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.

Investigation of B-atp6-orfH79 distributing in Chinese populations of Oryza rufipogon and analysis of its chimeric structure.

BACKGROUND: The cytoplasmic male sterility (CMS) of rice is caused by chimeric mitochondrial DNA (mtDNA) that is maternally inherited in the majority of multicellular organisms. Wild rice (Oryza rufipogon Griff.) has been regarded as the ancestral progenitor of Asian cultivated rice (Oryza sativa L.). To investigate the distribution of original CMS source, and explore the origin of gametophytic CMS gene, a total of 427 individuals with seventeen representative populations of O. rufipogon were collected in from Dongxiang of Jiangxi Province to Sanya of Hainan Province, China, for the PCR amplification of atp6, orfH79 and B-atp6-orfH79, respectively. RESULTS: The B-atp6-orfH79 and its variants (B-atp6-GSV) were detected in five among seventeen populations (i.e. HK, GZ, PS, TL and YJ) through PCR amplification, which could be divided into three haplotypes, i.e., BH1, BH2, and BH3. The BH2 haplotype was identical to B-atp6-orfH79, while the BH1 and BH3 were the novel haplotypes of B-atp6-GSV. Combined with the high-homology sequences in GenBank, a total of eighteen haplotypes have been revealed, only with ten haplotypes in orfH79 and its variants (GSV) that belong to three species (i.e. O. rufipogon, Oryza nivara and Oryza sativa). Enough haplotypes clearly demonstrated the uniform structural characteristics of the B-atp6-orfH79 as follows: except for the conserved sequence (671 bp) composed of B-atp6 (619 bp) and the downstream followed the B-atp6 (52 bp, DS), and GSV sequence, a rich variable sequence (VS, 176 bp) lies between the DS and GSV with five insertion or deletion and more than 30 single nucleotide polymorphism. Maximum likelihood analysis showed that eighteen haplotypes formed three clades with high support rate. The hierarchical analysis of molecular variance (AMOVA) indicated the occurrence of variation among all populations (FST = 1; P < 0.001), which implied that the chimeric structure occurred independently. Three haplotypes (i.e., H1, H2 and H3) were detected by the primer of orfH79, which were identical to the GVS in B-atp6-GVS structure, respectively. All seventeen haplotypes of the orfH79, belonged to six species based on our results and the existing references. Seven existed single nucleotide polymorphism in GSV section can be translated into eleven various amino acid sequences. CONCLUSIONS: Generally, this study, indicating that orfH79 was always accompanied by the B-atp6, not only provide two original CMS sources for rice breeding, but also confirm the uniform structure of B-atp-orfH79, which contribute to revealing the origin of rice gametophytic CMS genes, and the reason about frequent recombination of mitochondrial DNA.

GC-MS and LC-MS/MS metabolomics revealed dynamic changes of volatile and non-volatile compounds during withering process of black tea.

High-performance liquid chromatography (HPLC), headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) and ultra-high performance liquid chromatography-Q-Exactive HF-X mass spectrometer (UHPLC-Q-Exactive HF/MS) were carried out to reveal dynamic changes of volatile and non-volatile compounds during the withering process of black tea. A total of 118 volatile organic compounds (VOCs) and 648 metabolites were identified in fresh and withered tea-leaves, respectively. Among them, 47 VOCs (OAV > 1.0) for the aroma formation, and 46 characteristic metabolites (VIP > 1.50, p < 0.01) selected through orthonormal partial least squares-discriminant analysis, indicated the withering contribution during black tea processing. Overall, the withering promoted alcohols, aldehydes, phenols, heterocyclic oxygen, hydrocarbons and halogenated hydrocarbons through relevant hydrolyzation, decomposition, terpene synthesis, and O-methylation. The hydrolyzation, O-methylation, condensation and N-acylation of kaempferol glycosides, quercetin glycosides, ester catechins, and gallic acid generated the accumulation of methoxyl flavonoids and flavonoid glucosides, dihydrokaempferol, syringic acid, theaflavins, and N-acylated amino acids, respectively.

