Multi-omics analysis of the metabolism of phenolic compounds in tea leaves by Aspergillus luchuensis during fermentation of pu-erh tea.

Aspergillus fungi are extensively used in traditional food fermentation, so their functions, mechanisms, and safety risks are worth exploring. In this study, a dominant fungal strain (P1) was isolated from a fermented pu-erh tea and identified as A. luchuensis by phylogenetic analysis of fungal internally-transcribed spacer sequencing, partial ß-tubulin and calmodulin genes. A pure-strain fermentation of tea leaves was developed, and tea compounds were analyzed by widely-targeted metabolomics, using high-performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS). The mycotoxins, aflatoxin (B1, B2, M1 and M2), fumonisin B1 and B2, ochratoxin A, citrinin, were not detected in fermented tea leaves using methods in the National Standard of the Peoples' Republic of China. The genome of 36.60 Mb with 11,836 protein-coding genes was sequenced by PacBio sequencing and annotated. Expression of fungal genes during fermentation was analyzed by Illumina HiSeq 2500; genes encoding enzymes including glycoside hydrolases, phenolic acid esterases, laccases, tyrosinases, dehydrogenases, peroxidases, dioxygenases, monooxygenases, decarboxylases and O-methyltransferases were identified. These enzymes catalyze hydrolysis, oxidation, ring cleavage, hydroxylation, decarboxylation and O-methylation of phenolic compounds , significantly (p < 0.05) changing the phenolic compound composition. While, phenolic compounds were degraded through degradation of aromatic compounds pathways and xenobiotics biodegradation and metabolism pathways. These findings advance knowledge of the functions and mechanisms of action of Aspergillus in traditional food fermentation.

Unpruning improvement the quality of tea through increasing the levels of amino acids and reducing contents of flavonoids and caffeine.

Tea tree [Camellia sinensis var. sinensis or assamica (L.) O. Kuntze], an important crop worldwide, is usually pruned to heights of 70 to 80 cm, forming pruned tea tree (PTT) plantations. Currently, PTTs are transformed into unpruned tea tree (UPTT) plantations in Yunnan, China. This has improved the quality of tea products, but the underlying reasons have not been evaluated scientifically. Here, 12 samples of sun-dried green teas were manufactured using fresh leaves from an UPTT and the corresponding PTT. Using sensory evaluation, it was found that the change reduced the bitterness and astringency, while increasing sweetness and umami. Using high performance liquid chromatography detection showed that the contents of free amino acids (theanine, histidine, isoleucine and phenylalanine) and catechin gallate increased significantly (P < 0.05), whereas the content of alanine decreased significantly (P < 0.05). A liquid chromatography-mass spectrometry-based metabolomics analysis showed that the transformation to UPTT significantly decreased the relative levels of the majority of flavonols and tannins (P < 0.05), as well as γ-aminobutyric acid, caffeine and catechin (epigallocatechin, catechin, epigallocatechin gallate, gallocatechin gallate), while it significantly increased the relative contents of catechins (gallocatechin, epicatechin, epicatechin gallate and catechin gallate), phenolic acids and some amino acids (serine, oxidized glutathione, histidine, aspartic acid, glutamine, lysine, tryptophan, tyramine, pipecolic acid, and theanine) (P < 0.05). In summary, after transforming to UPTT, levels of amino acids, such as theanine increased significantly (P < 0.05), which enhanced the umami and sweetness of tea infusions, while the flavonoids (such as kaempferol, myricetin and glycosylated quercetin), and caffeine contents decreased significantly (P < 0.05), resulting in a reduction in the bitterness and astringency of tea infusions and an increase in tea quality.

Effect of inoculation with Penicillium chrysogenum on chemical components and fungal communities in fermentation of Pu-erh tea.

Developing an effective method to improve the quality of Pu-erh tea is of great scientific and commercial interest. In this work, Penicillium chrysogenum P1 isolated from Pu-erh tea was inoculated in sterilized or unsterilized sun-dreid green tea leaves to develop pure-culture fermentation (PF) and enhanced fermentation (EF) of Pu-erh tea. Spectrophotometry and high performance liquid chromatography determined that contents of free amino acids (FAA), total tea polyphenols and eight polyphenolic compounds in PF were significantly lower than these in non-inoculation control test (CK) (P < 0.05), whereas the contents of soluble sugars and theabrownins (TB) in PF were significantly higher (P < 0.05) than in CK. A total of 416 volatile compounds were detected by headspace solid-phase micro-extraction combined with gas chromatography-mass spectrometry. Comparison to CK, 124 compounds in PF were degraded or decreased significantly [Variable importance in projection [(VIP) > 1.0, P < 0.05, fold change (FC) < 0.5], whereas 110 compounds in PF were formed or increased significantly (VIP > 1.0, P < 0.05, FC > 2). Compared with normal fermentation (NF), the levels of gallic acid, (+)-catechin, (-)-epicatechin and 64 volatile compounds in EF were significantly lower (VIP > 1.0, P < 0.05, FC < 0.5), whereas the levels of FAA and 39 volatile compounds were significantly higher (VIP > 1.0, P < 0.05, FC > 2). Amplicon sequencing of fungal internal transcribed spacer 1 (ITS1) revealed that P. chrysogenum P1 didn't become the dominant fungus in EF; while the fungal communities in EF were different from those in NF, in that the relative abundances of Blastobotrys bambusae and P. chrysogenum in EF were higher, and the relative abundances of Aspergillus niger and Kluyveromyces marxianus in EF were lower. Overall, inoculation of P. chrysogenum in unsterilized sun-dreid green tea leaves changed the the fungal communities in fermentation of Pu-erh tea, and chemical compounds in fermented tea leaves, i.e., the levels of TB and the compounds responsible for the stale flavor, e.g., 2-amino-4-methoxybenzothiazole were increased, resulting in improvement of the sensory quality, including mellower taste and stronger stale flavor.