A high performance liquid chromatography method for simultaneous detection of 20 bioactive components in tea extracts.

Tea is the second most widely consumed beverage and contains various bioactive compounds. A simple method to analyze these compounds is of great scientific and commercial interest. In this work, a 30 min HPLC method was developed using a simple gradient elution system, and the mobile phases and elution gradients were optimized. This method separated 17 polyphenols and three alkaloid compounds in tea extracts, including catechins, alkaloids, phenolic acids, flavonols, and flavone, which are responsible for the bioactivity and flavor of tea. Excellent linearity was observed for all standard calibration curves, and correlation coefficients were above 0.9994. Heatmap analysis demonstrated significant separation between green, black, and pu-erh tea samples. The method described here is accurate and sensitive enough for the determination of active components in tea and could potentially be applied to other food products for the comprehensive investigation of their quality.

Influence of weeding methods on rhizosphere soil and root endophytic microbial communities in tea plants.

Introduction: Polyethylene mulch is a kind of inorganic mulch widely used in agriculture. The effects of plastic mulch debris on the structure of plant soil and root growth have been fully studied, but their effects on endophytic microbial communities have not been explored to a large extent. Methods: In this study, High-throughput sequencing of bacterial 16S rRNA genes and fungal ITS region sequences were used to analyze microbial community structure and composition in rhizosphere soil and root endophytic of tea plant under three different weeding methods: polyethylene mulching, hand weeding and no weeding (CK). Results: The results showed that the weeding methods had no significant effect on the rhizosphere and root endophytic microbial abundance, but the rhizosphere bacterial structure covered by polyethylene mulch was significantly different than hand weeding and CK. The rhizosphere fungal diversity was also significantly higher than the other two analyzed treatments. The community abundance of rhizosphere microorganisms Acidobacteria, Candidatus Rokubacteria and Aspergillus covered by polyethylene mulch decreased significantly, whereas Bradyrhizobium, Solirubrobacterales and Alphaproteobacteria increased significantly. The abundance of bacteria Ktedonobacter, Reticulibacter, Ktedonosporobacter and Dictyobacter communities covered by polyethylene mulch was significantly changed, and the abundance of Fusarium and Nitrobacteraceae was significantly increased. Rhizosphere dominant bacteria were negatively correlated with soil available nitrogen content, while dominant fungi were significantly correlated with soil pH, total nitrogen and total potassium. Discussion: Polyethylene mulch forms an independent micro-ecological environment. At the same time, the soil nutrient environment was enriched by affecting the nitrogen cycle, and the composition of microbial community was affected. This study elucidated the effects of polyethylene mulch on soil microbial community in tea garden and provided a new theoretical understanding for weed management.

Analysis of Metabolite Differences in Different Tea Liquors Based on Broadly Targeted Metabolomics.

To expand the development of characteristic extension products of Yunnan tea and improve the utilization rate of Yunnan tea resources, in this study, we compared the metabolite composition among raw Pu-erh tea, ripe Pu-erh tea prepared with glutinous rice (according to tea to glutinous rice ratio of 1:3), and ripe Pu-erh tea prepared with a mixture of sorghum, rice, glutinous rice, wheat, and corn as raw materials (according to a tea to glutinous rice ratio of 1:3). Rice flavor liquor prepared with 100% glutinous rice served as a control. The raw Pu-erh tea liquor (RAWJ), ripe Pu-erh tea liquor (RIPEJ), ripe Pu-erh tea mixed grain liquor (HHLSJ), and rice-flavor liquor (MJ) were all brewed by semi-solid fermentation. The non-volatile components of the liquor samples were analyzed by ultra-high-performance liquid chromatography-tandem mass spectrometry as a broadly targeted metabolomics technique. A total of 691 metabolites were identified from the four samples. Among them, 674, 671, 633, and 667 species were detected in RAWJ, RIPEJ, HHLSJ, and MJ samples, respectively. Venn diagram analysis demonstrated 19, 21, and 14 unique metabolites in RAWJ, RIPEJ, and HHLSJ, respectively, compared with the metabolite composition of MJ. Flavonoids are the most important differential metabolite between tea liquor and rice-flavor liquor. This study provides a theoretical basis for the development of tea liquor products and offers insight into the difference in non-volatile components between tea liquor and rice-flavor liquor.

Widely targeted metabolomics and HPLC analysis elaborated the quality formation of Yunnan pickled tea during the whole process at an industrial scale.