Geographical origin identification of Chinese white teas, and their differences in tastes, chemical compositions and antioxidant activities among three production regions.

The producing area of Chinese white tea has been expanded to Xinyang and Yunnan from Fuding region. In this study, six sensory tastes and fifty-one chemical components including seventeen phenolic compounds, three purine alkaloids and twenty amino acids were determined in eighteen Bai mudan sub-type of white tea by electronic tongue, high performance liquid chromatography and amino acid analyzer for geographical identification, respectively. Additionally, in vitro antioxidant activities were evaluated by five various assays. Multivariate statistical analyses such as PCA, HCA and PLS-DA, completely divided these white teas into Xinyang, Yunnan and Fuding groups, indicating the feasibility of white tea classification by the production region. Twelve characteristic compounds (VIP > 1.0, P < 0.05) like gallic acid, theaflavin and L-glutamic acid contributed to the geographical identification. In conclusion, this study explored the chemical, taste and antioxidant capacity differences among three main production regions, and revealed their potential relations in white tea.

Microbial community succession in the fermentation of Qingzhuan tea at various temperatures and their correlations with the quality formation.

With the aim to reveal the microbial community succession at various temperatures in the fermentation of Qingzhuan tea (QZT), the Illumina NovaSeq sequencing was carried out to analyze bacterial and fungal community structure in tea samples collected from the fermentation set at various temperatures, i.e., 25 °C, 30 °C, 37 °C, 45 °C, 55 °C, and room temperature. The results showed that fermentation temperature profoundly affected the microbial community succession in the QZT fermentation. Microbial richness and community diversity decreased along with the increase of fermentation temperature. Despite the differences between microorganisms and their metabolic types among various temperatures, most bacteria and fungi showed positive correlations at the genera level. Klebsiella, Paenibacillus, Cohnella, and Pantoea were confirmed as the main bacterial genera, and Aspergillus and Cyberlindnera were the main fungal genera in QZT fermentation. The microbial genera (i.e. Aspergillus, Rhizomucor, Thermomyces, Ralstonia, Castellaniella, and Vibrio) were positively correlated with fermentation temperature (P < 0.05), while Klebsiella, Paenibacillus, and Aspergillus had good adaptability at different temperatures. Conversely, Pantoea and Cyberlindnera were only suitable for low temperature (≤37 °C) growth, and Thermomyces was only suitable for high temperature (>37 °C) growth. Aspergillus had a significant positive correlation with tea aroma quality (r = 0.64, p < 0.05). This study would help to understand the formation mechanism of QZT from microflora perspective.

HPLC and high-throughput sequencing revealed higher tea-leaves quality, soil fertility and microbial community diversity in ancient tea plantations: compared with modern tea plantations.

BACKGROUND: Ancient tea plantations with an age over 100 years still reserved at Mengku Town in Lincang Region of Yunan Province, China. However, the characteristic of soil chemicophysical properties and microbial ecosystem in the ancient tea plantations and their correlation with tea-leaves chemical components remained unclear. Tea-leaves chemical components including free amino acids, phenolic compounds and purine alkaloids collected from modern and ancient tea plantations in five geographic sites (i.e. Bingdao, Baqishan, Banuo, Dongguo and Jiulong) were determined by high performance liquid chromatography (HPLC), while their soil microbial community structure was analyzed by high-throughput sequencing, respectively. Additionally, soil microbial quantity and chemicophysical properties including pH, cation exchange capacity (CEC), soil organic matter (SOM), soil organic carbon (SOC), total nitrogen (TN), total phosphorus (TP), total potassium (TK), alkali-hydrolyzable nitrogen (AN), available phosphorous (AP) and available potassium (AK) were determined in modern and ancient tea plantations. RESULTS: Tea-leaves chemical components, soil chemicophysical properties and microbial community structures including bacterial and fungal community abundance and diversity evaluated by Chao 1 and Shannon varied with geographic location and tea plantation type. Ancient tea plantations were observed to possess significantly (P < 0.05) higher free amino acids, gallic acid, caffeine and epigallocatechin (EGC) in tea-leaves, as well as soil fertility. The bacterial community structure kept stable, while fungal community abundance and diversity significantly (P < 0.05) increased in ancient tea plantation because of higher soil fertility and lower pH. The long-term plantation in natural cultivation way might significantly (P < 0.05) improve the abundances of Nitrospirota, Methylomirabilota, Ascomycota and Mortierellomycota phyla. CONCLUSIONS: Due to the natural cultivation way, the ancient tea plantations still maintained relatively higher soil fertility and soil microbial ecosystem, which contributed to the sustainable development of tea-leaves with higher quality.

