between the cup and the colon.

Subject of Research: Microbial and gut microbiota biotransformation of the tea catechin EGCG during dark tea fermentation and intestinal metabolism

Article Title: Aspergillus Species and Gut Microbiota-Mediated Biotransformation of Epigallocatechin Gallate: From Dark Tea Fermentation to Intestinal Metabolism

Article References: An, Z., Ma, R., Huang, X., Zhang, Y., Huang, J., Liu, Z., Wang, K., & Zhu, M. (2026). Aspergillus Species and Gut Microbiota-Mediated Biotransformation of Epigallocatechin Gallate: From Dark Tea Fermentation to Intestinal Metabolism. Current Research in Food Science, Article 101587. https://doi.org/10.1016/j.crfs.2026.101587

Image Credits: AI Generated

DOI: Not provided

Keywords: EGCG, dark tea, Aspergillus cristatus, Aspergillus niger, fermentation, gut microbiota, metabolomics, catechins, gallic acid, theaflavins, bioavailability, biotransformation

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