Yunnan pickled tea is produced from fresh tea-leaves through fixation, rolling, anaerobic fermentation and sun-drying. In this study, widely targeted metabolomics using UHPLC-QQQ-MS/MS and HPLC analysis were carried out to elaborate its quality formation during the whole process. Results confirmed the contribution of preliminary treatments and anaerobic fermentation to the quality formation. A total of 568 differential metabolites (VIP > 1.0, P < 0.05, FC > 1.50 or < 0.67) were screened through OPLS-DA. (-)-Epigallocatechin and (-)-epicatechin significantly (P < 0.05) increased from the hydrolyzation of ester catechins, such as (-)-epigallocatechin gallate and (-)-epicatechin gallate in anaerobic fermentation. Additionally, the anaerobic fermentation promoted vast accumulations of seven essential amino acids, four phenolic acids, three flavones and flavone glycosides, pelargonidin and pelargonidin glycosides, flavonoids and flavonoid glycosides (i.e. kaempferol, quercetin, taxifolin, apigenin, myricetin, luteolin and their glycosides) through relevant N-methylation, O-methylation, hydrolyzation, glycosylation and oxidation.

Multiomics analysis of the mechanisms behind flavonoid differences between purple and green tender shoots of Camellia sinensis var. assamica.

Flavonoids are rich in tea plants (Camellia sinensis), and responsible for the flavor and healthful benefits of tea beverage. The anthocyanin levels in the purple tender shoots are higher than in the general green leaves of tea plant, which provide special materials to search metabolic mechanisms of flavonoid enrichment in plant. In this work, flavonoid differences between purple and green shoots from tea cultivars "Zijuan" (ZJ) and "Yunkang10" (YK-10) were investigated through metabolomic analysis, and mechanisms for their difference were surveyed by comparative transcriptomic and proteomic analysis. Levels of 34 flavonoids were different between ZJ and YK-10 shoots. Among them, 8 and 6 were marker metabolites in ZJ and YK-10, respectively. The differentially expressed genes (DEGs), differentially expressed proteins (DEPs), and different-level metabolites (DLMs) between ZJ and YK-10 were researched, respectively; and interactions including DEG-DLM, DEP-DLM, DEG-DEP, and DEG-DEP-DLM were analyzed; the contents of 18 characteristic flavonoids in tea leaves and expressions of 34 flavonoid metabolic genes were measured to verify the omics results. Integrated above analyses, a proposed model of flavonoids biosynthesis in tea shoots were established. The differential expression of the leucoanthocyanidin reductase (LAR), anthocyanidin synthase (ANS), anthocyanidin reductase (ANR), UDPG-flavonoid glucosyltransferase (UGT) 75L12 and 94P1 at gene level, and the ANS, ANR, and UGT78A15 at protein level, were closely associated with differences in flavonoids between ZJ and YK-10 shoot. Together, this study provides new information on the flavonoid accumulation mechanism in tea plant.

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.

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.

Alternative Splicing Regulation of Anthocyanin Biosynthesis in Camellia sinensis var. assamica Unveiled by PacBio Iso-Seq.

Although the pathway and transcription factor regulation of anthocyanin biosynthesis in tea plants [Camellia sinensis (L.) O. Ktze] are known, post-transcriptional regulation mechanisms involved in anthocyanin accumulation have not been comprehensively studied. We obtained the full-length transcriptome of a purple cultivar ('Zijuan') and a normal green cultivar ('Yunkang 10#) of C. sinensis var. asssamica (Masters) showing different accumulation of anthocyanins and catechins through PacBio isoform sequencing (Iso-Seq). In total, 577,557 mapped full-length cDNAs were obtained, and 2,600 average-length gene isoforms were identified in both cultivars. After gene annotations and pathway predictions, we found that 98 key genes in anthocyanin biosynthesis pathways could have undergone alternative splicing (AS) events, and identified a total of 238 isoforms involved in anthocyanin biosynthesis. We verified expression of the C4H, CHS, FLS, CCOM, F3'5'H, LAR, PAL, CCR, CYP73A13, UDP75L12, UDP78A15/UFGT, UDP94P1, GL3, MYB113, ANR, ANS, F3H, 4CL1, CYP98A3/C3H, CHI, DFR genes and their AS transcripts using qRT-PCR. Correlation analysis of anthocyanin biosynthesis and gene expression results revealed that C4H1, FLS1, PAL2, CCR2, UDP75L122 and MYB113-1 are crucial AS transcripts for regulating anthocyanin biosynthesis in C. sinensis var. assamica Our results reveal post-transcriptional regulation of anthocyanin biosynthesis in tea plants, and provide more new insights into the regulation of secondary metabolism.