Impact of prolonged withering on phenolic compounds and antioxidant capability in white tea using LC-MS-based metabolomics and HPLC analysis: Comparison with green tea.

Contents of 20 bioactive compounds in 12 teas produced in Xinyang Region were determined by high performance liquid chromatography. Ultra-high performance liquid chromatography-quadrupole time of flight-mass spectrometry was developed for untargeted metabolomics analysis. Antioxidant activities were measured by 4 various assays. Those teas could be completely divided into green and white tea through principal component analysis, hierarchical cluster analysis and orthonormal partial least squares-discriminant analysis (R2Y = 0.996 and Q2 = 0.982, respectively). The prolonged withering generated 472 differentiated metabolites between white and green tea, prompted significant decreases (variable importance in the projection > 1.0, p-value < 0.05 and fold change > 1.50) of most catechins and 8 phenolic acids to form 4 theaflavins, and benefited for the accumulation of 17 flavonoids and flavonoid glycosides, 8 flavanone and their derivatives, 20 free amino acids, 12 sugars and 1 purine alkaloid. Additionally, kaempferol and taxifolin contributed to 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging ability of white tea.

Region identification of Xinyang Maojian tea using UHPLC-Q-TOF/MS-based metabolomics coupled with multivariate statistical analyses.

Xinyang Maojian tea is a kind of famous roasted green tea produced in the middle of China. Ultra-high performance liquid chromatography-quadrupole time of flight-mass spectrometry (UHPLC-Q-TOF/MS)-based metabolomics coupled with multivariate statistical analyses, including principal component analysis (PCA) and hierarchical cluster analysis (HCA), were carried out in XMMJTs collected from Luoshan, Shangcheng, and Shihe Counties, respectively. Additionally, seven catechins, four flavonoids, two purine alkaloids, and gallic acid contents were determined by HPLC. Differential metabolites were selected by p-value <0.05, and fold change >1.50 or < 0.66 among 745 detected metabolites in metabolomics analysis. The results showed significant (p < 0.05) differences of three catechins including (-)-epicatechin, (-)-epicatechin gallate, and (-)-gallocatechin gallate, four flavonoids (i.e. quercetin, kaempferol, myricetin, and rutin), and theobromine among three various regions, and significant (p < 0.05) differences of (-)-epicatechin gallate, (-)-epigallocatechin, (+)-catechin, gallic acid, and kaempferol between Shuchazao and Group cultivar. The HCA showed that, except for two samples (i.e. LS 2 and SH 2) of Shuchazao cultivar clustered together, others could be clustered completely according to production place. The 63 relevant differential metabolites could achieve the purpose of region identification through PCA. Kyoto encyclopedia of genes and genomes (KEGG) metabolic pathway analysis elaborated the impact of geographical origin and tea cultivar on physiological metabolism in tea tree. PRACTICAL APPLICATION: Ultra-high performance liquid chromatography-quadrupole time of flight-mass spectrometry (UHPLC-Q-TOF/MS)-based liquid chromatography-tendem mass spectrometry (LC-MS/MS) metabolomics coupled with multivariate statistical analyses, such as principal component analysis (PCA) and hierarchical cluster analysis (HCA), revealed 63 differential metabolites related to production place, which contributed to the region identification of Xinyang Maojian teas.

Comparison of characteristic components in tea-leaves fermented by Aspergillus pallidofulvus PT-3, Aspergillus sesamicola PT-4 and Penicillium manginii PT-5 using LC-MS metabolomics and HPLC analysis.

Microbiota influenced quality formation of ripened Pu-erh tea. To understand the effect of each tea-derived fungal strain, tea-leaves were fermented by Aspergillus pallidofulvus PT-3 (ApaPT), Aspergillus sesamicola PT-4 (AsePT) and Penicillium manginii PT-5 (PmaPT), respectively. 14 Phenolic compounds, 3 purine alkaloids, 19 free amino acids and γ-aminobutyric acid contents were determined by HPLC and amino acid analyzer analysis. Additionally, UHPLC-Q-TOF/MS method was developed for LC-MS metabolomics analysis. Multivariate statistical analyses, such as PCA and HCA, exhibited that the chemical profile of PmaPT fermentation was similar to biocidal treatment, but had significant differences with ApaPT and AsePT fermentation. The differentiated metabolites (VIP > 1, p < 0.05 and FC > 1.50 or < 0.66) and one-way ANOVA revealed the impact of three fungal strains in tea-leaves fermentation. APaPT and AsePT contributed to biosynthesis of gallic acid and several flavonoids, such as kaempferol, quercetin and myricetin in the metabolism of phenolic compounds.

3-Methylxanthine production through biodegradation of theobromine by Aspergillus sydowii PT-2.

BACKGROUND: Methylxanthines, including caffeine, theobromine and theophylline, are natural and synthetic compounds in tea, which could be metabolized by certain kinds of bacteria and fungi. Previous studies confirmed that several microbial isolates from Pu-erh tea could degrade and convert caffeine and theophylline. We speculated that these candidate isolates also could degrade and convert theobromine through N-demethylation and oxidation. In this study, seven tea-derived fungal strains were inoculated into various theobromine agar medias and theobromine liquid mediums to assess their capacity in theobromine utilization. Related metabolites with theobromine degradation were detected by using HPLC in the liquid culture to investigate their potential application in the production of 3-methylxanthine. RESULTS: Based on theobromine utilization capacity, Aspergillus niger PT-1, Aspergillus sydowii PT-2, Aspergillus ustus PT-6 and Aspergillus tamarii PT-7 have demonstrated the potential for theobromine biodegradation. Particularly, A. sydowii PT-2 and A. tamarii PT-7 could degrade theobromine significantly (p < 0.05) in all given liquid mediums. 3,7-Dimethyluric acid, 3-methylxanthine, 7-methylxanthine, 3-methyluric acid, xanthine, and uric acid were detected in A. sydowii PT-2 and A. tamarii PT-7 culture, respectively, which confirmed the existence of N-demethylation and oxidation in theobromine catabolism. 3-Methylxanthine was common and main demethylated metabolite of theobromine in the liquid culture. 3-Methylxanthine in A. sydowii PT-2 culture showed a linear relation with initial theobromine concentrations that 177.12 ± 14.06 mg/L 3-methylxanthine was accumulated in TLM-S with 300 mg/L theobromine. Additionally, pH at 5 and metal ion of Fe2+ promoted 3-methylxanthine production significantly (p < 0.05). CONCLUSIONS: This study is the first to confirm that A. sydowii PT-2 and A. tamarii PT-7 degrade theobromine through N-demethylation and oxidation, respectively. A. sydowii PT-2 showed the potential application in 3-methylxanthine production with theobromine as feedstock through the N-demethylation at N-7 position.

Correlation analysis between filamentous fungi and chemical compositions in a pu-erh type tea after a long-term storage.

Storage environment caused the difference between Jinhua Pu-erh tea (JPT) and General Pu-erh tea. In this study, fungal flora and chemical compositions were analyzed. The results showed that storage environment caused significant (p < .05) differences of theaflavins (TF), theabrownins (TB), tea polyphenols (TP), and water-soluble sugars (WSS), and a highly significant (p < .01) difference of thearubigins (TR). Aspergillus niger, Aspergillus pallidofulvus, Aspergillus sesamicola, Penicillium manginii, and Aspergillus tamarii were isolated from Pu-erh teas and identified based on colony characteristics and ITS, ß-tubulin, and calmodulin gene sequences, respectively. A. pallidofulvus, A. sesamicola, and P. manginii were dominant fungi in JPT and generated macroscopic yellow cleistothecia after a long-term storage. Correlation analysis showed that dominant fungi exhibited significantly (p < .05 or p < .01) positive or negative corrections with TF, TB, TP, WSS, TR, and gallic acid. This study revealed dominant fungi including A. pallidofulvus, A. sesamicola, and P. manginii and their effects on given chemical compositions.

Isolation, characterization and application of theophylline-degrading Aspergillus fungi.

BACKGROUND: Caffeine, theobromine and theophylline are main purine alkaloid in tea. Theophylline is the downstream metabolite and it remains at a very low level in Camellia sinensis. In our previous study, Aspergillus sydowii could convert caffeine into theophylline in solid-state fermentation of pu-erh tea through N-demethylation. In this study, tea-derived fungi caused theophylline degradation in the solid-state fermentation. The purpose of this study is identify and isolate theophylline-degrading fungi and investigate their application in production of methylxanthines with theophylline as feedstock through microbial conversion. RESULTS: Seven tea-derived fungi were collected and identified by ITS, ß-tubulin and calmodulin gene sequences, Aspergillus ustus, Aspergillus tamarii, Aspergillus niger and A. sydowii associated with solid-state fermentation of pu-erh tea have shown ability to degrade theophylline in liquid culture. Particularly, A. ustus and A. tamarii could degrade theophylline highly significantly (p < 0.01). 1,3-dimethyluric acid, 3-methylxanthine, 3-methyluric acid, xanthine and uric acid were detected consecutively by HPLC in A. ustus and A. tamarii, respectively. The data from absolute quantification analysis suggested that 3-methylxanthine and xanthine were the main degraded metabolites in A. ustus and A. tamarii, respectively. 129.48 ± 5.81 mg/L of 3-methylxanthine and 159.11 ± 10.8 mg/L of xanthine were produced by A. ustus and A. tamarii in 300 mg/L of theophylline liquid medium, respectively. CONCLUSIONS: For the first time, we confirmed that isolated A. ustus, A. tamarii degrade theophylline through N-demethylation and oxidation. We were able to biologically produce 3-methylxanthine and xanthine efficiently from theophylline through a new microbial synthesis platform with A. ustus and A. tamarii as appropriate starter strains.

LC-MS/MS-based metabolomic analysis of caffeine-degrading fungus Aspergillus sydowii during tea fermentation.

Natural microorganisms involved in solid-state fermentation (SSF) of Pu-erh tea have a significant impact on its chemical components. Aspergillus sydowii is a fungus with a high caffeine-degrading capacity. In this work, A. sydowii was inoculated into sun-dried green tea leaves for SSF. Metabolomic analysis was carried out by using UPLC-QTOF-MS method, and caffeine and related demethylated products were determined by HPLC. The results showed that A. sydowii had a significant (P < 0.05) impact on amino acids, carbohydrates, flavonoids, and caffeine metabolism. Moreover, A. sydowii could promote the production of ketoprofen, baclofen, and tolbutamide. Along with caffeine degradation, theophylline, 3-methylxanthine, 1,7-dimethylxanthine, 1-methylxanthine, and 7-methylxanthine were increased significantly (P < 0.05) during inoculated fermentation, which showed that demethylation was the main pathway of caffeine degradation in A. sydowii secondary metabolism. The absolute quantification analysis showed that caffeine could be demethylated and converted to theophylline and 3-methylxanthine. Particularly, about 93.24% of degraded caffeine was converted to theophylline, 27.92 mg/g of theophylline was produced after fermentation. PRACTICAL APPLICATION: Aspergillus sydowii could cause caffeine degradation in Pu-erh tea solid-state fermentation and produce theophylline through the demethylation route. Using a starter strain to ferment tea leaves offers a more controllable, reproducible, and highly productive alternative for the biosynthesis of theophylline.

Production of theophylline via aerobic fermentation of pu-erh tea using tea-derived fungi.

BACKGROUND: Caffeine is one of the most abundant methylxanthines in tea, and it remains stable in processing of general teas. In the secondary metabolism of microorganism, theophylline is the main conversion product in caffeine catabolism through demethylation. Microorganisms, involved in the solid-state fermentation of pu-erh tea, have a certain impact on caffeine level. Inoculating an appropriate starter strain that is able to convert caffeine to theophylline would be an alternative way to obtain theophylline in tea. The purpose of this study was to isolate and identify the effective strain converting caffeine to theophylline in pu-erh tea, and discuss the optimal conditions for theophylline production. RESULTS: Caffeine content was decreased significantly (p < 0.05) and theophylline content was increased significantly (p < 0.05) during the aerobic fermentation of pu-erh tea. Five dominant fungi were isolated from the aerobic fermentation and identified as Aspergillus niger, Aspergillus sydowii, Aspergillus pallidofulvus, Aspergillus sesamicola and Penicillium mangini, respectively. Especially, A. pallidofulvus, A. sesamicola and P. mangini were detected in pu-erh tea for the first time. All isolates except A. sydowii TET-2, enhanced caffeine content and had no significant influence on theophylline content. In the aerobic fermentation of A. sydowii TET-2, 28.8 mg/g of caffeine was degraded, 93.18% of degraded caffeine was converted to theophylline, and 24.60 mg/g of theophylline was produced. A. sydowii PET-2 could convert caffeine to theophylline significantly, and had application potential in the production of theophylline. The optimum conditions of theophylline production in the aerobic fermentation were 1) initial moisture content of 35% (w/w), 2) inoculation quantity of 8%, and 3) incubation temperature at 35 °C. CONCLUSIONS: For the first time, we find that A. sydowii PET-2 could convert caffeine to theophylline, and has the potential value in theophylline production through aerobic fermentation.

Biodegradation of caffeine by whole cells of tea-derived fungi Aspergillus sydowii, Aspergillus niger and optimization for caffeine degradation.

BACKGROUND: Pu-erh tea is a traditional Chinese tea and produced by natural solid-state fermentation. Several studies show that the natural microbiota influence caffeine level in pu-erh tea. Our previous research also found that the caffeine declined significantly (p < 0.05) in the fermentation, which suggested that the caffeine level could be influenced by specific strains. The purpose of this study was to isolate and identify microorganisms for caffeine degradation, and this research explored the degradation products from caffeine and optimal condition for caffeine degradation. RESULTS: 11 Fungi were isolated from pu-erh tea fermentation and 7 strains could survive in caffeine solid medium. Two superior strains were identified as Aspergillus niger NCBT110A and Aspergillus sydowii NRRL250 by molecular identification. In the substrate tests with caffeine, A. niger NCBT110A could use caffeine as a potential carbon source while glucose is absent, A. sydowii NRRL250 could degrade 600 mg/L caffeine completely in a liquid medium. During the degradation product analysis of A. sydowii NRRL250, theophylline and 3-methlxanthine were detected, and the level of theophylline and 3-methlxanthine increased significantly (p < 0.05) with the degradation of caffeine. The single factor analysis showed that the optimum conditions of caffeine degradation were 1) substrate concentration of 1200 mg/L, 2) reaction temperature at 30 °C, and 3) pH of 6. In the submerged fermentation of tea infusion by A. sydowii NRRL250, 985.1 mg/L of caffeine was degraded, and 501.2 mg/L of theophylline was produced. CONCLUSIONS: Results from this research indicate that Aspergillus sydowii NRRL250 was an effective strain to degrade caffeine. And theophylline and 3-methlxanthine were the main caffeine degradation products.